ENDOGENOUS RETROVIRUS-K fERVKl ENCODES AN ALTERNATE ENVELOPE

PROTEIN

CROSS REFERENCE TO RELATED APPLIATION

This application claims priority to United States Patent Application U.S. 62/573,290, filed October 17, 2017, the entire contents of which is hereby incorporated by reference.

FIELD

[0001] The present disclosure relates generally an endogenous Retrovirus-K (ERVK) alternate envelope protein.

BACKGROUND

[0002] Conotoxins are neurotoxic peptides found in the Conus genus of marine snails used to immobilize prey ²² . Conus species are distinct in their ability to produce hundreds of different toxic peptides ²³ . Conotoxins are disulfide-rich and are usually 10-30 amino acids in length ²² . Conotoxins act as antagonists to specific voltage and ligand-gated ion channels ²² . In humans, symptoms of conotoxin exposure include poor coordination, blurred vision, speech difficulties, and nausea ²³ . Conotoxins have also been associated with episodes of delirium and psychosis ²⁴ .

[0003] The O-superfamily of conotoxins exhibits an ICK fold. Members of the O- superfamily include μ-conotoxins, which inhibit voltage-gated sodium channels, and d- conotoxins, which delay sodium channel inactivation ²⁵ . K-Conotoxins are inhibitors of voltage-gated potassium channels; ω -conotoxins inhibit N-type voltage-gated calcium channels (VGCCs) ²⁵ . N-type VGCCs are located in presynaptic nerve terminals and are involved in neurotransmitter release ²⁶ , ω -Conotoxin's selectivity for N-type VGCCs has allowed for their development as therapeutic agents. The ω -conotoxin MVIIA has been developed into a drug for relief of chronic and inflammatory pain ²⁷ .

[0004] Genes encoding an ω-conotoxin-like protein (CTXLP) have also been identified in certain viruses. Nuclear polyhedrosis viruses (NPV) have been shown to secrete a small conotoxin-like peptide ²⁸ . NPVs are insect pathogens belonging to the family baculoviridae ²⁸ . Although NPV-CTXLP's function has not been elucidated, its structure was found to have a nearly identical structure to the conserved ω-conotoxin's cysteine motif ²⁸ . SUMMARY

[0005] In one aspect there is described an isolated polypeptide that comprises or consists of: an amino acid sequence having at least about 90% identity with the amino acid sequence set forth in SEQ ID NO:1 (CSDYGINCSHSYGCCSRSCIALFC).

[0006] In one example the isolated polypeptide comprises or consists of an amino acid sequence having at least about 95% identity with the amino acid sequence set forth in SEQ ID NO: 1.

[0007] In one example the isolated polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 1.

[0008] In one aspect there is described an isolated nucleic acid molecule comprising a nucleotide sequence encoding a peptide comprising or consisting of an amino acid sequence having at least about 90% identity with the amino acid sequence set forth in SEQ ID NO: 1.

[0009] In one example the isolated nucleic acid molecule comprises or consists of a nucleotide sequence having at least about 90% identity with the nucleotide acid sequence encoding the polypeptide of SEQ ID NO: 1.

[0010] In one example the isolated nucleic acid molecule comprises a nucleotide sequence having at least about 95% identity with the nucleotide acid sequence encoding the polypeptide of SEQ ID NO: 1.

[0011] In one aspect there is described a vector comprising the nucleic acid molecule according to any one of claims 4 to 6.

[0012] In one aspect there is described a mammalian cell comprising the nucleic acid molecule of any one of claims 4 to 6.

[0013] In one example said mammalian cell is a human cell or non-human primate cell.

[0014] In one aspect there is described a host cell comprising the nucleic acid molecule of any one of claims 4 to 6.

[0015] In one example said host cell is a mammalian cell, an insect cell (such as

Drosophila melanogaster), a bacteria cell, or a fungal cell.

[0016] In one aspect there is described a method for producing the peptide according to any one of claim 1 to 3, comprising: culturing a mammalian cell according to claim 8 or 9, or a host cell of claim 10 or 1 1 , in a culture medium; and isolating the peptide from the mammalian cell of claims 8 or 9, or host cell of claim 10 or 11 , or culture medium thereof. [0017] In one aspect there is described an antibody that specifically recognizes the peptide of any one claims 1 to 3.

[0018] In one example said antibody is a monoclonal antibody or a polyclonal antibody.

[0019] In one aspect there is described a method for treating or preventing conditions or disorders associated with CTXLP in a subject, comprising: administering to a subject in need thereof a therapeutically effective amount of active agent optionally in a physiological carrier, or a pharmaceutically acceptable salt thereof, wherein the active agent blocks or inhibits the CTXLP activity and/or CTXLP associated pathology.

[0020] In one aspect there is described a method for treating or preventing conditions or disorders associated with ERVK in a subject, comprising: administering to a subject in need thereof a therapeutically effective amount of an active agent optionally in a

physiological carrier, or a pharmaceutically acceptable salt thereof, wherein the active agent blocks or inhibits CTXLP activity and/or CTXLP associated pathology.

[0021] In one example said condition or disorder is an infectious disease.

[0022] In one example said infection disease is HSV infection, HIV infection, EBV infection, HTLV infection, toxoplasma gondii infection, HSV infection, or prion disease.

[0023] In one example said condition or disorder is a neurological disease.

[0024] In one example said neurological disease is amyotrophic lateral sclerosis

(ALS), bipolar disorder, Kennedy's disease, multiple sclerosis, or schizophrenia.

[0025] In one example said condition or disorder is a cancer.

[0026] In one example said cancer is breast cancer, chronic myelogenous leukemia, colon cancer, gastric cancer, germ cell tumours, germinogenic tongue tumours,

gonadoblastomas, hepatocellular carcinoma, adenocarcinoma, epithloid carcinoma, Acute T- cell leukemia, leukemia, lymphoma, T-cell lymphoma, Burkitt's lymphoma, neuroepithelioma, melanoma, myelodysplastic syndrome, nasopharyngeal carcinoma, ovarian cancer, pancreatic cancer, prostate cancer, testicular cancer, lung cancer, stomach cancer, skin cancer, trophoblastic tumours, tumorigenesis (e.g., via AR interaction), thyroid adenoma, or ERVK in cancerous tissues.

[0027] In one example said associated pathology is a change in CNS function of said subject, a developmental disorder, a stroke, Alzheimer's disease, spinal cord injury, cerebral ischemia, Huntington's disease, Parkinson's disease, a peripheral neuropathy, or epilepsy ocular disease. [0028] In one example said active agent a small molecule, an antibody, a nucleic acid, an aptamer, or a peptide.

[0029] In one example said active agent comprises a Michael acceptor electrophile (MAE).

[0030] In one example said active agent comprises gambogic acid.

[0031] In one example said active agent comprises celastrol.

[0032] In one example said active agent is a small molecule inhibitor of HIV Tat, for example a Michael acceptor electrophile (MAE) such as curcumin, rosmarinic acid, gambogic acid, celastrol (15-deoxy-A(12, 14)-prostaglandin J(2) (15d-PGJ(2)), cyclopentenone prostaglandins (CyPG), such as 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)), N- acetylcysteine amide (NACA), or D-penicillamine (also called Cuprimine); a sulfhydryl compound with chelating properties such as N-(2-Mercapto-propionyl)-glycin (MPG), 2,3- Dimercapto-propanol (DMP), 2,3-Dimercapto-propane-sulfonic acid (DMPS), Nitric oxide (NO), or sulphated polysaccharides; or a Thioredoxin reductase 1 (TRR1) inhibitor, such as B5 (curcumin analog).

[0033] In one example said active agent is a small molecule or antibody reversing

CTXLP blockade on oligodendrocyte precursor cell maturation and oligodendrocyte myelination, such as clemastine fumarate.

[0034] In one example further comprising administering a human anti-Nogo-A antibody.

[0035] In one example said active agent is a small molecule enhancer of CaV2.2 and its calcium channel associated transcription regulator (CaV2.2 CCAT) expression or activity, such as EGTA, or glutamate.

[0036] In one aspect there is described a use of a therapeutically effective amount of active agent optionally in a physiological carrier, or a pharmaceutically acceptable salt thereof, wherein the active agent blocks or inhibits the CTXLP activity and/or CTXLP associated pathology for treating or preventing conditions or disorders associated with CTXLP in a subject.

[0037] In one aspect there is described a use of a therapeutically effective amount of active agent optionally in a physiological carrier, or a pharmaceutically acceptable salt thereof, wherein the active agent blocks or inhibits the CTXLP activity and/or CTXLP associated pathology in the manufacture of a medicament for treating or preventing conditions or disorders associated with CTXLP in a subject. [0038] In one aspect there is described a use of a therapeutically effective amount of an active agent optionally in a physiological carrier, or a pharmaceutically acceptable salt thereof, wherein the active agent blocks or inhibits CTXLP activity and/or CTXLP associated pathology for treating or preventing conditions or disorders associated with ERVK in a subject.

[0039] In one aspect there is described a use of a therapeutically effective amount of an active agent optionally in a physiological carrier, or a pharmaceutically acceptable salt thereof, wherein the active agent blocks or inhibits CTXLP activity and/or CTXLP associated pathology in the manufacture of a medicament for treating or preventing conditions or disorders associated with ERVK in a subject.

[0040] In one example said condition or disorder is an infectious disease.

[0041] In one example said infection disease is HSV infection, HIV infection, EBV infection, HTLV infection, toxoplasma gondii infection, HSV infection, or prion disease.

[0042] In one example said condition or disorder is a neurological disease.

[0043] In one example said neurological disease is amyotrophic lateral sclerosis

(ALS), bipolar disorder, Kennedy's disease, multiple sclerosis, or schizophrenia.

[0044] In one example said condition or disorder is a cancer.

[0045] In one example said cancer is breast cancer, chronic myelogenous leukemia, colon cancer, gastric cancer, germ cell tumours, germinogenic tongue tumours,

gonadoblastomas, hepatocellular carcinoma, adenocarcinoma, epithloid carcinoma, Acute T- cell leukemia, leukemia, lymphoma, T-cell lymphoma, Burkitt's lymphoma, neuroepithelioma, melanoma, myelodysplastic syndrome, nasopharyngeal carcinoma, ovarian cancer, pancreatic cancer, prostate cancer, testicular cancer, lung cancer, stomach cancer, skin cancer, trophoblastic tumours, tumorigenesis (e.g., via AR interaction), thyroid adenoma, or ERVK in cancerous tissues.

[0046] In one example said associated pathology is a change in CNS function of said subject, a developmental disorder, a stroke, Alzheimer's disease, spinal cord injury, cerebral ischemia, Huntington's disease, Parkinson's disease, a peripheral neuropathy, or epilepsy ocular disease

[0047] In one example said active agent a small molecule, an antibody, a nucleic acid, an aptamer, or a peptide.

[0048] In one example said active agent comprises a Michael acceptor electrophile

(MAE). [0049] In one example said active agent comprises gambogic acid.

[0050] In one example said active agent comprises celastrol.

[0051] In one example said active agent is a small molecule inhibitor of HIV Tat, for example a Michael acceptor electrophile (MAE) such as curcumin, rosmarinic acid, gambogic acid, celastrol (15-deoxy-A(12, 14)-prostaglandin J(2) (15d-PGJ(2)), cyclopentenone prostaglandins (CyPG), such as 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)), N- acetylcysteine amide (NACA), or D-penicillamine (also called Cuprimine); a sulfhydryl compound with chelating properties such as N-(2-Mercapto-propionyl)-glycin (MPG), 2,3- Dimercapto-propanol (DMP), 2,3-Dimercapto-propane-sulfonic acid (DMPS), Nitric oxide (NO), or sulphated polysaccharides; or a Thioredoxin reductase 1 (TRR1) inhibitor, such as B5 (curcumin analog).

[0052] In one example said active agent is a small molecule or antibody reversing

CTXLP blockade on oligodendrocyte precursor cell maturation and oligodendrocyte myelination, such as clemastine fumarate.

[0053] In one example further comprising the use of a human anti-Nogo-A antibody.

[0054] In one example said active agent is a small molecule enhancer of CaV2.2 and its calcium channel associated transcription regulator (CaV2.2 CCAT) expression or activity, such as EGTA, or glutamate.

[0055] In one aspect there is described a method for transcriptional activation, comprising contacting a DNA molecule comprising a gene with a peptide of any one of claims 1 to 3.

[0056] In one aspect there is described a diagnostic reagent for use in the detection of CTXLP protein in a subject, comprising an antibody of claims 1 1 or 12.

[0057] In one aspect there is described a diagnostic reagent for use in the detection of CTXLP mRNA in a subject, comprising an isolated nucleic acid according to any one of claims 4 to 6.

[0058] In one aspect there is described a diagnostic reagent for use in the detection

CTXLP activity in a subject, comprising a peptide of any one of claims 1 to 3.

[0059] In one aspect there is described a method for treating or preventing conditions or disorders associated with CTXLP in a subject, comprising: measuring an amount of CTXLP polypeptide, or CTXLP activity, or CTXLP mRNA; and administering to a subject in need thereof a therapeutically effective amount of an active agent optionally in a

physiological carrier or a pharmaceutically acceptable salt thereof when the amount of CTXLP polypeptide, or CTXLP activity, or CTXLP mRNA, is high, optionally compared to a control, wherein the active agent blocks or inhibits the CTXLP activity and/or CTXLP associated pathology.

[0060] In one aspect there is described a method for treating or preventing conditions or disorders associated with ERVK in a subject, comprising: measuring an amount of CTXLP polypeptide, or CTXLP activity, or CTXLP mRNA; and administering to a subject in need thereof a therapeutically effective amount of an active agent optionally in a

physiological carrier or a pharmaceutically acceptable salt thereof when the amount of CTXLP polypeptide, or CTXLP activity, or CTXLP mRNA, is high, optionally compared to a control, wherein the active agent blocks or inhibits the CTXLP activity and/or CTXLP associated pathology.

[0061] In one example said condition or disorder is an infectious disease.

[0062] In one example said infection disease is HSV infection, HIV infection, EBV infection, HTLV infection, toxoplasma gondii infection, HSV infection, or prion disease.

[0063] In one example said condition or disorder is a neurological disease.

[0064] In one example said neurological disease is amyotrophic lateral sclerosis, bipolar disorder, Kennedy's disease, multiple sclerosis, or schizophrenia.

[0065] In one example said condition or disorder is a cancer.

[0066] In one example said cancer is breast cancer, chronic myelogenous leukemia, colon cancer, gastric cancer, germ cell tumours, germinogenic tongue tumours,

gonadoblastomas, hepatocellular carcinoma, adenocarcinoma, epithloid carcinoma, Acute T- cell leukemia, leukemia, lymphoma, T-cell lymphoma, Burkitt's lymphoma, neuroepithelioma, melanoma, myelodysplastic syndrome, nasopharyngeal carcinoma, ovarian cancer, pancreatic cancer, prostate cancer, testicular cancer, lung cancer, stomach cancer, skin cancer, trophoblastic tumours, tumorigenesis (e.g., via AR interaction), thyroid adenoma, or ERVK in cancerous tissues.

[0067] In one example said associated pathology is a change in CNS function of said subject, a developmental disorder, a stroke, Alzheimer's disease, spinal cord injury, cerebral ischemia, Huntington's disease, Parkinson's disease, a peripheral neuropathy, or epilepsy ocular disease

[0068] In one example said active agent a small molecule, an antibody, a nucleic acid, an aptamer, or a peptide. [0069] In one example said active agent comprises a Michael acceptor electrophile (MAE).

[0070] In one example said active agent comprises gambogic acid.

[0071] In one example said active agent comprises celastrol.

[0072] In one example said active agent is a small molecule inhibitor of HIV Tat, for example a Michael acceptor electrophile (MAE) such as curcumin, rosmarinic acid, gambogic acid, celastrol (15-deoxy-A(12, 14)-prostaglandin J(2) (15d-PGJ(2)), cyclopentenone prostaglandins (CyPG), such as 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)), N- acetylcysteine amide (NACA), or D-penicillamine (also called Cuprimine); a sulfhydryl compound with chelating properties such as N-(2-Mercapto-propionyl)-glycin (MPG), 2,3- Dimercapto-propanol (DMP), 2,3-Dimercapto-propane-sulfonic acid (DMPS), Nitric oxide (NO), or sulphated polysaccharides; or a Thioredoxin reductase 1 (TRR1) inhibitor, such as B5 (curcumin analog).

[0073] In one example said active agent is a small molecule or antibody reversing

CTXLP blockade on oligodendrocyte precursor cell maturation and oligodendrocyte myelination, such as clemastine fumarate.

[0074] In one example, further comprising administering a human anti-Nogo-A antibody.

[0075] In one example said active agent is a small molecule enhancer of CaV2.2 and its calcium channel associated transcription regulator (CaV2.2 CCAT) expression or activity, such as EGTA, or glutamate.

[0076] In one example the amount of CTXLP polypeptide is determined using an antibody of claim 13 or 14.

[0077] In one aspect there is described a kit comprising:(a) a container comprising a pharmaceutical composition containing the peptide of any one of claims 1 to 3, and/or a nucleic acid according to any one of claim 4 to 6, and/or a vector of claim 7, a mammalian cell of claims 8 or 9, a host cell of claims 10 or 11 , and/or an antibody of claim 13 or 14, in solution or in lyophilized form;(b) optionally, a second container containing a diluent or reconstituting solution for the lyophilized formulation; and(c) optionally, instructions for use.

[0078] In one example further comprising one or more of (iii) a buffer, (iv) a diluent,

(v) a filter, (vi) a needle, or (v) a syringe.

[0079] In one aspect there is described a method identifying CTXLP inhibitors, comprising: contacting a mammalian cell or a host cell, such as an insect cell (such as Drosophila melanogaster), a bacteria cell, or a fungal cell, with a test compound or test composition, and measuring an amount of CTXLP protein, CTXLP-mRNA, CTXLP- regulated gene, or CTXLP-associate biomarker.

[0080] In one aspect there is described a method identifying CTXLP inhibitors, comprising: contacting a organoid, with a test compound or test composition, and measuring an amount of CTXLP protein, CTXLP-mRNA, CTXLP- regulated gene, or CTXLP-associate biomarker

BRIEF DESCRIPTION OF THE DRAWINGS

[0081] Embodiments of the present disclosure will now be described, by way of example only, with reference to the attached Figures.

[0082] Figure 1 depicts two types of ERVK genomes. The ERVK genome consists of four main Retroviridae genes, which are from 5' to 3': gag, pro, pol, and env. These viral genes are flanked by long terminal repeats (LTRs) containing U3, R and U5 regions. Two types of ERVK genomes can be distinguished based on a 292 bp deletion in the env gene.

[0083] Figure 2 depicts open reading frames on both strands of the endogenous retrovirus K-113 genome. ORFs (yellow) on both the sense and antisense strands were predicted using CLCbio software. Any amino acid-encoding codon was accepted as an ORF start, although each ended with a stop codon. Note the overlapping ORFs within known ERVK genes, such as gag, protease, polymerase and envelope.

[0084] Figure 3 depicts alignment of cone snail and viral omega conotoxin domain sequences. Sequences from 3 Conus species (black), 1 conotoxin-like protein domain sequence from Autographa Californica Nuclear Polyhedrosis Virus (blue), the consensus sequence generated from the aforementioned sequences (red) and the sequence of the putative Endogenous Retrovirus K-113 conotoxin-like protein domain. Modified from ²⁸ . Note the characteristic C-C-CC-C-C knottin folding motif ²⁹

[0085] Figure 4 depicts an alignment and sequence logo of the putative endogenous retrovirus K-113 conotoxin-like protein domain and 10 Nuclear Polyhedrosis Virus conotoxin- like protein domain sequences. Sequences were aligned and sequence logo was assessed using Geneious v5 software ³⁰ . Note the conserved C-G-NC-Y-CCS-C-A-FC sequence logo in these viral conotoxin-like proteins. [0086] Figure 5 depicts an alignment of the ERVK CTXLP cysteine-rich motif which has strong similarity to both nuclear polyhedrosis virus (NPV, 46.2%) and Conus (45.8%) conotoxin proteins.

[0087] Figure 6 depicts the amino acid logo of the knottin domain from cluster representative CTXLP sequences.

[0088] Figure 7 depicts modeled 3-dimensional structure of the putative Endogenous

Retrovirus-K113 conotoxin-like protein domain. Protein tertiary structure was predicted using Knotter1 D3D software (gray sticks = carbon, green sticks = hydrogen, red sticks = oxygen, blue sticks = nitrogen and yellow spheres = sulfur). Note the interactions of the yellow cysteine residues, as they form disulfide bonds.

[0089] Figure 8 depicts aligned overlap of the predicted structures of viral conotoxin- like proteins from ERVK-113 and Ecotropis obliqua NPV. Knotter1 D3D was used to predict the structures of putative ERVK-113 CTXLP domain (blue) and Ecotropis obliqua NPV CTXLP domain (red). Structure alignment is based on sequence alignment and was prepared using UCSF Chimera software31.

[0090] Figure 9 depicts an aligned overlap of the predicted structures of viral conotoxin-like protein backbones from ERVK-1 13 and Ecotropis obliqua NPV. Knotter1 D3D was used to predict the structures of putative ERVK-1 13 CTXLP domain (blue) and Ecotropis obliqua NPV CTXLP domain (red). Structure alignment is based on sequence alignment and was prepared using UCSF Chimera software ³¹ .

[0091] Figure 10 depicts a predicted inhibitor cysteine knot fold of ERVK CTXLP cysteine-rich peptide. Disulfide bonds connect cysteine 1 to cysteine 4, cysteine 2 to cysteine 5, and cysteine 3 to cysteine 6, resulting in an inhibitor cysteine knot fold.

[0092] Figure 11 depicts an alignment and sequence logo of ERVK CTXLP cysteine- rich peptide and 12 spider toxin ICK peptides. Sequences were aligned and sequence logo generated using Geneious Software. A conserved C-C-CC-C-C motif is observed in all sequences. CTXLP, all Hainantoxin and one Guanxitoxin contained a conserved G between the first and second cysteine, as indicated by the star. Grey callouts indicate the cysteine motif spacing of each toxin.

[0093] Figure 12 depicts alignment and sequence logo of ERVK CTXLP cysteine-rich peptide and agouti-related peptide and agouti signalling protein. Sequences were aligned and sequence logo generated using Geneious Software. A conserved C-C^CC-C-C-C is observed in all sequences. [0094] Figure 13 depicts alignment and sequence logo of ERVK CTXLP cysteine-rich peptide to 7 VEGF Proteins. Sequences were aligned and sequence logo generated using Geneious Software. No significant conservation was identified between the VEGF proteins and CTXLP, due to the differences in spacing and total number of cysteine residues, as indicated by the grey bar cysteine spacing motif.

[0095] Figure 14 depicts alignment and sequence logo of ERVK CTXLP cysteine-rich peptide and HIV-1 and HIV-2 Tat proteins. Sequences were aligned and sequence logo generated using Geneious Software. Conservation of 6 of the 7 CTXLP cysteine residues are found in HIV Tat, as well as 1 lysine and 1 leucine residue in the C terminus, between amino acids 75 and 80.

[0096] Figure 15 depicts example Alignment of Chromosome 1 ERVK HML-2 insertions with the DNA sequences for Rec exon 1 and CTXLP. Sequences were aligned and sequence logo generated using Geneious software. Rec exon 1 aligned with bp 1 to 261 of env. CTXLP aligned with bp 1413 to 1505 of env.

[0097] Figure 16 depicts alignment of conotoxin-like peptides of 25 human ERVK

HML-2 insertions. Alignment and sequence logo generated using Geneious Software.

CTXLP-peptides showed variability in the amino acid sequence. Three distinct alleles were identified, as well as several unique sequences.

[0098] Figure 17 depicts CTXLP variants in the humans, based on genome build

GRCh38.

[0099] Figure 18 depicts schematic representation of CTXLP and amino acid sequence similarities found using NCBI-CDD and Pfam databases. The SU subunit of CTXLP is red and the omega conotoxin domain is in green. The wider portions of the diagram represent the ordered regions of the proteins and the narrow region represent the disordered region as predicted by ELM resource56.

[00100] Figure 19 depicts analysis of the ERVK envelope transcript reveals prototypic RNA secondary structures. Shown is the predicted IRES-like RNA hairpin structures in the ERVK env transcript. The first 350 bp of ERVK Env-encoding RNA contains numerous AUG (methionine) translational start sites. Two distinct IRES-like hairpins are identified at nucleotides 84-187 and 213-318. tRNA can potentially bind at the AUG start site identified in IRES-like hairpin to produce a smaller isoform of ERVK Env or CTXLP. Alos shown is the predicted RNA secondary structure for ERVK-4 env transcript upstream and including the CTXLP ORF. Directly upstream of the CTXLP ORF translational start is a conserved -1 programmed ribosomal frameshifting sequence, which contains three elements i) a slippery site containing an X-XXY-YYZ motif which after frameshifting by - 1 results in XXX- YYY reading, ii) a 5 to 10 nucleotide spacer sequence, and iii) a downstream hairpin-type pseudoknot. The ERVK env transcript slippery site is encoded by a U-UUA-AAU sequence, followed by a 5 nucleotide spacer. Structure prediction formed with RNAfold Software shows a strong probability of hairpin-loops forming within the CTXLP-coding region, likely providing a downstream hairpin-type pseudoknot.

[00101] Figure 20 depicts the predicted CTXLP isoforms derived from the ERVK envelope transcript. Ribosomal frameshifting event resulting in formation of CTXLP peptide fused to surface unit protein. At the RNA slippery site within the ERVK Env transcript, the ribosome translating the RNA may bounce back by 4 nucleotides and begin reading in an alternate frame. This introduces a canonical KRQK nuclear localization sequence before proceeding into the CTXLP peptide. The resulting protein is a modified SU-CTXLP fusion protein. We show that ERVK can also produce SU-CTXLP fusion proteins. These CTXLP isoforms contain a nuclear localization sequence (NLS) and an additional N-linked

glycosylation site at position 480.

[00102] Figure 21 depicts bioinformatic identification of CTXLP in the genome of endogenous retrovirus-K. ERVK113 was used as a template for the CTXLP domain in the ERVK envelope gene. The ERVK envelope polyprotein is cleaved by the cellular protease furin downstream of the R-X-R/K-R site. This splits the ERVK Env polyprotein into the surface unit (SU) and transmembrane (TM) proteins which interact to form the viral spike protein on the surface of virions. A -1 programmed ribosomal frameshift (-1 PRF) allows for the translation of the CTXLP cysteine-rich motif at the C-terminal end of the SU protein. Post- translational modification of ERVK SU protein includes glycosylation. N-linked N-X-S/T glycosylation sites are identified in red boxes.

[00103] Figure 22 depicts predicted post-translational modifications and protein interactions of CTXLP. (A) Schematic diagram of predicted glycosylation sites. (B)Schematic diagram of predicted phosphorylation and SUMOylation sites. (C) Schematic diagram of predicted protein cleavage sites. (D) Schematic diagram of predicted protein interaction sites.

[00104] Figure 23 depicts antigenic profile of the ERVK CTXLP domain, and predicted epitopes.

[00105] Figure 24 depicts rabbit immunization protocol for generation of a polyclonal antibody against the ERVK CTXLP domain. [00106] Figure 25 depicts Western blot of ERVK-expressing NCCIT whole cell extract and immunoprecipitated CTXLP-enriched fraction. The far-right lane in the image is an image of the left a-CTXLP lane that was over-exposed to bring out the details in the bands.

[00107] Figure 26 depicts PNGase treatment of IP-purified CTXLP protein results in a decrease in western blot band size associated with removal of N-linked glycosylation moieties.

[00108] Figure 27 depicts ERVK Env, and CTXLP expression in SVGA cells treated with 0.1 ng/mL TNFa, 1 ng/mL TNFa, and 1 ng/mL LIGHT. NCCIT cells were used as a positive control and β-actin as a loading control, n = 3.

[00109] Figure 28 depicts ERVK CTXLP is inducible in human neurons. ReNcell- derived neurons were treated with increasing doses of pro-inflammatory cytokines TNFa or LIGHT for 24 hours. CTXLP expression (90kDa form) was enhanced optimally with 1 ng/ml TNFa and 10ng/ml LIGHT treatment, n=1.

[00110] Figure 29 depicts CTXLP protein expression predominantly localizes with chromatin in NCCIT and SVGA cells. CTXLP expression was also identified in the cytosolic and nuclear fraction and soluble and insoluble whole cell lysates of NCCIT cells. Moreover, CTXLP appeared dispersed throughout NCCIT cells in confocal imaging. In contrast, SVGA cells exhibited expression of the small (32kDa) and large (90-110kDa) isoforms of CTXLP in association with the chromatin (A). This was supported by nuclear localization of CTXLP in SVGA cells as shown by confocal.

[00111] Figure 30 depicts ERVK CTXLP is inducible in astrocytes with proinflammatory cytokines. Confocal analysis of protein expression for ERVK CTXLP (red) and ERVK reverse transcriptase (RT, green) in cells treated with or without TNFa and LIGHT, n=2. Enhanced CTXLP expression precedes increases in RT expression. DAPI stain indicates nuclei (blue).

[00112] Figure 31 depicts the cellular localization of ERVK CTXLP and SU proteins in human astrocytes. Increased CTXLP protein and Env expression were identified (but not co- localized) in TNFa-treated cells compared to controls. Increased CTXLP expression indicated by arrows. Distinct puncta were identified within the 1) cytoplasm, 2) nucleus, and 3) surface membrane of the cell.

[00113] Figure 32 depicts CTXLP overexpression in soluble fractions of SVGA cells enhances the 32 kDa and 90 kDa forms of endogenous CTXLP. SVGA cells were transfected using Lipofectamine LTX with 5 μg Empty Vector, or 0.5, 2 and 5 μg CTXLP cysteine-rich construct for 48 hr.

[00114] Figure 33 depicts that CTXLP binds interferon response elements (ISREs) within the ERVK promoter (5' LTR). CTXLP may regulate ERVK gene expression, as well as other genes containing ISREs. Chromatin immunoprecipitation (ChIP) following 8 hours of 10 ng/ml TNFa or LIGHT treatment in human ReNcell-derived neurons (n=2) and human astrocytic cell line (SVGA) (n=3). Notable increase in CTXLP chromatin binding in neurons upon pro-inflammatory stimulation with the cytokine TNFa.

[00115] Figure 34 depicts ERVK Expression Multidimensional Scaling. Two dimensional plots were produced by reduction of the high dimensional RNA-seq expression data derived from the Sequence Read Archive (SRA) using the R package EdgeR. The plot labels correspond to SRA studies as follows: ALS (SRP064478), Bipolar Disorder

(SRP074904), Breast Cancer (SRP058722), HIV/HCV (SRP068424), Multiple Sclerosis (SRP1 10016), Prostate Cancer (ERP000550), Rheumatoid Arthritis (SRP102685), and Schizophrenia (SRP090259). In these plots each axis represents the leading log-2-scaled fold-change at one particular ERVK locus; the two loci chosen are those with the most extreme values in the majority of the clinical group's samples. Since these represent the biggest difference between samples, if no separation is apparent in these plots, there is no clear difference in transcript profiles. Each sample is represented by its SRA accession number, coloured red for disease-associated samples and black are the control samples.

[00116] Figure 35 depicts ERVK Multidimensional Scaling by CTXLP status in Human Disease. Each plot was produced as described in Figure 34, except with the modification that ERVK loci were subsetted into three states: "CTXLP+" which could produce CTXLP, "Disrupted" which cannot produce CTXLP but may have had an ancestral loci that produced CTXLP, and finally "CTXLP-" loci which do not and likely never did produce CTXLP from the ERVK env gene. In all cases, samples from the ALS, Bipolar Disorder, Breast Cancer, HIV- 1/HCV co-infection, Multiple Sclerosis and Rheumatoid Arthritis cohorts expressed all three types of ERVK env transcripts.

[00117] Figure 36 depicts Per-Locus Differential ERVK Expression. Each panel shows in detail the expression of individual ERVK loci encoding envelope in each human disease condition. ERVK loci (black) are plotted against CTXLP+ (red), CTXLP- (blue) and disrupted (grey) loci. The plot labels correspond to SRA studies as follows: ALS (ALS: SRP064478), Bipolar Disorder (BP: SRP074904), Breast Cancer (BC: SRP058722), HIV/HCV (HV; HIV/HCV + interferon (HI): SRP068424), Multiple Sclerosis (MS: SRP1 10016), Prostate Cancer (PC: ERP000550), Rheumatoid Arthritis (RA: SRP102685), and Schizophrenia (SZ: SRP090259). For each study, controls (C) and indicated to the right of cases. Panel A exclude ERVK loci with very low expression; only loci with a median expression greater than 0 and a mean expression greater than 0.1 are plotted. Panel B shows only loci which were highly expressed; only ERVK loci which had a maximum expression higher than 2 are plotted.

[00118] Figure 37 depicts ERVK CTXLP encoding transcripts and CTXLP protein are present in Amyotrophic Lateral Sclerosis (ALS). Re-analysis of RNAseq data6 in sporadic ALS and control spinal cords for expression of disrupted non-coding (black), Env+/CTXLP- (blue) and Env+/CTXLP+ (red) env transcripts. Principle component analysis (PCA) reveals ALS patient clustering in terms of CTXLP+ transcript expression, with most frequently expressed CTXLP encoding loci indicated.

[00119] Figure 38 depicts ERVK CTXLP levels are enhanced in autopsy spinal cord and brain tissues of patients with ALS, as measured by western blot analysis. Bar graph represents total CTXLP (A) and CX3CL1 (B) quantification in NN (n=9) and ALS (n=15) motor cortex specimens, as measured by western blot. Bar graph represents total CTXLP (C), CX3CL1 (D), ERVK Env SU (E) and CaV2.2 (F) quantification in NN (n=6) and ALS (n=13) cervical spinal cord specimens, as measured by western blot.

[00120]

[00121] Figure 39 depicts confocal micrographs of ERVK CTXLP levels being enhanced in autopsy spinal cord and brain tissues of patients with ALS. (A) Representative 10x confocal micrographs of ERVK CTXLP expression in ex vivo cervical spinal cord of a neuronormal control (NN, n=3) and patient with ALS (n=3). DAPI stain depicts nuclei. High magnification reveals staining in cells surrounding MAP2+ axons, suggesting CTXLP+ oligodendrocytes. CTXLP+ rings ranged from 6-16μΜ in diameter. (B) Representative 40x confocal micrographs of ERVK CTXLP, voltage-gated calcium channel CaV2.2 (C-terminal antibody) and neuronal MAP2 expression in Brodmann area 6 (BA6) motor cortex tissue of a NN control (n=3) and patient with ALS (n=3). Note the translocation of nuclear CTXLP to cytoplasmic aggregates in neurons from an ALS patient, as well as an overall decrease in CaV2.2 expression. DAPI stain depicts nuclei.

[00122] Figure 40 depicts micrographs showing that ERVK CTXLP levels are enhanced in autopsy cervical and lumbar spinal cord tissues from patients with ALS, as measured by light and confocal microscopy. Representative 10x confocal micrographs of ERVK CTXLP expression in ex vivo cervical (CC) and lumbar (LC) spinal cord of a neuronormal control (NN, n=3) and patients with ALS (n=3). Solochrome cyanine (SC) stain (purple) with eosin counterstain (pink) depicts tissue myelination; pale lesions appear in ALS tissues. These lesioned areas exhibit increased CTXLP expression is in red.

Oligodendrocyte precursor marker TCF4 is in green. DAPI stain depicts cellular nuclei. Note that CTXLP expression occurs in either lateral and/or anterior cortical spinal tracts.

