Peptides comprising an Rpt1 domain of an INI1/hSNF5 which inhibit HIV-1 production in a human cell, and vectors encoding those peptides are provided. Also provided are methods of inhibiting HIV-1 production in a cell, or spread of the HIV-1 to another cell, by treating the cells with the above peptides or vectors. Other methods of inhibiting HIV-1 production in a cell, or spread of the HIV-1 to another cell, by inhibiting production of INI1/hSNF5 are provided. Additionally, methods of determining whether a test compound inhibits HIV-1 virion production in a mammalian cell, or spread of the HIV-1 to another cell, are provided. Those methods comprise determining whether the test compound inhibits the production of INI1/hSNF5 or disrupts the interaction of HIV-1 integrase with INI1/hSNF5.

Description of the Invention

SUMMARY OF THE INVENTION

The present invention is based on the discovery that fragments of INI/hSNF5 that comprise the Rpt1 domain, aa 183-245, inhibit HIV-1 replication, particle production, and cell-to-cell spread. The HIV-1 inhibitory effect is more pronounced with shorter fragments, but longer fragments, including fragments aa 1-246 and 141-385, also inhibit HIV-1.

The invention is also based on the discovery that inhibiting production of INI1/hSNF5 by a cell inhibits HIV.

Accordingly, in some embodiments the present invention is directed to peptides comprising an Rpt1 domain of an INI1/hSNF5, which inhibit HIV-1 virion production in a human cell.

In other embodiments, the invention is directed to cells expressing the above peptides, vectors encoding the above peptides, and cells transfected with those vectors.

The present invention is also directed to methods of inhibiting replication or virion production of an HIV-1 in a mammalian cell, or spread of the HIV-1 to another cell. The methods comprise treating the cell with the above peptides.

Additionally, the invention is directed to related methods of inhibiting replication or virion production of HIV-1 in a mammalian cell, or spread of HIV-1 to another cell. The methods comprise treating the cell with the above-described vectors.

The invention is additionally directed to alternative methods of inhibiting replication or virion production of HIV-1 in a cell, or spread of the HIV-1 to another cell. The methods comprise inhibiting production of an INI1/hSNF5 by the cell.

The invention is also directed to methods of evaluating whether a test compound inhibits replication or virion production of HIV-1 in a cell, or cell-to-cell spread of HIV-1. The methods comprise determining whether the test compound inhibits the production of INI1/hSNF5 in the cell.

Additionally, the invention is directed to methods of evaluating whether a test compound inhibits replication or virion production of HIV-1 in a human cell, or cell-to-cell spread of HIV-1. The methods comprise determining whether the test compound disrupts the interaction of HIV-1 integrase with INI1/hSNF5.

In still other embodiments, the invention is directed to additional methods of inhibiting replication or virion production of an HIV-1 in a cell, or spread of the HIV-1 to another cell. The methods comprise treating the cell with a compound, where the HIV-1 inhibitory activity of the test compound was determined by the above-described evaluation methods. The invention is also directed to the test compounds themselves.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the discovery, first described in Yung et al., 2001, that fragments of INI/hSNF5 that comprise the Rpt1 domain, exemplified herein as SEQ ID NO:2 (aa 183-245 of SEQ ID NO:1), inhibit HIV-1 replication, particle production, and cell to cell spread. The HIV-1 inhibitory effect is more pronounced with shorter fragments, such that the fragment consisting of aa 183-294, called the s6 fragment, completely inhibits HIV-1 particle production. However, longer fragments, including the fragment 1-245, have some inhibitory activity (see Example 1).

Without being bound by any particular mechanism, it is believed that the inhibitory fragments inhibit the interaction of INI1/hSNF5 by directly interacting with integrase within the context of Gag-Pol (see Example 1). Thus, the longer fragments are believed to inhibit HIV-1 production less than shorter fragments by providing partial INI1/hSNF5 functionality for HIV-1 replication. Since the Rpt1 domain interaction with HIV-1 integrase is the inhibitory aspect, it is expected that any peptide comprising an Rpt1 domain, other than a substantially complete INI1/hSNF5, would inhibit HIV-1 replication, particle production, and cell to cell spread.

Thus, in some embodiments, the invention is directed to peptides comprising an Rpt1 domain of an INI1/hSNF5, which inhibit HIV-1 virion production in a human cell.

