Integrase-dirived HIV-inhibiting agents

Abstract

The present invention relates to agents based on integrase of HIV-1, for inhibiting the proliferation of HIV-1. The agents are derived from the C-terminal domain of HIV-1 integrase, comprising at least one of the regions identified as being important for interaction between integrase and imp7 or impβ, and/or for nuclear localization of the HIV PIC, replication of HIV, or infection of HIV.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will be further understood from the following description with reference to the drawings, in which:

FIG. 1 shows the interaction of HIV-1 IN and importin 7.

1A) Schematic representation of constructs of IN-YFP, T7-imp7 and T7-imp8. For IN-YFP, a full-length wild-type HIV-1 IN was fused in frame to the N-terminus of EYFP. For T7-imp7 and imp8, a T7-tag (9 amino acids) was fused in frame to the N-terminus of imp7 and imp8.

1B) Expression of IN-YFP and T7-imp7 and T7-imp8. Cell lysates from about 6×10⁵ 293T cells transfected with CMV-YFP, CMV-IN-YFP or indicated importin expressors was analyzed with immunoprecipitation with rabbit anti-GFP antibody followed by western blotting using mouse anti-GFP antibody (lanes 1 to 3) or immunoprecipitation with mouse anti-T7 antibody followed by western blotting using the same antibody (lanes 4 to 5).

1C) The in vivo co-IP assay. CMV-IN-YFP was co-transfected with plasmids for T7-imp7 (lane 3) or T7-imp8 (lane 4) into 2×10⁶ 293T cells. As a control, CMV-YFP also was co-transfected with each importin expressing plasmid (lane 1, 2). After 48 hr of transfection, cells were lysed by 0.5% CHAPS buffer and immunoprecipitated with rabbit anti-GFP antibody. Then, immunoprecipitated complexes were resolved by 12.5% SDS-PAGE and immunoblotted with either mouse anti-T7 antibody (upper panel) or mouse anti-GFP antibody (middle panel). The unbound T7-imp7 and T7-imp8 were also checked by sequential immunoprecipitation with anti-T7 antibody followed by immunoblotting with the same antibody (lower panel).

FIG. 2 shows that HIV-1 IN interacts with endogenous imp7 and that the interaction between IN and impβ takes place in the cells.

2A) The IN-YFP and T7-imp7 plasmids were co-transfected (lane 2) or transfected individually (lane 3) into 293T cells. After 48 hrs, cells were mixed accordingly, lysed and analyzed with co-immunoprecipitation using the same procedure as FIG. 1C. Upper panel: co-precipitated imp7 detected by western blot with anti-T7 antibody; Middle panel: The expression of IN-YFP detected by western blot with mouse anti-GFP antibody; Lower panel: the unbound imp7 visualized by immunoprecipitation and western blot by anti-T7 antibody.

2B) IN interacts with endogenous imp7. 10×10⁶ 293T cells were mock-transfected (lane 1) or transfected with CMV-YFP (lane 2) and CMV-IN-YFP (lane 3). After 48 hours of transfection, cells were lysed by 05%. CHAPS buffer and immunoprecipitated with rabbit polyclone anti-GFP antibody. Then, immunoprecipitates were separated in 10% SDS-PAGE followed by immunoblotting with rabbit anti-importin 7 (upper panel) or monoclonal anti-GFP antibody (lower panel). In parallel, 2×10⁶ of non-transfected 293T cells were lysed with the same lysis buffer and 10% of celllysates were loaded in SDS-PAGE as positive control (PC).

FIG. 3 shows in vitro interaction between IN and imp7.

3A) GST (lane 1) and GST-imp7 (lane 2) were expressed in E coli and affinity-purified on amylose resin. The similar amount of purified protein was directly loaded on a 12.5% SDS-PAGE followed by the Coomassie Blue staining.

3B) Equal amount of GST (lane 1) and GST-imp7 (lane 2) was incubated with a purified recombinant HIV-1 IN in 199 medium (containing 0.1% CHAPS) for 2 hours at 4° C. Then, the glutathione-sepharose 4B beads were added and incubate for additional one hour. After incubation, the beads were washed five times with the same lysis buffer and the protein complexes bound to glutathione-sepharose 4B beads were eluted with 10 mM glutathione buffer and loaded onto a 12.5% SDS-PAGE followed by western blot analysis with rabbit anti-IN specific antibodies.

