Inhibition of Early Steps of HIV-1 Replication by SNF5/Ini1
2006; Elsevier BV; Volume: 281; Issue: 32 Linguagem: Inglês
10.1074/jbc.m604849200
ISSN1083-351X
AutoresMarlène Maroun, Olivier Delelis, Gaël Coadou, Thomas Bader, Emmanuel Ségéral, Gladys Mbemba, Caroline Petit, P. Sonigo, Jean‐Christophe Rain, Jean‐François Mouscadet, Richard Bénarous, Stéphane Emiliani,
Tópico(s)Advanced biosensing and bioanalysis techniques
ResumoTo replicate, human immunodeficiency virus, type 1 (HIV-1) needs to integrate a cDNA copy of its RNA genome into a chromosome of the host cell, a step controlled by the viral integrase (IN) protein. Viral integration involves the participation of several cellular proteins. SNF5/Ini1, a subunit of the SWI/SNF chromatin remodeling complex, was the first cofactor identified to interact with IN. We report here that SNF5/Ini1 interferes with early steps of HIV-1 replication. Inhibition of SNF5/Ini1 expression by RNA interference increases HIV-1 replication. Using quantitative PCR, we show that both the 2-long terminal repeat circle and integrated DNA forms accumulate upon SNF5/Ini1 knock down. By yeast two-hybrid assay, we screened a library of HIV-1 IN random mutants obtained by PCR random mutagenesis using SNF5/Ini1 as prey. Two different mutants of interaction, IN E69G and IN K71R, were impaired for SNF5/Ini1 interaction. The E69G substitution completely abolished integrase catalytic activity, leading to a replication-defective virus. On the contrary, IN K71R retained in vitro integrase activity. K71R substitution stimulates viral replication and results in higher infectious titers. Taken together, these results suggest that, by interacting with IN, SNF5/Ini1 interferes with early steps of HIV-1 infection. To replicate, human immunodeficiency virus, type 1 (HIV-1) needs to integrate a cDNA copy of its RNA genome into a chromosome of the host cell, a step controlled by the viral integrase (IN) protein. Viral integration involves the participation of several cellular proteins. SNF5/Ini1, a subunit of the SWI/SNF chromatin remodeling complex, was the first cofactor identified to interact with IN. We report here that SNF5/Ini1 interferes with early steps of HIV-1 replication. Inhibition of SNF5/Ini1 expression by RNA interference increases HIV-1 replication. Using quantitative PCR, we show that both the 2-long terminal repeat circle and integrated DNA forms accumulate upon SNF5/Ini1 knock down. By yeast two-hybrid assay, we screened a library of HIV-1 IN random mutants obtained by PCR random mutagenesis using SNF5/Ini1 as prey. Two different mutants of interaction, IN E69G and IN K71R, were impaired for SNF5/Ini1 interaction. The E69G substitution completely abolished integrase catalytic activity, leading to a replication-defective virus. On the contrary, IN K71R retained in vitro integrase activity. K71R substitution stimulates viral replication and results in higher infectious titers. Taken together, these results suggest that, by interacting with IN, SNF5/Ini1 interferes with early steps of HIV-1 infection. The replication cycle of human immunodeficiency virus, type 1 (HIV-1) 4The abbreviations used are: HIV-1, human immunodeficiency virus, type 1; PIC, preintegration complex; IN, integrase; LTR, long terminal repeat; siRNA, short interference RNA; m.o.i., multiplicity of infection; GST, glutathione S-transferase; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; WT, wild type; HTRF, homogenous time-resolved fluorescence assay; GFP, green fluorescent protein; VSR-G, vesicular stomatitis virus-glycoprotein. involves the insertion of a DNA copy of its RNA genome into a chromosome of the host cell. Following retrovirus entry, a large nucleoprotein complex called preintegration complex (PIC) is formed in the cytoplasm with components of the virion core and cellular factors. In addition to viral cDNA, PICs contain several viral proteins: matrix, nucleocapsid, reverse transcriptase, VPR protein, and integrase (IN), which carries out DNA-cutting and -joining reactions (1Bukrinsky M.I. Sharova N. McDonald T.L. Pushkarskaya T. Tarpley W.G. Stevenson M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6125-6129Crossref PubMed Scopus (392) Google Scholar, 2Miller M.D. Farnet C.M. Bushman F.D. J. Virol. 