Other than the Rpt1 domain, the peptide sequence is not narrowly limited and can include non-Rpt1 sequences, provided the non-Rpt1 sequences allow the Rpt1 domain to be available for interacting with HIV-1 integrase. The skilled artisan would be able to identify numerous peptides that would meet this criteria. Additionally, any peptide comprising an Rpt1 domain could be easily tested for anti-HIV-1 activity, e.g., by using the methods described in Example 1.

Thus, the non-Rpt1 regions can be another part of the INI1/hSNF5 protein, such that the peptide is a fragment of the INI1/hSNF5. The non-Rpt1 regions can also be a sequence not found in INI1/hSNF5, for example a functional protein, e.g., hemagglutinin (see Example 1). Additionally or alternately, the Rpt-1 containing peptide can be linked to a non-peptide molecule. Non-limiting examples include a nucleic acid molecule or a label such as a fluorescent molecule, a radioactive molecule, or a hapten or antigen that is subject to specific binding by a labeled antibody.

The portion of the peptide that is part of the INI1/hSNF5 protein preferably consists of amino acids 183-294 (SEQ ID NO:3) or a smaller portion which includes the entire Rpt1 domain (amino acids 183-245)(SEQ ID NO:2), however larger portions of the INI1/hSNF5 protein also are effective in inhibiting HIV-1 production. Nevertheless, as the size of the peptide that is homologous with INI1/hSNF5 increases in side, its ability to inhibit HIV-1 production decreases. For example, a peptide consisting of amino acids 141-395 of INI1/hSNF5 (SEQ ID NO:5) inhibits HIV-1 less effectively than the 183-294 peptide (SEQ ID NO:3), and a peptide consisting of amino acids 1-245 (SEQ ID NO:4) has less inhibitory than the 141-395 peptide (SEQ ID NO:5) (see Example 1).

The peptides of these embodiments prevent HIV-1 virion production in any cell capable of supporting HIV-1 replication. Preferred cells are those relevant to natural HIV-1 infection, i.e., human T cells, most preferably T-helper cells (CD4.sup.+).

In other embodiments, the present invention is directed to cells which comprise any of the above peptides that are capable of inhibiting HIV-1 virion production in a human cell. Preferably, the cells are mammalian cells, more preferably human cells, even more preferably human T cells. In most preferred embodiments, the cells are human T-helper cells. In other embodiments, the cell further comprises HIV-1. The peptide is preferably present in the cell in an amount sufficient to inhibit replication or virion production of HIV-1 in the cell, or spread of HIV-1 to another cell. Such cells will not support HIV-1 production. The peptide can be present due to treatment of the cell with the peptide. Alternatively, the peptide cen be present due to expression of the peptide (i.e., translation of genetic material present in the cell that encodes the peptide).

In additional embodiments, the invention is directed to vectors encoding peptides comprising an Rpt1 domain of an INI1/hSNF5, which inhibit HIV-1 virion production in a human cell. The peptides that can be encoded in the vectors are fully described above. In some aspects of these embodiments, the portion of the vector encoding the peptide comprises a fragment of the INI/hSNF5 gene, e.g., as provided herein as SEQ ID NO:6.

In preferred embodiments, the vector can be expressed in a mammalian cell that has been treated with the vector. Preferably, the cell is a human cell, most preferably a cell capable of being infected with HIV-1 (i.e., T-helper cells).

The vector of these embodiments are not narrowly limited to any particular form, and can be a viral vector, a plasmid vector, a cosmid vector, a linear naked DNA vector, or any other type of vector useful for any particular purpose. In aspects of the invention where the vector is used to transfect human cells to prevent HUV-1 production in those cells, preferred types of vectors are viral vectors and naked DNA vectors. In those aspects, the vector preferably causes the cell to express the truncated INI1/hSNF5 in amounts sufficient to inhibit replication or virion production of HIV-1 in the cell, or spread of HIV-1 to another cell.

Thus, in related embodiments, the invention is directed to cells transfected with the above vectors. These cells can be from any species, including bacteria or yeast (useful for storing and increasing the quantity of the vector by well-known methods) and mammalian cells (e.g., to prevent production of HIV-1 by the cell, were the cell to become infected with HIV-1). The cell can be in vitro or in vivo (e.g., a T cell in a human). Additionally, the cell can be removed from a human, transfected with the vector, then reintroduced into the human (ex vivo treatment). The cell can also further comprise HIV-1, wherein the transfection of the cell with the vector preferably causes the truncated INI1/hSNF5 to be expressed in amounts sufficient to inhibit replication or virion production of the HIV-1 in the cell, or spread of the HIV-1 to another cell.