[00123] Figure 41 depicts confocal micrographs of ERVK CTXLP levels are associated with demyelination and CTXLP is enhanced in TCF4+Olig1 + oligodendrocyte precursors in cervical spinal cord tissues from patients with ALS. Representative 20x confocal

micrographs of ERVK CTXLP expression in ex vivo cervical (CC) spinal cord of a

neuronormal control (NN, n=3) and a patient with ALS (n=3). Notably decreased myelin stain as measure my MAG protein expression (green) is evident in ALS tissue as compared to control. In ALS tissue, CTXLP expression (red) co-localizes with TCF4 (green) or Olig1 (green) markers indicative of oligodendrocyte precursor cells. DAPI stain depicts cellular nuclei.

[00124] Figure 42 depicts confocal micrographs showing ERVK CTXLP+

oligodendrocyte precursors express myelin inhibitory protein Nogo-A, or are in close proximity to Nogo-A positive cells in spinal cord tissues of patients with ALS. Human ex vivo cervical spinal cord tissues were stained for ERVK CTXLP (red), TCF4 (green), Nogo-A (grey) and nuclei (blue) in neuro-normal controls (n=3) and patients with ALS (n=3). Image merging for CTXLP and TCF4 indicate that oligodendrocyte precursors express CTXLP in ALS. Image merging for CTXLP and Nogo-A indicate that oligodendrocyte precursors can express myelin inhibitor protein Nogo-A (left panel) or alternately are in proximity to Nogo-A expressing cells in ALS (right panel). White stars indicate areas that are magnified to depict overlapping protein expression in CTXLP+ rings.

[00125] Figure 43 depicts cancer cells express greater levels of CTXLP as compared to non-cancer cells. Prototypic cell lines for teratocarcinoma (NCCIT) and breast cancer (T47D) were examined for CTXLP expression as compared to astrocytic SVGA cells using confocal microscopy. No antibody negative control is to show that specificity of CTXLP (red) staining requires an antibody targeting ERVK CTXLP. Nuclei are shown in blue using a DAPI stain. [00126] Figure 44 depicts CTXLP expression in G-Bioscience Ready-to-screen cancer tissue and cell line blots. TB56-I (A), TB55 (B) and TB56-II (C) blots were screen for CTXLP expression (blue bars, top blot) normalized to β-actin loading control (lower blot). Enhanced CTXLP expression in noted is several cancer types, including T cell lymphoma,

neuroepithelioma, prostate, ovary, testis and skin cancers. Figure 45 depicts changes in the gene expression of pro-inflammatory NF-κΒ p65 and anti-viral IRF7 in response to CTXLP and SU expression. 293T cells were transfected with plasmids encoding empty vector, ERVK CTXLP or ERVK SU for 24 hours. Cell pellets were collected, RNA extracted and cDNA produced for use in Q-PCR experiments to evaluate relative gene expression of RELA and IRF7. Analysis performed using AACt method and 18S RNA as a calibrator.

[00127] Figure 46 depicts confocal images of control, CTXLP-expressing and SU- expressing 293T cells. Cells were stained with DAPI nuclear stain, and antibodies against Env SU (green) fluorescent dye, NF-KB p65 (red) fluorescent dye and CTXLP (grey / white) fluorescent dye. Only CTXLP expressing cells exhibit upregulated NF-KB p65 expression. Representative micrographs of n=3 experiments.

[00128] Figure 47 depict western blot and confocal micrographs of ERVK CTXLP, but not ERVK Env SU, depleting CaV2.2 calcium channel-associated transcription regulator (CCAT). CaV2.2 expression in SVGA cells treated with 5μΙ of immunoprecipitation (IP) products from NCCIT cell lysates extracted using rabbit pre-immune serum, custom rabbit anti-CTXLP antibody or custom rabbit anti-ERVK Env SU antibody for 24 hours.

Quantification of CaV2.2 depletion in IP-product treated astrocytes (2hrs), based on confocal quantification (****p<0.0001 , 80-100 cells per condition quantified). Confocal imaging of CaV2.2 (N-terminal antibody) and CaV2.2 CCAT (C-terminal antibody) illustrates that CTXLP depletes nuclear CaV2.2 CCAT within 2 hours, but not the membrane-associated CaV2.2 channel (n=2).

[00129] Figure 48 depicts Caspase-3 expression in SVGA cells in control and CTXLP- treated conditions after 24 hours. (A) Images of live cells after 24 hours taken using EVOS imager, top image depicts untreated control cells and bottom image depicts cells treated with 5μΙ of CTXLP immunoprecipitated solution. (B) Graphical depiction of caspase-3 expression in control and CTXLP-treated cell after 24 hours. .

[00130] Figure 49 depicts CTXLP induces caspase-3 activation and apoptosis, which can be blocked by excess extracellular calcium. Cell survival 1 hour and 24 hours post treatment with 5μΙ of buffer, calcium chloride, CTXLP or CTXLP and calcium chloride. Cells were examined using an EVOS microscopy for caspase-3 activation (green) or nuclei (blue), n=2). Excess calcium chloride is known to block the cellular effects of conotoxin proteins ⁸¹ .

[00131] Figure 50 depicts cell survival and confluency 24 hours post treatment with 5μΙ of pre-immune serum, pre-immune serum and calcium chloride, CTXLP, CTXLP and calcium chloride, SU, or SU and calcium chloride. (A) Graphical depiction of caspase-3 expression in SVGA cells 24 hours post treatment. (B) Graphical depiction of cell confluency of SVGA cells 24 hours post-treatment. Note that pre-immune serum was used as a negative control as this is the component of immunoprecipitation product.

[00132] Figure 51 depicts Live cell images of SVGA cells in control, SU-treated and CTXLP-treated conditions stained for caspase-3 using EVOS live cell imaging. Cells in each condition were imaged after 5 days. Control cells were imaged at 7 days and SU and

CTXLP-treated cells were imaged after 8 days. Controls cells express greater amounts of the apoptotic marker caspase-3, and have not proliferated to the extent observed in CTXLP and Env treatments.

[00133] Figure 52 depicts percentage of control and CTXLP-transfected SVGA cells expressing caspase-3 after 24 hours. Graphical depiction of apoptosis marker caspase-3 in control and CTXLP-transfected SVGA cell after 24 hours.

[00134] Figure 53 depicts abnormal cellular morphology in cells exposed to CTXLP. (A) Fluorescent image of CTXLP-treated SVGA cell expressing caspase-3 (green) and stained with DAPI nuclear stain (blue). (B) Confocal image of Env SU expression (green) in a CTXLP-expressing 293T cell.

[00135] Figure 54 depicts CTXLP-limiting drug screen in human NCCIT

teratocarcinoma cells. The ERVK-expressing NCCIT teratocarcinoma cell line was grown in a monolayer in RMPI 1640 media supplemented with FetalGro. Cells were treated for 24 (data not shown) and 48 hours with known IC50 concentrations of drugs (NCCIT cells alone, 1 % DMSO as drug carrier control, 100μΜ Curcumin, 50μΜ Rosmarinic acid, 0.25μΜ

Gambogic acid, 0.25μΜ Celastrol, 200μΜ D-Penicillamine and 50μΜ Tetramethyl

Nordihydroguaiaretic acid/TMNGA). Cells were collected, protein extracted, and western blot performed to measure the expression of ERVK CTXLP, as compared to β-actin loading control. Results demonstrate that select MAEs can reduce CTXLP expression in NCCIT cells.

[00136] Figure 55 depicts that MAE drug Celastrol abrogates CTXLP expression in human NCCIT teratocarcinoma cells. The ERVK-expressing NCCIT teratocarcinoma cell line was grown in a monolayer in RMPI 1640 media supplemented with FetalGro. Cells were treated for 24 hours with increasing doses of celastrol (Cel, 0.1 , 0.25, 1 and 2.5μΜ). Cells were collected, protein extracted, and western blot performed to measure the expression of ERVK CTXLP, as compared to β-actin loading control. Results demonstrate that Cel dose- dependently reduces CTXLP expression in NCCIT cells.

[00137] Figure 56 depicts that MAE drug Gambogic acid abrogates CTXLP expression in human NCCIT teratocarcinoma cells. The ERVK-expressing NCCIT teratocarcinoma cell line was grown in a monolayer in RMPI 1640 media supplemented with FetalGro. Cells were treated for 24 hours with increasing doses of gambogic acid (GA, 0.1 , 0.25, 1 and 2.5μΜ). Cells were collected, protein extracted, and western blot performed to measure the expression of ERVK CTXLP, CaV2.2 C-terminal calcium channel-associated transcriptional regulator (CaV2.2 CCAT) and NOGO-A, as compared to β-actin loading control. Results demonstrate that GA dose-dependently reduces CTXLP and NOGO-A expression in NCCIT cells. CaV2.2 CCAT is inversely correlated with the expression of ERVK CTXLP in this culture system.

[00138] Figure 57 depicts that endogenous levels of CTXLP in human astrocytes can be depleted in the presence of gambogic acid. Human astrocytic cell line SVGA were treated with increasing doses of gambogic acid in the low micromolar range (0.25 and 0.5μΜ).

[00139] Figure 58 depicts that Gambogic acid blocks TN Fa-induced CTXLP expression in human neurospheres. The ReNcell CX neuroprogenitor cell line was grown in suspension to produce human neurospheres (approximately 0.5mm diameter).

Neurospheres were treated for 24 hours in neurobasal media alone, or with or without 1 ng/ml TNFa and/or 0.5μΜ gambogic acid (GA). Cells were collected, protein extracted, and western blot performed to measure the expression of soluble (standard lysis) and insoluble (RIPA lysis) proteins for ERVK CTXLP, CaV2.2 C-terminal calcium channel-associated transcriptional regulator (CaV2.2 CCAT) and NOGO-A, as compared to β-actin loading control. Results demonstrate that TNFa enhances CTXLP and NOGO-A expression in neurons, whereas in the presence of GA this effect is blocked. CaV2.2 CCAT is inversely correlated with the expression of ERVK CTXLP in this neuronal culture system.

[00140] Figure 59 depicts alignments of orthologous and non-orthologous ERVK loci with at least one Toxin_18+ ORF. Examination of CTXLP encoding loci in three non-human primate genomes, Pan troglodytes (Common chimpanzee), Gorilla gorilla gorilla (Western lowland gorilla), and Cercocebus atys (Sooty Mangabey), as well as humans reveals orthologous loci and conservation of the Toxin_18 cysteine motif (yellow). Orthology was determined by pairwise best BLAST matches of whole Retroexplorer retroelement entries and their flanking 1000bp, which mostly correspond to entire ERVs, but which sometimes were fragments. Three and four-way orthology was determined from pairwise orthology. Some loci do not have any species-specific sequence in the alignment, as the orthologous region in that species did not return a tBLASTx result. Non-orthologous sequences represent entries for which no orthologue was identified (possible paralogues or unique insertions). Representative sequences from each from each primate species highlights the degree of conservation in the Toxin_18 cysteine motif (yellow).

[00141] Figure 60 depicts different mutational patterns between orthologues and paralogues of ERVK env genes. Represented is a combined set of heatmaps generated by superheat from frames 0 (CTXLP) and 1 (Envelope) of the human and gorilla orthologues ORFs, which where both are positive for Toxin_18 positive (CTXLP). The sequences which are orthologues are indicated by black squares in the center of each space (paralogues do not have black squares). Blue is an ω (dN/dS ratio) less than 1 , indicating purifying selection and similarity between the sequences. Yellow is an ω more than 1 , indicating diversifying selection and dissimilarity between the sequences. Grey indicates that ω could not be computed (in all cases dS = 0, indicating no synonymous differences between the two sequences). Sequences which are identical along the diagonal are blacked out. The cytological bands are based on the human genome, with Gorilla designations indicating their respective human orthologue/paralogue coordinate. It is notable that orthologous sequences have a low ω when it is defined (when undefined it still has a low dN value). This suggests that there is more conservation between orthologues in different species than between paralogues from the same species. This pattern is much more apparent for CTXLP than for Env reading frame, where differences are smaller if present at all.

[00142] Figure 61 depicts that the transgene employed in the generation of ERVK envelope transgenic mice encodes CTXLP. The viral gene insert for the vector used in the generation of ERVK envelope transgenic mice8, was analysed for the potential to encode and produce ERVK CTXLP. The red annotation indicates the location of CTXLP in the transgene insert. [00143] Figure 62 depicts an alignment of ERVK113 CTXLP sequence and the ERVK Consensus sequence found in the ERVK envelope transgenic mice. Note the similarity and retention of key cysteine motif.

[00144] Figure 63 depicts the ERVK Env and CTXLP proteins. Env is composed of the SU and TM subunits and is the prototypical gene product of the env gene. CTXLP is composed of the SU subunit and a C-terminal omega conotoxin domain and is encoded in an alternate open reading frame and may be produced due to ribosomal frameshifting.

[00145] Figure 64 depicts illustration of the disruption of voltage-gated calcium channel CaV2.2 by ERVK CTXLP. Both canonical endogenous retrovirus-K (ERVK) envelope protein and conotoxin-like protein (CTXLP) can be produced from the ERVK env gene. ERVK CTXLP disrupts CaV2.2 on multiple levels, by decreasing CaV2.2 channel expression, as well as depleting the CaV2.2 calcium channel-associated transcription regulator (CCAT) in the nucleus. CTXLP inhibition of voltage gated calcium ion channel CaV2.2 may preventing neurotransmitter release and the continuation of signal transduction in the postsynaptic neurons.

[00146] Figure 65 depicts the putative pathological implications of CTXLP expression in oligodendrocyte precursor cells. CTXLP expression is enhanced in the presence of proinflammatory cytokines, including TNFa. Increased CTXLP protein levels in ex vivo human spinal cord tissue coincides with an increase in oligodendrocyte precursor cell (OPC) markers, transcription factor 4 (TCF4) and Olig1 , along with elevated neurite outgrowth inhibitor A (Nogo-A) levels in adjacent cells and decreased myelin associated glycoprotein (MAG) axonal/myelin expression, which all suggest oligodendrocyte (OL) pathology. Nogo-A is a regulator of OPC differentiation, inhibitor of OL myelination, and axonal growth cone collapse during axon regeneration in the CNS. MAG is a marker for differentiated OLs and highly expressed in myelinating OLs. Based on expression patterns of OPC and OL markers, this suggests that heightened CTXLP expression in ALS is associated with OPCs being arrested in an immature state and inhibition of OL myelination. Treatment with CTXLP inhibitors, including gambogic acid, may reduce inflammatory signalling and decrease OPC and OL pathology by inhibiting increased Nogo-A expression.

DETAILED DESCRIPTION

[00147] In one aspect, there is described herein the identification of a region in the ERVK provirus DNA which encodes a conotoxin-like polypeptide, and which may have significance in ERVK pathogenesis. In a specific example, the polypeptide is CTXLP

(CSDYGINCSHSYGCCSRSCIALFC) (SEQ ID NO: 1).

[00148] In one example, there is described an isolated polypeptide that comprises or consists of: an amino acid sequence having at least about 70% identity with the amino acid sequence set forth in SEQ ID NO: 1. In another example, the isolated polypeptide comprises or consists of an amino acid sequence having at least about 75% identity with the amino acid sequence set forth in SEQ ID NO: 1. In another example, the isolated polypeptide comprises or consists of an amino acid sequence having at least about 80% identity with the amino acid sequence set forth in SEQ ID NO: 1. In another example, the isolated polypeptide comprises or consists of an amino acid sequence having at least about 85% identity with the amino acid sequence set forth in SEQ ID NO: 1. In another example, the isolated polypeptide comprises or consists of an amino acid sequence having at least about 90% identity with the amino acid sequence set forth in SEQ ID NO: 1. In another example, the isolated polypeptide comprises or consists of an amino acid sequence having at least about 95% identity with the amino acid sequence set forth in SEQ ID NO: 1. In another example, the isolated polypeptide comprises or consists of an amino acid sequence having at least about 99% identity with the amino acid sequence set forth in SEQ ID NO: 1. In another example, the isolated polypeptide comprises or consists of an amino acid sequence having at least about 100% identity with the amino acid sequence set forth in SEQ ID NO: 1. In another example, the isolated polypeptide comprises or consists of an amino acid sequence having at least about 70% identity to about 100% identify with the amino acid sequence set forth in SEQ ID NO:1.

[00149] In one example, there is described an isolated nucleic acid molecule comprising a nucleotide sequence encoding a peptide comprising or consisting of an amino acid sequence having at least about 70% identity with the amino acid sequence set forth in SEQ ID NO: 1. In another example the isolated nucleic acid encoding a peptide comprising or consisting of an amino acid sequence having at least about 75% identity with the amino acid sequence set forth in SEQ ID NO: 1. In another example the isolated nucleic acid encoding a peptide comprising or consisting of an amino acid sequence having at least about 80% identity with the amino acid sequence set forth in SEQ ID NO: 1. In another example the isolated nucleic acid encoding a peptide comprising or consisting of an amino acid sequence having at least about 85% identity with the amino acid sequence set forth in SEQ ID NO: 1. In another example the isolated nucleic acid encoding a peptide comprising or consisting of an amino acid sequence having at least about 90% identity with the amino acid sequence set forth in SEQ ID NO:1. In another example the isolated nucleic acid encoding a peptide comprising or consisting of an amino acid sequence having at least about 95% identity with the amino acid sequence set forth in SEQ ID NO: 1. In another example the isolated nucleic acid encoding a peptide comprising or consisting of an amino acid sequence having at least about 99% identity with the amino acid sequence set forth in SEQ ID NO: 1. In another example the isolated nucleic acid encoding a peptide comprising or consisting of an amino acid sequence having at least about 100% identity with the amino acid sequence set forth in SEQ ID NO: 1. In another example the isolated nucleic acid encoding a peptide comprising or consisting of an amino acid sequence having at least about 70% to about 100% identity with the amino acid sequence set forth in SEQ ID NO: 1.

[00150] The term "isolated", as used herein, refers to altered or removed from the natural state. For example, a polypeptide or nucleic acid naturally present in a living animal is not "isolated," but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is "isolated." An isolated nucleic acid or polypeptide can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.

[00151] Unless otherwise specified, a "nucleotide sequence encoding a polypeptide" (and the like) includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence that encodes a polypeptide protein or an RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some version contain an intron(s).

[00152] The terms "peptide," "polypeptide," and "protein", as used herein are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein's or peptide's sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. "Polypeptides" include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. The polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.

[00153] In some examples, there is described a vector comprising the nucleic acid molecule described above and herein.

[00154] The term "vector" or "expression vector" as used herein refers to a

recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide. In on example, the vector is a pcDNA3.1 vector.

[00155] The term "homologous" as used herein refers to the sequence similarity or sequence identity between two polypeptides or between two nucleic acid molecules. When a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percent of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared* 100. For example, if 6 of 10 of the positions in two sequences are matched or homologous then the two sequences are 60% homologous. By way of example, the DNA sequences ATTGCC and TATGGC share 50% homology. Generally, a comparison is made when two sequences are aligned to give maximum homology.

[00156] "Similarity", for example between two peptides, may be determined by comparing the amino acid sequence and its conserved amino acid substitutes of one polypeptide to a sequence of a second polypeptide. Variants are defined to include peptide sequences different from the original sequence, for example, different from the original sequence in less than 40% of residues per segment of interest, different from the original sequence in less than 25% of residues per segment of interest, different by less than 10% of residues per segment of interest, or different from the original protein sequence in just a few residues per segment of interest and at the same time sufficiently homologous to the original sequence to preserve the functionality of the original sequence. [00157] The term "sequence identity" of a polypeptide or polynucleotide as used herein refers to a degree of sameness in an amino acid residue or a base in a specific region of two sequences that are aligned to best match each other for comparison. The sequence identity is a value obtained via alignment and comparison of the two sequences in the specific region for comparison, in which a partial sequence in the specific region for comparison may be added or deleted with respect to a reference sequence. The sequence identity represented in a percentage may be calculated by, for example, comparing two sequences that are aligned to best match each other in the specific region for comparison, determining matched sites with the same amino acid or base in the two sequences to obtain the number of the matched sites, dividing the number of the matched sites in the two sequences by a total number of sites in the compared specific regions (i.e., a size of the compared region), and multiplying a result of the division by 100 to obtain a sequence identity as a percentage. The sequence identity as a percentage may be determined using a known sequence comparison program, for example, BLASTP or BLASTN (NCBI), CLC Main Workbench (CLC bio), or MegAlign™ (DNASTAR Inc).

[00158] A polypeptide of may be synthesized by conventional techniques. For example, the peptides may be synthesized by chemical synthesis using solid phase peptide synthesis. These methods employ either solid or solution phase synthesis methods.

Automated synthesis may be used.

[00159] In some example, a polypeptide may be produced by culturing a cell comprising a nucleic acid which encoded the polypeptide, and isolating the polypeptide from the host cell or culture medium thereof.

[00160] The peptides of the invention can be post-translationally modified. For example, post-translational modifications that fall within the scope of the present invention include signal peptide cleavage, glycosylation, acetylation, isoprenylation, proteolysis, myristoylation, protein folding and proteolytic processing, etc. Some modifications or processing events require introduction of additional biological machinery. For example, processing events, such as signal peptide cleavage and core glycosylation, are examined by adding canine microsomal membranes or Xenopus egg extracts to a standard translation reaction.

[00161] In some examples, the polypeptides described herein may include unnatural amino acids formed by post-translational modification or by introducing unnatural amino acids during translation. A variety of approaches are available for introducing unnatural amino acids during protein translation.

[00162] A "cell" or "host cell" refers to an individual cell or cell culture that can be or has been a recipient of any recombinant vector(s), isolated polynucleotide, or polypeptide. Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or in total DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation and/or change. A host cell includes cells transfected or infected in vivo or in vitro with a recombinant vector or a polynucleotide of the invention. A host cell which comprises a recombinant vector of the invention is a recombinant host cell.

[00163] In one example, the host cell is a cell obtained or derived from a subject.

[00164] The term "subject" or "patient" as used herein, refers to an animal, and can include, for example, domesticated animals, such as cats, dogs, etc., livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), laboratory animals (e.g., mouse, rabbit, rat, guinea pig, etc.), mammals, non-human mammals, primates, non-human primates, rodents, birds, reptiles, amphibians, fish, and any other animal. The subject may be an infant, a child, an adult, or elderly. In a specific example, the subject is a human.

[00165] In one example the cell host is a human cell.

[00166] In one example, the cell is SVGA (astrocytes), RenCell CX (neuroprogenitor cells), or NCCIT (teratocarcinoma).

[00167] In some examples, there is described an antibody that specifically binds to a polypeptide as described herein. In one example, the polypeptide comprises or consists of the sequence of SEQ ID NO: 1.

[00168] The term "antibody" or "antibodies" is used herein refers to both polyclonal and monoclonal antibodies. In addition to intact or "full" immunoglobulin molecules, also included in the term "antibodies" are fragments (e.g., CDRs, Fv, Fab and Fc fragments) or polymers of those immunoglobulin molecules and humanized versions of immunoglobulin molecules, as long as they exhibit any of the desired properties according to the description.

[00169] Antibodies of the description may also be generated using well-known methods.

[00170] In some examples, a polypeptide may be used for generating an antibody of the description may be partially or fully purified from a natural source, or may be produced using recombinant DNA techniques. [00171] In some examples, the antibodies may be purchased commercially.

[00172] In some examples, the generation of two or more different sets of monoclonal or polyclonal antibodies may maximize or increase the likelihood of obtaining an antibody with the specificity and affinity required for its intended use.

[00173] The antibodies produced may tested for their desired activity by known methods, in accordance with the purpose for which the antibodies are to be used (e.g., Immunoblooting, ELISA, immunohistochemistry, immunotherapy, etc).

[00174] For example, antibodies may be tested in ELISA assays or, Western blots, immunohistochemical staining of formalin-fixed or frozen tissue sections. After their initial in vitro characterization, antibodies intended for therapeutic or in vivo diagnostic use are tested according to known clinical testing methods.

[00175] The term "monoclonal antibody" as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e.; the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. The monoclonal antibodies herein specifically include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired antagonistic activity.

[00176] Monoclonal antibodies may be prepared using hybridoma methods. In a hybridoma method, a mouse or other appropriate host animal is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes may be immunized in vitro.

[00177] The monoclonal antibodies may also be made by recombinant DNA methods.

[00178] In some example, the antibodies are humanized antibodies. Methods for humanizing non-human antibodies are well known in the art.

[00179] In some examples, antibodies may be labeled with probes suitable for detection by various imaging methods. Methods for detection of probes include, but are not limited to, fluorescence, light, confocal and electron microscopy; magnetic resonance imaging and spectroscopy; fluoroscopy, computed tomography and positron emission tomography.

[00180] Examples of probes may include, but are not limited to, fluorescein, rhodamine, eosin and other fluorophores, radioisotopes, gold, gadolinium and other lanthanides, paramagnetic iron, fluorine-18 and other positron-emitting radionuclides.

Antibodies may be directly or indirectly labeled with said probes. Attachment of probes to the antibodies includes covalent attachment of the probe, incorporation of the probe into the antibody, and the covalent attachment of a chelating compound for binding of probe, amongst others well recognized in the art.

[00181] In one example, there is described a method for treating or preventing conditions or disorders associated with CTXLP in a subject, comprising: administering to a subject in need thereof a therapeutically effective amount of an active agent or a

pharmaceutically acceptable salt thereof, wherein the active agent blocks or inhibits the CTXLP activity.

[00182] In one example, there is described a method for treating or preventing conditions or disorders associated with ERVK in a subject, comprising: administering to a subject in need thereof a therapeutically effective amount of an active agent or a

pharmaceutically acceptable salt thereof, wherein the active agent blocks or inhibits the CTXLP activity.

[00183] In one example, the active agent is a CTXLP inhibitors.

[00184] In one example, a CTXLP inhibitors inhibits or reduces the activity of CTXLP polypeptide.

[00185] In one example, a CTXLP inhibitors inhibits or reduces the level or amount of CTXLP polypeptide.

[00186] In one example, a CTXLP inhibitors inhibits or reduces the level or amount of of CTXLP mRNA.

[00187] In some example, a CTXLP inhibitor may be, without being limiting thereto, a small molecule, an antibody, a nucleic acid, an aptamer, a peptide.

[00188] The term "small molecule" as used herein refers to a molecule of less than about 1 ,000 daltons, in particular organic or inorganic compounds.

[00189] In one example, the small molecule may be a small molecule inhibitor of HIV Tat. In one example, the small molecule inhibitor of HIV Tat is a Michael acceptor electrophile (MAE). In one example, the MAE is curcumin, rosmarinic acid, gambogic acid, celastrol (15-deoxy-A(12,14)-prostaglandin J(2) (15d-PGJ(2)), cyclopentenone prostaglandins (CyPG), such as 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)), N- acetylcysteine amide (NACA), or D-penicillamine (also called Cuprimine). In one example, the small molecule inhibitor of HIV Tat is a sulfhydryl compound with chelating properties. In one example, the sulfhydryl compound with chelating properties is N-(2-Mercapto-propionyl)- glycin (MPG), 2,3-Dimercapto-propanol (DMP), 2,3-Dimercapto-propane-sulfonic acid (DMPS), Nitric oxide (NO), or sulphated polysaccharides. In one example the small molecule inhibitor of HIV Tat is a Thioredoxin reductase 1 (TRR1) inhibitor. In one example, the Thioredoxin reductase 1 (TRR1) inhibitor is B5 (curcumin analog).

[00190] In one example, the CTXLP inhibitor is a nucleic acid molecule interfering specifically with CTXLP expression. In some example, the nucleic acid CTXLP inhibitor may be an antisense against CTXLP, a siRNA against CTXLP, a shRNA against CTXLP, or a ribozyme.

[00191] The term "RNAi" or "interfering RNA" refers an RNA, which is capable of down-regulating the expression of the targeted polypeptide, such as CTXLP. It

encompasses small interfering RNA (siRNA), double-stranded RNA (dsRNA), single- stranded RNA (ssRNA), micro-RNA (miRNA), and short hairpin RNA (shRNA) molecules. RNA interference, designates a phenomenon by which dsRNA specifically suppresses expression of a target gene at post-translational level. In normal conditions, RNA interference is initiated by double-stranded RNA molecules (dsRNA) of several thousand base pairs in length. In vivo, dsRNA introduced into a cell is cleaved into a mixture of short dsRNA molecules called siRNA. The enzyme that catalyzes the cleavage, Dicer, is an endo-RNase that contains RNase III domains

[00192] siRNA are usually designed against a region 50-100 nucleotides downstream the translation initiator codon, whereas 5'UTR (untranslated region) and 3'UTR are usually avoided. The chosen siRNA target sequence should be subjected to a BLAST search against EST database to ensure that the only desired gene is targeted. Various products are commercially available to aid in the preparation and use of siRNA. In a preferred

embodiment, the RNAi molecule is a siRNA of at least about 15-50 nucleotides in length, preferably about 20-30 base nucleotides.

[00193] RNAi can comprise naturally occurring RNA, synthetic RNA, or recombinantly produced RNA, as well as altered RNA that differs from naturally-occurring RNA by the addition, deletion, substitution and/or alteration of one or more nucleotides. Such alterations can include addition of non-nucleotide material, such as to the end of the molecule or to one or more internal nucleotides of the RNAi, including modifications that make the RNAi resistant to nuclease digestion.

[00194] RNAi may be administered in free (naked) form or by the use of delivery systems that enhance stability and/or targeting, e.g., liposomes, or incorporated into other vehicles, such as hydrogels, cyclodextrins, biodegradable nanocapsules, bioadhesive microspheres, or proteinaceous vectors. They may also be administered in the form of their precursors or encoding DNAs.

[00195] Antisense nucleic acid can also be used to down-regulate the expression of CTXLP. The antisense nucleic acid can be complementary to all or part of a sense nucleic acid encoding CTXLP e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence, and is thought to interfere with the translation of the target mRNA.

[00196] An antisense nucleic acid can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length. An antisense nucleic acid can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art. Particularly, antisense RNA can be chemically synthesized, produced by in vitro transcription from linear (e.g. PCR products) or circular templates (e.g., viral or non-viral vectors), or produced by in vivo transcription from viral or non-viral vectors.

[00197] Antisense nucleic acid may be modified to have enhanced stability, nuclease resistance, target specificity and improved pharmacological properties. For example, antisense nucleic acid may include modified nucleotides designed to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides.

[00198] Ribozyme molecules can also be used to block the expression of CTXLP. Ribozymes are catalytic RNA molecules with ribonuclease activity which are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a

complementary region. Thus, ribozymes can be used to catalytically cleave mRNA transcripts to thereby inhibit translation of the protein encoded by the mRNA. Ribozyme molecules specific for CTXLP can be designed, produced, and administered by methods commonly known to the art.

[00199] The term "aptamer" refers to a molecule of nucleic acid or a peptide able to bind specifically to CTXLP polypeptide. [00200] The term "administering" as used herein includes all means of introducing the compounds and compositions described herein to the subject, including, but are not limited to, oral (po), intravenous (iv), intramuscular (im), subcutaneous (sc), transdermal, inhalation, and the like. The compounds and compositions described herein may be administered in unit dosage forms and/or formulations containing conventional nontoxic pharmaceutically- acceptable carriers, adjuvants, and vehicles.

[00201] Non limiting examples of oral administration include tablets, capsules, elixirs, syrups, and the like.

[00202] Non limiting examples of parenteral administration include intravenous, intraarterial, intraperitoneal, epidurial, intraurethral, intrasternal, intramuscular and subcutaneous, as well as any other art recognized route of parenteral administration.

[00203] Non limiting examples of means of parenteral administration include needle (including microneedle) injectors, needle-free injectors and infusion techniques, as well as any other means of parenteral administration recognized in the art. Parenteral formulations are typically aqueous solutions which may contain excipients such as salts, carbohydrates and buffering agents (preferably at a pH in the range from about 3 to about 9), but, for some applications, they may be more suitably formulated as a sterile non-aqueous solution or as a dried form to be used in conjunction with a suitable vehicle such as sterile, pyrogen-free water. The preparation of parenteral formulations under sterile conditions, for example, by lyophilization, may readily be accomplished using standard pharmaceutical techniques well known to those skilled in the art. Parenteral administration of a compound is illustratively performed in the form of saline solutions or with the compound incorporated into liposomes. In cases where the compound in itself is not sufficiently soluble to be dissolved, a solubilizer such as ethanol can be applied.

[00204] The dosage of each compound(s) depends on several factors, including: the administration method, the condition to be treated, the severity of the condition, whether the condition is to be treated or prevented, and the age, weight, and health of the person to be treated. Additionally, pharmacogenomic (the effect of genotype on the pharmacokinetic, pharmacodynamic or efficacy profile of a therapeutic) information about a particular patient may affect the dosage used.

[00205] In one example, examples of ERVK associated diseases may include but are not limited to infectious diseases, autoimmune diseases, neurological diseases, cancer, and other conditions such as idiopathic nephrotic syndrome. [00206] In one example, examples of CTXLP associated diseases may include but are not limited to infectious diseases, autoimmune diseases, neurological diseases, cancer, and other conditions such as idiopathic nephrotic syndrome.

[00207] Infectious disease, including but not limited to HSV infection, HIV infection, EBV infection, HTLV infection, toxoplasma gondii infection, HSV infection, or prion disease.

[00208] Autoimmune disease, including but not limited to, Insulin dependent diabetes mellitus, morphea, Psoriasis, rheumathoid arthritis, systemic lupus erythematosus, Type 2 diabetes mellitus (in people with SCZ).

[00209] Neurological disease, including but not limited to, amyotrophic lateral sclerosis, bipolar disorder, Kennedy's disease, multiple sclerosis, Schizophrenia

[00210] Cancer, including but not limited to, Breast cancer, Chronic myelogenous leukemia, Colon cancer, Gastric cancer, Germ cell tumours, Germinogenic tongue tumours, Gonadoblastomas, Hepatocellular carcinoma, Leukemia, Lymphoma, Melanoma,

Myelodysplastic syndrome, Nasopharyngeal carcinoma, Ovarian cancer, Pancreatic cancer, Prostate cancer, Trophoblastic tumours, Tumorigenesis (via AR interaction), Thyroid adenoma, ERVK in cancerous tissues.

[00211] Other, including but not limited to, Idiopathic nephrotic syndrome.

[00212] In one example, there is described a method for transcriptional activation, comprising contacting a DNA molecule comprising a gene with a polypeptide as described herein.

[00213] In one example, there is described a diagnostic reagent for use in the detection of CTXLP polypeptide in a subject, comprising an antibody specific for CTXLP polypeptide.

[00214] In one example, there is described a diagnostic reagent for use in the detection of CTXLP mRNA in a subject, comprising an isolated nucleic acid specific for CTXLP.

[00215] In one example, there is described a diagnostic reagent for use in the detection CTXLP activity in a subject, comprising a polypeptide as described herein.

[00216] In one example, there is described a method for treating or preventing conditions or disorders associated with CTXLP in a subject, comprising: measuring an amount of CTXLP polypeptide, or CTXLP activity, or CTXLP mRNA; and administering to a subject in need thereof a therapeutically effective amount of an active agent or a

pharmaceutically acceptable salt thereof when the amount of CTXLP polypeptide, or CTXLP activity, or CTXLP mRNA, is high, optionally compared to a control, wherein the active agent blocks or inhibits the CTXLP activity.

[00217] In one example, there is described a method for treating or preventing conditions or disorders associated with ERVK in a subject, comprising: measuring an amount of CTXLP polypeptide, or CTXLP activity, or CTXLP mRNA; and administering to a subject in need thereof a therapeutically effective amount of an active agent or a

pharmaceutically acceptable salt thereof when the amount of CTXLP polypeptide, or CTXLP activity, or CTXLP mRNA, is high, optionally compared to a control, wherein the active agent blocks or inhibits the CTXLP activity.