1997; 71: 5382-5390Crossref PubMed Google Scholar). HIV-1 IN consists of three functional domains: the N-terminal domain (residues 1-49), the catalytic core domain (residues 50-212), and the C-terminal domain (residues 213-288) (3Chen J.C. Krucinski J. Miercke L.J. Finer-Moore J.S. Tang A.H. Leavitt A.D. Stroud R.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8233-8238Crossref PubMed Scopus (383) Google Scholar, 4Wang J.Y. Ling H. Yang W. Craigie R. EMBO J. 2001; 20: 7333-7343Crossref PubMed Scopus (313) Google Scholar). The N-terminal domain contains an HHCC motif that binds one Zn2+ atom and is involved in the multimerization of the protein (4Wang J.Y. Ling H. Yang W. Craigie R. EMBO J. 2001; 20: 7333-7343Crossref PubMed Scopus (313) Google Scholar). The C-terminal domain binds DNA non-specifically and plays a role in the formation of an active multimer of IN (3Chen J.C. Krucinski J. Miercke L.J. Finer-Moore J.S. Tang A.H. Leavitt A.D. Stroud R.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8233-8238Crossref PubMed Scopus (383) Google Scholar). The catalytic core domain contains the canonical 3-amino acid motif, D, D(35)E, that is essential for the catalytic activity of the protein (4Wang J.Y. Ling H. Yang W. Craigie R. EMBO J. 2001; 20: 7333-7343Crossref PubMed Scopus (313) Google Scholar). These residues coordinate a divalent metal ion (Mg2+) and are highly conserved among all integrases and retrotransposases. Integration proceeds in three steps, 3′-processing, strand transfer, and gap repair. 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Cell. 2003; 115: 135-138Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). SNF5/Ini1 was the first host protein identified as an IN-interacting factor by two-hybrid screenings (8Kalpana G.V. Marmon S. Wang W. Crabtree G.R. Goff S.P. Science. 1994; 266: 2002-2006Crossref PubMed Scopus (462) Google Scholar). SNF5/Ini1 is one of the core subunits of the ATP-dependent chromatin remodeling complex SWI/SNF that regulates expression of numerous eukaryotic genes by altering DNA/histone interactions (26Kingston R.E. Bunker C.A. Imbalzano A.N. Genes Dev. 1996; 10: 905-920Crossref PubMed Scopus (404) Google Scholar, 27Kingston R.E. Narlikar G.J. Genes Dev. 1999; 13: 2339-2352Crossref PubMed Scopus (610) Google Scholar). This complex was recently shown to be directly involved in Tat-mediated activation of HIV-1 transcription (28Treand C. du Chene I. Bres V. Kiernan R. Benarous R. Benkirane M. Emiliani S. 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Chem. 2002; 277: 22330-22337Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar). The exact role of SNF5/Ini1 in HIV-1 replication remains unclear. Recombinant SNF5/Ini1 stimulates IN catalytic activity in vitro (8Kalpana G.V. Marmon S. Wang W. Crabtree G.R. Goff S.P. Science. 1994; 266: 2002-2006Crossref PubMed Scopus (462) Google Scholar). When overexpressed, a cytoplasmic fragment of SNF5/Ini1 was able to interact with IN in the context of the Gag-pol precursor and in addition was reported to inhibit viral particle production, suggesting a role during the late stage of HIV-1 replication (38Yung E. Sorin M. Pal A. Craig E. Morozov A. Delattre O. Kappes J. Ott D. Kalpana G.V. Nat. Med. 2001; 7: 920-926Crossref PubMed Scopus (125) Google Scholar). Furthermore, SNF5/Ini1 was shown to be packaged in HIV-1, but not HIV-2 or simian immunodeficiency virus, viral particles (39Yung E. Sorin M. Wang E.J. Perumal S. Ott D. Kalpana G.V. J. Virol. 