BACKGROUND OF THE INVENTION FIELD OF THE INVENTION

The present disclosure relates to the ssRNA-specific ribonuclease activity of SAMHDl (sterile alpha motif (SAM) domain and HD domain-containing protein 1) and its use. DESCRIPTION OF THE RELATED ART

Ribonucleases are enzymes that catalyze the degradation of RNA. A well studied ribonuclease is bovine pancreatic ribonuclease A (RNase A), the putative biological function of which is to break down the large amount of RNA that accumulates in 'the ruminant gut. Ribonucleases can be divided into endoribonucleases and exoribonucleases, and comprise several sub-classes within the EC 2.7 (for the phosphorolytic enzymes) and 3.1 (for the hydrolytic enzymes) classes of enzymes.

In the meantime, recent studies have reported that SAMHDl possesses deoxynucleoside triphosphohydrolase (dNTPase) activity {Nature 480, 379-382). However, ribonuclease activity has not yet been described for this protein.

Throughout this application, various patents and publications are referenced and citations are provided in parentheses. The disclosure of these patents and publications in their entities are hereby incorporated by references into this application in order to more - fully describe this invention and the state of the art to which this invention pertains.

SUMMARY OF THE INVENTION

The present inventors have made intensive researches on biological activity related with a virus replication of SAMHDl. As a result, we have found that the SAMHDl had ability to degrade viral genomic single stranded RNA (ssRNA), and that this protein was able to inhibit viral replications.

Accordingly, it is an object of this invention to provide a pharmaceutical composition for preventing or treating a ssRNA viral infection.

It is an additional object of this invention to provide a method of preventing or treating a ssRNA viral infection in a subject in need thereof.

It is another object of this invention to provide a composition for degrading a viral genomic ssRNA.

It is still another object of this invention to provide a method of degrading a viral genomic ssRNA in a cell infected by a ssRNA virus.

It is still further another object of this invention to provide use of SAMHDl or a nucleic acid molecule encoding the SAMHDl for degrading a viral genomic ssRNA, or preventing or treating a ssRNA viral infection. Other objects and advantages of the present invention will become apparent from the detailed description to follow taken in conjugation with the appended claims and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 shows that SAMHDl is a single-stranded RNA-specific ribonuclease. [a]

Purified GST-SAMHD1 (150 nM) was incubated for 30 min at 37°C with various synthetic nucleic acid substrates (20-mers) that were 5'-end labeled with [γ-32Ρ]ΑΤΡ (indicated by an asterisk). The products were analyzed by electrophoresis and autoradiography. RNA and DNA are represented by red and blue lines, respectively, [b] GST-SAMHD1 was assayed for ribonuclease activity in time course experiments, [c] Ribonuclease activity of wild-type and putative catalytic mutants toward a 20-mer single-stranded RNA substrate labeled at the 5'-end.

Fig. 2 shows that SAMHDl degrades RNA with a preference for a 3'-end containing a free hydroxyl group in a 3' to 5' direction, [a and b] GST-SAMHDl was assayed for exoribonuclease activity in time course experiments. The RNA substrates were labeled with 32P at 3'-end. The 3'-end of the 30-mer substrate carried a hydroxyl group (a) or a phosphate group (b). The reaction products were separated by PAGE and visualized by autoradiography.

Fig. 3 shows that ribonuclease and dNTPase are dual and separable functions of SAMHD1. [a] A dGTPase activity assay was performed essentially as described previously(/V<3f Immunol 13, 223-228 (2012)). The reaction products generated upon incubation of GST-SAMHDl proteins with dGTP were analyzed by polyethyleneimine cellulose thin layer chromatography. Unlabeled dGTP, dGDP, and dGMP were visualized using 254 nm ultraviolet and served as migration standards. PPP, triphosphate; PPP%, PPP/(dGTP + PPP) expressed as a percentage, [b] The ribonuclease activity assay was performed as described in Fig. lc using ssRNA substrates that were 5'-end labeled with 32P.

Fig. 4 shows that the ribonuclease function of SAMHD1 is essential for HIV- 1 restriction, [a] U937 cells stably expressing mock, wild-type or mutant SAMHD1 proteins with substitutions at the indicated positions were treated with PMA, followed by infection with HIV-l-GFP. After 48 hrs, these cells were subjected to whole-cell extraction before immunoblot analysis using the indicated antibodies, [b] The cells from (a) were harvested and then analyzed to determine the percentage of GFP-positive cells by flow cytometry, [b] The relative percentage of GFP-positive cells from (b) was calculated relative to the number of GFP-positive cells among the mock-transfected cells. Graphs show the mean ± S.D. from three independent experiments. Two asterisks reflect a p value of <0.005. [d] The synthesis of viral cDNA intermediates was determined 24 hrs after infection by RT-PCR using R/U5 (early products, left panel) and US/ψ primers (late products, right panel).

Fig. 5 shows that SAMHD1 directly degrades HIV-1 genomic RNA during infection,

[a] The experimental design for time course analysis. SAMHDl-expressing U937 cells were infected with HIV-l-GFP and total cellular RNA was extracted at different time points post-infection, [b] Northern blot analysis of tRNA ^Lys3 levels using 18-mer nucleotide probe specific for tRNA ^Lys3 as a probe (upper panel). Equal loading of RNA samples is shown by ethidium bromide stain of gel before Northern blotting (lower panel), [c] HIV-1 genomic RNA content was quantified by qRT-PCR using HIV-1 gag-specific primer. The data were normalized to an internal control gene (β-actin). Similar results were obtained in three independent experiments, [d] U937 cells expressing vector alone, SAMHDlwr or SAMHD1 _D 207N were infected for 90 min with HIV-1-GFP and crosslinked by formaldehyde. Cell lysates were immunoprecipitated with anti-HA antibody. Purified RNAs from the immunoprecipitates were quantified by qRT-PCR using HIV-1 gag-specific primer, [e] Role of SAMHD1 in HIV-1 restriction and AGS-related autoimmune diseases. Once HIV-1 genomic RNA is released from the core in the cytoplasm, SAMHDl recognizes and directly degrades the HIV-1 genomic RNA. Effective clearance of HIV-1 by SAMHD1 may require the combined action of the ribonuclease and dNTPase activities (left). In human cells with missense mutations in SAMHDl, the RNA debris of endogenous retroviruses accumulates in the cytoplasm and is detected by sensors of antiviral innate immunity. The aberrant activation of innate immune response could cause pro-inflammatory diseases such as AGS (right).

DETAILED DESCRIPTION OF THIS INVETNION

In one aspect of the present invention, there is provided a pharmaceutical composition for preventing or treating an RNA viral infection comprising a therapeutically effective amount of an RNA-degrading molecule as an active ingredient, wherein the RNA-degrading molecule is selected from SAMHD1 and a nucleic acid molecule encoding the SAMHDl.

