IMMUNODEFICIENCY VIRUS VIF

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. Provisional Patent Application No. 62/186,256, filed June 29, 2015, which is incorporated herein by reference.

GOVERNMENT FUNDING

This invention was made with government support under Grant Numbers AI064046 and GM091743, awarded by the National Institutes of Health. The government has certain rights in the invention.

SEQUENCE LISTING

This application contains a Sequence Listing electronically submitted via EFS-Web to the United States Patent and Trademark Office as an ASCII text file entitled "SequenceListing-VIF- 493_ST25.txt" having a size of 4 kilobytes and created on June 27, 2016. The information contained in the Sequence Listing is incorporated by reference herein.

BACKGROUND

APOBEC3B (A3B) is a member of a family of APOBEC3 (A3) proteins that deaminate DNA cytosine bases to produce the pro-mutagenic lesion, uracil. APOBEC3B is a source of mutations in breast, lung, head/neck, bladder, cervical, and several other types of cancer.

SUMMARY OF THE INVENTION

In one aspect, this disclosure describes a polynucleotide that includes SEQ ID NO:2.

In another aspect, this disclosure describes a method that includes providing tumor cells, providing a polynucleotide encoding at least a portion of a simian immunodeficiency virus (SIV) Vif gene, and introducing the polynucleotide into the tumor cells.

In some embodiments, the polynucleotide encodes a protein having the amino acid sequence of SEQ ID NO:3. In some of these embodiments, the polynucleotide includes SEQ ID NO:2. In some embodiments, the polynucleotide encodes a sequence that includes a conserved SLQ tri-residue peptide motif.

In some embodiments, the polynucleotide encodes a sequence that includes a conserved TLQ tri-residue peptide motif.

In some embodiments, the polynucleotide encodes at least a portion of a simian

immunodeficiency virus (SIV) Vif protein. In some of these embodiments, the SIV Vif protein can include SIVmac239 Vif, SIVsmCFU212 Vif, SIVsmPG Vif, SIVsmPBj Vif, SIVsmE041 Vif, SIVstm Vif, SIVmacl42 Vif, SIVmfal86 Vif, SIVmne07 Vif, or SIVsmE543 Vif. In some of these embodiments, the polynucleotide is codon-optimized for expression in a human cell.

In another aspect, this disclosure describes a method that includes introducing into a cell a polynucleotide encoding a simian immunodeficiency virus (SIV) Vif, wherein expression of the SIV Vif in the cell results in modulation of the functional activity of APOBEC3B in the cell.

In some embodiments, the cell is a cancer cell. In some of these embodiments, the cell is a breast cancer cell.

In some embodiments, the cell is a precancerous cell.

In some embodiments, the cell is a human papilloma virus (HPV)-infected cervical cell.

In some embodiments, the cell is a human cell.

In some embodiments, the polynucleotide encodes a protein comprising SEQ ID NO:3. In some of these embodiments, the polynucleotide includes SEQ ID NO:2.

In some embodiments, the polynucleotide encodes a conserved SLQ tri-residue peptide motif or a conserved TLQ tri-residue peptide motif.

In some embodiments, the polynucleotide encodes at least a portion of a simian

immunodeficiency virus (SIV) Vif protein. In some of these embodiments, the SIV Vif protein can include SIVmac239 Vif, SIVsmCFU212 Vif, SIVsmPG Vif, SIVsmPBj Vif, SIVsmE041 Vif, SIVstm Vif, SIVmacl42 Vif, SIVmfal86 Vif, SIVmne07 Vif, or SIVsmE543 Vif. In some of these embodiments, the polynucleotide is codon-optimized for expression in a human cell.

In some embodiments, the method further includes introducing into the cell a vector that includes the polynucleotide. In some of these embodiments, the vector is a viral vector.

In some embodiments, the polynucleotide is RNA. In some of these embodiments, the RNA is delivered in a small particle or a viral particle.

As used herein, the term "gene" refers to a region of a deoxyribonucleic acid that encodes a protein. A gene can include certain non-coding sequences (e.g., one or more introns) or may be intronless, whether natively intronless or derived from cDNA. The transfer of a gene from one organism to another can include the transfer of expression control sequences native to the gene being transferred. Alternatively, the gene may be placed under the control of heterologous expression control sequences.

As used herein, "operably linked" refers to a functional linkage between a first nucleic acid sequence and second nucleic acid sequence in such a manner as to allow general functions. For example, a nucleic acid sequence coding for a target protein or RNA may be operably linked to a nucleic acid expression control sequence, in such a manner that the expression control sequence affects expression of the coding nucleic acid sequence.

The terms "comprises" and variations thereof do not have a limiting meaning where these terms appear in the description and claims.

Unless otherwise specified, "a," "an," "the," and "at least one" are used interchangeably and mean one or more than one.

Also herein, the recitations of numerical ranges by endpoints include all numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).

For any method disclosed herein that includes discrete steps, the steps may be conducted in any feasible order. And, as appropriate, any combination of two or more steps may be conducted simultaneously.

The above summary of the present invention is not intended to describe each disclosed embodiment or every implementation of the present invention. The description that follows more particularly exemplifies illustrative embodiments. In several places throughout the application, guidance is provided through lists of examples, which examples can be used in various

combinations. In each instance, the recited list serves only as a representative group and should not be interpreted as an exclusive list.