The present invention is also directed to methods of inhibiting replication or virion production of an HIV-1 in a mammalian cell, or spread of the HIV-1 to another cell. The methods comprise treating the cell with any of the truncated INI1/hSNF5 peptides discussed above. Since peptides alone are generally unable to enter a cell, the peptides of these methods are preferably formulated in a composition that facilitates entry of the INI1/hSNF5 into the cell, such as a liposome composition, as are well-known in the art. In preferred embodiments the cell is a human cell, more preferably a human T cell, most preferably a human T-helper cell. The methods encompass in vitro, ex vivo, or in vivo treatments.

In similar embodiments, the invention is directed to other methods of inhibiting replication or virion production of an HIV-1 in a mammalian cell, or spread of the HIV-1 to another cell. These methods comprise treating the cell with any of the vectors previously discussed. Preferably, these methods also utilize human T-helper cells, and encompass in vitro, ex vivo, or in vivo treatments.

It has also been discovered that inhibiting the production of INI1/hSNF5 in the cell inhibits replication, virion production, and cell-to-cell spread of HIV-1 (see Example 1). This finding enables methods of inhibiting HIV-1 using specific inhibitory molecules such as ribozymes, antisense oligonucleotides, triplex-forming oligonucleotides and interfering RNAs, e.g. siRNAs. Techniques for the production and use of such molecules are well known to those of skill in the art.

Thus, in some embodiments, the present invention is directed to oligonucleotides comprising at least six nucleotides complementary to a contiguous sequence of a coding region of an INI1/hSNF5 gene. In these embodiments, the oligonucleotides inhibit expression of the INI1/hSNF5 gene in a cell.

An oligonucleotide sequence "complementary" to a portion of an RNA or DNA, as referred to herein, means a sequence having sufficient complementarity to be able to hybridize with the RNA or DNA, forming a stable duplex. The ability to hybridize depends on both the degree of complementarity and the length of the oligonucleotide. Generally, the longer the hybridizing oligonucleotide, the more base mismatches it may contain and still form a stable duplex (or triplex, as the case may be). One skilled in the art can ascertain a tolerable degree of mismatch by use of standard procedures to determine the melting point of the hybridized complex.

The oligonucleotides of the present invention should be at least six nucleotides in length, and are preferably ranging from 6 to about 50 nucleotides in length. In specific aspects, the oligonucleotide is at least 10 nucleotides, at least 15 nucleotides, at least 20 nucleotides or at least 50 nucleotides.

The oligonucleotides can also comprise a non-nucleotide moiety, such as a hapten, a fluorescent molecule, or a radioactive moiety, useful, e.g., to detect or quantify the amount of oligonucleotide that has entered the cell.

Oligonucleotides of the invention may be synthesized by standard methods known in the art, e.g., by use of an automated DNA synthesizer (such as are commercially available from Biosearch, Applied Biosystems, etc.). As examples, phosphorothioate oligonucleotides may be synthesized by the method of Stein, et al. (1988), methylphosphonate oligonucleotides can be prepared by use of controlled pore glass polymer supports (Sarin, et al., 1988), etc.

The oligonucleotides can be DNA or RNA or chimeric mixtures or derivatives or modified versions thereof, single-stranded or double-stranded, depending on the purpose intended. The oligonucleotide can be modified at the base moiety, sugar moiety, or phosphate backbone, for example, to improve stability of the molecule, hybridization, etc. The oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell markers in vivo, such as CD4, to improve the specificity of the oligonucleotide to cells likely to be infected with HUV-1), or agents facilitating transport across the cell membrane (see, e.g., Letsinger, et al., 1989; Lemaitre, et al., 1987; PCT Publication No. WO 88/09810) or the blood-brain barrier (see, e.g., PCT Publication No. WO 89/10134), hybridization-triggered cleavage agents (see, e.g., Krol et al., 1988) or intercalating agents (see, e.g., Zon, 1988). To this end, the oligonucleotide may be conjugated to another molecule, e.g., a peptide, hybridization triggered cross-linking agent, transport agent, hybridization-triggered cleavage agent, etc.