In another aspect of this invention, there is provided a method of preventing or treating an RNA viral infection in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising an RNA-degrading molecule selected from SAMHDl and a nucleic acid molecule encoding the SAMHDl.

In still another aspect of this invention, there is provided use of SAMHDl or a nucleic acid molecule encoding the SAMHDl as an RNA-degrading molecule for preventing or treating an RNA viral infection.

The present inventors have made intensive researches on biological activity related with a virus replication of SAMHDl. As a result, we have found that the SAMHDl had ability to degrade viral genomic single stranded RNA (ssRNA), and that this protein was able to inhibit viral replications. The present invention is based on the new findings.

The term used herein "prevention" refers to the complete prevention of infection, the prevention of occurrence of symptoms in an infected subject, the prevention of recurrence of symptoms in an infected subject, or a decrease in severity or frequency of symptoms of viral infection, condition or disease in the subject.

The term "treatment" as used herein, refers to the partial or total elimination of symptoms or decrease in severity of symptoms of viral infection, condition or disease in the subject, or the elimination or decrease of viral presence in the subject.

The term used herein "subject" is intended to encompass human or non-human mammal or animal. Non-human mammals include livestock animals and companion animals, such as cattle, sheep, goats, equines, swine, dogs and cats.

As used herein, the term "therapeutically effective amount" means an amount of an RNA-degrading molecule which is sufficient, in the subject to which it is administered, to treat or prevent the symptoms, condition or disease related with viral infection.

The term "RNA-degrading molecule" as used herein refers to SAMHDl and SAMHDl-encoding nucleic acids which are capable of degrading viral genomic ssRNAs.

The term used herein "RNA viral infection" refers to the entry of an ssRNA virus into a cell and the subsequent replication of the virus in the cell.

According to the present invention, the treatment strategies of the present invention are divided into two categories: (i) protein therapy or (ii) gene therapy.

According to the protein therapy strategy of this invention, SAMHDl (protein or polypeptide) is applied to the pharmaceutical composition as an active ingredient. As shown in Fig. 4b, SAMHD1 is non-toxic to the cells in which the SAMHD1 is introduced or overexpressed, indicating the SAMHD1 is ability to degrade specifically viral genomic ssRNA.

According to the gene therapy strategy of this invention, SAMHDl-encoding nucleic acid molecule is applied to the pharmaceutical composition as an active ingredient.

According to an embodiment, the nucleic acid molecule is a naked DNA or a component of gene delivery system. In addition, the pharmaceutical composition of this invention can be applied to a living body by various gene delivery methods known to gene therapy field. The term "gene delivery system" refers to any forms of carriers that harbor and transport exogenous nucleic acid molecules to a target cell. Specifically, the SAMHDl-encoding nucleotide sequence may be applied to a multitude of gene delivery systems useful in gene therapy, such as plasmid, adenovirus (Lockett LJ, et al., Clin. Cancer Res. 3:2075-2080(1997)), adeno-associated virus (AAV, Lashford LS., et al., Gene Therapy Technologies, Applications and Regulations Ed. A. Meager, 1999), retrovirus (Gunzburg WH, et al., Retroviral vectors. Gene Therapy Technologies, Applications and Regulations Ed. A. Meager, 1999), lentivirus (Wang G. et al., J. Clin. Invest. 104(11):R55-62(1999)), herpes simplex virus (Chamber R., et al., Proc. Natl. Acad. Sci USA 92:1411-1415(1995)), vaccinia virus (Puhlmann M. et al., Human Gene Therapy 10:649-657(1999)), liposome (Methods in Molecular Biology, Vol 199, S.C. Basu and M. Basu (Eds.), Human Press 2002), neosome or nanoparticles.

(i) Adenovirus

Adenovirus has been usually employed as a gene delivery system because of its mid-sized genome, ease of manipulation, high titer, wide target-cell range, and high infectivity. The nucleotide sequence of interest to be delivered is inserted into either the deleted El region (E1A region and/or E1B region, preferably, EIB region) or the deleted E3 region, preferably, the deleted El region. Furthermore, the inserted sequences may be incorporated into the deleted E4 region.

According to an embodiment, the adenoviral gene delivery system of this invention comprises both "promoter-nucleotide sequence of interest-poly A sequence" and "promoter- SAMHD1 gene-poly A sequence". The promoter-nucleotide sequence of interest-poly A sequence is present in either the deleted El region (EIA region and/or E1B region, preferably, EIB region) or the deleted E3 region, preferably, the deleted El region. The promoter-relaxin gene-poly A sequence is present in either the deleted El region (EIA region and/or EIB region, preferably, EIB region) or the deleted E3 region, preferably, the deleted E3 region. In addition, the adenoviral gene delivery system may comprise a bicistronic expression system in which the nucleotide sequence of interest and SAMHDl-encoding nucleotide sequence are linked each other by IRES (internal ribosome entry site) to form "promoter-nucleotide sequence of interest-poly A sequence- SAMHD1 gene-poly A sequence.

The foreign genes delivered by the present adenoviral gene delivery system are episomal, and therefore, have low genotoxicity to host cells. Therefore, gene therapy using the adenoviral gene delivery system of this invention may be considerably safe.

(ii) Retrovirus

Retroviruses capable of carrying relatively large exogenous genes have been used as viral gene delivery vectors in the senses that they integrate their genome into a host genome and have broad host spectrum.

In order to construct a retroviral vector, the SAMHDl-encoding nucleotide sequences and the nucleotide sequence of interest to be transferred are inserted into the viral genome in the place of certain viral sequences to produce a replication-defective virus. To produce virions, a packaging cell line containing the gag, pol and env genes but without the LTR (long terminal repeat) and Ψ components is constructed (Mann et al., Cell, 33:153-159(1983)).

A successful gene transfer using the second-generation retroviral vector has been reported. Kasahara et al. {Science, 266:1373-1376(1994)) prepared variants of moloney murine leukemia virus in which the EPO (erythropoietin) sequence is inserted in the place of the envelope region, consequently, producing chimeric proteins having novel binding properties. Likely, the present gene delivery system can be constructed in accordance with the construction strategies for the second-generation retroviral vector. (iii) AAV vector

Adeno-associated viruses are capable of infecting non-dividing cells and various types of cells, making them useful in constructing the gene delivery system of this invention. The detailed descriptions for use and preparation of AAV vector are found in U.S. Pat. No. 5,139,941 and 4,797,368.