[00218] Method are conveniently practiced by providing the compounds and/or compositions used in such method in the form of a kit. Such kit preferably contains the composition. Such a kit preferably contains instructions for the use thereof.

[00219] In one example, there is described a kit comprising:(a) a container comprising a pharmaceutical composition containing a polypeptide as described herein, and/or a nucleic acid as described herein, and/or an expression vector, and/or a host cell, and/or an antibody as described herein, in solution or in lyophilized form;(b) optionally, a second container containing a diluent or reconstituting solution for the lyophilized formulation; and(c) optionally, instructions for use.

[00220] In one example, the kit further comprising one or more of (iii) a buffer, (iv) a diluent, (v) a filter, (vi) a needle, or (v) a syringe.

[00221] To gain a better understanding of the invention described herein, the following examples are set forth. It should be understood that these examples are for illustrative purposes only. Therefore, they should not limit the scope of this invention in anyway.

[00222] EXAMPLES

[00223] Endogenous retroviruses (ERVs) are host genetic elements originating from prior infection of host germ-line cells that are subsequently inherited through the germline. ERVs represent approximately 8% of human genomic DNA. ERVs can benefit their host, or in other contexts are proposed to be involved in pathogenesis and disease. Notably, our interest in ERVK CTXLP lies in its association to motor neuron conditions such as Amyotrophic Lateral Sclerosis (ALS), as well in cancers.

[00224] ERVK CTXLP Bioinformatics: Endogenous retrovirus-K (ERVK) conotoxin- like protein (CTXLP) is produced following a ribosomal frameshifting event and is subject to post-translational modifications (PTMs). PTMs and alternative start sites allow for a variety of CTXLP isoforms which may drive distinct pathogenic mechanisms. The prevalence and polymorphic variability of ERVK CTXLP-encoding insertions suggests that CTXLP is a pervasive and conserved ERVK protein. The molecular characterization of CTXLP revealed a conotoxin domain which predicts that it acts as antagonist to specific voltage-gated calcium channels. CTXLP also contains a cysteine motif that aligned to multiple cone snail, spider and viral toxins, which are known to function as antagonists to voltage-gated ion channels. This intrinsic capacity to interfere with calcium channels through these motifs suggests a putative mechanism by which ERVK can act in the pathogenesis of motor neuron diseases such as ALS.

[00225] CTXLP biological characterization: CTXLP protein isoform expression in NCCIT and SVGA cells was elucidated by Western blots which indicated presumed isoform sizes of 32 kDa, 51 kDa, and 90/110 kDa. In NCCIT cells, endogenous CTXLP is ubiquitously expressed in the nucleus, and also identified in the cytoplasm and cell membrane, based on cell fractionation and confocal experiments. In contrast, in SVGA cells basal CTXLP levels are limited, but highly inducible by pro-inflammatory stimuli. In addition, CTXP expression is almost exclusively in the chromatin fraction and demonstrates a prominence in the nucleus upon confocal imaging. The notable exception is that after pro-inflammatory activation for 24 hours CTXLP puncta appear in the cytoplasm and on cellular membranes reminiscent of pathogenic protein aggregates. Moreover, the localization pattern in response to proinflammatory activators resulting in a prominence in the nucleus ability to bind chromatin suggests that CTXLP may be involved in viral transcription. A primary candidate as a viral transcription factor is the 32 kDa CTXLP isoform, as small cysteine-rich proteins have previously been identified as transcriptional activators, as per HIV-1 Tat (15 kDa) and HTLV Tax (40 kDa) role as viral transcription co-activators.

[00226] CTXLP Expression in disease states: ERVK CTXLP localized to the motor cortex in spinal cord sections from autopsy samples of patients with ALS, but not neuro-normal controls. Concomitantly, CTXLP expression was substantially enhanced in diseased ALS tissues, aligning with oligodendrocytes, Nogo-A expression and demyelinated lesions. In addition, cancer cell lines and tissue expressed greater levels of CTXLP relative to normal controls. Together, these findings provide significant evidence for the activity of CTXLP in ALS and certain cancers.

[00227] Pathological consequences of CTXLP expression: ERVK CTXLP has the capacity to enhance NF-κΒ p65 and p50 proteins that play a critical role in ALS pathogenesis. In addition, CTXLP administration or transfection induced significant levels of capase-3. The induction of caspase-3 activation and apoptosis by CTXLP was inhibited by excess extracellular calcium pointing to a calcium channel mediated activation of toxicity.

Remarkably, despite the initial die off of cells, cells remaining in the cultures appeared to demonstrate appreciable cellular proliferation relative to control suggesting the induction of a carcinogenic process. CTXLP also had a notable effect on the depletion of CaV2.2 voltage- gated calcium channel-associated transcriptional regulator (CaV2.2 CCAT) from the nucleus.

[00228] ERVK CTXLP can be targeted by small molecule therapeutics: A drug screen revealed that celastrol and gambogic acid have the capacity to inhibit endogenous CTXLP expression in NCCIT cancer cell line. Moreover, gambogic acid was able to reduce inducible CTXLP expression the presence of TNFa and ameliorate the concomitant expression of pathogenic marker Nogo-A. This strongly suggests that therapeutic targeting of CTXLP in human disease could be an agent in the efforts to ameliorate the devastation of ALS.

[00229] Development of cell and animal models to investigate CTXLP

pathogenesis: Human tissue and animal models for the study of CTXLP in ALS and cancer are needed. We are actively working to further develop our human tissue culture models. In addition, together with Dr. Alberto Civetta, we are in the process of developing a model in Drosophilia at the University of Winnipeg. Importantly, we will continue to pursue mammalian models with our collaborators which offer an opportunity to explore multiple features of pathogenesis as we continue to elucidate the processes involved in CTXLP pathogenesis.

[00230] ERVK CTXLP is a novel pathological target for the development of therapeutics for inflammatory, neurological and oncogenic diseases.

ABBREVIATIONS

[00231] Abbreviations used in text.

AGRP Agouti-related peptide

ALS Amyotrophic lateral sclerosis

ASIP Agouti-signalling protein

BA6 Brodmann area 6

BLAST Basic local alignment search tool

BMAA Beta-N-methylamino-L-alanine

CC Cervical spinal cord

CCAT Calcium channel-associated transcription regulator

cDNA Complimentary deoxyribonucleic acid

CEL Celastrol

ChIP Chromatin immunoprecipitation CNS Central nervous system

CTXLP Conotoxin-like protein

CX3CL1 Chemokine (C-X3-C motif) ligand 1

DAPI 4',6-diamidino-2-phenylindole

DNA Deoxyribonucleic acid

Env Envelope

ERV Endogenous retrovirus

ERVH Endogenous retrovirus-H

ERVK Endogenous retrovirus-K

ERVW Endogenous retrovirus-W

GA Gambogic acid

HAART Highly active antiretroviral therapy

HAUSP/USP7 Herpesvirus-associated ubiquitin-specific protease/Ubiquitin-specific- processing protease 7

HCV Hepatitis C virus

HIV Human Immunodeficiency virus

HTLV Human T-lymphotrophic virus

HML Human Mouse mammary tumour virus-like

ICK Inhibitor cysteine knot

IP Immunoprecipitation

IRES Internal ribosomal entry site

IRF7 Interferon regulatory factor 7

ISRE Interferon response element

LATS Large tumor suppressor kinase

LC Lumbar spinal cord

LIGHT Homologous to lymphotoxin, exhibits inducible expression and competes with

HSV glycoprotein D for binding to herpesvirus entry mediator, a receptor expressed on T lymphocytes

LTR Long terminal repeat

MAE Michael acceptor electrophile

MAG Myelin-associated glycoprotein

MAP2 Microtubule-associated protein 2

MAPK Mitogen-activated protein kinases

MC1 R Melanocortin receptor 1

MM TV Mouse mammary tumour virus

MOG Myelin oligodendrocyte glycoprotein

mRNA Messenger ribonucleic acid

MS Multiple sclerosis

MUSCLE Multiple Sequence Comparison by Log- Expectation

NCCIT National Cancer Center Institute Tokyo, teratocarcinoma cell line

NF-KB Nuclear factor κΒ

NCBI National Centre for Biotechnology Information

NEC-1/2 Necrostatin-1/2

NgR1 Nogo-A receptor

NN Neuronormal

NLS Nuclear localization signal

NPV Nuclear polyhedrosis virus

Olig1/2 Oligodendrocyte transcription factor 1/2

OPC Oligodendrocyte precursor cell

ORF Open reading frame PCA Principle component analysis

PLP Proteolipid protein

PRF Programmed ribosomal frameshift

PTM Post-translational modification

Q-PCR Quantitative polymerase chain reaction

RA Rheumatoid arthritis

RelA REL-associated protein

RNA Ribonucleic acid

RT Reverse transcriptase

RTN4R Reticulon 4 receptor

SC Solochrome cyanine

SRA Sequence Read Archive

SU Surface unit

SVGA SV40 T antigen glial astrocytes

Tat Trans-activator of transcription

Tax Transactivator from the X-gene region

TCF4 Transcription factor 4

TDP-43 TAR DNA-binding protein 43

TM Transmembrane

TMEV Theiler's Murine Encephalomyelitis Virus

TNFa Tumour necrosis factor a

TRAF-2/6 TNF receptor associated factor

VEGF Vascular endothelial growth factors

VGCC Voltage gated calcium channel

WCE Whole cell extract

[00232] Endogenous retroviruses

[00233] Retroviruses are single-stranded RNA viruses that replicate through reverse transcription ¹ . Retroviruses use the enzyme reverse transcriptase to convert their genomic RNA to DNA, and then use a viral integrase to insert itself into a host genome ² . Retroviruses are categorized as being either exogenous or endogenous ³ . Examples of exogenous retroviruses include Human-lmmunodeficiency virus (HIV) and Human T-lymphotropic virus (HTLV). Alternatively, endogenous retroviruses (ERVs) are genetic elements originating from prior infection of host germ-line cells, allowing them to be inherited through Mendelian genetics ³ . ERVs represent approximately 8% of human genomic DNA ⁴ . ERVs can benefit their host ⁵ , or in other contexts are proposed to be involved in pathogenesis and disease ⁶ .

[00234] Endogenous Retrovirus-K (ERVK) is the most recently endogenated retrovirus in the human genome ¹ . ERVK is a group of similar viruses that are categorized into 10 clades (sub-groups). ERVK (HML-2 clade) first entered the human genome approximately 28 million years ago, occurring before the divergence of hominids and old-world monkeys ⁷ . More recent insertions of ERVK occurred up to 200,000 years ago, and are specific to the human lineage. This has resulted in several human-specific ERVK insertions ⁸ . Approximately 1000 ERVK loci have been identified in the human genome ⁹ . Although the majority of ERVK insertions have been silenced through mutations and negative selection, there are an estimated 24 fixed loci capable of producing viral proteins ^{3 6} . ERVs are also found to be highly polymorphic between individuals and different ethnic groups ⁷ . ERVK expression has been detected in several tissues throughout the body at varying levels between

individuals ^{3 10} .

[00235] ERVK genome

[00236] The ERVK genome consists of the essential retroviral genes gag-pro-pol-env, along with its own accessory genes ¹ (Figure 1). The group specific antigen (gag) gene encodes structural proteins including the viral capsid ^{8 11} . The protease (pro) gene encodes a protease which cleaves newly synthesized viral proteins ¹ . The polymerase (pol) gene encodes for proteins including reverse transcriptase and integrase ^{2 11} . The envelope (env) gene encodes the glycoproteins of the viral envelope ¹¹ . The ERVK genome is flanked by long terminal repeats (LTRs), which were assistive in retroviral DNA insertion into the host ¹² . Once inserted into the host genome, the virus is considered a provirus. LTRs contain elements of enhancers and promoters, including transcription factor binding-sites and interferon- stimulated response elements that regulate both retroviral and host gene expression ^{6 11} .

[00237] ERVK can be organized into two types based on their genome. Type 1 proviruses contain a 292 base-pair deletion near the 5' end of env not found in type 2 proviruses ¹³ . The presence or absence of this deletion affects the accessory proteins the provirus produces ^{13 14} .

[00238] ERVK envelope protein

[00239] The ERVK envelope (Env) protein is initially translated as a large, inactive polyprotein ¹⁵ ' ¹⁶ . The polyproteins dimerizes or trimerizes and are then cleaved by the cellular protease furin, forming a surface unit (SU) and transmembrane (TM) subunit ¹⁵ . Like other retroviral envelope proteins, the assembled Env trimer is heavily glycosylated and is expressed on the viral capsid membrane, as well as infected host cell membranes, allowing for incorporation of the virus into host cells ^{15 17} .

[00240] Cysteine knot proteins

[00241] Cysteine knots are protein structural motifs found throughout animals, fungi and plants ¹⁸ . Cysteine knot proteins are known for their stability, attributed to their 3 disulfide bonds; two of the disulfide bonds and their peptide backbone form a ring that the third bond goes through, thus forming a "knot" structure^. Cysteine knot proteins are categorized as cyclic cysteine knots, growth factor cysteine knots, or inhibitor cysteine knots (ICK). Cyclic cysteine knots are found in plants and often have defense functions as bactericides and insecticides ¹⁸ . Growth factor cysteine knots are found in extracellular signaling molecules and are involved in various functions including cell-cell communication and embryonic

development ¹⁹ . Examples include the vascular endothelial growth factors (VEGFs), and nerve growth factor ¹⁹ . ICK proteins are found in fungi, plants and animals and act as antagonists to a variety of receptors and ion channels ¹⁸ .

[00242] ICK proteins include a vast array of peptides found in various living organisms. The ICK structure consists of six conserved (connected as Cys1-CyslV, Cysll-CysV, and Cyslll-CysVI) cysteine residues and an otherwise variable peptide backbone ¹⁸ (Figure 3). Within the animal kingdom, ICK peptides are found in the venoms of spiders, scorpions, and marine snails, and function either as pore-blockers or gate-modifiers of ion channels ¹⁸ .

Mammalian ICK peptides have also been identified, including agouti-signalling protein (ASIP) and agouti-related peptide (AGRP) ²⁰ .

[00243] Animal ICKs are proposed to be a result of divergent evolution ²¹ . Functional constraints during evolution have resulted in spider, snail, and scorpion ICKs maintaining a similar gene structure, protein fold, and target receptor, which are all evidence for a common ancestor ²¹ . Alternatively, plant and fungi ICK do not have these similarities to animal ICKs, suggesting they are a product of convergent evolution ²¹ . In certain baculoviruses, a cysteine- rich ORF has been detected, that potentially translates into an ICK fold ²¹ . The putative ICK motif resembles the animal ICKs, suggesting that viruses may have obtained this genetic sequence by a gene transfer event after infecting an ICK-carrying host ²¹ .

[00244] Conotoxins

[00245] Conotoxins are neurotoxic peptides found in the Conus genus of marine snails used to immobilize prey ²² . Conus species are distinct in their ability to produce hundreds of different toxic peptides ²³ . Conotoxins are disulfide-rich and are usually 10-30 amino acids in length ²² . Conotoxins act as antagonists to specific voltage and ligand-gated ion channels ²² . In humans, symptoms of conotoxin exposure include poor coordination, blurred vision, speech difficulties, and nausea ²³ . Conotoxins have also been associated with episodes of delirium and psychosis ²⁴ .

[00246] The O-superfamily of conotoxins exhibits an ICK fold. Members of the O- superfamily include μ-conotoxins, which inhibit voltage-gated sodium channels, and δ- conotoxins, which delay sodium channel inactivation ²⁵ . K-Conotoxins are inhibitors of voltage-gated potassium channels; ω -conotoxins inhibit N-type voltage-gated calcium channels (VGCCs) ²⁵ . N-type VGCCs are located in presynaptic nerve terminals and are involved in neurotransmitter release ²⁶ , ω -Conotoxin's selectivity for N-type VGCCs has allowed for their development as therapeutic agents. The ω -conotoxin MVIIA has been developed into a drug for relief of chronic and inflammatory pain ²⁷ .

[00247] Genes encoding an ω-conotoxin-like protein (CTXLP) have also been identified in certain viruses. Nuclear polyhedrosis viruses (NPV) have been shown to secrete a small conotoxin-like peptide ²⁸ . NPVs are insect pathogens belonging to the family baculoviridae ²⁸ . Although NPV-CTXLP's function has not been elucidated, its cysteine bridges were found to have a nearly identical structure to the conserved w-conotoxin's cysteine motif ²⁸ . We have discovered a novel CTXLP ORF in the envelope gene of ERVK. The full pathogenic potential of ERVK CTXLP domain remains unknown.

[00248] ERVK CTXLP Bioinformatics

[00249] Identification of a conotoxin-like domain in the ERVK genome

[00250] Splicing and Conserved Domains in the ERVK Genome (Start Codon-Biased

Analysis)

[00251] NetGene2 splice site prediction yielded a large number of predicted splice junctions (105-1 19 per ERVK sequence). However, after exhaustive analysis, none of these splice junctions resulted in the creation of domains that could be identified using the

Conserved Domains Database. However, the predicted splicing patterns resulted in the identification of between 27 and 46 newly created ORFs per ERVK sequence.

[00252] Conserved Domains (Start Codon-Unbiased Analysis)

[00253] After finding no conserved domains in the initial analysis, the requirement for a start codon (ATG, CTG, TTG, GTG or ATT) at the beginning of each ORF was removed, because a start codon could be introduced through splicing and thus was not strictly necessary. The removal of this requirement resulted in slightly different ORFs, which can be seen in Figure 2. Analysis of these ORFs identified a previously undescribed region that would generate a peptide with significant homology to known proteins. This ORF occurred in both type 1 and type 2 genomes (that is, it was not affected by the 292-base pair deletion). DNA with the potential to encode a peptide containing a domain with homology to the O- conotoxin superfamily was identified in a region of env, but in a different reading frame, from nucleotide 7863 to nucleotide 7934 in the 5'-3' direction. This ORF did not contain the typical methionine codon that is often used as a start codon for translation.

[00254] Conotoxin-like Domain

[00255] The putative conotoxin-like domain contained six characteristic cysteine residues and one characteristic glycine residue, indicating that it is most similar to the ω- conotoxin family. Another group of viruses, Nuclear Polyhedrosis Viruses, which are insect- infecting Baculoviruses, produce a similar conotoxin-like protein (NPV CTXLP). The putative ERVK CTXLP showed the greatest similarity to these viral proteins. Figure 3 shows the sequences of several ω-conotoxins produced by 3 cone snail species, as well as the sequence of a Nuclear Polyhedrosis Virus conotoxin-like domain. Although these sequences differ from each other in notable ways, 7 conserved residues (6 cysteines and 1 glycine) are found in all of them. These residues are also observed in the ERVK CTXLP domain.

[00256] The ERVK CTXLP sequence showed the greatest homology to NPV CTXLP sequences (E-value = 1.09 x 10 ^"5 ). Figure 4 shows the sequence logo for ERVK CTXLP and 10 NPV sequences, in which several more amino-acid residues (in addition to the 7 described above) are conserved.

[00257] Figure 5 summarizes the Conus and NPV sequences with greatest similarity and centrality to ERVK CTXLP. Figure 6 shows the logo of the knotin domain of CTXLP and its amino acid composition.

[00258] Three-Dimensional Modeling of the ERVK Conotoxin-like Protein

[00259] Conotoxins adopt a knot-like conformation, called a knottin domain, which is important for their action. Omega-conotoxin and NPV CTXLP knottins include 3 disulfide bonds. Tertiary structure prediction of the ERVK-1 13 CTXLP protein using Knotter 1 D3D software resulted in the conclusion that it too could form these characteristic features. The predicted 3-dimensional structure of the ERVK-1 13 CTXLP domain is shown in Figure 7.

[00260] This predicted structure was then superimposed on the predicted structure of an NPV CTXLP domain to examine the similarity between the two. This structure alignment (Figure 8) is based on sequence alignment and was prepared using UCSF Chimera software ³¹ .

[00261] The root mean square deviation between 24 atom pairs in this alignment is 0.426 angstroms. However, it can be difficult to see how similar the predicted structure of these two protein domains are from this image (Figure 8). As such, the structures were reduced to their respective peptide backbones. The resultant image can be seen in Figure 9. [00262] Conotoxin-Like Proteins are Not Encoded by Other Retroviruses

[00263] After identifying that these two distantly related groups of viruses (ERVK and NPVs) both contain conotoxin-like protein coding capacity, we also searched for conotoxin- like domains within translations of all three reading frames of the env region of several other retroviral genomes (HIV-1 , HTLV-1 , MMTV, ERVW, ERVH). No conotoxin-like domains were identified in any of these retroviruses from our analysis.

[00264] Alignments of ERVK C TXLP and other cysteine-rich proteins

[00265] The ERVK CTXLP ORF is 39 amino acids long, with the cysteine-rich motif accounting for 30/39 amino acids (CSDYGINCSHSYGCCSRSCIALFCSVSKLC). The CTXLP cysteine-rich sequence was aligned to inhibitor cysteine knot (ICK) proteins and other cysteine-rich proteins using Geneious software (Version R8) ³⁰ . A sequence logo was generated from the alignment to assess amino acid conservation between CTXLP and known cysteine-rich proteins (Figure 10). Geneious alignment software was used to compare ERVK CTXLP's cysteine-rich amino acid sequence to other cysteine-rich proteins (Table 1) to further understand CTXLP's structure and potential function.

Table 1: Proteins and peptides from various organisms and their respective accession numbers compared to CTXLP cysteine-rich motif found using Geneious software.

[00266] Several spiders are known to utilize ICK peptides in their toxins ³² . The putative ERVK CTXLP cysteine motif was aligned to 12 spider toxins from various species of spiders. Figure 11 shows ERVK CTXLP peptide had significant similarity to the Hainantoxin-I and Hainantoxin-IV ICK motifs, with a pairwise identity above 25% suggesting conserved protein function (NCBI).

[00267] The sequence logo generated showed that the cysteine knot (C-C-CC-C-C) motif is conserved in ERVK CTXLP and the spider toxins examined. Although there was significant sequence diversity in other amino acids, each sequence contained the essential 6 cysteine residues for an ICK. Five of the 12 spider toxins also contained a glycine residue in an identical position to ERVK CTXLP's characteristic glycine. The overall cysteine spacing of the CTXLP motif was unique when compared with spider toxins, suggesting that despite forming an ICK fold, the overall protein conformations are likely divergent. This could explain receptor binding specificity of each toxin species. Spider toxins and CTXLP were then examined for conserved motifs (Table 2). Overall, there was little conservation outside of the ICK motif, with the most significant conservation found in Hainantoxin-I, a voltage gated sodium channel inhibitor.

Table 2: Spider toxins, host species and target receptors show similarity and conserved motifs with ERVK CTXLP.

[00268] When ERVK CTXLP peptide was aligned to the human ICK proteins agouti- signalling protein (ASIP) and agouti-related peptide (AGRP), significant similarity was found in the conserved cysteine domain (both with a pairwise identity of 21.9% with CTXLP), suggesting structural similarity and possible functional overlap (Figure 12). [00269] Seven of ten cysteine residues found in the agouti family peptides aligned with ERVK CTXLP. Agouti proteins use 8 cysteines to form an ICK structure ³³ , whereas CTXLP only has 7 cysteines and is likely to take on a simpler ICK fold. Agouti-like proteins are the only known ICK domain containing protein in humans; however, these findings suggest that CTXLP may also be a human-derived ICK protein.

[00270] ERVK CTXLP was also aligned to 7 VEGF proteins, which utilize a growth factor cysteine knot. Although there was some alignment between the cysteine residues and some similar motifs (ex. GCC) identified, the large gaps in spacing and the different spacing of cysteine residues in the VEGF proteins suggests that there is no significant similarity to ERVK CTXLP (all with identity≤ 7.7 %) (Figure 13). The dissimilarity suggests that CTXLP does not take on a growth factor cysteine knot structure.

[00271] When ERVK CTXLP was aligned to the retroviral accessory protein Tat from HIV-1 and HIV-2, some degree of similarity was detected (identity 19.4% and 16.1 %, respectively) (Figure 14). Tat (HIV-1 and HIV-2) contains a conserved C-C-CC-C-C motif that has a much tighter spacing than ERVK CTXLP. In contrast to HIV-1 Tat and CTXLP, HIV-2 Tat did not contain a central "CC" pair of cysteine residues. ERVK CTXLP was also aligned with Tax proteins from HTLV-1 , HTLV-2, and HTLV-3 along with envelope from HTLV-4 and envelope from Jaagsiekte retrovirus, and no homology was detected (data not shown). The conservation of a cysteine motif and central "CC" cysteine pair in HIV-1 Tat and CTXLP is a potential basis for conserved structure and function of these viral proteins.

[00272] Aligning ERVK CTXLP to several cysteine-rich peptides provided insight into the potential function of the CTXLP protein domain. ERVK CTXLP showed the greatest similarity to ICK peptides. The cysteine knot motif was conserved in all of the spider toxins examined, and Hainaintoxin-I showed the greatest similarity to CTXLP with an identity of 26.5%, suggesting similarity in function (NCBI). All other amino acid residues were highly variable, suggesting that the conservation of the cysteine residues and the tertiary structure are more important for peptide function rather than the primary amino acid sequence. The spider toxins function as antagonists to voltage gated ion channels, suggesting CTXLP may have a similar function ¹⁸ . Hainantoxin-I is a voltage-gated sodium channel inhibitor ³⁴ ; thus, CTXLP may function as a voltage-gated sodium channel inhibitor. Although the ERVK CTXLP had significant similarity to Hainantoxin-I, ERVK CTXLP still had the greatest similarity (25.9- 33.3%) to the cone snail ω-conotoxins, suggesting CTXLP functions as a VGCC inhibitor. Previous studies have also shown that ω-conotoxin's amino acid residues threonine 11 , tyrosine 13, lysine 2, lysine 4, and arginine 22 are important for calcium channel receptor binding ³⁵ . CTXLP has some similar conserved residues including a tyrosine in position 12 and an arginine in position 17. CTXLP may alternatively utilize different amino acid residues to bind to cognate VGCC targets.

[00273] ERVK CTXLP also showed significant identity to the human agouti-family proteins, specifically ASIP and AGRP peptides. ASIP and AGRP are mammalian ICK peptides that both function as antagonists to melanocortin receptors 1 , 3 and 4 (MC1 R, MC3R, and MC4R) ³⁶ . ASIP is produced in the skin to promote pigment production, while AGRP is involved in metabolism ³⁶ . The agouti-family of peptides contains a unique ICK pattern ³³ .

CTXLP is only capable of forming 3 of the 4 cysteine bridges identified in agouti, suggesting that CTXLP takes on the basic ICK fold. The similarity between ERVK CTXLP to the agouti family of peptides provides further support for CTXLP's structure as an ICK peptide, along with first evidence for the presence of viral ICK peptides in humans.

[00274] Vascular endothelial growth factors (VEGFs) contain a growth factor cysteine knot motif, and are signalling molecules involved in angiogenesis ¹⁹ . ERVK CTXLP did not show significant similarity (≤ 7.7%) to the VEGF proteins. A dissimilar cysteine motif with a different spacing of cysteine residues and a significant difference in overall protein size (12-47 kDa for VEGF versus 32 and 51 kDa for CTXLP), suggests that ERVK CTXLP does not function in a growth factor or cytokine manner ¹⁹ . CTXLP's similarity to the ICK peptides and dissimilarity to VEGF suggests that ERVK CTXLP likely functions as a receptor antagonist via an ICK motif.

[00275] Cysteine-rich peptides have also been identified in exogenous retroviruses. ERVK CTXLP has some sequence similarity with the Tat accessory protein of HIV-1 and HIV- 2. Although Tat is not an ICK peptide and has a slightly different cysteine spacing pattern to ERVK CTXLP, a similar cysteine rich motif was identified in both proteins (19.6%). The cysteine-rich motif of Tat endows this protein with neurotoxic properties ³⁷ . Tat expression in the brains of HIV-1 infected patients has been associated with neuronal apoptosis via caspase activation and calcium accumulation ³⁸ . The structural similarities between Tat and ERVK CTXLP may suggest that they both use similar mechanisms for pathogenicity. HIV-2 is known to be a less pathogenic than HIV-1 ³⁹ . A partial explanation to this decreased pathogenicity may lie in the structural differences between their respective Tat proteins. HIV-2 Tat has a deletion of one cysteine, losing the "CC" motif. Interestingly, all ERVK CTXLP domains examined contained a "CC" motif. The mechanisms surrounding HIV Tat neurotoxicity are diverse and manifold ³⁸ ' ⁴⁰⁴¹ , suggesting that substantial research may be required to address potential ERVK CTXLP cellular toxicity in the CNS. The cysteine motif in HIV Tat has also been associated with increased HIV transactivation, by translocating to the nucleus and interacting with transcriptional machinery ³⁸ . HIV Tat can also transactivate ERVK ⁴² . Thus, the multiple functions of HIV-1 and HIV-2 Tat suggest that CTXLP's conserved cysteine motif may also contribute to neurotoxicity and retroviral transcription. Other retroviral proteins examined (HTLV Tax) did not show any homology to ERVK CTXLP, suggesting that this pathogenic mechanism is not conserved among all retroviruses.

[00276] Identification of CTXLP-encoding ERVK loci in the human genome

[00277] Nomenclature for each ERVK loci is based on their common names, as well as their chromosome location. Geneious was used to align both ERVK rec exon 1 and the predicted CTXLP DNA sequence with 95 ERVK HML-2 insertions identified in the human genome. After the ERVK insertions were aligned, many insertions (33) were excluded from further analysis due to the absence of an intact env. The ERVK insertions with an intact env were then aligned to both the rec gene and CTXLP cysteine motif nucleotide sequences. Figure 15 shows an example of the rec exon 1 and CTXLP nucleotide alignments against intact ERVK envelope genes. The CTXLP sequence was found at bp 1413 of env, just at the 3' end of the coding region for the envelope SU protein. This location is the cleavage site of the envelope polyprotein, where there is a junction between the SU and TM proteins. The aligned nucleotide sequences were then translated into an amino acid sequence and examined for intact Rec and CTXLP coding sequences. Table 3 shows that there are 25 ERVK insertions (out of the 62 examined) capable of producing a CTXLP protein, with 5 proviruses also capable of producing Rec.

Table 3: Sixty-two ERVK HML-2 human insertions and their chromosomal location examined for an intact Rec and intact CTXLP ORF along with any known disease associations with Multiple Sclerosis, Cancer or Schizophrenia. JN675073 15q25.2 ERVK_HML-2_15q25.2_84829020 No No

JN675074 16pll.2 ERVK_HML-2_16pll.2_ 34231474 No Yes

JN675075 17pl3.1 ERVK_HML-2_17pl3.1_7960357 No No

JN675076 19pl2a ERVK52 ^b _HML-2_ 19pl2a _20387400 No No

JN675077 19pl2b ERVK113 ^b _HML-2_19pl2b_21841536 Yes Yes

JN675078 19pl2c ERVK51 ^b _HML-2_19pl2c_22757824 No Yes

J N 675080 19qll ERVK-19 ^b _HML-2_19qll_228128498 Yes No

JN675081 19ql3.12a ERVK_HML-2_19ql3.12a_36063207 No No

JN675082 19ql3.12b ERVKOLD12309_HML-2_19ql3.12b_37597549 No No

JN675083 19ql3.41 ERVK3-6 ^a _HML-2_19ql3.41_53248274 No No

J N 675084 19ql3.42 LTR13 ^b _HML-2_19ql3.42_53862348 No No

JN675085 20qll.22 ERVK59 ^b _HML-2_20qll.22_32714750 No Yes

JN675086 21q21.1 ERVK-23 ^a _HML-2_21q21.1_19933916 No Yes

JN675087 22qll.21 ERVK-24 ^a _H M L-2_22q 11.21_18926187 No Yes

JN675088 22qll.23 ERVK-KOLD345b_HML-2_22qll.23_23879930 No No

JN675090 Xqll.l ERVK_HML-2_Xqll.l_61959549 No No

JN675094 Ypll.2 ERVK_HML-2_ Ypll.2_6 826441 No No

Disease associations: MS (yellow,) MS (No CTXLP; pale yellow), Cancer (Green), Cancer (No CTXLP; dark green), Schizophrenia (blue).

a) Mayer, J., Blomberg, J., & Seal, R. L. (2011). A revised nomenclature for transcribed human endogenous retroviral loci. Mobile DNA, 2(1), 7.

b) Subramanian, R. P., Wildschutte, J. H., Russo, C, & Coffin, J. M. (2011). Identification, characterization, and comparative genomic distribution of the HERV-K (HML-2) group of human endogenous retroviruses. Retrovirology, 8(1), 90.

[00278] Of the 95 ERVK DNA sequences, 33 were excluded due to an incomplete env sequence. The remaining 62 sequences were then translated and examined for an intact CTXLP in the appropriate reading frame. The resulting CTXLP peptide sequences were aligned and a sequence logo and consensus sequence were generated to assess amino acid conservation and detect polymorphisms (Figure 16).

[00279] In total, 25 ERVK insertions containing the CTXLP cysteine motif were analysed for overall conservation of the peptide sequence and to identify specific variants. Figure 16 shows that although each CTXLP peptide has a nearly identical amino acid sequence (identity 95.6%), there are distinct CTXLP polymorphisms identified.

[00280] Ten distinct CTXLP polymorphisms were detected. The most prevalent polymorphism is found in ERVK-3, ERVK-104, ERVK-10, ERVK-9, ERVK-14, ERVK-14(b), ERVK-8, ERVK-103, ERVK-25, ERVK-7, ERVK-21 , ERVK-16p11.2, and ERVK-113 (Allele 1). The second most prevalent substitutes glycine for serine, relative to the consensus, at two alignment positions (Ser16Gly, Ser27Gly) and is found in ERVK-5 and ERVK-20 (Allele 2). ERVK-HML-2_2q21.2 differs only at the latter Serine (Ser27Gly). ERVK-18 differs only at the former Serine (Ser16Gly). ERVK-51 has the former variation as well as a valine in position 20 (Ser16Gly, Ne20Val). ERVK-1 has phenylalanine at position 22 (Leu22Phe). ERVK-4 contains arginine at position 5 (Gly5Arg). ERVK-HML-2_12q13.2 contained an asparagine at position 16 (Ser16Asn). ERVK-23 shows three polymorphisms at positions 5, 17, and 27 (Gly5Arg, Arg17Lys, Val26Glu). The prevalence and polymorphic variability of ERVK CTXLP-encoding insertions suggests that CTXLP is a pervasive and conserved ERVK protein.

[00281] Out of the identified CTXLP encoding proviruses, 20 of 25 ERVK insertions were human-specific ⁴³ . ERVK-18, ERVK-5, ERVK-69, ERVK-20, ERVK-HML-2_16p21 , and ERVK- 51 are found in other primates including orangutan, chimpanzee, and rhesus monkey, demonstrating that ERVK CTXLP is an evolutionarily conserved protein, which either entered the genome of a common primate ancestor or through cross-species infection with a specific CTXLP-encoding ERVK virus ¹⁶ (See Figure 59 below in "primate models" section). The evolutionary age and clade of ERVK retroviruses that encode CTXLP suggests that CTXLP is unique to primates ⁴³⁴⁴ , and that the human genome has an enrichment of this type of ERVK proviruses.

[00282] A re-analysis of CTXLP variants in the human genome using a different methodology resulted in similar conclusion regarding the polymorphic nature of CTXLP+ ERVK genomes (Figure 17).