FIG. 4 shows differential binding ability of HIV-1 MAp17 and IN to cellular importins Rch1 and imp7.

4A) HIV-I MAp17_G2A, but not IN, binds to T7-Rch1. 293T cells were co-transfected by CMV-T7-Rch1 with YFP (lane 2), IN-YFP (lane 3) or MAp17_G2A-YFP expressor (lane 4). After 48 hrs of transfection, cells were lysed by CHAPS lysis buffer and immunoprecipitated with rabbit anti-GFP antibody followed by western blot with either anti-T7 or mouse anti-GFP antibodies, as described in the legend for FIG. 1C. Upper panel shows the co-precipitated T7-Rch1 protein. The middle panel shows the expression of YFP, IN-YFP or Map17_G2A-YFP and the lower panel reveals the unbound T7-Rch1.

4B) HIV-1 IN, but not MAp17_G2A, binds to imp7. 293T cells were co-transfected with YFP (lane 2), IN-YFP (lane 3) or MAp17_G2A-YFP (lane 4) plasmid with T7-imp7 expressor. Upper panel indicates the co-precipitated T7-imp7; the middle panel shows the expression of YFP, IN-YFP or MAp17_G2A-YFP and the lower panel reveals the unbound T7-imp7 in each cell lysate sample.

FIG. 5 indicates the region(s) of HIV-1 IN that interact with imp7.

5A) Schematic representation of IN-YFP and YFP-IN truncated proteins used for binding assay. The IN sequence shown corresponds to amino acids 210-288 of SEQ ID NO:1.

5B) The N-terminal domain is dispensable for IN:imp7 interaction. The YFP (lane 3), IN-YFP (lane 4) and IN_50-288-YFP (lane 5) were co-expressed with T7-imp7 in 293T cells. In parallel, YFP and IN-YFP were expressed alone in 293T cells as control (lanes 1 and 2). At 48 hrs of transfection, cells were lysed and the interaction between IN-YFP mutants and imp7 was analyzed using anti-GFP immunoprecipitation and subsequently western blot with anti-T7 or anti-GFP antibodies, as described in FIG. 1C. The upper panel reveals the co-precipitated T7-imp7 and the middle panel shows the expression of YFP, IN-YFP, IN_50-288-YFP, as indicated. The lower panel shows the detection of unbound T7-imp7 by anti-T7 immunoprecipitation and western blot.

5C) The C-terminal domain is required for IN:imp7 interaction. The IN full-length protein (lane 3), IN_1-212 (lane 4), IN_1-240 (lane 5) and IN_1-260 (lane 6) were assayed for the interaction with imp7 as described before. Upper panel: co-precipitated T7-imp7. Middle panel: Expression of YFP, YFP-IN and YFP-IN mutants. Lower panel: unbound T7-imp7.

FIG. 6 shows the effect of different IN C-terminal substitutions on IN:imp7 interaction.

6A) Diagram of HIV-1 IN domain structure and introduced mutations at the C-terminal domain of the protein. The position of introduced mutation is shown at the bottom of sequence. The IN sequence shown corresponds to amino acids 210-288 of SEQ ID NO:1.

6B) Both of KK240,4 and RK263,4 of IN are involved in Imp7 interaction. The YFP (lanes 2 and 7), YFP-INwt (lanes 3 and 8) and different YFP-IN mutant expressors were co-transfected with T7-Imp7 expressor in 293T cells and after 48 h of infection, cells were lysed with CHAPS lysis buffer and the IN/Imp7 interaction for each IN mutant was analyzed by using the same protocol as described in FIG. 1C. Upper panel: co-precipitated T7-Imp7. Middle panel: Expression of YFP, YFP-INwt and YFP-IN mutants. Lower panel: unbound T7-Imp7. The position of each immunoprecipitated and co-precipitated proteins were indicated on the right side of the gel.

6C) The YFP-IN mutants as in B) were transfected into cells and visualized by fluorescence.

FIG. 7 shows interaction of HIV-1 IN with T7-impβ in co-transfected 293T cells. 293T cells were mock-transfected or transfected with SVCMVin-YFP, SVCMVin-IN-YFP or SVCMVin-YFP-IN expressors. Cells were lysed with 199 medium containing 0.25% NP-40 and a protease inhibitor cocktail (Roche), and clarified by centrifugation at 13,000 rpm for 30 min at 4° C.