BRIEF DESCRIPTION OF THE FIGURES FIG. 1. Sequence alignment of reference and codon optimized SIVmac239 virion infectivity factor (Vif). (A) Nucleotide alignment of Vif coding DNA sequence from AY588946 (positions 4436-5080) (SEQ ID NO: l) and a codon optimized SIVmac239 Vif coding DNA sequence (SEQ ID NO:2). Identical residues are shaded grey; these two sequences are 76% identical. (B) Amino acid alignment of the translated Vif coding DNA sequence described above (SEQ ID NO:3). These two protein sequences are 100% identical. FIG 2. SIVmac239 Vif efficiently degrades human APOBEC3B (huA3B). (A) Immunoblot showing Vif from HIV-l _n i _B (50 ng, 100 ng, 200 ng), SIVmac239 (50 ng, 100 ng, 200 ng), BIV (100 ng, 200 ng, 400 ng), FIV (50 ng, 100 ng, 200 ng) and MVV lentiviruses (100 ng, 200 ng, 400 ng) was expressed in 293T cells at comparable ranges. (B) Immunoblot demonstrating the varying abilities of the lentiviral Vif proteins to degrade huA3B, and the increased degradation observed in the presence of SIVmac239 Vif. The ly sates were blotted for myc to detect Vif, HA to detect huA3B, and tubulin (TUB) as a loading control.

FIG 3. Degradation of huA3B by diverse SIV Vif proteins. Representative immunoblot demonstrating the abilities of Vif from SIVsmCFU212, SIVsmPG, SIVsmPBj, SIVsmE041, SIVstm, SIVmacl42, SIVmfal86, SIVmne07, SIVsmE543, and SIVagmTAN lentiviruses to degrade huA3B, in comparison to HIV- I _HIB and SIVmac239 Vif proteins. With the exception of Vif from SIVagmTAN, the ability to mediate degradation of huA3B appears conserved in SIV Vif proteins. The lysates were blotted for myc to detect Vif, HA to detect huA3B, and tubulin (TUB) as a loading control.

FIG 4. A3B restricts HIV-1 and is counteracted by SIVmac239 Vif. (A) 293T cells were transfected with Vif-deficient HIV- I _HIB and with either empty expression constructs or the indicated A3 expression constructs (huA3B-HA, rhA3B-HA, or huA3G-HA) and Vif expression constructs (HIV-lniB Vif, or SIVmac239 Vif). After 48 hours, virus-containing supernatants from the 293 T cells were purified and used to infect CEM-GFP reporter cells. The percentage of infected reporter cells, as indicated by expression of GFP, was determined by flow cytometry 48 hours later, (n = 3; mean and SD shown) (B) Representative immunoblots for each infection condition (vector control, HIV-l niB Vif, or SIVmac239 Vif; and vector control, huA3B-HA expression construct, rhA3B-HA expression construct, or huA3G-HA expression construct) are shown. 293T producer cells lysates were blotted for HA to detect A3, for Myc to detect Vif, and for Tubulin (TUB) as a loading control. Purified viral particles were blotted for HA to detect A3 and for p24 (Gag) as a loading control.

FIG 5. SIVmac239 Vif-mediated degradation of huA3B is analogous to HIV- Ι _ΠΙΒ Vif- mediated degradation of huA3G. (A) 293T cells were transfected with the indicated constructs, allowed to incubate for 32 hours, then treated with 5 uM of the proteasomal inhibitor MG132 or vehicle (40% acetonitrile in water) for 16 hours, processed into soluble lysates, and subjected to immunoblotting. These representative immunoblots demonstrate that MG132 treatment prevents Vif-mediated degradation of A3 proteins. (B) Amino acid alignment of the ElonginC (ELOC)- binding SLQ region of the HIV-1 and SIV Vif proteins used in this study. Conserved residues are shaded, with underlining identifying more conserved positions. The residue positions included in the alignment are indicated. The SLQ tri-residue motif is underlined. (C) Representative

immunoblots demonstrating that SIVmac239 Vif-mediated degradation of huA3B is dependent on the SLQ amino acid motif, as is HIV- I _HIB Vif-mediated degradation and SIVmac239 Vif-mediated degradation of huA3G. Mutation of the SLQ Vif residues to AAA abolishes degradation of A3. 293T cells were transfected with the indicated A3 and Vif constructs, allowed to incubate for 48 hours, processed into soluble lysates, and subjected to immunoblotting. Cell lysates were blotted for MYC to detect Vif, for HA to detect A3, and for tubulin (TUB) as a loading control.

FIG 6. SIVmac239 Vif-mediated rescue of cells from huA3B-mediated DNA damage and cytotoxicity. (A) Clonogenic assay for T-REx 293 cells expressing A3B-GFP with doxycycline induction and either stably expressing vector (circles), HIV-I _HIB Vif (diamonds), or SIVmac239 Vif (triangles). Relative viability indicates the ratio of clones that grew in increasing doxycycline, compared to no doxycycline induction (n = 3, mean and SD shown). The lysates (inset) were blotted for myc to detect Vif and tubulin (TUB) as a loading control. (B) Representative

immunoblot for cells as described in part (A) at each doxycycline concentration shows induction of A3B in the presence of the indicated Vif constructs. The lysates were blotted for GFP to detect A3B and Hsp90 as a loading control. (C) Clonogenic assay for T-REx 293 cells expressing GFP with doxycycline induction, and stably expressing vector (squares), HIV- I _HIB Vif (triangles), or

SIVmac239 Vif (circles), as described above (n = 3, mean and SD shown). The lysates (inset) were blotted for myc to detect Vif and tubulin (TUB) as a loading control. (D) Representative

immunoblot for cells as described in part (C) at each doxycycline concentration shows induction of GFP in the presence of the indicated Vif constructs. The lysates were blotted for GFP and Hsp90 as a loading control.