[00283] ERVK C TXLP domain and disease associations

[00284] CTXLP was identified in both type 1 and type 2 ERVK (Figure 1). The prevalence of CTXLP in both types of ERVK suggests that CTXLP originated early on in ERVK evolution, being present before the divergence of ERVK Δ env genomes ⁴⁵ . Select CTXLP+ loci had known disease associations (Table 3). Out of the 25 CTXLP-encoding ERVK insertions examined, no insertions were associated with ALS - most likely due to a lack of research on this topic. Two of the 25 insertions (ERVK-18 and ERVK-10) were associated with the psychiatric condition schizophrenia. Two out of 3 insertions associated with MS contained CTXLP-encoding insertions. ERVK expression has previously been associated with

neurological disease ⁶ , suggesting that CTXLP may be one mechanism for neurotoxicity.

Surprisingly, 9 CTXLP-encoding insertions were associated with cancer. ERVK env expression has previously been identified in several cancers ⁴⁶ . As we demonstrate below (see "CTXLP and disease" section, Figures 25, 36, 43 and 44), there is a notable association between CTXLP and cancer, suggesting that currently identified (Table 3) and other CTXLP-encoding loci with no known disease association may serve as future biomarkers for cancer. The different polymorphisms of CTXLP identified did not associate with any specific disease conditions. Any effects these polymorphisms have on CTXLP function remains unknown.

[00285] Identification of ERVK CTXLP - an alternate form of the ERVK envelope protein

[00286] Predicted full ER VK CTXLP protein sequence

[00287] The results of both the Pfam and NCBI-CDD databases indicated that the predicted CTXLP amino acid sequence (used to produce the CTXLP plasmids described below) shares similarities with both ERVK Rec, an oncogenic alternate splice product of the env gene, and ERVK Env. These results support the prediction that CTXLP is partially composed of the SU unit of the Env glycoprotein. The NCBI-CDD database also indicated the presence of a surface glycoprotein signal peptide domain. Lastly, the C-terminal portion of the CTXLP sequence was found to share similarities with the O-conotoxin superfamily, which ω- conotoxins are a part of. Lastly, the DUF4408 domain corresponds to a domain of unknown function which is primarily found in plants. Together, these results suggest that CTXLP is composed of the ERVK Env SU unit with a C-terminal ω-conotoxin domain (Figure 18).

[00288] Programmed frameshifting and internal ribosomal entry site

[00289] Since the reading frames of ERVK env (frame +1) and CTXLP (frame +3) differed by -1 , the ERVK env transcript (Figure 19) was examined for evidence of -1 programmed ribosomal frameshifting (-1 PRF), using ERVK-1 , ERVK-18, ERVK-7, ERVK_HML-2_2q21.1 , ERVK-5, ERVK-3, ERVK-4, ERKV-104, and ERVK-10 insertions.

[00290] RNAfold analysis of RNA structures in the ER VK env transcript

[00291] Our biomedical experiments suggested that there were CTXLP isoforms of different sizes, therefore we examined whether the conventional and alternative methionine start sites could be used to make both long and short CTXLP proteins. The first 350 bp of the ERVK Env-encoding RNA was inserted into RNAfold software to predict RNA secondary structure (http://rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi). Figure 19B suggests that the CTLXP proteins can originate from the conventional Env methionine start or potentially use IRES-like RNA hairpins to initiate translation at a downstream methionine.

[00292] This alternative mechanism for CTXLP expression is the use of internal ribosomal entry site (IRES), using an alternative translational start site (Figure 19B). The 51 and 32 kDa isoforms of CTXLP likely form using the start of the env ORF (nucleotide position 1) or an IRES in the env reading frame, starting from one of its many methionine start codons (specifically amino acid position 200). Translating env from alternative start codons would result in different sized isoforms of CTXLP. Certain viruses, including HIV, use IRES to allow for mRNA translation to begin in the middle of the transcript ⁴⁸⁴⁹ . The mRNA forms a hairpin structure that allows the binding of the 40S RNA subunit followed by protein translation ⁵⁰ . Using RNAfold software to predict mRNA secondary structure upstream of CTXLP, an RNA fold structure similar to HIV IRES was predicted (nucleotide 84 to 187, and 213 to 318 in env transcript), that could take advantage of alternate methionine start codon.

[00293] ERVK env nucleotide sequences were also inserted into RNAfold starting from 150 base pairs upstream of the CTXLP ORF to predict RNA secondary structure. RNA secondary structure was examined for evidence of -1 programmed ribosomal frameshifting motifs, including a slippery site with the X-XXY-YYZ form, a 5-10 nucleotide spacer and a downstream pseudoknot or hairpin structure (Figure 19C). The 1-350 bp region of the ERVK env gene was also examined for RNA secondary structure motifs, as it contains numerous alternative methionine start sites. RNA secondary structure was then examined for potential internal ribosomal entry site (IRES) binding sites, which take the form of complex RNA hairpins ⁴⁸ . The likelihood that these ERVK RNA secondary structures represent an IRES was determined using IRES prediction software called IRESite (http://iresite.org/IRESite_web.php), and reported as a similarity with known cellular and viral IRES 2D structures.

[00294] PRF can occur when three elements are combined: i) a slippery site containing an X-XXY-YYZ motif which after frameshifting by -1 results in XXX-YYY reading, ii) a 5 to 10 nucleotide spacer sequence, and iii) a downstream hairpin-type pseudoknot. ERVK CTXLP- encoding insertions contained an appropriate U-UUA-AAU slippery site to allow for -1 frameshifting to UUU-AAA. After the slippery site there was a 5 nucleotide spacer sequence before the CTXLP ORF. All sequences examined showed a strong probability of forming a hairpin-type pseudoknot within the RNA sequence encoding the CTXLP cysteine-rich motif (Figure 19C). Paradoxically, if the envelope protein translates past the CTXLP ORF start and frameshifts by -4, this introduces a conserved KRQK nuclear localization sequence (NLS) into the hypothetical protein (Figure 20). Another transcription factor that contains a KRQK NLS is N F-KB p50 ⁵¹ . There are no other known NLS in either the ERVK envelope protein or the predicted ERVK CTXLP proteins.

[00295] If CTXLP encoding originates from the conventional start site in the envtranscript, followed by a -1 PRF then it may produce a 51 kDa CTXLP protein. Alternatively, if CTXLP encoding originates from an IRES site using an alternate methionine in the env transcript (Figure 20), followed by a -1 PRF then it may produce a 32 kDa CTXLP protein. The insertions that showed the highest probability of forming a potential IRES binding site upstream of the CTXLP ORF were ERVK-113 and ERVK-4. These sequences analysed by IRESite software predict that the IRES-like RNA hairpins most resemble those found in HIV, Theiler's Murine Encephalomyelitis Virus (TMEV), and Hepatitis C virus (HCV).

[00296] We had previously hypothesized that ERVK CTXLP be produced via alternative splicing of the Rec transcript. Although alternative splicing is a common mechanism in retroviruses, only 4 of 25 CTXLP-encoding insertions also had intact Rec protein. The prevalence of CTXLP-encoding insertions in the absence of Rec suggests that alternative splicing is not the mechanism of CTXLP formation. Upstream and downstream of the CTXLP ORF are several stop codons. One mechanism that retroviruses use to compensate for stop codons or a lack of methionine starts is called programmed minus-one ribosomal frameshifting ⁵² . This involves the formation of an H-type pseudoknot in the RNA transcript at the site of frameshifting. The H-type pseudoknot would likely halt the ribosome from continuing translation, leading to the -1 PRF ⁵² . The slippery site UUU-AAA-U would re-establish ribosomal tRNA and mRNA base pairing and allow for the continuation of translation after frameshifting ⁵² . We predict that this mechanism could be used to extend the ORF of the ERVK SU protein by adding on a C-terminal CTXLP domain (Figures 19 & 20). Although the predicted structural stability of the ERVK env RNA H- type pseudoknot (Figure 18) was stronger in some ERVK insertions than others, the presence of this RNA secondary structure was conserved in all sequences examined using RNAfold. To our knowledge, frameshifting under conditions of inflammation has not been previously described. Enhanced frameshifting due to inflammation may be a unique mechanism in retroviruses, particularly upon exposure to TNFa (Figures 27 and 28).

[00297] Together, this data suggests that ERVK CTXLP is likely expressed as a cryptic peptide through frameshifted translation of the env transcript (Figures 19 & 20). Alternative forms of viral env proteins (CTXLP proteins may be considered isoforms of env) may be translated under specific physiological conditions ⁵³⁵⁴ . When ERVK env is translated in this proposed alternative ORF, the CTXLP peptide would be expressed within the translated env as a cryptic peptide. Cryptic peptides are proposed to have significantly different functions than their precursor protein ⁵⁵ . Cryptic epitopes within modified HIV proteins have been shown to be immunogenic ⁵⁶ ' ⁵⁷ .

[00298] Therefore, ERVK CTXLP is likely formed from a -4 PRF occurring slightly upstream of the furin cleavage site in env. An IRES sequence likely allows for a shorter ERVK CTXLP protein isoform to be produced, explaining the distinct isoforms of CTXLP identified. [00299] Prediction of post-translational modifications for CTXLP

[00300] ERVK CTXLP is predicted to have the following post-translational modifications, including, but not limited to phosphorylation, SUMOylation, glycosylation and lipid addition (Figures 20 & 21, Tables 4-8) using ELM software ⁴⁷ and other resources.

Table 4: Predicted phosphorylation sites within ERVK CTXLP protein

Predicted

Motif

Kinase phosphorylation ELM NetPhos3.1

Scan

sites

CDC2 41 •

213 •

279 •

281 •

321 •

374 •

376 •

417 •

438 •

475 •

482 •

CDK 184-191 •

CDK5 72 •

187 •

288 •

329 •

CK1 187-193 •

190-196 • •

198-204 •

235-241 •

276-282 •

281-287 •

293-299 •

329-335 •

373-379 •

CK2 32-35 • • ·

116-119 •

260-263 •

293 • ·

331-334 • • ·

411-414 •

DNAPK 298 •

374

400 •

EGF 272 •

GSK3 54-61

82-89

210-217 •

281-288

350-357

397-404

409-416

INSR 160 •

LATS 304-310 •

MAPK/PDK 69-75

184-190 •

285-291

326-332

443-349

NEK2 64-69

82-87 •

86-91

371-376

460-465

495-500

PIKK 54-60 •

67-79 276-282 •

397-403 •

PKA 54-60 •

155 •

190-196 •

250 •

340-346 •

353 •

491 •

PKC 24-26 • ·

41-43 • ·

57 •

117 •

193 •

200-203 • ·

216-218 • ·

279 •

281 •

298 •

309 •

321-324 • ·

343 •

360 •

376 •

381 •

412 •

417 •

462-464 • ·

PLK 414-420 •

p38 MAPK 72 •

187 •

329 •

Table 5: Predicted SUMOylation and SUMO interaction sites within ERVK CTXLP protein

G PS- Predicted SUMOylation sites ELM

¹ SUMO

143-146

397

Predicted SUMO interaction sites

123-127

180-184

367-374

424-428

Table 6: Predicted glycosylation sites within ERVK CTXLP protein

Predicted

Motif

Sugar attachment glycosylation ELM Netglyc4.0

Scan sites

Fucose 275-282

408-413

487-492

Glycosaminoglycan 197-200

249-252

275-278

283-286

331-334

352-355

N-glycosylation 99-102

127-130

152-155

273-276

354-357

371-374

460-463

472-475

479-482

O-glycosylation 213

217

235

284

288

321

Table 7: Predicted lipid attachment sites within ERVK CTXLP protein

CSS- GPS-

Lipid attachment Predicted lipid addition sites

Palm Lipid

S-Farnesylation 497 •

503 •

S-

Geranylgeranylation 503 •

S-palmitoylation 140 •

141 •

227 •

275 •

382 •

408 •

487 • ·

488 •

[00301] The N-linked glycosylation of ERVK CTXLP has been verified experimentally using PNGase treatment of CTXLP protein fractions, followed by western blot analysis for shifts in high molecular weight protein banding patterns (Figure 26, n=3). Phosphorylation is postulated to occur to ERVK CTXLP when bound to chromatin, as seen in the 32kDa band shift in Figure 30.

[00302] Moreover, bioinformatic predictions also predicted protein cleavage and interaction sites within CTXLP (Table 8) using the PROSPER website

(https://prosper.erc.monash.edu.au/home.html). Among the proteins predicted to cleave CTXLP were HIV protease, furin, NEC1 , and NEC2. The predicted furin cleavage site is consistent with the location of the known furin cleavage site that typically cleaves the Env polyprotein into discrete SU and TM peptide chains that are then assembled into multimer proteins. However, it is unclear how an overlapping NRS N-linked glycosylation site would impact the ability of furin the cleave the site. As well, the predicted cleavage by HIV protease is interesting as ERVK interactions with HIV proteins have been previously reported ⁴² ' ^{58 59} . Of note, cleavage predictions only take into account primary amino acid sequence only, and do not account for how viral protein tertiary structure and cellular factors come into play. Table 8: Predicted protease cleavage sites within CTXLP, as predicted by PROSPER.

N- C- fragment fragment Cleavage

Merops ID Protease Name Position P4-P4' site (kDa) (kDa) score

A02.001 HIV-1 retropepsin SEQ ID NO: 139 TEVL|WEEC 15.57 30.67 1.24

A02.001 HIV-1 retropepsin SEQ ID NO: 140348 KGVLI IQKI 41.54 4.69 1.05

A02.001 HIV-1 retropepsin SEQ ID NO: 141 277 PYML| VVGN 33.14 13.09 0.99

A02.001 HIV-1 retropepsin ^{SEQ 10 N0: 142} 71 WLVE|VPTV 8.5 37.74 0.94

C01.036 cathepsinK SEQ ID NO: 143268 VPLQISCVK 32.12 14.11 1.15

C01.036 cathepsinK SEQ ID NO: 144201 FYLW| EWEE 23.97 22.26 1.15

C01.036 cathepsinK SEQ ID NO: 145164 HNCS|GQTQ 19.72 26.51 1.14

C01.036 cathepsin K SEQ ID NO: 146196 KKLQISFYL 23.28 22.96 1.13

C01.036 cathepsinK SEQ ID NO: 147 VILQl NNEF 17.04 29.2 1.12

C01.036 cathepsinK SEQ ID NO: 148 78 VSPN|SRFT 9.19 37.04 1.1

C01.036 cathepsinK SEQ ID NO: 149341 HILT I EILK 40.67 5.56 1.09

C01.036 cathepsinK SEQ ID NO: 150247 QTLE |TRYR 29.6 16.63 1.08

C01.036 cathepsin K SEQ ID NO: 151199 QSFY| LWEW 23.68 22.56 1.08

C01.036 cathepsinK SEQ ID NO: 152238 HHIR|IWSG 28.46 17.78 1.06

C01.036 cathepsinK SEQ ID NO: 153 23 VWVP|GPTD 2.67 43.57 0.98

C01.036 cathepsinK SEQ ID NO: 154223 SGPE I HPEL 26.62 19.61 0.97

C01.036 cathepsinK SEQ ID NO: 155209 KGIS|TPRP 25.05 21.18 0.92

M10.003 matrix metallopeptidase-^ ^{0 ID N0: 156} 88 MVSG I MSLR 10.47 35.76 1.15

M10.004 matrix metallopeptidase ID NO: 15784 FTYH I MVSG 9.98 36.25 1.33

M10.004 matrix metallopeptidase ID NO: NPIE|VYVN 1.51 44.72 1.28

SEQ ID NO:

M10.004 matrix metallopeptidase-9 159 276 PPYM I LVVG 33.03 13.21 1.26

M10.004 matrix metallopeptidase ID NO: 65 MPAVIQNWL 7.73 38.51 1.19

M10.004 matrix metallopeptidase-9 SE ^Q I ₆ D NO: i _Ο RPKG I KTCP 13.6 32.64 1.15

M10.004 matrix metallopeptidase-9 SE ^Q ₁ I ₆ D ₂ NO: ₁ ! -> CPSAIQVSP 20.69 25.54 1.14

„ SEQ ID NO: _Λ

M10.004 matrix metallopeptidase-9 163 -L" NTEV| LWEE 15.45 30.78 1.13

SEQ ID NO:

M10.004 matrix metallopeptidase-9 164 213 TPRP I KIIS 25.5 20.73 1.12

SEQ ID NO:

M10.004 matrix metallopeptidase-9 165 00 MVSG I MSLR 10.47 35.76 1.11

M10.004 matrix metallopeptidase-9 157 APRG IQFYH 18.84 27.39 1.09

M10.004 matrix metallopeptidase-9 6 TPVT|WMDN 0.62 45.61 1.08

M10.004 matrix metallopeptidase-9 ^SEQ ,6 ^D ₈ ^NO: 90 SGMS| LRPR 10.69 35.54 1.08

M10.004 matrix metallopeptidase-9 SE ^Q ID NO: ₁ - ₃ >o _Q VANS|VVIL 16.48 29.75 1.08

. , „ SEQ ID NO:

M10.004 matrix metallopeptidase-9 170 2/5 KPPY|MLVV 32.89 13.34 1.08

SEQ ID NO:

M10.004 matrix metallopeptidase-9 171 182 VDSD| LTES 21.59 24.64 1.07

SEQ ID NO:

M10.004 matrix metallopeptidase-9 ⁷² 38 EEEG 4.48 41.75 1.06

SEQ ID NO: I MMIN

M10.004 matrix metallopeptidase-9 ⁷³ 339 SIHI I LTEI 40.46 5.78 1.05 SEQ ID NO:

M10.004 matrix metallopeptidase-9 174 4 MVTP|VTWM 0.42 45.81 1.05

„ SEQ ID NO:

M10.004 matrix metallopeptidase-9 175 274 VKPP|YMLV 32.73 13.5 1.05

SEQ ID NO:

M10.004 matrix metallopeptidase-9 176 362 SDYG|INCS 43.37 2.86 1.04

SEQ ID NO:

M10.004 matrix metallopeptidase-9 177 281 VVGN|IVIK 33.62 12.61 1.04

„ SEQ ID NO:

M10.004 matrix metallopeptidase-9 178 351 LIQK 11 HFY 41.91 4.32 1.03

M10.004 matrix metallopeptidase-9 SEQ ₁ I ₇ D ₉ NO: 266 LTVP 1 LQSC 31.88 14.35 1.03

SEQ ID NO

M10.004 matrix metallopeptidase-9 180 313 H IL| LV A 37.36 8.88 1.02

SEQ ID NO:

M10.004 matrix metallopeptidase-9 181 347 LKGV| LIQK 41.43 4.81 1.02

SEQ ID NO

M10.004 matrix metallopeptidase-9 182 39 EEGM 1 MINI 4.61 41.62 1.01

M10.004 matrix metallopeptidase-9 SEQ ID NO

i ₈₃ 385 SVSK 1 LC 46.02 0.21 0.99

M10.005 matrix metallopeptidase SEQ ID NO:

-3 134 173 CPSAIQVSP 20.69 25.54 1.16

SEQ ID NO:

M10.005 matrix metallopeptidase-3 185 137 CVAN|SVVI 16.4 29.84 1.02

SEQ ID NO:

M10.005 matrix metallopeptidase-3 iee 288 KPAS 1 QTIT 34.33 11.9 0.99

SEQ ID NO:

M10.005 matrix metallopeptidase-3 187 276 PPYM 1 LVVG 33.03 13.21 0.96 chymotrypsin A

S01.001 (cattle-type) SEQ ID NO: 188 142 VVILIQNNE 16.91 29.33 1.02 chymotrypsin A

S01.001 (cattle-type) SEQ ID NO: 189 83 RFTY| HMVS 9.85 36.39 1.01 chymotrypsin A

S01.001 (cattle-type) SEQ ID NO: 190 228 PELW| RLTV 27.29 18.95 0.97

S01.131 elastase-2 SEQ ID NO: 191 219 ISPV|SGPE 26.14 20.1 1.41

S01.131 elastase-2 SEQ ID NO: 192 352 IQKI 1 HFYF 42.02 4.21 1.37

S01.131 elastase-2 SEQ ID NO: 193 140 NSVV|ILQN 16.68 29.55 1.21

S01.131 elastase-2 SEQ ID NO: 194 377 RSCI|ALFC 45.19 1.05 1.18

S01.131 elastase-2 SEQ ID NO: 195 52 YPPI|CLGR 6.23 40.01 1.18

S01.131 elastase-2 SEQ ID NO: 196 20 NDSV|WVPG 2.29 43.95 1.17

S01.131 elastase-2 SEQ ID NO: 197 315 ILLV| RARE 37.57 8.66 1.16

S01.131 elastase-2 SEQ ID NO: 198 43 MINI ISIGY 5.08 41.15 1.12

S01.131 elastase-2 SEQ ID NO: 199 278 YMLV| VGNI 33.24 13 1.11

S01.131 elastase-2 SEQ ID NO: 200 86 YHMV|SGMS 10.21 36.02 1.09

S01.131 elastase-2 SEQ ID NO: 201 237 SHHI|RIWS 28.3 17.93 1.09

S01.131 elastase-2 SEQ ID NO: 202 349 GVLI IQKIH 41.65 4.58 1.09

S01.131 elastase-2 SEQ ID NO: 203 65 MPAVIQNWL 7.73 38.51 1.09

S01.131 elastase-2 SEQ ID NO: 204 41 GMMI|NISI 4.86 41.38 1.08

S01.131 elastase-2 SEQ ID NO: 205 5 VTPV|TWMD 0.52 45.71 1.07

S01.131 elastase-2 SEQ ID NO: 206 233 LTVA|SHHI 27.83 18.41 1.07

S01.131 elastase-2 SEQ ID NO: 207 151 GTII|DWAP 18.04 28.19 1.07

S01.131 elastase-2 SEQ ID NO: 208 45 NISI 1 GYHY 5.28 40.95 1.04

S01.131 elastase-2 SEQ ID NO: 209 129 NTEV| LWEE 15.45 30.78 1.02

S01.131 elastase-2 SEQ ID NO: 210 325 WIPV|STDR 38.88 7.35 1

S01.133 cathepsinG SEQ ID NO: 211 214 PRPK 1 MSP 25.63 20.61 1.33 S01.133 cathepsin G SEQ ID NO: 212 160 GQFY | HNCS 19.28 26.96 1.24

S01.133 cathepsin G SEQ ID NO: 213 40 EGMM 11 N IS 4.74 41.49 1.18

S01.133 cathepsin G SEQ ID NO: 214 277 PYML | VVGN 33.14 13.09 1.17

S01.133 cathepsin G SEQ ID NO: 215 260 IDLN I SILT 31.27 14.96 1.11

S01.133 cathepsin G SEQ ID NO: 216 15 IEVY | VNDS 1.77 44.46 1.1

S01.133 cathepsin G SEQ ID NO: 217 240 I IW | SGNQ 28.76 17.48 1.09

S01.133 cathepsin G SEQ ID NO: 218 8 VTWM | DNPI 0.94 45.29 1.06

S01.133 cathepsin G SEQ ID NO: 219 83 RFTY | HMVS 9.85 36.39 1.05

S01.133 cathepsin G SEQ ID NO: 220 47 SIGY | HYPP 5.62 40.61 1.04

S01.133 cathepsin G SEQ ID NO: 221 313 HRIL| LVRA 37.36 8.88 1.03

S01.133 cathepsin G SEQ ID NO: 222 276 PPYM 1 LVVG 33.03 13.21 1.01

S01.133 cathepsin G SEQ ID NO: 223 199 QSFY | LWEW 23.68 22.56 0.99

S01.133 cathepsin G SEQ ID NO: 224 321 REGM | WIPV 38.38 7.85 0.99

S01.133 cathepsin G SEQ ID NO: 225 39 EEGM 1 MINI 4.61 41.62 0.96

S01.133 cathepsin G SEQ ID NO: 226 361 CSDY | GINC 43.2 3.04 0.94

S01.133 cathepsin G SEQ ID NO: 227 7 PVTW | MDNP 0.81 45.42 0.93

S01.133 cathepsin G SEQ ID NO: 228 89 VSGM | SLRP 10.6 35.63 0.92

S01.217 thrombin SEQ ID NO: 229 214 PRPK I MSP 25.63 20.61 1.03

S01.269 glutamyl peptidase |SEQ ID NO: 230 319 RARE | GMWI 38.08 8.15 1.04

S01.269 glutamyl peptidase |SEQ ID NO: 231 185 DLTE | SLDK 21.94 24.3 1 thylakoidal processing

S26.008 peptidase SEQ ID NO: 232 102 QDFS | YQRS 12.12 34.11 0.94 signalase (anima I) 21 kDa

S26.010 component SEQ ID NO: 233 317 LVRA | REGM 37.8 8.44 0.95

[00303] Lastly, the predicted protein interactions of cellular proteins and CTXLP were equally intriguing (Table 9). Among the predicted interactions were proteins involved in cell cycle regulation such as MAPK and l_ATS. As well, interactions were also predicted with proteins involved in innate immunity such as UPS-7/HAUSP, TRAF-2 and TRAF-6, which are upstream of NF-κΒ in inflammatory signalling.

Table 9: Predicted protein interaction partners of CTXLP using ELM software.

Predicted protein interaction

Predicted CTXLP interaction sites partner

B CA1 216-220

Calcineurin 90-93

375-378

Cyclin 216-220

423-427

Dyenein 66-72

MAPKs 233-242

361-371

363-371

428-437

462-470

464-470

MAPKs (ERK1/2 and p38

subfamilies) 425-437

427-439

Pinl 69-74

184-189

285-290

326-331

443-448

Protein phosphatase 1

(catalytic subunit) 112-119

214-221

465-471

STAT5 63-66

101-104

106-109

126-129

208-211

367-370

471-474

SUMO 179-185

367-374

TRAF2 32-35

145-148 T AF6 310-318

USP7/HAUSP 219-223

225-229

229-233

232-236

290-294

301-305

378-382

445-449

[00304] Design of a custom ERVK CTXLP antibody

[00305] Pierce Custom antibody services has produced a CTXLP-specific polyclonal rabbit antibody, used in all the experiments described below. The predicted epitopes are listed in Figure 22. The rabbit protocol and immunization plan is stated in Figure 23.

[00306] Design of a custom ERVK CTXLP vector, and complementary ERVK Env and ERVK SU vector

[00307] GenScript custom plasmid services has produced an ERVK CTXLP- expressing vector within a pcDNA3.1 backbone, used in all the experiments described below.

We also synthesized a matching ERVK SU vector as a complementary plasmid devoid of the

CTXLP domain. The sequences used to produce the vectors are listed below:

[00308] ERVK SU + ERVK CTXLP (Based on ERVK113): 57.14 kDa [SEQ ID NO:2]

[00309] MNPSEMQRKAPPRRRRHRNRAPLTHKMNKMVTSEEQMKLPSTKKAEPPT

WAQLKKLTQLATKYLENTKVTQTPESMLLAALMIVSMVVSLPMPAGAAAANYTYWAY VPFP

PLIRAVTWMDNPIEIYVNDSVWVPGPTDDCCPAKPEEEGMMINISIGYRYPPICLGR APGCL

MPAVQNWLVEVPTVSPISRFTYHMVSGMSLRPRVNYLQDFSYQRSLKFRPKGKPCPK EIP

KESKNTEVLVWEECVANSAVILQNNEFGTLIDWAPRGQFYHNCSGQTQSCPSAQVSP AVD

SDLTESLDKHKHKKLQSFYPWEWGEKGISTARPKIISPVSGPEHPELWRLTVASHHI RIWSG

NQTLETRDRKPFYTIDLNSSLTVPLQSCVKPPYMLVVGNIVIKPDSQTITCENCRLL TCIDSTF

NWQHRILLVRAREGVWIPVSMDRPWEASPSVHILTEVLKGVLNRSKRQKIHF

YFNCSDYGINCSHSYGCCSRSCIALFCSVSKLC*

[00310] First portion is ERVK Env SU, aa residues 1-465 (furin cleavage site) of ERVK 113 (19p12b) with normal frame. Second portion is ERVK CTXLP, aa residues 463-500 of ERVK 113 (19p12b) with +3 reading frame and no start codon bias. Env SU ends at KR before bolded font indicating where Env CTXLP starts (QK). Bolded portion of sequence represents the allele 1 portion of Env CTXLP. [00311] NRS is an N-linked glycosylation site, RSKR is the furin cleavage site, KRQK is the nuclear localization sequence (NLS). NRS may mask furin site allowing for NLS function.

[00312] ERVK Env (Based on ERVK113): 79.2 kDa [SEQ ID NO. 234]

[00313] MNPSEMQRKAPPRRRRHRNRAPLTHKMNKMVTSEEQMKLPSTKKAEPPT

WAQLKKLTQLATKYLENTKVTQTPESMLLAALMIVSMVVSLPMPAGAAAANYTYWAY VPFP

PLIRAVTWMDNPIEIYVNDSVWVPGPTDDCCPAKPEEEGMMINISIGYRYPPICLGR APGCL

MPAVQNWLVEVPTVSPISRFTYHMVSGMSLRPRVNYLQDFSYQRSLKFRPKGKPCPK EIPK

ESKNTEVLVWEECVANSAVI LQN N EFGTLI DWAPRGQFYH NCSGQTQSCPSAQVSPAVDS

DLTESLDKHKHKKLQSFYPWEWGEKGISTARPKIISPVSGPEHPELWRLTVASHHIR IWSGN

QTLETRDRKPFYTIDLNSSLTVPLQSCVKPPYMLVVGNIVIKPDSQTITCENCRLLT CIDSTFN

WQHRILLVRAREGVWIPVSMDRPWEASPSVHILTEVLKGVLNRSKRFIFTLIAVIMG LIAVTAT

AAVAGVALHSSVQSVNFVNDWQNNSTRLWNSQSSIDQKLANQINDLRQTVIWMGDRL MSL

EHRFQLQCDWNTSDFCITPQIYNESEHHWDMVRCHLQGREDNLTLDISKLKEQIFEA SKAHL

NLVPGTEAIAGVADGLANLNTVTWVKTIGSTTIINLILILVCLFCLLLVYRCTQQLR RDSDHRER

AMMTMVVLSKRKGGNVGKSKRDQIVTVSV*

[00314] -^aa residues 1-465 (furin cleavage site- R-X-K/R-Rj) of ERVK 113 (19p12b).

[00315] -^aa residues 466-699 (furin cleavage site to end of aa sequence) of ERVK 1 13

(19p12b). Bolded portion of sequence represent the ISU domain of ERVK TM.

[00316] ERVK SU (Based on ERVK113): 52.86 kDa [SEQ ID NO: 3]

[00317] MNPSEMQRKAPPRRRRHRNRAPLTHKMNKMVTSEEQMKLPSTKKAEPPT

WAQLKKLTQLATKYLENTKVTQTPESMLLAALMIVSMVVSLPMPAGAAAANYTYWAY VPFP

PLIRAVTWMDNPIEIYVNDSVWVPGPTDDCCPAKPEEEGMMINISIGYRYPPICLGR APGCL

MPAVQNWLVEVPTVSPISRFTYHMVSGMSLRPRVNYLQDFSYQRSLKFRPKGKPCPK EIP

KESKNTEVLVWEECVANSAVI LQN N EFGTLI DWAPRGQFYH NCSGQTQSCPSAQVSPAVD

SDLTESLDKHKHKKLQSFYPWEWGEKGISTARPKIISPVSGPEHPELWRLTVASHHI RIWSG

NQTLETRDRKPFYTIDLNSSLTVPLQSCVKPPYMLVVGNIVIKPDSQTITCENCRLL TCIDSTF

NWQH Rl LLVRAREGVWI PVSM DRPWEASPSVH I LTEVLKGVLN RSKR*

[00318] -^aa residues 1-465 (furin cleavage site- R-X-K/R-Rj) of ERVK 113 (19p12b).

[00319] CTXLP biological characterization

[00320] The ERVK CTXLP domain is found in several distinct proteinaceous forms

[00321] Western blot analysis of ERVK-expressing NCCIT cells [00322] Whole cell extract of ERVK-expressing NCCIT cells and an immunoprecipitated (IP) fraction enriched for CTXLP were analyzed by Western blot (Figure 25). In the whole cell extract, major bands were identified at 90 and 110 kDa, suggesting this may be the predominant form of CTXLP found in the cell. Unmodified CTXLP would be expected to correspond to a 51 kDa band; thus, is it possible that heavier bands are due to PTM such as glycosylation, phosphorylation or sumoylation. It should be noted that higher molecular weight bands are also observed in other cell types (see below). It is likely that these heavier bands reflect PTM including phosphorylation (~ 2 kDa) and glycosylation (≥ 2 kDa) (Figures 21 and 26) or sumoylation. The possibility that the higher molecular weights reflect post-translational modification of the 32 and 51 kDa CTXLP isoforms through glycosylation, would be in accordance with the results of the bioinformatic analysis which indicate that ERVK Env and CTXLP proteins are heavily glycosylated (Figure 21, Table 6, Figure 26) ¹⁵ ' ⁶⁰ · ⁶¹ . Another possibility for observing larger than expected protein bands is detergent-resistant multimerization, as retroviral envelope proteins form trimers ¹⁵ . Light bands were also observed at 51 kDa and 32 kDa, the latter form possibly due to an alternative start site within or cleavage of CTXLP protein.

[00323] In the CTXLP-enriched fraction, the most heavily enriched band was detected at 51 kDa. In this fraction, CTXLP-reactive bands were also found at 110 and 142 kDa, which are also possible results of protein PTM or multimerization. Lighter bands were found at 26 and 29 kDa, again these bands suggest CTXLP cleavage or alternative methionine start site products.

[00324] ERVK CTXLP is inducible through the action of pro-inflammatory signalling

[00325] Astroctye expression of ERVK CTXLP in the presence of pro-inflammatory agents

[00326] NCCIT, used as control cells, were cultured along side- SVGA cells. In addition to higher molecular weight bands (not shown) NCCIT cells demonstrated a 51 kDa band and 32 kDa band. Unlike NCCIT cells, in the astrocytic cell line SVGA ERVK CTXLP protein is spontaneously expressed at low levels, with a minor 32 kDa protein being apparent (Figure 25). However, CTXLP expression in SVGA cells was upregulated upon treatment with proinflammatory cytokines tumor neurosis factor (TNFa) and LIGHT (lymphotoxin-like inducible member of the TNF superfamily protein) (Figure 27). In contrast to CTXLP expression, Env protein expression was not induced by pro-inflammatory stimulus. [00327] As with NCCIT cells, larger 90/110 kDa CTXLP reactive bands were also observed upon TNFa or LIGHT treated SVGA andReNcell neurons (Figures 28, 30, 56 and 58). As detailed above, these larger bands may represent post-translational modification (PTM) of CTXLP isoforms.

[00328] The localization of ERVK CTXLP expression is cell type and inflammation dependent

[00329] Ubiquitous expression of ER VK CTXLP expression in NCCIT cells

[00330] The localization of CTXLP protein expression was examined through Western blot analysis of cellular fractions and confocal imaging. In NCCIT cells, endogenous CTXLP protein appeared ubiquitously expressed and localizes to the cytoplasm, nucleus and chromatin enriched fractions. CTXLP protein was also found in both the soluble and insoluble NCCIT whole cell lysates (Figure 29 A and B). The latter further suggests that there is interaction of CTXLP with cell membranes. The ubiquitous nature of CTXLP was supported by confocal imaging (Figure 27 C).