2004; 78: 2222-2231Crossref PubMed Scopus (63) Google Scholar). Interestingly, it has been observed that HIV-1 infection induces the cytoplasmic relocation of SNF5/Ini1 along with PML, leading to their association with incoming PIC before nuclear migration (40Turelli P. Doucas V. Craig E. Mangeat B. Klages N. Evans R. Kalpana G. Trono D. Mol. Cell. 2001; 7: 1245-1254Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar). However, the cytoplasmic accumulation of PML observed after retroviral infection is independent of the presence of SNF5/Ini1 (41Boese A. Sommer P. Gaussin A. Reimann A. Nehrbass U. FEBS Lett. 2004; 578: 291-296Crossref PubMed Scopus (14) Google Scholar). Furthermore, a direct effect of PML on HIV-1 infectivity was recently challenged (42Berthoux L. Towers G.J. Gurer C. Salomoni P. Pandolfi P.P. Luban J. J. Virol. 2003; 77: 3167-3180Crossref PubMed Scopus (73) Google Scholar). It has also been postulated that SNF5/Ini1 could target PICs to regions of the genome that are enriched for the SWI/SNF complex (43Greene W.C. Peterlin B.M. Nat. Med. 2002; 8: 673-680Crossref PubMed Scopus (210) Google Scholar). Using siRNA-mediated silencing of SNF5/Ini1 expression, we found that SNF5/Ini1 impairs early steps of HIV-1 replication by inhibiting formation of 2-LTR circle and integrated forms of viral DNA. We show that a single amino acid change, K71R, in integrase that reduces its ability to interact with SNF5/Ini1 leads to an increase in viral infectivity. Our results highlight the role of the interaction between SNF5/Ini1 and the incoming IN during early steps of the HIV-1 life cycle. Integrase Mutant Library—Yeast two-hybrid screening procedures were performed as previously described (14Emiliani S. Mousnier A. Busschots K. Maroun M. Van Maele B. Tempe D. Vandekerckhove L. Moisant F. Ben-Slama L. Witvrouw M. Christ F. Rain J.C. Dargemont C. Debyser Z. Benarous R. J. Biol. Chem. 2005; 280: 25517-25523Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar). Plasmids—The GFP-INs expression vector was generated as previously described (14Emiliani S. Mousnier A. Busschots K. Maroun M. Van Maele B. Tempe D. Vandekerckhove L. Moisant F. Ben-Slama L. Witvrouw M. Christ F. Rain J.C. Dargemont C. Debyser Z. Benarous R. J. Biol. Chem. 2005; 280: 25517-25523Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar). Mutations were incorporated into the HIV-1Bru molecular clones using PCR-directed mutagenesis as previously described (14Emiliani S. Mousnier A. Busschots K. Maroun M. Van Maele B. Tempe D. Vandekerckhove L. Moisant F. Ben-Slama L. Witvrouw M. Christ F. Rain J.C. Dargemont C. Debyser Z. Benarous R. J. Biol. Chem. 2005; 280: 25517-25523Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar). To generate the envelope-deleted NL4-3 vector (NL4-3Δenv), a frameshift was introduced in the env gene. The NdeI site (nt6399) in pNL4-3 was digested, filled with Klenow, and religated. Wild-type and mutant integrases were inserted into bacterial expression vector pET15b (Novagene). GST-SNF5/Ini1 was constructed by PCR amplification from the SNF5/Ini1 expression vector (28Treand C. du Chene I. Bres V. Kiernan R. Benarous R. Benkirane M. Emiliani S. EMBO J. 2006; 25: 1690-1699Crossref PubMed Scopus (137) Google Scholar) and subcloning into the pGEX4T1 expression plasmid (GE Healthcare). Cells, Viruses, Transfections, and Infections—Human embryonic kidney 293, HeLa, and HeLa P4.2 cells were grown