The supernatant was subjected to immunoprecipitation with rabbit anti-GFP antibody and immunoprecipitates were resolved by 10% SDS-PAGE gel followed by western blot using mouse anti-T7 (upper panel) or mouse anti-GFP antibodies (middle panel), respectively. Also, the total T7-Impβ expression in cell lysates was sequentially immunoprecipitated with mouse anti-T7 antibody followed by western blot using the same antibody (lower panel).

FIG. 8 shows an immunocomplex of IN-YFP and endogenous impβ and imp7 in 293T cells. 293T cells were transfected with SVCMVin-YFP, SVCMVin-IN-YFP or SVCMVin-YFP-IN expressor. After 48 hours of transfection, cells were lysed by 199 medium with 0.25% NP-40 and immuno-precipitated with anti-GFP followed by western blot with a rabbit anti-human Impβ antibody (Cat# SC-11367, Santa Cruz Biotechnology Inc) (shown in middle panel) and anti-GFP antibody (shown in the lower panel). Then, the nitrocellular membrane from the middle panel was stripped with glycine/HCl buffer (0.1M glycine, pH. 2.7) and re-processed with western blot with anti-imp7 antibody (shown in upper panel). The positions of different proteins are shown at the right side of the gel.

FIG. 9 shows interaction of HIV-1 IN with impβ in vitro.

9A) Protein expression. Left panel: [³⁵S]methionine-labeled CAT, T7-IN and T7-Ran protein were expressed in vitro using TnT T7 coupled reticulocyte lysate system, extracts were separated on a SDS-PAGE and expressed proteins were detected by autoradiography. Right panel: Purified GST (Control), GST-Impα, and GST-impβ were verified by directly loading on a 12.5% SDS-PAGE followed by the Coomassie Blue staining.

9B) Impβ interacts with T7-IN and with T7-Ran, but not with T7-CAT. A GST pull-down assay was conducted as described above. Following extensive washing in 199 medium containing 0.25% NP40, the bound protein complexes were eluted with 50 mM glutathione and separation on a SDS-PAGE followed by autoradiography.

9C) Direct binding of GST-impβ and imp7 with purified HIV-1 IN. Left panel: as above, GST, GST imp7 and impβ were expressed and purified. Purified proteins were separated on an SDS-PAGE and detected by Coomassie Blue staining. Right panel: A GST pull-down assay was conducted as described above, with purified HIV-1 IN protein. Following extensive washing in 199 medium containing 0.25% NP40, the bound protein complexes were eluted with 50 mM glutathione and separation on a SDS-PAGE and finally pulled-down protein was detected by a western anti-IN antibody.

FIG. 10 shows that the C-terminal domain of HIV-1 IN interacts with impβ in co-transfected 293T cells. YFP-IN and different mutant expressors, as indicated, were co-transfected with T7-impβ in 293T cells. After 48 hours, cells were lysed, immunopreciptated with anti-GFP followed by western blot with anti-T7 antibody (upper panel) and anti-GFP antibody (middle panel). The total amount of T7-impβ was analyzed by sequential IP with anti-T7 antibody followed by western blot with anti-T7 antibody.

FIG. 11 shows that the HIV-1 IN C-terminal domain alone is sufficient for binding to imp7 and to inhibit HIV-1 infection.

11A) Schematic representation of CMV-YFP and CMV-YFP-INc205 (IN amino acids 205-288) expressors.

11B) intracellular localization of YFP or YFP-INc205 in HeLa cells.

11C) The HIV-1 IN C-terminal domain alone is sufficient for binding to imp7. Plasmids expressing YFP, IN-YFP, or YFP-INc205 were cotransfected with CMV-T7-imp7. After 48 hrs of transfection, imp7-binding was analyzed using anti-GFP immunoprecipitation and subsequently western blot with anti-T7 or anti-GFP antibodies.

11D) Over-expression of the HIV-1 IN C-terminal domain alone is sufficient for inhibiting infection of VSV-G-pseudotyped HIV-1 virus in 293T cells. To test the effect of YFP-INc205 on HIV-1 infection, each 293T cell line, including parental 293T cells, was infected with equal amounts of VSV-G pseudotyped pNLlucΔBgII virus (at 5 cpm of RT activity/cell). Since viruses contain a luciferase (luc) gene in place of the nef gene, viral infection can be monitored by using a sensitive luc assay which could efficiently detect viral gene expression After 48 hours of infection, equal amounts of cells (1×10⁶ cells) were lysed in 50 μl of luc lysis buffer and then, 10 μl of cell lysates was used for measurement of luc activity.