The oligonucleotide may comprise at least one modified base moiety including but not limited to 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) 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-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, and 2,6-diaminopurine.

The oligonucleotides may also comprise at least one modified sugar moiety including, but not limited to, arabinose, 2-fluoroarabinose, xylose, and hexose.

In other embodiments, the oligonucleotides comprise at least one modified phosphate backbone known in the art, for example a phosphorothioate, a phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a methylphosphonate, an alkyl phosphotriester, a formacetal, or analog thereof.

In additional embodiments, the oligonucleotide is an .alpha.-anomeric oligonucleotide. An .alpha.-anomeric oligonucleotide forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual .beta.-units, the strands run parallel to each other (Gautier, et al., 1987). The oligonucleotide is a 2'-O-methylribonucleotide (Inoue, et al., 1987a), or a chimeric RNA-DNA analogue (Inoue, et al., 1987b).

In some aspects of these embodiments, the oligonucleotides of the present invention are antisense nucleic acids. Antisense nucleic acid molecules act to directly block the translation of mRNA by hybridizing to targeted mRNA and preventing protein translation. Antisense approaches involve the design of oligonucleotides which are complementary to a portion of an INI1/hSNF5 mRNA. The antisense oligonucleotides will bind to the complementary protective sequence mRNA transcripts and prevent translation. Absolute complementarity, although preferred, is not required.

Antisense molecules are preferably capable of being delivered to cells that are susceptible to HIV-1 infection. A number of methods have been developed for delivering antisense DNA or RNA to cells; e.g., antisense molecules can be injected directly into the tissue site, or modified antisense molecules, designed to target the desired cells (e.g., antisense linked to peptides or antibodies which specifically bind receptors or antigens expressed on the target cell surface) can be administered systemically.

A preferred approach to achieve intracellular concentrations of the antisense sufficient to suppress translation of endogenous INI1/hSNF5 mRNAs utilizes a recombinant DNA construct in which the antisense oligonucleotide is placed under the control of a strong promoter such as a pol III or pol II promoter. The use of such a construct to transfect target cells in a patient would be expected to result in the transcription of sufficient amounts of single stranded RNAs to form complementary base pairs with the endogenous INI1/hSNF5 transcripts and thereby prevent translation of the INI1/hSNF5 mRNA. For example, a vector can be introduced such that it is taken up by a cell and directs the transcription of an antisense RNA. Such a vector can remain episomal or become chromosomally integrated, as long as it can be transcribed to produce the desired antisense RNA. Such vectors can be constructed by recombinant DNA technology methods standard in the art. Vectors can be plasmid, viral, or others known in the art, used for replication and expression in mammalian cells.

Expression of the sequence encoding the INI1/hSNF5 antisense RNA can be by any promoter known in the art to act in mammalian, preferably human cells. Such promoters can be inducible or constitutive. Such promoters include but are not limited to: the SV40 early promoter region (Bemoist and Chambon, 1981), the promoter contained in the 3'-long terminal repeat of Rous sarcoma virus (Yamamoto, et al., 1980), the herpes thymidine kinase promoter (Wagner, et al., 1981), and the regulatory sequences of the metallothionein gene (Brinster, et al., 1982). Any type of suitable plasmid, cosmid, YAC or viral vector can be used to prepare the recombinant DNA construct that can be introduced directly into the tissue site. Alternatively, viral vectors can be used that selectively infect the desired tissue, in which case administration may be accomplished by another route (e.g., systemically).

Ribozyme molecules designed to catalytically cleave INI1/hSNF5 mRNA transcripts can also be used to prevent translation of INI1/hSNF5 mRNA and, therefore, expression of the INI1/hSNF5 protein. See, e.g., PCT Publication WO 90/11364; Sarver, et al., 1990.

Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA. For a review, see Rossi, 1994. The mechanism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA, followed by an endonucleolytic cleavage event. The composition of ribozyme molecules must include one or more sequences complementary to the INI1/hSNF5 mRNA, and must include the well known catalytic sequence responsible for mRNA cleavage. For this sequence, see, e.g., U.S. Pat. No. 5,093,246.