[00331] CTLXP expression was also associated with indicators of autophagy in NCCIT cells (Figure 29B). Autophagy occurs when dysfunctional proteins and damaged organelles accumulate within a cell, and has been associated with cell dysfunction in neurodegenerative disease ⁶² . NCCIT cells exhibited insoluble caspase-3, which is an indicator for autophagy ⁶³ . There was also evidence of LC3B cleavage (data not shown) which occurs during autophagy.

[00332] Nuclear Localization of ERVK C TXLP expression in S VGA cells

[00333] In contrast to NCCIT cells, in SVGA cells endogenous CTXLP protein localization occurred predominantly in the chromatin cellular fraction (Figure 29A) and was poorly detected by confocal imaging (Figure 29C). However, CTXLP protein levels were strongly elevated in the nucleus upon treatment of 0.1 and 1 ng/mL TNFa when compared to cells alone (Figure 30). This increase in CTXLP expression upon treatment of astroctyes with low levels of TNFa or LIGHT, suggests that chronic, low level inflammation may augment CTXLP levels physiologically. Moreover, with pro-inflammatory cytokine exposure, CTXLP puncta formed within the nucleus, but staining was excluded from the nucleoli (Figure 31). Higher resolution images of CTXLP expressing astrocytes showed that CTXLP puncta also formed in the cytoplasm and on the cell surface, suggesting that potential isoforms of CTXLP may have location-specific functions (Figure 31 B).

[00334] Enhanced CTXLP expression was also associated with enhanced cytoplasmic RT expression (Figure 30), suggesting regulation of global ERVK gene expression may be linked. Pro-inflammatory cytokines have been shown to induce ERVK expression ^64-67 . During inflammatory events, ERVK Env has been shown to be either neuroprotective ⁶⁸ and neuropathologicaP. Indeed, chronic exposure to TNFa may facilitate frameshifting in ERVK env translation leading to CTXLP being produced as a cryptic protein. TNFa expression and its subsequent downstream signalling cascade products may result in enhanced IRES-dependant translation ⁷⁰ , promoting the formation of the smaller CTXLP isoform. Nonetheless, the observation that ERVK CTXLP did not co-localize with ERVK Env protein which localized (Figure 31), suggesting that these proteins may have different localization sequences and/or patterns. Thus, the relationship between CTXLP and Env in ERVK gene regulation remain unclear.

[00335] Overexpression of the CTXLP cysteine-rich domain in SVGA cells also resulted in increased CTXLP 32 kDa and 90/110 kDa protein bands (Figure 32), suggesting that this protein domain is sufficient to enhance its own expression.

[00336] ERVK CTXLP binds chromatin

[00337] Consistent with our observation that CTXLP is enriched in the chromatin fraction (Figure 29), DNABIND prediction (http://dnabind.szialab.org/) ⁷¹ predicts that CTXLP binds DNA. Prediction parameters were as follows: false positive rate of 6%, expected sensitivity of 58.7%, expected Matthews correlation coefficient of 0.55, score threshold is set to 1.577 (threshold probability of 0.8288). Prediction results of the CTXLP sequence were a score of 1.771 and a probability of DNA binding of 0.8546.

[00338] An analysis is DNA interactions revealed that CTXLP bound the interferon response elements (ISREs) within the ERVK viral promoter (5' LTR) (Figure 33). This binding is enhanced in the presence of inflammatory cytokine stimuli, and is distinct in select cell types (Figure 33). This suggests that CTXLP, and specifically the 32kDa form of CTXLP (Figure 24), may bind DNA and regulate gene expression. Indeed, preliminary data suggest that CTXLP can alter both ERVK expression (Figure 32) and the transcription of NF-κΒ transcripts (see below). Further, CTXLP may alter the gene expression patterns of numerous viral and cellular genes containing an ISRE elements in their promoters ^{72 73} . Thus, CTXLP may regulate ERVK gene expression, as well as other genes containing ISREs.

[00339] In summary, CTXLP protein isoform expression in NCCIT and SVGA cells was elucidated by Western blots which indicated presumed isoform sizes of 32 kDa, 51 kDa, and 90/110 kDa. In NCCIT cells, endogenous CTXLP is ubiquitously expressed being present in the nucleus and also identified in the cytoplasm and cell membrane, based on cell fractionation and confocal experiments. In contrast, in SVGA cells basal CTXLP levels are limited, but highly inducible by pro-inflammatory stimuli. In addition, CTXP expression in almost exclusively in the chromatin fraction and demonstrates a prominence in the nucleus upon confocal imaging. The notable exception is that after pro-inflammatory activation for 24 hours CTXLP puncta appear in the cytoplasm and on cellular membranes reminiscent of pathogenic protein aggregates. Moreover, the localization pattern in response to pro-inflammatory activators resulting in a prominence in the nucleus (Figures 29-31), ability to bind chromatin (Figures 29 and 33) and absence from the nucleoli (Figure 31) suggests that CTXLP may be involved in viral transcription. A primary candidate as a viral transcription factor is the 32 kDa CTXLP isoform, as small cysteine-rich proteins have previously been identified as transcriptional activators ⁷⁴ - ⁷⁵ , as per HIV-1 Tat (15 kDa) and HTLV Tax (40 kDa) role as viral transcription co-activators ⁷⁶ - ⁷⁷ . Additionally, low basal CTXLP staining in non-diseased cells suggests that it might have a role in normal physiology and gene regulation processes.

[00340] CTXLP Expression in Disease States

[00341] CTXLP is expressed in vivo in humans

[00342] RNAseq analysis of CTXLP+ transcripts in disease states

[00343] To evaluate the significance of CTXLP in disease, we evaluated the expression of CTXLP encoding ERVK loci in publicly available RNA-Seq datasets in the Sequence Read

Table 10: RNA-Seq datasets in the Sequence Read Archive (SRA) used for the analysis of ERVK expression.

Archive (SRA) (Table 10). This analysis is summarized in Table 11 and Figures 34-37. These loci were identified by searching the SRA by disease affiliation and then evaluating each potential study based on samples size, tissue and sequencing quality. Preference was given to studies with large sample sizes, autologous controls, ex vivo disease-relevant tissue, and high sequencing quality. Paired-end reads were preferred to single-end. We focused on studies with fewer measures selecting for particular RNA sub-populations, which could have depleted ERVK RNA from the input. [00344] The studies examine included breast cancer, prostate cancer, Amyotrophic Lateral Sclerosis (ALS), Multiple Sclerosis (MS), Rheumatoid Arthritis (RA), schizophrenia, bipolar disorder and HIV+/HCV+ infection. ERVK expression by HML group is summarized in Table 11. We found that the overall expression of ERVK in these disease states was low, and all HML groups were expressed. ERVK expression was highest in blood and cancerous tissue. In addition, we found loci with significantly different expression between patients and controls, but these were different for each study. Breast cancer, prostate cancer, and Multiple Sclerosis datasets contained expression patterns which could potentially distinguish patients from controls. These patterns were driven by differences in expression of CTXLP- loci and loci with inactivating Env mutations.

Table 11: ERVK expression by HML group in RNAseq datasets

study hml Minimum Median Mean SD Maximum

HML1 0 0.0125001 0.1179662 0.3213379 11.465101

HML 10 0 0.0389112 0.1801432 0.3599358 5.855237

HML2 0 0.0214437 1.0386104 28.9656795 2800.179981

HML3 0 0.0085536 0.0772390 0.1934535 5.249185

HML4 0 0.0274862 0.0863698 0.1811918 3.655131

HML5 0 0.0000000 0.0859580 0.2350117 4.212269

HML6 0 0.0000000 0.1133778 0.3016479 4.628222

SRP090259 HML7 0 0.0128447 0.0602345 0.1412095 3.037872

HML8 0 0.0193117 0.2915583 11.7006947 1289.465670

K14C 0 0.0000000 0.0542510 0.1667601 3.168318

LTR22 B 1 #LTR/ E RVK 0 0.0000000 0.1161527 0.3674700 8.431398

LTR22B2#LTR/ERVK 0 0.0000000 0.1241151 0.3284346 2.537287

LTR22C2#LTR/ERVK 0 0.0000000 0.0826217 0.2326312 2.358517

LTR22 E#LTR/E RVK 0 0.0000000 0.1423927 0.3607523 3.823776

LTR3B_#LTR. ^' ERVK 0 0.0000000 0.0855171 0.2227743 3.975364

HML 1 0 0.0000000 0.1061961 0.3018500 3.824492

HML 10 0 0.0000000 0.1789209 0.4687300 7.527670

HML2 0 0.0072385 0.1691623 1.8123759 197.784008

HML3 0 0.0000000 0.0917908 0.3355194 12.499732

HML4 0 0.0000000 0.1252037 0.2851307 5.880441

HML5 0 0.0000000 0.0909087 0.3553731 9.824726

HML6 0 0.0000000 0.1070234 0.2950223 3.474829

SRP074904 HML7 0 0.0000000 0.0786869 0.2005897 2.337186

HML8 0 0.0000000 1.0845542 56.4008441 5752.139071

K14C 0 0.0000000 0.0566177 0.1889185 2.725355

LTR22 B 1 #LTR/E RVK 0 0.0000000 0.0929150 0.2638750 4.531172

LTR22 B2#LTR: E RVK 0 0.0000000 0.1643443 0.5538800 6.122761

LTR22C ^' 2#LTR/E RVK 0 0.0000000 0.0697017 0.2267312 3.086240

LTR22 E#LTR.E RVK 0 0.0000000 0.1307125 0.3980359 4.137923

LTR3B_#LTR/ERVK 0 0.0000000 0.1079325 0.4247868 11.348338

SRP110016 HML 1 0 0.0000000 0.0939917 0.4084061 6.709148

HML 10 0 0.0246443 0.1977949 0.6106722 8.991545

HML2 0 0.0748145 0.1923879 0.7013925 25.584209

HML3 0 0.0207453 0.0850121 0.4052984 14.993567

HML4 0 0.0676325 0.2055983 0.5833871 17.361348

HML5 0 0.0000000 0.0627547 0.2304348 4.292697

HML6 0 0.0000000 0.1176631 0.3652445 4.943406

HML7 0 0.0165461 0.0571758 0.1534795 2.665017 HML8 0 0.0237677 0.0779974 0.5586158 31.923344 K14C 0 0.0000000 0.0319094 0.1170643 1.377055

LT 22B1#LTR/ERV 0 0.0000000 0.0744216 0.2581293 2.729679

LTR22B2#LTR.ERVK 0 0.0000000 0.1462319 0.6989967 4.71 645

LTR22C2#LTR. ^' ERVK 0 0.0000000 0.0341152 0.1244866 1.373369

LTR22 E#LTR E RVK 0 0.0000000 0.0483235 0.1773740 1.849842

LTR3B_#LTR.ERV 0 0.0000000 0.0478708 0.1685803 2.643292

H Ll 0 0.0000000 0.1268767 0.3353742 4.052137

H L10 0 0.0304281 0.1950971 0.3455298 2.487003

HML2 0 0.0229832 0.1938948 1.8896213 219.947046

HML3 0 0.0000000 0.1223329 0.3815171 10.135958

HML4 0 0.0237879 0.1731327 0.3562414 5.434816

H L5 0 0.0000000 0.1089093 0.3043274 7.381214

H L6 0 0.0000000 0.1585309 0.3921706 4.257929

SRP102685 HML7 0 0.0000000 0.0912298 0.2878545 7.654548

HML8 0 0.0081154 2.0901303 98.5146905 8515.001781 K14C 0 0.0000000 0.0706536 0.2391837 3.356916

LTR22B1#LTR/ERV 0 0.0000000 0.1234718 0.3168124 5.835195

LTR22B2#LTR.ERVK 0 0.0000000 0.1937394 0.7134805 5.204694

LTR22C ^' 2#LTR. ^' ERV 0 0.0000000 0.0902775 0.2336134 2.321173

LTR22 E#LTR E RVK 0 0.0000000 0.1764475 0.4852693 4.627528

LTR3B_#LTR/ERVK 0 0.0000000 0.1258778 0.3518004 4.700366

HMLl 0 0.0000000 0.1546103 0.9128325 32.738476

HMLIO 0 0.0000000 0.1910084 0.5156409 8.690614

HML2 0 0.0000000 0.2408015 2.7415319 195.191293

HML3 0 0.0000000 0.1016756 0.7212861 32.342361

HML4 0 0.0000000 0.3064975 1.5651 06 56.983694

HML5 0 0.0000000 0.1737037 0.966825 I 22.605106

HML6 0 0.0000000 0.1513285 0.7387770 15.850478

SRP068424 HML7 0 0.0000000 0.0647419 0.2488850 5.277685

HML8 0 0.0000000 0.2023157 6.0970185 705.290157 K14C 0 0.0000000 0.0794524 0.4541036 8.772424

LTR22B1#LTR/ERV 0 0.0000000 0.1325097 0.6447649 13.163549 LTR22B2#LTR.ERVK 0 0.0000000 0.0693838 0.2707259 2.985159 LTR22C2#LTR.ERVK 0 0.0000000 0.0440100 0.1820465 2.502976 LTR22 E#LTR E RVK 0 0.0000000 0.0989893 0.3236325 4.761848 LTR3B_#LTR.ERVK 0 0.0000000 0.1085489 0.5031198 8.617390 SRP064478 HMLl 0 0.0000000 0.0601878 0.1912765 3.648491

HMLIO 0 0.0046214 0.1085939 0.2846323 3.347339 HML2 0 0.0114172 0.1068918 1.0920575 60.595378

HML3 0 0.0000000 0.0663053 0.2855648 11.555712

H L4 0 0. 122422 0.0818699 0.2004683 2.824911

H L5 0 0.0000000 0.0544799 0.1695744 3.303590

H L6 0 0.0000000 0.0687535 0.1899200 1.844958

H L7 0 0.0000000 0.0453627 0.1214706 1.274142

HML8 0 0.0000000 1.6252062 86.0880409 9238.633200 14C 0 0.0000000 0.0356487 0.1248000 2.091446

LTR22B1#LTR/ERV 0 0.0000000 0.0603075 01706618 3.324758

LTR22B2#LTR/ERV 0 0.0000000 0.0890397 0.3238018 2.795000

LTR22C2#LTR/ERV 0 0.0000000 0.0532044 0.1480141 1.725137

LTR22E#LTR/ERVK 0 0.0000000 0.0890193 0.2572100 2.633204

LTR3B_#LTR,ERV 0 0.0000000 0.0548118 0.1628587 2.074693

HMLl 0 0.0000000 0.1099929 0.5374086 19.771974

HML10 0 0.0000000 0.3120755 4.0788949 209.074420

H L2 0 0.0000000 0.3083341 7.0141530 716.132793

H L3 0 0.0000000 0.0819258 0.5721171 44.512233

H L4 0 0.0055612 0.1631802 0.5669 11 15.160832

HML5 0 0.0000000 0.0740519 0.29915 1 8.570903

HML6 0 0.0000000 0.1508548 0.7572169 35.954051

SRP058722 HML7 0 0.0000000 0.0721502 0.2653196 7.406958

HML8 0 0.0000000 0.1347603 5.6620404 1058.007060 14C 0 0.0000000 0.0942404 1.3013323 77.419996

LTR22B 1 #LTR/ERV 0 0.0000000 0.1381469 1.3368361 54.714245

LTR22B2#LTR/ERV 0 0.0000000 0.1202672 0.6000457 6.857656

LTR22C2#LTR/ERVK 0 0.0000000 0.0524224 0.3472964 9.886468

LTR22E#LTR/ERVK 0 0.0000000 0.1179183 0.5986011 19.318676

LTR3B_#LTRERV 0 0.0000000 0.0871452 0.4722096 17.621088

ERP000550 HMLl 0 0.0000000 0.0913613 0.6721108 25.754908

HMLIO 0 0.0000000 0.5265347 3.7211529 104.449407

HML2 0 0.0000000 0.3829747 9.4737435 494.573463

HML3 0 0.0000000 0.0758083 0.6113529 24.861316

HML4 0 0.0000000 0.1226314 0.5223547 39.400311

HML5 0 0.0000000 0.0545070 0.3573734 15.142432

HML6 0 0.0000000 0.1665188 1.1979633 46.109402

HML7 0 0.0000000 0.0653212 0.5217105 11.243053

HML8 0 0.0000000 0.0792274 2.3560620 344.064632 14C 0 0.0000000 0.0208334 0.0993372 1.730934

LTR22B 1 #LTR/ERV 0 0.0000000 0.0496653 0.2114473 3.282185 LTR22 B2#LTR Έ RVK 0 0.0000000 0. 1243072 0.6755299 6.64595 I

LT R22 C2 # LT R Έ RV 0 0.0000000 0.0542908 0.3050922 6.0 I I 783

LTR.22 E#I_TR/E RVK 0 0.0000000 0.0386306 0. 139 1434 2.082330

LTR3 B_#LTR. E RVK 0 0.0000000 0.0649027 0.4006698 8.956652

[00345] The lack of differential total RNA expression in controls versus the ALS cohort (Figure 37), which is intriguing given data from protein immunostaining showing obvious differences between clinical groups (see below, Figures 38-42). PCA analysis reveals that expression of select ERVK CTXLP+ loci cluster in controls versus the ALS cohort (Figure 37), suggesting that specific CTXLP loci may drive the expression of CTXLP protein in ALS.

[00346] ERVK CTXLP expression is enhanced in CNS tissues from patients with Amyotrophic Lateral Sclerosis (ALS)

[00347] ALS pathology involves degeneration of upper (brain) and lower (spinal cord) motor neurons, leading to muscle weakness and paralysis (reviewed in ^78"80 ). Brain and spinal cord inflammation is a hallmark of ALS (reviewed in ⁸¹ · ⁸² ). The majority of ALS cases are sporadic, and the cause of this disease remains unknown. Here, we focus on the connection between neuropathology associated with ALS and ERVK CTXLP, such as proteinopathy ^{83 84} , aberrant calcium signalling ⁸⁵ , demyelination ⁸⁶ , and oligodendrocyte dysfunction ⁸⁷ .

[00348] To show that CTXLP protein is not only expressed in in vitro cell cultures, but also in ex vivo (autopsy) human tissues, spinal cord and brain tissues from neuro-normal controls and patients with ALS were assayed for CTXLP by western blot (Figure 38) and confocal microscopy (Figures 39-42). Western blot analysis of motor cortex specimens from neuronormal controls and patients with ALS reveals significantly enhanced CTXLP expression in ALS (Figure 38A, p<0.05). CTXLP was concomitantly expressed with inflammation and tissue injury marker CX3CL1 (Figure 38B) ⁸⁸ . Analysis of cervical spinal cord tissues also demonstrates elevated CTXLP and CX3CL1 expression in ALS as compared to controls, alongside a modest decrease in levels of voltage-gated calcium channel CaV2.2 in ALS (Figure 38, C-F). Together, these results point to tissue injury and inflammation in CTXLP+ tissues from patients with ALS.

[00349] In addition, confocal microscopy of cervical spinal cord (Figure 39A) and motor cortex specimens (Figure 39B) from neuro-normal controls and patients with ALS reveals substantially enhanced CTXLP expression in ALS. In the motor cortex, CTXLP+ cells were neurons (based on MAP2 neuronal marker). This is consistent with previous observations of ERVK proteins present in the motor cortex of patients with ALS ⁶⁷ ' ⁸⁹⁹⁰ . Notably, basal CTXLP expression was mostly nuclear in neuronormal tissues, whereas CTXLP exhibited a pattern of cytoplasmic aggregation in motor cortex tissues from patients with ALS (Figure 39B). Enhanced MAP2 staining in the axon hillock of CTXLP+ pyramidal neurons may be an indicator of virus activity, as seen during rabies infection ^{91 92} . This pattern of CTXLP expression coincided with a notable decrease in CaV2.2 expression in ALS as compared to controls. Based on staining pattern, this decrease may represent a loss of CaV2.2 expressing pyramidal neurons, as well as smaller CaV2.2+ cells ⁹³ . Remarkably, CTXLP patterning in the cervical spinal cord exhibited a ring pattern surrounding MAP2+ neurons (MAP2 marks neuronal axons in grey, Figure 39A, far right panel).

[00350] Our evidence further indicates that CTXLP can alter oligodendrocyte behavior. In the CNS, highly specialized cells called oligodendrocytes protect neuronal axons by wrapping them in an extensive plasma membrane compacted to produce the myelin sheath ⁹⁴ . Oligodendrocyte precursor cells (OPCs) are a pool of immature oligodendrocytes, which express characteristic markers such TCF4, Oligl and Olig2 ⁹⁵⁹⁶ . Upon differentiation into mature oligodendrocytes, they begin to express myelin proteins such as PLP, MOG and MAG ⁹⁵ . Oligodendrocytes must myelinate early post-differentiation and myelination occurs within a short timeframe (12-18 hours), where their extended processes ensheath 50-60 axonal segments simultaneously ⁹⁷ . Some CNS regions (spinal cord, brainstem and visual cortex) exhibit early myelination during human development, whereas other regions undergo myelination into adulthood (prefrontal cortex and association fibers). Pools of OPCs can remain in tissues and are capable of migration and later differentiation into mature oligodendrocytes, often in response to brain injury ⁹⁸ . However, in many disease states, an attempt at remyelination is most often unsuccessful ⁹⁸ . A prevailing theory surrounding defects in remyelination is that despite increased numbers of OPCs in injured tissue, these precursor cells become stalled in an immature state and fail to properly differentiate into mature oligodendrocytes ⁹⁶⁹⁸ . Alterations in OPC markers, such as enhanced TCF4 and Oligl occurs in tissue lesions from patients with MS ⁹⁹ ' ¹⁰⁰ .

[00351] Our observations show that CTXLP expression occurs in either lateral and/or anterior cortical spinal tracts in ALS (Figure 40). Strong CTXLP+ staining coincides with demyelinating lesions, as shown by solochrome cyanine staining of adjacent tissues (Figure 40). Increased TCF4 (oligodendrocyte precursor marker) is also evident in association with CTXLP expression in tissue from patients with ALS (Figure 40). [00352] Figure 41 depicts increased numbers of TCF4+ and Olig1 + cells expressing CTXLP in cervical spinal cord tissue from patients with ALS, as compared to controls. This indicates that CTXLP expression in the spinal cord of patients with ALS does indeed occur in oligodendrocytes (Figures 39-41), specifically in cells expressing OPC markers.

[00353] Neurite outgrowth inhibitor (Nogo-A) is a key regulator of oligodendrocyte precursor cell (OPC) differentiation; when OPCs express Nogo-A they are unable to progress towards a mature oligodendrocyte phenotype, which is capable of myelination ^{101 102} . Thus, enhanced expression of Nogo-A in OPCs in the context of inflammation and disease states prevents axonal regeneration by restricting OPC maturation ^103-105 . As an example, demyelinated MS lesions show an increased abundance of Nogo-A+ OPCs, yet the inability of OPCs to mature is proposed as the mechanism driving a non-permissive environment leading to remyelination failure ^{103 106 107} . In mature oligodendrocytes, Nogo-A expression prevents axonal sprouting and is expressed in these cells until the initiation of active myelination.

[00354] Nogo-A is implicated in a variety of neurological conditions, such as spinal cord injury, peripheral neuropathies, stroke, temporal lobe epilepsy, Alzheimer's disease, ALS, MS and schizophrenia ^{101 108"110} . Nogo-A has been identified as a prognostic marker and therapeutic target in ALS due to its substantial expression in muscle tissue from patients with motor neuron disease ^{111 112} . Mechanistically, Nogo-A expression destabilizes neuromuscular junctions ^113-116 . Indeed, clinical trials using human anti-Nogo-A antibodies have been performed (ATI 355 from Novartis Pharma and Ozanezumab and GSK1223249 from GlaxoSmithKline) ¹⁰¹ ' ¹¹⁷ ' ¹¹⁸ . These therapies were designed to target Nogo-A expression in the periphery (intravenous infusions), but may fail to block Nogo-A expression in the CNS (Figure 42), thus explaining the negative results in Phase II clinical ALS trials with Ozanezumab ^{119 120} . Of note, genetic polymorphism reticulon 4 receptor (RTN4R) gene encoding the Nogo-A receptor (NgR1), is associated with sporadic ALS ¹²¹ .

[00355] Figure 42 demonstrates that CTXLP expression in the spinal cord of patients with ALS is associated with elevated Nogo-A expression, specifically in OPCs and other cell types. This specifically occurs in areas of myelin depletion (see Figure 41). It has been demonstrated in human spinal cord, that select myelin protein rings (PLP, MOG, but not MAG) are detectable by immunohistochemistry even 3 years after injury in degenerating fibre tracts exhibiting the absence of intact axons ¹²² . Nogo-A expression also persists in degenerating spinal tissue and may create a non-permissive environment for axon regeneration ¹²² . Furthermore, it has been shown that Nogo-A favours a pro-inflammatory context ¹²³ , one that would promote ERVK expression via modulation of NF-κΒ and pro-inflammatory cytokine secretion ⁶⁷ .

[00356] ERVK C TXLP expression is enhanced in cancer cells

[00357] To further evaluate the potential pathogenic activity of CTXLP, we examined CTXLP levels in cancer to follow-up on our observation that NCCIT human embryonic carcinoma line spontaneously expressed CTXLP. The localization pattern that included the cytoplasm also suggested that this represented a stage in course of aberrant CTXLP expression. Thus, we assayed prototypic teratocarcinoma (NCCIT) and breast cancer cells (T47D) for CTXLP expression as compared to the karyotypically normal, non-cancerous, cell lines astrocytic SVGA cells (Figure 43). Cancer cells clearly show higher levels of ERVK CTXLP as compared to non-cancerous cells. A cancer screen also reveals several cancers exhibiting enhanced CTXLP levels, including T cell lymphoma, Acute T-cell leukemia, epithelioid carcinoma, Burkitt's lymphoma, neuroepithelioma, prostate, breast, ovary, testis and skin cancers (Figure 44).

[00358] In summary, ERVK CTXLP localized to the motor cortex and spinal cord sections from autopsy samples of patients with ALS, but not neuronormal controls. Concomitantly, CTXLP expression was substantially enhanced in diseased ALS tissues aligning with oligodendrocytes, Nogo-A expression and demyelinated lesions. In addition, cancer cell lines and tissue expressed greater levels of CTXLP relative to normal controls. Together, these findings provide significant evidence for the activity of CTXLP in ALS and certain cancers.

[00359] Pathological consequences of CTXLP expression

[00360] ERVK CTXLP enhances NF-κΒ protein expression, whereas ERVK Env does not

[00361 ] Real Time PCR analysis of transfected 293T cells

[00362] To investigate how cells may react to the expression of CTXLP and SU, RT- PCR analysis was used to measure the expression of the pro-inflammatory NF-κΒ p65 subunit and the anti-viral response protein IRF7. This analysis showed that both CTXLP and SU triggered a marked increase in the mRNA expression of NF-κΒ p65. Conversely, neither protein was able to trigger an upregulation of IRF7 (Figure 45). The upregulation of N F-KB p65 may be beneficial to the ERVK provirus as it is able to 1) act as a direct transcriptional activator of the ERVK LTR, and 2) trigger inflammatory conditions that are conducive to ERVK activation ^{64 67} . [00363] Confocal microscopy analysis of transfected 293T cells

[00364] SU and CTXLP transfected 293T cells were also stained to determine whether the presence of either of these proteins was sufficient to trigger the expression of NF-κΒ p65. It was observed that CTXLP was able to trigger NF-κΒ p65 protein expression, whereas SU was unable (Figure 46). This was in stark contrast to the RT-PCR results in Figure 45, where both ERVK SU and CTXLP induced NF-κΒ p65 transcription. As expected, there was a marked increase in SU expression in CTXLP-expressing cells, as the epitope for the SU antibody binds to either SU or CTXLP as both proteins contain the SU amino acid sequence.

[00365] To further confirm whether the effect of NF-κΒ induction by CTXLP is a general phenomenon occurring the multiple cell types, astrocytic SVGA cells were also transfected as described above and evaluated for NF-κΒ protein expression. Interestingly, both NF-κΒ p65 and p50 proteins were induced by CTXLP, but not ERVK SU overexpression (n=4). This finding is notable, considering we have shown that ERVK transcription is mediated by IRF1 , p50 and p65 transcription factors, and impacts ERVK expression in ALS ⁶⁷ . It is also intriguing considering TRAF proteins were predicted to be interacting partners of CTXLP, and may alter NF-κΒ signalling ^{124 125} .

[00366] ERVK CTXLP depletes CaV2.2 CCAT protein expression

[00367] A surprizing feature of several voltage-gated calcium channels (VGCCs) is the ability of their C-terminal fragments to translocate to the nucleus and impact gene expression. Termed calcium channel-associated transcription regulator (CCAT) by Gomez-Ospina et al. in 2006 ¹²⁶ , these novel gene products encoded within the VGCC sequences. In most cases, an antibody targeting a C-terminal CACNA1 epitope will identify an approximately 75 kDa CCAT fragment with a cellular distribution within the nucleus (or nuclear fractions), unlike the intact channel protein localized to the cytoplasm and membrane ^{126 127} . VGCC CCAT proteins can be regulated by cell signalling events. For example, cellular signals that promote CaV1.2 CCAT nuclear localization include treatment of neurons with 2.5mM EGTA (a chelator which reduces free extracellular calcium), whereas 65mM KCI treatment (mimicking tonic activity of VGCC) decreased nuclear CCAT levels ¹²⁶ . Several signals which drive high intracellular calcium levels in neurons, including 100μΜ glutamate, depolarization and NMDA signalling, lead to decreased nuclear CCAT levels ¹²⁶ .

[00368] We have previously demonstrated an inverse correlation between CTXLP and voltage-gated calcium channel CaV2.2 expression in ALS brain and spinal cord tissues (Figures 38 and 39). To further extend this observation, we performed in vitro experiments of CTXLP and ERVK SU exposure by overlaying immunoprecipitation products on human astrocytes (Figure 47). Treatment of SVGA cells with CTXLP, but not ERVK SU, resulted in the depletion of Cav2.2 CCAT (75 kDa), as measured by western blot analysis and confocal microscopy. A decreased in the full size CaV2.2 channel (220 kDa) was also observed (n=2, data not shown). Thus, CTXLP in the CNS may lead to an overall decrease in CaV2.2 channel and CaV2.2 CCAT expression, thus explaining the observed decreased expression of CaV2.2 in ALS (Figures 38 and 39). The regulatory role of CaV2.2 on cellular transcription is currently unknown.

[00369] ERVK CTXLP is toxic via mechanisms that differ from ERVK Env- mediated toxicity

[00370] Treatment of SVGA astrocyte cells with ERVK Env proteins isolated via immunoprecipitation

To determine the neurotoxicity of CTXLP, SVGA astrocyte cells were treated with CTXLP proteins isolated from NCCIT cells via immunoprecipitation (IP). This simulates conditions wherein CTXLP would enter the cell from the outside and possibly exert its effects by binding to cell surface receptors (such as calcium channels). There was considerable variation in the neurotoxicity assays, which may be due in part to the fact that cells were dosed by volume of CTXLP. There was no reliable way to measure the concentration of the protein in the IP product, as protein concentration was well below sensitivity of our in-house BCA assay (20 μg/ml). However, a much higher number of cells treated with CTXLP expressed caspase-3 (apoptosis marker used in the toxicity assays) than control cells demonstrating that CTXLP was toxic to astrocytes, even at unmeasurably low concentrations (Figure 48).

[00371] A separate toxicity assay was performed by treating astrocytes with SU and CTXLP (respectively) in the presence and absence of calcium. Theoretically, if CTXLP does in fact contain an ω-conotoxin domain, by flooding cells with calcium and thus saturating calcium channels, it's ability to exert toxic effects on cells via calcium channel binding should be blocked. This is what was seen in CTXLP, but not SU, toxicity assays. In the presence of calcium, the levels of caspase expression in CTXLP-treated cells were similar to controls conditions. They were also less than those of cells treated with CTXLP in the absence of calcium. Further, cells treated with SU in the presence and absence of calcium expressed similar levels of caspase-3 in comparison to CTXLP alone (Figures 49 and 50).

[00372] Cells in this same toxicity assay were also analyzed at later time points. It was observed that CTXLP and SU-treated cells appeared to be able to continue to replicate despite high levels of caspase expression. After 8 days, cells in both conditions increased in cell density despite high levels of caspase-3 expression and without the addition of media. These observations suggest that these cells may have been transformed oncogenically ^129"131 . Conversely, control cells were not viable after 8 days in culture (Figure 51). It is interesting to note that some viruses, such as the influenza virus, require caspase expression to replicate efficiently ¹³² . Moreover, HIV Tat, the viral transactivator, induces caspase activation as part of its neurotoxic mechanism ³⁸ .

[00373] It is notable that the trend of enhanced caspase-3 positivity is seen in both treated (Figures 48-51) and transfected cells (Figure 52), suggesting that exposure to CTXLP and/or cellular production of CTXLP in vivo may be toxic to cells.

[00374] ERVK CTXLP expression drives morphological changes in cells

[00375] ERVK CTXLP-treated SVGAs and 293T cells transfected with ERVK CTXLP- encoding plasmids were imaged to observe the cellular morphology. Though many of the cells looked normal in appearance, it was observed that an increased number of the cells produced long filipodia (Figure 53). As well, the formation of syncytia (multinucleated cells) was also observed amongst cells exposed to CTXLP. These features are characteristic of retrovirus- infected cells and are known to promote virus transfer between cells ¹³³ .

[00376] This data indicates that ERVK CTXLP expression has the capacity to enhance N F-KB p65/p50 and CaV2.2 proteins that play a critical role in ALS pathogenesis. In addition, CTXLP administration or transfection induced significant levels of capase-3. The induction of caspase-3 activation and apoptosis by CTXLP was inhibited by excess extracellular calcium pointing to a calcium channel mediated activation of toxicity. Remarkably, despite the initial die off of cells, cells remaining in the cultures appeared to demonstrate appreciable cellular proliferation relative to control suggesting the induction of a carcinogenic process.

[00377] ERVK CTXLP can be targeted by small molecule therapeutics

[00378] Taken together this data strongly indicates that targeting CTXLP would have significant therapeutic value in ALS. To this end, we have began investigating A small molecule inhibitors to capable of counteracting the pathological effects associated with CTXLP expression is of therapeutic value. A drugs screen in ERVK CTXLP-expressing NCCIT cells was performed to evaluate potential efficacity against CTXLP (Figure 54).

Michael acceptor electrophile (MAE) compounds are known to inhibit HIV Tat-dependent transcription by interfering with thiols in its cysteine-rich domain ^{134 135} . As CTXLP and HIV Tat share commonality in their cysteine-rich domains (Figure 14), we evaluated a series of MAE compounds including curcumin, rosmarinic acid, gambogic acid and celastrol. Two MAE drugs, celastrol (Cel) and gambogic acid (GA), were identified as suppressing CTXLP expression in NCCIT cells in the low micromolar range (Figure 54).

[00379] Derived as an active compound from the Thunder God vine (Tripterygium wilfordii Hook F), celastrol (https://pubchem.ncbi.nlm.nih.gov/compound/celastrol) is a plant- derived triterpene with antioxidant, anti-viral and anti-inflammatory activity ¹³⁴ ' ^{136 137} . Celastrol is currently used as a therapeutic agent for rheumatoid arthritis (RA) and lupus in China ^{136 138} . Celastrol has also been shown to impact pathological outcomes and symptoms in animal models of RA ¹³⁹ , as well as inflammatory markers in activated fibroblast-like synoviocytes from patients with rheumatoid arthritis ¹⁴⁰ . Celastrol has been shown to limit beta-amyloid pathology and neuronal degeneration in Alzheimer's disease models ¹⁴¹ . Its anti-cancer properties are also under investigation ¹⁴² .

