WO2013052947A1 - Junctional retroviral peptides and their use in identifying novel targets for antiretroviral drugs - Google Patents

Abstract

Provided in the present invention are novel targets for use in identifying drugs useful for prevention of HIV infectivity in mammalian cells based on the N and C terminal of junctional peptides of HIV, known as matrix, capsid, SP1, nucleocapsid, SP2 and p6. Methods and systems for identifying novel active agents which prevent the interaction of these junctional peptides with proteins in the plasma membrane of mammalian cells are also provided. Uses of the agents identified using such methods in the prevention and treatment of HIV are also provided.

Description

JUNCTIONAL RETROVIRAL PEPTIDES AND THEIR USE IN IDENTIFYING NOVEL TARGETS FOR ANTIRETRO VIRAL DRUGS

REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 61/544,060, filed on October 6, 2011, which is hereby incorporated by reference for all purposes as if fully set forth herein.

STATEMENT OF GOVERNMENTAL INTEREST

[0002] This invention was made with U.S. government support under grant no.

R01DK45787. The U.S. government has certain rights in the invention.

BACKGROUND OF THE INVENTION

[0003] Animal cells release small single-membrane vesicles (-50-250 nm) that have the same topology as the cell. These vesicles mediate the release of specific proteins, lipids, mRNAs, and microRNAs; transmit signals to neighboring cells; and traffic molecules from the cytoplasm and membranes of one cell to the cytoplasm and membranes of others. In addition, there is increasing evidence that these secreted vesicles play important roles in both normal physiological processes, such as immune signaling and development, as well as in diseases such as cancer, viral infections, and amyloidopathies.

[0004] It has been proposed that cells secrete small vesicles by two separate mechanisms, microvesicle biogenesis and exosome biogenesis. In this view, microvesicles bud from the plasma membrane, whereas exosomes are secreted by a two-step process in which vesicles 1) bud at endosomes to form multivesicular bodies (MVBs) and 2) are subsequently released after the fusion of MVBs with the plasma membrane. The budding of human

immunodeficiency virus (HIV) and other retroviruses shares many features with EMV biogenesis, including the ability to bud from the surface of some cell types but via MVB-like structures in others.

[0005] One approach to understanding the biogenesis of secreted vesicles is to identify the cis-acting signals that target proteins to EMVs. The inventors previously reported that the combination of plasma membrane binding and higher-order oligomerization is sufficient to target proteins to EMVs as well as their enrichment at sites of EMV budding. In addition, the inventors found that these same signals are sufficient to target proteins to HIV particles and are the primary budding signals in HIV Gag, lending further support to the EMV model of retrovirus budding. Although the budding of HIV has attracted a great deal of attention, there is still no clear consensus about the regions of HIV Gag that are sufficient for its vesicular release.

[0006] Therefore, there exists a need to understand the mechanism of the budding signals of the Gag protein and identify targets that could inhibit HIV budding could be useful in the study and treatment of HIV.

SUMMARY OF THE INVENTION

[0007] HIV and many other viruses synthesize structural proteins that are subsequently cleaved into smaller polypeptides. For example, HIV synthesizes a 55 kDa Gag protein that drives the budding of HIV from the cell. However, once the budding reaction is complete, the viral protease cleaves Gag at 5 positions, generating 6 polypeptides known as matrix, capsid, SP 1, nucleocapsid, SP2, and p6. The standard view is that the small spacer peptides serve primarily as just that- spacer peptides that presumably exist to facilitate the production of the larger, functional Gag proteins. The inventors now show here that the actual role of these small viral spacer peptides is to promote viral infection by altering cellular physiology at the site of virus:cell fusion. This activity involves interaction of spacer peptides with specific cellular factors, for example, by peptide-protein binding. As such, these spacer peptides and their interaction with their ligand(s) represent an ideal target for the development of anti-viral drugs. For example, the N- and C-termini of the HIV capsid protein are only generated during virus maturation, after budding, and thus, they have the potential for novel functions in newly infected cells.

[0008] In accordance with an embodiment, the present invention provides a system for identifying compounds which modulate HIV infection in a mammalian cell comprising: a) obtaining a population of mammalian cells in vitro capable of being infected by HIV; b) infecting the population of cells with a HIV virus that has been mutated such that the virus cannot produce at least one or more of the following junctional peptides cleaved from the GAG protein: matrix, capsid, SP1, nucleocapsid, SP2 and p6; c) adding to the population of cells from b) the at least one peptides and a target agent or control agent; d) allowing the population to grow for a sufficient period of time; and e) assaying the population of cells for the quantity of cells infected with HIV was increased or decreased in the presence of the target agent when compared with the control agent.

[0009] In accordance with another embodiment, the present invention provides the use of a target agent identified using the methods described herein, in the preparation of a medicament, comprising the target agent and a pharmaceutically acceptable carrier, suitable for use in treating a subject infected with HIV.

[0010] In accordance with another embodiment, the present invention provides the use of a target agent identified using the methods described herein, in the preparation of a medicament, comprising the target agent, and at least one other antiretroviral agent, and a pharmaceutically acceptable carrier, suitable for use in treating a subject infected with HIV.

[0011] In accordance with a further embodiment, the present invention provides one or more target agents identified using the methods described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1. Identification of an inhibitory budding signal. (A) Line diagram of Gag proteins. (B-D) Immunoblots of EMV and cell lysates from 293T cells expressing WT and mutant Gag proteins, probed with antibodies specific for HIV Gag. As with all experiments in this study, the cells were transfected with equal amounts of plasmid, and EMVs and cells were loaded at the same ratio. Furthermore, bar graphs of relative budding (in arbitrary units) display the average (solid bar) and SD (error brackets) from at least three trials, relative to WT control, where *** denotes a p value < 0.0005, ** denotes a p value < 0.005, and * denotes a p value < 0.05. (B) Immunoblot of EMV and cell lysates from 293T cells expressing WT HIV Gag, Gag(p49), Gag(p6PTAP-LIRL), Gag(p49)-3xmyc, and Gag(p49)- SF. The bar graph to the right displays the relative budding in arbitrary units. (C) Immunoblot of EMV and cell lysates from 293T cells expressing WT HIV Gag, Gag-SP2, Gag(ASP2), Gag(ASP2)-SP2, Gag(p49)-SF, and Gag(p49)-SF-SP2, with the bar graph to the right showing the relative budding from multiple trials. (D) Immunoblot of EMV and cell lysates from 293T cells expressing WT HIV Gag, Gag(p49), and Gag(p47). As above, the bar graph shows the relative budding efficiency of each protein, expressed as described herein.

[0013] Figure 2. The IBS inhibits the budding of heterologous proteins. (A) Immunoblots of EMV and cell lysates from 293T cells expressing (left blots) EIAV Gag-SF and EIAV Gag-SF-SP2*, (center blots) RSV Gag-SF and RSV Gag-SF-SP2*, and (right blots) HTLV-1 Gag-SF and HTLV-1 Gag-SF-SP2*, probed with anti-Flag antibodies specific for the SF tag. Asterisks denote the primary translation product, and the bar graph displays the average and SD of their relative budding. (B) Immunoblots of EMV and cell lysates from 293T cells expressing (left blots) CD63-SF and CD63-SF-SP2*, and (right blots) Acyl-LZ-DsRED and Acyl-LZ-DsRED-SP2*, probed with antibodies specific for the SF tag or DsRED, respectively. CD63 is heavily and heterogeneous ly glycosylated, giving rise to proteins with varying Mr that are highlighted by brackets. The bar graph to the right displays the relative budding data from a minimum of three trials, as described above. (C) Anti-Flag immunoblots of EMV and cell lysates from 293T cells expressing 1) CD63-SF and GFP or 2) CD63-SF and Gag-SP2. CD63 is once again highlighted with brackets, and the data from multiple trials are presented in the bar graph to the right.

[0014] Figure 3. Mutational analysis of the IBS. (A, B) Deletion analysis of the IBS. (A) Line diagram displaying the organization of Gag, Gag-SP2, and the amino acid sequences of the SP2 deletion mutants generated in the Gag-SP2 protein. (B) Anti-Gag immunoblots of EMV and cell lysates from 293T cells expressing WT HIV Gag, Gag-SP2, and the ΔΝ and AC mutations of the C-terminal SP2 peptide. (C) Alanine scanning mutagenesis of the IBS. Anti-Gag immunoblots were performed on EMV and cell lysates from 293T cells expressing WT HIV Gag, Gag-SP2, and alanine substitution mutants of the 12 amino acids that lie within the functional domain of SP2. The bar graph to the right shows the relative budding of each protein ascertained from three independent trials. (D) Anti-Gag immunoblots of EMV and cell lysates from 293T cells expressing WT HIV Gag, Gag-SP2, and four variants of Gag-SP2 that carried virus-compatible mutations of critical residues of the IBS. (E) Anti-Flag immunoblots of EMV and cell lysates from 293T cells expressing CD63-SF-SP2* and a variant of CD63-SF-SP2* that has the four-amino acid substitution mutation in the SP2 peptide, RPNF to KLSS.