FIG 7. SIVmac239 Vif can degrade endogenous huA3B in cancer cells. Immunoblot of

HCC1569 (breast cancer cell line), JSQ3 (head and neck cancer cell line), and OVCAR-5 (ovarian cancer cell line) cells engineered to express empty vector, HIV- I _HIB Vif, or SIVmac239 Vif. The lysates were blotted for endogenous A3B using rabbit polyclonal anti-A3B (10-87-13), myc to detect Vif, and tubulin (TUB) as a loading control. DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS APOBEC3B can be a source of mutations in breast and several other types of cancer.

Several members of the APOBEC3 family can restrict the replication of human immunodeficiency virus HIV-1. The HIV-1 protein virion infectivity factor (Vif) counteracts the effect of APOBEC3 protein restriction at least in part by targeting the APOBEC3 proteins for ubiquitin ligase-mediated proteasomal degradation. However, APOBEC3B does not restrict HIV-1 and is not targeted by HIV-1 Vif. This disclosure describes the use of simian immunodeficiency virus (SIV) Vif, including modifications, derivatives and conjugates thereof, for use in neutralizing and/or degrading APOBEC3B. This disclosure also describes vectors including nucleic acids encoding SIV Vif. This disclosure further describes the use of SIV Vif as a therapeutic agent, for example, to treat cancers, precancerous conditions, growth of tumors, or inhibit genetic evolution of tumors.

APOBEC3B, unlike other APOBEC3 family members does not restrict HIV-1 and is not targeted by HIV-1 Vif. To determine if a different lentiviral Vif protein could degrade APOBEC3B, a diverse panel of Vif proteins were tested for their ability to degrade APOBEC3B, and Vif from SIVmac239 was identified as a potent APOBEC3B antagonist. The ability of Vif to rescue cells overexpressing APOBEC3B from cell death was tested, and SIVmac239 Vif was found to be able to effectively counteract APOBEC3B-mediated cytotoxicity. The APOBEC3B degradation potential of SIVmac239 Vif may be an effective strategy for efficiently neutralizing the cancer genomic DNA deaminase APOBEC3B.

In one aspect, the present disclosure describes a simian immunodeficiency virus (SIV) Vif that may be used to inhibit, neutralize, degrade and/or modulate the functional activity of

APOBEC3B.

A modulation in a functional activity can be quantitatively measured and described as a percentage of the functional activity of a comparable control. The functional activity of

APOBEC3B includes a modulation that is at least 5%, at least 10%, at least 15%, at least 20%, at least 25%), at least 30%>, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 100%, at least 110%, at least 125%, at least 150%, at least 200%, or at least 250%) of the activity of a suitable control.

For example, the stimulation of a functional activity of an APOBEC3B polypeptide can be quantitatively measured and described as a percentage of the functional activity of a comparable control. Stimulation of a functional activity of an APOBEC3B polypeptide includes a stimulation that is at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 100%, at least 110%, at least 125%, at least 150%, at least 200%, or at least 250% greater than the activity of a suitable control.

For example, inhibition of a functional activity of an APOBEC3B polypeptide can be quantitatively measured and described as a percentage of the functional activity of a comparable control. Inhibition of a functional activity of an APOBEC3B polypeptide includes an inhibition that is no more than 5%, no more than 10%, no more than 15%, no more than 20%, no more than 25%, no more than 30%, no more than 35%, no more than 40%, no more than 45%, no more than 50%, no more than 55%, no more than 60%, no more than 65%, no more than 70%, no more than 75%, no more than 80%, no more than 85%, no more than 90%, no more than 95%, no more than 99%, or no more than 100% of the activity of a suitable control.

In one embodiment, the simian immunodeficiency virus (SIV) Vif may be, for example, SIVmac239 Vif, SIVsmCFU212 Vif, SIVsmPG Vif, SIVsmPBj Vif, SIVsmE041 Vif, SIVstm Vif, SIVmacl42 Vif, SIVmfal86 Vif, SIVmne07 Vif, and/or SIVsmE543 Vif, etc. In one embodiment, the SIV Vif may be SIVmac239 Vif. In one embodiment, the SIV Vif may include the polypeptide sequence SEQ ID NO:3. In some aspects, the disclosure provides polynucleotides that encode any of the SIV Vif polypeptides or portions of the SIV Vif polypeptides described herein, and the polynucleotide sequences that can encode such polypeptide sequences. Given the amino acid sequence of any one of the SIV Vif polypeptides described herein, a person of ordinary skill in the art can determine the full scope of polynucleotides that encode that amino acid sequence using conventional, routine methods.