[00380] Gambogic acid (https://pubchem.ncbi.nlm.nih.gov/compound/16072310) is an active compound from the Gamboge tree (Garcinia hanburyi), with antioxidant, anti-viral and anti-inflammatory properties) ^{134 143"145} . It has been shown to be neuroprotective ¹⁴⁶ , and inhibit spinal cord injury and inflammation in a rat model ¹⁴⁷ . Both celastrol and gambogic acid can prevent mutant huntingtin protein aggregation and its neuronal toxicity ¹⁴⁸ . The anti-cancer properties of gambogic acid are also under investigation ^{144 145} .

[00381] Improvements on drug efficacy, toxicity and tissue-targeting are possible by using MAE-derivatives, related compounds (Table 12), soluble analogues and nanosystem delivery to the brain ^{149 150} . Here we provide proof-of-concept that CTXLP is druggable using small molecules (Figures 54-58); further drug development may improve upon the anti-CTXLP effects of celastrol and gambogic acid.

Table 12: Compounds with similar structures and/or bioactivities with celastrol and gambogic acid found using PubChem open chemistry database.

KQJSQWZMSAGS H N- PACOHS D FSA-N 16757868 Gambogic acid 134129562

20alpha-Hydroxytingenone 10717799 Gambogenic Acid 9895478

J FAC ETXYABV H F D- LQS RW FAZS A- N 16757909 Gambogic acid 9852185

20beta-Hydroxytingenone 44559597 Gambogic acid 11599836

ZTCAJ LZRROIDHU-WAFCJU BUSA-N 44572792 Gambogic amide 25252739

FXLVCCDIUNLGKU-BRUCSKOJSA-N 5701992 Gambogellic Acid 52945437

Remangilone D 44558996 Morellic acid 54580250

ZKJXU KU IPQCU MI-QSZQYENJSA-N 10624078 Gambogic acid 3451

AIXBDINLSQYZGS-JWNVRJNBSA-N 45482038 Acetyl isogambogic acid 6857789

GSIDIGLXCWJQPN-YMEQZMBDSA-N 25424418 Gambogin 11753679

DMHJWMODPYSFCD-DUSJ HGMNSA-N 71498413 Gambogic amide 56951189

PDMJ IWXURDBAOZ-CIFOREJBSA-N 45482047 Gambogic acid 91668478

21-Oxopristimerine 50907761 Gambogic acid 5281632

Dispermoquinone 53320376 Gambogic acid 5380091

QHYPSOWPDMYTTQ-U NNRZNSMSA-N 71498452 Gambogic acid 5353639

QIRU FAFQGKOTKA-YKUCPAPWSA-N 389017 Isomorellic acid 9915833

OQLD DXD MTOPTDO-QRARIYCASA- N 10695614 Gambogenic Acid 10794070

QGWDYPREORDRIT-DGRUGRQQSA-N 16745529 Gambogic acid 20054919

QGWDYPREORDRIT-DSIOGZMYSA-N 229868 Isogambogenic acid 70639870

ZNFSSQAJGMMWBY-WXPPGMDDSA-N 25197280 Gambogic acid 70639872

W H D KOW N 1 OG JX H K- PTRAYG LTS A- N 45482045 10-Hydroxygambogic Acid 71450485

OQLDDXDMTOPTDO-PEKWGEHZSA-N 44289021 BLDWFKHVHHINGR-U HFFFAOYSA-N 125071

GAPWCQHXCIXKLV-RKHSXEAASA-N 6708798 Deoxymorellin 635828

QGWDYPREORDRIT-U HFFFAOYSA-N 3129312 Morellic acid 5319893

RRRZQVJZDVPAJ N-ZRCCSVPJSA-N 25197172 Isomorellin 5364585

Polpunonic acid 169521 Isomorellic acid 5366120

KZCZQJ NPWZPAEJ-ZRCCSVPJSA-N 25195610 DRRWWKSGTSQOON-DXMWQDMHSA-N 52947871 Dihydrocelastrol 10411574 7-Methoxyepigambogic acid 45270567

FATJTRUVRFSESL-HZYNXAPGSA-N 44566365 U ONS IJYWYKP E D K- NXWWLH KBSA- N 45272194

Isoiguesterinol 10477355 BIGAHFWHALQTRA-TYAVBBKTSA-N 45272195

Wilfolic acid C 44559659 GJFGYTWPU IBTJN-UOCU BHIGSA-N 45272197

HVRSOVWJUJGHSI-JHGSJXKWSA-N 44558965 UUARHDVFLLQMF-KELXBUOKSA-N 45272281

11-Oxoursonic acid 22210052 BMPKGNCYXQRU MA-BLACGAOQSA-N 52941797

6-Oxopristimerol 11754914 JEWKRGWMKMU BBF-JFDIIJRYSA-N 52943022

MAAVNXPPBHQXNL-INMPMWFSSA-N 6710688 ORRQVYBMKAECLD-KMIBLQPDSA-N 52946647

GZKMFYLBPHPWEI-DGRUQNUSA-N 363631 QQQN F 1 ABW PXS FD-PUYFONRISA-N 52946668

LUSKWXDBHNQVEL-JXZITWAVSA-N 53323497 JTTOKKDLXZMVBA-IHECDDROSA-N 52947870

QGWDYPREORDRIT-NFGWGJPRSA-N 73758286 XSDFUWMDPU DYHN-CLKBEGISSA-N 52948934

CDOKUYLTAYCBST-GKMFGOQDSA-N 9869964 Gaudichaudione H 57345562

C P F U J A KVJ BYWJ A-BTRWLOMLSA-N 10411393 6-Hydroxycluvenone 57390441

YXEPIOPYEDGEEP-IITIDZKMSA-N 71498373 KCALBYYRHOKKBO-BCFMDSCZSA-N 57395879

KGQO H ECLTO NQOT- H KFG FSCZSA- N 10645721 CSIZKMXLDPOBKM-PQIHDOCZSA-N 57395880

QEACUYQTRMGOSD-MZRSIZMESA-N 14465806 XZPYBDPGHSLINX-OHTINKFRSA-N 71720741

CVAILKMOFONEDU-KRJMWWH ISA-N 15765122 KCYKVBCVFBSOKZ-RWPPGCTJSA-N 118737824

2-Picenecarboxylic acid 23757062 PVRDWAUIIJ ESEC-WFSGSTODSA-N 118737833

QLTFHGMEDZMMTF-VUQPYPCZSA-N 44298352 OIZCJCKDTJMDFV-CVTCU NNCSA-N 118737835

OKOGABAEYJ RJOB-JSJVQHDDSA-N 46184390 KSEYPOHNDKJEPF-ILHGWRPKSA-N 45267055

UZEJIMRGSSLBIV-SAQIBKBSSA-N 25197281 Dimethyl gambogate 6857785

Fupenzic acid 12045007 Garcinolic acid 6857794

WZAU FGYINZYCKH-AQCDROMSSA-N 6708673 Methyl gambogate 12113746

Amazoquinone 44559090 Decahydrogambogic acid 5149276

Dihydrocelastryl diacetate 9828620 REDMIYQFNIRTDF-WIKVJIRTSA-N 16758035

20-Epi-isoiguesterinol 21575471 VZXLWEWYBUGUA-SAABBMRESA-N 44449775

TTWPKNPRMGVGJO-QZLVDJ LTSA-N 49797930 FJRORJDZZLUAPP-BUJCIKCXSA-N 25208438 23-Nor-Blepharodol 53320826 Dimethyl-Ga 44449753

GSIDIGLXCWJQPN-BRLXHVQISA-N 5336986 7-Methoxygambogic acid 45268014

SAOOBRU HTPONGX-UANCAJPASA-N 6708713 7-Methoxygambogellic acid 25208761

PLXMKOYILCBYGS-UXDHXBHYSA-N 6710689 VZIUOBFNNRU PAK-QBEUZEMSA-N 71717656

QGWDYPREORDRIT-VLYMFKARSA-N 44435792 JDGCU RVTMMXMDY-TYTBCFIUSA-N 71718886

DQHHRVQZU PBARM-LRRZNWEGSA-N 90233240 FTTRVECHPLAERQ-UZTNAXEXSA-N 71717668

YQUGEUGWFBESNQ-WXPPGMDDSA-N 118408942 U C U DAO AF D H F E A-XYBO N B LRS A- N 71717669

QOGSXJ H N N DQXSS-YNJ 1 RTJXSA- N 88303297 PU DIAMZKKXFSOI-UZTNAXEXSA-N 71717674

SKMCTU IWOMRTKB-RLLZTQLFSA-N 68198583 CDIIOQLRYIQLOZ-JSJFTWSHSA-N 71717675

GAVQRDLYJ PTRLX-NGXGXHCGSA-N 68028960

KKPBMYHMXYLZMU-KDBWZQHXSA-N 71718259

QGWDYPREORDRIT-XYMLVXMESA-N 60103590

PYCHDQHGAJKLEB-ALPBHPBSSA-N 71718272

XLZGVFDYBDKLSJ-H KUCNJ HLSA-N 59492093 XTQBIQOZQZIJJW-HQCJEUBQSA-N 71718866

INHU ILMVLIXIOP-LLYXLEJJSA-N 59428287 CTSIBWDVPNXDDY-WSOPLZEESA-N 71718874

OZKBTRIBGQYSPM-VRAWMDIMSA-N 59350382 GSU LQLPVU MJ WJ N-YWJ DO DG HSA- N 71718876

PNZQDIUZNCHSAL-ZRCCSVPJSA-N 58338790 OSDWBDHHKBVHAI-CNADFBRCSA-N 71717053

ILAWPU DRPIJSHC-CDKFMWKUSA-N 58338787 BZRJTOIKOJHZGQ-ALPBHPBSSA-N 71716428

QGWDYPREORDRIT-OUMYWODESA-N 57321836 QSJ AAIXXD PZJ D l-QVZJ NGHZSA-N 71717051

FGU PEMLTKNSPDS-WYUYVVTISA-N 56847557 KLEWBU IXEFWIOX-QBEUZEMSA-N 71717031

TZYINTOVRMOTDT-WXPPGMDDSA-N 56847496 NPPZU PS AZVYWJ X-RGAU DQMMSA-N 71716434

J KZDPNSGTLVKFS-JSJVQHDDSA-N 25197171 CRZXNKVWVIOGLG-KCJGJVMNSA-N 71717027

PPBNDNNFAYLU KF-SUNMMQDUSA-N 25197168 MGBNLVNNMKFWHO-MNDRQJQGSA-N 71717026

XAWKZQBU EYU MJQ-WXPPGMDDSA-N 118404337 PJ RSCTLUXRMVLG-LPIQBNQASA-N 71717025

LMRBFTMNXRSIDU-ANCVDJAISA-N 56846808 YRQDRRMNQYXEQF-TYTBCFIUSA-N 71716450

GKBQZTNFLYSBDI-HKUCNJHLSA-N 90922624 LKTCFJ PAKUCNIB-QBEUZEMSA-N 71716443

KTHCLZLOFJSMHN-NGXGXHCGSA-N 68029427 YKSIZBBLYFQODP-YWJ DODGHSA-N 71716435

XFFJOOGKLFMNFN-NOZRFFRFSA-N 68029424 FJYQZSO KE M DM H D-RGAU DQM MSA- N 71720740

MEVHISVZZRNRNQ-HJJLTIBASA-N 67406451 VOYHKSWHADONRT-U HFFFAOYSA-N 117591472 QGWDYPREORDRIT-GWEJSANNSA-N 60103581 VJXSSTZKBZWPPU-U HFFFAOYSA-N 117591470

