WO2008076370A2 - P21 compositions and methods for the treatment of hiv - Google Patents

Abstract

The invention features methods and compositions for preventing or treating a viral infection, such as an HIV-1 infection, in a cell by increasing the expression or biological activity of a p21 polypeptide or nucleic acid molecule, and screening assays for the identification of such agents.

Description

P21 COMPOSITIONS AND METHODS FOR THE TREATMENT OF HIV

RELATED APPLICATION

This application claims priority to U.S. provisional application Ser. No. 60/875,446, filed December 18, 2006, the entire disclosure of which is incorporated herein by this reference. STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY

SPONSORED RESEARCH

This work was supported by the following grants from the National Institutes of Health, Grant Nos: AI55313, HL044851, and HL71859. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION AIDS is a chronic, life-threatening condition caused by the human immunodeficiency virus (HIV). By damaging or destroying the cells of the immune system, HIV interferes with the body's ability to effectively fight off viruses, bacteria and fungi that cause disease. This increases infected individuals susceptibility to certain types of cancers and to opportunistic infections. The virus and the infection itself are known as HIV. The term AIDS (acquired immunodeficiency syndrome) is used to mean the later stages of HIV infection, hi the more than two decades since the first reports of the disease, AIDS has become a global epidemic. Worldwide, an estimated 38 million people are living with HIV. An estimated 950,000 Americans are currently living with HIV/AIDS, up from 900,000 in 2001. None of the currently available anti-viral therapeutics is capable of curing or preventing HIV-I or AIDS. In view of the burgeoning HIV-I epidemic, improved therapeutic methods and compositions are urgently required.

SUMMARY OF THE INVENTION

As described below, the present invention features methods and compositions for preventing or treating an HIV-I infection in a cell, and related screening methods for the identification of prophlactic and therapeutic agents.

In one aspect, the invention generally features a method for preventing or reducing a viral infection in a cell (e.g., a human cell in vitro or in vivo), the method comprising contacting a cell with an effective amount of an agent that increases the expression or biological activity of a p21 nucleic acid molecule or polypeptide, thereby preventing or reducing the viral infection (e.g., HIV-I infection). In one embodiment, the agent is any one or more of Beta-escin, (-)-Epigallocatechin-3 gallate, Iressa, 6- anilino-5,8-quinolinequinone, mevastatin, Oncostatin M, 7 hydroxyl-staurosporine, 1,25-Dihydroxy vitamin D3, Pentagalloglucose, indole-3-carbenol, 3,5-diaryl- oxadiazoles, high molecular weight fractions of green or black tea, casuarinin, 5- azacytidine, aresenic trioxide, Notch ligand, WAFl, synthetic peptide KRRQTSMTDFYHSKRRLIFS, and retinoic acid, or analogs, or derivatives thereof. In other embodiments of the above aspects, the cell is a descendant of a hematopoietic stem cell, a myeloid or a lymphoid cell. hi another aspect, the invention generally features a method for preventing or reducing HIV-I infection in a cell (e.g., a human cell in vivo), the method comprising contacting the cell with an effective amount of retinoic acid, or an analog or derivative thereof, wherein the contacting increases p21 nucleic acid molecule or polypeptide expression, thereby preventing or reducing HIV-I infection. hi yet another aspect, the invention features a method for preventing HIV-I viral infection in a cell, the method comprising contacting a cell with an effective amount of an agent that increases p21 biological activity, thereby preventing HIV-I viral infection in the cell. hi yet another aspect, the invention provides a method for preventing or reducing a viral infection in a cell, the method comprising the steps of contacting the cell with an expression vector comprising a p21 nucleic acid molecule operably linked to a promoter positioned for expression in the cell; and expressing a p21 polypeptide in the cell, thereby preventing or reducing a viral infection in the cell. hi various embodiments of the above aspects, the agent reduces viral integration or viral replication, hi other embodiments of the above aspects, the cell is in vitro, in vivo or ex vivo. In still other embodiments, the method further comprises the step of obtaining the agent, hi still other embodiment, the viral infection is an HIV-I infection or AIDS. In another embodiment, the cell is a descendant of a hematopoietic stem cell, or is a myeloid or lymphoid cell. In another embodiments of the above aspects, the agent reduces viral integration or viral replication, hi yet other embodiments of the above aspects, the method further comprises the step of obtaining the agent. In yet another aspect, the invention provides a method for preventing or reducing an HIV-I infection in a subject in need thereof, the method comprising administering to the subject an effective amount of an agent that increases p21 nucleic acid molecule or polypeptide expression, thereby preventing or reducing HIV-I infection in the subject. hi yet another aspect, the invention provides a method for preventing or reducing

HIV-I infection in a subject in need thereof, the method comprising administering to the subject an effective amount of an agent that increases p21 biological activity, thereby preventing or reducing HIV-I infection in the subject.

In yet another aspect, the invention provides a method for preventing or reducing an HIV-I infection in a subject in need thereof, the method comprising the steps of contacting a cell of the subject with an expression vector comprising a p21 nucleic acid molecule or fragment thereof operably linked to a promoter suitable for expression in the cell; and expressing a p21 polypeptide in the cell, thereby preventing or reducing HIV-I infection in the subject. hi various embodiments of the above aspects, the method ameliorates an HIV-I infection or ATDS in the subject. In still other embodiments of the above aspects, the agent is any one or more of Beta-escin, (-)-Epigallocatechin-3 gallate, Iressa, 6-anilino- 5,8-quinolinequinone, mevastatin, Oncostatin M, 7 hydroxyl-staurosporine, 1,25- Dihydroxy vitamin D3, Pentagalloglucose, indole-3-carbenol, 3,5-diaryl-oxadiazoles, high molecular weight fractions of green or black tea, casuarinin, 5-azacytidine, aresenic trioxide, Notch ligand, WAFl, synthetic peptide KRRQTSMTDFYHSKRRLIFS, and retinoic acid, or analogs or derivatives thereof, hi yet other embodiments of the above aspects, the agent reduces viral integration or viral replication. In still other embodiments of the above aspects, the subject is a human patient, such as a human patient diagnosed as having HIV-I or AIDS. In still other embodiments, the method ameliorates HIV-I or AIDS.

In yet another aspect, the invention features a pharmaceutical composition for the treatment or prevention of a viral infection (e.g., HIV-I or AIDS), the composition comprising an effective amount of an expression vector comprising a p21 nucleic acid molecule or fragment thereof operably linked to a promoter suitable for expression in a hematopoietic stem cell, cell of the myeloid or lymphoid lineages, or dendritic cell or fragment thereof, and a pharmacologically acceptable excipient. In yet another aspect, the invention features a pharmaceutical composition for the treatment of HIV-I or AIDS, the composition comprising an effective amount of any one or more of Beta-escin, (-)-Epigallocatechin-3 gallate, Iressa, 6-anilino-5,8- quinolinequinone, mevastatin, Oncostatin M, 7 hydroxyl-staurosporine, 1,25-Dihydroxy vitamin D3, Pentagalloglucose, indole-3-carbenol, 3,5-diaryl-oxadiazoles, high molecular weight fractions of green or black tea, casuarinin, 5-azacytidine, aresenic trioxide, Notch ligand, WAFl, synthetic peptide KRRQTSMTDFYHSKRRLIFS, and retinoic acid, or analogs or derivatives thereof and a pharmaceutical excipient.

In yet another aspect, the invention features a pharmaceutical composition for the treatment of HIV- 1 or AIDS , the composition comprising an effective amount of a p21 polypeptide, p21 inducer, or p21 mimetic, in a pharmaceutical excipient.

In yet another aspect, the invention features a method for identifying an agent that prevents or reduces a viral infection (e.g., HIV-I) in a cell, the method comprising the steps of contacting a cell expressing a p21 nucleic acid molecule or polypeptide with a candidate agent; and comparing the level of p21 biological activity in the cell with the level present in an untreated control cell, wherein an increase in p21 biological activity in the contacted cell thereby identifies the candidate agent as preventing or reducing a viral infection in a cell. In one embodiment, p21 biological activity is assayed by detecting a reduction in the level of HIV-I infection in the cell. In one embodiment, the level of HIV-I infection is measured by measuring HIV-I replication in the cell or by measuring HIV-I integration into the genome of the cell.

In yet another aspect, the invention provides a method for identifying an agent that prevents or reduces a viral infection in a cell, the method comprising the steps of contacting a cell expressing a p21 nucleic acid molecule with an agent; and comparing the level of p21 expression in the contacted cell relative to an untreated control cell, wherein an increase in p21 expression thereby identifies the agent as preventing or reducing a viral infection in the cell. In another embodiment, the agent that increases p21 transcription or increases translation of an mRNA transcribed from a p21 nucleic acid molecule. In yet another aspect, the invention provides a method for identifying an agent that prevents or reduces a viral infection in a cell, the method comprising the steps of contacting a cell expressing a p21 polypeptide with a candidate agent; and comparing the level of p21 polypeptide in the contacted cell with the level in an untreated control cell, wherein an increase in the level of p21 polypeptide in the contacted cell relative to an untreated control cell thereby identifies the agent as preventing or reducing a viral infection in a cell.

In yet another aspect, the invention provides a method for identifying an agent that prevents or reduces an HIV-I infection in a cell, the method comprising the steps of contacting a cell expressing a p21 promoter operably linked to a detectable reporter with a candidate agent; and comparing the level of reporter expression in the contacted cell relative to an untreated control cell, wherein an increase in reporter expression in the contacted cell thereby identifies the candidate agent as preventing or reducing an HIV-I infection.

In various embodiments of the above aspects, the agent enhances the resistance of the cell to HIV-I infection. In other embodiments of the above aspects, the agent reduces HIV-I replication or integration in the cell. In still other embodiments, the cell is a CD34 positive cell, is descended from a hematopoietic stem cell precursor, is a cell of the myeloid or lymphoid lineages or a dendritic cell.

In yet another aspect, the invention provides a packaged pharmaceutical comprising an effective amount of a p21 nucleic acid molecule or polypeptide, p21 inducer, or p21 mimetic; and instructions for using the p21 nucleic acid molecule or polypeptide, inducer, or mimetic to ameliorate HIV-I or AIDS. In yet another aspect, the invention provides a kit for ameliorating HIV-I or

AIDS in a subject comprising a p21 polypeptide, p21 inducer, or p21 mimetic, and instructions for use thereof.

Other features and advantages of the invention will be apparent from the detailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

Figures 1 A-IC show that p21 expression is inhibited by p21 siRNA. Figure IA is a graph showing the inhibition of p21 mRNA expression by RNAi. p21 mRNA levels were decreased after transfection of chemically synthesized siRNA (Si-I, -2) and by cell-expressed shRNA (p2.3, 3.2) specific for p21. The decrease of p21 mRNA did not occur in control cells treated with siRNA specific for HPRT, the vector only (pU/B), or in mock treated cells (or a mutated form of Si-2 shown in ref.15). Figure IB is a graph that shows the standard curve of real time quantitative RT-PCR assay for analyzing cellular mRNA levels. p21 mRNA was detected with a Taqman real time RT-PCR assay. This RT-PCR assay is able to detect as few as 10 copy numbers of p21 mRNA in 50 ng of total cellular RNA. Error bars are not visible when standard deviation (SD) is < 2.0%. RNA copy number in 50 ng of total cellular RNA was detected by a Taqman real time RT-PCR assay performed in quadruplicate. Figure 1C is a Western blot showing that p21 expression is inhibited by p21 siRNA. The same cell aliquots used in Figure IA was examined for p21 protein expression after RNAi treatment. The lanes are designated as follows: lane M shows p21 protein detected in cells mock treated with siRNA; the pU6/B lane shows p21 protein in cells treated with vector only; the p2.3 lane shows p21 protein in cells treated with p21 shRNA; the H lane shows p21 protein in cells treated with HPRT siRNA; the Si-2 lane in cells treated with p21 siRNA; the N lane shows p21 levels in a nuclear extract, which served as a positive control. Results are representative of three independent experiments.