Research results for AAV as gene delivery systems are disclosed in LaFace et al,

Viology, 162:483486(1988), Zhou et al., Exp. Hematol. (NY), 21:928-933(1993), Walsh et al, J. Clin. Invest, 94:1440-1448(1994) and Flotte et al Gene Therapy, 2:29-37(1995). Recently, an AAV vector has been approved for Phase I human trials for the treatment of cystic fibrosis.

Typically, a recombinant AAV virus is made by cotransfecting a plasmid containing the gene of interest {i.e. SAMHD1 gene and nucleotide sequence of interest to be delivered) flanked by the two AAV terminal repeats (McLaughlin et al., 1988; Samulski et al., 1989) and an expression plasmid containing the wild type AAV coding sequences without the terminal repeats (McCarty et al., J. Virol., 65:2936-2945(1991)).

(iv) Other viral vectors

Other viral vectors may be employed as a gene delivery system in the present invention. Vectors derived from viruses such as vaccinia virus (Puhlmann M. et al., Human Gene 777^/7/ 10:649-657(1999); Ridgeway, "Mammalian expression vectors," In: Vectors: A survey of molecular cloning vectors and their uses. Rodriguez and Denhardt, eds. Stoneham: Butterworth, 467-492(1988); Baichwal and Sugden, "Vectors for gene transfer derived from animal DNA viruses: Transient and stable expression of transferred genes," In: Kucherlapati R, ed. Gene transfer. New York: Plenum Press, 117-148(1986) and Coupar et al., Gene, 68:1-10(1988)), lentivirus (Wang G. et al., J. Clin. Invest 104(11):R55-62(1999)) and herpes simplex virus (Chamber R., et al., Proc. Natl. Acad. Sci USA 92: 1411-1415(1995)) may be used in the present delivery systems for transferring both the SAMHD1 gene and nucleotide sequence of interest into cells.

(v) Liposome

Liposomes are formed spontaneously when phospholipids are suspended in an excess of aqueous medium. Liposome-mediated nucleic acid delivery has been very successful as described in Nicolau and Sene, Biochim. Biophys. Acta, 721: 185-190(1982) and Nicolau et al., Methods Enzymol., 149:157-176(1987). Example of commercially accessible reagents for transfecting animal cells using liposomes includes Lipofectamine (Gibco BRL). Liposomes entrapping the SAMHD1 gene and nucleotide sequence of interest interact with cells by mechanism such as endocytosis, adsorption and fusion and then transfer the sequences into cells.

According to an embodiment, the SAMHDl-encoding nucleotide sequence is contained in a suitable expression construct. The term "expression construct" refers to any type of genetic construct comprising a nucleic acid coding for SAMHD1. According the expression construct, the SAMHDl-encoding nucleotide sequence is operatively linked to a promoter. The term "operatively linked" refers to functional linkage between a nucleic acid expression control sequence (such as a promoter, signal sequence, or array of transcription factor binding sites) and a second nucleic acid sequence, wherein the expression control sequence affects transcription and/or translation of the nucleic acid corresponding to the second sequence. According to the present invention, the promoter linked to the SAMHD1 gene is operable in, e.g. animal and mammalian cells, to control transcription of the SAMHD1 gene, including the promoters derived from the genome of mammalian cells or from mammalian viruses, for example, CMV (cytomegalovirus) promoter, the adenovirus late promoter, the vaccinia virus 7.5K promoter, SV40 promoter, HSV tk promoter, RSV promoter, EF1 alpha promoter, metallothionein promoter, beta-actin promoter, human IL-2 gene promoter, human IFN gene promoter, human IL-4 gene promoter, human lymphotoxin gene promoter and human GM-CSF gene promoter.

In an embodiment, the expression construct used in this invention comprises a polyadenylation sequence {e.g. bovine growth hormone terminator and SV40-derived polyadenylation sequence).

According to an embodiment, the expression construct for the SAMHDl-encoding nucleotide sequence has a structure of "promoter-SAMHDl-encoding nucleotide sequence-polyadenylation sequence.

According to an embodiment, the SAMHDl-encoding nucleic acid molecule is operatively linked to a promoter in an expression cassette. The term "expression cassette" refers to a nucleic acid molecule capable of directing expression of the SAMHDl-encoding nucleotide sequence in an appropriate host cell {e.g. human cells ¹ such as ssRNA virus-infected cells), comprising a promoter operatively linked to the nucleotide sequence of interest which is operatively linked to termination signals. It can be also include sequences required for proper translation of the nucleotide sequence.

According to an embodiment, SAMHDl or SAMHDl-encoding nucleic acid molecule is derived from human. Their amino acid sequences and nucleotide sequences are disclosed in NCBI (National Center for Biotechnology Information). Examples of accession numbers of the SAMHDl amino acid sequences are NM_015474, EAW76090 and CAI42293. Examples of accession numbers of the SAMHDl nucleotide sequences are NM_015474. The human SAMHDl has an amino acid sequence of SEQ ID NO: 17.

According to the present invention, the RNA-degrading molecule is intended to include functional equivalents of SAMHDl or SAMHDl-encoding nucleic acid molecule. As used herein, the term "functional equivalent" refers to amino acid sequence variants having amino acid substitutions, additions or deletions in some of the amino acid sequence of wild-type SAMHDl while simultaneously having similar or improved biologically activity when compared to the SAMHDl, and refers to the amino acid sequence variant-encoding nucleotide sequences. The amino acid substitutions may be conservative substitutions. Examples of the conservative substitutions of naturally occurring amino acids include aliphatic amino acids (Gly, Ala, and Pro), hydrophobic amino acids (He, Leu, and Val), aromatic amino acids (Phe, Tyr, and Trp), acidic amino acids (Asp, and Glu), basic amino acids (His, Lys, Arg, Gin, and Asn), and sulfur-containing amino acids (Cys, and Met). The deletions of amino acids are located in a region which is not involved directly in the activity of SAMHD1.

According to the present invention, the amino acid sequences and nucleotide sequences of SAMHD1 available to the present invention are intended to include polynucleotide and polypeptide sequences having substantial identity to wild-type human SAMHD1 sequences. The term "substantial identity" as used herein means that the two nucleic acid or amino acid sequences, when optimally aligned, such as by the program BLAST, GAP or BESTF1T, or by visual inspection, share at least about 60%; 70%, 80%, 85%, 90% or 95% sequence identity or sequence similarity. Methods of alignment of sequences for comparison are well-known in the art. Various programs and alignment algorithms are described in: Smith and Waterman, Adv. Appl. Math. 2:482 (1981), Needleman and Wunsch, J. Mol. Bio. 48:443 (1970), Pearson and Lipman, Methods in Mol. Biol. 24: 307-31 (1988), Higgins and Sharp, Gene 73:237-44 (1988), Higgins and Sharp, CABIOS 5: 151-3 (1989), Corpet et al., Nuc. Acids Res. 16:10881-90 (1988), Huang et al., Comp. Appl. BioSci. 8: 155-65 (1992) and Pearson et al., Meth. Mol. Biol. 24:307-31 (1994) presents a detailed consideration of sequence alignment methods and homology calculations.