CW N G KYC BO AG DTO- M IZXVQKXS A- N 59546528 FWO BTO PXT H 1 FAR- U H F F FAOYS A- N 117591530

HMWZFAJRZJAGAL-CDKFMWKUSA-N 58338783 PEVFZCIVSYJXJJ-U HFFFAOYSA-N 117591589

RZBXUSAFLXVWGM-ANCVDJAISA-N 56847608 YPFPFCJFKUOQMA-U HFFFAOYSA-N 117592388

WDJQPABVFANMAQ-XIRGYHLMSA-N 118408890 DQPKYEHRFQDOLK-RWPPGCTJSA-N 118737825

QGWDYPREORDRIT-BPTQZAATSA-N 54227450 ZYUWYMMXGSCUQT-U HFFFAOYSA-N 117591047

YGLDJGRHKCVIQN-ZRCCSVPJSA-N 46184627 YANZBSJ NCIQKSX-ZFHAUAHYSA-N 118737828

DHGOQCNNFIELMK-ABJUJWBKSA-N 42630196 KJBDNAFUXXSLAD-U HFFFAOYSA-N 117590910

HKRKU MPPXYIXCF-JSJVQHDDSA-N 25197278 AICGDFMIRHRSPB-MMEAOPOPSA-N 118737829

ZEXSHDSVCZQLCO-WFVGHVPHSA-N 25197169 CXFIQFGADOTDPF-CMLKYFHXSA-N 76311369

QGWDYPREORDRIT-ZUZOHOKMSA-N 21118757 XUYFKRZDNDQABJ-XDSJHDTISA-N 71718887

11-Oxooleanonic acid 11576389 BAHZVPGPIWTLLZ-RGAU DQMMSA-N 71720723

KQJSQWZMSAGSHN-AHWKKIARSA-N 58636951 U EMVXKVCUBRCBG-KCJGJVMNSA-N 71720719

KQJSQWZMSAGSHN-WDVYODBHSA-N 118655639 XAUUFNQHVHEWRB-RGAU DQMMSA-N 71720718

YDUSFXWVWNIQDZ-IGTSBU IGSA-N 118404346 YGVBIDJABJLVOL-DGTITLQCSA-N 71720708

HCAYAMHXSDCPTR-MRZDQBBQSA-N 118404343 CO EJ N DYM OAU U K-J CJ N IYC KS A- N 71720089

KQJSQWZMSAGS H N- M RH U JCAJSA- N 90233238 JESNVXLQVMHXLZ-JSJ FTWSHSA-N 71720088

FESQODXDXOHLEO-JNEFGXKCSA-N 71249962 XTBWCIYKONYIDG-AQOIPSNVSA-N 71720068

SMYCYEXFRJ MQLW-ANQVMFJUSA-N 71167315 WSCCRZWUYZCJAZ-KAJKVYITSA-N 71719495

FMTPU LGTIHBJRT-LGVWSNLESA-N 69574940

LWIGRTRTVVPXOZ-XDSJHDTISA-N 71719493

UAJBCGCAPNHLHM-QUYLDEAFSA-N 68028959

ZMMPPBWNSJCZGR-QVZJ NGHZSA-N 71719492

LLKPYONQSFCTMG-IGTSBU IGSA-N 123598084

XCRBRZWMQVMPIY-KENSWSBLSA-N 23629033

UQSBWMQFUNJXTI-AZUGTCGHSA-N 58338788

BIMUUWRNJBALEP-AWNOVZCOSA-N 45268895

REKHQMDGAPXWJP-ANCVDJAISA-N 56847556

DBWZFQAU MYEWML-WWZJDETNSA-N 45267910

GBQYERWLNHTHAH-ZRCCSVPJSA-N 56847495

34-Hydroxy-gambogic acid 45267909

VMVBZDHQRFGSLA-FAWNWTIBSA-N 56846810

DXSGQRROBDYCSJ-YVMJPLCQSA-N 45267154

AQKDBFWJOPNOKZ-MTWWMYJUSA-N 53656716

XRBAWHATHDIBFU-XOSCNRPVSA-N 45266948 MXM N 1 XPSWDE ED-GTKRWHGSSA-N 25197170

XCRBRZWMQVMPIY-OQOGLVOPSA-N 44583737

RPGDRWMPFKEMPI-JSJVQHDDSA-N 25177624

JBRMVLBIZUTECR-FXRUCJ BFSA-N 44403668

QGWDYPREORDRIT-OHHDNCQJSA-N 16401165

VZQQLPACAVHZQT-OLZUXEKSSA-N 44403667

GYUVZGGERRSPQY-UHKCKZGUSA-N 66583327

Gambogenific acid 25208911

SY LI RT RY LBYO BO-J J WQI E BTS A- N 91408393

UQHRXFAVXKZFRU-RGAU DQMMSA-N 71716426

VDDPQVQCXWFZJM-LDLRJHFFSA-N 91367949

Rl FZOYVQO F H OCS- W 1 KVJ 1 RTS A- N 16758013

LSSXGFNMUHCIAI-WXPPGMDDSA-N 91190512

REDMIYQFNIRTDF-LARDOQITSA-N 5469880

SYLIRTRYLBYOBO-AHWKKIARSA-N 91065125

VLAD F N OT LYCW M W- U H F F FAOYS A- N 52916109

WNCVCMKJU FWTFA-OWDORJPTSA-N 71249923

XZPYBDPGHSLINX-UHFFFAOYSA-N 52916002

QGWDYPREORDRIT-AVJZJKPBSA-N 70532695

Tetrahydrogambogic acid 9917275

DNLGFGXSBOIDNV-ZRCCSVPJSA-N 67421869

Dihydrogambogic acid 6857793

U RDWIVHPIKIQNK-JJWQIEBTSA-N 66583411

HHOLRSVIZVDOIV-OXFPAKKDSA-N 52949100

WJ LHDYQXCJXZGV-WXPPGMDDSA-N 91463431

XUYFKRZDNDQABJ-DLPQTZSGSA-N 57400963

QGWDYPREORDRIT-WBTUSMEDSA-N 60103570

HHVDVNJHGHGGHI-LDNSNGAXSA-N 57394941

QGWDYPREORDRIT-VPHAENBISA-N 57301674

YTIQONQLSSBXHE-MOWCMFFRSA-N 70691871

QOGSXJ H N N DQXSS-ZH ITZLKESA- N 57051700

LWIGRTRTVVPXOZ-DLPQTZSGSA-N 57393978

LMHNQDYMADJAAM-CDKFMWKUSA-N 56847555

TZXRZTWZRWZRQS- WSO P LZE ESA-N 71716427

GICPBFRHOLICHK-UHKCKZGUSA-N 56847494

LPYYTLGGS MEQM H-ZSJJ N DTGSA- N 45269643

WWKHRRYBBUOLCO-CDKFMWKUSA-N 25197277

Cochinchinoxanthone 53355017

QGWDYPREORDRIT-MQLBBMOOSA-N 18637982

BKRLQHWNGLIVCW-NFWYAXIXSA-N 52945436

33-Chlorogambogellic Acid 52943021

GBQLXOPZKHBGOY-SCWSFWMSSA-N 46886397

DRRWWKSGTSQOON-WIRZGQEJSA-N 46886396

QYRPARUSU FWOPG-HBWLMKOJSA-N 45272280

G ITYG ECAVAWXHS- FZGW 1 H BJSA-N 45272279

(9,10)-Dihydroxy-gambogic acid 45270476

7-Methoxyisomorellinol 45269745

O N KM N KXX FSJ VS P- U H F F FAOYS A- N 117591965

PLPLFPMHLSHHDS-UHFFFAOYSA-N 117593100

MUQLGQHFYINEFE-U HFFFAOYSA-N 117592428

YHHMSCSXVOQXAF-U HFFFAOYSA-N 117592536

1 VZP D DZYGYQLH F- U H F F FAOYS A- N 117592735

RZRZWSHHU HKZRH-UHFFFAOYSA-N 117592761 XCNLXYWAJHGEPU-U HFFFAOYSA-N 117592772

HAZSRZGEUAECEB-U HFFFAOYSA-N 117592892

PWVDTBGVDGFEKA-U HFFFAOYSA-N 117592295

1 U Y F DS GSZVXF L- LQE U QG N QS A- N 118753349

XXHKTHKJENJGLT-U HFFFAOYSA-N 117592031

DQLSLPKMHSAVGY-U HFFFAOYSA-N 117592271

HEZLRSFHGIYFNV-UHFFFAOYSA-N 117592219

WVBHHKPTCIYZRW-U HFFFAOYSA-N 117592147

ANXHWYZDFGXCSL-U HFFFAOYSA-N 117592105

FEU KMNUTHOBIFJ-U HFFFAOYSA-N 117592081

JEHWFPNSWRPRFY-U HFFFAOYSA-N 117593116

N BOTX P P F MTZDJ X- U H F F FAOYS A- N 117593375

RIZDOCXODMLFMH-UHFFFAOYSA-N 117593544

RJ ZOS QYZ DIYLOD-U HFFFAOYSA-N 117593652

CIMLDIMAIQTAAF-U HFFFAOYSA-N 117593685

SLOASZPUTSRJOS-U HFFFAOYSA-N 117594239

KQB IZWO EXAC M RJ -U H F F FAOYS A- N 117594907

HQPAKWNGDAIVMM-U HFFFAOYSA-N 117594911

XITFXYOJRIAYLM-UHFFFAOYSA-N 117595036

UUNPNKKRLMOBNZ-U HFFFAOYSA-N 117595578

XWFNYKWKDWAAMZ-QKBJRNKPSA-N 118707564

ZISRIFHOONSTEW-XQUYNDDWSA-N 118753102

MQXZYU NEWMQRJ D-MIRSFJNZSA-N 118753301

WCBINTABDRSBOM-BXQNXPOQSA-N 118753302

TZPROOSLUNQHV-YBSJ KGMBSA-N 118753348

Scortechinone A 44559179

9,10-Dihydrogambogic Acid 71459533

GEZHEQNLKAOMCA-UOONSFDBSA-N 58209843

Gambogic acid amide 16725080

REDMIYQFNIRTDF-UCQKPKSFSA-N 5475311

Acetyl isoallogambogic acid 6857765

Gamboginic acid, methyl ester 23806091

REDMIYQFNIRTDF-OZWPVNNZSA-N 44449776

Decahydro-Ga 44449798

Tetrahydro-Ga 44449824

UYPYPAISERHQAO-PBBIOFTGSA-N 44452392

F N J G RU CXYD W BQQ- U H F F FAOYS A- N 117591871

Scortechinone B 44559180

Scortechinone 1 44559181

Scortechinone-Q 44559270

Scortechinone R 44559271

Scortechinone S 44559272

Bractatin 44583731

1-O-Methylbractatin 44583733

Methyl 8, 8a-dihydromorellate 45268013

YTIQONQLSSBXHE-OGRXGPJ BSA-N 70687664

OLVQCRKEVCKWSS-WNDU KFFSA-N 71717041

U PJGOGQG KKP FQF-VJ E MJ KLZSA- N 71720078

WNQJCSUJ MQRMEE-KWXZSCLYSA-N 76315019 FBNIACMJ HDKGKH-U HFFFAOYSA-N 117591045

MRFFDQCGXSUJFO-U HFF FAOYS A- N 117591692

KG P H 0 VCV H POW BR- U H F F FAOYS A- N 117591869

GEZHEQNLKAOMCA-U BYIDDGGSA-N 91332450

QOZHTUAZXBIGBU-WRXOINPPSA-N 117647595

GEZHEQNLKAOMCA-KSZVLNGESA-N 91351716

GEZHEQNLKAOMCA-IGPPFNQUSA-N 91356587

GEZHEQNLKAOMCA-WQMCTBSRSA-N 91395869

REDMIYQFNIRTDF-CLWCGEPSSA-N 91421299

DVT LN R RWC RGS E B-QLM U F R IZS A- N 91507797

DCU BEADPOQPJCP-WWYBWCOQSA-N 91525565

30-Hydroxygambogic acid 102004804

CCEGWRPYDCDELZ-J DHSLWBYSA-N 117640037

REDMIYQFNIRTDF-UVYBHTOASA-N 117640050

CXFIQFGADOTDPF-VAVNHFACSA-N 91116286

GEZHEQNLKAOMCA-QSNZZALHSA-N 91081424

MNNVIONVHRRQPF-IGPPFNQUSA-N 90998876

CGTWSSOGZQAVN l-YUTXI DHZSA- N 90956184

MNNVIONVHRRQPF-QSNZZALHSA-N 90904334

GEZHEQNLKAOMCA-BMAVOULBSA-N 90837811

MFUIGIDUBRLEU-RHDRSXQYSA-N 90802529

F A E QAX FMLPWRFS-U HFFFAOYSA-N 90793624

XA P L N RWTXV BXJQ-U HFFFAOYSA-N 89737453

AAEQTEKIFSEBLF-JCDNVTHQSA-N 123214118

IQDYCICYXWCEEX-QTFYU PPWSA-N 89593387

PHWVEYPUZJUGEV-OAWOWVGUSA-N 121241349

IWZRSTD KYHZSQF-S D RU QS ECSA- N 88870689

AORIIYVDTXBCHV-YCVDEPICSA-N 123197254

JTEORTUOYDVEOM-FOQNCPQJSA-N 123187933

GCHJONZZLLRAEM-IGQYWBJASA-N 123186661

LPPVILAKSVOTHC-VABJNMDGSA-N 123168249

DUZIVTBZXZNFKW-LGYDYSPQSA-N 123153387

XDJCBNWCKIBHCH-J LZOOELISA-N 123146135

IWZRSTDKYHZSQF-FBFXOJ LPSA-N 123143901

UJPXBLUJNQMYIY-VRJU NHGMSA-N 122542892

FHJLQIOSAMVKBE-UTKNUOMGSA-N 121241350

MPVLKYHKVPLU BC-WRXOINPPSA-N 117649446

RUOIRPONLNREJT-IXTCAIOQSA-N 121241347

RUOIRPONLNREJT-GAZVMYCTSA-N 121241346

WVBSIKJNTVCEQJ-NBQSLMHUSA-N 121241345

RCWNBHCZYXWDOV-WPKINVRVSA-N 121241344

WTHZNKDLVYBPIU-XKZIYDEJSA-N 121241343

VZXLWEWYBUGUA-KCZYMQEJSA-N 118218885

KWSMUTWPBWYJTJ-GBFWCEHUSA-N 118215929

YTINOMMNLVRQDZ-ODZJVPPQSA-N 118215928

PNVQU HOJEOSPST-CZHHEZJISA-N 117649448

AORIIYVDTXBCHV-NTXDIHSUSA-N 88870699

TZPSXPDSUISGI-JSTMFIRTSA-N 88870710

SYPMLUQDIRBAJH-DPGBVESVSA-N 88870709 TZPSXPDSUISGI-SMCHVARRSA-N 88870708

KIIICIKEPTYGHX-HQTPSEOASA-N 88870707

Kl 11 C 1 KE PTYG HX-W LVTU AKAS A- N 88870706

SYPMLUQDIRBAJH-GKPRHQBLSA-N 88870705

MPUTYJ MBORRTBJ-BJGVHYDOSA-N 88870704

GCHJONZZLLRAEM-ZWNPRXLMSA-N 88870703

AORIIYVDTXBCHV-XENTYZTMSA-N 88870702

LPPVILAKSVOTHC-MGXSGBCKSA-N 88870701

XTMOYKJZVWYKPJ-BEYSKSGQSA-N 88870711

XDJCBNWCKIBHCH-USNSJPIISA-N 88870698

PVG H FW KFADSYSJ- MU U FOGJZSA- N 88870697

TZPSXPDSUISGI-FZVGPGJDSA-N 88870696

DUZIVTBZXZNFKW-DJXAADCISA-N 88870695

FMCZWXSKQSSEHP-DJXAADCISA-N 88870694

DUZIVTBZXZNFKW-HAAYKU LCSA-N 88870693

KIIICIKEPTYGHX-LNYPENFMSA-N 88870692

KLX F RVJTZKO F KV-SXQTYU KPS A- N 88870691

TZPSXPDSUISGI-OU IKJMRCSA-N 88870690

XIDKYIKGGBTU PH-LFVJCYFKSA-N 126602554

PVG H FW KFADSYSJ-VS BO KRG HS A-N 88870721

AFLHWBGXZWEU LQ-PKAZHMFMSA-N 89410167

DU EZXUMGZFEZCZ-OYKKKHCWSA-N 89409368

KFTSCWCFQNHXEF-OYKKKHCWSA-N 89409366

MPUTYJ MBORRTBJ-ZRJUZVLNSA-N 88870748

IWZRSTDKYHZSQF-SAJNXLGZSA-N 88870747

AORIIYVDTXBCHV-PKRKJABKSA-N 88870746

KLX F RVJTZKO F KV- PQI N OV KQS A- N 88870744

MVRLZFHWUTWTPZ-OAWOWVGUSA-N 88870743

IWZRSTD KYHZSQF- WAS DJ RS KSA- N 88870742

XDJCBNWCKIBHCH-FXXVQEQYSA-N 88870740

VJGFCQXTEBDXCL-XYGWBWBKSA-N 89410235

FJTHIYKOKGKDFM-YECKHLLKSA-N 88870720

MPUTYJ MBORRTBJ-ZFGQNZLVSA-N 88870719

GCHJONZZLLRAEM-RHZAVJPWSA-N 88870718

FQCIAFWZLWMCNQ-HSU LCKAZSA-N 88870717

XTMOYKJZVWYKPJ-REPUEAQBSA-N 88870716

SYPMLUQDIRBAJ H-VG RXJTFRSA- N 88870715

N H N BZYVAEYGXJ D- PYOCCJ RJSA- N 88870714

KKKVOYLLLKUGN-MWJHYMAZSA-N 88870713

FMCZWXS KQSS E H P-XE NTYZTMS A- N 88870712

FJTHIYKOKGKDFM-WHJBVOODSA-N 123809343

GFZFBIVRU FQXDE-RAKWAVLCSA-N 123867307

JTEORTUOYDVEOM-AKJUZXHISA-N 123858966

KLX F RVJTZKO F KV- RWJ QYVG MS A- N 123858384

XTMOYKJZVWYKPJ-IIIU NIONSA-N 123851264

COVMVPHACFXMAX-NJEUQTODSA-N 123849167

PVGHFWKFADSYSJ-QFSWFWNDSA-N 123845773

TZPSXPDSUISGI-OWMZLRFQSA-N 123844233

DUZIVTBZXZNFKW-YCVDEPICSA-N 123821973 AORIIYVDTXBCHV-UOVBMFSZSA-N 123819800

IWZRSTDKYHZSQF-OWDHWTJPSA-N 123811651

KWSMUTWPBWYJTJ-GTU NQJGZSA-N 123867860

XTMOYKJZVWYKPJ-ZPHHXPIHSA-N 123801882

IWZRSTDKYHZSQF-STFZBRADSA-N 123799471

F M CZWXS KQSSEH P- LGYDYS PQS A- N 123783895

M PUTYJ M BO RRTBJ-U IVVF IOZSA- N 123768632

F M CZWXS KQSS E H P- N RBG CZKAS A- N 123764889

TZPSXPDSUISGI-HHTAKYNCSA-N 123747944

FJTHIYKOKGKDFM-VABJNMDGSA-N 123730782

COQAPWLZSHQTKA-WQXODUOJSA-N 123725925

SYPMLUQDIRBAJH-QHKZOBPHSA-N 123713443

GCHJONZZLLRAEM-MNTUFJQYSA-N 123689815

GEZHEQNLKAOMCA-MAKUZWOISA-N 123970099

MPUTYJ MBORRTBJ-GQPSOBIZSA-N 124083343

MVRLZFHWUTWTPZ-PLUQQRNKSA-N 124083342

TZNZFVPLCOBOHE-SRHIZFSVSA-N 124083341

KRGVLKLMSZLNJ D-CZBSRG PZSA-N 124083340

U N PJ U MTRSO E BG-YTJXBEJ ASA- N 124083339

MPUTYJ MBORRTBJ-ORYAWNIFSA-N 124011815

N H N BZYVAEYGXJ D- WQXO D U OJ S A- N 124009406

FMCZWXSKQSSEHP-UOVBMFSZSA-N 124004813

XDJCBNWCKIBHCH-PCCITZADSA-N 123995047

IWZRSTDKYHZSQF-IVPRPU KMSA-N 123970404

PVGHFWKFADSYSJ-AHWIIWHVSA-N 123672510

REDMIYQFNIRTDF-SNSZSSRMSA-N 123962837

NFVXKLYWFCNBCO-BIDYHREASA-N 123946655

FQCIAFWZLWMCNQ-MZQQFRDZSA-N 123931583

FMCZWXSKQSSEHP-YCVDEPICSA-N 123926824

SYPMLUQDIRBAJH-J KFUFCISA-N 123921560

SYPMLUQDIRBAJH-MXTXYYSDSA-N 123903462

Kl 11 C 1 KE PTYG HX- KAX RJ KLWS A- N 123902138

KLXFRVJTZKOFKV-XLPBWHEJSA-N 123898922

KKKVOYLLLKUGN-QHQAMMJOSA-N 123885028

GEZHEQNLKAOMCA-RAKWAVLCSA-N 123307604

TZPSXPDSUISGI-GTHFLSHASA-N 123415116

KIIICIKEPTYGHX-FEHYYDPSSA-N 123392802

KIIICIKEPTYGHX-QGNRMNGYSA-N 123385862

FBJVPBMWPVODRO-PCCITZADSA-N 123363376

GEZHEQNLKAOMCA-ADCYJAEYSA-N 123352107

DUZIVTBZXZNFKW-UOVBMFSZSA-N 123351208

SYPMLUQDIRBAJH-KUWKLSGISA-N 123340327

AORIIYVDTXBCHV-NRBGCZ KAS A- N 123338137

TZPSXPDSUISGI-OYYJVTFHSA-N 123324331

COVMVPHACFXMAX-WJQTU EGHSA-N 123322435

AORIIYVDTXBCHV-NWODWGALSA-N 123437820

N H N BZYVAEYGXJ D-RN LYVTM ESA-N 123307294

XDJCBNWCKIBHCH-MEXQKCLWSA-N 123306297

QOZ HTU AZXB IG BU - U H F F FAOYS A- N 123299953 SYPMLUQDI BAJH-CFCC WGCSA-N 123290623

UWZMGTSPGQXAAP-WQXODUOJSA-N 123272237

NFVXKLYWFCNBCO-VDMQVCGESA-N 123263934

BYS LEZZCJZXNQG- RWJQYVG MSA- N 123248797

DU EZXUMGZFEZCZ-U HFFFAOYSA-N 123248044

HWTUJOKAWVHJCJ-OPUOJSSUSA-N 123242824

RCWNBHCZYXWDOV-KBGSTRFQSA-N 123550305

XDJCBNWCKIBHCH-RAKWAVLCSA-N 123661979

SYPMLUQDIRBAJ H-XZG WO 1 PS A- N 123658150

UWZMGTSPG QXAA P- R N LYVT M ES A- N 123650727

KFTSCWCFQNHXEF-U HFFFAOYSA-N 123649704

KIIICIKEPTYGHX-ILHXXLRDSA-N 123628892

GEZHEQNLKAOMCA-VRYKAPMJSA-N 123626409

AO Rl IYVDTXBCH V- LGYDYS PQSA- N 123615972

DUZIVTBZXZNF KW- N R BG CZ KAS A- N 123581184

GEZHEQNLKAOMCA-CCZYIYSKSA-N 123570917

XTMOYKJZVWYKPJ-NVDUSELSA-N 123554777

BYSLEZZCJZXNQG-TYTNRNEYSA-N 123234071

PVGHFWKFA DSYSJ-KF M J CXDSS A- N 123549300

PVGHFWKFADSYSJ-JTFGTGAKSA-N 123542499

AAEQTEKIFSEBLF-DZGHJPMGSA-N 123522015

FQCIAFWZLWMCNQ-MLFAQU DHSA-N 123514172

XTMOYKJZVWYKPJ-GMLGDLCKSA-N 123504656

MPUTYJ MBORRTBJ-WHGJWNQISA-N 123494893

RCWNBHCZYXWDOV-NWPLKAIBSA-N 123475850

YTINOMMNLVRQDZ-MCGXLDAJSA-N 123469661

GEZHEQNLKAOMCA-BIDYHREASA-N 123464815

IWZRSTDKYHZSQF-GATNSQFVSA-N 58545200

RCWNBHCZYXWDOV-YZYLOZNXSA-N 58545211

SYPMLUQDIRBAJ H-HYTANOMUSA-N 58545210

XTMOYKJZVWYKPJ-ZOLZCLEESA-N 58545209

GEZHEQNLKAOMCA-BSGXHHMHSA-N 58545208

AORIIYVDTXBCHV-NTJOPWEGSA-N 58545207

UWZMGTSPGQXAAP-CAQBGEGQSA-N 58545206

VZQQLP ACAV HZQT- 1 YYXI F P BS A- N 58545204

DMEVOSRJ MNDQOB-XKZIYDEJSA-N 58545203

DUZIVTBZXZNFKW-SMSNCEFTSA-N 58545202

COVMVPHACFXMAX-PWZQPALBSA-N 58545201

KLXFRVJTZKOFKV-XFTJLXKISA-N 58545213

AAEQTEKIFSEBLF-CEGNIYDJSA-N 58545199

KIIICIKEPTYGHX-FBQUOBMKSA-N 58545198

IWZRSTDKYHZSQF-HJBKNYNFSA-N 58545197

FJT H IYKO KG KD F M- 1 D AC PZ N ES A- N 58545196

PVGHFWKFA DSYSJ-SZYQW LCWSA- N 58545195

KIIICIKEPTYGHX-ASZVOQTMSA-N 58545193

AORIIYVDTXBCHV-SMSNCEFTSA-N 58545192

SYPMLUQDIRBAJ H-WVJIZLELSA-N 58545190

GEZHEQNLKAOMCA-ITMOWBSKSA-N 58545188

TZPSXPDSUISGI-IOHWOTBESA-N 58545187 LPPVILAKSVOTHC-IDACPZNESA-N 58545228

CXFIQFGADOTDPF-DLLRBFTDSA-N 59248849

RAWMYNQUVMHIBX-IADYIPOJSA-N 59060966

MFUIGIDUBRLEU-SJZKZEADSA-N 58802103

MKRYDGCYDKJGSE-OMRLFU BUSA-N 58802102

MVRLZFHWUTWTPZ-UZAZBKDBSA-N 58554586

KIIICIKEPTYGHX-MGTQMBPOSA-N 58554585

MPUTYJ MBORRTBJ-QMNYGIFOSA-N 58554584

KKKVOYLLLKUGN-JTZZIZQTSA-N 58545231

PHWVEYPUZJUGEV-UZAZBKDBSA-N 58545230

SYPMLUQDIRBAJH-GUGAKOSKSA-N 58545229

FQCIAFWZLWMCNQ-PJ EVLYNISA-N 58545186

HWTUJOKAWVHJCJ-OFNKHKRGSA-N 58545227

MPUTYJ MBORRTBJ-UCUGODPPSA-N 58545224

N H N BZYVAEYGXJ D-SO RCPTHGS A- N 58545221

XTMOYKJZVWYKPJ-JBZGCEAXSA-N 58545220

BYSLEZZCJZXNQG-PSRDIXOWSA-N 58545219

XDJCBNWCKIBHCH-ASCSEZEHSA-N 58545218

TZPSXPDSUISGI-HEERNYNQSA-N 58545217

COQAPWLZSHQTKA-CAQBGEGQSA-N 58545215

DUZIVTBZXZNFKW-NTJOPWEGSA-N 58545214

VDSCKSOYNLTQSY-KKQCBWBVSA-N 6710618

MNNVIONVHRRQPF-HMMDVQROSA-N 16750435

MNNVIONVHRRQPF-JDFKUOOISA-N 16750413

GEZHEQNLKAOMCA-ZEYIWNDBSA-N 11556381

KCALBYYRHOKKBO-QDTIIGTASA-N 11468283

DWYQBZCXZCGVHI-YSMPRRRNSA-N 11422617

KVXKEBWQNIIKMB-MTJSOVHGSA-N 11308419

AANGJSKPBIKCLU-ITYLOYPMSA-N 9896558

MFUIGIDUBRLEU-UCQKPKSFSA-N 9895689

KYPSMUUXSFJTAR-HEEAUFFFSA-N 9851944

LFSCNWNADRU BLS-U HFFFAOYSA-N 6710687

GEZHEQNLKAOMCA-DTWORVFFSA-N 16750462

COQAPWLZSHQTKA-FRMWRBSQSA-N 6419330

VDSCKSOYNLTQSY-KKQCBWBVSA-N 6710618

Isomorellic acid 6419329

VZQQLP ACAV HZQT- R N LYVTM ES A- N 6325059

CXFIQFGADOTDPF-LPYMAVHISA-N 6284659

VZXLWEWYBUGUA-U HFFFAOYSA-N 5205218

CXFIQFGADOTDPF-U HFFFAOYSA-N 5149277

COVMVPHACFXMAX-U HFFFAOYSA-N 550587

COQAPWLZSHQTKA-RNLYVTMESA-N 442607

GEZHEQNLKAOMCA-PCCITZADSA-N 442595

REDMIYQFNIRTDF-U HFFFAOYSA-N 421874

PZOHDYPLDDMKLL-BRXPKNJNSA-N 56595878

FMCZWXSKQSSEHP-NTJOPWEGSA-N 58545185

FMCZWXSKQSSEHP-SMSNCEFTSA-N 58545184

GCHJONZZLLRAEM-VSNLTSTASA-N 58545183

XDJCBNWCKIBHCH-KKCBTXSASA-N 58545182 PVG H FW KFADSYSJ-QE DXCBQSSA- N 58545181

NFVXKLYWFCNBCO-UOONSFDBSA-N 58209844

CGTWSSOGZQAVNI-NXHYFTOVSA-N 57941599

GEZHEQNLKAOMCA-AOJNUVNQSA-N 57845639

RE DM IYQFN 1 TD F-YOXD LC KMSA- N 57586028

QHQBTUGYLFRMGB-U HFFFAOYSA-N 57332076

GEZHEQNLKAOMCA-WOMUXGJCSA-N 59248851

AAEQTEKIFSEBLF-CPNRCEQSSA-N 56595835

DRCNCMDYOLGEQM-BRXPKNJNSA-N 54764387

PVRRTDHRPRHFPD-BRXPKNJNSA-N 54764299

JTEORTUOYDVEOM-LMZOBULNSA-N 25134602

UYPYPAISERHQAO-XKZIYDEJSA-N 23391922

YXDVFXVYFZUHNH-OYKKKHCWSA-N 23391915

WCBINTABDRSBOM-BKUYFWCQSA-N 23391867

CAZOJ RLTSYFYH R- H M APJ EAMSA- N 23391861

IQYGG NGWJ AGS BX-QTSGYQI KSA-N 21603452

BZFVSJCAEZAUPC-U HFFFAOYSA-N 76658597

GAIPRNHDISFSOS-U HFFFAOYSA-N 78056261

JMMQRHVWNJXTCK-U HFFFAOYSA-N 78056259

ZOKLYUGLUUVLW-U HFFFAOYSA-N 78056187

SUOOENMABGOQCH-U HFFFAOYSA-N 78056180

KCYKVBCVFBSOKZ-U HFFFAOYSA-N 77153183

JAWDBXPEU RIBJV-U HFFFAOYSA-N 77152099

G U AS LO U LTW PT KR- U H F F FAOYS A- N 76658664

SU FYIU RU DYGNLI-U HFF FAOYS A- N 76658661

WCVGFLPSZFYRCL-U HFFFAOYSA-N 76658621

KRU KCSS BDGDWBK-U HFF FAOYS A- N 76658617

FSSXEBRQIMPUGN-U HFFFAOYSA-N 78056262

YHKGUOGCUACMHC-U HFFFAOYSA-N 76658582

BVWTXTU LIJ KQBW-U HFFFAOYSA-N 76658577

UYPYPAISERHQAO-U HFFFAOYSA-N 74047027

YXDVFXVYFZUHNH-U HFFFAOYSA-N 74047023

WCBINTABDRSBOM-U HFF FAOYS A- N 74046984

CAZOJ R LTSY FYH R- U H F F FAOYS A- N 74046983

RC W N B H CZYXW DOV- U H F F FAOYS A- N 73008268

KCALBYYRHOKKBO-U HFFFAOYSA-N 72973410

DWYQBZCXZCG VH l-U H FF FAOYSA- N 72955606

KVX KE BWQN 11 KM B- U H F F FAOYS A- N 72795177

AORIIYVDTXBCHV-H AAY KU LCS A- N 88870677

IWZRSTDKYHZSQF-SEXUYDOESA-N 88870687

NHNBZYVAEYGXJD-BUZUJXMISA-N 88870686

KIIICIKEPTYGHX-GWVHQLHWSA-N 88870685

SYPMLUQDIRBAJ H-VWXYWCCPSA- N 88870684

XTMOYKJZVWYKPJ-XPIJMDORSA-N 88870683

SYPMLUQDIRBAJ H-QNAMZJFPSA-N 88870682

XTMOYKJZVWYKPJ-KZHARUQXSA-N 88870681

HWTUJOKAWVHJCJ-KJ LFQBLLSA-N 88870680

PHWVEYPUZJUGEV-PLUQQRNKSA-N 88870679

AO Rl IYVDTXBCH V- DJXAADC ISA- N 88870678 RAWMYNQUVMHIBX-U HFFFAOYSA-N 72503918

XDJCBNWCKIBHCH-NDZGPYJESA-N 88870676

PVG H FW KFADSYSJ-SQH FVPGGSA- N 88870675

PVGHFWKFADSYSJ-CZBSRGPZSA-N 88870674

DUZIVTBZXZNFKW-XENTYZTMSA-N 88870673

FMCZWXSKQSSEHP-NTXDIHSUSA-N 88870672

FMCZWXS KQSS E H P-HAAYKU LCS A- N 88870671

DUZIVTBZXZNFKW-NTXDIHSUSA-N 88870670

FJTHIYKOKGKDFM-MGXSGBCKSA-N 88870669

AANGJSKPBIKCLU-U HFFFAOYSA-N 85062448

GEZHEQNLKAOMCA-FSLBZXJLSA-N 66603456

GEZHEQNLKAOMCA-DDBFOHIYSA-N 70639874

Deoxymorellin 70639873

BYSLEZZCJZXNQG-FEOFYTQISA-N 70639871

GEZHEQNLKAOMCA-ZESOCHHDSA-N 70639869

Isomorellinol 70639868

AAEQTEKIFSEBLF-HABQCU FLSA-N 70639867

CXFIQFGADOTDPF-DXJQLTJMSA-N 70235747

GEZHEQNLKAOMCA-WFPDQCIUSA-N 70235746

KCYKVBCVFBSOKZ-KPKJ PENVSA-N 66612138

JAWDBXPEU RIBJV-WUXMJOGZSA-N 66603468

UWZMGTSPGQXAAP-XVQCHYCYSA-N 70639876

LWIGRTRTVVPXOZ-FNUJ IKEJSA-N 66561235

GEZHEQNLKAOMCA-UTDYKPHNSA-N 66509103

COQAPWLZSHQTKA-XVQCHYCYSA-N 59895966

LWIGRTRTVVPXOZ-BVLCDU KVSA-N 59895965

QDXKAHJQAXFABR-JQJUWNFSA-N 59895964

GEZHEQNLKAOMCA-HWOJJXKDSA-N 59895963

DCU BEADPOQPJCP-YGJDYZIVSA-N 59607534

HNEQMSDU EKZLSM-HGSFHZCQSA-N 59248855

U FULGOYVXFMAL-SSQGKGTLSA-N 59248854

QGMGFU LXKBKTCL-KIXMQPMBSA-N 70641345

MFUIGIDUBRLEU-U HFFFAOYSA-N 72428343

MKRYDGCYDKJGSE-U HFFFAOYSA-N 72428342

NTOPU RIAG NZM KL-IM RQLAEWSA-N 71262488

FSSXEBRQIMPUGN-UCQKPKSFSA-N 71261269

GAIPRNHDISFSOS-HMAPJEAMSA-N 71261268

JMMQRHVWNJXTCK-UCQKPKSFSA-N 71261266

ZOKLYUGLUUVLW-ITYLOYPMSA-N 71261175

SUOOENMABGOQCH-ITYLOYPMSA-N 71261168

MFUIGIDUBRLEU-LOSVMQNTSA-N 71215924

WCILAHSZCPIJPY-NTDTWHTNSA-N 70968535

FQCIAFWZLWMCNQ-JTWMGGOBSA-N 88870688

BLDWFKHVHHINGR-BBHBLYOZSA-N 70641343

NPTGLFQRDIIEBF-IZWSUVCCSA-N 70641230

NPTGLFQRDIIEBF- BXIW ITQHSA- N 70641229

J EGOO EVCLQI 1 NX- IWBBQFJ ISA- N 70641225

IXIIUJBIELTYTO-WECGGMARSA-N 70641219

FGVLMIINZOOWDO-BRWU PGPDSA-N 70641217 AAEQTEKIFSEBLF-WXZUYDNESA-N 70639883

Desoxygambogenin 70639878

COVMVPHACFXMAX-GJFVWJTOSA-N 70639877

[00382] Gambogic acid remedies the levels of CTXLP-associated pathological markers Ca V2.2 CCA T and Nogo-A

[00383] Figure 56 highlights than not only CTXLP is reduced in the presence of gambogic acid, but that the drug treatment also restores CaV2.2 CCAT expression (known to be suppressed by CTXLP, Figure 47). Furthermore, we demonstrate show that gambogic acid is a potent inhibitor of Nogo-A (which inhibits remyelination) expression in cancerous NCCIT cells (Figure 56). As the decrease in Nogo-A expression directly correlates with a drop in CTXLP expression, this therapeutic strategy may also reduce pathogenic Nogo-A expression in ALS (Figure 42). Nogo-A inhibitors have been previously identified, such as green tea polyphenols and other natural product extracts ¹⁵¹ . Proteolytic turnover of Nogo-A is a physiological mechanism to reduce Nogo-A levels ¹⁵² . Gambogic acid may reduce Nogo-A expression by having an effect of CTXLP-driven pathology or a more direct effect on specific protein turnover ^{153 154} .

[00384] Development of cell and animal models to investigate CTXLP pathogenesis

[00385] Identification of C TXLP-encoding ER VK loci in primate genomes and their human homologues

[00386] Our close relatives also encode ERVK, but some ERVK loci are unique to humans. Examination of the ERVK content of three non-human primate genomes, Pan troglodytes (Common chimpanzee), Gorilla gorilla gorilla (Western lowland gorilla), and Cercocebus atys (Sooty Mangabey) shows that CTXLP is not limited to humans.

[00387] The most recent genomic assembly for each primate species was searched for CTXLP in the same manner as the human genome (Table 13). panTro5 and gorGor5 were retrieved from UCSC, and Caty_1.0 was retrieved from NCBI. Chimpanzee ERVK were identified using UCSC table panTro5.nestedRepeats, but no such table exists for the Gorilla or Sooty Mangabey. Gorilla and Mangabey ERVK were identified directly from

RepeatMasker output. To reduce the number of small ERV fragments to be BLASTed and to increase the accuracy of orthology predictions by including flanking genomic regions, the loci annotated in RepeatMasker were extended by 1000 bp to either side and then any less than 10 bp apart were merged.

Table 13: ERVK and CTXLP loci in primates.

Orthologous ERVK fragments identified by BLAST Number of PF080S7* and PF13804* Loci by Species

. sapiens P. troglodytes 0. gorilla C. atys Species CTXLP Env

H. sapiens 7358 6060 SS55 2¾8S H. sapiens 28 3S3

P. troglodytes 6060 7389 5S12 2¾81 P. troglodytes 33 402

G gorilla 5S53 5812 6504 2562 G gorilla 39 31S

C otys 25S5 25S1 2565 7411 C otys 31 379

[00388] The expected relationship between the four species is displayed by the number of orthologs recognized. Humans are most closely related to Chimpanzees, then to Gorillas, and most distantly to the Sooty Mangabey. We can see in the second table that the number of CTXLP and Env positive ERVK loci varies minimally between species.

[00389] Only two human CTXLP+ loci are present in all four species. There is also 1 mangabey locus present in all four. No loci were CTXLP+ and present in all four species. Figure 59 depicts MUSCLE alignments of tBLASTx results from loci in human, chimpanzee, gorilla, and mangabey where an orthologue in at least one species encodes a Toxin_18+ ORF. The first four alignments are split in two, where the top half contains sequences belonging to orthologous sets aligned horizontally, and the bottom half contains the remaining sequences from each species. The fifth alignment is tBLASTx results for the cd-hit cluster representative sequence of the largest cluster of Toxin_18+ ORFs from each species. Only tBLASTx results for loci which encoded a representative sequence of a cluster containing more than one member were included. This alignment was generated by

MUSCLE, then curated so that the Cys residues of chimpanzee_108932 aligned better with the other sequences. Figure 60 suggests that there is more conservation between orthologues in different species than between paralogues from the same species. This pattern is much more apparent for CTXLP than for Env reading frame, where CTXLP appear to be under diversifying selection as indicated by increased dissimilarity between the CTXLP reading frame as compared to the Env reading frame of given sequences, differences are smaller if present at all. Taken together, this suggests that non-human primates are viable models for CTXLP research.

[00390] Murine model of ERVK CTXLP

[00391] Avindra Nath's group has successfully developed a murine model which supports the neurotoxic potential of the ERVK envelope gene towards motor neurons ⁶⁹ .

ERVK env gene transgenic mice exhibit progressive motor dysfunction and hallmark pathology associated with ALS such decreased motor cortex volume and injury to pyramidal neurons and anterior horn cells in the spinal cord. This murine model is a solid platform for ERVK research, yet it remains unclear whether pathology and clinical outcomes were driven by canonical retroviral envelope proteins or CTXLP. This is because the insert used to generate the transgenic mice has the capacity to encode CTXLP (Figures 57 and 58).

However, these mice do represent a putative model of CTXLP-driven neuropathology and neurodegeneration.

[00392] Drosophila models of ERVK Env gene products, including CTXLP

[00393] Drosophila (fruit flies) are a widely-used model organism, often used to study the cellular effects of pathogenic human viruses ¹⁵⁵ . Moreover, TDP-43 null and TDP-43 mutant flies develop measurable motor deficits ^156-158 , making this model an exceptional tool to evaluate the impact of ERVK on ALS-like neuropathology and clinical outcomes. In collaboration with Dr. Alberto Civetta (University of Winnipeg), have designed an animal model system in which the ERVK proteins are transgenically expressed in Drosophila melanogaster.

[00394] ERVK Env, SU, TM and CTXLP open reading frames have been cloned (by GenScript, USA) into a pUAST vector (Drosophila Genomic Resource Center, #1000), allowing for Gal4 control of transgene expression patterns (see section above on design of custom CTXLP, SU and Env vectors). Generation of an ERVK protein transgenic flies is outsourced to BestGene Inc. (Chino Hills, CA). Flies will be crossed with neuronal (ELAV ¹⁵⁶ ), glia (repo ¹⁵⁹ ) or astrocyte (alrm ¹⁶⁰ )-restricted Gal4 fly strains (Bloomington Drosophila Stock Center 8760, 7415 & 67032, respectively) to generate flies selectively expressing cell-type specific ERVK proteins. For each experimental group, lifespan analysis and locomotor impairment (# of walks/focal) will be monitored as previously described ^{156 161 162} ENREF 80. To perform pathological examinations, flies will be cold-sacrificed, heads removed, and tissue either flash frozen for western blot or fixed for immunohistochemical analyses. Biological readouts will be correlated with survival and motor-impairment metrics, as to assess how pathological events track with clinical outcomes. In a second series of experiments, fly models exhibiting clinical impairment will be used to assess the efficacy of a panel of CTXLP inhibitors, such as celastrol and gambogic acid (Figures 54-58). Inhibitors will be dissolved in DMSO and spiked into standard fly food just before solidification, at therapeutic concentrations of drug. Impact of drug administration on neuropathology and clinical outcomes will be evaluated.

[00395] Human tissue culture models of CTXLP expression

[00396] CTXLP was detectable in all human cell lines assayed (SVGA, ReNcell CX, NCCIT, T47D, cancer cell line panel), with varying degrees of expression. Based on the data shown above (Figures 54-58), ERVK CTXLP-expressing NCCIT teratocarcinoma cells are a viable model for drug screening applications. Additionally, we have developed a transient vector (pcDNA3.1 , Figures 46 and 52) and a drug (cumate)-inducible lentiviral system (SBI SparQ QM812B-1 ¹⁶³ ) allowing for stable overexpression of ERVK CTXLP, SU and Env. By using the feeder-independent pluripotent stem cell line WA09 (WiCell, mTeSR™1/Matrigel™ Platform) we can establish cerebral organoids, as previously described ¹⁶⁴ . The above described protocols will form the foundation for generating ERVK CTXLP-expressing cerebral organoids, a human, druggable, three-dimensional brain model. Lentivirus transduction and flow cytometry selection will be used to generate WA09 stem cells containing the previously described cumate-inducible CTXLP vector. To establish a model of ERVK CTXLP-mediated pathology in intact cerebral tissue, these genetically modified WA09 cells will be grown to maximally sized cerebral organoids in the absence of cumate. Both wild-type and CTXLP- inducible cerebral organoids will be treated with varying doses of cumate, to allow for dose- response and time course experiments. Other potential human systems to study CTXLP in the future includes patient-derived human induced pluripotent stem cells systems ^165"167 .

[00397] In summary, Human tissue and animal models for the study of CTXLP in ALS and cancer are needed. We are actively working to further develop our human tissue culture models. In addition, together with Dr. Alberto Civetta, we are in the process of developing a model in Drosophilia at the University of Winnipeg. Importantly, we will continue to pursue mammalian models with our collaborators which offer an opportunity to explore multiple features of pathogenesis as we continue to elucidate the processes involved in CTXLP pathogenesis.

[00398] Discussion

[00399] Endogenous retroviruses (ERVs) are host genetic elements, representing approximately 8% of human genomic DNA. ERV activation can benefit their host, or in other contexts are proposed to be involved in pathogenesis and disease ⁶ . Our interest in ERVK and the CTXLP protein lies in its association to motor neuron conditions such as Amyotrophic Lateral Sclerosis (ALS), as well in cancers.

[00400] ERVK is known to be up-regulated in the neurons of many individuals with ALS ⁶⁷ ' ^{8990 168} . The motor impairment in ALS is linked to calcium channel dysfunction, which is considered a viable therapeutic target. Thus, we were particularly intrigued by the CTXLP region of the ERVK genome when we discovered that it encoded a conotoxin-like peptide indicative of a pathogenic mechanism in ERVK associated disease. O-superfamily conotoxins are known to inhibit voltage gated calcium ion channels ¹⁶⁹ . These channels are predominantly expressed at the presynaptic terminal of neural synapses ¹⁷⁰ . When an impulse reaches this region, they allow calcium ions into the neuron, thereby increasing calcium ion concentration. This increase in concentration leads to fusion of synaptic vesicles containing neurotransmitters with the presynaptic membrane. The neurotransmitters are then released into the synaptic cleft where they bind to receptors on the postsynaptic terminal and stimulate downstream signaling. Inhibition of these channels with conotoxins can lead to tremors and an inhibition of motor function ^{23 24} . Our findings indicate that ERVK CTXLP may likewise be able to inhibit VGCCs and their calcium channel-associated transcriptional regulator (CCAT) and elicit similar responses resulting in impaired motor function.

[00401] It may be that ERVK CTXLP was previously implicated in ALS pathology. Notably, a 1997 study found that sera from 5 out of 6 ALS patients was able to reduce calcium ion currents when applied to mouse dorsal root ganglia ¹⁷¹ . The sera from a variety of disease control groups did not exhibit any effect on calcium ion currents. The authors concluded that "serum factors" from ALS patients can be passively transferred to affect calcium ion channel activity. It is possible that ERVK CTXLP may be the mediator of this effect. If this was the case, the protein would either have to be produced in non-brain cells or tissues. ERVK reverse transcriptase has been detected in the serum of many ALS patients ^{172 173} . If this enzyme originates from ERVK, it would demonstrate that ERVK proteins enter the serum during ALS. Thus, it would be possible that CTXLP could be present in the serum as well. In addition, a compromised blood-brain barrier observed in ALS and other neurology inflammatory events would allow CTXLP to cross into the serum ¹⁷⁴ .

[00402] Conotoxins are able to specifically inhibit ion channels of certain types of neurons ¹⁶⁹ , which may correlate with the loss of motor function and neurocognitive decline that is observed in ALS ¹⁷⁵ . Other neurotoxin models have been proposed as etiological agents of ALS. The most prominent example is the suspected link between beta-N-methylamino-L- alanine (BMAA), a neurotoxin produced by a group of terrestrial cyanobacterial symbionts in cycad plants, and ALS (or an ALS-like syndrome) ¹⁷⁶ . However, large scale spatial clustering of individuals with ALS has been inconsistent with the range of BMAA-producing cyanobacteria and other suspected environmental risk factors (although small, regional concordances have been identified) ¹⁷⁶ . That is, no yet-proposed neurotoxin etiology has been able to explain the vast majority of cases of ALS. Therefore, although an environmental neurotoxin model for ALS makes sense at a physiological level, a genetic-based model (with environmental/epigenetic influence) seems more likely at an epidemiological level. A genetically-encoded neurotoxin etiology of ALS, such as ERVK CTXLP, would be consistent with both of these approaches. This model would not rely on the requirement to identify unique genes in individuals with ALS, as the different phenotypes (having ALS or not having it) could be caused by differential expression of the same genetic material. Additionally, two active ERVK loci unique to ALS patients have been identified ⁹⁰ . It is possible that the CTXLP proteins of these loci are more functional than other ERVK sequences. Another alternative possibility is that insertional polymorphisms or single nucleotide polymorphisms result in differential ERVK CTXLP production or function. Thus, it is possible that sequence variation in ALS patients leads to differentially functional ERVK CTXLP proteins.

[00403] Additionally, there are several seemingly disparate features of conotoxin toxicity and ALS pathophysiology that would have to be resolved in order for such a link to be possible. For instance, calcium ion concentrations in the neurons of many ALS patients are elevated ^{175 177} . Since omega-conotoxins inhibit calcium influx into neurons ¹⁶⁹ , elevated calcium levels are the opposite of what would normally be expected in an ERVK CTXLP etiology of ALS ¹⁷⁵ . However, it may be possible that these two features are not inconsistent, as many factors (aside from VGCCs) control neuronal calcium ion concentrations ¹⁷⁰ . For instance, calcium-binding proteins such as calbindin-D28K and parvalbumin are absent in motor neurons lost early in ALS ^{170 177} . These proteins were present in significantly higher concentrations in healthy motor neurons, and in those affected later in the course of the disease ¹⁷⁰ . Impaired mitochondrial calcium buffering has also been observed in ALS neurons ¹⁷⁰ ' ¹⁷⁷ .

[00404] Apart from pathology associated with VGCC disruption, we have also shown that CTXLP expression is correlated with elevated Nogo-A in the spinal cord of patients with ALS. Nogo-A is a key regulator of oligodendrocyte precursor cell (OPC) differentiation, ultimately negatively impacting remyelination and tissue repair. Demyelinated spinal cord lesions show an increased abundance of Nogo-A+ OPCs, yet the inability of OPCs to mature is proposed as the mechanism driving a non-permissive environment leading to remyelination failure ^103-107 . Additionally, Nogo-A favours a pro-inflammatory context ¹²³ , one that would promote ERVK expression via modulation of NF-κΒ and pro-inflammatory cytokine secretion ⁶⁷ .

[00405] Nogo-A is implicated in a variety of neurological conditions, such as spinal cord injury, peripheral neuropathies, stroke, temporal lobe epilepsy, Alzheimer's disease, ALS, MS and schizophrenia ^{101 108"110} . Nogo-A has been identified as a prognostic marker and therapeutic target in ALS due to its substantial expression in muscle tissue from patients with motor neuron disease ^{111 112} . Mechanistically, Nogo-A expression destabilizes neuromuscular junctions ^113"116 . Indeed, clinical trials using human anti-Nogo-A antibodies have been performed (ATI 355 from Novartis Pharma and Ozanezumab and GSK1223249 from GlaxoSmithKline) ¹⁰¹ ' ¹¹⁷ ' ¹¹⁸ . These therapies were designed to target Nogo-A expression in the periphery (intravenous infusions), but may fail to block Nogo-A expression in the CNS, thus explaining the negative results in Phase II clinical ALS trials with Ozanezumab ^{119 120} . Yet, anti- Nogo-A and remyelination-based therapies may be of value in the treatment of CTXLP+ disease states.

[00406] As ERVK CTXLP is present in the tissues of ALS patients, it may be used as a biomarker for the disease. This is significant given that ALS is often difficult to diagnose in its initial stages ¹⁷⁵ . Furthermore, if it is found to be an etiological agent of disease, ERVK CTXLP levels could be useful in assessing disease progression or prognosis. Perhaps most importantly, therapeutics could be designed to target it in order to reduce motor function deficits and increase longevity. For instance, a humanized monoclonal antibody could be designed against ERVK CTXLP for intravenous immunoglobulin (IVIG) therapy. Alternatively, small molecule inhibitors, such as MAEs celastrol and gambogic acid, could be used to target ERVK CTXLP DNA binding, gene transactivation effects, enhancement of NF-κΒ expression and modulation of pathogenic biomarkers. If ERVK CTXLP is found to play pathological roles in other diseases, for example spinal cord injury, multiple sclerosis, schizophrenia or cancers to name a few, this avenue of research could have implications on the diagnosis and treatment of these diseases as well.

[00407] ERVK expression is up-regulated in schizophrenia and bipolar disorders ¹⁷⁸ (unpublished data). This may be worth investigating further if ERVK CTXLP production is confirmed, given the fact that omega-conotoxins can cause emotional distress and prolonged delirium with psychotic features ^{24 179} . [00408] Additionally, HIV and HTLV infections are known to lead to up-regulation of ERVK expression ^{180 181} . Both of these infections are associated with poorly understood, reversible ALS-like syndromes in a small number of patients ^182-184 . HIV-associated ALS can be treated effectively with highly active anti retroviral therapy (HAART) ^{182 183} . It is possible that ERVK CTXLP is a pathological contributor to exogenous retrovirus infections and these ALS- like diseases.

[00409] Many cancers are associated with ERVK expression ¹⁸⁵ . Evidence that increased ERVK CTXLP expression occurs in cancers cells implicates this viral protein in oncogenesis and possibly metastasis. Specifically, the induction of NF-κΒ is likely a key feature of ERVK CTXLP activity which may facilitate cancer development and progression ^{186 187} .

[00410] Together, the results of this analysis provide a basis for further research into the ERVK genome and the relationship between ERVK and inflammatory disease. Given the possible correlations between ERVK CTXLP and disease pathology, this line of research deserves further study.

[00411] Figures 63-65 summarize possible implications of our discoveries.

[00412] References

[00413] 1. Hohn, O., Hanke, K. & Bannert, N. HERV-K(HML-2), the Best

Preserved Family of HERVs: Endogenization, Expression, and Implications in Health and Disease. Frontiers in oncology 3, 246 (2013).

[00414] 2. Lesbats, P., Engelman, A.N. & Cherepanov, P. Retroviral DNA

Integration. Chemical reviews (2016).

[00415] 3. Christensen, T. HERVs in neuropathogenesis. J Neuroimmune

Pharmacol 5, 326-335 (2010).

[00416] 4. Weiss, R.A. The discovery of endogenous retroviruses. Retrovirology 3, 67 (2006).

[00417] 5. Lokossou, A.G., Toudic, C. & Barbeau, B. Implication of human endogenous retrovirus envelope proteins in placental functions. Viruses 6, 4609-4627 (2014).

[00418] 6. Manghera, M., Ferguson, J. & Douville, R. Endogenous retrovirus-K and nervous system diseases. Curr Neurol Neurosci Rep 14, 488 (2014).

[00419] 7. Macfarlane, C. & Simmonds, P. Allelic variation of HERV-K(HML-2) endogenous retroviral elements in human populations. J Mol Evol 59, 642-656 (2004). [00420] 8. Shin, W., et al. Human-specific HERV-K insertion causes genomic variations in the human genome. PLoS One 8, e60605 (2013).

[00421] 9. Marchi, E., Kanapin, A., Magiorkinis, G. & Belshaw, R. Unfixed endogenous retroviral insertions in the human population. J Virol 88, 9529-9537 (2014).

[00422] 10. Seifarth, W., et al. Comprehensive analysis of human endogenous retrovirus transcriptional activity in human tissues with a retrovirus-specific microarray. J Virol 79, 341-352 (2005).

[00423] 11. Griffiths, D.J. Endogenous retroviruses in the human genome sequence. Genome Biol 2, REVIEWS1017 (2001).

[00424] 12. Buzdin, A., Kovalskaya-Alexandrova, E., Gogvadze, E. & Sverdlov, E. At least 50% of human-specific HERV-K (HML-2) long terminal repeats serve in vivo as active promoters for host nonrepetitive DNA transcription. J Virol 80, 10752-10762 (2006).

[00425] 13. Ruggieri, A., et al. Human endogenous retrovirus HERV-K(HML-2) encodes a stable signal peptide with biological properties distinct from Rec. Retrovirology 6, 17 (2009).

[00426] 14. Denne, M., et al. Physical and functional interactions of human endogenous retrovirus proteins Np9 and rec with the promyelocytic leukemia zinc finger protein. J Virol 81 , 5607-5616 (2007).

[00427] 15. Contreras-Galindo, R., et al. Characterization of human endogenous retroviral elements in the blood of H I V-1 -infected individuals. J Virol 86, 262-276 (2012).

[00428] 16. Dewannieux, M., Blaise, S. & Heidmann, T. Identification of a functional envelope protein from the HERV-K family of human endogenous retroviruses. J Virol 79, 15573-15577 (2005).

[00429] 17. Julien, J. P., et al. Crystal structure of a soluble cleaved HIV-1 envelope trimer. Science 342, 1477-1483 (2013).

[00430] 18. Daly, N.L. & Craik, D.J. Bioactive cystine knot proteins. Curr Opin Chem Biol 15, 362-368 (2011).

[00431] 19. Iyer, S. & Acharya, K.R. Tying the knot: the cystine signature and molecular-recognition processes of the vascular endothelial growth factor family of angiogenic cytokines. FEBS J 278, 4304-4322 (201 1).

[00432] 20. McNulty, J.C., et al. Structures of the agouti signaling protein. J Mol Biol 346, 1059-1070 (2005). [00433] 21. Zhu, S., Darbon, H., Dyason, K., Verdonck, F. & Tytgat, J. Evolutionary origin of inhibitor cystine knot peptides. FASEB J 17, 1765-1767 (2003).

[00434] 22. Becker, S. & Terlau, H. Toxins from cone snails: properties, applications and biotechnological production. Appl Microbiol Biotechnol 79, 1-9 (2008).

[00435] 23. Anderson, P.D. Bioterrorism: toxins as weapons. Journal of pharmacy practice 25, 121-129 (2012).

[00436] 24. Obafemi, A. & Roth, B. Prolonged delirium with psychotic features from omega conotoxin toxicity. Pain Med 14, 447-448 (2013).

[00437] 25. Norton, R.S. & Olivera, B.M. Conotoxins down under. Toxicon 48, 780- 798 (2006).

[00438] 26. Su, S.C., et al. Regulation of N-type voltage-gated calcium channels and presynaptic function by cyclin-dependent kinase 5. Neuron 75, 675-687 (2012).

[00439] 27. Adams, D.J. & Berecki, G. Mechanisms of conotoxin inhibition of N- type (Ca(v)2.2) calcium channels. Biochim Biophys Acta 1828, 1619-1628 (2013).