Figures 2A-2D are graphs showing HIV-I replication levels in p21 siRNA- treated cells. Figure 2 A shows HIV-I replication in p21 siRNA-treated CD34⁺ primary cells as measured using the HIV-I p24 antigen. HIV-I p24 levels were detected at day 4, 7, 10, and 14 post-HIV-1 infection. Cells were treated with chemically synthesized p21 siRNA (Si-I, -2), or control siRNA (HPRT), or mock treated (M). Culture supernatants collected at each time point were analyzed by HIV-I p24 antigen assay. HIV-I growth kinetics in mock-treated cells (M) were indistinguishable from HPRT siRNA treated cells. Each point represented the p24 level from two separate experiments run in duplicate (SD ± 0.2-4.7). Figure 2B shows HIV-I replication in p21 siRNA treated CMK cells. HIV-I p24 levels were detected at day 4, 7, 10, and 14 post viral infection. Cells were treated with chemically synthesized p21 siRNA (Si-2), control siRNA (HPRT), p21shRNA (p2.3), vector without shRNA (pU/B), or mock treated (Mock). Each point represented the p24 level from two separate experiments run in duplicate. Figures 2C and 2D show that TPA-induced p21 expression inhibited HIV- 1 replication in CMK cells. Cells were treated with TPA for one day (Tl), two days (T2), three days (T3), or mock treated (M) before HIV-I infection. Figure 2C shows that p21 mRNA levels were examined at day 12 compared to mock treated control (M). Figure 2D shows that HIV-I p24 levels determined at day 1, 7, 10 and 12. Each bar represented the p24 level from two separate experiments run in duplicate (SD ± 0.3-2.6). Figures 3A-3C are graphs showing the detection of HIV-I in 2-LTR circle and provirus levels in CD34⁺ primary cells using quantitative PCR. Figure 3A shows that HIV-I 2-LTR circles were detected at 16 hours post-infection. HIV-I integration was increased significantly in p21 siRNA treated cells (Si-2) in contrast to control siRNA (HPRT) or mock treated cells (M). Each bar of the graph represents the mean from four individual tests with each sample run in triplicate. Each sample was tested using an equal amount of total cellular DNA from 6 x 10³ CD34⁺ primary cells. Figure 3B shows the standard curve of real time quantitative AIu-LTR PCR assay for detecting HIV-I integrated DNA in the cellular genome. Figure 3 C is a graph showing that HIV-I integrated DNA was examined by AIu-LTR PCR in cells treated with p21 siRNA or control siRNA. This was a two-step strategy, with the first set of primers amplifying the HIV-I 3'-LTR to the nearest AIu element within 1.8 kb and the second set of primers and probe further amplifying the 3'-LTR to the nearest AIu element within 250-bp (Butler et al., Nat Med. 7: 631-4, (2001); O'Doherty et al., J. Virol. 76: 10942-50, (2002); Zhang et al., Gene Therapy 12: 1444-52, (2002)). The copy number of provirus was calculated according to the standard curve and adjusted by two reference curves that have known numbers of provirus per cell (Butler et al., Nat Med.l: 631-4, (2001); O'Doherty et al., J. Virol. 76: 10942-50, (2002); Zhang et al., Gene Therapy 12: 1444- 52, (2002)). Figures 4A, 4B, and 4C are Western blots showing the detection of p21 polypeptide in HIV-I preintegration complexes (PICs). CMK (restrictive) and H9 (permissive) cells were infected with HIV-IIHB and cytoplasmic PICs isolated at seven hours post infection. Viral matrix protein is a component of PIC (Bukrinsky et al., Nature 365: 666-9, (1993); Von Schwedler et al., Proc. Acad. Natl. Sci. USA 91: 6992-6, (1994)). Figure 4A shows p21 polypeptide present in crude cytoplasmic PICs that were isolated from CMK cells. Antibodies against p21 or HIV-I matrix (MA)(Lin et al., 2003) recovered similar levels of integrase (IN) (lanes 1, 2). Purified integrase served as a marker (lane 3). Figure 4B shows Western blot analyses following PIC purification by spin column chromatography. Antibody against p21 co-immunoprecipitated p21 with integrase (IN) in CMK cells (lane 4). Antibody against p21 failed to recover integrase when CMK cells were treated with p21 siRNA (lane 6), but not when cells were treated with control siRNA (lane 7). Isotype controls IgG3k (for HIV-I matrix) and IgGl (for p21) failed to recover integrase (lanes 2, 8). Purified integrase served as a marker (lane 1). Figure 4C shows that anti-HIV-1 matrix antibodies coimmunoprecipitated p21 in preintegration complexes (PICs) made from CMK cells (lane 1), but not from H9 cells (lane 2). p21 -containing nuclear extract served as a marker (lane 5).

Figures 5A-5D show cell cycle analyses of CD34⁺ primary cells. Figures 5 A and 5B show flow cytometric estimation of the cell cycle status of CD34⁺ primary cells (Figure 5A) and CMK cells (Figure 5B) at 42 hours post siRNA treatment. Figure 5A shows that CD34⁺ cells treated with p21 siRNA and control siRNAs were examined by Hoechst (DNA dye) and Pyronin (RNA dye) staining. Cell cycle status was examined at both 10 hours and 42 hours post-siRNA treatment. No significant change in the cell cycle status was observed in CD34⁺ primary cells that were treated with p21 siRNA, or control siRNA (HPRT), or mock treated at these time points. Figure 5B shows the cell cycle status of CMK cells at 42 hours post siRNA treatment. No significant change was observed in the cell cycle status in megakaryoblasts treated with p21 siRNA (Si-I, -2), control siRNA (HPRT), p21 shRNA (ρ2.3), vector only (pU6/B), or mock treated. Figures 5C and 5D show BrdU incorporation in CD34⁺ cells. Figure 5C shows the cell cycle status of CD34⁺ primary cells that were examined at both 10 hours and 42 hours post-siRNA treatment. No significant change was observed between cells treated with p21 siRNA (Si-2) and cells treated with control siRNA (HPRT). Cells incorporating BrdU (UL + UR) consisted of 23.75% and 23.72% in p21 siRNA or control siRNA treated cells, respectively. Figure 5D is a graph showing that levels of BrdU incorporation remained constant in cells treated with p21 siRNA, HPRT, or mock treated. These results show the mean results of four independent assays.

Figures 6 A and 6B are graphs showing that RNAi silencing of pi 8 or p27 did not increase HIV-I replication. Figure 6 A shows a quantitation of the effect of siRNA specific for pi 8 or p27 in CD34⁺ cells on pi 8 and p27 expression. Expression levels were analysed using a real time Taqman RT-PCR assay. CT values from cells treated with siRNA-specific for pi 8 or p27 were compared to levels present in cells treated with control siRNA (HPRT). Samples run in quintuplicate were normalized to endogenous GAPDH levels. Figure 6B shows HIV-I replication in CD34⁺ cells treated with siRNA specific for pi 8 or p27. HIV-I replication levels were determined at days 4, 7, 10, and 14 post HIV- 1 infection (shown at day 4 and 14) using p24 antigen assays. No active replication was detected in cells treated with pi 8, p27, or control (HPRT) siRNA in contrast to cells treated with p21 siRNA (Si-2) (SD ± 0.22-2.1). Figures 7A and 7B are graphs showing SIV replication levels in cells treated with p21 siRNA or under control conditions. SIVmac-251 infection of CD34⁺ cells and CD34^~ cells. Figure 7 A shows that silencing of p21 expression in CD34⁺ cells did not induce SIVmac-251 replication. SIV antigen p27 was detected in CD34⁺ cells treated with p21 siRNA (Si-2), control siRNA (HPRT), or mock treated (SIV infection only). Figure 7B shows that SIVmac-251 replicated in CD34^~ cells of the same donor. SIV antigen p27 was detected in CD34^~ cells infected with virus (SIV), but not in mock infected CD34^~ cells (M-). Assays were run in duplicate (SD +/- 0.01-0.66).

Figure 8 is the amino acid sequence of p21.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

By "agent " is meant any small molecule chemical compound, antibody, nucleic acid molecule, or polypeptide, or fragments thereof. By "AIDS" is meant an acquired human immunodeficiency disorder or symptom thereof.

By "alteration" is meant a change (increase or decrease) in the expression levels of a gene or polypeptide as detected by standard art known methods such as those described above. As used herein, an alteration includes a 10% change in expression levels, preferably a 25% change, more preferably a 40% change, and even more preferably a 50% or greater change in expression levels.

By "ameliorate" is meant decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease.

In this disclosure, "comprises," "comprising," "containing" and "having" and the like can have the meaning ascribed to them in U.S. Patent law and can mean " includes," "including," and the like; "consisting essentially of or "consists essentially" likewise has the meaning ascribed in U.S. Patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments.

By "detectable" is meant a composition that when linked to a molecule of interest renders the latter detectable, via spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful labels include radioactive isotopes, magnetic beads, metallic beads, colloidal particles, fluorescent dyes, electron- dense reagents, enzymes (for example, as commonly used in an ELISA), biotin, digoxigenin, or haptens.

By "an effective amount" is meant the amount of an agent needed to ameliorate the symptoms of a disease relative to an untreated patient. The effective amount of active agent(s) used to practice the present invention for therapeutic treatment of HIV-I or AIDS varies depending upon the manner of administration, the age, body weight, and general health of the subject. Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen. Such amount is referred to as an "effective" amount.

An "expression vector" is a nucleic acid construct, generated recombinantly or synthetically, bearing a series of specified nucleic acid elements that enable transcription of a particular gene in a host cell. Typically, gene expression is placed under the control of certain regulatory elements, including constitutive or inducible promoters, tissue- preferred regulatory elements, and enhancers.

By "fragment" is meant a portion of a polypeptide or nucleic acid molecule. This portion contains, preferably, at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference nucleic acid molecule or polypeptide. A fragment may contain about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids. Desirably, a fragment of a polypeptide or nucleic acid molecule is of a length sufficient to carry out at least one biological activity of the polypeptide or nucleic acid molecule from which it is derived. By "isolated nucleic acid molecule" is meant a nucleic acid (e.g., a DNA) that is free of the genes, which, in the naturally occurring genome of the organism from which the nucleic acid molecule of the invention is derived, flank the gene. The term therefore includes, for example, a recombinant DNA that is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or that exists as a separate molecule (for example, a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. In addition, the term includes an RNA molecule, which is transcribed from a DNA molecule, as well as a recombinant DNA, which is part of a hybrid gene encoding additional polypeptide sequence.

By "reporter gene" is meant a gene encoding a polypeptide whose expression may be assayed; such polypeptides include, without limitation, glucuronidase (GUS), luciferase, chloramphenicol transacetylase (CAT), and beta-galactosidase.

By "HIV-I infection" is meant the viral transformation of at least one cell of a host with a human immunodeficiency virus as characterized by viral replication or integration.

By "obtaining" as in "obtaining the agent" is meant synthesizing, purchasing, or otherwise acquiring the agent.

By "operably linked" is meant that a first polynucleotide is positioned adjacent to a second polynucleotide that directs transcription of the first polynucleotide when appropriate molecules (e.g., transcriptional activator proteins) are bound to the second polynucleotide.

By "reduces" or "increases" is meant a negative or positive change, respectively, of at least about 5%, 10%, 25%, 50%, 75%, 80%, 90%, or 100%.

By "p21 biological activity" is meant inhibition of HIV-I infection, inhibition of viral integration into a host genome, inhibition of viral replication, inhibition of cyclin- dependent kinase activity, or chromatin modifying scaffold activity or apoptosis modifying activity or cell cycle regulatory activity.

By "p21 polypeptide" is meant an amino acid sequence having at least about 85%, 90%, or 95% identity to GenBank Accession No. P38936, or a fragment thereof, and having at least one p21 biological activity.

By "p21 nucleic acid molecule" is meant a nucleic acid sequence that encodes a p21 polypeptide. Exemplary p21 nucleic acid sequences are described by Zhang et al., Gene Therapy. 12:1444-52 (2005).

By "p21 inducer" is meant an agent that increases the expression or a biological activity of a p21 nucleic acid molecule or polypeptide."

By "p21 mimetic" is meant an agent that carries out at least one biological activity of p21.

Exemplary p21 inducers and mimetics include, but are not limited to, beta-escin or aescin, epigallocatechin-3 gallate, Iressa, LY83583, mevastatin, oncostatin M, 7- hydroxyl-staurosporine, 1,25-Dihydroxyvitamin D3, pentagalloglucose, indole-3- carbenol, 3,5-diaryl-oxadiazoles, high molecular weight fractions of green or black tea, casuarinin, 5-azacytidine, aresenic trioxide, and Notch ligand, a 20 amino-acid p21 (Wafl) peptide, a truncated eight amino acid peptide of p21 (WAFl) (see, Ball KL et. al. (1997) Curr. Biol. 7(1): 71-80) a synthetic peptide KRRQTSMTDFYHSKRRLIFS, which is based on the sequence of the 141-160 amino acids from p2 IWAFl /CIPl and fused with a carrier peptide, AA V ALLP A VLLALLA (see, Goulvestre C. et. al. (2005) J. Immunol. 175: 6959-6967) By "polypeptide" is meant any chain of amino acids, regardless of length or post- translational modification.

By "positioned for expression" is meant that the polynucleotide of the invention (e.g., a DNA molecule) is positioned adjacent to a DNA sequence that directs transcription and translation of the sequence (i.e., facilitates the production of, for example, a recombinant polypeptide of the invention, or an RNA molecule). By "reference" is meant a standard or control condition. As used herein, the terms "treat," treating," "treatment," and the like refer to reducing or ameliorating a disorder and/or symptoms associated therewith. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated.

As used herein, the terms "prevent," "preventing," "prevention," "prophylactic treatment" and the like refer to reducing the probability of developing a disorder or condition in a subject, who does not have, but is at risk of or susceptible to developing a disorder or condition.

By "promoter" is meant a polynucleotide sufficient to direct transcription. By "enhances the cell's resistance to HIV-I infection" is meant decreases by at least 5-10% the susceptibility of the cell to HIV-I transformation, viral integration, or viral replication relative to a control cell. Other definitions appear in context throughout this disclosure.