in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal calf serum (Invitrogen) and antibiotics (100 units/ml penicillin G, 100 mg/ml streptomycin; Invitrogen). HeLa P4.2 cells were grown in the presence of 100 μg/ml geneticin (Invitrogen). Jurkat and A3.01 cells were grown in RPMI 1640 (Invitrogen) supplemented with 10% fetal calf serum (Invitrogen) and antibiotics (100 units/ml penicillin G, 100 μg/ml streptomycin; Invitrogen). A3.01 cells were transfected by nucleofection using the AMAXA cell line, Nucleofector Kit V, and program T014 following the manufacturer's instructions. Virus stocks were produced by transfecting human embryonic kidney 293 cells using the calcium phosphate method with pBru-derived molecular clone. Single-round virus stocks were produced by co-transfecting pNL4-3Δenv with vesicular stomatitus virus-glycoprotein (VSV-G) envelope expression vector. Supernatants were collected 2 days after transfection, and levels of HIV-1 p24 antigen were monitored by enzyme-linked immunoabsorbent assay (BD Biosciences). Jurkat cells were infected with viral doses corresponding to 30 ng of HIV-1 p24 antigen/106 cells. siRNA Knockdown Experiments—SNF5/Ini1 siRNAs (SNF5.1, GAGAUACCCCUCACUCUGGTT, and SNF5.3, GAACUCACCAGAGAAGUUUTT) and control siRNAs (SNF5inv, GGUCUCACUCCCCAUAGAGTT, and GL2, CGUACGCGGAAUACUUCGATT) were synthesized by Eurogentec and annealed following the manufacturer's instructions. Jurkat T cells (2 × 106) were washed with PB-sucrose (phosphate-buffered sucrose buffer: 7 mm sodium phosphate, 272 mm sucrose, 1 mm MgCl2) and then electroporated with siRNA at 1 mm with a Bio-Rad Gene Pulser II electroporator with an RF module (100 V, modulation 50%, 25 kHz, 10 bursts of 2 ms, burst interval 100 ms). HeLa or HeLa P4.2 cells were transfected twice at 24 h intervals with 10 or 30 nm siRNA using Oligofectamine reagent (Invitrogen). Western Blot Analysis—Cells were lysed in radioimmune precipitation buffer containing 1 mm dithiothreitol and protease inhibitors. Proteins were separated by SDS-PAGE using the NuPAGE Bis-Tris Electrophoresis System (Invitrogen) and revealed by Western blotting. Antibodies—Rabbit polyclonal anti-SNF5 (raised against a synthetic peptide representing amino acids 370-386 of SNF5/Ini1 (HRNTRRMRRLANTGPAW), mouse monoclonal anti-tubulin (clone DM 1A; Sigma). Secondary peroxidase-conjugated antibodies against mouse or rabbit immunoglobulins were purchased from Dako. Quantification of Total HIV-1 DNA, 2-LTR Circles, and Integrated HIV-1 DNA—HeLa cells (2 × 105) were transfected with 30 nm siRNA. 24 h later, cells were washed three times with phosphate-buffered saline and infected with VSV-G-pseudotyped Bru virus (multiplicity of infection (m.o.i.) corresponding to 0.1). At different times postinfection cells were harvested, washed in phosphate-buffered saline, and treated for 1 h at 37 °C with 500 units of DNaseI (Roche Diagnostics) prior to DNA extraction using a QIAamp blood DNA mini kit (Qiagen). Quantifications were performed by real-time PCR on a LightCycler instrument (Roche Diagnostics). Sequences of primers and probes have been described previously (14Emiliani S. Mousnier A. Busschots K. Maroun M. Van Maele B. Tempe D. Vandekerckhove L. Moisant F. Ben-Slama L. Witvrouw M. Christ F. Rain J.C. Dargemont C. Debyser Z. Benarous R. J. Biol. Chem. 2005; 280: 25517-25523Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar). Copy numbers of total DNA, 2-LTR circle forms, and integrated DNA were determined in reference to standard curves prepared by amplification of cloned DNA with matching sequences (44Brussel A. Sonigo P. J. Virol. 