[0015] Figure 4. Inactivation of the IBS suppresses the budding defect of p6-deficient HIV and reduces HIV infectivity. (A) Sequences of HIV and relevant HIV mutants. Top line is the deduced amino acid sequence of the Gag ORF in the vicinity of the SP2/p6 junction, denoted by the vertical line between GNF and LQS. The next line shows the positions of the DNA sequence changes in the corresponding mutant proviruses. (B) Anti-Gag immunoblot of virus and cell lysates from 293T cells expressing control HIV (NL4.3*), p6-deficient HIV (NL4-3*[p6Llter]), and variants of p6-deficient HIV that have inactivating mutations in the IBS (NL4-3*[p6Llter/SP2R12K], NL4-3*[p6Llter/SP2P13L], NL4-3*[p6Llter/SP2N15S], and NL4-3*[p6Llter/SP2RPNF-KLSS]). The p55 and p49 Gag proteins are the primary translation products of the control and p6-deficient proviruses, respectively. The p41 products are generated by Gag cleavage at either of the PR sites that lie between CA and NC. The p24/25 CA products are generated by additional cleavage of p41 between MA and CA. Note that most of the Gag protein in viruses produced by WT HIV is fully cleaved (p24) whereas most of the Gag protein detected in the viruses produced by p6-deficient HIV is either uncleaved (p49) or cleaved at one or two of the sites that lie between CA and NC (p41). The relative budding efficiencies of each virus are presented in the bar graph to the right, and in all cases the p value was calculated relative to WT control. (C) Relative infectivity of the same control and mutant viruses as in (B). The p values for the infectivity of the doubly mutant viruses are in relation to the infectivity of NL4-3*(p6Llter). (D) BLAST alignment of the HIV SP2 domain with the SIV Gag protein shows the conservation of many IBS residues. (E) Relative infectivity ofNL4.3* and NL4.3*(SP2P 13L), presented as the average ± 1 SD, with *** denoting a p value < 0.0005. Anti-Gag immunoblot of virus and cell lysates from 293T cells expressing control and mutant viruses. (F) Anti-Gag immunoblot of virus and cell lysates of 293T cells expressing NL4.3* and NL4.3*(SP2P 13L), with the averages ± 1 SD presented graphically at the right.

[0016] Figure 5. Gag-Pol and the IBS are required for the budding defect of PTAP- deficient HIV. (A) Sequences of HIV and relevant HIV mutants. Top line is the deduced amino acid sequence of the Gag ORF in the vicinity of the SP2/p6 junction, denoted by the vertical line between GNF and LQS. The next line shows the positions of the DNA sequence changes in the corresponding mutant proviruses. (B) Anti-Gag immunoblot of virus and cell lysates from 293T cells expressing control HIV (NL4.3*), p6-deficient HIV (NL4- 3*[p6Llter]), and PTAP-deficient HIV (NL4-3*[p6PTAP-LIRL]). (C) Anti-Gag immunoblot of virus and cell lysates from 293T cells expressing NL4.3*(TFter), NL4.3*(TFter/p6PTAP- LIRL), NL4.3*(TFter/p6Llter), and NL4.3*(TFter/SP2I5ter), with the averages and SD plotted to the right. (D) Anti-Gag immunoblot of virus and cell lysates from 293T cells expressing NL4.3*, NL4.3*(TFter/p6PTAP-LIRL), and NL4.3*(SP2RPNF-KLSS/p6PTAP- LIRL), with the averages and SD plotted to the right.

[0017] Figure 6. The IBS does not impair membrane binding. (A-C) Anti-Gag immunoblots of fractions from sucrose density flotation gradients that were carried out on (A) cell lysates from 293T cells expressing (top) HIV Gag and (bottom) Gag-SP2, (B) cell lysates from 293T cells expressing (top) HIV Gag and (bottom) Gag-SP2 that had been preincubated with 0.25% Triton X-100 for 20 min at 37°C, and (C) cell lysates from 293T cells expressing (top) HIV Gag and (bottom) Gag-SP2 that had been preincubated with 0.25% Triton X-100 for 20 min at 4°C. Fractions were collected from the top of the gradient, with fraction 1 having the lowest density and fraction 10 having the highest density.

[0018] Figure 7. The IBS does not block plasma membrane localization. (A-P)

Fluorescence and phase microscopy of 293T cells expressing (A-D) WT HIV Gag, (E-H) HIV Gag-SP2, (I, J) HIV Gag(p49), (K, L) HIV Gag(p47), (M, N) CD63-SF, and (O, P) CD63-SF-SP2*. Bar, 10 μιη. The images shown here are representative of what was seen in greater than 90% of expressing cells, with at least 100 expressing cells examined for each protein tested. (Q-X) Transmission electron microscopy of 293T cells expressing (Q, R) WT HIV Gag, (S-V) HIV Gag-SP2, or (W, X) HIV Gag(p6PTAP-LIRL). Bar, 1 μηι for Q-T, V, and W; bar, 100 nm for U and X.

[0019] Figure 8. The IBS does not impair Gag-Gag oligomerization. (A) Anti-Flag immunoblot of EMV and cell lysates from 293T cells expressing AcylSF-Gag and AcylSF- Gag-SP2. (B) Top, anti-Flag immunoblot of anti-myc IPs generated from 293T cells expressing Gag-3xmyc and either AcylSF-Gag or AcylSF-Gag-SP2. Bottom, anti-Flag immunoblot of the input, 27% of what was used in the IP reaction. The + lanes were IPs with anti-GFP antibodies; the - lanes were IPs with nonimmune IgG. (C) Gel filtration eluates of 293T cell lysates expressing either HIV Gag or Gag-SP2 were processed for immunoblot using antibodies specific for (top two panels) HIV Gag or (bottom two panels) β-tubulin.

[0020] Figure 9. The IBS impairs an interaction between EMV cargoes and VPS4B. (A- C) Immunoblots of (top) coimmunoprecipitations and (bottom) cell lysates generated from 293T cells coexpressing GFP-VPS4B and (A) CD63-SF or CD63-SF-SP2*, (B) CD81-SF or CD81-SF-SP2*, or (C) Gag(p47) or Gag(p49). The + lanes were IPs with anti-GFP antibodies, the - lanes were IPs with nonimmune IgG. Immunoblots were with 1% of IP input.

DETAILED DESCRIPTION OF THE INVENTION

[0021] In accordance with an embodiment, the present invention provides a system for identifying compounds which modulate HIV infection in a mammalian cell comprising: a) obtaining a population of mammalian cells in vitro capable of being infected by HIV; b) infecting the population of cells with a HIV virus that has been mutated such that the virus cannot produce at least one or more of the following junctional peptides cleaved from the GAG protein: matrix, capsid, SP1, nucleocapsid, SP2 and p6; c) adding to the population of cells from b) the at least one peptides and a target agent or control agent; d) allowing the population to grow for a sufficient period of time; and e) assaying the population of cells for the quantity of cells infected with HIV was increased or decreased in the presence of the target agent when compared with the control agent.

[0022] It will be understood by those of skill in the art that the novel junctional peptide N and C terminal regions can be used as targets for development of antiviral drugs, especially agains HIV.

[0023] In one or more embodiments of the present invention the target agent binds to the N or C terminus of the at least one or more junctional peptides cleaved from the GAG protein: matrix, capsid, SP1, nucleocapsid, SP2 and p6.

[0024] 3 In one or more embodiments of the present invention the target agent inhibits the interaction of the N or C terminus of the at least one or more junctional peptides cleaved from the GAG protein: matrix, capsid, SP1, nucleocapsid, SP2 and p6 with a membrane protein in the plasma membrane of the mammalian cell or population of cells.

[0025] In one or more embodiments of the present invention the membrane protein is actin.

[0026] In one or more embodiments of the present invention when the population of cells for the quantity of cells infected with HIV was decreased in the presence of the target agent, the target agent is identified as an inhibitor of infectivity.

[0027] In one or more embodiments of the present invention the target agent is selected from the group consisting of antibodies, oligonucleotides such as siRNA or microRNA, small molecules, peptides and derivatives thereof.

[0028]

[0029] In accordance with another embodiment, the present invention provides the use of a target agent identified using the methods described herein, in the preparation of a medicament, comprising the target agent and a pharmaceutically acceptable carrier, suitable for use in treating a subject infected with HIV.

[0030] In accordance with a further embodiment, the present invention provides one or more target agents identified using the methods described herein. [0031] By "nucleic acid" as used herein includes "polynucleotide," "oligonucleotide," and "nucleic acid molecule," and generally means a polymer of DNA or RNA, which can be single-stranded or double-stranded, synthesized or obtained (e.g., isolated and/or purified) from natural sources, which can contain natural, non-natural or altered nucleotides, and which can contain a natural, non-natural or altered internucleotide linkage, such as a phosphoroamidate linkage or a phosphorothioate linkage, instead of the phosphodiester found between the nucleotides of an unmodified oligonucleotide. It is generally preferred that the nucleic acid does not comprise any insertions, deletions, inversions, and/or substitutions. However, it may be suitable in some instances, as discussed herein, for the nucleic acid to comprise one or more insertions, deletions, inversions, and/or substitutions.

[0032] In an embodiment, the nucleic acids of the invention are recombinant. As used herein, the term "recombinant" refers to (i) molecules that are constructed outside living cells by joining natural or synthetic nucleic acid segments to nucleic acid molecules that can replicate in a living cell, or (ii) molecules that result from the replication of those described in (i) above. For purposes herein, the replication can be in vitro replication or in vivo replication.