Compositions and Methods of the Invention

The invention provides methods and compositions for the prevention or treatment of a viral infection (e.g., HIV-1/AIDS) infection. The invention is based, at least in part, on the discovery that p21 restricts viral DNA integration into a host cell genome and reduces HIV-I replication.

Hematopoietic stem cells (HSC) are a self-replenishing source of all blood and immune cells that are naturally resistant to HIV-I. In contrast, the differentiated progeny cells of HSCs, including cells of the myeloid or lymphoid lineages, are highly susceptible to infection by HIV-I. (Aiuti et al., Blood 94: 62-73, (1999); Lee et al., Blood 93: 1145-55, (1999); Louache et al., Blood 84: 3344-55, (1994); Shen et al., J Virol. 73: 728-37, (1999); von Laer et al., Blood 76: 1281-6, (1990); Weichold et al., Blood 91 : 907-15 (1998)). A characteristic distinguishing feature of adult stem cells is their relative cell cycle quiescence. Cyclin-dependent kinase inhibitors (CKI), p21 and pi 8 ^mK4C (pi 8) serve as G₁ checkpoint regulators and play important roles in stem cell physiology (Cheng et al., Science 287: 1804-8, (2000); Yuan et al., Nat Cell Biol. 6: 436-42, (2004)). These proteins affect the size and self-renewal capacity of the HSC pool in vivo (Cheng et al., Science 287: 1804-8, (2000); Yuan et al., Nat Cell Biol. 6: 436-42, (2004)). One of the regulatory proteins, p21 , is known to be highly expressed in stem cells, but not in stem cell descendants (Cheng et al., Science 287: 1804-8, (2000); 2001; Steinman et al., Oncogene 21: 3403-13, (2002); Stier et al., Blood 102: 1260-6, (2003)). p21 plays a role in maintaining the intrinsic cellular defense against HIV-I infection in HSC. Of note, the effect of p21 on HIV susceptibility reported herein was not due to an effect on cell cycle entry and was independent of the known mediators of HIV-I resistance, Trim5α, PML, Murrl, or IFN-α (Ganesh et al., Nature 426: 853-7, (2003); Marcello et al., EMBOJ. 22: 2156-66, (2003); Stremlau et al., Nature All: 848- 53, (2004)). Without wishing to be bound by theory, p21 's efficacy is likely associated with restricting viral DNA integration into the host cell genome. Cells that are susceptible to HIV-I infection include cells descended from a hematopoeitic stem cell, and other cells of the hematopoietic lineage, including cells within the myeloid and lymphoid lineage and dendritic cells. Myeloid stem cells typically develop into granulocytes, macrophages, megakaryocytes, and erythrocytes. Lymphoid stem cells typically develop into lymphocytes, which include T cells, B cells, and natural killer cells. p21 Polynucleotide Therapy

As reported herein, increased p21 expression reduces HIV-I integration into the genome of a cell and also reduces HIV-I replication. Accordingly, polynucleotide therapy featuring a polynucleotide encoding an p21 protein, variant, or fragment thereof is one therapeutic approach for treating or preventing viral infections (e.g., HIV-

1/AIDS). Such nucleic acid molecules can be delivered to cells of a subject having a viral infection (e.g., HIV-1/AIDS). The nucleic acid molecules should be delivered to the cells of a subject in a form in which they can be taken up so that therapeutically effective levels of an p21 protein or fragment thereof can be produced.

Transducing viral (e.g., retroviral, adenoviral, and adeno-associated viral) vectors can be used for somatic cell gene therapy, especially because of their high efficiency of infection and stable integration and expression (see, e.g., Cayouette et al., Human Gene Therapy 8:423-430, (1997) ; Kido et al., Current Eye Research 15:833-844, (1996) ; Bloomer et al., Journal of Virology 71:6641-6649, (1997); Naldini et al., Science 272:263-267, (1996); and Miyoshi et al., Proc. Natl. Acad. Sci. U.S.A. 94:10319, (1997)). For example, a polynucleotide encoding a p21 protein, variant, or a fragment thereof, can be cloned into a retroviral vector and expression can be driven from its endogenous promoter, from the retroviral long terminal repeat, or from a promoter specific for a target cell type of interest (e.g., hematopoietic stem cells, CD34⁺ primary cells, myeloid and lymphoid progenitors, and differentiated cells of the myeloid and lymphoid lineage or dendritic cells. Other viral vectors that can be used include, for example, a vaccinia virus, a bovine papilloma virus, or a herpes virus, such as Epstein- Barr Virus (also see, for example, the vectors of Miller, Human Gene Therapy 15-14, (1990); Friedman, Science 244:1275-1281, (1989); Eglitis et al., BioTechniques 6:608- 614, (1988); Tolstoshev et al., Current Opinion in Biotechnology 1:55-61, (1990); Sharp, The Lancet 337:1277-1278, (1991); Cornetta et al., Nucleic Acid Research and Molecular Biology 36:311-322, (1987); Anderson, Science 226:401-409, (1984); Moen, Blood Cells 17:407-416, (1991); Miller et al., Biotechnology 7:980-990, (1989); Le Gal La Salle et al., Science 259:988-990, (1993); and Johnson, Chest 107:77S-83S, (1995)). Retroviral vectors are particularly well developed and have been used in clinical settings (Rosenberg et al., N. Engl. J. Med 323:370, (1990); Anderson et al., U.S. Pat. No. 5,399,346). In one embodiment, a viral vector is used to administer a p21 polynucleotide to a cell susceptible to a viral infection, such as HIV-1/AIDS (e.g., cells of the myeloid or lymphoid lineages, dendritic cells).

Non- viral approaches can also be employed for the introduction of therapeutic to a cell of a patient requiring modulation of an immune response. For example, a nucleic acid molecule can be introduced into a cell by administering the nucleic acid in the presence of lipofection (Feigner et al., Proc. Natl. Acad. Sci. U.S.A. 84:7413, (1987); Ono et al., Neuroscience Letters 17:259, (1990); Brigham et al., Am. J. Med. Sci. 298:278, (1989); Staubinger et al., Methods in Enzymology 101:512, (1983)), asialoorosomucoid-polylysine conjugation (Wu et al., Journal of Biological Chemistry 263:14621, (1988)); Wu et al., Journal of Biological Chemistry 264:16985, (1989)), or by micro-injection under surgical conditions (Wolff et al., Science 247:1465, (1990)). Preferably the nucleic acids are administered in combination with a liposome and protamine.

Gene transfer can also be achieved using non-viral means involving transfection in vitro. Such methods include the use of calcium phosphate, DEAE dextran, electroporation, and protoplast fusion. Liposomes can also be potentially beneficial for delivery of DNA into a cell. Transplantation of normal genes into the affected tissues of a patient can also be accomplished by transferring a normal nucleic acid into a cultivatable cell type ex vivo (e.g., an autologous or heterologous primary cell or progeny thereof), after which the cell (or its descendants) are injected into a targeted tissue. cDNA expression for use in polynucleotide therapy methods can be directed from any suitable promoter (e.g., the human cytomegalovirus (CMV), simian virus 40 (SV40), or metallothionein promoters), and regulated by any appropriate mammalian regulatory element. For example, if desired, enhancers known to preferentially direct gene expression in specific cell types can be used to direct the expression of a nucleic acid. Promoters such as CD34, ScI, GATA-2, that are active in primitive cells or those such as PAX-5, CD19, Lck or CD14 that may be expressed in lineage committed cells. The enhancers used can include, without limitation, those that are characterized as tissue- or cell-specific enhancers. Alternatively, if a genomic clone is used as a therapeutic construct, regulation can be mediated by the cognate regulatory sequences or, if desired, by regulatory sequences derived from a heterologous source, including any of the promoters or regulatory elements described above. p21 nucleic acid molecules useful in the methods of the invention include any nucleic acid molecule that encodes a p21 polypeptide or fragement thereof. Such nucleic acid molecules need not be 100% identical with an endogenous p21 nucleic acid sequence, but will typically exhibit substantial identity. Polynucleotides having substantial identity to an endogenous sequence are typically capable of hybridizing with at least one strand of a p21 double-stranded nucleic acid molecule. By "hybridize" is meant pair to form a double-stranded molecule between complementary polynucleotide sequences (e.g., a p21 gene), or portions thereof, under various conditions of stringency. (See, e.g., Wahl, G. M. and S. L. Berger (1987) Methods Enzymol. 152:399; Kimmel, A. R. (1987) Methods Enzymol. 152:507).

For example, stringent salt concentration will ordinarily be less than about 750 mM NaCl and 75 mM trisodium citrate, preferably less than about 500 mM NaCl and 50 mM trisodium citrate, and more preferably less than about 250 mM NaCl and 25 mM trisodium citrate. Low stringency hybridization can be obtained in the absence of organic solvent, e.g., formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide, and more preferably at least about 50% formamide. Stringent temperature conditions will ordinarily include temperatures of at least about 30° C, more preferably of at least about 37° C, and most preferably of at least about 42° C. Varying additional parameters, such as hybridization time, the concentration of detergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art. Various levels of stringency are accomplished by combining these various conditions as needed. In a preferred: embodiment, hybridization will occur at 30° C C. in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. hi a more preferred embodiment, hybridization will occur at 37° C C. in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 .mu.g/ml denatured salmon sperm DNA (ssDNA). In a most preferred embodiment, hybridization will occur at 42° C C. in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 μg/ml ssDNA. Useful variations on these conditions will be readily apparent to those skilled in the art.

For most applications, washing steps that follow hybridization will also vary in stringency. Wash stringency conditions can be defined by salt concentration and by temperature. As above, wash stringency can be increased by decreasing salt concentration or by increasing temperature. For example, stringent salt concentration for the wash steps will preferably be less than about 30 mM NaCl and 3 mM trisodium citrate, and most preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate. Stringent temperature conditions for the wash steps will ordinarily include a temperature of at least about 25° C, more preferably of at least about 42° C, and even more preferably of at least about 68° C. In a preferred embodiment, wash steps will occur at 25° C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 42.degree. C. in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 68° C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additional variations on these conditions will be readily apparent to those skilled in the art. Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196:180, (1977)); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72:3961, (1975)); Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, (2001)); Berger and Kimmel (Guide to Molecular Cloning Techniques, (1987), Academic Press, New York); and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York. Another therapeutic approach included in the invention involves administration of a recombinant therapeutic, such as a recombinant p21 protein, variant, or fragment thereof, either directly to the site of a potential or actual disease-affected tissue or systemically (for example, by any conventional recombinant protein administration technique). The dosage of the administered protein depends on a number of factors, including the size and health of the individual patient. For any particular subject, the specific dosage regimes should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions. Screening Assays As reported herein, increasing expression of a p21 polypeptide inhibits HIV-I replication. Accordingly, agents that increase p21 expression or biological activity are useful for the treatment or prevention of HIV-I or AIDS. Other agents useful in the methods of the invention include those that indirectly increase p21 through the action of p53 or FOXO for example; or decrease p21 through the action of PTEN or Akt. Agents that change the stability of p21 , such as Notch ligands or other activities of ubiquitin ligases. Compositions of the invention are useful for the high-throughput low-cost screening of candidate agents that increase p21 expression or biological activity or that prevent or inhibit an a viral infection (e.g., HIV-1/AIDS) in a cell.

In one example, candidate agents are added at varying concentrations to the culture medium of cultured cells. The level of p21 polypeptide or gene expression in the contacted cell is subsequently measured. A candidate agent that increases p21 polypeptide or gene expression in a contacted cell relative to a control cell not contacted with the candidate agent is identified as useful in a method of the invention. In another example, a candidate agent is added at varying concentrations to the culture medium of cultured cells. The level of p21 polypeptide or gene expression in a contacted cell is subsequently measured. A candidate agent that increases p21 polypeptide or gene expression in a contacted cell relative to a control cell is identified as useful in a method of the invention. hi one particular embodiment, a p21 nucleic acid described herein is expressed as a transcriptional or translational fusion with a detectable reporter, and expressed in an isolated cell (e.g., mammalian or insect cell) under the control of an endogenous or a heterologous promoter. The cell expressing the fusion protein is then contacted with a candidate agent, and the expression of the detectable reporter in that cell is compared to the expression of the detectable reporter in an untreated control cell. A candidate agent that increases the expression of the detectable reporter is an agent that is useful for the treatment or prevention of an HIV-I infection. In preferred embodiments, the candidate agent increases the expression of a reporter gene fused to a p21 nucleic acid molecule. In another example, candidate agents are added at varying concentrations to a cell prior to, concurrent with, or following contact of the cell with HTV-I under conditions suitable for establishing an HIV-I infection. The level of cell infection is then measured using standard methods. The level of infection in the presence of the candidate agent is compared to the level measured in a control culture medium lacking the candidate molecule. An compound that decreases or prevents a viral infection (e.g., HIV-1/AIDS) is considered useful in the invention; such a candidate agent may be used, for example, as a therapeutic agent to prevent, delay, ameliorate, stabilize, or treat a viral infection . Methods of assaying a viral infection (e.g., HIV-1/AIDS) are known in the art and are described in the Examples, herein. In some embodiments, an compound that promotes an increase in the biological activity of a p21 polypeptide of the invention is considered useful. Such agents are identified in assays to determine their effect on p21 biological activity. For example, a candidate agent is contacted with a cell that expresses a p21 polypeptide. The effect of the agent on a p21 biological activity is then measured and compared to the level of p21 biological activity in a corresponding control cell in the absence of the candidate agent. Candidate agents that increase a p21 biological activity are identified as useful in the methods of the invention. One skilled in the art appreciates that the effects of a candidate agent on p21 expression or biological activity are typically compared to the expression or activity of p21 in the absence of the candidate agent. Thus, the screening methods include comparing the value of a cell modulated by a candidate agent to a reference value of an untreated control cell.