FIG. 12 shows that mutations in the C-terminal domain of IN inhibit HIV single-cycle replication and affect reverse transcription and nuclear import.

12A) 293T cells were transfected with a RT, IN and Env deleted HIV-1 provirus NLlucΔBglΔRI with different Vpr-RT-IN(WT/Mutant) expressors and a VSV-G expresser. Produced viruses (lane 1 to 3) were lysed and directly loaded in 12% SDS-PAGE and analyzed by Western blot with human anti-HIV serum. The positions of HIV-1 Gag, RT and IN proteins are indicated.

12B) The CD4+ C8166 cells were infected with viruses vWT, vD64E, and vKKRK viruses. At different time intervals after infection, the equal amount (1×10⁶) of cells was collected and cell-associated luciferase activity was measured by luciferase assay.

12C) Effects of Imp7-binding defect mutants on HIV-1 reverse transcription and DNA nuclear import. At 24 hours post-infection, 2×10⁶ cells were gently lysed and fractionated into the cytoplasmic and the nuclear fractions. The amount of viral DNA in both fractions were analyzed by PCR using HIV-1 LTR-Gag primers and Southern blot. Nuc. nuclear fraction; Cyt. cytoplasmic fraction, The purity and DNA content of each subcellular fraction were monitored by PCR detection of human globin DNA and visualized by specific Southern blot (lower panel).

12D) The total amounts of viral DNA (right panel) and the percentage of nucleus-associated viral DNA relative to the total amount of viral DNA (left panel) for each mutant was also quantified by laser densitometry. Means and standard deviations from two independent experiments are shown.

FIG. 13 shows siRNA-mediated silencing of Imp7 inhibits HIV-1 infection.

13A) A schematic depiction of the method steps shown as an example.

13B) siRNA-mediated silencing of Imp7 in 293T and HeLa-β-Gal-CD4/CCR5 cells. Cells were transfected with 20 nM of siRNA at 0 and 18 hours. After 48, 72 and 96 hours post initial transfection, the Imp7 expression levels in the cells were verified by Western blot with anti-Imp7 antibody (upper panel). Meanwhile, the expression of α-tubulin was also verified (lower panel).

13C) 293T cells were treated with sc-RNA or si-imp7 once a day for two days and used to produced VSV-G-pseudotyped HIV-1 4.3 virus (sc-virus and si-virus). Both viruses were then used to infect HeLa-β-Gal-CD4/CCR5 cells that have been treated with Imp7 siRNA or scramble RNA for 72 h. Luciferase activity was measured at 48 h post-infection.

13D) sc-RNA or si-imp7 treated HeLa-β-Gal cells were infected with wild-type enveloped HxBru virus produced from sc-RNA- or si-imp7-treated HeLa cells. Viral Infection was evaluated by MAGI assay.

FIG. 14 shows subcellular localization of the wild-type and truncated HIV integrase fused with YFP.

14A) Schematic structure of HIV-1 integrase-YFP fusion proteins. Full-length (1-288aa) HIV-1 integrase, the N-terminus-truncated mutant (51-228aa) or the C-terminus-truncated mutant (1-212aa) was fused in frame at the N-terminus of YFP protein. The cDNA encoding for each IN-YFP fusion protein was inserted in a SVCMV expression plasmid.

14B) Expression of different IN-YFP fusion proteins in 293T cells. 293T cells were transfected with each IN-YFP expressor and at 48 hours of transfection, cells were lysed, immunoprecipitated with anti-HIV serum and resolved by electrophoresis through a 12.5% SDS-PAGE followed by Western blot with rabbit anti-GFP antibody. The molecular weight markers are indicated at the left side of the gel.

14C) Intracellular localization of different IN-YFP fusion proteins. HeLa cells were transfected with each HIV-1 IN-YFP fusion protein expressor and at 48 hours of transfection, cells were fixed and subjected to indirect immuno-fluorescence using rabbit anti-GFP and then incubated with FITC-conjugated anti-rabbit antibodies. The localization of each fusion protein was viewed by Fluorescence microscopy with a 50× oil immersion objective. Upper panel is fluorescence images and bottom panel is DAPI nucleus staining.