Preferred types of ribozymes for the present invention are hammerhead ribozymes. In these embodiments the hammerhead ribozymes cleave INI1/hSNF5 mRNA at locations dictated by flanking regions which form complementary base pairs with the mRNA. The sole requirement of the hammerhead ribozyme is that the mRNA have the two base sequence 5'-UG-3', which occurs numerous times in the INI1/hSNF5 gene (see SEQ ID NO:6). The construction and production of hammerhead ribozymes is well known in the art and is described more fully in Myers, 1995, Molecular Biology and Biotechnology: A Comprehensive Desk Reference, VCH Publishers, New York, (see especially FIG. 4, page 833) and in Haseloff and Gerlach, 1988.

The ribozymes of the present invention also include RNA endoribonucleases (hereinafter "Cech-type ribozymes") such as the one which occurs naturally in Tetrahymena thermophila (known as the IVS, or L-19 IVS RNA) and which has been extensively described by Thomas Cech and collaborators (Been and Cech, 1986; Zaug, et al., 1984; Zaug and Cech, 1986; Zaug, et al., 1986; WO 88/04300, Cell, 47:207-216). The Cech-type ribozymes have an eight base pair active site that hybridizes to the INI1/hSNF5 mRNA sequence wherever cleavage of the INI1/hSNF5 RNA is desired. The invention encompasses those Cech-type ribozymes that target eight base-pair sequences that are present in the INI1/hSNF5 gene.

As with the antisense approach, the ribozymes can be composed of modified oligonucleotides (e.g., for improved stability, targeting, etc.) and should be delivered to cells that are susceptible to HIV infection in vivo, preferably T-helper cells. A preferred method of delivery involves using a DNA construct "encoding" the ribozyme under the control of a strong constitutive pol III or pol II promoter, so that transfected cells will produce sufficient quantities of the ribozyme to destroy endogenous INI1/hSNF5 gene messages and inhibit translation. Because ribozymes, unlike antisense molecules, are catalytic, a lower intracellular concentration is required for efficiency.

Alternatively, endogenous target gene expression can be reduced by targeting deoxyribonucleotide sequences complementary to the regulatory region of the INI1/hSNF5 gene (i.e., the INI1/hSNF5 gene promoter and/or enhancers) to form triple helical structures which prevent transcription of the INI1/hSNF5 gene in target cells in the body. See generally, Helene, 1991; Helene, et al., 1992; Maher, 1992.

Nucleic acid molecules to be used in triple helix formation for the inhibition of transcription should be single stranded and composed of deoxynucleotides. The base composition of these oligonucleotides must be designed to promote triple helix formation via Hoogsteen base pairing rules, which generally require sizable stretches of either purines or pyrimidines to be present on one strand of a duplex. Nucleic acids may be pyrimidine-based, which will result in TAT and CGC.sup.+ triplets across the three associated strands of the resulting triple helix. The pyrimidine-rich molecules provide base complementarity to a purine-rich region of a single strand of the duplex in a parallel orientation to that strand. In addition, nucleic acid molecules may be chosen which are purine-rich, for example, contain a stretch of G residues. These molecules will form a triple helix with a DNA duplex that is rich in GC pairs, in which the majority of the purine residues are located on a single strand of the targeted duplex, resulting in GGC triplets across the three strands in the triplex. Several such GC-rich areas are available for targeting in the INI1/hSNF5 gene (SEQ ID NO:6).

Alternatively, the potential sequences that can be targeted for triple helix formation may be increased by creating a so-called "switchback" nucleic acid molecule. Switchback molecules are synthesized in an alternating 5'-3', 3'-5' manner, such that they base pair with first one strand of a duplex and then the other, eliminating the necessity for a sizable stretch of either purines or pyrimidines to be present on one strand of a duplex.

In other embodiments, the oligonucleotide can be a small interfering RNA (siRNA), known in the art to be double stranded RNAs, complementary to the target mRNA (here INI1/hSNF5), that interacts with cellular factors to bind to the target sequence, which is then degraded. The siRNA sequence can be complementary to any portion of the INI1/SNF5. The siRNA is preferably 21-23 nt long, although longer sequences will be processed to that length. References include Caplen et al., 2001; Elbashir et al., 2001; Jarvis and Ford, 2002; and Sussman and Peirce, 2002.