[00440] 28. Eldridge, R., Li, Y. & Miller, L.K. Characterization of a baculovirus gene encoding a small conotoxinlike polypeptide. J Virol 66, 6563-6571 (1992).

[00441] 29. Gracy, J., et al. KNOTTIN: the knottin or inhibitor cystine knot scaffold in 2007. Nucleic Acids Res 36, D314-319 (2008).

[00442] 30. Kearse, M., et al. Geneious Basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics 28, 1647-1649 (2012).

[00443] 31. Pettersen, E.F., et al. UCSF Chimera-a visualization system for exploratory research and analysis. Journal of computational chemistry 25, 1605-1612 (2004).

[00444] 32. Daly, N.L., Rosengren, K.J., Henriques, S . & Craik, D.J. NMR and protein structure in drug design: application to cyclotides and conotoxins. Eur Biophys J 40, 359-370 (201 1).

[00445] 33. Jiang, L, et al. 1 11 In-labeled cystine-knot peptides based on the Agouti-related protein for targeting tumor angiogenesis. J Biomed Biotechnol 2012, 368075 (2012).

[00446] 34. Li, D., et al. Function and solution structure of hainantoxin-l, a novel insect sodium channel inhibitor from the Chinese bird spider Selenocosmia hainana. FEBS Lett 555, 616-622 (2003). [00447] 35. Sato, K., et al. Binding of Ala-scanning analogs of omega-conotoxin

MVIIC to N- and P/Q-type calcium channels. FEBS Lett 469, 147-150 (2000).

[00448] 36. Jackson, P. J., et al. Structural and molecular evolutionary analysis of

Agouti and Agouti-related proteins. Chem Biol 13, 1297-1305 (2006).

[00449] 37. Bagashev, A. & Sawaya, B.E. Roles and functions of HIV-1 Tat protein in the CNS: an overview. Virol J 10, 358 (2013).

[00450] 38. Kruman, II, Nath, A. & Mattson, M.P. HIV-1 protein Tat induces apoptosis of hippocampal neurons by a mechanism involving caspase activation, calcium overload, and oxidative stress. Exp Neurol 154, 276-288 (1998).

[00451] 39. Popper, S.J., et al. Lower human immunodeficiency virus (HIV) type 2 viral load reflects the difference in pathogenicity of HIV-1 and HIV-2. J Infect Dis 180, 1 116- 1121 (1999).

[00452] 40. Dhamija, N., Choudhary, D., Ladha, J.S., Pillai, B. & Mitra, D. Tat predominantly associates with host promoter elements in HIV-1-infected T-cells - regulatory basis of transcriptional repression of c-Rel. FEBS J 282, 595-610 (2015).

[00453] 41. Kim, J., Yoon, J.H. & Kim, Y.S. HIV-1 Tat interacts with and regulates the localization and processing of amyloid precursor protein. PLoS One 8, e77972 (2013).

[00454] 42. Gonzalez-Hernandez, M.J., et al. Regulation of the human

endogenous retrovirus K (HML-2) transcriptome by the HIV-1 Tat protein. J Virol 88, 8924- 8935 (2014).

[00455] 43. Subramanian, R.P., Wildschutte, J.H., Russo, C. & Coffin, J.M.

Identification, characterization, and comparative genomic distribution of the HERV-K (HML-2) group of human endogenous retroviruses. Retrovirology 8, 90 (2011).

[00456] 44. Gifford, R. & Tristem, M. The evolution, distribution and diversity of endogenous retroviruses. Virus Genes 26, 291-315 (2003).

[00457] 45. Barbulescu, M., et al. Many human endogenous retrovirus K (HERV-K) proviruses are unique to humans. Curr Biol 9, 861-868 (1999).

[00458] 46. Schmitt, K., Reichrath, J., Roesch, A., Meese, E. & Mayer, J.

Transcriptional profiling of human endogenous retrovirus group HERV-K(HML-2) loci in melanoma. Genome biology and evolution 5, 307-328 (2013).

[00459] 47. Dinkel, H., et al. ELM 2016-data update and new functionality of the eukaryotic linear motif resource. Nucleic Acids Res 44, D294-300 (2016). [00460] 48. Vagner, S., et al. Alternative translation initiation of the Moloney murine leukemia virus mRNA controlled by internal ribosome entry involving the p57/PTB splicing factor. J Biol Chem 270, 20376-20383 (1995).

[00461] 49. Buck, C.B., et al. The human immunodeficiency virus type 1 gag gene encodes an internal ribosome entry site. J Virol 75, 181-191 (2001).

[00462] 50. Lopez-Lastra, M., Rivas, A. & Barria, M.I. Protein synthesis in eukaryotes: the growing biological relevance of cap-independent translation initiation. Biol Res 38, 121-146 (2005).

[00463] 51. Boulikas, T. Putative nuclear localization signals (NLS) in protein transcription factors. J Cell Biochem 55, 32-58 (1994).

[00464] 52. Chang, K.C. Revealing -1 programmed ribosomal frameshifting mechanisms by single-molecule techniques and computational methods. Comput Math Methods Med 2012, 569870 (2012).

[00465] 53. Touriol, C, et al. Generation of protein isoform diversity by alternative initiation of translation at non-AUG codons. Biol Cell 95, 169-178 (2003).

[00466] 54. Buee, L, Bussiere, T., Buee-Scherrer, V., Delacourte, A. & Hof, P.R. Tau protein isoforms, phosphorylation and role in neurodegenerative disorders. Brain Res Brain Res Rev 33, 95-130 (2000).

[00467] 55. Ueki, N., Someya, K., Matsuo, Y., Wakamatsu, K. & Mukai, H.

Cryptides: functional cryptic peptides hidden in protein structures. Biopolymers 88, 190-198 (2007).

[00468] 56. Ho, O. & Green, W.R. Cytolytic CD8+ T cells directed against a cryptic epitope derived from a retroviral alternative reading frame confer disease protection. J Immunol 176, 2470-2475 (2006).

[00469] 57. Garrison, K.E., et al. Transcriptional errors in human immunodeficiency virus type 1 generate targets for T-cell responses. Clin Vaccine Immunol 16, 1369-1371 (2009).

[00470] 58. Brinzevich, D., et al. HIV-1 interacts with human endogenous retrovirus K (HML-2) envelopes derived from human primary lymphocytes. J Virol 88, 6213-6223 (2014).

[00471] 59. Terry, S.N., et al. Expression of HERV-K108 envelope interferes with HIV-1 production. Virology 509, 52-59 (2017). [00472] 60. Hanke, K., et al. Reconstitution of the ancestral glycoprotein of human endogenous retrovirus k and modulation of its functional activity by truncation of the cytoplasmic domain. J Virol 83, 12790-12800 (2009).

[00473] 61. Lemaitre, C, Harper, F., Pierron, G., Heidmann, T. & Dewannieux, M. The HERV-K human endogenous retrovirus envelope protein antagonizes Tetherin antiviral activity. J Virol 88, 13626-13637 (2014).

[00474] 62. Kesidou, E., Lagoudaki, R., Touloumi, O., Poulatsidou, K.N. &

Simeonidou, C. Autophagy and neurodegenerative disorders. Neural Regen Res 8, 2275- 2283 (2013).

[00475] 63. Jang, G.Y., et al. Transglutaminase 2 suppresses apoptosis by modulating caspase 3 and NF-kappaB activity in hypoxic tumor cells. Oncogene 29, 356-367 (2010).

[00476] 64. Manghera, M. & Douville, R.N. Endogenous retrovirus-K promoter: a landing strip for inflammatory transcription factors? Retrovirology 10, 16 (2013).

[00477] 65. Manghera, M., Ferguson, J. & Douville, R. ERVK polyprotein processing and reverse transcriptase expression in human cell line models of neurological disease. Viruses 7, 320-332 (2015).

[00478] 66. Manghera, M., Ferguson-Parry, J. & Douville, R.N. TDP-43 regulates endogenous retrovirus-K viral protein accumulation. Neurobiol Dis 94, 226-236 (2016).

[00479] 67. Manghera, M., Ferguson-Parry, J., Lin, R. & Douville, R.N. NF-kappaB and IRF1 Induce Endogenous Retrovirus K Expression via Interferon-Stimulated Response

Elements in Its 5' Long Terminal Repeat. J Virol 90, 9338-9349 (2016).

[00480] 68. Bhat, R.K., et al. Human Endogenous Retrovirus-K(ll) Envelope

Induction Protects Neurons during HIV/AIDS. PLoS One 9, e97984 (2014).

[00481] 69. Li, W., et al. Human endogenous retrovirus-K contributes to motor neuron disease. Science translational medicine 7, 307ra153 (2015).

[00482] 70. Zhou, J., Callapina, M., Goodall, G.J. & Brune, B. Functional integrity of nuclear factor kappaB, phosphatidylinositol 3'-kinase, and mitogen-activated protein kinase signaling allows tumor necrosis factor alpha-evoked Bcl-2 expression to provoke internal ribosome entry site-dependent translation of hypoxia-inducible factor 1 alpha. Cancer

Res 64, 9041-9048 (2004).

[00483] 71. Szilagyi, A. & Skolnick, J. Efficient prediction of nucleic acid binding function from low-resolution protein structures. J Mol Biol 358, 922-933 (2006). [00484] 72. Marsili, G., et al. On the role of interferon regulatory factors in HIV-1 replication. Ann N Y Acad Sci 1010, 29-42 (2003).

[00485] 73. Williams, B.R. Transcriptional regulation of interferon-stimulated genes. Eur J Biochem 200, 1-1 1 (1991).

[00486] 74. Garcia, J.A., Harrich, D., Pearson, L, Mitsuyasu, R. & Gaynor, R.B. Functional domains required for tat- induced transcriptional activation of the HIV-1 long terminal repeat. EMBO J 7, 3143-3147 (1988).

[00487] 75. Keller, G., Gross, C, Kelleher, M. & Winge, D.R. Functional independence of the two cysteine-rich activation domains in the yeast Mad transcription factor. J Biol Chem 275, 29193-29199 (2000).

[00488] 76. Vocero-Akbani, A., Lissy, N.A. & Dowdy, S.F. Transduction of full- length Tat fusion proteins directly into mammalian cells: analysis of T cell receptor activation- induced cell death. Methods Enzymol 322, 508-521 (2000).

[00489] 77. Sheehy, N., et al. Functional analysis of human T lymphotropic virus type 2 Tax proteins. Retrovirology 3, 20 (2006).

[00490] 78. Hardiman, O., et al. Amyotrophic lateral sclerosis. Nat Rev Dis Primers 3, 17071 (2017).

[00491] 79. Eisen, A., et al. Cortical influences drive amyotrophic lateral sclerosis. J Neurol Neurosurg Psychiatry 88, 917-924 (2017).

[00492] 80. Cappello, V. & Francolini, M. Neuromuscular Junction Dismantling in Amyotrophic Lateral Sclerosis. Int J Mol Sci 18(2017).

[00493] 81. Moisse, K. & Strong, M.J. Innate immunity in amyotrophic lateral sclerosis. Biochim Biophys Acta 1762, 1083-1093 (2006).

[00494] 82. McCombe, P.A. & Henderson, R.D. The Role of immune and inflammatory mechanisms in ALS. Curr Mol Med 11 , 246-254 (2011).

[00495] 83. Scotter, E.L., Chen, H.J. & Shaw, C.E. TDP-43 Proteinopathy and ALS: Insights into Disease Mechanisms and Therapeutic Targets. Neurotherapeutics 12, 352-363 (2015).

[00496] 84. Kwong, L.K., Neumann, M., Sampathu, D.M., Lee, V.M. & Trojanowski, J.Q. TDP-43 proteinopathy: the neuropathology underlying major forms of sporadic and familial frontotemporal lobar degeneration and motor neuron disease. Acta Neuropathol 1 14, 63-70 (2007). [00497] 85. Leal, S.S. & Gomes, CM. Calcium dysregulation links ALS defective proteins and motor neuron selective vulnerability. Frontiers in cellular neuroscience 9, 225 (2015).

[00498] 86. Zhou, T., et al. Implications of white matter damage in amyotrophic lateral sclerosis (Review). Mol Med Rep 16, 4379-4392 (2017).

[00499] 87. Tognatta, R. & Miller, R.H. Contribution of the oligodendrocyte lineage to CNS repair and neurodegenerative pathologies. Neuropharmacology 110, 539-547 (2016).

[00500] 88. Poniatowski, L.A., et al. Analysis of the Role of CX3CL1 (Fractalkine) and Its Receptor CX3CR1 in Traumatic Brain and Spinal Cord Injury: Insight into Recent Advances in Actions of Neurochemokine Agents. Mol Neurobiol 54, 2167-2188 (2017).

[00501] 89. Manghera, M., Ferguson-Parry, J. & Douville, R.N. TDP-43 regulates endogenous retrovirus-K viral protein accumulation. Neurobiol Dis 94, 226-236 (2016).

[00502] 90. Douville, R., Liu, J., Rothstein, J. & Nath, A. Identification of active loci of a human endogenous retrovirus in neurons of patients with amyotrophic lateral sclerosis. Ann Neurol 69, 141-151 (2011).

[00503] 91. Hurtado, A. P., Rengifo, A.C. & Torres-Fernandez, O.

Immunohistochemical Overexpression of MAP-2 in the Cerebral Cortex of Rabies-Infected Mice. International Journal of Morphology 33, 465-470 (2015).

[00504] 92. Monroy-Gomez, J., Santamaria, G. & Torres-Fernandez, O.

Overexpression of MAP2 and NF-H Associated with Dendritic Pathology in the Spinal Cord of Mice Infected with Rabies Virus. Viruses 10(2018).

[00505] 93. Timmermann, D.B., Westenbroek, R.E., Schousboe, A. & Catterall,

W.A. Distribution of high-voltage-activated calcium channels in cultured gamma-aminobutyric acidergic neurons from mouse cerebral cortex. J Neurosci Res 67, 48-61 (2002).

[00506] 94. McTigue, D.M. & Tripathi, R.B. The life, death, and replacement of oligodendrocytes in the adult CNS. J Neurochem 107, 1-19 (2008).

[00507] 95. Othman, A., et al. Olig1 is expressed in human oligodendrocytes during maturation and regeneration. Glia 59, 914-926 (201 1).

[00508] 96. Chong, S.Y. & Chan, J.R. Tapping into the glial reservoir: cells committed to remaining uncommitted. J Cell Biol 188, 305-312 (2010).

[00509] 97. Watkins, T.A., Emery, B., Mulinyawe, S. & Barres, B.A. Distinct stages of myelination regulated by gamma-secretase and astrocytes in a rapidly myelinating CNS coculture system. Neuron 60, 555-569 (2008). [00510] 98. de Faria, O., Jr., Gonsalvez, D., Nicholson, M. & Xiao, J. Activity- dependent central nervous system myelination throughout life. J Neurochem (2018).

[00511] 99. Fancy, S.P., et al. Dysregulation of the Wnt pathway inhibits timely myelination and remyelination in the mammalian CNS. Genes Dev 23, 1571-1585 (2009).

[00512] 100. Arnett, H.A., et al. bHLH transcription factor Olig1 is required to repair demyelinated lesions in the CNS. Science 306, 211 1-21 15 (2004).

[00513] 101. Schmandke, A., Schmandke, A. & Schwab, M.E. Nogo-A: Multiple Roles in CNS Development, Maintenance, and Disease. Neuroscientist 20, 372-386 (2014).

[00514] 102. Pernet, V., Joly, S., Christ, F., Dimou, L. & Schwab, M.E. Nogo-A and myelin-associated glycoprotein differently regulate oligodendrocyte maturation and myelin formation. J Neurosci 28, 7435-7444 (2008).

[00515] 103. Miron, V.E., Kuhlmann, T. & Antel, J. P. Cells of the oligodendroglial lineage, myelination, and remyelination. Biochim Biophys Acta 1812, 184-193 (201 1).

[00516] 104. Talbott, J.F., et al. Endogenous Nkx2.2+/Olig2+ oligodendrocyte precursor cells fail to remyelinate the demyelinated adult rat spinal cord in the absence of astrocytes. Exp Neurol 192, 11-24 (2005).

[00517] 105. Cafferty, W.B. & Strittmatter, S.M. The Nogo-Nogo receptor pathway limits a spectrum of adult CNS axonal growth. J Neurosci 26, 12242-12250 (2006).

[00518] 106. Kuhlmann, T., et al. Differentiation block of oligodendroglial progenitor cells as a cause for remyelination failure in chronic multiple sclerosis. Brain 131 , 1749-1758 (2008).

[00519] 107. Theotokis, P., et al. Time course and spatial profile of Nogo-A expression in experimental autoimmune encephalomyelitis in C57BL/6 mice. J Neuropathol Exp Neurol 71 , 907-920 (2012).

[00520] 108. Wojcik, S., Engel, W.K. & Askanas, V. Increased expression of Noga-A in ALS muscle biopsies is not unique for this disease. Acta myologica : myopathies and cardiomyopathies : official journal of the Mediterranean Society of Myology / edited by the Gaetano Conte Academy for the study of striated muscle diseases 25, 116-1 18 (2006).

[00521] 109. Teng, F.Y. & Tang, B.L. Nogo signaling and non-physical injury- induced nervous system pathology. J Neurosci Res 79, 273-278 (2005).

[00522] 110. McDonald, C.L., Bandtlow, C. & Reindl, M. Targeting the Nogo receptor complex in diseases of the central nervous system. Curr Med Chem 18, 234-244 (201 1). [00523] 11 1. Dupuis, L, et al. Nogo provides a molecular marker for diagnosis of amyotrophic lateral sclerosis. Neurobiol Dis 10, 358-365 (2002).

[00524] 112. Pradat, P.F., et al. Muscle Nogo-A expression is a prognostic marker in lower motor neuron syndromes. Ann Neurol 62, 15-20 (2007).

[00525] 113. Sui, Y.P., Zhang, X.X., Lu, J.L & Sui, F. New Insights into the Roles of Nogo-A in CNS Biology and Diseases. Neurochem Res 40, 1767-1785 (2015).

[00526] 114. Bruneteau, G., et al. Endplate denervation correlates with Nogo-A muscle expression in amyotrophic lateral sclerosis patients. Annals of clinical and

translational neurology 2, 362-372 (2015).

[00527] 115. Bros-Facer, V., et al. Treatment with an antibody directed against Nogo-A delays disease progression in the SOD1 G93A mouse model of Amyotrophic lateral sclerosis. Hum Mol Genet 23, 4187-4200 (2014).

[00528] 116. Jokic, N., et al. The neurite outgrowth inhibitor Nogo-A promotes denervation in an amyotrophic lateral sclerosis model. EMBO Rep 7, 1 162-1 167 (2006).

[00529] 117. Ineichen, B.V., et al. Nogo-A Antibodies for Progressive Multiple Sclerosis. CNS Drugs 31 , 187-198 (2017).

[00530] 118. Meininger, V., et al. Safety, pharmacokinetic, and functional effects of the nogo-a monoclonal antibody in amyotrophic lateral sclerosis: a randomized, first-inhuman clinical trial. PLoS One 9, e97803 (2014).

[00531] 119. Meininger, V., et al. Safety and efficacy of ozanezumab in patients with amyotrophic lateral sclerosis: a randomised, double-blind, placebo-controlled, phase 2 trial. Lancet Neurol 16, 208-216 (2017).

[00532] 120. Wills, A.M. Blockade of the neurite outgrowth inhibitor Nogo-A in amyotrophic lateral sclerosis. Lancet Neurol 16, 175-176 (2017).

[00533] 121. Amy, M., et al. A common functional allele of the Nogo receptor gene, reticulon 4 receptor (RTN4R), is associated with sporadic amyotrophic lateral sclerosis in a French population. Amyotrophic lateral sclerosis & frontotemporal degeneration 16, 490-496 (2015).

[00534] 122. Buss, A., et al. Sequential loss of myelin proteins during Wallerian degeneration in the human spinal cord. Brain 128, 356-364 (2005).

[00535] 123. Fang, Y., et al. The Nogo/Nogo Receptor (NgR) Signal Is Involved in Neuroinflammation through the Regulation of Microglial Inflammatory Activation. J Biol Chem 290, 28901-28914 (2015). [00536] 124. Ghosh, S. & Dass, J.F. Study of pathway cross-talk interactions with NF-kappaB leading to its activation via ubiquitination or phosphorylation: A brief review. Gene 584, 97-109 (2016).

[00537] 125. Yang, X.D. & Sun, S.C. Targeting signaling factors for degradation, an emerging mechanism for TRAF functions. Immunol Rev 266, 56-71 (2015).

[00538] 126. Gomez-Ospina, N., Tsuruta, F., Barreto-Chang, O., Hu, L. &

Dolmetsch, R. The C terminus of the L-type voltage-gated calcium channel Ca(V)1.2 encodes a transcription factor. Cell 127, 591-606 (2006).

[00539] 127. Gomez-Ospina, N., et al. A promoter in the coding region of the calcium channel gene CACNA1 C generates the transcription factor CCAT. PLoS One 8, e60526 (2013).

[00540] 128. Nielsen, K.J., Schroeder, T. & Lewis, R. Structure-activity relationships of omega-conotoxins at N-type voltage-sensitive calcium channels. J Mol Recognit 13, 55-70 (2000).

[00541] 129. Takata, T., et al. Clinical significance of caspase-3 expression in pathologic-stage I, nonsmall-cell lung cancer. Int J Cancer 96 Suppl, 54-60 (2001).

[00542] 130. Pu, X., et al. Caspase-3 and caspase-8 expression in breast cancer: caspase-3 is associated with survival. Apoptosis 22, 357-368 (2017).

[00543] 131. Chen, H., et al. Prognostic value of Caspase-3 expression in cancers of digestive tract: a meta-analysis and systematic review. Int J Clin Exp Med 8, 10225-10234

(2015).

[00544] 132. Wurzer, W.J., et al. Caspase 3 activation is essential for efficient influenza virus propagation. EMBO J 22, 2717-2728 (2003).

[00545] 133. Do, T., et al. Three-dimensional imaging of HIV-1 virological synapses reveals membrane architectures involved in virus transmission. J Virol 88, 10327-10339 (2014).

[00546] 134. Narayan, V., et al. Celastrol inhibits Tat-mediated human

immunodeficiency virus (HIV) transcription and replication. J Mol Biol 410, 972-983 (201 1).

[00547] 135. Kalantari, P., Narayan, V., Henderson, A.J. & Prabhu, K.S. 15-Deoxy- Delta12, 14-prostaglandin J2 inhibits HIV-1 transactivating protein, Tat, through covalent modification. FASEB J 23, 2366-2373 (2009).

[00548] 136. Cascao, R., Fonseca, J.E. & Moita, L.F. Celastrol: A Spectrum of Treatment Opportunities in Chronic Diseases. Front Med (Lausanne) 4, 69 (2017). [00549] 137. Salminen, A., Lehtonen, M., Paimela, T. & Kaarniranta, K. Celastrol: Molecular targets of Thunder God Vine. Biochem Biophys Res Commun 394, 439-442 (2010).

[00550] 138. Tao, X., Sun, Y. & Zhang, N. [Treatment of rheumatoid arthritis with low doses of multi-glycosides of Tri pterygium wilfordii]. Zhong Xi Yi Jie He Za Zhi 10, 289- 291 , 261-282 (1990).

[00551] 139. Venkatesha, S.H., Astry, B., Nanjundaiah, S.M., Yu, H. & Moudgil,

K.D. Suppression of autoimmune arthritis by Celastrus-derived Celastrol through modulation of pro-inflammatory chemokines. Bioorg Med Chem 20, 5229-5234 (2012).

[00552] 140. Fang, Z., et al. High-Throughput Study of the Effects of Celastrol on

Activated Fibroblast-Like Synoviocytes from Patients with Rheumatoid Arthritis. Genes

8(2017).

[00553] 141. Paris, D., et al. Reduction of beta-amyloid pathology by celastrol in a transgenic mouse model of Alzheimer's disease. J Neuroinflammation 7, 17 (2010).

[00554] 142. Kashyap, D., et al. Molecular targets of celastrol in cancer: Recent trends and advancements. Critical reviews in oncology/hematology 128, 70-81 (2018).

[00555] 143. Reutrakul, V., et al. Cytotoxic and anti-HIV-1 caged xanthones from the resin and fruits of Garcinia hanburyi. Planta Med 73, 33-40 (2007).

[00556] 144. Banik, K., et al. Therapeutic potential of gambogic acid, a caged xanthone, to target cancer. Cancer Lett 416, 75-86 (2018).

[00557] 145. Kashyap, D., Mondal, R., Tuli, H.S., Kumar, G. & Sharma, A.K.

Molecular targets of gambogic acid in cancer: recent trends and advancements. Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine 37, 12915-12925 (2016).

[00558] 146. Jang, S.W., et al. Gambogic amide, a selective agonist for TrkA receptor that possesses robust neurotrophic activity, prevents neuronal cell death. Proc Natl Acad Sci U S A 104, 16329-16334 (2007).

[00559] 147. Fu, Q., Li, C. & Yu, L. Gambogic acid inhibits spinal cord injury and inflammation through suppressing the p38 and Akt signaling pathways. Mol Med Rep 17, 2026-2032 (2018).

[00560] 148. Wang, J., Gines, S., MacDonald, M.E. & Gusella, J.F. Reversal of a full-length mutant huntingtin neuronal cell phenotype by chemical inhibitors of polyglutamine- mediated aggregation. BMC Neurosci 6, 1 (2005). [00561] 149. Chen, S.R., et al. A Mechanistic Overview of Triptolide and Celastrol, Natural Products from Tripterygium wilfordii Hook F. Frontiers in pharmacology 9, 104 (2018).

[00562] 150. Saini, P., Ganugula, R., Arora, M. & Kumar, M.N. The Next Generation Non-competitive Active Polyester Nanosystems for Transferrin Receptor-mediated Peroral Transport Utilizing Gambogic Acid as a Ligand. Scientific reports 6, 29501 (2016).

[00563] 151. Fan, T.K., Gundimeda, U., Mack, W.J. & Gopalakrishna, R.

Counteraction of Nogo-A and axonal growth inhibitors by green tea polyphenols and other natural products. Neural Regen Res 11 , 545-546 (2016).

[00564] 152. Sepe, M., et al. Proteolytic control of neurite outgrowth inhibitor NOGO-A by the cAMP/PKA pathway. Proc Natl Acad Sci U S A 1 11 , 15729-15734 (2014).

[00565] 153. Yu, X.J., Zhao, Q., Wang, X.B., Zhang, J.X. & Wang, X.B.

Gambogenic acid induces proteasomal degradation of CIP2A and sensitizes hepatocellular carcinoma to anticancer agents. Oncol Rep 36, 3611-3618 (2016).

[00566] 154. Wang, J., et al. Gambogic acid-induced degradation of mutant p53 is mediated by proteasome and related to CHIP. J Cell Biochem 112, 509-519 (2011).

[00567] 155. Hughes, T.T., et al. Drosophila as a genetic model for studying pathogenic human viruses. Virology 423, 1-5 (2012).

[00568] 156. Krug, L, et al. Retrotransposon activation contributes to

neurodegeneration in a Drosophila TDP-43 model of ALS. PLoS Genet 13, e1006635 (2017).

[00569] 157. Chang, J.C., Hazelett, D.J., Stewart, J.A. & Morton, D.B. Motor neuron expression of the voltage-gated calcium channel cacophony restores locomotion defects in a Drosophila, TDP-43 loss of function model of ALS. Brain Res 1584, 39-51 (2014).

[00570] 158. Estes, P.S., et al. Motor neurons and glia exhibit specific individualized responses to TDP-43 expression in a Drosophila model of amyotrophic lateral sclerosis. Dis Model Mech 6, 721-733 (2013).

[00571] 159. Ghosh, A., et al. Targeted ablation of oligodendrocytes triggers axonal damage. PLoS One 6, e22735 (201 1).

[00572] 160. Huang, Y., Ng, F.S. & Jackson, F.R. Comparison of larval and adult Drosophila astrocytes reveals stage-specific gene expression profiles. G3 (Bethesda) 5, 551- 558 (2015).

[00573] 161. Civetta, A. & Clark, A.G. Correlated effects of sperm competition and postmating female mortality. Proc Natl Acad Sci U S A 97, 13162-13165 (2000). [00574] 162. Civetta, A., Montooth, K.L. & Mendelson, M. Quantitative trait loci and interaction effects responsible for variation in female postmating mortality in Drosophila simulans and D. sechellia introgression lines. Heredity (Edinb) 94, 94-100 (2005).

[00575] 163. Mullick, A., et al. The cumate gene-switch: a system for regulated expression in mammalian cells. BMC biotechnology 6, 43 (2006).

[00576] 164. Lancaster, M.A., et al. Cerebral organoids model human brain development and microcephaly. Nature 501 , 373-379 (2013).

[00577] 165. Rosati, J., et al. Establishment of stable iPS-derived human neural stem cell lines suitable for cell therapies. Cell death & disease 9, 937 (2018).

[00578] 166. Dimos, J.T., et al. Induced pluripotent stem cells generated from patients with ALS can be differentiated into motor neurons. Science 321 , 1218-1221 (2008).

[00579] 167. Egawa, N., et al. Drug screening for ALS using patient-specific induced pluripotent stem cells. Science translational medicine 4, 145ra104 (2012).

[00580] 168. Douville, R.N. & Nath, A. Human Endogenous Retrovirus-K and TDP- 43 Expression Bridges ALS and HIV Neuropathology. Frontiers in microbiology 8, 1986 (2017).

[00581] 169. Olivera, B.M. & Teichert, R.W. Diversity of the neurotoxic Conus peptides: a model for concerted pharmacological discovery. Mol Interv 7, 251-260 (2007).

[00582] 170. Marambaud, P., Dreses-Werringloer, U. & Vingtdeux, V. Calcium signaling in neurodegeneration. Molecular neurodegeneration 4, 20 (2009).

[00583] 171. Yan, H.D., Lim, W., Lee, K.W. & Kim, J. Sera from amyotrophic lateral sclerosis patients reduce high-voltage activated Ca2+ currents in mice dorsal root ganglion neurons. Neurosci Lett 235, 69-72 (1997).

[00584] 172. MacGowan, D.J., Scelsa, S.N. & Waldron, M. An ALS-like syndrome with new HIV infection and complete response to antiretroviral therapy. Neurology 57, 1094- 1097 (2001).

[00585] 173. McCormick, A.L., Brown, R.H., Jr., Cudkowicz, M.E., Al-Chalabi, A. & Garson, J. A. Quantification of reverse transcriptase in ALS and elimination of a novel retroviral candidate. Neurology 70, 278-283 (2008).

[00586] 174. Garbuzova-Davis, S. & Sanberg, P.R. Blood-CNS Barrier Impairment in ALS patients versus an animal model. Frontiers in cellular neuroscience 8, 21 (2014).

[00587] 175. Mitchell, J.D. & Borasio, G.D. Amyotrophic lateral sclerosis. Lancet 369, 2031-2041 (2007). [00588] 176. Caller, T.A., et al. Spatial clustering of amyotrophic lateral sclerosis and the potential role of BMAA. Amyotroph Lateral Scler 13, 25-32 (2012).

[00589] 177. Appel, S.H., Beers, D., Siklos, L, Engelhardt, J.I. & Mosier, D.R.

Calcium: the Darth Vader of ALS. Amyotroph Lateral Scler Other Motor Neuron Disord 2 Suppl 1 , S47-54 (2001).

[00590] 178. Frank, O., et al. Human endogenous retrovirus expression profiles in samples from brains of patients with schizophrenia and bipolar disorders. J Virol 79, 10890- 10901 (2005).

[00591] 179. Thompson, J.C., Dunbar, E. & Laye, R.R. Treatment challenges and complications with ziconotide monotherapy in established pump patients. Pain physician 9, 147-152 (2006).

[00592] 180. Toufaily, C, Landry, S., Leib-Mosch, C, Rassart, E. & Barbeau, B.

Activation of LTRs from different human endogenous retrovirus (HERV) families by the

HTLV-1 tax protein and T-cell activators. Viruses 3, 2146-2159 (201 1).

[00593] 181. Vincendeau, M., et al. Modulation of human endogenous retrovirus

(HERV) transcription during persistent and de novo HIV-1 infection. Retrovirology 12, 27

(2015).

[00594] 182. Verma, A. & Berger, J.R. ALS syndrome in patients with HIV-1 infection. J Neurol Sci 240, 59-64 (2006).

[00595] 183. Alfahad, T. & Nath, A. Retroviruses and amyotrophic lateral sclerosis. Antiviral Res 99, 180-187 (2013).

[00596] 184. Matsuzaki, T., et al. HTLV-l-associated myelopathy (HAM)/tropical spastic paraparesis (TSP) with amyotrophic lateral sclerosis-like manifestations. J Neurovirol 6, 544-548 (2000).

[00597] 185. Anwar, S.L., Wulaningsih, W. & Lehmann, U. Transposable Elements in Human Cancer: Causes and Consequences of Deregulation. Int J Mol Sci 18(2017).

[00598] 186. Vlahopoulos, S.A., et al. Dynamic aberrant NF-kappaB spurs tumorigenesis: a new model encompassing the microenvironment. Cytokine Growth Factor Rev 26, 389-403 (2015).

[00599] 187. Bradford, J.W. & Baldwin, A.S. IKK/nuclear factor-kappaB and oncogenesis: roles in tumor-initiating cells and in the tumor microenvironment. Adv Cancer Res 121 , 125-145 (2014).

[00600] References [00601]

[00602] 1. Eldridge, R., Li, Y. & Miller, L.K. Characterization of a baculovirus gene encoding a small conotoxinlike polypeptide. J Virol 66, 6563-6571 (1992).

[00603] 2. Gracy, J., et al. KNOTTIN: the knottin or inhibitor cystine knot scaffold in 2007. Nucleic Acids Res 36, D314-319 (2008).

[00604] 3. Kearse, M., et al. Geneious Basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics 28, 1647-1649 (2012).

[00605] 4. Pettersen, E.F., et al. UCSF Chimera-a visualization system for exploratory research and analysis. Journal of computational chemistry 25, 1605-1612 (2004).

[00606] 5. Dinkel, H., et al. ELM 2016-data update and new functionality of the eukaryotic linear motif resource. Nucleic Acids Res 44, D294-300 (2016).

[00607] 6. Brohawn, D.G., O'Brien, L.C. & Bennett, J. P., Jr. RNAseq Analyses Identify Tumor Necrosis Factor-Mediated Inflammation as a Major Abnormality in ALS Spinal Cord. PLoS One 1 1 , e0160520 (2016).

[00608] 7. Nielsen, K.J., Schroeder, T. & Lewis, R. Structure-activity relationships of omega-conotoxins at N-type voltage-sensitive calcium channels. J Mol Recognit 13, 55-70 (2000).

[00609] 8. Li, W., et al. Human endogenous retrovirus-K contributes to motor neuron disease. Science translational medicine 7, 307ra153 (2015).

[00610] The embodiments described herein are intended to be examples only.

Alterations, modifications and variations can be effected to the particular embodiments by those of skill in the art. The scope of the claims should not be limited by the particular embodiments set forth herein, but should be construed in a manner consistent with the specification as a whole.

[00611] All publications, patents and patent applications mentioned in this

Specification are indicative of the level of skill those skilled in the art to which this invention pertains and are herein incorporated by reference to the same extent as if each individual publication patent, or patent application was specifically and individually indicated to be incorporated by reference.

[00612] The invention being thus described, it will be obvious that the same may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the invention, and all such modification as would be obvious to one skilled in the art are intended to be included within the scope of the following claims.