FIG. 15 shows the effect of different IN C-terminal substitution mutants on IN-YFP intracellular localization.

15A) Diagram of HIV-1 IN domain structure and introduced mutations at the C-terminal domain of the protein. The position of lysines in two tri-lysine regions and introduced mutations are shown at the bottom of sequence. The IN sequence shown corresponds to amino acids 210-288 of SEQ ID NO:1.

15B) The expression of the wild-type and mutant IN-YFP fusion proteins were detected in transfected 293T cells by using immunoprecipitation with anti-HIV serum and Western blot with rabbit anti-GFP antibody, as described in FIG. 1. The molecular weight markers are indicated at the left side of the gel.

15C) Intracellular localization of different HIV-1 IN mutant-YFP fusion proteins in HeLa cells were analyzed by fluorescence microscopy with a 50× oil immersion objective. The nucleus of HeLa cells was simultaneously visualized by DAPI staining (lower panel).

FIG. 16 shows the production of different single-cycle replicating viruses and their infection in HeLa-CD4-CCR5-β-Gal cells.

16A) To evaluate the trans-incorporation of RT and IN in VSV-G pseudotyped viral particles, viruses released from 293T cells transfected with NLlucΔBglΔRI provirus alone (lane 6) or cotransfected with different Vpr-RT-IN expressors and a VSV-G expresser (lane 1 to 5) were lysed, immunoprecipitated with anti-HIV serum. Immunoprecipitates were run in 12% SDS-PAGE and analyzed by Western blot with rabbit anti-IN antibody (middle panel) or anti-RT and anti-p24 monoclonal antibody (upper and lower panel).

16B) The infectivity of trans-complemented viruses produced in 293 T cells was evaluated by MAGI assay. HeLa-CD4-CCR5-LTR-β-Gal cells were infected with equal amounts (at 10 cpm/cell) of different IN mutant viruses and after 48 hours of infection, numbers of β-Gal positive cells (infected cell) were monitored by X-gal staining. Error bars represent variation between duplicate samples and the data is representative of results obtained in three independent experiments.

FIG. 17 shows the effect of IN mutants on viral infection in dividing and nondividing C8166 T cells. To test the effect of different IN mutants on HIV-1 infection in CD4+ T cells, dividing (panel A) and non-dividing (aphidicolin-treated, panel B) C8166 T cells were infected with equal amount of VSV-G pseudotyped IN mutant viruses (at 5 cpm/cell). For evaluation of the effect of different IN mutants on HIV-1 envelope-mediated infection in CD4+ T cells, dividing C8166 T cells were infected with equal amount of HIV-1 envelope competent IN mutant viruses (at 10 cpm/cell) (panel C). After 48 hours of infection, HIV-1 DNA-mediated luciferase induction was monitored by luciferase assay. Briefly, the same amount (10⁶ cells) of cells was lysed in 50 ul of luciferase lysis buffer and then, 10 μl of cell lysate was subjected to the luciferase assay. Error bars represent variation between duplicate samples and the data is representative of results obtained in three independent experiments.

FIG. 18 shows the effects of different IN mutants on HIV-1 reverse transcription and DNA nuclear import.

Dividing C8166 T cells were infected with equal amounts of different HIV-1 IN mutant viruses.

18A) At 12 hours post-infection, 1×10⁶ cells were lysed and the total viral DNA was detected by PCR using HIV-1 LTR-Gag primers and Southern blot.

18B) Levels of HIV-1 late reverse transcription products detected in panel A were quantified by laser densitometry and viral DNA level of the wt virus was arbitrarily set as 100%. Means and standard deviations from two independent experiments are presented.

18C) At 24 hours post-infection, 2×10⁶ cells were fractionated into cytoplasmic and nuclear fractions as described in Materials and Methods. The amount of viral DNA in cytoplasmic and nuclear fractions were analyzed by PCR using HIV-1 LTR-Gag primers and Southern blot (upper panel, N. nuclear fraction; C. cytoplasmic fraction). Purity and DNA content of each subcellular fraction were monitored by PCR detection of human globin DNA and visualized by specific Southern blot (lower panel).

18D). The percentage of nucleus-associated viral DNA relative to the total amount of viral DNA for each mutant was also quantified by laser densitometry. Means and standard deviations from two independent experiments are shown.