Expression levels can be compared by procedures well known in the art such as RT-PCR, Northern blotting, Western blotting, flow cytometry, immunocytochemistry, binding to magnetic and/or antibody-coated beads, in situ hybridization, fluorescence in situ hybridization (FISH), flow chamber adhesion assay, and ELISA, microarray analysis, or colorimetric assays, such as the Bradford Assay and Lowry Assay.

In yet another working example, candidate agents are screened for those that specifically bind to a p21 polypeptide expressed by a cell susceptible to HIV-I infection. The efficacy of such a candidate agent is dependent upon its ability to interact with the p21 polypeptide, or with functional equivalents thereof. Such an interaction can be readily assayed using any number of standard binding techniques and functional assays (e.g., those described in Ausubel et al., supra), hi one embodiment, an agent that binds a p21 polypeptide is assayed in a cell in vitro that expresses a p21 polypeptide. Desirably, the candidate agent enhances p21 expression or biological activity as measured by a decrease in an HIV-I infection. hi one particular working example, a candidate agent that binds to a p21 polypeptide is identified using a chromatography-based technique. For example, a recombinant polypeptide of the invention may be purified by standard techniques from cells engineered to express the polypeptide (e.g., those described above) and may be immobilized on a column. A solution of candidate agent is then passed through the column, and an agent specific for a p21 polypeptide is identified on the basis of its ability to bind to the polypeptide and be immobilized on the column. To isolate the agent, the column is washed to remove non-specifically bound molecules, and the agent of interest is then released from the column and collected. Similar methods may be used to isolate an compound bound to a polypeptide microarray. Agents identified using such methods are then assayed for their effect on a viral infection (e.g., HIV-1/AIDS) as described herein. hi another example, the agent, e.g., the substrate, is coupled to a radioisotope or enzymatic label such that binding of the agent, e.g., the substrate, to the p21 polypeptide can be determined by detecting the labeled agent, e.g., substrate, in a complex. For example, agents can be labeled with ¹²⁵1, ³⁵S, ¹⁴C, or ³H, either directly or indirectly, and the radioisotope detected by direct counting of radioemmission or by scintillation counting. Alternatively, agents can be enzymatically labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product. In yet another embodiment, a cell- free assay is provided in which a p21 polypeptide or a biologically active portion thereof is contacted with a test agent and the ability of the test agent to bind to the polypeptide thereof is evaluated. The interaction between two molecules (e.g., a p21 polypeptide and a candidate agent) can also be detected, e.g., using fluorescence energy transfer (FET) (see, for example, Lakowicz et al, U.S. Patent No. 5,631,169; Stavrianopoulos et al, U.S. Patent No. 4,868,103). A fluorophore label on the first, 'donor' molecule is selected such that its emitted fluorescent energy will be absorbed by a fluorescent label on a second, 'acceptor' molecule, which in turn is able to fluoresce due to the absorbed energy. Alternately, the 'donor' protein molecule may simply utilize the natural fluorescent energy of tryptophan residues. Labels are chosen that emit different wavelengths of light, such that the 'acceptor' molecule label may be differentiated from that of the 'donor'. Since the efficiency of energy transfer between the labels is related to the distance separating the molecules, the spatial relationship between the molecules can be assessed, hi a situation in which binding occurs between the molecules, the fluorescent emission of the 'acceptor' molecule label in the assay should be maximal. An FET binding event can be conveniently measured through standard fluorometric detection means well known in the art (e.g., using a fluorimeter). In another embodiment, determining the ability of a test agent to bind to a p21 polypeptide can be accomplished using real-time Biomolecular Interaction Analysis (BIA) (see, e.g., Sjolander, S. and Urbaniczky, C, Anal. Chem. 63:2338-2345, (1991); and Szabo et al, Curr. Opin. Struct. Biol. 5:699-705, (1995)). "Surface plasmon resonance" or "BIA" detects biospecific interactions in real time, without labeling any of the interactants (e.g., BIAcore). Changes in the mass at the binding surface (indicative of a binding event) result in alterations of the refractive index of light near the surface (the optical phenomenon of surface plasmon resonance (SPR)), resulting in a detectable signal that can be used as an indication of real-time reactions between biological molecules.

It may be desirable to immobilize either the candidate agent or its p21 polypeptide target to facilitate separation of complexed from uncomplexed forms of one or both of the proteins, as well as to accommodate automation of the assay. Binding of a candidate agent to a p21 polypeptide can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtiter plates, test tubes, and micro-centrifuge tubes. In one embodiment, a fusion protein can be provided which adds a domain that allows one or both of the proteins to be bound to a matrix. For example, glutathione-S-transferase/ p21 polypeptide fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, MO) or glutathione derivatized microtiter plates, which are then combined with the test agent or the test compound and a sample comprising the GST-tagged p21 polypeptide, and the mixture incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtiter plate wells are washed to remove any unbound components, the matrix immobilized in the case of beads, complex determined either directly or indirectly, for example, as described above. Other techniques for immobilizing a complex of a candidate agent and a p21 polypeptide on matrices include using conjugation of biotin and streptavidin. For example, biotinylated proteins can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, IL), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical).

In order to conduct the assay, the non-immobilized component is added to the coated surface containing the anchored component. After the reaction is complete, unreacted components are removed (e.g., by washing) under conditions such that any complexes formed will remain immobilized on the solid surface. The detection of complexes anchored on the solid surface can be accomplished in a number of ways. Where the previously non-immobilized component is pre-labeled, the detection of label immobilized on the surface indicates that complexes were formed. Where the previously non-immobilized component is not pre-labeled, an indirect label can be used to detect complexes anchored on the surface; e.g., using a labeled antibody specific for the immobilized component (the antibody, in turn, can be directly labeled or indirectly labeled with, e.g., a labeled anti-Ig antibody). Alternatively, cell free assays can be conducted in a liquid phase. In such an assay, the reaction products are separated from unreacted components, by any of a number of standard techniques, including but not limited to: differential centrifugation (see, for example, Rivas, G., and Minton, A.P., Trends Biochem Sci 18:284-7, (1993)); chromatography (gel filtration chromatography, ion-exchange chromatography); electrophoresis and immunoprecipitation (see, for example, Ausubel, F. et al, eds. (1999) Current Protocols in Molecular Biology, J. Wiley: New York). Such resins and chromatographic techniques are known to one skilled in the art (see, e.g., Heegaard, N.H., JMoI Recognit 11:141-8, (1998); Hage. D.S., and Tweed, S.A., J Chromatogr B Biomed Sci Appl. 699:499-525, (1997)). Further, fluorescence energy transfer may also be conveniently utilized, as described herein, to detect binding without further purification of the complex from solution. Preferably, cell free assays preserve the structure of the p21 polypeptide.

Agents isolated by this method (or any other appropriate method) may, if desired, be further purified (e.g., by high performance liquid chromatography). In addition, these candidate agents may be tested for their ability to increase the biological activity of a p21 polypeptide (e.g., as described herein). Agents isolated by this approach may also be used, for example, as therapeutics to treat or prevent an a viral infection (e.g., HIV-1/AIDS) in a subject. Agents that are identified as binding to a polypeptide of the invention with an affinity constant less than or equal to 10 mM are considered particularly useful in the invention. Alternatively, any in vivo protein interaction detection system, for example, any two-hybrid assay may be utilized.

Molecules that increase p21 expression or activity include organic molecules, peptides, peptide mimetics, polypeptides, nucleic acids, and antibodies that bind to a p21 nucleic acid sequence or polypeptide and increase its expression or biological activity are preferred.

Each of the DNA sequences listed herein may also be used in the discovery and development of a therapeutic agent for the treatment of HIV-I infection. The encoded protein, upon expression, can be used as a target for the screening of drugs. Additionally, the DNA sequences encoding the amino terminal regions of the encoded protein or Shine-Delgarno or other translation facilitating sequences of the respective mRNA can be used to construct sequences that promote the expression of the coding sequence of interest. Such sequences may be isolated by standard techniques (Ausubel et al., supra).

Small molecules of the invention preferably have a molecular weight below 2,000 daltons, more preferably between 300 and 1,000 daltons, and even more preferably between 400 and 700 daltons. It is preferred that these small molecules are organic molecules. Test Agents and Extracts

In general, agents capable of increasing the expression or biological activity of a p21 polypeptide are identified from large libraries of both natural product or synthetic (or semi-synthetic) extracts or chemical libraries or from polypeptide or nucleic acid libraries, according to methods known in the art. Those skilled in the field of drug discovery and development will understand that the precise source of test extracts or agents is not critical to the screening procedure(s) of the invention. Agents used in screens may include known agents (for example, known therapeutics used for other diseases or disorders). Alternatively, virtually any number of unknown chemical extracts or agents can be screened using the methods described herein. Examples of such extracts or agents include, but are not limited to, plant-, fungal-, prokaryotic- or animal- based extracts, fermentation broths, and synthetic agents, as well as modification of existing agents. Numerous methods are also available for generating random or directed synthesis

(e.g., semi-synthesis or total synthesis) of any number of chemical agents, including, but not limited to, saccharide-, lipid-, peptide-, and nucleic acid-based agents. Synthetic agent libraries are commercially available from Brandon Associates (Merrimack, N.H.) and Aldrich Chemical (Milwaukee, Wis.). Alternatively, chemical agents to be used as candidate agents can be synthesized from readily available starting materials using standard synthetic techniques and methodologies known to those of ordinary skill in the art. Synthetic chemistry transformations and protecting group methodologies (protection and deprotection) useful in synthesizing the agents identified by the methods described herein are known in the art and include, for example, those such as described in R. Larock, Comprehensive Organic Transformations, VCH Publishers (1989); T. W. Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, 2nd ed., John Wiley and Sons (1991); L. Fieser and M. Fieser, Fieser and Fieser's Reagents for Organic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995), and subsequent editions thereof. Alternatively, libraries of natural agents in the form of bacterial, fungal, plant, and animal extracts are commercially available from a number of sources, including Biotics (Sussex, UK), Xenova (Slough, UK), Harbor Branch Oceangraphics Institute (Ft. Pierce, FIa.), and PharmaMar, U.S.A. (Cambridge, Mass.). In addition, natural and synthetically produced libraries are produced, if desired, according to methods known in the art, e.g., by standard extraction and fractionation methods. Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt et al, Proc. Natl. Acad. ScL U.S.A. 90:6909, (1993); Erb et al, Proc. Natl. Acad. ScL USA 91:11422, (1994); Zuckermann et al, J. Med. Chem. 37:2678, (1994); Cho et al., Science 261:1303, (1993); Carrell et al. , Angew. Chem. Int. Ed. Engl. 33:2059, (1994); Carell et al., Angew. Chem. Int. Ed. Engl. 33:2061, (1994); and Gallop et al, J. Med. Chem. 37:1233, (1994). Furthermore, if desired, any library or agent is readily modified using standard chemical, physical, or biochemical methods.

Libraries of agents may be presented in solution (e.g., Houghten, Biotechniques 13:412-421, (1992)), or on beads (Lam, Nature 354:82-84, (1991)), chips (Fodor, Nature 364:555-556, (1993)), bacteria (Ladner, U.S. Patent No. 5,223,409), spores (Ladner U.S. Patent No. 5,223,409), plasmids (Cull et al, Proc Natl Acad Sci USA 89:1865-1869, (1992)) or on phage (Scott and Smith, Science 249:386-390, (1990); Devlin, Science 249:404-406, (1990); Cwirla et α/. Proc. Natl. Acad. ScL 87:6378-6382, (1990); Felici, J. MoI. Biol 222:301-310, (1991); Ladner supra.). hi addition, those skilled in the art of drug discovery and development readily understand that methods for dereplication (e.g., taxonomic dereplication, biological dereplication, and chemical dereplication, or any combination thereof) or the elimination of replicates or repeats of materials already known for their activity should be employed whenever possible.

When a crude extract is found to increase the biological activity of a p21 polypeptide, or to increase the expression of a p21 polypeptide, further fractionation of the positive lead extract is optimal to isolate chemical constituents responsible for the observed effect. Thus, the goal of the extraction, fractionation, and purification process is the careful characterization and identification of a chemical entity within the crude extract that increases the expression or activity of a p21 polypeptide, for example, by preventing or reducing a viral infection (e.g., HIV-1/AIDS) in a cell. Methods of fractionation and purification of such heterogenous extracts are known in the art. If desired, agents shown to be useful as therapeutics for the treatment or prevention of a viral infection (e.g., HIV-1/AIDS) are chemically modified according to methods known in the art.