[0033] The nucleic acids can be constructed based on chemical synthesis and/or enzymatic ligation reactions using procedures known in the art. See, for example, Sambrook et al. (eds.), Molecular Cloning, A Laboratory Manual, 3rd Edition, Cold Spring Harbor Laboratory Press, New York (2001) and Ausubel et al, Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, NY (2007). For example, a nucleic acid can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed upon hybridization (e.g.,

phosphorothioate derivatives and acridine substituted nucleotides). Examples of modified nucleotides that can be used to generate the nucleic acids include, but are not limited to, 5- fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4- acetylcytosine, 5-(carboxyhydroxymethyl) uracil, 5-carboxymethylaminomethyl-2- thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1 -methylguanine, 1 -methylinosine, 2,2-dimethylguanine, 2- methyladenine, 2 -methylguanine, 3-methylcytosine, 5-methylcytosine, N6-substituted adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio- N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2- thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5- oxyacetic acid methylester, 3-(3-amino-3-N-2-carboxypropyl) uracil, and 2,6-diaminopurine. Alternatively, one or more of the nucleic acids of the invention can be purchased from companies, such as Macromolecular Resources (Fort Collins, CO) and Synthegen (Houston, TX).

[0034] The nucleic acids of the invention can be incorporated into a recombinant expression vector. In this regard, the invention provides recombinant expression vectors comprising any of the nucleic acids of the invention. For purposes herein, the term

"recombinant expression vector" means a genetically-modified oligonucleotide or polynucleotide construct that permits the expression of an mRNA, protein, polypeptide, or peptide by a host cell, when the construct comprises a nucleotide sequence encoding the mRNA, protein, polypeptide, or peptide, and the vector is contacted with the cell under conditions sufficient to have the mRNA, protein, polypeptide, or peptide expressed within the cell. The vectors of the invention are not naturally-occurring as a whole. However, parts of the vectors can be naturally-occurring. The inventive recombinant expression vectors can comprise any type of nucleotides, including, but not limited to DNA and RNA, which can be single-stranded or double-stranded, synthesized or obtained in part from natural sources, and which can contain natural, non-natural or altered nucleotides. The recombinant expression vectors can comprise naturally-occurring, non-naturally-occurring internucleotide linkages, or both types of linkages. Preferably, the non-naturally occurring or altered nucleotides or internucleotide linkages do not hinder the transcription or replication of the vector.

[0035] The recombinant expression vectors of the invention can be prepared using standard recombinant DNA techniques described in, for example, Sambrook et al, supra, and Ausubel et al, supra. Constructs of expression vectors, which are circular or linear, can be prepared to contain a replication system functional in a prokaryotic or eukaryotic host cell. Replication systems can be derived, e.g., from ColEl, 2 μ plasmid, λ, SV40, bovine papilloma virus, and the like.

[0036] Desirably, the recombinant expression vector comprises regulatory sequences, such as transcription and translation initiation and termination codons, which are specific to the type of host (e.g., bacterium, fungus, plant, or animal) into which the vector is to be introduced, as appropriate and taking into consideration whether the vector is DNA or RNA based. [0037] The recombinant expression vector can include one or more marker genes, which allow for selection of transformed or transfected hosts. Marker genes include biocide resistance, e.g., resistance to antibiotics, heavy metals, etc., complementation in an auxotrophic host to provide prototrophy, and the like. Suitable marker genes for the inventive expression vectors include, for instance, LacZ, green fluorescent protein (GFP), luciferase, neomycin/G418 resistance genes, hygromycin resistance genes, histidinol resistance genes, tetracycline resistance genes, and ampicillin resistance genes.

[0038] The heterologous nucleic acid can be a nucleic acid not normally found in the target cell, or it can be an extra copy or copies of a nucleic acid normally found in the target cell. The terms "exogenous" and "heterologous" are used herein interchangeably.

[0039] The invention further provides a host cell comprising any of the recombinant expression vectors described herein. As used herein, the term "host cell" refers to any type of cell that can contain the inventive recombinant expression vector. The host cell can be an animal cell. Preferably, in an embodiment, the host cell is a mammalian cell. The host cell can be a cultured cell or a primary cell, i.e., isolated directly from an organism, e.g., a human. The host cell can be an adherent cell or a suspended cell, i.e., a cell that grows in suspension. Most preferably, the host cell is a human cell. The host cell can be of any cell type, can originate from any type of tissue, and can be of any developmental stage. Most preferably the host cells can include, for instance, muscle, lung, and brain cells, and the like.

[0040] The host referred to in the inventive methods can be any host. Preferably, the host is a mammal. As used herein, the term "mammal" refers to any mammal, including, but not limited to, mammals of the order Rodentia, such as mice and hamsters, and mammals of the order Logomorpha, such as rabbits. It is preferred that the mammals are from the order Carnivora, including Felines (cats) and Canines (dogs). It is more preferred that the mammals are from the order Artiodactyla, including Bovines (cows) and Swines (pigs) or of the order Perssodactyla, including Equines (horses). It is most preferred that the mammals are of the order Primates, Ceboids, or Simoids (monkeys) or of the order Anthropoids (humans and apes). An especially preferred mammal is the human.

[0041] Also provided by the invention is a population of cells comprising at least one host cell described herein. The population of cells can be a heterogeneous population comprising the host cell comprising any of the recombinant expression vectors described, in addition to at least one other cell, e.g., a host cell (e.g., a lung cell), which does not comprise any of the recombinant expression vectors, or a cell other than a lung cell, e.g., a skin cell, a neutrophil, an erythrocyte, a hepatocyte, an endothelial cell, an epithelial cell, a muscle cell, a brain cell, etc. Alternatively, the population of cells can be a substantially homogeneous population, in which the population comprises mainly of host cells (e.g., consisting essentially of) comprising the recombinant expression vector. The population also can be a clonal population of cells, in which all cells of the population are clones of a single host cell comprising a recombinant expression vector, such that all cells of the population comprise the recombinant expression vector. In one embodiment of the invention, the population of cells is a clonal population comprising host cells comprising a recombinant expression vector as described herein.

[0042] The terms "treat," and "prevent" as well as words stemming therefrom, as used herein, do not necessarily imply 100% or complete treatment or prevention. Rather, there are varying degrees of treatment or prevention of which one of ordinary skill in the art recognizes as having a potential benefit or therapeutic effect. In this respect, the inventive methods can provide any amount of any level of treatment or prevention of a disease in a mammal.

Furthermore, the treatment or prevention provided by the inventive method can include treatment or prevention of one or more conditions or symptoms of the disease being treated or prevented. Also, for purposes herein, "prevention" can encompass delaying the onset of the disease, or a symptom or condition thereof.

[0043] The target compounds identified using the methods described herein can be administered as a combination of the target compound and one or more antiviral compounds in a pharmaceutically acceptable carrier.

[0044] For use in medicines, the salts of the target compounds identified using the methods of the present invention should be pharmaceutically acceptable salts. Other salts may, however, be useful in the preparation of the compounds according to the invention or of their pharmaceutically acceptable salts.

[0045] As used herein, the term "antiviral compound" includes classes of drugs suitable for use in treating viral infections in vivo and/or in vitro. In particular, the term "antiviral compound" in the present invention, also means an "antiretroviral compound" suitable for use in treating retrovirus infections in vivo and/or in vitro. Examples of classes of antiviral compounds include NRTIs, RTIs, protease inhibitors, fusion or entry inhibitors, and integrase inhibitors. [0046] In accordance with the present invention, examples of NRTIs include, but are not limited to, for example, lamivudine, abacavir, zidovudine, stavudine, didanosine,

emtricitabine, and tenofovir.

[0047] In accordance with the present invention, examples of NNRTIs include, but are not limited to, for example, delavirdine, efavirenz, etravirine, rilpilvirine and nevirapine.

[0048] In accordance with the present invention, examples of protease inhibitors include, but are not limited to, for example, amprenavir, fosamprenavir, atazanavir, darunavir, indinavir, lopinavir, nelfinavir, ritonavir, saquinavir, and tipranavir.

[0049] In accordance with the present invention, examples of fusion or entry inhibitors include, but are not limited to, for example, enfuvirtide and maraviroc.

[0050] In accordance with the present invention, an examples of integrase inhibitors include, but are not limited to raltegravir, elvitegravir, and dolutegravir.

[0051] As defined herein, in one or more embodiments, "contacting" means that the one or more compounds of the present invention are introduced into a sample having at least one retrovirus, including for example, and appropriate enzymes or reagents, in a test tube, flask, tissue culture, chip, array, plate, microplate, capillary, or the like, and incubated at a temperature and time sufficient to permit binding of the at least one compounds of the present invention to interact with the junctional peptides.

[0052] In a further embodiment, the present invention provides a method of treating a retroviral infection in a subject, the method comprising administering to the subject, a pharmaceutical composition comprising at least one compound of the present invention, and at least one other compound suitable for use in treating a retroviral infection, with a pharmaceutically acceptable carrier, in an effective amount to inhibit, suppress or treat symptoms of the retroviral infection.