The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215:403-10 (1990)) is available from several sources, including the NCBI and on the internet, for use in connection with the sequence analysis programs blastp, blasm, blastx, tblastn and tblastx. It can be accessed at http://www.ncbi.nlm.nih.gov/BLAST/. A description of how to determine sequence identity using this program is available at http://www.ncbi.nlm.nih.gov/BLAST/blast_help.html.

According to the present invention, the RNA virus is a ssRNA virus selected from a group consisting of a retrovirus, a positive-sense ssRNA virus, a negative-sense ssRNA virus and an ambisense ssRNA virus. Animal RNA viruses are classified into two distinct groups depending on their genome and mode of replication: double-stranded RNA viruses and single stranded RNA viruses. The ssRNA viruses can be further classified according to the sense or polarity of their RNA into negative-sense and positive-sense, or ambisense RNA viruses. Positive-sense viral RNA is similar to mRNA and thus can be immediately translated by the host cell. Negative-sense viral RNA is complementary to mRNA and thus must be converted to positive-sense RNA by an RNA polymerase before translation. Ambisense RNA viruses resemble negative-sense RNA viruses, except they also translate genes from the positive strand. Retroviruses have also a ssRNA genome. The term "retrovirus" as used herein is defined as an RNA virus of the Retroviridae family.

Examples of retroviruses include, but are not limited to, members of the Lentivirus, Alpharetrovirus, Betaretrovirus, Gam ma retrovirus, Deltaretrovirus, Spumavirus and Epsilonretrovirus.

According to an embodiment, the retroviruses include, but are not limited to,

Human immunodeficiency virus such as HIV-1 and HIV-2, Simian immunodeficiency virus, Caprine arthritis encephalitis virus, Visna/maedi virus, Feline immunodeficiency virus, Puma lentivirus, Equine infectious anemia virus, Bovine immunodeficiency virus, Jembrana disease virus, Avian leukosis virus, Rous sarcoma virus, Jaagsiekte sheep retrovirus, Simian retrovirus, Mouse mammary tumor virus, Feline leukemia virus, Bovine leukemia virus, Walleye epidermal hyperplasia virus 1 and Walleye epidermal hyperplasia virus 2.

Examples of RNA viruses with positive-sense single stranded genome include, but are not limited to, members of the Arteriviridae, Coronaviridae, Roniviridae, Dicistroviridae, Iflaviridae, Marnaviridae, Picornaviridae, Secoviridae, Alphaflexiviridae, Betaflexiviridae, Gammaflexiviridae, Tymoviridae, Astrovirus, Barnaviridae, Bromoviridae, Caliciviridae, Closteroviridae, Flaviviridae, Leviviridae, Luteoviridae, Namaviridae, Nodaviridae, Potyviridae, Togaviridae, Tombusviridae, Virgaviridae, Benyvirus, Cilevirus, Hepevirus, Idaeovirus, Ourmiavirus, Sobemovirus and Umbravirus. According to an embodiment, the positive-sense ssRNA viruses include, but are not limited to, Equine arteritis virus, Lactate dehydrogenase elevating virus, Porcine reproductive and respiratory syndrome virus, Simian hemorrhagic fever virus, Coronavirus, Severe acute respiratory syndrome (SARS) coronavirus, Cripavirus, Deformed wing virus, Poliovirus, Human rhinoviruses, Hepatitis A virus, Avian nephritis virus, Norwalk virus, Yellow fever virus, West Nile virus, Hepatitis C virus, Dengue fever virus, Barley yellow dwarf virus, Rubella virus, Ross River virus, Sindbis virus, Chikungunya virus, Hepatitis E virus, Beet necrotic yellow vein virus, Beet soil-borne mosaic virus, Cocksfoot mottle virus, Lucerne transient streak virus and Rice yellow mottle virus.

Examples of RNA viruses with negative-sense single stranded genome include, but are not limited to, members of the Bornaviridae, Filoviridae, Paramyxoviridae, Rhabdoviridae, Arenaviridae, Bunyaviridae, Ophioviridae, Orthomyxoviridae, Deltavirus, Nyavirus and Tenuivirus.

According to an embodiment, the negative-sense ssRNA viruses include, but are not limited to, Borna disease virus, Ebola virus, Marburg virus, Measles virus, Mumps virus,

Nipah virus, Hendra virus, Rabies virus, Lassa virus, Hantavirus, Citrus psorosis virus,

Influenzavirus A, Influenzavirus B, Influenzavirus C and hepatitis D virus.

Examples of RNA viruses with ambisense genome include, but are not limited to, members of the bunyaviridae and arenaviridae.

According to an embodiment, the ambisense ssRNA viruses include, but are not limited to, Nairovirus, Orthobunyavirus, Phlebovirus, Tospovirus, Lymphocytic choriomeningitis (LCM) virus and Lassa fever virus.

According to an embodiment, the RNA-degrading molecule is exoribonuclease. In an embodiment, the RNA-degrading molecule is able to digest ssRNA processively in the 3' to 5' direction, i.e. 3'-5' exoribonuclease. Accordingly, where the RNA-degrading molecule is introduced into cells infected by the ssRNA virus, it degrades the viral genomic ssRNA present in the cell, thereby inhibiting virus replication and eventually preventing or treating the viral infection. According to an embodiment, the degradation of viral genomic ssRNA by the RNA-degrading molecule occurs in a cell cytoplasm.

According to an embodiment, the RNA viral ssRNA are degraded into 5 to 8-mer oligonucleotides by the RNA-degrading molecule.

According to an embodiment, the RNA-degrading molecule is able to exhibit the ssRNA-specific ribonuclease activity without dGTP as a cofactor that is essential for the dNTPase activity. As shown in Examples below, we have first found that wild-type SAMHD1 has dual catalytic functions which are ribonuclease activity and dNTPase activity. It has been reported that the dNTPase function of SAMHD1 is allosterically activated by dGTP. However, as demonstrated in Fig. 1, unlike the dNTPase activity, the ribonuclease activity of SAMHD1 did not require the cofactor dGTP.

According to an embodiment, the SAMHD1 is lack of dNTPase activity.

According to an embodiment, the lack of dNTPase activity of SAMHD1 is achieved by conferring a mutation to the human SAMHD1.