Treatment of HIV-I

Increasing the expression or biological activity of a p21 nucleic acid molecule or polypeptide in a cell (e.g., myeloid or lymphoid cell), tissue (e.g., bone marrow), or organ of a patient is one therapeutic approach for preventing, treating, or ameliorating a viral infection (e.g., HIV-1/AIDS). In one approach, agents that increase p21 expression, such as retinoic acid, are suitable for use as therapeutics for the treatment of HIV-I or AIDS. Other agents that increase p21 expression or activity are shown in Table 1 (below).

Table 1

Methods for administering such agents are known in the art, and are described for example, in the references listed in Table 1, the contents each of which are expressly incorporated herein by reference. Other agents useful in the methods of the invention include indole-3-carbenol (Aggerwal et al., Cell Cycle 4(9):1201-15, 2005), 3,5-diaryl- oxadiazoles (Jessen et al., MoI Cancer Ther. 4(5):761-71, (2005)), high molecular weight fractions of green or black tea (Ohata M Biomed Res 1:1-7, (2005)), casuarinin (Kuo et al., Anticancer Drugs. 16(4):409-15, (2005)), 5-azacytidine (Schneider-Stock et al., J. Pharmacol Exp Ther. Feb;312(2):525-36 (2005)), aresenic trioxide (Liu et al., Blood 101, 4078, (2003)), and Notch ligand (Rangarajan et al., EMBO Journal 20:3427- 3436, (2001)).

Biological agents that act as p21 mimics or inducers are also useful for the treatment of HIV-I or AIDs. Such biological agents include, for example, a 20 amino- acid and a truncated eight amino acid peptide based on the sequence of p21 (WAFl) that binds to and inhibits cyclin Dl-Cdk4 (see, Ball KL et. al. (1997) Curr. Biol. 7(1): 71-80, which is incorporated by reference); also, the synthetic peptide KRRQTSMTDFYHSKRRLIFS, which is based on the sequence of the 141-160 amino acids from p21 WAF 1 /CIP 1 and fused with a carrier peptide, AAV ALLP AVLLALLA (see, Goulvestre C. et. al. J. Immunol. 175: 6959-6967 (2005)). IN specific embodiments, p21 mimics also include cyclized or pegylated P21 polypeptides, hi another approach, the invention features a small molecule mimetic of p21 that functions to inhibit HIV infection or HIV integrase. hi another approach, a nucleic acid molecule encoding p21 is delivered to cells that need an increase in p21 protein expression or biological activity. In one embodiment, a p21 nucleic acid molecule is delivered to cells having or to cells at risk of developing an HIV-I infection. The nucleic acid molecules is delivered to those cells in a form in which it can be taken up by the cells such that sufficient levels of protein can be produced to decrease an HIV-I infection. P21 Polypeptides

Another therapeutic approach included in the invention involves administration of a recombinant therapeutic, such as a p21 polypeptide, either directly to a site or systemically (for example, by any conventional recombinant protein administration technique). The dosage of the administered protein depends on a number of factors, including the size and health of the individual patient. For any particular subject, the specific dosage regimes should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions. Generally, between 0.1 mg and 100 mg, is administered per day to an adult in any pharmaceutically acceptable formulation. hi one embodiment, the invention provides a modified p21 polypeptide having a protein transduction domain that allows it to enter a cell directly. Recombinant polypeptides of the invention are produced using virtually any method known to the skilled artisan. Typically, recombinant polypeptides are produced by transformation of a suitable host cell with all or part of a polypeptide-encoding nucleic acid molecule or fragment thereof in a suitable expression vehicle.

Those skilled in the field of molecular biology will understand that any of a wide variety of expression systems may be used to provide the recombinant protein. The precise host cell used is not critical to the invention. A polypeptide of the invention may be produced in a prokaryotic host (e.g., E. colϊ) or in a eukaryotic host (e.g., Saccharomyces cerevisiae, insect cells, e.g., Sf21 cells, or mammalian cells, e.g., NIH 3T3, HeLa, or preferably COS cells). Such cells are available from a wide range of sources (e.g., the American Type Culture Collection, Rockland, Md.; also, see, e.g., Ausubel et al., Current Protocol in Molecular Biology, New York: John Wiley and Sons, (1997)). The method of transformation or transfection and the choice of expression vehicle will depend on the host system selected. Transformation and transfection methods are described, e.g., in Ausubel et al. (supra); expression vehicles may be chosen from those provided, e.g., in Cloning Vectors: A Laboratory Manual (P. H. Pouwels et al., (1985), Supp. 1987).

A variety of expression systems exist for the production of the polypeptides of the invention. Expression vectors useful for producing such polypeptides include, without limitation, chromosomal, episomal, and virus-derived vectors, e.g., vectors derived from bacterial plasmids, from bacteriophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations thereof. One particular bacterial expression system for polypeptide production is the E. coli pET expression system (e.g., pET-28) (Novagen, Inc., Madison, Wis). According to this expression system, DNA encoding a polypeptide is inserted into a pET vector in an orientation designed to allow expression. Since the gene encoding such a polypeptide is under the control of the T7 regulatory signals, expression of the polypeptide is achieved by inducing the expression of T7 RNA polymerase in the host cell. This is typically achieved using host strains that express T7 RNA polymerase in response to EPTG induction. Once produced, recombinant polypeptide is then isolated according to standard methods known in the art, for example, those described herein.

Another bacterial expression system for polypeptide production is the pGEX expression system (Pharmacia). This system employs a GST gene fusion system that is designed for high-level expression of genes or gene fragments as fusion proteins with rapid purification and recovery of functional gene products. The protein of interest is fused to the carboxyl terminus of the glutathione S-transferase protein from Schistosoma japonicum and is readily purified from bacterial lysates by affinity chromatography using Glutathione Sepharose 4B. Fusion proteins can be recovered under mild conditions by elution with glutathione. Cleavage of the glutathione S-transferase domain from the fusion protein is facilitated by the presence of recognition sites for site- specific proteases upstream of this domain. For example, proteins expressed in pGEX- 2T plasmids may be cleaved with thrombin; those expressed in pGEX-3X may be cleaved with factor Xa.

Alternatively, recombinant polypeptides of the invention are expressed in Pichia pastoris, a methylotrophic yeast. Pichia is capable of metabolizing methanol as the sole carbon source. The first step in the metabolism of methanol is the oxidation of methanol to formaldehyde by the enzyme, alcohol oxidase. Expression of this enzyme, which is coded for by the AOXl gene is induced by methanol. The AOXl promoter can be used for inducible polypeptide expression or the GAP promoter for constitutive expression of a gene of interest. Once the recombinant polypeptide of the invention is expressed, it is isolated, for example, using affinity chromatography. In one example, an antibody (e.g., produced as described herein) raised against a polypeptide of the invention may be attached to a column and used to isolate the recombinant polypeptide. Lysis and fractionation of polypeptide-harboring cells prior to affinity chromatography may be performed by standard methods (see, e.g., Ausubel et al., supra). Alternatively, the polypeptide is isolated using a sequence tag, such as a hexahistidine tag, that binds to nickel column. Once isolated, the recombinant protein can, if desired, be further purified, e.g., by high performance liquid chromatography (see, e.g., Fisher, Laboratory Techniques In Biochemistry and Molecular Biology, eds., Work and Burdon, Elsevier, (1980)). Polypeptides of the invention, particularly short peptide fragments, can also be produced by chemical synthesis (e.g., by the methods described in Solid Phase Peptide Synthesis, 2nd ed., (1984) The Pierce Chemical Co., Rockford, 111.). These general techniques of polypeptide expression and purification can also be used to produce and isolate useful peptide fragments or analogs (described herein). p21 Polypeptides and Analogs

Also included in the invention are p21 polypeptides or fragments thereof that are modified in ways that enhance their ability to ameliorate an HIV- 1/ATDs infection. The invention provides methods for optimizing a p21 amino acid sequence or nucleic acid sequence by producing an alteration in the sequence. Such alterations may include certain mutations, deletions, insertions, or post-translational modifications. The invention further includes analogs of any naturally-occurring polypeptide of the invention. Analogs can differ from a naturally-occurring polypeptide of the invention by amino acid sequence differences, by post-translational modifications, or by both.

Analogs of the invention will generally exhibit at least 85%, more preferably 90%, and most preferably 95% or even 99% identity with all or part of a naturally-occurring amino, acid sequence of the invention. The length of sequence comparison is at least 5, 10, 15 or 20 amino acid residues, preferably at least 25, 50, or 75 amino acid residues, and more preferably more than 100 amino acid residues. Again, in an exemplary approach to determining the degree of identity, a BLAST program may be used, with a probability score between e^"3 and e^"100 indicating a closely related sequence. Modifications include in vivo and in vitro chemical derivatization of polypeptides, e.g., acetylation, carboxylation, phosphorylation, pegylation, cyclization or glycosylation; such modifications may occur during polypeptide synthesis or processing or following treatment with isolated modifying enzymes. Analogs can also differ from the naturally- occurring polypeptides of the invention by alterations in primary sequence. These include genetic variants, both natural and induced (for example, resulting from random mutagenesis by irradiation or exposure to ethanemethylsulfate or by site-specific mutagenesis as described in Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual (2d ed.), CSH Press, (1989), or Ausubel et al., supra). Also included are cyclized peptides, molecules, and analogs which contain residues other than L-amino acids, e.g., D-amino acids or non-naturally occurring or synthetic amino acids, e.g., β or γ amino acids.

In addition to full-length polypeptides, the invention also includes fragments of any one of the polypeptides of the invention. As used herein, the term "a fragment" means at least 5, 10, 13, or 15. In other embodiments a fragment is at least 20 contiguous amino acids, at least 30 contiguous amino acids, or at least 50 contiguous amino acids, and in other embodiments at least 60 to 80 or more contiguous amino acids. Fragments of the invention can be generated by methods known to those skilled in the art or may result from normal protein processing (e.g., removal of amino acids from the nascent polypeptide that are not required for biological activity or removal of amino acids by alternative mRNA splicing or alternative protein processing events).

Non-protein p21 analogs have a chemical structure designed to mimic the biological activity of the p21 protein. Such analogs are administered according to methods of the invention. P21 protein analogs may exceed the physiological activity of the original p21 polypeptide. Methods of analog design are well known in the art, and synthesis of analogs can be carried out according to such methods by modifying the chemical structures such that the resultant analogs have an increased biological activity relative to a naturally occurring p21 polypeptide. These chemical modifications include, but are not limited to, substituting alternative R groups and varying the degree of saturation at specific carbon atoms of a reference p21 polypeptide. Preferably, the protein analogs are relatively resistant to in vivo degradation, resulting in a more prolonged therapeutic effect upon administration. Assays for measuring functional activity include, but are not limited to, those described in the Examples below. Pharmaceutical Therapeutics The invention provides a simple means for identifying compositions (including nucleic acids, peptides, small molecule inhibitors, and mimetics) capable of acting as therapeutics for the treatment or prevention of a viral infection (e.g., HIV-1/AIDS) or to prevent or treat the progression of HIV-I to full blown AIDS. In one embodiment, the invention provides a pharmaceutical composition comprising an effective amount of a p21 inducer or mimetic. Accordingly, a chemical entity discovered to have medicinal value using the methods described herein is useful as a drug or as information for structural modification of existing agents, e.g., by rational drug design. Such methods are useful for screening agents that prevent or treat a viral infection (e.g., HIV-1/AIDS) in a subject.

For therapeutic uses, the compositions or agents identified using the methods disclosed herein may be administered systemically, for example, formulated in a pharmaceutically acceptable buffer such as physiological saline. Preferable routes of administration include, for example, subcutaneous, intravenous, interperitoneally, intramuscular, or intradermal injections that provide continuous, sustained levels of the drug in the patient. Treatment of human patients or other animals will be carried out using a therapeutically effective amount of a therapeutic agent in a physiologically acceptable carrier. Suitable carriers and their formulation are described, for example, in Remington's Pharmaceutical Sciences by E. W. Martin. The amount of the therapeutic agent to be administered varies depending upon the manner of administration, the age and body weight of the patient, and with the clinical symptoms of HIV-I or AIDS. Generally, amounts will be in the range of those used for other agents used in the treatment of other diseases associated with a retroviral infection. A agent is administered at a dosage that controls the clinical or physiological symptoms of HIV-I or AIDS as determined by a diagnostic method known to one skilled in the art, or using any that assay that measures the expression or the biological activity of a p21 polypeptide. Formulation of Pharmaceutical Compositions

The administration of an agent for the treatment or prevention of HIV-I or AIDS may be by any suitable means that results in a concentration of the therapeutic that, combined with other components, is effective in ameliorating, reducing, or stabilizing or prevention of HIV-I or ADDS. The agent may be contained in any appropriate amount in any suitable carrier substance, and is generally present in an amount of 1-95% by weight of the total weight of the composition. The composition may be provided in a dosage form that is suitable for parenteral (e.g., subcutaneously, intravenously, intramuscularly, or intraperitoneally) administration route. The pharmaceutical compositions may be formulated according to conventional pharmaceutical practice (see, e.g., Remington: The Science and Practice of Pharmacy (20th ed.), ed. A. R.