FIG. 19 shows the effect of IN mutants on HIV-1 proviral DNA integration. Dividing C8166 T cells were infected with equal amounts of different HIV-1 IN mutant viruses. At 24 hours post-infection, 1×10⁶ cells were lysed and serial-diluted cell lysates were analyzed by two-step Alu-PCR and Southern blot for specific detection of integrated proviral DNA from infected cells (Upper panel). The DNA content of each lysis sample was also monitored by PCR detection of human β-globin DNA and visualized by specific Southern blot (middle panel). The serial-diluted ACH-2 cell lysates were analyzed for integrated viral DNA and as quantitative control (lower panel). The results are representative for two independent experiments.

FIG. 20 shows that expression of HIV-1 integrase C-terminal domain in viral producer cells inhibits subsequent HIV-1 infection in HeLa-β-Gal-CD4-CCR5 cells and in CD4⁺ T-lymphoid MT4 cells. 293T cells were transfected with HIV-1 provirus NL4.3-Nef+/GFP+ and SVCMVin-T7 or SVCMVin-T7-IN_C205-288 expressor (the IN sequence shown as SEQ ID NO:2). After 48 hours of transfection, viruses were collected from the supernatant through an ultracentrifugation, and virus titers were quantified by HIV-1 RT activity assay. Equal amounts of viruses, as measured by virion-associated reverse transcriptase activity (A), were used to infect HeLa-β-Gal-CD4/CCR5 cells (B) or MT4 cells (C). At 48 h post-infection, the viral infection levels were evaluated by MAGI assay (B) or by counting of GFP-positive cells (C).

FIG. 21 shows the amino acid sequence of HIV-1 integrase (SEQ ID NO:1 derived from HIV-1 pNL4.3 strain) shown as an example. The C-terminal domain of IN and the two tri-lysine regions and an arginine/lysine region involved in IN/imp7 and IN/impβ interactions are indicated.

FIG. 22 shows fusions of Tat peptide (SEQ ID NO:9) with IN peptides (SEQ ID NOs 10-15) as examples.

FIG. 23 shows the siRNA target regions of IN as examples. The HIV-1 IN RNA sequence from nt 628 to 801 is shown (SEQ ID NO:16); this sequence encodes amino acids 210 to 267 of integrase. Also indicated are the RNA sequence encoding the two tri-lysine regions and the arginine/lysine rich region. These sequences (siRNA #1-4; SEQ ID NOs 17-20) can be used for siRNA silencing of IN protein expression during viral replication.

FIG. 24 is an ELISA scheme based on INc205-288 as an example, for screening of compounds that inhibit IN interaction with imp7 and/or with impβ.

FIG. 25 is a schematic depiction of a live cell BRET assay used as an example for detecting interaction of the C-terminal domain of HIV-1 IN with impβ and imp7 in live cells.

Claims

1. An isolated peptide comprising at least 8 and no more than 83 consecutive amino acids from residues 205 to 288 of an HIV-1 integrase sequence, wherein the at least 8 and no more than 83 consecutive amino acids of integrase comprises at least one of the following regions of SEQ ID NO:1: amino acids #211-219, amino acids #236-244, amino acids #235-250, amino acids #259-266, amino acids #258-266, and amino acids #261-268.

2. The peptide according to claim 1 wherein the at least 8 and no more than 83 consecutive amino acids of integrase comprises at least one of the following regions of SEQ ID NO:1: amino acids #210-225, amino acids #233-250, amino acids #261-273, amino acids #211-245, amino acids #236-266, and amino acids #211-266.

3. The peptide according to claim 1 wherein the at least 8 and no more than 83 consecutive amino acids of integrase comprises amino acids #211-219 of SEQ ID NO:1 or amino acids #236-244 of SEQ ID NO:1, or both.

4. The peptide according to claim 1 wherein the at least 8 and no more than 83 consecutive amino acids of integrase comprises amino acids #236-244 of SEQ ID NO:1 or amino acids #259-266 of SEQ ID NO:1 or both.

5. The peptide according to claim 1 wherein the at least 8 and no more than 83 consecutive amino acids of integrase comprises amino acids #211-219 of SEQ ID NO:1, amino acids #236-244 of SEQ ID NO:1, and amino acids #259-266 of SEQ ID NO:1.