2003; 77: 10119-10124Crossref PubMed Scopus (184) Google Scholar). Results were normalized by the number of cells and the amount of cellular DNA quantified by PCR of the β-globin gene according to the manufacturer's instructions (Roche Diagnostics). Expression and Purification of Recombinant Proteins and in Vitro Integration Assays—Recombinant GST-SNF5/Ini1 and N-terminal His-tagged IN were produced in Escherichia coli BL21. E. coli transformed with GST-SNF5/Ini1 expression plasmid were induced with 0.4 mm isopropyl-1-thio-β-d-galactopyranoside for 3 h to induce protein expression. The bacterial pellet was resuspended in GST-lysis buffer A (20 mm Tris-HCl, pH 8.0, 500 mm KCl, 1 mm MgCl2, 0.5% IGEPAL, 1 mm dithiothreitol, protease inhibitor mixture) (Sigma), and 1 mg/ml lysozyme was added. Cells were lysed by three cycles of freeze and thaw and then sonicated for 30 s. After centrifugation, the supernatant was incubated with glutathione-Sepharose for 1 h at 4 °C. The resin was washed four times with GST-lysis buffer and then twice with buffer B (20 mm Tris-HCl, pH 8.0, 20 mm KCl, 1 mm MgCl2, 17% glycerol, 1 mm dithiothreitol). GST-SNF5/Ini1 was eluted from the resin in buffer B containing 25 mm reduced glutathione for 10 min at room temperature. His-IN mutants were purified by nickel affinity as previously described (45Leh H. Brodin P. Bischerour J. Deprez E. Tauc P. Brochon J.C. LeCam E. Coulaud D. Auclair C. Mouscadet J.F. Biochemistry. 2000; 39: 9285-9294Crossref PubMed Scopus (121) Google Scholar). Oligonucleotide substrates for IN reaction assays were as follows: U5B (5′-GTGTGGAAAATCTCTAGCAGT-3′), U5B-2 (5′-GTGTGGAAAATCTCTAGCA-3′), and U5A (3′-CACACCTTTTAGAGATCGTCA-5′). U5B or U5B-2 oligonucleotides were 32P labeled using polynucleotide kinase and annealed to the complementary U5A oligonucleotide. IN activity reactions were carried out in a buffer containing 20 mm Hepes, pH 7.2, 1 mm dithiothreitol, and 10 mm MgCl2. 3′-processing reactions were performed in the presence of 1.25 nm blunt IN substrate U5B/U5A. Strand transfer reactions were performed in the presence of 6.25 nm U5B-2/U5A substrate. 32P-labeled duplex DNAs were incubated in 20 μl of reaction buffer with 200, 400, or 600 nm integrase at 37 °C for 1 h. Reactions were stopped by adding 80 μlof a stop solution (7 mm EDTA, 0.3 m sodium acetate, 10 mm Tris-HCl, pH 8). IN was extracted with phenol/chloroform. DNA fragments were ethanol precipitated, suspended in a loading dye, and separated on 18% polyacrylamide denaturing gels. Gels were analyzed on a STORM 840® PhosphorImager (GE Healthcare). Homogenous Time-resolved Fluorescence Assays (HTRF)— Assays were carried out in a black 384-halfwell microplate (Greiner) using the following assay buffer: 100 mm phosphate buffer, pH 7.0, 800 mm KF, 0.44 mm CHAPS, 10 μm ZnCl, and 5 mm MgCl. Anti-GST cryptate (lot 49F) and anti-His-XL (lot 33F) from CisBio International were reconstituted as recommended. Protein concentrations and buffer conditions were previously optimized to result in an optimal signal. Consequently, recombinant His-tagged integrases were used at a final concentration of 50 μg/ml, whereas GST-SNF5/Ini1 was at 0.25 μg/ml. After addition of the interacting proteins and both antibodies on ice, the microplate was kept at 4 °C and read every 30 min in a Pherastar (BMG) at 665 and 620 nm after excitation at 337 nm. All points were in quadruplicate. The specific HTRF signal was expressed as a percentage of Δ F and calculated as follows: Δ F (%) = {[(665/620) sample - (665/620) blank]/(665/620) blank} × 100. Transient Inhibition of SNF5/Ini1 Expression Stimulates HIV-1 Replication—To evaluate the importance of SNF5/Ini1 for viral replication, we first used RNA interference to knock down SNF5/Ini1 expression in different cell lines before infection with HIV-1 was performed. Jurkat cells were