In some embodiments, the coding sequence of SIV Vif may be modified to increase expression in a mammal including, for example, a mouse, a rat, a human, etc. In some

embodiments, the coding sequence of SIV Vif may be codon optimized. In some embodiments, the coding sequence of SIV Vif may be codon optimized using OPTIMUMGE E (GenScript USA, Inc., Piscataway, NJ). The optimization for expression including, for example, codon optimization, may take into account factors such as, for example, codon adaptability, mRNA structure, various c/5-elements in transcription and translation, etc. In some embodiments, the coding sequence of SIV Vif may include SEQ ID NO: 1 or SEQ ID NO:2. In some embodiments, the polypeptide sequence of SIV Vif includes a conserved SLQ tri- residue peptide motif and/or a conserved TLQ tri-residue peptide motif. The SLQ tri-residue peptide motif and/or a conserved TLQ tri-residue peptide motif may be involved in binding to an E3 ubiquitin ligase or E3 ubiquitin ligase complex. In some embodiments, the SLQ tri-residue motif and/or TLQ tri-residue motif of SIV Vif may be involved in binding to ElonginC (ELOC).

In some embodiments, a polynucleotide that encodes an SIV Vif polypeptide or portions of an SIV Vif polypeptide may be introduced into a cell. In some embodiments, the polynucleotide or portions of the polynucleotide may be introduced into the cell by transfection, transduction, and/or infection. In some embodiments, the polynucleotide may be in a vector, a small particle (e.g., a virus-like particle), a viral particle, etc.

A vector containing a polynucleotide encoding SIV Vif may be used to transfect, transduce, and/or infect cells, including, for example, cancerous or pre-cancerous cells. In some embodiments, cells into which the polynucleotide is introduced are human cells including, for example, human tumor cells. In some embodiments, the expression of the SIV Vif in the transfected, transduced, or infected cell may result in neutralizing and/or degrading APOBEC3B in the transfected, transduced, or infected cell. In some embodiments, expression of the SIV Vif in the transfected, transduced, or infected cell may result in abated mutagenesis of the cell's DNA.

While described above in the context of an exemplary embodiment in which the vector is a based on a lentivirus, the technology described herein can be implemented using any suitable virus- based vector. Exemplary vectors that may be designed to contain a polynucleotide encoding an SIV Vif polypeptide or a portion of an SIV Vif polypeptide include, for example, plasmids, cosmids, bacteriophages, viral vectors, etc. The vector may contain a selectable marker for propagation in a host. In some embodiments, the vector may be a gene therapy -type viral vector including, for example, a retrovirus, a lentivirus, an adenovirus, an adeno-associated virus, a pox virus, an alphavirus, a herpes virus, a measles virus, an influenza virus, etc.

A vector that contains a polynucleotide encoding an SIV Vif may include one or more expression regulatory elements, such as a promoter, an operator, an initiation codon, a stop codon, a polyadenylation signal and/or an enhancer. Each of these elements may be operably linked. The vector may further contain a site for transcription initiation, a site for transcription, a site for termination and/or, in the transcribed region, a ribosome binding site for translation. The coding portion of a mature transcript expressed by the constructs may include a translation initiation at the beginning and/or a termination codon appropriately positioned at the end of the polypeptide to be translated.

A viral particle including a polynucleotide that encodes an SIV Vif polypeptide or portions of an SIV Vif polypeptide may include, for example, a viral particle derived from an enveloped- virus, for example a retroviruses, including, for example, a rous sarcoma virus, a human and/or bovine T-cell leukemia virus (HTLV and BLV), a lentivirus such as human and simian

immunodeficiency viruses (HIV and SIV), Mason-Pfizer monkey virus; a foamy virus; a herpes virus, including for example, HSV, varicella-zoster, vaccinia; a Pox virus; an orthomyxovirus including, for example, influenza; a paramyxovirus, including, for example, a parainfluenza virus, a respiratory syncytial virus, a Sendai virus, a mumps virus, a measles virus; a corona virus, a flavivirus; an alphavirus; a rhabdovirus including, for example, a vesicular stomatitis virus, a rabies virus; a vaccinia virus; a bunyavirus, and/or an RNA virus, including, for example, a Rhabdovirus VSV (vesicular stomatitis virus).

This disclosure further provides a method that includes administering a polynucleotide encoding an SIV Vif as an active ingredient in an anticancer composition.

In addition to the active ingredient, the anticancer composition can include a

pharmaceutically acceptable carrier, and can be prepared into various dosage forms including tablets, troches, capsules, elixirs, suspensions, creams, syrups, wafers, injections, etc. As long as it is pharmaceutically acceptable, any form of solvent, dispersing media, antibacterial agent, antifungal agent, isotonic, absorption retardant, or any combination thereof may be used as a carrier. These media and materials for effective use in pharmaceutically active ingredients are well known in the art.

The composition can be formulated into a suitable pharmaceutical preparation. In a one embodiment, a vector, a viral particle, or a small particle containing a polynucleotide encoding an SIV Vif may be administered via an oral route or a non-oral route such as, for example, an intramuscular, intravenous, peritoneal, subcutaneous, or intradermal route. An injection may be, for example, an intravenous injection, a subcutaneous injection, an intradermal injection, an intramuscular injection, instillation, or an intratumoral injection. The composition may be administered in a single dose or in multiple doses. The composition may be administered either locally— e.g., to the location of a tumor— or systemically.

The anticancer composition may be administered in a pharmaceutically effective amount. The pharmaceutically effective amount may depend, at least in part, on the route of administration, frequency of administration, the kind of the cancer being treated, the severity of the cancer being treated, the age of the patient, the gender of the patient, the condition of the patient, and/or other factors well-known in the pharmaceutical art. The pharmaceutically effective amount may be administered once or in multiple doses.