[0053] Embodiments of the invention include a process for preparing pharmaceutical products comprising the compounds, salts, solvates or stereoisomers thereof. The term "pharmaceutical product" means a composition suitable for pharmaceutical use

(pharmaceutical composition), as defined herein. Pharmaceutical compositions formulated for particular applications comprising the RNase H inhibitors of the present invention are also part of this invention, and are to be considered an embodiment thereof.

[0054] As used herein, the term "treat," as well as words stemming therefrom, includes preventative as well as disorder remitative treatment. The terms "reduce", "suppress" and "inhibit," as well as words stemming therefrom, have their commonly understood meaning of lessening or decreasing. These words do not necessarily imply 100% or complete treatment, reduction, suppression, or inhibition.

[0055] With respect to pharmaceutical compositions described herein, the

pharmaceutically acceptable carrier can be any of those conventionally used, and is limited only by physico-chemical considerations, such as solubility and lack of reactivity with the active compound(s), and by the route of administration. The pharmaceutically acceptable carriers described herein, for example, vehicles, adjuvants, excipients, and diluents, are well- known to those skilled in the art and are readily available to the public. It is preferred that the pharmaceutically acceptable carrier be one which is chemically inert to the active agent(s), and one which has little or no detrimental side effects or toxicity under the conditions of use. Examples of the pharmaceutically acceptable carriers include soluble carriers such as known buffers which can be physiologically acceptable (e.g., phosphate buffer) as well as solid compositions such as solid-state carriers or latex beads.

[0056] The carriers or diluents used herein may be solid carriers or diluents for solid formulations, liquid carriers or diluents for liquid formulations, or mixtures thereof.

[0057] Solid carriers or diluents include, but are not limited to, gums, starches (e.g., corn starch, pregelatinized starch), sugars (e.g., lactose, mannitol, sucrose, dextrose), cellulosic materials (e.g., microcrystalline cellulose), acrylates (e.g., polymethylacrylate), calcium carbonate, magnesium oxide, talc, or mixtures thereof.

[0058] For liquid formulations, pharmaceutically acceptable carriers may be, for example, aqueous or non-aqueous solutions, suspensions, emulsions or oils. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, and injectable organic esters such as ethyl oleate. Aqueous carriers include, for example, water, alcoholic/aqueous solutions, cyclodextrins, emulsions or suspensions, including saline and buffered media.

[0059] Examples of oils are those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, mineral oil, olive oil, sunflower oil, fish-liver oil, sesame oil, cottonseed oil, corn oil, olive, petrolatum, and mineral. Suitable fatty acids for use in parenteral formulations include, for example, oleic acid, stearic acid, and isostearic acid. Ethyl oleate and isopropyl myristate are examples of suitable fatty acid esters.

[0060] Parenteral vehicles (for subcutaneous, intravenous, intraarterial, or intramuscular injection) include, for example, sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's and fixed oils. Formulations suitable for parenteral administration include, for example, aqueous and non-aqueous, isotonic sterile injection solutions, which can contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.

[0061] Intravenous vehicles include, for example, fluid and nutrient replenishers, electrolyte replenishers such as those based on Ringer's dextrose, and the like. Examples are sterile liquids such as water and oils, with or without the addition of a surfactant and other pharmaceutically acceptable adjuvants. In general, water, saline, aqueous dextrose and related sugar solutions, and glycols such as propylene glycols or polyethylene glycol are preferred liquid carriers, particularly for injectable solutions.

[0062] In addition, in an embodiment, the compounds of the present invention may further comprise, for example, binders (e.g., acacia, cornstarch, gelatin, carbomer, ethyl cellulose, guar gum, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, povidone), disintegrating agents (e.g., cornstarch, potato starch, alginic acid, silicon dioxide,

croscarmelose sodium, crospovidone, guar gum, sodium starch glycolate), buffers (e.g., Tris- HC1, acetate, phosphate) of various pH and ionic strength, additives such as albumin or gelatin to prevent absorption to surfaces, detergents (e.g., Tween 20, Tween 80, Pluronic F68, bile acid salts), protease inhibitors, surfactants (e.g. sodium lauryl sulfate), permeation enhancers, solubilizing agents (e.g., cremophor, glycerol, polyethylene glycerol, benzlkonium chloride, benzyl benzoate, cyclodextrins, sorbitan esters, stearic acids), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite, butylated hydroxyanisole), stabilizers (e.g.,

hydroxypropyl cellulose, hyroxypropylmethyl cellulose), viscosity increasing agents (e.g., carbomer, colloidal silicon dioxide, ethyl cellulose, guar gum), sweetners (e.g., aspartame, citric acid), preservatives (e.g., thimerosal, benzyl alcohol, parabens), lubricants (e.g., stearic acid, magnesium stearate, polyethylene glycol, sodium lauryl sulfate), flow-aids (e.g., colloidal silicon dioxide), plasticizers (e.g., diethyl phthalate, triethyl citrate), emulsifiers (e.g., carbomer, hydroxypropyl cellulose, sodium lauryl sulfate), polymer coatings (e.g., poloxamers or poloxamines), coating and film forming agents (e.g., ethyl cellulose, acrylates, polymethacrylates), and/or adjuvants.

[0063] The choice of carrier will be determined, in part, by the particular compound, as well as by the particular method used to administer the compound. Accordingly, there are a variety of suitable formulations of the pharmaceutical composition identified using the methods of the present invention. The following formulations for parenteral, subcutaneous, intravenous, intramuscular, intraarterial, intrathecal and interperitoneal administration are exemplary, and are in no way limiting. More than one route can be used to administer the compounds of the present invention, and in certain instances, a particular route can provide a more immediate and more effective response than another route. Injectable formulations are in accordance with the invention. The requirements for effective pharmaceutical carriers for injectable compositions are well-known to those of ordinary skill in the art (see, e.g., Pharmaceutics and Pharmacy Practice, J.B. Lippincott Company, Philadelphia, PA, Banker and Chalmers, eds., pages 238-250 (1982), and ASHP Handbook on Injectable Drugs, Trissel, 15th ed., pages 622-630 (2009)).

[0064] For purposes of the invention, the amount or dose of the identified target agent or compound of the present invention, or a salt, solvate or stereoisomer thereof, administered should be sufficient to effect, e.g., a therapeutic or prophylactic response, in the subject over a reasonable time frame. The dose will be determined by the efficacy of the particular compound and the condition of a human, as well as the body weight of a human to be treated.

[0065] The dose of the compound of the present invention, or a salt, solvate or stereoisomer thereof, also will be determined by the existence, nature and extent of any adverse side effects that might accompany the administration of a particular compound. Typically, an attending physician will decide the dosage of the compound with which to treat each individual patient, taking into consideration a variety of factors, such as age, body weight, general health, diet, sex, compound to be administered, route of administration, and the severity of the condition being treated. By way of example, and not intending to limit the invention, the dose of the compound can be about 0.001 to about 1000 mg/kg body weight of the subject being treated/day, from about 0.01 to about 10 mg/kg body weight/day, about 0.01 mg to about 1 mg/kg body weight/day.

[0066] The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope.

EXAMPLES

[0067] Cell culture, transfection, and microscopy. 293T cells were maintained in DMEM supplemented with 10% fetal bovine serum and transfected by electroporation (Chang et al, 1997 >) (for immunoblot and IP experiments) using a BTX ECM 600 electroporator, or by lipofection (for immunofluorescence and electron microscopy) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA). 293T cells were processed for immunofluorescence microscopy (Booth et al, 2006 &>) using rabbit polyclonal antibodies specific for HIV Gag, mouse monoclonal antibodies specific for the Flag tag, and fluorescein isothiocyanate- or Texas Red-labeled secondary antibodies. Immunofluorescence images were obtained at room temperature on a BH2-RFCA microscope (Olympus, Center Valley, PA) equipped with an Olympus S-Plan-Apo 60x 0.40 oil objective and a Sensicam QE (Cooke, Romulus, MI) digital camera using IPLab 3.6.3 software (Scanalytics, Reutlingen, Germany). Images were converted to TIFF files, imported into Photoshop CS, and assembled into figures using Illustrator CS (Adobe Systems, San Jose, CA). For transmission electron microscopy, 293T cells were fixed, processed, and examined as described previously for Jurkat T-cells (Booth et al, 2006; Fang et al., 2007).

[0068] EMV/virus preparations, immunoblot, coimmunoprecipitation, density gradient analysis, and gel filtration.

[0069] EMVs and viruses were collected from the medium as described previously (Booth et al, 2006; Fang et al, 2007). For immunoblot experiments, cells were transfected and incubated for 2 d, EMVs/viruses were collected, and cells and EMVs/viruses were lysed in SDS-PAGE sample buffer. Each sample was separated by SDS-PAGE, transferred to PVDF membranes, and processed for immunoblot using specific primary antibodies and HRP-conjugated secondary antibodies, followed by chemiluminescent detection and detection of proteins by exposure of x-ray film. Films were scanned and converted to TIFF files, and the signal for each band was quantified using ImageJ software. Budding efficiencies were calculated from extent of budding (vesicle/vesicle + cell), relative to that of the positive control. For those experiments that were performed three or more times, the data are also presented as the average ± 1 SD, along with the p value from a Student's t test.