According to an embodiment, the SAMHD1 whose dNTPase activity is inactivated has the substitution mutation of Asn or Ala for Asp at position 137 of SEQ ID NO: 17. As demonstrated in Fig. 3b, the mutation of amino acid 137 (D137N) in the aliosteric site of SAMHD1 did not affect the ribonuclease activity, and the ribonuclease activity of this SAMHDl _D i37N mutant was very similar to that of the wild-type SAMHD1.

According to the present invention, the pharmaceutical composition may contain pharmaceutically acceptable carriers. The pharmaceutically acceptable carrier may be conventional one for formulation, including lactose, dextrose, sucrose, sorbitol, mannitol, starch, rubber arable, potassium phosphate, arginate, gelatin, potassium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrups, methyl cellulose, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate, and mineral oils, but not limited to. The pharmaceutical composition according to the present invention may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, and a preservative. Details of suitable pharmaceutically acceptable carriers and formulations can be found in Remington's Pharmaceutical Sciences (19th ed.,

1995), which is incorporated herein by reference.

The pharmaceutical composition according to the present invention may be administered via the oral or parenterally. When the pharmaceutical composition of the present invention is administered parenterally, it can be done by intravenous, subcutaneous, intramuscular, abdominal or transdermal administration.

A suitable dose of the pharmaceutical composition of the present invention may vary depending on pharmaceutical formulation methods, administration methods, the patient's age, body weight, sex, severity of diseases, diet, administration time, administration route, an excretion rate and sensitivity for a used pharmaceutical composition. the pharmaceutical composition of the present invention is administered with a daily dose of

0.0001-1,000 mg/kg (body weight).

According to the conventional techniques known to those skilled in the art, the pharmaceutical composition may be formulated with pharmaceutically acceptable carrier and/or vehicle as described above, finally providing several forms including a unit dose form and a multi-dose form. Formulation may be oil or aqueous media, resuspension or emulsion, extract, powder, granule, tablet and capsule and further comprise dispersant or stabilizer.

In yet another aspect of this invention, there is provided a composition for degrading a viral genomic ssRNA comprising an RNA-degrading molecule selected from SAMHD1 and a nucleic acid molecule encoding the SAMHD1.

In still yet another aspect of this invention, there is provided a method of degrading a viral genomic ssRNA in a cell infected by a ssRNA virus, comprising introducing to the cell an RNA-degrading molecule selected from SAMHD1 and a nucleic acid molecule encoding the SAMHD1, thereby degrading the viral genomic ssRNA.

In further another aspect of this invention, there is provided use of SAMHD1 or a nucleic acid molecule encoding the SAMHD1 as an RNA-degrading molecule for degrading a viral genomic ssRNA. According to an embodiment, the virus is selected from a group consisting of a retrovirus, a positive-sense ssRNA virus, a negative-sense ssRNA virus and an ambisense ssRNA virus.

According to an embodiment, the nucleic acid molecule encoding SAMHD1, which is to be introduced into the cell, is a component of the expression construct mentioned above.

According to the present invention, any methods or techniques known in the art for delivering proteins or nucleic acids into cells {i.e. protein delivery system or gene delivery system) is utilized to introduce the RNA-degrading molecule into the cell infected by the ssRNA virus. Examples of the methods or techniques include, but are not limited to, microinjection method (Capecchi, M.R., Cell, 22:479(1980)), calcium phosphate precipitation method (Graham, F.L et al., Virology, 52:456(1973)), electroporation (Neumann, E. et al., EMBO J., 1:841(1982)), liposome-mediated transformation method (Wong, T.K. et al., Gene, 10:87(1980)), DEAE-dextran treatment method (Gopal, Mol. Cell Biol., 5:1188-1190(1985)), and gene bombardment (Yang et al., Proc. Natl. Acad. Sci., 87:9568-9572(1990)).

The features and advantages of this invention will be summarized as follows:

(i) The present invention is directed to a single stranded RNA-degrading activity of SAMHD1 and its use.

(ii) It is noteworthy that SAMHD1 has dual catalytic functions which are ribonuclease activity and dNTPase activity: however unlike the dNTPase activity, the ribonuclease activity of SAMHD1 did not require the cofactor dGTP.

(iii) The present invention can be employed to degrade viral genomic ssRNA and to prevent or treat a ssRNA viral infection. The present invention will now be described in further detail by examples. It would be obvious to those skilled in the art that these examples are intended to be more concretely illustrative and the scope of the present invention as set forth in the appended claims is not limited to or by the examples. EXAMPLES

Experimental methods

Methods summary

GST and GST-tagged proteins were expressed in E. coli Rosetta (ADE3) and purified using glutathione-agarose. After incubation of the recombinant proteins with synthetic 32P-labeled nucleic acid substrates, the reaction products were resolved using 15% polyacrylamide gels and were analyzed by autoradiography. For the dNTPase activity assay, recombinant proteins were incubated with [a-32P]dGTP and 200 μΜ cold dGTP, and the hydrolysis products were resolved by polyethyleneimine (PEI) cellulose thin-layer chromatography. Prior to HIV-1-GFP infection, U937 cells were differentiated overnight with 30 ng/ml PMA. Flow cytometry analysis of GFP expression was performed 2 days post-infection. The levels of HIV-1 genomic RNA were determined by quantitative RT-PCR.

Plasm ids

Human SAMHD1 was amplified by PCR from cDNA generated by the reverse transcription of RNA from HeLa cells, and the PCR product was inserted into pGEX-4T-l vector and pMSCV-puro vector. The D207N, D311A and D137N mutants were generated using the nPfu-forte DNA Polymerase Kit (Enzynomics). HIV-1-GFP and HCMV-VSV-G were gift from Dan R. Littman (New York University School of Medicine).

Protein expression and purification

Recombinant GST-fusion proteins were expressed in E coli Rosetta (ADE3) (Novagen). Rosetta cells were grown in Terrific broth containing ampicillin (100 pg/ml) at 37 °C until an optical density (OD) ₆₀ o of 2.0 was reached, after which point the cells were quickly cooled on ice to 16°C. After induction with 0.1 mM isopropyl-p-D-thiogalacto-pyranoside (IPTG; Ducheba), the cells were allowed to grow for 16 hr at 16°C. The £ coli pellet containing the GST fusion protein was lysed with PBS, and the protein was purified using glutathione-Sepharose column chromatography, as previously described (Rohman, M. & Harrison-Lavoie, KJ. Protein Expr Purif 20, 45-47 (2000)).