Gennaro, Lippincott Williams & Wilkins, (2000) and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan, (1988-1999), Marcel Dekker, New York). Pharmaceutical compositions according to the invention may be formulated to release the active agent substantially immediately upon administration or at any predetermined time or time period after administration. The latter types of compositions are generally known as controlled release formulations, which include (i) formulations that create a substantially constant concentration of the drug within the body over an extended period of time; (ii) formulations that after a predetermined lag time create a substantially constant concentration of the drug within the body over an extended period of time; (iii) formulations that sustain action during a predetermined time period by maintaining a relatively, constant, effective level in the body with concomitant minimization of undesirable side effects associated with fluctuations in the plasma level of the active substance (sawtooth kinetic pattern); (iv) formulations that localize action by, e.g., spatial placement of a controlled release composition adjacent to or in the central nervous system or cerebrospinal fluid; (v) formulations that allow for convenient dosing, such that doses are administered, for example, once every one or two weeks; and (vi) formulations that treat or prevent HIV-I or AIDS are delivered using carriers or chemical derivatives to deliver the therapeutic agent to a particular cell type (e.g., a cell having or at risk of developing an HIV-I infection). In one embodiment, p21 function is perturbed in a cell infected with HIV-I. For some applications, controlled release formulations obviate the need for frequent dosing during the day to sustain the plasma level at a therapeutic level.

Any of a number of strategies can be pursued in order to obtain controlled release in which the rate of release outweighs the rate of metabolism of the agent in question. In one example, controlled release is obtained by appropriate selection of various formulation parameters and ingredients, including, e.g., various types of controlled release compositions and coatings. Thus, the therapeutic is formulated with appropriate excipients into a pharmaceutical composition that, upon administration, releases the therapeutic in a controlled manner. Examples include single or multiple unit tablet or capsule compositions, oil solutions, suspensions, emulsions, microcapsules, microspheres, molecular complexes, nanoparticles, patches, and liposomes. Patient Monitoring

The disease state or treatment of a patient having or at risk of developing HIV-I or ADDS can be monitored using standard clinical methods known to one skilled in the art. In one embodiment, patient monitoring includes measuring the level of HIV-I infection, HIV-I replication, HIV-I integration into the genome of a host cell in a cell or biological sample derived from an HIV-I infected patient and treated with a composition or method of the invention. In another embodiment, patient monitoring (e.g., monitoring of a patient receiving a composition of the invention) includes measuring the level of p21 expression or activity in a patient sample. Such monitoring may be useful, for example, in assessing the efficacy of a particular drug in a patient or in assessing patient compliance with a treatment regimen. Therapeutics that increase the expression of a p21 nucleic acid molecule or polypeptide are taken as particularly useful in the invention. Kits or Pharmaceutical Systems The present compositions may be assembled into kits or pharmaceutical systems for use in ameliorating HIV-I or AIDS. Kits or pharmaceutical systems according to this aspect of the invention comprise a carrier means, such as a box, carton, tube or the like, having in close confinement therein one or more container means, such as vials, tubes, ampoules, bottles and the like. The kits or pharmaceutical systems of the invention may also comprise associated instructions for using the agents of the invention (e.g., p21, p21 inducers or mimetics). In various embodiments, such kits are labeled for use in HIV-I or AIDS, or include directions for the use of the polypeptides of the invention to ameliorate HIV-I or AIDS.

EXAMPLES

As reported in more detail below, knocking down p21 by siRNA enabled HIV-I infection and induction of p21 expression blocked HIV-I replication. Analysis of p21 siRNA-treated cells revealed that the enhanced infection preceded a change in cell cycle. Silencing p27¹^¹ and pi 8 ^INK4C, cyclin dependent kinase inhibitors that relate to p21 and affect cell cycle, revealed no impact on HIV-I infection. p21 significantly decreased viral DNA integration into the cellular genome, and resulted in a marked increase in 2- LTR circles, a surrogate marker of abortive integration. Further, p21 coimmunoprecipitated with HIV-I integrase and both were detected in the preintegration complex (PIC). Knocking down p21 did not affect gene expression of other known mediators of HIV-I infection including Trim5α, PML, Murrl, and IFN-α. The effect of p21 was specific to HIV-I, as a closely related dual-tropic lentivirus with a distinct integrase, SIVmac-251, was unaffected. These studies identify p21 as an endogenous cellular component that restricts HIV-I infection and viral DNA integration within primitive hematopoietic cells. Based on these findings, it is likely that methods and agents that increase the expression or biological activity of p21 would similarly protect other cells from HIV-I infection. Example 1: p21 restricted HIV-I replication in primitive hematopoietic cells To assess the role of p21 in HIV-I replication in primitive hematopoietic cells, p21 expression was knocked down in cells using two types of siRNA, an in vitro synthesized siRNA and an in vivo plasmid transcribed shRNA, each targeting distinct sequences of p21 (Zhang et al., 2005, supra). Both forms of siRNA suppressed p21 mRNA expression in cells in the range of 62 - 98% (Figure IA), as measured by real time RT-PCR that detected as few as ten copies of target mRNA in a sample of 50 ng total cellular RNA (Figure 1). Western blot analysis confirmed that p21 protein expression was decreased in p21 siRNA-treated cells but not in control cells (Figure 1C).

The CD34⁺ fraction of adult human bone marrow mononuclear cells is enriched for hematopoietic stem cells that resist HIV-I infection (Shen et al., J. Virol 73: 728-37, (1999); von Laer et al., Blood 76: 1281-6, (1990); Weichold et al., Blood 91: 907-15, (1998)). Markedly increased active HIV-I replication was observed in primary human bone marrow CD34⁺ cells after treatment with p21 siRNA. At 14 days post infection, HIV-I Gag p24 levels were 50-fold higher in p21 siRNA treated cells compared with controls (Figure 2A). HIV-I replication occurred only in cells that were treated with siRNA specific for p21. Cells that were mock treated or treated with control siRNA had only a background level of HIV-I detected (Figure 2A).

To assess the knockdown of p21, HIV-I replication was tested in CMK cells, a p53-deficient human megakaryoblastic cell line with an established ability to modulate p21 (Matsumura et al., MoI Cell Biol. 17: 2933-43, (1997)). CMK cells are derived from megakaryoblasts that are precursor cells of megakaryocytes (MK) and platelets

(Vainchenker et al., Crit Rev Oncol Hematol. 20: 165-92, (1995). Treating these cells with p21 siRNA increased HIV-I replication up to 14-fold in comparison with controls (Figure 2B). Further, expression of p21 in CMK cells can be stimulated by 12-0- tetradecanoylphorbol-13-acetate (TPA) or retinoic acid without affecting the levels of other Cyclin Kinase Inhibitors (CKI) p27^Kipl , p 16^INK4A, p 15^⁴⁸ and p 1 S^mKAC

(Matsumura et al., MoI Cell Biol. 17: 2933-43, (1997)). To examine whether stimulation of p21 expression inhibited HIV-I replication, CMK cells were treated with 12-0- tetradecanoylphorbol- 13 -acetate (TPA) at different time points prior to infection with a high dose of HIV-I (10⁴ TCID₅0/I0⁶ cells). The expression of p21 mRNA was increased in a dose-responsive manner after TPA treatment. The HIV-I replication was blocked in all TPA treated cells, even in cells treated with TPA for as little as 24 hours. In contrast, mock treated cells, which had low levels of p21 expression, had high levels of HIV-I replication (Figure 2C and D). p21 restricted viral DNA integration into the host cell genome

To elucidate the mechanism by which p21 affects HIV-I susceptibility in primary hematopoietic cells, the HIV-I replication steps in the viral life cycle was examined. First, HIV-I co-receptors CD4 and CXCR4 expression on the cell surface was measured using flow cytometric analysis. No change in the receptor expression level was detected between cells treated with p21 siRNA and those treated with control siRNA.

Soon after the HIV-I virus enters a cell, viral reverse transcriptase converts HIV- 1 RNA into double-strand cDNA, and viral integrase integrates the viral cDNA into a host cell chromosome (Coffin et al., Retroviruses (1997)). To determine whether p21 inhibited viral integration, the levels of HIV-I integrated DNA and 2-LTR circular DNA in primitive CD34⁺ cells as well as in CMK cells was examined using real time, quantitative AIu-LTR PCR and 2-LTR circle PCR assays. At 16 hours post infection, primitive CD34⁺ cells treated with control siRNA had significantly higher levels of 2- LTR circles than cells treated with p21 siRNA (2,721.8 ± 142.6 copies vs. 21.7 ± 1.46 copies/6 x 10³ cells) (Figure 3A). Examining the HIV-I provirus at 18 days postinfection revealed that primitive CD34⁺ cells treated with p21 siRNA had significantly increased levels of viral integrated DNA in the cell genome in contrast to control cells. An average of 2.9 integrated copies per cell was detected in p21 siRNA treated cell. In contrast, no integrated copies were detected per cell in control siRNA treated cells (Figure 3B). The standard curve used to calculate the copy number of provirus is shown in Figure 3C. hi CMK cells, an average of 2.4 integrated copies per cell were detected in cells treated with p21 siRNA. Only 0.07 copies per cell in cells treated with control siRNA. These results indicate that knocking down p21 expression dramatically increased the levels of viral integration in both preliminary primitive CD34⁺ cells and in the CMK cell line. The high level of 2-LTR circles detected in cells that express p21 represented the end products of aborted HIV-I integration, similar to the phenotype of cells infected with integrase mutant viruses blocked in their capacity to achieve integration or in cells treated with specific inhibitors that inhibit integrase function (Engelman, Adv Virus Res. 52: 411-26, (1999); Hazuda et al., Science 287:646-50,(2000)).

HIV- 1 preintegration complexes are the functional unit needed for integration of the retroviral genome into the host genome. To determine whether p21 interacts with the viral integration machinery, preintegration complexes were isolated from control and siRNA-treated CMK cells, and the interaction of p21 with preintegration complex components was examined by coimmunoprecipitation- Western blot assays. The viral matrix protein is a preintegration complex component (Bukrinsky et al., Nature 365: 666- 9, (1993); Von Schwedler et al., Proc. Natl. Acad. Sci.USA 91- 6992-6, (1994)), and anti- matrix antibodies co-immunoprecipitated integrase from the initial cytoplasmic extracts (Figure 4A, lane 2) as well as from preintegration complexes purified by spin column chromatography (Figure 4B, lane 5). Similarly, anti-p21 monoclonal antibody co- immunoprecipitated integrase from starting and purified preintegration complexes (Figure 4A, lane 1 and 4B, lane 4). Interestingly, no co-immunoprecipitation of integrase was observed in purified preintegration complexes samples from p21 siRNA treated cells (Figure 4B, lanes 6 and 7). This effect was not observed in control cells. In addition, p21 was not detected in preintegration complexes derived from H9 cells, a T-cell line highly susceptible to HIV-I_11IB infection (Figure 4C, lane 2). Taken together, these results demonstrate that p21 in primitive primary cells or CMK cells interacted with the HIV-I integration machinery and that the presence of p21 in these complexes was associated with an inability of viral DNA to integrate into cellular DNA. p21 restricted HIV-I infection before cell cycling

To address the relationship between cell cycle and viral replication in p21 siRNA treated cells, cell cycle status was analyzed using flow cytometry. Cells were stained with Hoechst 33342 for DNA and pyronin Y for RNA content at 10 hours and 42 hours post siRNA treatment. Up to the time of HIV-I infection (42 hours post siRNA treatment), there was no significant change in cycle status between cells treated with p21 siRNA or with control siRNA, in either primitive CD34⁺ cells (Figure 5A) or in CMK cells (Figure 5B). A BrdU incorporation assay was conducted to provide a sensitive and temporally integrated analysis of cycle status in cells treated with p21 siRNA or with control siRNA. At 42 hours post silencing, no appreciable difference in BrdU incorporation was detected between cells treated with p21 siRNA and with control siRNA (Figure 5C and D). After 68 hours post siRNA treatment, more dividing cells were detected in siRNA treated cells relative to control cells. This change was noted at 26 hours post- viral infection, and is consistent with the observation that HIV-I integration occurs prior to cell division (Bukrinsky et al., Nature 365: 666-9, (1993); Li et al., (1993); Von Schwedler et al., Proc. Natl. Acad. ScL USA 91: 6992-6, (1994); Zack et al., Adv. Exp. Med. Biol. 374: 27-31, (1995); Zhou et al., J. Virol. 79: 2199-210, (2005)).

To determine whether other CKI affects HIV-I infection, the effects of silencing p27^Kipl (p27, another member of the CIP/KIP sub-family) and pi 8 (a member of the INK4 sub-family) expression on HIV-I replication in CD34⁺ cells. Neither p27 nor pi 8 silencing increased HIV-I replication in CD34⁺ cells (Figure 6A and B).

Inhibition of HIV-I infection is specific and independent of other restriction factors

To determine whether expression of other host genes known to inhibit HIV-I could account for the differences seen in susceptibility after silencing p21, expression of Trim5a, PML, Murrl, or IFN-a was examined using real time RT-PCR assays, hi cells with or without silencing of p21 , no changes in mRNA expression of any these cellular virus inhibitors was detected (Table 2).