The disclosure further provides a therapeutic method of treating a subject suffering from a cancer or a precancerous condition. Generally, the method includes administering a vector including a polynucleotide encoding a SIV Vif to the subject. Therapeutic treatment is initiated after the development of cancer or a precancerous condition.

Administering a vector including a polynucleotide encoding a SIV Vif may be particularly advantageous for treating a cancer or a precancerous condition wherein the cancerous or precancerous cells are known to express APOBEC3B. Expression of APOBEC3B may be determined using various methods including, for example, quantitative PCR and/or detection with anti-A3B antibodies (including those described in U.S. Provisional Patent Application No. 62/186, 109, filed June 29, 2015).

The therapeutic method can be used to treat a variety of cancerous or precancerous conditions, including tumors or dysplasia. A tumor can be a solid tumor, such as a carcinoma, a sarcoma, or a lymphoma, and can be present, for example, in the bone, brain, breast, cervix, larynx, lung, pancreas, prostate, skin, spine, stomach, bladder, uterus, etc. The cancer treated can also be a blood cancer, such as leukemia or lymphoma. The dysplasia can be an epithelia dysplasia. The tumor can made up of tumor cells, including lymphoid and myeloid cancers; multiple myeloma; cancers of the bone, breast, prostate, stomach, colon, pancreas, or thyroid; melanoma; head and neck squamous cell carcinoma; ovarian carcinoma; or cervical carcinoma.

In some embodiments, a precancerous condition may include infection with a virus that increases APOBEC3 expression (Vieira et al., 2014, mBio. 5(6): e02234-14). In some

embodiments, a precancerous condition may include infection with human papilloma virus (HPV). In some embodiments, a precancerous condition may include a cervical abnormality. In one embodiment, a precancerous cell may include a HPV-infected cervical cell.

Administration of a vector including a polynucleotide encoding a SIV Vif can occur before, during, and/or after other treatments. Such combination therapy can involve the administration of vector including a polynucleotide encoding a SIV Vif before, during and/or after the use of other anti-cancer agents, for example, chemotherapeutic agents or radiation or both. It is expected that a vector including a polynucleotide encoding a SIV Vif may potentiate the effects of cytokines, chemotherapeutic agents, or gamma radiation. The administration of vector including a polynucleotide encoding a SIV Vif can be separated in time from the administration of other anticancer agents by hours, days, or even weeks. Additionally or alternatively, the administration of a vector including a polynucleotide encoding a SIV Vif can be combined with other biologically active agents or modalities such as, but not limited to, an antineoplastic agent, and non-drug therapies, such as, but not limited to, surgery.

The invention is illustrated by the following examples. It is to be understood that the particular examples, materials, amounts, and procedures are to be interpreted broadly in accordance with the scope and spirit of the invention as set forth herein.

EXAMPLES

Materials and Methods

APOBEC3 expression constructs. The APOBEC3 proteins huA3B (GenBank Accession No. (gb) NM_004900), huA3G (gb NM021822), and rhA3B (gb JF714485, but with the asparagine at amino acid residue 316 corrected to aspartic acid (McDougle et al. Virology 2013, 441 :31-39) were expressed with carboxy -terminal HA tag in the pcDNA3.1(+) vector (Invitrogen Corp., Carlsbad, CA). RhA3B cDNA was generously provided by Dr. Theodora Hatziioannou (Aaron Diamond AIDS Research Center, New York) (Virgen et al., 2007, J. Virol. 81 : 13932-13937). Additionally, huA3B was expressed with a carboxy-terminal eGFP tag in the pcDNA5TO vector (Clontech Laboratories, Inc., Mountain View, CA).

Vif expression constructs. The coding DNA sequences of lentiviral Vif proteins from HIV- I _IIIB (protein sequence matches gb EU541617), SIVmac239 (protein sequence matches gb

AY588946), BIV _BIMI27 (protein sequence matches gb M32690), MVV ₁₅₁₄ (protein sequence matches gb M60610), and FIV _N SCU (protein sequence matches gb m25381) were codon optimized (GenScript USA, Inc., Piscataway, NJ) and expressed with carboxy-terminal myc tag in the pVR1012 vector (LaRue et al., 2010, J. Virol. 84(16): 8193-8201). The codon optimized

SIVmac239 Vif has a cDNA sequence that does not exist in nature (FIG. 1 A), but the encoded protein matches gb AY588946 (FIG. IB). The codon optimized coding DNA sequences of lentiviral Vif proteins from HIV- IHIB, SIVmac239, BIVBIMI27, MVV1514 , and FIVNSCU were used in this Example and FIGS. 2-7. Vif expression constructs from SIVsmCFU212 (gb JX860407),

SIVsmPG (gb AAC68657), SIVsmPBj (gb AAB22996), SIVsmE041 (gb HM059825), SIVstm (gb AAA91941), SIVmacl42 (gb Y00277), SIVmfal86 (gb KF030930), SIVmne027 (gb U70412), SIVsmE543 (gb U72748), and SIVagmTAN (gb AAC57053) were a generous gift of Dr. Welkin Johnson (Boston College, Boston, MA) and are described in Krupp et al. (PLoS Pathog.