Substrate preparation

Synthetic oligonucleotides were 5'-end labeled with ³² P using T4 polynucleotide kinase and [γ- ³² Ρ]ΑΤΡ. Duplex substrates were prepared by incubating a 5'-end labeled oligonucleotide with the nonradioactive complementary oligonucleotide in a 1:1.2 molar ratio in the presence of 10 mM Tris-HCI (pH 8.0) and 20 mM KCI. The mixture was heated at 95 °C for 5 min and then allowed to cool slowly to room temperature. For 3' labeling, an RNA oligonucleotide was incubated with [5'- ³² P]pCp and T4 RNA ligase. The 3' phosphate was removed by incubation with calf intestinal phosphatase. Following phenol/chloroform extraction, the RNA was precipitated. The sequences for all nucleic acid substrates are outlined in Table 1.

[Table 1]

Oligomer Sequence

20-bp ssRNA 5'-CUCCAUCACCCUCCAUCACC-3' (SEQ ID NO:l)

30-bp ssRNA 5'-ACCCACUGAAUCAGAUAAAGAGUUGUGUCA-3'

(SEQ ID O:2)

20-bp dsRNA 5'-CUCCAUCACCCUCCAUCACC-3' (SEQ ID NO:3)

3'-GAGGUAGUGGGAGGUAGUGG-5' (SEQ ID NO:4)

20-bp ssDNA y-CTCCATCACCCTCCATCACC-S' (SEQ ID NO:5)

20-bp dsDNA 5'-CTCCATCACCCTCCATCACC-3 (SEQ ID NO:6)

3 -GAGGTAGTGGGAGGT AGTGG-5' (SEQ ID NO:7)

20-bp RNA:DNA duplex 5'-CUCCAUCACCCUCCAUCACC-3' (SEQ ID NO:8)

3'-GAGGTAGTGGGAGGTAGTGG-5' (SEQ ID NO: 9) In vitro nuclease assay

Assays were carried out in 20 μΙ reaction mixtures containing PBS supplemented with 5 mM MgCI ₂ , 2 mM DTT, 10% glycerol, 0.01% NP-40, ³² P-labeled nucleic acid substrates, and purified recombinant proteins at 37°C for the indicated time. RNA substrates labeled at the 3'-end with [5'- ³ P]pCp were either untreated or treated with calf intestine phosphatase (CIP). Reactions were stopped with the addition of an equal volume of formamide loading buffer and then boiled. The products were separated in 15% polyacrylamide gels containing 8 M urea and buffered with 0.5x Tris-borate-EDTA (TBE) and then analyzed by autoradiography with a phosphorimager (BAS2500, Fujifilm). dGTP-triphosphohydrolase assay

The enzymatic assay based on thin layer chromatography was performed as described previously (Lahouassa, H. et a/. Nat Immunol 13, 223-228(2012)). In brief, the purified recombinant protein was incubated in 50 mM Tris-HCI (pH 8.0), 20 mM KCI, 5 mM MgCI ₂ , 0.1 pCi of the [c- ³² P]dGTP, and 200 μΜ cold dGTP for 3 h at 37°C. The reactions were stopped by heat-inactivation at 70 °C for 10 minutes. The reaction mixtures were spotted together with standards of dGMP, dGDP, and dGTP onto polyethyleneimine (PEI)-cellulose plates (Sigma-Aldrich) and subsequently separated with a mobile phase of 1.2 M LiCI. Following separation, the a- ³² P-labeled reaction products were visualized using a phosphorimager, and the migration indicators were detected by UV-C (254 nm).

Cell lines

U937 cells were cultured in RPMI 1640 supplemented with 10% fetal bovine serum

(Hyclone), 2 mM GlutaMAX-I, 100 U/ml penicillin and 100 pg/ml streptomycin (Invitrogen). 293T cells and Phoenix Ampho cells were cultured in DMEM supplemented as described for RPMI 1640. Stable U937 cells expressing SAMHD1 proteins were obtained by retroviral infection. The pMSCV-puro construct was transfected into Phoenix Ampho cells by the calcium phosphate method. Two days later the retrovirus-containing supernatant was used to transduce U937 cells by spin infection in the presence of 8 pg/ml polybrene. Transduced cells were selected in 1 g/ml puromycin. Virus production and infection

293T cells were transfected with 10 pg of HIV-1-GFP and 2 pg of HCMV-VSV-G using the calcium phosphate method. Virus-containing media was collected and filtered at 0.45 pm at 48 h after transfection. U937 cells (0.5 10 ⁶ cells/ml) were seeded in 12-well plates and differentiated for 20 h in PMA (30 ng/ml), and the viruses were added to target cells and incubated for 2 hrs. The cells were washed and then cultured for 48 h. Infected cells were analyzed by flow cytometry.

Quantitative real-time PCR

RNA isolation from HIV-l-GFP-infected cells was performed at various time points according to the manufacturer's instructions (Invitrogen). A total of 1 g RNA was reverse transcribed using random primers and real-time PCR was performed using the iCycler iQ real-time PCR detection system (BioRad) using the HIV-l-specific primers: R/U5 [forward (SEQ ID NO: 10), reverse (SEQ ID NO:ll)]; ^ [forward (SEQ ID NO: 12), reverse (SEQ ID NO: 13)]; [forward (SEQ ID NO: 14), reverse (SEQ ID NO: 15)].

RNA immunoprecipitation

HIV-1-GFP infected cells were crosslinked by 1% formaldehyde for 10 min at room temperature. Crosslinking reactions were stopped by the addition of glycine (1 M, pH 7.0) to a final concentration of 0.25 M followed by incubation at room temperature for 5 min. The cells were washed with ice-cold PBS and resuspended in RIPA buffer (50 mM Tris-HCI, pH 7.4/1% NP40/0.5% sodium deoxycholate/0.05% SDS/1 mM EDTA/150 mM NaCI) containing protease inhibitors and RNase inhibitor. The cell suspensions were sonicated and centrifuged at 13,000 rpm at 4°C for 10 min to remove insoluble material. The remaining supernatants were precleared by incubation with protein G-agarose beads. The precleared supernatants were incubated with anti-HA antibody conjugated beads at 4°C for 2 h. The beads were washed with RIPA buffer and resuspended with reversal buffer (50 mM Tris-HCI, pH 7.0/5 mM EDTA/10 mM OTT/1% SDS) followed by incubation at 70°C for 45 min to reverse the crosslinks. The immunoprecipitated RNAs were isolated according to the manufacturer's protocol (Invitrogen).