[0070] For co-IP experiments, 293T cells were transfected with plasmids encoding both test proteins, at a 1 : 1 ratio, and incubated for 2 d. Cells were then resuspended in RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% NP-40, 1 mM EDTA, and a protease inhibitor cocktail; Roche, Basel, Switzerland) at 4°C, lysed by freeze-thaw (two cycles), and clarified by centrifugation at 16,000 x g for 10 min. The resulting supernatant was precleared by incubating with protein A beads (Sigma) and nonimmune rabbit IgG for 2 h at 4°C. The beads were removed, each sample was split in half, one-half was incubated O/N at 4°C with nonimmune rabbit IgG, and the other half was incubated O/N at 4°C with rabbit polyclonal antibodies to GFP. Each sample was incubated with protein A beads for 2 h at 4°C, and the beads were washed six times with RIPA buffer and boiled in SDS-PAGE loading buffer. Samples were then separated by SDS-PAGE, transferred to PVDF membranes, and processed for immunoblot using mouse monoclonal antibodies specific for either the flag tag or HIV Gag.

[0071] For density gradient centrifugation experiments, we transfected 293T cells with plasmids designed to express Gag or Gag-SP2, incubated the cells for 2 d, washed the cells in cold phosphate-buffered saline (PBS) (4°C), scraped from the tissue culture dish, and pelleted by centrifugation at 600 x g for 5 min. Cell pellets were washed once with 10 mM Tris-HCl (pH 7.4) containing 1 mM ethylene glycol tetraacetic acid and once with 10 mM Tris-HCl (pH 7.4) containing 1 mM EDTA and then resuspended in TE buffer (10 mM Tris-HCl containing 1 mM EDTA), 10% (wt/vol) sucrose, and Complete Protease Inhibitor Cocktail (Roche). Cells were then lysed by sonication. Lysates were pelleted by spinning at 2000 rpm for 3 min in an Eppendorf Micro fuge to remove unbroken cells and nuclei. Each sample was resuspended in TE buffer, and 0.25 ml of each was added to 1.5-ml microcentrifuge tubes. One was untreated, one was adjusted to 0.25% Triton X-100 and incubated at 37°C for 20 min, and one was adjusted to 0.25% Triton X-100 and incubated at 4°C for 20 min. Then 1.25 ml cold 85.5% (wt/vol) sucrose/TE buffer was added to each of the three tubes, and these were placed on the bottom of a centrifuge tube. To each we layered 7 ml 65% (wt/vol) sucrose/TE and then 3.25 ml 10% (wt/vol) sucrose/TE. The gradients were then spun at 100,000 x g for 18 h at 4°C in a Beckman SW41 rotor. At the conclusion of the spin, we collected 10 1.2-ml fractions from the top of the centrifuge tube and processed each for immunoblot using antibodies specific for Gag.

[0072] For gel filtration experiments, we transfected 293T cells with plasmids designed to express Gag or Gag-SP2, incubated the cells for 2 d, washed the cells in cold PBS (4°C), lysed the cells in RIPA buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40, protease inhibitor cocktail [Roche]) by two freeze-thaw cycles, and clarified the lysates by passing them through a 0.22-μιη filter. Before fractionation of the clarified lysates, we generated a pair of Sephacryl-500 HR resin (GE Healthcare, Piscataway, NJ) columns (1 cm diameter) by loading two columns each with a slurry of 15 ml Sephacryl-500 HR resin and 4 ml gel filtration buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA). Columns were packed by flowing gel filtration buffer through the resin for 2 h at 1 ml/min and then for 1 h at 2.3 ml/min. Then 0.5 ml of each lysate was loaded at the top of the column and

fractionated by flowing gel filtration buffer at 1.5 ml/min. Fifteen 1-ml fractions were collected from each column, and each fraction was examined by immunoblot using antibodies specific for HIV Gag and β-tubulin.

[0073] HIV infectivity measurements. To determine the relative infectivity of different HIV proviruses, we adapted an established assay (Zhang et al, 2004) as follows. First, 6.7 μg of each HIV provirus plasmid was cotransfected with 3.3 μg of pVSV-G into 7.5 x 106 293T cells by electroporation. Two days later the tissue culture supernatants were collected, and the cells were fixed and examined by fluorescence microscopy to determine the percentage of cells expressing the provirus-encoded GFP protein (NL4.3* encodes a modified form of GFP that is localized to the lumen of the endoplasmic reticulum [Zhang et al, 2004 *]). The tissue culture supernatants were processed by passage through a 0.2-μιη filter and pelleted by centrifugation at 70,000 x g for 1 h. Each pellet was resuspended in 30 μΐ RPMI medium supplemented with 20% fetal calf serum (FCS), and 1 μΐ of each was mixed with 5 x 105 CD4+ T-cells (Jurkat). The cell/virus mixtures were then spun at 1200 x g for 2 h at room temperature. Each cell/virus mixture was then resuspended in 1 ml RPMI supplemented with 20% FCS and incubated for an additional 2 d. The cells were then fixed and examined by fluorescence microscopy to determine the number of cells expressing the virus-encoded GFP. These experiments included an initial titration of control virus preparations to ensure the percentage of Jurkat cells infected by the WT control provirus was -15%, within the linear range of the assay (Zhang et al, 2004). Each experiment was performed a minimum of three times, and the data are presented as the average ± 1 SD, along with the p value from a Student's t test.

EXAMPLE 1

[0074] C-terminal exposure of spacer peptide 2 blocks the budding of HIV Gag. The precise removal of the p6 domain from HIV Gag can cause a severe defect in HIV budding from 293T and certain other cell types. On the basis of these (and other) observations, it has been proposed that the p6 domain represents the positive budding signal in HIV and HIV Gag. However, it has also been demonstrated that loss of p6 has little or no effect on the budding of HIV from human T-cells, a result that argues against a primary role for the p6 domain in Gag and virus budding. As a first step toward resolving this paradox, we tested whether HIV Gag displayed a similar dependence on its p6 domain for budding from 293T cells. Specifically, we transfected 293T cells with plasmids designed to express wild-type (WT) HIV Gag or a p6-deficient form of HIV Gag, Gag(p49). We then incubated the cells for 2 d, collected cells and secreted vesicles (EMVs), separated the samples by SDS-PAGE, and processed them for immunoblot using antibodies specific for HIV Gag. Loss of the p6 domain caused an approximately sevenfold decrease in budding (Figure IB), to 15 ± 3.0% (average ± 1 SD) of WT HIV Gag (n = 3; p = 4.3 x 10-4). Thus deletion of the p6 domain causes a severe defect in the budding of Gag from 293T cells.

[0075] The severe budding defect caused by loss of the p6 domain is thought to be caused by the concomitant loss of specific ESCRT -binding motifs that lie within the p6 domain and, in particular, to the loss of the PTAP motif that binds the ESCRT protein tumor suppressor gene 101 (TSG101). If this model is correct, elimination of the PTAP motif, by mutating it to the amino acids LIRL, should also cause a severe defect in budding. However, when we compared the budding of Gag(p6PTAP-LIRL) to that of Gag(p49) and WT HIV Gag (Figure IB), we found that Gag(p6PTAP -LIRL) budded just as well as WT HIV Gag (1 10 ± 26%; n = 3; p = 0.56). Thus the severe budding defect of p6-deficient HIV Gag was not caused by loss of the PTAP motif. As for whether the budding defect might be caused by loss of an Alix-binding motif, the Gag protein we worked with in these studies lacks the p6-localized Alix-binding site found in some other HIV strains.

[0076] Given that the severe budding defect of Gag(p49) is not caused by loss of the sole ESCRT-binding site (PTAP) in p6, we considered the possibility that some other difference was responsible for the severe budding defect of p6-deficient Gag. One obvious difference is that Gag(p49) has a new C terminus composed of the spacer peptide 2 (SP2) domain (SP2 has the sequence FLGKIWPSHKGRPGNFCOOH (SEQ ID NO: 1) and lies between the NC and p6 domains of the Gag translation product; Figure 1A). To determine whether C-terminal exposure of SP2 might contribute to the budding defect of Gag(p49), we examined the effect of masking its C terminus. Specifically, we appended epitope tags to the C terminus of Gag(p49) and followed the budding of the resulting proteins (Figure IB). Gag(p49)-3xmyc and Gag(p49)-SF budded far better than Gag(p49) and no differently from WT HIV Gag (80 ± 15% and 109 ± 28%, respectively; n = 3; p = 0.15 and 0.61, respectively). The SF tag is a 61 amino acid-long tag carrying four copies of the Strep tag and two copies of the Flag tag.

[0077] To further explore the idea that C-terminal exposure of SP2 might inhibit Gag budding, we followed the budding of Gag-SP2, a full-length HIV Gag protein containing an additional copy of the SP2 peptide appended to its C terminus. 293T cells were transfected with plasmids designed to express either WT HIV Gag or Gag-SP2 and incubated for 2 d, and then cell and EMV lysates were collected. Immunoblot analysis of these samples using anti- Gag antibodies revealed a severe budding defect for Gag-SP2 (Figure 1C), only 4.2 ± 3% relative to the WT control (n = 4; p = 6.3 x 10-6). This result demonstrates that C-terminal exposure of SP2 has a potent inhibitory effect on Gag budding. Additional experiments revealed that the internal copy of SP2 in WT HIV Gag did not inhibit its budding and did not contribute significantly to the poor budding of Gag-SP2 (Figure 1C). The potent effect of exposing SP2 at the C terminus was also observed in the context of Gag(p49)-SF (Figure 1C), as Gag(p49)-SF-SP2 budded very poorly, only 1.7 ± 0.8% in comparison to Gag(p49)-SF (n = 3; p = 2.3 10-5).