9:el003641, 2013). These constructs were individually cloned into the pVR1012 vector with a carboxy-terminal myc tag. The Vif expression construct pVR1012 was a generous gift of Dr. Xiao- Fang Yu (John Hopkins University, Baltimore, MD) and is described in LaRue et al. (J. Virol.

84(16): 8193-8201, 2010). For transient expression in HCC1569 and JSQ3 cells, the constructs were transfected with TransIT-2020 (Minis Bio LLC, Madison, WI) and TransIT-X2 (Minis Bio LLC, Madison, WI), respectively. For stable expression in OVCAR-5 cells, HlV-lum and

SIVmac239 Vif were subcloned into the pLenti4-Hygro-TO backbone, transduced into OVCAR-5 cells, and a stably expressing pool was selected with hygromycin.

HIV constructs. The Vif proficient and Vif deficient (X26X27) HIV-I _B A200C proviral expression constructs (gb EU541617) have been reported previously (Hache et al., 2008, Curr. Biol. 18:819-824).

Cell lines. 293T cells, T-REx 293 (Invitrogen Corp., Carlsbad, CA) cells, and JSQ3 cells (generous gift of Dr. Mark Herzberg, University of Minnesota- Twin Cities, Minneapolis, MN; Weichselbaum et al., 1988, Int. J. Radial Oncol. Biol. Phys. 15:575-579) were maintained in Dulbecco's modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS) and 0.5% penicillin-streptomycin (P/S). CEM-GFP cells (obtained from the NIH AIDS Reagent Program) (Gervaix et al., 1997, Proc Natl Acad Sci USA 94:4653-4658), HCC1569 cells (ATCC CRL-2330, ATCC, Manassas, VA), and OVCAR-5 cells (obtained from the Mayo Clinic ovarian cell line repository; Monks et al., 1991, J. Natl. Cancer Inst. 83 :757-766) were maintained in RPMI medium with 10% FBS and 0.5% P/S.

Immunoblotting. Cell lysates were prepared by resuspending washed cell pellets directly in 2.5 X Laemmli sample buffer. Viral particles were purified from the filtered supernatant by centrifugation prior to resuspension in 2.5X Laemmli sample buffer. A3-HA was detected with monoclonal mouse anti-HA (BioLegend, San Diego, CA), Vif-myc was detected with monoclonal rabbit anti-myc (Sigma-Aldrich, St. Louis, MO), tubulin (TUB) was detected with monoclonal mouse anti-a- Tubulin (Covance Inc., Princeton, NJ), HIV-1 Gag was detected with monoclonal mouse anti-HIV-1 p24 (NIH AIDS Reagent Program) (Chesebro et al., 1992, J. Virol. 66:6547- 6554), A3-GFP was detected with monoclonal mouse anti-GFP (Clontech Laboratories, Inc., Mountain View, CA), Hsp90 was detected with mouse anti-HSP90 (BD Biosciences, Franklin Lakes, NJ). A3B was detected with rabbit polyclonal anti-A3B (10-87-13) (described in U.S.

Provisional Patent Application No. 62/186, 109, filed June 29, 2015).

Vif degradation. 293T cells were transfected in triplicate with pVR1020-Vif-myc or empty vector, at levels normalized by immunoblot, and pcDNA3.1 A3-HA, or empty vector, as indicated, using PEI (polyethyleneimine) (Polysciences, Inc., Warrington, PA). After 48 hours, the cells were harvested for immunoblot analysis. To inhibit proteasomal degradation, MG132 (American Peptide, Sunnyvale, CA) was added at 5 μΜ, 16 hours before harvesting the cells.

HIV-1 single cycle infection with replication-proficient virus. The single cycle infectivity assays were performed as previously reported (Hultquist et al., 2011, J. Virol. 85: 11220-11234) by transfecting 293T cells (TransIT-LTl Transfection Reagent, Minis Bio LLC, Madison, WI) in triplicate with 1 μg of a Vif deficient HIV-1 proviral expression construct along with 25 ng of A3- HA expression construct or empty vector, and 25 ng of myc-tagged Vif expression construct or empty vector. After 48 hours, purified virus-containing supernatants were used to infect the CEM- GFP HIV-1 reporter cells, and cell and viral particle lysates were prepared for immunoblotting. Infectivity was normalized to the vector control.

Flow cytometry. HIV-infected CEM-GFP cells were prepared for flow cytometry by fixation in 4% paraformaldehyde. GFP fluorescence was measured on a BD FACS Canto II flow cytometer (BD Biosciences, Franklin Lakes, NJ). All data were analyzed using FlowJo flow cytometry analysis software (version 8.8.7). GFP fluorescence was quantified from the gated live cell population.