Quantification of tRNA ^Lys3 by Northern blot analysis

Approximately 1 pg of total RNA was analyzed on 15% polyacrylamide gel containing 7 M urea after ethidium bromide staining. For Northern blot analysis, an oligomer probe specific for tRNA ^Lys3 (SEQ ID NO: 16) was used, which is complementary to 18 nucleotide sequences at the 3' end of the tRNA ^Lys3 . Briefly, the gel was electroblotted onto a positively charged nylon membrane at 20V for 2 hrs. Following cross-linking RNA to nylon membrane by using EDC (l-ethyl-3-(3-dimethylaminopropyl)carbodiimide), the membrane was baked in an oven at 80°C overnight. Pre-hybridization and hybridization were subsequently carried out in the presence of 32P-5'-labelled nucleotide probes at 42°C for 24 hrs. Following hybridization, the membrane was washed five times in 2x SSC (three times in 2x SSC containing 0.05% SDS and twice in 2x SSC containing 0.1% SDS) before autoradiography.

Experimental Results

SAMHD1 is a single-stranded RNA-specific 3'-5' exoribonuclease

We explored the potential role of SAMHDl's nuclease activity in HIV-I restriction. The N-terminal GST-tagged full-length human SAMHD1 protein (GST-SAMHD1) was purified from £ coli and incubated with various types of 20-mer nucleic acid substrates. The substrates were labeled at 5'-end with ³² P and nuclease activity directed against nucleic acid substrates was assessed using gel electrophoresis. As shown in Fig. la, wild-type (WT) SAMHD1 specifically hydrolyzed single-stranded RNA (ssRNA), yielding a 5-8-mer as the major product. No nuclease activity was detected for double-stranded RNA (dsRNA), single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), or RNA-DNA hybrid substrates (sequence information is provided in Table 1). This result indicates that SAMHD1 acts as an ssRNA-specific ribonuclease. To characterize the ribonuclease function of SAMHD1 more precisely, SAMHD1 was incubated for different periods of time with the same RNA labeled at its 5'-end with P. This incubation resulted in the loss of the substrate and in the accumulation of 5- to 8-mer oligonucleotide products with a direct substrate/product relationship. No fragments of intermediate length were observed (Fig. lb). This is a distinct feature of a processive rather than a distributive mode of hydrolysis. The HD domains possess putative nucleotidase and phophodiesterase activities, and the highly conserved His and Asp residues are critical for catalytic activity Nature 480, 379-382 (2011)). We investigate whether these catalytic residues are required for ribonuclease activity of SAMHD1. An in vitro ribonuclease assay showed that the mutations at amino acids 207 and 311 in the catalytic site abolished the ribonuclease activity of the protein, indicating that these residues have the critical roles in the dNTPase and ribonuclease activities of SAMHD1 (Fig. lc).

Next, to determine the direction , of the degradation of ssRNA, SAMHD1 was incubated with another 30-mer ssRNA labeled with 32P at its 3'-end instead of its 5'-end. This incubation led to the loss of the signal, with no intermediate, and the signal of one nucleotide size was increased (Fig. 2a, right panel). As a control, the catalytically inactive SAMHD1 _D 207N mutant failed to degrade the substrate (Fig. 2a, left panel). By contrast, the substrate with a phosphate group at its 3'-end was not degraded (Fig. 2b). These results show that the SAMHDl-mediated degradation of ssRNA occurs in the 3' to 5' direction and displays a strong preference for a 3'-end containing a free hydroxy! group. The SAMHDl-mediated degradation of ssRNA is unlikely to be sequence specific based on the observation that both 20- and 30-mer "random" sequence ssRNAs were effectively targeted by SAMHD1. Taken together, our data demonstrate that SAMHD1 is a single-stranded RNA-specific exoribonuclease that processively digests RNA in the 3' to 5' direction. Ribonuclease and dNTPase are dual and separate functions ofSAMHDl

The results of this and previous works {Nature 480, 379-382 (2011); J Biol Chem 286, 43596-43600 (2011)) suggest that ribonuclease activity and dNTPase activity are dual catalytic functions of SAMHD1. The dNTPase function of SAMHD1 is allosterically activated by dGTP. Mutations of Asp 137 and Arg 145 in the allosteric site or Asp 207 and Asp 311 in the catalytic site in the HD domain abolish dNTPase activity entirelyl2. Because, unlike the dNTPase activity, the ribonuciease activity did not require the cofactor dGTP (Fig. 1), we proposed that these two catalytic functions of SAMHDl might be distinguishable. To test this possibility, mutant with an Asn substitution in the allosteric site of SAMHDl was constructed. GST-SAMHDl _D i37N protein was produced in E coli and purified by affinity chromatography. For unknown reasons, the production of another allosteric mutant substituted at Arg 145 failed despite numerous trials. The analysis of the dNTPase activity using dGTP as a substrate revealed that the mutations at amino acids 207, 311 and 137 abolished the dNTPase activity of SAMHDl (Fig. 3a), consistent with the results of a previous studyl2. Most interestingly, the mutation of amino acid 137 in the allosteric site did not abolish the ribonuciease activity (Fig. 3b). In contrast to the dNTPase activity, the ribonuciease activity of SAMHDl appears not to involve the dGTP-mediated allosteric activation mechanism. These findings indicate that the ribonuciease activity of SAMHDl is distinguishable from its dNTPase activity.

The ribonuciease function of SAMHDl is critical for HIV-1 restriction

The identification of a SAMHDl _D i3 _7N mutant that exhibits ribonuciease but no dNTPase activity enabled us to determine the discrete contribution of the ribonuciease activity of SAMHDl to HIV-1 restriction. For this analysis, we stably expressed SAMHDlwr, SAMHD1 _d207 N, SAMHDl _D 3iiA or SAMHDl _D i _37N in U937 monocytic cells that do not express endogenous SAMHDl. After treatment of these cells with phorbol myristate acetate (PMA), they were infected with VSV-G pseudotyped HIV-1-GFP (hereafter HIV-1-GFP). The wild-type and mutant proteins were expressed at comparable levels in the HIV-l-GFP-infected cells (Fig. 4a). The SAMHDIWT effectively inhibited HIV-I infection, whereas the catalytically inactive mutants (SAMHD1 _d207 N and SAMHD1 _D 3UA) were essentially inactive for the restriction of HIV-1 infection (Fig. 4b and 4c), and the results for the cells expressing these mutants were indistinguishable from the result for the mock-transfected control cells. Intriguingly, the SAMHD1 _D137N allosteric mutant, which is devoid of dNTPase activity, still retained potent anti-HIV-1 restriction activity (79.7% of the SAMHDI _WT activity) (Fig. 4c). This result is in contrast to the complete loss of the restriction activity exhibited upon mutation of the two catalytic residues, D207 and D311. The SAMHDl _D i _37N displayed a modest but consistent 20.3% reduction relative to the SAMHDIW _T in its ability to restrict HIV-1-GFP (Fig. 4c), across a broad range of HIV-1-GFP doses. The lack of dNTPase activity may account for this 20.3% reduction in the anti-HIV-1 activity observed for the SAMHD1 _D137N . Thus, we conclude, under our experimental conditions, that the ribonuclease function of SAMHDl plays a major role in HIV-1 restriction. Quantification of viral cDNA intermediates by RT-PCR using the primers specific to the stages of HIV-1 reverse transcription showed that SAMHDI _D B™ inhibited the synthesis of both the early and late viral cDNA products to a comparable extent as SAMHDIWT (Fig- 4d), indicating that the ribonuclease function of SAMHDl operates at the early step of HIV-1 reverse transcription. SAMHDl associates with and induces destabilization of the HIV-1 genomic RNA