[0078] These results support the methods of the present invention that the severe budding defect displayed by Gag(p49) is also caused by C-terminal exposure of SP2. As such, a Gag protein with a slightly larger deletion, one that removes the p6 domain and also the inhibitory elements within SP2, should bud from 293T cells nearly as well as WT HIV Gag. To test this prediction, we compared the budding of Gag(p49) to that of Gag(p47), which lacks all of p6 and also the C-terminal 12 amino acids of SP2 (Figure ID). In each of three trials we observed that this shorter protein, Gag(p47), budded as well as WT HIV Gag (103 ± 7%; p > 0.05) and far better than Gag(p49) (14 ± 3% of WT; n = 3; p < 0.0005). These data demonstrate that the budding defect of p6-deficient HIV Gag was caused primarily by C- terminal exposure of SP2 and not by loss of any positive budding information that may reside within the p6 domain. Thus, this is a potential target for anti-HIV and antiretroviral therapy.

EXAMPLE 2

[0079] C-terminal exposure of SP2 activates a cis-acting inhibitory budding signal. The inhibitory effect of C-terminal SP2 exposure could be restricted to just HIV Gag.

Alternatively, it could reflect the existence of an inhibitory budding signal (IBS) that can impair the budding of heterologous proteins. To distinguish between these possibilities, we appended the C-terminal 12 amino acids of SP2 (IWPSHKGRPGNFCOOH (SEQ ID NO: 2), which we refer to as SP2*) to the C terminus of several other budding-competent proteins and assayed their budding from 293T cells. Gag proteins from retroviruses typically bud well from animal cells, and SF-tagged versions of the equine infectious anemia virus (EIAV), Rous sarcoma virus (RSV), and human T-lymphotropic virus 1 (HTLV-1) Gag proteins all budded at high levels from 293T cells (Figure 2A). Moreover, we found that addition of SP2* to the C terminus of these proteins impaired their budding from 293T cells (Figure 2A), to 24 ± 2% in the case of EIAV Gag-SF-SP2* (n = 3; p = 0.0002), 5.2 ± 7% in the case of RSV Gag-SF-SP2* (n = 3; p = 0.002), and 5.7 ± 5% in the case of HTLV-1 Gag-SF-SP2* (n = 3; p = 8 10-6). Given that the HIV, EIAV, RSV, and HTLV-I Gag proteins lack significant amino acid sequence similarity and share only a single, short motif

(QXXXEXXXXXOOXRO) within their CA domains, these results indicate that C-terminal exposure of SP2* activates an IBS that can block the budding of heterologous proteins.

[0080] To test whether the activity of the IBS extended to nonviral EMV proteins, we created plasmids designed to express SF-tagged forms of CD63 and CD63-SP2*, transfected these into 293T cells, and assayed EMV and cell lysates for the relative budding of each protein. CD63 is an integral membrane protein that is secreted in EMVs by numerous cell types and is the most commonly used marker of EMVs. CD63-SF budded well from 293T cells (Figure 2B), demonstrating that this C-terminally tagged form of human CD63 is an EMV cargo protein. The budding of CD63-SF-SP2* was substantially less than CD63-SF (3.0 ± 3%; n = 3; p = 0.0004), supporting the hypothesis that C-terminal exposure of SP2* activates an IBS. We also examined the effect of the IBS on Acyl-LZ-DsRED (Fang et al, 2007 Acyl-LZ-DsRED budded well from 293T cells whereas Acyl-LZ-DsRED-SP2* displayed no detectable budding from these cells (Figure 2B).

[0081] We next tested whether the IBS impaired the budding of all EMV cargoes from the cell or whether its effect was limited to the protein to which it was attached. To do this we cotransfected 293T cells with plasmids designed to express 1) the EMV marker CD63-SF and 2) either green fluorescent protein (GFP) or Gag-SP2. After incubating the cells for 2 d, we collected cell and EMV lysates and processed them for immunoblot using antibodies specific for the SF tag. The budding of CD63-SF was no less in cells expressing Gag-SP2 than in cells expressing GFP (Figure 2C), indicating that expression of an IBS-containing cargo does not impair EMV budding in general. In fact, expression of Gag-SP2 led to a slight increase in the budding of CD63-SF, to 140 ± 14% (n = 3; p = 0.047).

EXAMPLE 3

[0082] Mutational analysis of the IBS. To better understand the structural basis for IBS function, we performed a series of mutational studies, using Gag-SP2 as the test protein. Deletion of just the C-terminal amino acid, F 16, resulted in a Gag-SP2 protein that budded quite well from 293T cells, and loss of additional residues from the SP2 C terminus had similar effects (Figure 3, A and B). Removing the N-terminal 2 or 4 amino acids of SP2 had little if any effect on IBS activity, while deletion of the N-terminal 7 amino acids impaired IBS activity, and larger deletions seemed to eliminate IBS function altogether (Figure 3, A and B). These observations are consistent with 1) the ability of the C-terminal 12 amino acids of SP2 (SP2*, amino acids 5-16 of SP2) to block the budding of viral and nonviral EMV cargoes, and 2) the observation that removing SP2* is what allowed Gag(p47) to bud much better than Gag(p49) and nearly as well as WT HIV Gag (see Figure ID).

[0083] On the basis of these results, it appeared that the IBS was located within the 12 amino acids from Ile-5 to Phe-16. To identify the amino acids within this region that are critical to IBS function, we replaced each of them with alanine, again in the context of the Gag-SP2 protein (Figure 3C). Consistent with our previous observations, Gag-SP2 budded poorly, 4.2 ± 3% (n = 4; p < 0.0005) relative to full-length HIV Gag. Similar results were observed for Gag-SP2 variants in which alanine had been substituted for Pro-7 (5.8 ± 3%), Ser-8 (9.9 ± 4%), His-9 (8.2 ± 3%), Lys-10 (4.8 ± 3%), Gly-11 (6.6 ± 2%), or Gly-14 (4.2 ± 2%), indicating that these amino acids do not play essential roles in IBS function (numbers are from four trials, and in all cases there was no significant difference from the budding of Gag-SP2 (p > 0.05). In contrast, substituting alanine for several other residues effectively eliminated IBS activity, seen here by the high levels of budding detected for the Ile-5A (190 ± 16%, relative to full-length HIV Gag), Trp-6A (210 ± 8%), Arg-12A (190 ± 22%), Pro-13A (200 ± 22%), Asn-15A (240 ± 25%), and Phe-16A (130 ± 13%) variants of Gag-SP2 (n = 4; p < 0.005). These mutants also budded slightly more than WT HIV Gag (p < 0.05).

[0084] Although the single alanine substitutions identified several critical residues within the IBS, these particular alanine substitution mutations cannot be introduced into an HIV proviral clone without also changing the amino acid sequence of the Gag-Pol polyprotein (the ribosomal frameshift in the translation of the Gag open reading frame [ORF] to generate the Gag-Pol protein occurs just upstream of the SP2 coding region). We therefore examined the relative budding of four additional substitution mutations in SP2, three single amino acid substitutions (SP2R12K, SP2P13L, and SP2N15S) and a four-amino acid substitution (SP2RPNF-KLSS). Each of these mutations impaired IBS function in the context of the Gag- SP2 protein (Figure 3D).

[0085] We also tested whether one of these mutations could inactivate the IBS in the context of a nonviral EMV cargo. For this we compared the budding of CD63-SF-SP2* to that of CD63-SF-SP2*RPNF-KLSS. The budding of CD63-SF-SP2*RPNF-KLSS was higher than that of CD63-SF-SP2* (Figure 3E), consistent with the hypothesis that these residues play an important role in IBS activity.

EXAMPLE 4

[0086] IBS inactivation suppresses the budding defect of p6-deficient HIV. The preceding observations demonstrated that the severe budding defect of p6-deficient HIV is effectively phenocopied by p6-deficient HIV Gag protein and that this budding defect is caused primarily by C-terminal exposure of SP2 and the concomitant activation of an IBS. However, our studies were carried out on a Gag protein from a different strain (type C) than that used in most studies on HIV budding (type B). Moreover, they were carried out on Gag and not virus. To determine whether our findings were relevant to a type B HIV strain, we tested whether the IBS was responsible for the severe budding defect of a p6-deficient from of NL4.3, a commonly used type B clone. NL4.3* is our designation for NL4-3-AE-GFP, a variant of NL4.3 designed for measuring HIV infectivity and the parental clone we used for all experiments. To eliminate p6 expression, we used the p6Llter mutation, which terminates the HIV Gag ORF at the first codon (Leu) of p6 and causes a severe budding defect in 293T cells.