Table 2: The mRNA levels of Trim5α, PML, Murrl, IFN-α in p21-silenced CD34⁺ cells

Treatment p21 Trimδα PML Murrl IFN-α

CT CT CT C_x CT

p21 siRNA 37.3 27.5 20.9 24.1 22.1

HPRT siRNA 19.8 27.6 21.1 24.5 22.4

*NTC 40 40 40 39.3 38.5 *NTC: No Template Control, tested in duplicate.

C_T value represents mean of quadruplets for each gene tested (SD ± 0.001-0.004).

Table 2 shows that knockdown of p21 did not affect mRNA expression of four other cellular factors known to inhibit HIV-I. Direct CT values were used for comparison. RT-PCR condition was the same as described previously. This indicates that the role of p21 in HIV-I replication inhibition in primitive cells was not mediated by increased expression of four other known modulators of innate cell resistance to HIV- 1. To assess whether the effect of p21 on HIV-I replication was virus specific, the infectivity of SIVmac-251, a dual tropic lentivirus related to HIV-I (Schmitz et al., (2005)), in both p21 siRNA treated primary cells and in CMK cells was studied. Silencing p21 expression in primary and CMK cells did not induce or increase SIV infection, although the more mature bone marrow CD34^" primary cells from the same donors was actively infected by SIVmac-251 (SIV capsid protein p27 level >3.92 ng/ml at day 7, Figure 7B). A low level of viral antigen p27 was detected (0.103 ± 0.001ng/ml) in culture supernatant of CD34⁺ cells at day 7, but this dropped to background levels by day 10 (<0.05 ng/ml, Figure 7A). Thus, silencing of p21 expression did not induce SIVmac-251 replication. This is in marked contrast to what was observed with HIV-I.

The foregoing results were obtained using the following methods and materials. Cells and viruses

Human CD34⁺ primary cells were obtained from the bone marrow of healthy adult volunteers. Mononuclear cells from bone marrow were selected by density gradient centrifugation using Ficoll/Paque Plus (Amersham Pharmacia Biotech, Buckinghamshire, UK). CD34⁺ cells were enriched by immunomagnetic selection using a kit provided by Miltenyi Biotec GmbH (Bergisch Gladbach, Germany). 96% purity was obtained. Human megakaryoblastic leukemia cells (CMK) were grown in RPMI cell culture media with 20% fetal bovine serum (FBS) and cells in log growth were used in experiments (Matsumura et al., MoI Cell Biol. 17: 2933-43, (1997)). The viral pool of HIV-I_111B was harvested from H9/HTLV-IIIB cell culture (NIH AIDS Research & Reference Program) and viral stocks were titrated with the Tissue Culture Infecting Dose (TCID₅o) assay in H9 and in human peripheral blood mononuclear cells (Hollinger, ACTG Virology Manual for HIV laboratories, (1994)). HIV-I infection and detection

One million cells were infected with 1 x 10³ TCID₅₀ of HIV-I _ΠIB, at 42 hours post siRNA treatment. Cell number and viability after siRNA treatment were determined using trypan blue exclusion. Virus and cells were incubated at 37⁰C for 2 hours, and them unadsorbed viruses were washed out with phosphate-buffered saline (PBS). Cells were cultured at 37⁰C and the culture supernatant or cells were harvested at indicated time points. A commercially available ELISA assay, the Coulter p24 antigen assay (Beckman Coulter, Inc, Fullerton, CA) was used to detect HIV-I levels in culture supernatant.

SIV infection and detection

SIVmac-251 is described by Schmitz et al., (2005). Infection was performed as described previously (Schmitz et al., (2005)). Coulter SIV p27 antigen ELISA assay (Beckman Coulter, Inc, Fullerton, CA) was used to detect SFV replication in the supernatant of cell cultures. AIu-LTR PCR assay

The AIu-LTR PCR assay (Butler et al., Nat Med. 7: 631-4, (2001)) was used to measure levels of HIV-I DNA integration. This assay was adapted with the following modifications. Briefly, a nested PCR assay was carried out using two sets of AIu-LTR primers and a probe. The sequences of the primers for the first step PCR were the same as that of AIu forward (MH535) and AIu reverse (SB704) (Butler et al., Nat Med. 7: 631-4, (2001)). The PCR was performed with Taq polymerase (Gibco BRL, Rockville, MD) and a DNA thermal cycler (Perkin Elmer, Foster City, CA). Viral DNA was detected using 500 ng of cellular genomic DNA, subjected to denaturation at 94⁰C for 4 minutes, then 20 cycles of denaturation at 94⁰C for 0.5 minutes. Annealing was carried out at 55⁰C for 0.5 minutes and extension was carried out at 72⁰C for 3 minutes, with a last cycle extension at 72⁰C for 7 minutes. Following the initial PCR step, real-time PCR was performed using an aliquot equivalent to 1/20 of the 20-cycle PCR product, with second round AIu-LTR primers and a Taqman probe. The sequences of the second set of primer and probe are as follows: AIu forward (3' of LTR) 5'GGTAACTAGAGATCCCTCAGAC CCT-3¹; AIU reverse (5¹ of AIu) 5'- GCGTGAGCCACCGC-3'; and AIu probe 5'-TTAGTCAGTG TGGAAAATCTCT AGCAGGCCG-3¹. The copy numbers of integrated viral DNA in samples was derived from the IS cell line (O'Doherty et al., J. Virol. 76: 10942-50, (2002)), adjusted with the known copy number of SM2 plasmid spiked with H9 cellular DNA (Butler et al., Nat Med. 7: 631-4, (2001); Zhang et al., Gene Therapy 14: 1444-52, (2002)). The gel purified genomic DNA of OM10.1 cells was used as a reference standard (Zhang et al., Gene Therapy 14: 1444-52, (2002)). Each sample was tested in triplicate. The copy number of viral DNA was read directly from the result spreadsheet that is converted from the threshold cycles (C_T value < 36 with FAM, 6- carboxyfluorescein) (O'Doherty et al., J. Virol. 76: 10942-50, (2002); Zhang et al., Gene Therapy 14: 1444-52, (2002)) in the function of the standard curves with sequence analysis softward, SEQUENCE DETECTOR V 1.7 software from Applied Biosystems (Foster City, CA). 2-LTR circle PCR assays The 2-LTR circle PCR assays was performed as previously described (Butler et al, Nat Med. 7: 631-4, (2001); Zhang et al., Gene Therapy 14: 1444-52, (2002)). The PCR assay was performed as described and the primers and probe are the same as described (Butler et al., Nat Med. 7: 631-4, (2001); Zhang et al., Gene Therapy 14: 1444-52, (2002). Viral 2-LTR circles were detected from 500 ng cellular genomic DNA in quintuplicate.

Coimmunoprecipitation - Western blot assay

P21 protein in preintegration complex samples was detected following immunoprecipitated using an anti-matrix antibody (MAb 3H7, Lin et al., 2003; Niedrig et al., J. Virol. 63: 3525-3528, (1989); Nilsen et al., J. Virol. 70: 1580-1587, (1996) or anti-p21 antibody (MAb SC-187, Santa Cruz Biotechnology, Inc., Santa Cruz, CA)

(Zhang et al., Gene Therapy 12: 1444-52, (2005). HIV-I Gag protein matrix (MA) is a preintegration complex component. MAb 3H7 has been previously used to immunoprecipitate preintegration complexes (Lin et al., (2003); Niedrig et al., J. Virol. 63: 3525-2538, (1989); Nilsen et al., J. Virol. 70: 1580-1587, (1996)). Antibody (10 μl) was prebound to 15 μl protein AJG- Agarose (Santa Cruz) in 100 μl of buffer K for 1 h at 4⁰C, and 200 μl of CL-4B column purified cytoplasmic preintegration complex were added. The mixture was agitated for 4 hours at 4⁰C, washed and the immune complexes were examined in 12% Tris-glycine gel. Integrase and p21 protein were detected by using MAb 8E5 (anti-integrase) and SC-187, respectively. Western blot analysis of p21 post RNAi treatment was performed as previously described (Zhang et al., Gene Therapy 12: 1444-52, (2005)). Briefly, samples containing identical amounts of lysate protein, as determined using a commercially available assay for total protein, the BCA protein assay, (Pierce) were separated on 16% SDS PAGE (Invitrogen). Following transfer, the membrane was blocked with 5% milk and protein was detected with MAb SC-187, a mouse-anti-human p21 monoclonal antibody. The second antibody was a HRP labeled rabbit anti-mouse IgG (Jackson Immuno). The image was obtained by immediately exposing the membrane to film for 30 sec. - 10 minutes a flat bed scanner, the Hewlett-Packard ScanJet Iicx, (Palo Alto, CA) was used to scan the gel photographs and density of each band was measured and analyzed with image analysis software, ImageQuant (Molecular Dynamics, Sunnyvale, CA).

Cell culture and TPA assay CD34⁺ primary cells were cultured in commercially available media, StemSpan media (StemCell Technologies,. Vancouver, BC, Canada) supplemented with 300 ng/ml stem cell factor (SCF), Flt-3-ligand (Flt-3-L), and 60 ng/ml interleukin-3 (IL-3) (R & D Systems, Minneapolis, MN) at 37⁰C under 5% CO₂ (Zhang et al., Gene Therapy 12: 1444-52, (2005); Zandstra et al., Blood 96: 1215-22, (2000)). The cell culture supernatants post viral infection were collected at indicated time points and replaced back with same amount of fresh media. CMK cells post HIV-I infection were cultured in RPMIl 640 media containing 10% FBS, 2 mM L-glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin. Cells were cultured in 37⁰C under 5% CO₂. hi TPA experiments, cells were cultured in the presence or absence of 10 nM of TPA for 1, 2 and 3 days. The cell number and viability were examined with the trypan blue exclusion assay, and the cells were washed with PBS before being infected with HIV- I_HIB at 1 X 10⁴ TCID₅₀AO⁶ CeIIs. Real-time quantitative RT-PCR

Real time quantitative reverse transcription polymerase chain reaction was performed as previously described (Steir et al., Blood 102: 1260-6, (2003); Zhang et al., Gene Therapy 12: 1444-52, (2005). Briefly, the sequences of the primer set for detection of human p27 and pi 8 mRNA were as follows: p27 forward: 5'- CGGTGGACCACGAAGAGTTAA-3'; reverse: 5'-GGCTCGCCTCTTCCATGTC-S' ; and probe: 5'-(FAM)CCGGGACTTGGAGAAGCACTGCA-S(BHQ)' . P18 forward: 5'- CCCCACAAAAC CGGGAA-3 ' ; reverse: 5 ' -GCTGTAGGC ACTCATTGAGCTG-3 ' ; and probe: 5'-(FAM)AAGGAAAG GACAGCGGCGGCA(BHQ)-3'. The sequences of the primer set for detection of human Trim5α, PML, Murr land INF-α mRNA were as follows: Trim5α forward: 5'-ACCGTGGTCACCACAGGT T-3'; reverse: 5'-TG CCTGGAGCTTCACTTGGTA-3'; and probe: 5'-(FAM)CCCACAGAGGAGG TTGCCCAGGAG-3(BHQ)'. PML forward: 5'-CCGCCCTGGATAACGTCTTT-S'; reverse: 5'-CCA CAATCTGCCGGTACACC-3'; and probe: 5'- (FAM)TCGAGAGTCTGCAGCGGCGC-S(BHQ)'. Murrl forward: 5'- CAAAAATCCGTGAGAGCCTCA-3'; reverse: 5'-CAGCTCAGGCCCCGAAG-S'; and probe: 5'-(FAM)AACCAGAGCCGCTGGAATAGCGG-S(BHQ)'. IFN-α forward: 5'-CCTCGCC CTTTGCTTTACTG-S'; reverse: 5' -GCCC AGAGAGC AGCTTGACT- 3'; and probe: 5'-(FAM)TGG TCCTGGTGGTGCTCAGCTGC-S(BHQ)'. Human cellular GAPDH mRNA was used as the endogenous control in each RT-PCR reaction, and the primers and probe to detect GAPDH mRNA were from a commercially available RT-PCR kit, TaqMan GAPDH Control Reagent kit (Perkin-Elmer, Wellesley, MA). Total cellular RNA from cells was isolated using a commercially available silica-based membrane, the RNeasy kit (QIAGEN GmbH, Hilden, Germany) and purified with DNase following the RNeasy clean up protocol (QIAGEN GmbH, Hilden, Germany). An equal amount of purified RNA (50 ng or 100 ng dependent on each assay) in quadruplicate from each sample was examined in each experiment. A standard curve of the amplicon being measured was run in duplicate from 1 to 1 x 10¹⁰ copies in each assay, with two no template controls. The amplicons from each reaction were analyzed using the Sequence Detector Vl .7 software. The RNA copy number in each sample was converted according to the standard curve and the C_T value of the amplicons in each sample (Figure IB). siRNAs and Transfection of siRNA into cells.

Both synthetic siRNA duplexes and the plasmid-derived small hairpin RNA (shRNA) were used in this study and the sequences of siRNA and shRNA were as previously described (Zhang et al., Gene Therapy 12: 1444-52, 2005). The transfection of siRNA and shRNA into cells was carried out as previously described (Zhang et al., Gene Therapy 12: 1444-52, 2005). Cell-cycle analysis.