We investigated the mechanism by which SAMHDl exerts its ribonuclease activity to inhibit HIV-1 replication. The experimental design is outlined in Figure 5a. HIV-1 reverse transcription involves two RNA components, tRNA ^Lys3 as a primer and HIV-1 genomic RNA as a template for the synthesis of cDNA. Total cellular RNA was isolated from U937 cells infected with HIV-1-GFP at the early time points post-infection. Analysis by Northern blot revealed that SAMHDl had no effect on the level of tRNA ^Lys3 (Fig. 5b, upper panel). In contrast, the quantification of viral RNA content by quantitative RT-PCR (qRT-PCR) using the HIV-1 gag-specific primer showed that at 3 and 6 hrs after infection, HIV-1-GFP genomic RNA was significantly reduced only in cells expressing SAMHDIWT or SAMHD1 _D I3 ₇ N (Fig- 5c). An analysis using the primers specific for different regions of HIV-1 genome showed essentially the same results. In particular, the SAMHD1 _D137N allosteric mutant was able to reduce HIV-1 genomic RNA to levels almost comparable to the SAMHDlwrv which correlates with the results obtained by FACS analysis (Fig. 4). Expression of SAMHD1 _D 207N or SAMHD1D3UA did not reduce HIV-1 genomic RNA compared with mock control. At 1 hr after infection, both SAMHDIWT and SAMHDl _D i _37N had no impact on the viral RNA content (Fig. 5c), presumably due to the inaccessibility of SAMHDl to the HIV-1 genomic RNA in the cores at this early time post-infection. To provide further evidence that SAMHDl directly targets HIV-1 genomic RNA for degradation, we assessed whether SAMHDl associated with HIV-1 genomic RNA during infection. We infected U937 cells expressing SAMHDlwr-HA, SAMHD1 _D207 N-HA or vector alone with HIV-1-GFP for 90 min. The whole cell extracts were immunoprecipitated using anti-HA antibody. After RNA purification from the precipitates, qRT-PCR was performed to quantitate HIV-1 RNA. There was significant enrichment of HIV-1 RNA in cells expressing SAMHDlwr-HA. The HIV-1 RNA enrichment was more pronounced for the SAMHD1 _D207N -HA catalytic mutant with a 6- to 8-fold increase (Fig. 5d). Essentially the same results were obtained when quantification of the results was performed using the different primers (R/U5 and egfp), indicating that SAMHDl associates with HIV-1 genomic RNA. Given the localization of substantial amounts of SAMHDl to the cytoplasm, our data indicate that SAMHDl binds and directly degrades HIV-1 genomic RNA in the cytoplasm where HIV-1 reverse transcription occurs.

Discussion

Our results demonstrate that SAMHDl possesses a long-sought ribonuclease activity. Although the in vitro enzymatic analysis showed that SAMHDl specifically degrades ssRNA, it remains elusive how SAMHDl can recognize and degrade the secondary structured HIV-1 genomic RNA with distinct hairpin motifs in vivo. The putative helicase may be able to disrupt the secondary structure of the viral RNA for SAMHDl ribonuclease function. Our findings, along with the previous findings for the dNTPase function of SAMHDl, indicate that SAMHDl functions both as a ribonuclease and as a dNTPase. Interestingly, these two enzyme activities are separable as judged by the observations that the mutation of amino acid in the allosteric site abolishes the dNTPase but not the ribonuclease activity, and that the ribonuclease activity does not require a cofactor dGTP that is essential for the dNTPase activity. An intriguing question is why SAMHDl evolves to have dual catalytic functions of SAMHDl in a single enzyme towards restriction of HIV-1. HIV-1 can still replicate in non-cycling cells with low levels of dNTPs, such as macrophages and dendritic cells, albeit slowly and inefficiently (J Virol 68, 1258-1263 (1994); Proc Natl Acad Sci U S A 90, 8925-8928 (1993)), most likely due to the ability of HIV-l's reverse transcriptase to bind dNTPs with high affinity (J Biol Chem 279, 51545-51553 (2004)). It was also known that reverse transcriptase of HIV-1 can use rNTP as substrate instead of dNTP in macrophage (J Biol Chem 285, 39380-39391). These studies suggest that it might not be sufficient to inhibit HIV-1 replication by the dNTPase activity alone of SAMHDl. Given the key roles of macrophages and dendritic cells as virus reservoirs in HIV-1 infection, the slow HIV-1 replication may be of physiological importance. Slowly replicating HIV-1 could sneak past immune surveillance and may be sufficient to allow long-term transmission to CD4+ T cells {Science 257, 383-387 (1992)). Effective clearance of HIV-1 by SAMHDl may require the combined action of the ribonuclease and dNTPase activities.

Based on our data and the links between the accumulation of nucleic acids and the inappropriate triggering of innate immune responses, the natural function of SAMHDl is likely to clean up dysfunctional cellular RNAs or RNA species derived from endogenous retroelement, thus preventing an unwanted inflammatory response. Human endogenous retroviruses make up nearly 8% of the human genome and have been implicated in some autoimmune diseases. In line with the specific recognition of HIV-1 RNA by SAMHDl (Fig. 5) and the ability of SAMHDl to restrict a wide range of retroviruses, the byproducts of human endogenous retroviruses are potentially major targets of SAMHDl. In addition, this mechanism may also explain the molecular basis of AGS and related lupus-like diseases in patients with loss-of-function mutations in SAMHDl. The failure to degrade endogenous RNA debris is likely a principle pathway causing the aberrant activation of innate immune responses in individuals with SAMHDl-mediated autoimmune diseases (Fig. 5e). The unique property of SAMHDl that affects the balance between HIV-1 infection and innate immune response could be exploited to develop a new class of intervention for retroviruses and autoimmune diseases associated with nucleic acid metabolism.

Having described a preferred embodiment of the present invention, it is to be understood that variants and modifications thereof falling within the spirit of the invention may become apparent to those skilled in this art, and the scope of this invention is to be determined by appended claims and their equivalents.