[0087] To explore the contribution of the IBS to the budding defect of p6-deficient HIV, we introduced IBS-inactivating mutations into NL4.3*(p6Llter), transfected control and mutant proviruses into 293T cells, collected cells and viruses, and processed cell and virus lysates for immunoblot using anti-Gag antibodies. As expected, NL4.3* budded well from 293T cells, whereas the budding of NL4.3*(p6Llter) was significantly reduced, only 23 ± 3% of control (n = 3; p = 5.5 x 10-4) (Figure 4B). In contrast, forms of NL4.3*(p6Llter) that carried inactivating mutations in the IBS budded at nearly WT levels, 94 ± 2% for

NL4.3*(SP2R12K/p6Llter), 1 10 ± 7% for NL4.3*(SP2P13L/p6Llter), 100 ± 2% for NL4.3*(SP2N15S/p6Llter), and 96 ± 3% for NL4.3*(SP2RPNF-KLSS/p6Llter) (n = 3; p = 0.024, 0.18, 0.96, and 0.12, respectively). These data demonstrate that inactivation of the IBS suppresses the budding defect of p6-deficient HIV. As such, these observations provide strong evidence that the budding defect of p6-deficient virus is caused primarily by the C- terminal exposure of SP2 and the concomitant activation of the IBS, and not by loss of any positive budding information that might reside within the p6 domain. EXAMPLE 5

[0088] The IBS data shows that the N and C termini of junctional peptides contributes to HIV infectivity. We also assessed the relative infectivity of these viruses. For this we took advantage of the fact that L4.3* allows one to measure the infectivity of mutant HIV viruses by direct visualization of transfected and infected cells (NL4.3* encodes a form of GFP that is localized to the endoplasmic reticulum. In brief, we 1) cotransfected HIV proviruses into 293T cells together with a plasmid designed to express VSV-G, a fusogenic ENV protein; 2) incubated the cells for 2 d; 3) collected virus samples; 4) used them to infect CD4+ T-cells; and 5) scored the relative infectivity of each provirus by counting the number of NL4.3*-expressing (GFP-positive) T-cells, normalized to the number of NL4.3*- expressing 293T cells in the first phase of the assay. These experiments revealed that the p6Llter mutation reduced HIV infectivity ~50-fold (Figure 4C), to 1.4 ± 0.2% of WT HIV (n = 4; p < 0.0005). In contrast, we observed a three- to fivefold increase in the infectivity of p6- deficient HIV when the IBS was also inactivated, to 7.6 ± 0.9% for NL4- 3*(p6Llter/SP2RPNF-KLSS), 4.5 ± 0.6% for NL4-3*(p6Llter/SP2R12K), 5.1 ± 0.8% for NL4-3*(p6Llter/SP2P13L), and 5.2 ± 1% for NL4-3*(p6Llter/SP2N15S).

[0089] On the basis of these observations, it appears that IBS inactivation fully suppresses the budding defect of p6-deficient HIV but only partially suppresses its infectivity defect. This is not surprising when one considers that 1) the HIV accessory protein Vpr plays critical postentry roles in the HIV life cycle, and 2) loss of p6 eliminates the Vpr-binding site that HIV uses to recruit Vpr into nascent virions. Moreover, the fact that the IBS has potent biological activity and is conserved between simian immunodeficiency virus (SIV) and HIV (Figure 4D) raises the possibility that the IBS might itself contribute to HIV infectivity. To test this hypothesis, we introduced the SP2P 13L mutation into NL4.3* and determined its effect on HIV infectivity. The P13L mutation reduced HIV infectivity approximately sevenfold (Figure 4D), to 15 ± 2% of control (n = 3; p < 0.0005). This effect was not caused by a drop in HIV budding (Figure 4E) as this virus budded at the same level as WT control (96 ± 5%; n = 4; p = 0.27). These data demonstrate that the IBS and other junctional peptides of the present invention play a positive role in the HIV life cycle.

EXAMPLE 6 [0090] The budding defect of PTAP-deficient HIV requires Gag-Pol and the IBS. We next attempted to understand why PTAP-deficient HIV Gag buds at nearly WT levels (see Figure 1, A and B) whereas PTAP-deficient virus has a budding defect as severe as p6- deficient HIV, seen here by the poor budding of both NL4.3*(p6PTAP-LIRL) and

NL4.3*(p6Llter) (Figure 5, A and B). Intriguingly, cells expressing PTAP-deficient HIV generated significantly higher levels of Gag(p49) than the control virus. This raised the possibility that loss of the PTAP motif results in the activation of the IBS, via the aberrant, PR-mediated cleavage of Gag at the SP2/p6 junction.

[0091] If this hypothesis is correct, it should be possible to suppress the budding defect of PTAP-deficient HIV by eliminating expression of the Gag-Pol polyprotein (the viral protease, PR, is generated only via expression of Gag-Pol). To test this prediction, we created a form of HIV, NL4.3*(TFter), that is unable to express the Gag-Pol polyprotein due to a nonsense mutation in the transframe region of the Gag-Pol ORF (the TFter mutation blocks Gag-Pol translation but does not alter the predicted translation product of the Gag ORF [Figure 5 A]). Following transfection of 293T cells with the corresponding proviruses, we found that the TFter mutation suppressed most of the budding defect caused by loss of the PTAP motif: NL4.3*(TFter/p6PTAP-LIRL) budded from cells at 83 ± 2% (n = 4; p = 0.0054) of that seen for NL4.3*(TFter). In contrast, the TFter mutation did not suppress the budding defect of p6- deficient HIV (Figure 5C), as NL4.3*(TFter/p6Llter) budded at only -7% the level of NL4.3*(TFter) (6.9 ± 1%; n = 4; p = 9.9 ^χ 10-6). The budding defect of

NL4.3*(TFter/p6Llter) could, however, be suppressed by elimination of SP2, shown here by the nearly normal budding of NL4.3*(TFter/SP2I5ter) (92 ± 3% relative to control; n = 4; p = 0.012). This virus has a stop codon in place of Ile-5 of the SP2 domain, expresses neither the IBS nor the p6 domain, and budded ~13-fold more than NL4.3*(TFter/p6Llter).

[0092] If the budding defect of PTAP-deficient HIV involves the PR-mediated activation of the IBS, it should also be suppressed by inactivating mutations in the IBS. To test this prediction, we compared the budding of NL4.3*, NL4.3*(p6PTAP-LIRL), and

NL4.3*(SP2RPNF-KLSS/p6PTAP-LIRL). Following the transfection of 293T cells with the corresponding viruses, we found that NL4.3*(p6PTAP-LIRL) budded at 8.7 ± 0.4% of control virus (n = 3; p = 5.3 x 10-6) and that inactivation of the IBS largely suppressed this budding defect (Figure 5D), elevating it eightfold to 70 ± 3% of control (n = 3; p = 0.001). EXAMPLE 7

[0093] The IBS does not block membrane binding. In an effort to understand how the IBS functions, we first examined its effect on the membrane-binding activity of HIV Gag. 293T cells were transfected with plasmids designed to express HIV Gag or HIV Gag-SP2, incubated for 2 d, and lysed in hypotonic buffer to generate membrane fragments. The lysates were adjusted at a high concentration of sucrose (73%) and split into several tubes, and then one portion was fractionated by sucrose density flotation gradient centrifugation. Under these conditions, free proteins remain at the bottom of the gradient whereas membrane-associated proteins float to upper fractions due to the low density of the membranes to which they are attached. When the resulting fractions were assayed for the presence of Gag proteins by immunoblot, we found that both Gag and Gag-SP2 floated out of the initial, high-density fraction into the lower density fractions, as expected for membrane-associated proteins (Figure 6A). The only difference in the behavior of Gag-SP2 was that it was more highly enriched in the membrane-associated fractions (140 ± 18%; n = 3; p = 0.050).

[0094] To test whether the flotation of these proteins was truly due to their association with membranes, we preincubated an aliquot of each lysate with 0.25% Triton X-100 for 20 min at 37°C, a treatment that is known to solubilize biological membranes. These samples were also subjected to sucrose density gradient centrifugation, and the resulting fractions were analyzed by immunoblot using anti-Gag antibodies. Following this treatment, neither protein floated out of the initial high-density fractions (Figure 6B). In contrast to treatment with warm Triton X-100, treatment with cold Triton X-100 fails to fully solubilize membrane domains that are enriched in cholesterol and sphingolipids, and HIV Gag is enriched in these detergent-resistant membrane (DRMs) fractions. To determine whether the IBS might impair this aspect of Gag activity, we added cold Triton X-100 to yet another portion of the lysates and then subjected them also to sucrose density gradient centrifugation. Gag and Gag-SP2 were both enriched in DRMs (Figure 6C), though Gag-SP2 showed a slightly greater enrichment in DRM fractions (140 ± 3%; n = 3; p = 0.002) than WT Gag.

EXAMPLE 8

[0095] The IBS does not prevent plasma membrane localization. We next tested whether the IBS might act by preventing its trafficking to the plasma membrane. Immunofluorescence microscopy revealed that WT HIV Gag was distributed throughout the cell (Figure 7, A-D), consistent with prior reports on the steady-state distribution of this protein (Freed, 1998 *·; Freed and Martin, 2006 ; Morita and Sundquist, 2004 In contrast, Gag-SP2 was highly enriched at the plasma membrane and in many cells was concentrated at large patches of the plasma membrane (Figure 7, E-H). Thus the IBS did not prevent the trafficking of Gag to the plasma membrane. This conclusion is also supported by the trafficking of Gag(p49), which was also enriched at the plasma membrane (Figure 7, 1 and J). In contrast, Gag(p47) displayed an intracellular distribution that more closely resembled that of WT HIV Gag, with no apparent enrichment at the plasma membrane (Figure 7, K and L). Perhaps the simplest interpretation of these data is that the inability of IBS-activated proteins to bud from cells merely causes them to accumulate at the prior step in their journey, the plasma membrane.