The siRNA treated CD34⁺ cells and CMK cells were stained with CD34⁺ FITC (Becton Dickinson, Franklin Lakes, NJ) followed by an incubation with a DNA-dye

Hoechst 33342 (Ho) (Sigma Chemical, St. Louis, Missouri) and an RNA dye Pyronin Y (PY). The proportion of cells in G₀ (PY ^low Hoechst ^low) and in activated status (PY ^high Hoechst ^hlgh) was measured from the CD34⁺ or CMK cells, representing quiescent and activated cells at 10 hours and 42 hours post siRNA treatment, respectively. BrdU incorporation assay.

The siRNA treated CD34⁺ cells were incubated with BrdU in a final concentration of 10 μM. BrdU-pulsed cells were fixed in 70% ethanol at 20°C overnight and denatured by 2 N HCl, 0.5% Triton X-100 for 30 minutes at room temperature, followed by neutralization with borate buffer (pH 8.5). Treated cells were subjected to dual-color staining with anti-BrdU MoAb (Becton Dickinson) followed by goat anti-mouse FITC-conjugated and 5 μg/mL propidium iodide. Analyses were performed using flow cytometer, FACSCalibur flow cytometer, and flow cytometry software, Cell Quest and Modefit software (Becton Dickinson). Other Embodiments

From the foregoing description, it will be apparent that variations and modifications may be made to the invention described herein to adopt it to various usages and conditions. Such embodiments are also within the scope of the following claims.

The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each independent patent and publication was specifically and individually indicated to be incorporated by reference.

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Incorporation by Reference

The contents of all references (including literature references, issued patents, published patent applications, and co-pending patent applications) cited throughout this application are hereby expressly incorporated herein in their entireties by reference. Equivalents Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents of the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

What is claimed is:

1. A method for preventing or reducing a viral infection in a cell, the method comprising contacting a cell with an effective amount of an agent that increases the expression or biological activity of a p21 nucleic acid molecule or polypeptide, thereby preventing or reducing the viral infection.

2. The method of claim 1, wherein the viral infection is an HIV-I infection.

3. The method of claim 1, wherein the agent is selected from the group consisting of Beta-escin, (-)-Epigallocatechin-3 gallate, Iressa, 6-anilino-5,8-quinolinequinone, mevastatin, Oncostatin M, 7 hydroxyl-staurosporine, 1,25-Dihydroxy vitamin D3, Pentagalloglucose, indole-3-carbenol, 3,5-diaryl-oxadiazoles, high molecular weight fractions of green or black tea, casuarinin, 5-azacytidine, aresenic trioxide, Notch ligand, WAF 1 , synthetic peptide KRRQTSMTDFYHSKRRLIFS, and retinoic acid, or analogs, or derivatives thereof.

4. The method of claim 1, wherein the cell is in vivo.

5. The method of claim 5, wherein the cell is a descendant of a hematopoietic stem cell.

6. The method of claim 6, wherein the cell is a myeloid or lymphoid cell.

7. The method of claim 1, wherein the agent reduces viral integration.

8. The method of claim 1, wherein the agent reduces viral replication.

9. The method of claim 1, wherein the cell is ex vivo.

10. The method of any one of claims 1-9, wherein the method further comprises the step of obtaining the agent.

11. A method for preventing or reducing HIV-I infection in a cell, the method comprising contacting the cell with an effective amount of retinoic acid, or an analog or derivative thereof, wherein the contacting increases p21 nucleic acid molecule or polypeptide expression, thereby preventing or reducing HIV-I infection.

12. The method of claim 11, wherein the cell is contacted in vivo.

13. A method for preventing HIV-I viral infection in a cell, the method comprising contacting a cell with an effective amount of an agent that increases p21 biological activity, thereby preventing HIV-I viral infection in the cell.

14. The method of claim 13, wherein the cell is in vivo or ex vivo.

15. The method of claim 13, wherein the wherein the agent is selected from the group consisting of Beta-escin, (-)-Epigallocatechin-3 gallate, Iressa, 6-anilino-5,8- quinolinequinone, mevastatin, Oncostatin M, 7 hydroxyl-staurosporine, 1,25-Dihydroxy vitamin D3, Pentagalloglucose, indole-3-carbenol, 3,5-diaryl-oxadiazoles, high molecular weight fractions of green or black tea, casuarinin, 5-azacytidine, aresenic trioxide, Notch ligand, WAFl, synthetic peptide KRRQTSMTDFYHSKRRLIFS, and retinoic acid, or analogs or derivatives thereof.

16. The method of claim 14, wherein the cell is a descendant of a hematopoietic stem cell.

17. The method of claim 15, wherein the cell is a myeloid or lymphoid cell.

18. The method of claim 12, wherein the agent reduces viral integration or viral replication.

19. The method of any one of claims 13-18, wherein the method further comprises the step of obtaining the agent.

20. A method for preventing or reducing a viral infection in a cell, the method comprising the steps of:

(a) contacting the cell with an expression vector comprising a p21 nucleic acid molecule operably linked to a promoter positioned for expression in the cell; and (b) expressing a p21 polypeptide in the cell, thereby preventing or reducing a viral infection in the cell.

21. The method of claim 20, wherein the viral infection is an HIV-I infection or AIDS.

22. The method of claim 20, wherein the cell is a descendant of a hematopoietic stem cell.

23. The method of claim 22, wherein the cell is a myeloid or lymphoid cell.

24. The method of claim 20, wherein the agent reduces viral integration or viral replication.

25. The method of claim 20, wherein the cell is in vitro or in vivo.

26. A method for preventing or reducing an HIV-I infection in a subject in need thereof, the method comprising administering to the subject an effective amount of an agent that increases p21 nucleic acid molecule or polypeptide expression, thereby preventing or reducing HIV-I infection in the subject.

27. The method of claim 26, wherein the agent is selected from the group consisting of Beta-escin, (-)-Epigallocatechin-3 gallate, Iressa, 6-anilino-5,8-quinolinequinone, mevastatin, Oncostatin M, 7 hydroxyl-staurosporine, 1,25-Dihydroxy vitamin D3, Pentagalloglucose, indole-3-carbenol, 3,5-diaryl-oxadiazoles, high molecular weight fractions of green or black tea, casuarinin, 5-azacytidine, aresenic trioxide, Notch ligand, WAFl, synthetic peptide KRRQTSMTDFYHSKRRLIF S, and retinoic acid, or analogs or derivatives thereof.

28. The method of claim 26, wherein the agent reduces viral integration.

29. The method of claim 26, wherein the agent reduces viral replication.

30. The method of claim 26, wherein the subject is a human patient.

31. The method of claim 30, wherein the human patient is diagnosed as having HIV-I or AIDS.

32. The method of claim 31, wherein the method ameliorates HIV-I or AIDS.

33. A method for preventing or reducing HIV-I infection in a subject in need thereof, the method comprising administering to the subject an effective amount of an agent that increases p21 biological activity, thereby preventing or reducing HIV-I infection in the subject.

34. The method of claim 33, wherein the agent is selected from the group consisting of Beta-escin, (-)-Epigallocatechin-3 gallate, Iressa, 6-anilino-5,8-quinolinequinone, mevastatin, Oncostatin M, 7 hydroxyl-staurosporine, 1,25-Dihydroxy vitamin D3, Pentagalloglucose, indole-3-carbenol, 3,5-diaryl-oxadiazoles, high molecular weight fractions of green or black tea, casuarinin, 5-azacytidine, aresenic trioxide, Notch ligand, WAFl, synthetic peptide KRRQTSMTDFYHSKRRLIFS, and retinoic acid, or analogs or derivatives thereof.

35. A method for preventing or reducing an HIV-I infection in a subject in need thereof, the method comprising the steps of:

(a) contacting a cell of the subject with an expression vector comprising a p21 nucleic acid molecule or fragment thereof operably linked to a promoter suitable for expression in the cell; and

(b) expressing a p21 polypeptide in the cell, thereby preventing or reducing HIV- 1 infection in the subject.

36. The method of claim 33 or 35, wherein the method ameliorates an HIV-I infection or AIDS in the subject.

37. A pharmaceutical composition for the treatment or prevention of a viral infection, the composition comprising an effective amount of an expression vector comprising a p21 nucleic acid molecule or fragment thereof operably linked to a promoter suitable for expression in a hematopoietic stem cell, cell of the myeloid or lymphoid lineages, or dendritic cell or fragment thereof, and a pharmacologically acceptable excipient.

38. The pharmaceutical composition of claim 36, wherein the viral infection is HIV-I .

39. A pharmaceutical composition for the treatment of HIV-I or AIDS, the composition comprising an effective amount of retinoic acid, an analog, or a derivative thereof and a pharmaceutical excipient.

40. A pharmaceutical composition for the treatment of HIV-I or AIDS, the composition comprising an effective amount of a p21 polypeptide, p21 inducer, or p21 mimetic, in a pharmaceutical excipient.

41. A method for identifying an agent that prevents or reduces a viral infection in a cell, the method comprising the steps of: (a) contacting a cell expressing a p21 nucleic acid molecule or polypeptide with a candidate agent; and

(b) comparing the level of p21 biological activity in the cell with the level present in an untreated control cell, wherein an increase in p21 biological activity in the contacted cell thereby identifies the candidate agent as preventing or reducing a viral infection in a cell.

42. The method of claim 41, wherein the viral infection is HIV-I.

43. The method of claim 41, wherein p21 biological activity is assayed by detecting a reduction in the level of HIV-I infection in the cell.

44. The method of claim 41, wherein the level of HIV-I infection is assayed by measuring HIV-I replication in the cell.

45. The method of claim 41, wherein the level of HIV-I infection is assayed by measuring HIV-I integration into the genome of the cell.

46. The method of claim 41 , wherein the agent enhances the resistance of the cell to HIV-I infection.

47. The method of claim 41, wherein the agent reduces HIV-I replication or integration in the cell.

48. The method of claim 41 , wherein the cell is a CD34 positive cell.

49. The method of claim 40, wherein the cell is descended from a hematopoietic stem cell precursor.

50. The method of claim 41, wherein the cell is a cell of the myeloid or lymphoid lineages or a dendritic cell.

51. A method for identifying an agent that prevents or reduces a viral infection in a cell, the method comprising the steps of:

(a) contacting a cell expressing a p21 nucleic acid molecule with an agent; and

(b) comparing the level of p21 expression in the contacted cell relative to an untreated control cell, wherein an increase in p21 expression thereby identifies the agent as preventing or reducing a viral infection in the cell.

52. The method of claim 51, wherein the viral infection is HIV-I.

53. The method of claim 51, wherein the method identifies an agent that increases p21 transcription.

54. The method of claim 51, wherein the method identifies an agent that increases translation of an mRNA transcribed from the p21 nucleic acid molecule.

55. The method of claim 51, wherein the agent reduces HIV-I replication in the cell.

56. The method of claim 51, wherein the agent reduces HIV-I integration into the genome of the cell.

57. The method of claim 51, wherein the agent enhances the resistance of the cell to

HIV-I infection.

58. The method of claim 51, wherein the cell is a CD34 positive cell.

59. The method of claim 51, wherein the cell is descended from a hematopoietic stem cell precursor.

60. The method of claim 51, wherein the cell is a cell of the myeloid or lymphoid lineages or a dendritic cell.

61. A method for identifying an agent that prevents or reduces a viral infection in a cell, the method comprising the steps of:

(a) contacting a cell expressing a p21 polypeptide with a candidate agent; and (b) comparing the level of p21 polypeptide in the contacted cell with the level in an untreated control cell, wherein an increase in the level of p21 polypeptide in the contacted cell relative to an untreated control cell thereby identifies the agent as preventing or reducing a viral infection in a cell.

62. The method of claim 61, wherein the viral infection is HIV-I.

63. The method of claim 61, wherein the agent enhances the resistance of the cell to HIV-I infection.

64. The method of claim 61, wherein the cell is a CD34 positive cell.

65. The method of claim 61, wherein the cell is descended from a hematopoietic stem cell precursor.

66. The method of claim 61, wherein the cell is a cell of the myeloid or lymphoid lineages or a dendritic cell.

67. A method for identifying an agent that prevents or reduces an HIV-I infection in a cell, the method comprising the steps of:

(a) contacting a cell expressing a p21 promoter operably linked to a detectable reporter with a candidate agent; and

(b) comparing the level of reporter expression in the contacted cell relative to an untreated control cell, wherein an increase in reporter expression in the contacted cell thereby identifies the candidate agent as preventing or reducing an HIV-I infection.

68. The method of claim 67, wherein the cell is a CD34 positive cell.

69. The method of claim 67, wherein the cell is descended from a hematopoietic stem cell precursor.

70. The method of claim 67, wherein the cell is a cell of the myeloid or lymphoid lineages or a dendritic cell.

71. A packaged pharmaceutical comprising: a) an effective amount of a p21 polypeptide, p21 inducer, or p21 mimetic; and b) instructions for using said p21 polypeptide, inducer, or mimetic to ameliorate HIV-l or AIDS.

72. A kit for ameliorating HIV-I or AIDS in a subject comprising a p21 polypeptide, p21 inducer, or p21 mimetic, and instructions for use thereof.