6. The peptide according to claim 1 comprising at least 13 and no more than 83 consecutive amino acids from residues 205 to 288 of an HIV-1 integrase sequence, wherein the at least 13 and no more than 83 consecutive amino acids of integrase comprises at least one of the following regions of SEQ ID NO:1: amino acids #211-219, amino acids #236-244, amino acids #235-250, amino acids #259-266, amino acids #258-266, and amino acids #261-268.

7. The peptide according to claim 1 further comprising a heterologous sequence which is fused with the sequence derived from residues 205 to 288 of HIV-1 integrase.

8. The peptide according to claim 7 wherein the heterologous sequence is a membrane-translocating sequence.

9. The peptide according to claim 8 wherein the membrane-translocating sequence is the HIV Tat membrane-translocating sequence (SEQ ID NO:9).

10. The peptide according to claim 7 wherein the heterologous sequence is a reporter sequence.

11. The peptide according to claim 1 that, when expressed with HIV-1 provirus, renders HIV-1 replication-defective or infection-defective.

12. A variant polypeptide of HIV-1 integrase having a substitution or deletion in at least one of the following positions of HIV-1 integrase: K211, K215, K219, K236, K240, K244, V249, V250, K258, R262, R263, K264, K266, and K273.

13. A variant polypeptide of HIV-1 integrase having at least one of the following regions of SEQ ID NO:1 deleted: amino acids #211-219, amino acids #236-244, amino acids #235-250, amino acids #259-266, amino acids #258-266, and amino acids #261-268.

14. A fusion polypeptide comprising the variant polypeptide of claim 12 fused to a heterologous sequence.

15. An isolated polynucleotide encoding the peptide defined in claim 1.

16. An isolated polynucleotide encoding the variant polypeptide defined in claim 12.

17. A monoclonal antibody specifically immunoreactive against at least one of the following regions of SEQ ID NO:1: amino acids #211-219, amino acids #236-244, amino acids #235-250, amino acids #259-266, amino acids #258-266, amino acids #261-268, amino acids #210-225, amino acids #233-250, amino acids #261-273, amino acids #211-245, amino acids #236-266, and amino acids #211-266.

18. The monoclonal antibody according to claim 17 which is a single chain monoclonal antibody.

19. A chemically synthesized double stranded short interfering nucleic acid (siNA) molecule that directs cleavage via RNA interference (RNAi) of a HIV RNA encoding amino acids 205 to 288 of HIV-1 integrase, wherein a) each strand of said siNA molecule is about 18 to about 23 nucleotides in length; and b) one strand of said siNA molecule comprises nucleotide sequence having sufficient complementarity to said HIV RNA for the siNA molecule to direct cleavage of the HIV RNA via RNA interference.

20. A method of inhibiting HIV-1 replication in a cell, comprising transporting into the cell the peptide defined in claim 1.

21. A method of inhibiting HIV-1 replication in a cell, comprising expressing in the cell the polynucleotide defined in claim 15.

22. A method of inhibiting HIV-1 infection in a human comprising administering to the human the peptide defined in claim 1.

23. A method for screening for a compound that affects HIV-1 replication or infection, the method comprising: (a) incubating, in the presence of a candidate agent, the peptide defined in claim 1 with imp7 or impβ, under conditions suitable for binding to occur between the peptide and imp7 or impβ; (b) determining the level of binding between the peptide and imp7 or impβ, wherein detecting a change in the level of binding between the peptide and imp7 or impβ in the presence of the candidate agent, compared to the level of binding in the absence of the candidate agent, indicates that said agent is a compound that affects HIV-1 replication or infection.

24. A method for screening for a compound that affects HIV-1 replication or infection, the method comprising: (a) providing a cell that expresses (i) the peptide defined in claim 1 and (ii) imp7 or impβ; (b) providing the cell with a candidate agent; and (c) determining the level of binding between the expressed peptide and the expressed imp7 or impβ, wherein detecting a change in the level of binding between the peptide and imp7 or impβ in the presence of the candidate agent, compared to the level of binding in the absence of the candidate agent, indicates that said agent is a compound that affects HIV-1 replication or infection.

25. The method according to claim 23 for screening for a compound that inhibits HIV-1 replication or infection, and wherein detecting a decrease in the level of binding between the peptide and imp7 or impβ in the presence of the candidate agent, compared to the level of binding in the absence of the candidate agent, indicates that said agent is a compound that inhibits HIV-1 replication or infection.