Viability assay. T-REx 293 cells (Invitrogen Corp., Carlsbad, CA), which stably express the tetracycline repressor, were transfected with pcDNA5TO-A3B-eGFP and pcDNA5TO-eGFP constructs using TransIT-LTl (Minis Bio LLC, Madison, WI). Stable clones were selected with hygromycin and blasticidin. These T-REx 293 huA3B and T-REx 293 GFP stable clones were further engineered to stably express HIV-I _HIB Vif-myc, SIVmac239 Vif-myc or vector by transfection of pcDNA3.1 expression constructs and selection with G418. To assess viability, equal numbers of cells were plated in triplicate in increasing doxycycline concentrations and clones were allowed to form. The clones were quantified using ImageJ (1.42q) software. In parallel, these cells were plated in increasing doxycycline concentrations and harvested after 48 hours for

immunoblotting. TCID ₅₀ was calculated in Prism 5 (Graphpad Software Inc., La Jolla, CA). Results

SIVmac239 Vif potently counteracts human A3B. We, and others, have previously demonstrated that HIV- I _HIB Vif does not mediate degradation of huA3B. To determine if huA3B is susceptible to degradation by the Vif from other lentiviruses, the ability of a panel of Vif constructs derived from HIV- I _B , SIVmac239, BIV, MLV and FIV to mediate degradation of huA3B was tested. These Vif constructs were transfected into 293T cells at equivalent levels, based on immunoblot, along with a constant amount of huA3B or vector control (FIG. 1). HIV-I _HIB Vif was able to mediate degradation of huA3B at the highest levels of expression, but did not have any effect on huA3B at the lower levels. SIVmac239 Vif was able to mediate degradation of huA3B at all tested expression levels, with the lowest level of SIVmac239 Vif mediating a similar level of huA3B degradation as the highest level of HIV- I _HIB Vif, and the highest level of SIVmac239 Vif rendering huA3B barely detectable by immunoblot (FIG. IB). huA3B cotransfected with BIV Vif showed moderately lower levels of expression, regardless of the amount of BIV Vif co-transfected. FIV Vif and MVV Vif did not have any effect on huA3B, regardless of expression level.

To determine if degradation of huA3B is an effect mediated by Vif from multiple SIV strains, a panel of SIV Vif expression constructs, including lentiviruses that infect sooty

mangabeys, rhesus macaques, stump tailed macaques, pig tailed macaques, cynomolgus macaques, and African green monkeys (described in Krupp et al., 2013, PLoS Pathog. 9:el003641) were expressed in 293T cells (FIG. 2A). The constructs were expressed at equivalent levels based on immunoblot (FIG. 2A). Cellular lysates were immunoblotted to assess the level of Vif-mediated degradation of huA3B (FIG. 2B). In general, the different SIV Vif proteins mediated degradation of huA3B at similar levels as SIVmac239 Vif, and considerably better than HIV-I _OB Vif (FIG. 2B). A notable exception was the Vif from SIVagmTAN, which did not mediate degradation of huA3B (FIG. 2B).

SIVmac239 Vif mediates degradation of huA3B in a manner analogous to HIV- I _HIB Vif mediated degradation of huA3G. To determine if SIVmac239 Vif mediates degradation of huA3B in a conserved manner to HIV- I _HIB Vif degradation of huA3G - an interaction that has been extensively studied - Vif mediated relief of HIV restriction in a single cycle assay was tested. Additionally, rhA3B susceptibility to SIVmac239 Vif was examined, as rhA3B is the cis-species target of SIVmac239. Vif. huA3B, rhA3B, huA3G, and vector control constructs were transfected into 293T cells with Vif-deficient full-length molecular clone HIV- I _IUB . HIV- I _HIB Vif and SIVmac239 Vif were also transfected into the cells on separate expression vectors. As has been previously reported, huA3G restricted the viral infectivity in the absence of any Vif protein, but not when HIV-I _IIIB Vif was present (FIG. 4A). The ability of huA3G to restrict HIV replication was also counteracted by SIVmac39 Vif (FIG. 4A). Furthermore, huA3G was degraded in the presence of both HIV- I _IIIB and SIVmac239 Vif (FIG. 4B). In contrast, huA3B restricted HIV replication both in the absence of Vif protein, and in the presence of HIV-I _HIB Vif. SIVmac239 Vif alone was able to relieve huA3B restriction of HIV replication (FIG. 4 A), and only SIVmac239 Vif mediated degradation of huA3B (FIG. 4B). The rhA3B protein showed a similar restriction profile to huA3B. rhA3B was restrictive in the absence of Vif, and in the presence of HIV- I _HIB Vif. (FIG. 4A) SIVmac239 Vif moderately restored viral infectivity in the presence of rhA3B (FIG. 4 A), and had a small effect on rhA3B degradation (FIG. 4B).

To further characterize similarities between huA3G, huA3B and rhA3B degradation by HIV-I _IIIB Vif and SIVmac239 Vif, whether the degradation observed occurred through a ubiquitin- mediated proteasomal degradation pathway, as is the case for HIV-1 Vif mediated degradation of huA3G, was tested by inhibiting proteasomal degradation with proteasomal inhibitor MG132.

huA3G was degraded in the presence of HIV-1 _HIB Vif and SIVmac239 Vif in the absence of MG132, but in the presence of proteasomal inhibition, huA3G degradation was inhibited (FIG. 5A). SIVmac239 Vif, but not HIV-I _HIB Vif, mediated degradation of huA3B, and this degradation was also inhibited by MG132. Like huA3B, rhA3B was degraded in the presence of SIVmac239 Vif, while HIV-I _HIB Vif was not observed to mediate degradation of rhA3B. Inhibition of the

proteasome with MG132 decreased SIVmac239 Vif mediated degradation of rhA3B (FIG. 5A).