Antisense RNA and DNA, ribozyme, triple helix, and siRNA molecules of the invention may be prepared by any method known in the art for the synthesis of DNA and RNA molecules, as discussed above. These include techniques for chemically synthesizing oligodeoxyribonucleotides and oligoribonucleotides well known in the art such as for example solid-phase phosphoramidite chemical synthesis. Alternatively, RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences encoding the antisense RNA molecule. Such DNA sequences may be incorporated into a wide variety of vectors that incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters. In another alternative, antisense cDNA constructs that synthesize antisense RNA constitutively or inducibly, depending on the promoter used, can be introduced stably into cell lines.

In related embodiments, the present invention is directed to additional methods of inhibiting replication or virion production of HIV-1 in a mammalian cell, or spread of the HIV-1 to another cell. These methods comprise inhibiting production of an INI1/hSNF5 by the cell. Preferably, production of the INI1/hSNF5 is inhibited with any of the above-described antisense, ribozyme, triple helix, and/or siRNA oligonucleotides described above.

For these embodiments, the cell is also preferably a human cell that can support HIV-1 infection and/or multiplication, such as a T-helper cell. In some embodiments, the cell is treated in vitro, then preferably implanted into a human at risk for HIV-1 infection. In other embodiments, the cell is treated in vivo.

The present invention is also directed to methods of evaluating whether a test compound inhibits replication or virion production of HIV-1 in mammalian cells, or cell-to-cell spread of HIV-1. The methods comprise determining whether the test compound inhibits the production of INI1/hSNF5 in the cell. The compound can be a nonoligonucleotide compound such as a nonpeptide molecule, or a peptide. However, in preferred embodiments, the test compound is an oligonucleotide, preferably complementary to a contiguous sequence of a coding region of an INI1/hSNF5 gene. Nonlimiting examples of such oligonucleotides are oligonucleotides designed to be antisense RNA and DNA, ribozymes, triple helix, or siRNAs.

In some preferred embodiments, the determination of the ability of the test compound to inhibit production of INI1/hSNF5 is made by measuring INI1/hSNF5 protein production by the cell after treatment of the cell with the compound. In other preferred embodiments, the determination is made by measuring INI1/hSNF5 mRNA production by the cell after treatment of the cell with the compound. Preferably, the cell is a human cell that can support HIV-1 infection and/or multiplication, such as T helper cells.

Other embodiments of the present invention include additional methods of evaluating whether a test compound inhibits replication or virion production of HIV-1 in a human cell, or cell-to-cell spread of HIV-1. The methods comprise determining whether the test compound disrupts the interaction of HIV-1 integrase with INI1/hSNF5. The disruption can be determined using a fragment of the INI1/hSNF5 that also interacts, such as a peptide comprising an Rpt1 domain, as previously described. The INI1/hSNF5 or fragment, or integrase used in these methods can also comprise a non-peptide component, for example a label (e.g., a radioactive or flourescent label, or a hapten or antigen that allows binding of a labeled antibody).

Methods of inhibiting replication or virion production of the HIV-1 in a mammalian cell, or spread of the HIV-1 to another cell, by treating the cell with a test compound, where the HIV-1 inhibitory activity of the test compound was determined by the above-described methods, are also envisioned as within the scope of the invention, as are the test compounds themselves.

Preferred embodiments of the invention are described in the following Examples. Other embodiments within the scope of the claims herein will be apparent to one skilled in the art from consideration of the specification or practice of the invention as disclosed herein. It is intended that the specification, together with the Examples, be considered exemplary only, with the scope and spirit of the invention being indicated by the claims which follow the Examples.

In accordance with the present invention there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Maniatis, Fritsch & Sambrook, "Molecular Cloning: A Laboratory Manual" (1989); "Current Protocols in Molecular Biology" Volumes I-IV, Ausubel, R. M., ed. (1997); Myers, "Molecular Biology and Biotechnology: A Comprehensive Desk Reference" (1995) and "Cell Biology: A Laboratory Handbook" Volumes I-III, J. E. Celis, ed. (1994).
 

Claim 1 of 21 Claims

1. A purified peptide comprising an Rpt1 domain of an INI1/hSNF5, the Rpt1 domain having the sequence of SEQ ID NO:2, wherein the peptide inhibits HIV-1 virion production in a human cell and wherein the peptide consists of SEQ ID NO:5 or is a fragment of SEQ ID NO:5.

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