[0096] We also tested the effect of the IBS on the subcellular localization of CD63-SF. This protein buds well from 293T cells (see Figure 2) and localizes to the plasma membrane, and addition of the IBS did not prevent its plasma membrane localization (Figure 7, M-P). These images are representative of >90% of expressing cells, and each protein was examined in 100 or more expressing cells.

[0097] The plasma membrane enrichment of Gag-SP2 should also be apparent in transmission electron micrographs, as the highly oligomeric Gag complex forms an electron- dense lamina under membranes to which it is attached. 293T cells expressing WT HIV Gag were surrounded by numerous vesicles containing the electron-dense lamina (Figure 7, Q and R). Cells expressing Gag-SP2 differed in that they failed to secrete Gag-containing vesicles and instead contained a Gag-SP2 lamina underlying large patches of the plasma membrane (Figure 7, S-U). Although we occasionally detected cell profiles in which the Gag-SP2 lamina extended from the plasma membrane (Figure 7V), these were "headless" extensions that bore no resemblance to the budding intermediates that accumulate in cells expressing the classic late-domain mutant Gag(p6PTAP-LIRL) (Figure 7, W and X). Given that the accumulation of trapped budding intermediates is the defining characteristic of a "late" budding defect, the IBS appears to block Gag budding at an earlier stage in the budding process. EXAMPLE 9

[0098] The IBS does not prevent Gag-Gag oligomerization. Protein budding, including the budding of HIV and HIV Gag, is driven by a combination of plasma membrane targeting and higher-order oligomerization (Fang et al, 2007 Given that Gag-Gag oligomerization is required for the formation of an electron-dense lamina such as that seen in 293T cells expressing Gag-SP2, it is unlikely that the IBS impairs cargo budding by preventing its oligomerization. Furthermore, it is unclear how such a short peptide (12 amino acids) could impair the budding of so many structurally unrelated cargoes (retroviral Gag proteins, CD63, Acyl-LZ-DsRED). Nevertheless, we assessed the effect of the IBS on Gag-Gag

coimmunoprecipitation. As a prelude to such experiments, we tested whether Gag and Gag- SP2 proteins carrying an N-terminal AcylSF tag (specifying N-terminal acylation and containing multiple Strep and Flag tags) retained the relative budding activities of untagged Gag and Gag-SP2. They did (Figure 8A). Next, we cotransfected 293T cells with plasmids designed to express 1) Gag-3xmyc and 2) either AcylSF-Gag or AcylSF-Gag-SP2. Two days later we lysed the cells in detergent buffer, immunoprecipitated Gag-3xmyc, and probed the resulting immunoprecipitates for the presence of AcylSF-Gag or AcylSF-Gag-SP2, respectively. These experiments revealed that the IBS did not reduce Gag-Gag

coimmunoprecipitation (Figure 8B).

[0099] HIV Gag forms oligomeric complexes with upward of 1000 Gag subunits and a size of 50 MDa, well above the resolution of size exclusion chromatography. Nevertheless, Gag assembly in vivo proceeds from small oligomers, and thus it is formally possible that gel filtration chromatography might nevertheless expose some differences in the oligomerization profile of Gag and Gag-SP2. Toward this end we transfected 293T cells with plasmids designed to express either WT Gag or Gag-SP2, lysed the cells in radio immunoprecipitation assay (RIPA) buffer, fractionated the lysates on a Sephacryl-500 gel filtration resin (size exclusion limit of -500 kDa), and assayed each fraction by immunoblot using antibodies specific for HIV Gag and β-tubulin (Figure 8C). The elution profiles of Gag and Gag-SP2 were very similar, but there appeared to be a slightly higher proportion of Gag-SP2 in the late void volume fractions, 5 and 6. As with the enhanced plasma membrane localization of Gag- SP2, this difference might be a relatively simple consequence of the severe budding defect of Gag-SP2 because the higher-order oligomerization of WT Gag leads to its release from the cell. EXAMPLE 10

[0100] IBS activation impairs an EMV cargo-VPS4 interaction. The ESCRT proteins comprise the only machinery that is known to catalyze outward vesicle budding. This role has been demonstrated in vitro and is consistent with the observation that ESCRT dysfunction inhibits MVB biogenesis, cytokinesis, and HIV budding. Although the inhibition of ESCRT function does not prevent EMV budding, a role for the ESCRT machinery in EMV biogenesis cannot be excluded. We therefore explored the possibility that EMV cargoes might interact with the ESCRT machinery. Specifically, we explored the possibility that EMV cargoes interacted with vacuolar protein sorting 4 (VPS4), an ESCRT-associated ATPase that binds and disassembles complexes of ESCRT proteins and their cargoes. 293T cells were cotransfected with plasmids designed to express GFP-VPS4B together with various EMV cargoes, incubated for 2 d, and then lysed. GFP-VPS4B and associated proteins were subjected to immunoprecipitation (IP) using anti-GFP or control antibodies, and the resulting IPs were examined by SDS-PAGE and immunoblot using antibodies specific for the EMV cargoes. These experiments revealed that there is a specific interaction between GFP-VPS4B with CD63-SF (Figure 9A). Furthermore, we found that the presence of the IBS at the C terminus of CD63-SF significantly impaired this interaction. We also detected a specific interaction between GFP-VPS4B and CD81-SF, and this too was significantly reduced by the presence of the IBS (Figure 9B). Similar results were observed for Gag(p47), which buds from cells and showed a significant interaction with GFP-VPS4B. In contrast, the 12 amino acid-longer Gag(p49) protein, which exposes the IBS at its C terminus and buds poorly from cells, showed little if any specific interaction with GFP-VPS4B (Figure 9C). These data represent the first evidence that there is an interaction between the ESCRT machinery and budding-competent proteins. Furthermore, they demonstrate that the IBS attenuates this interaction. These data do not, however, demonstrate a direct physical interaction between VPS4B and EMV cargoes or that the IBS impairs protein budding by impairing this cargo-ESCRT interaction.

[0101] The present invention demonstrates the existence of the IBS, and that junctional peptides have a role on protein and virus budding. Thee ability of IBS-inactivating mutations to suppress the budding defects of p6-deficient and PTAP-deficient HIV show that these peptides are significant new drug targets for HIV treatment. In its place, our data suggest a new paradigm in which the budding defects of these particular mutant viruses are caused by activation of the IBS, either directly in the case of p6-deficient HIV or indirectly in the case of PTAP-deficient HIV. Our data also support the hypothesis that the main source of positive budding information in Gag lies not in p6 but instead in the MA-CA-NC region of the protein.

[0102] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[0103] The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to,") unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[00100] Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

Claims:

1. A system for identifying compounds which modulate HIV infection in a mammalian cell comprising:

a) obtaining a population of mammalian cells in vitro capable of being

infected by HIV;

b) infecting the population of cells with a HIV virus that has been mutated such that the virus cannot produce at least one or more of the following junctional peptides cleaved from the GAG protein: matrix, capsid, SPl, nucleocapsid, SP2 and p6;

c) adding to the population of cells from b) the at least one peptides and a target agent or control agent;

d) allowing the population to grow for a sufficient period of time; and e) assaying the population of cells for the quantity of cells infected with HIV was increased or decreased in the presence of the target agent when compared with the control agent.

2. The method of claim 1, wherein the target agent binds to the N or C terminus of the at least one or more junctional peptides cleaved from the GAG protein: matrix, capsid, SPl, nucleocapsid, SP2 and p6.

3. The method of claim 1, wherein the target agent inhibits the interaction of the N or C terminus of the at least one or more junctional peptides cleaved from the GAG protein: matrix, capsid, SPl, nucleocapsid, SP2 and p6 with a membrane protein in the plasma membrane of the mammalian cell or population of cells.

4. The method of claim 3, wherein the membrane protein is actin.

5. The method of any of claims 1 to 4, wherein when the population of cells for the quantity of cells infected with HIV was decreased in the presence of the target agent, the target agent is identified as an inhibitor of infectivity.

6. The method of any of claims 1 to 5, wherein the target agent is selected from the group consisting of antibodies, oligonucleotides such as siRNA or microR A, small molecules, peptides and derivatives thereof.

7. The method of claim 6, wherein the target agent is a small molecule.

8. Use of a target agent identified using the methods of any of claims 1 to 7, in the preparation of a medicament, comprising the target agent and a pharmaceutically acceptable carrier, suitable for use in treating a subject infected with HIV.

9. Use of a target agent identified using the methods of any of claims 1 to 7, in the preparation of a medicament, comprising the target agent, and at least one other antiretroviral agent, and a pharmaceutically acceptable carrier, suitable for use in treating a subject infected with HIV.

10. A target agent identified using the methods of any of claims 1 to 7.