To further characterize the pathway of proteasomal degradation, the role of the SLQ motif of Vif, which mediates interaction with ElonginC of the E3 ubiquitin ligase complex, on mediating degradation of huA3B was examined. The SLQ motif in HIV-1 Vif has previously been shown to be important for degradation of huA3G, and other APOBEC3 proteins. HIV- I _HIB Vif and

SIVmac239 Vif are only 30% identical at the amino acid level, but the SLQ motif is conserved. In fact, this motif is conserved in all the SIV Vif strains used in this study (FIG. 5B). Cells were transfected with APOBEC3 and Vif constructs as shown in FIG. 5C. Mutation of the SLQ region to AAA in HIV- I _IIIB Vif abrogated its ability to mediate degradation of huA3G. Similarly, mutation of the SLQ region to AAA in SIVmac239 Vif also abolished degradation of huA3G (FIG. 5C).

Neither wild type nor SLQ->AAA versions of HIV-1 _111B Vif mediated degradation of huA3B.

However, the SLQ->AAA mutation in SIVmac239 Vif prevented Vif-mediated degradation of huA3B (FIG. 5C), indicating that this Vif protein interacts with the E3 ligase complex in a similar manner to HIV-1 Vif to mediate degradation of huA3B.

SIVmac239 Vif rescues cells from huA3B mediated cytotoxicity. When overexpressed, huA3B is toxic and kills cells in a dose-dependent manner (Burns et al., 2013, Nature 494:366- 370). To determine if SIVmac239 Vif could rescue cells from huA3B mediated cytotoxicity, huA3B-GFP or GFP alone was stably expressed under the control of a doxycycline-inducible promoter in T-REx 293 cells, allowing for titratable expression of the protein. Vector control, HIV- I _IIIB Vif, and SIVmac239 Vif were stably transfected into the inducible huA3B and GFP cells, and their expression was confirmed by immunoblot (FIG. 6A, inset, and FIG. 6B, inset). These cells were plated for clones in increasing concentrations of doxycycline to assess viability in the presence of Vif and the inducible presence of huA3B.

The cells that inducibly expressed huA3B alone {i.e. A3B + vector) showed a marked decrease in viability that correlated with increased huA3B expression, with an IC ₅₀ value of 5 ^χ 10 ^"1 pg/mL doxycycline (FIG. 6 A). Stable expression of HIV-1 _111B Vif moderately counteracted the decreased viability associated with huA3B, as demonstrated by party restored cell viability, increasing the IC ₅₀ value to 4.4 x 10 ³ pg/mL doxycycline. Additionally, as seen by immunoblot, stable expression of HIV-I _HIB Vif decreased detected huA3B protein levels compared to no Vif (FIG. 6B). Stable expression of SIVmac239 Vif robustly counteracted huA3B-induced loss of viability, as evidenced by almost fully restored cell viability. Stable expression of SIVmac239 Vif also decreased cellular huA3B levels, as measured by immunoblotting. When SIVmac239 Vif was stably expressed, cellular viability was affected at only the high concentrations of doxycycline tested, and the maximal loss in viability observed was only 30%, disallowing the determination of an IC ₅₀ value. The amount of huA3B detectable by immunoblot when SIVmac239 Vif was stably expressed is less than that with no Vif, or HIV-I _HIB , but still discernable. These levels of expression may indicate that Vif binding to huA3B may counteract its activity, even in the absence of degradation.

For comparison, the cells that inducibly expressed GFP, GFP+SIV mac239 (circles) Vif GFP+HIV-l iiiB (squares), or Vif GFP+vector (triangles), showed a constant level of viability with increasing GFP expression (FIGS. 6C, 6D). The viability was independent of HIV-1 _HIB or

SIVmac239 Vif expression, indicating that in the absence of huA3B, these Vif proteins have no effect on viability. SIVmac239 Vif degrades endogenously expressed huA3B. To determine the feasibility of using SIVmac239 Vif or a derivative to counteract endogenous huA3B as an anti-cancer therapeutic, the effect of SIVmac239 Vif in three cancer cell lines that endogenously express high levels of huA3B: HCC1569 cells, a human breast cancer cell line; JSQ3, a human head and neck cancer cell line; and OVCAR5, a human ovarian cancer cell line. HIV- I _HIB and SIVmac239 Vif were stably expressed and cell lysates were immunoblotted for huA3B. Cells expressing vector and HIV-I _IIIB Vif exhibited comparable levels of endogenous huA3B (FIG. 7). In contrast, all three cell lines engineered to express SIVmac239 Vif showed lower levels of huA3B, indicating that

SIVmac239 Vif is capable of mediating degradation of endogenous huA3B in cancer cells (FIG. 7).

The complete disclosure of all patents, patent applications, and publications, and

electronically available material (including, for instance, nucleotide sequence submissions in, e.g., GenBank and RefSeq, and amino acid sequence submissions in, e.g., SwissProt, PIR, PRF, PDB, and translations from annotated coding regions in GenBank and RefSeq) cited herein are incorporated by reference. In the event that any inconsistency exists between the disclosure of the present application and the disclosure(s) of any document incorporated herein by reference, the disclosure of the present application shall govern. The foregoing detailed description and examples have been given for clarity of understanding only. No unnecessary limitations are to be understood therefrom. The invention is not limited to the exact details shown and described, for variations obvious to one skilled in the art will be included within the invention defined by the claims.

Unless otherwise indicated, all numbers expressing quantities of components, molecular weights, and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about." Accordingly, unless otherwise indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. All numerical values, however, inherently contain a range necessarily resulting from the standard deviation found in their respective testing measurements. All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so specified.