transiently transfected with siRNA directed against SNF5/Ini1 (SNF5.1)or control siRNA (GL2). Expression of SNF5/Ini1 was greatly reduced 48 h after treatment with SNF5.1 siRNA but not with GL2 siRNA, whereas the expression of tubulin was similar in both conditions (Fig. 1A, lower panel). When compared with cells treated with control siRNA, HIV-1 replication was stimulated up to 3-fold when SNF5/Ini1 expression was inhibited (Fig. 1A, upper panel). Because of the transient effect of siRNA, the increase in HIV-1 replication upon SNF5/Ini1 knock down was optimal at day 3. In a single-round assay using HeLa P4.2 reporter cells, silencing of SNF5/Ini1 gene expression using two different siRNA (SNF5.1 or SNF5.3, Fig. 1B, lower panel) enhanced infection of NL4-3Δenv virus pseudotyped with VSV-G by 3- to 4-fold (Fig. 1B, upper panel). These results indicate that inhibition of SNF5/Ini1 expression enhances early steps of HIV-1 replication. As expected, HeLa cells transfected with two different doses of SNF5.3 siRNA also showed a 2-fold increase in transduction efficiency of an HIV-1 GFP reporter gene vector (Fig. 1C). Furthermore, the converse effect was observed. When SNF5/Ini1 was transiently overexpressed in A3.01 cells, HIV-1 replication was reduced ∼3-fold 24 h postinfection. Overexpression of SNF5/Ini1 was confirmed by immunoblot analysis (Fig. 4C). All together, these data suggested that SNF5/Ini1 could negatively regulate an early step of HIV-1 replication.FIGURE 4K71R mutation increases viral infectivity. A, infectivity of HIV-1WT, HIV-1E69G, and HIV-1K71R was measured in a single-cycle assay on HeLa P4.2 cells and was normalized for the amount of p24 viral antigen of the virus stocks. Values are expressed as percentages of HIV-1WT and represent the means and standard deviation of three independent experiments. B, Jurkat cells were infected with HIV-1WT, HIV-1E69G, and HIV-1K71R, (25 ng of p24 antigen/106 cells), and viral accumulation was quantified by measuring p24 antigen in the supernatant of the cells. Values represent the means and standard deviation of three independent experiments. C, transient overexpression of SNF5/Ini1 impairs HIV-1 replication. A3.01 cells were transfected with control or HA-SNF5/Ini1 expression plasmids. Cells were infected with HIV-1WT or HIV-1K71R (m.o.i. 10−3), and 24 h postinfection viral accumulation was quantified by measuring p24 antigen in the supernatant of the cells. Errors bars represent standard deviation of three experiments. Ratio of p24 in HA-SNF5/Ini1 versus control cells is indicated for each virus. SNF5/Ini1 overexpression in these cells was detected 24 h after transfection by Western blotting of cell lysates using anti-HA antibody, or anti-tubulin antibody as a control.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Accumulation of 2-LTR Circles and Integrated HIV DNA Forms in SNF5/Ini1 Knockdown Cells—Because we observed that inhibition of SNF5/Ini1 expression led to an increase in HIV-1 replication, we next assessed which step in the virus life cycle is affected by SNF5/Ini1. We monitored the levels of total DNA, 2-LTR circles, and integrated forms of proviral HIV-1 DNA by quantitative PCR on cell extract from HeLa cells transfected with either siRNA control or siRNA against SNF5/Ini1 (SNF5.3). SNF5/Ini1 expression was efficiently inhibited 24 h after siRNA transient transfection (Fig. 2A). Cells were then infected with HIV-1 Bru pseudotyped with VSV-G envelope (m.o.i. 0.1) and harvested at 3, 9, 24, and 48 h postinfection. In HeLa cells, HIV-1 replication was restricted to a single round of infection. Levels of total HIV cDNA synthesis peaked at 9 h postinfection and were similar in cells treated with either control or SNF5.3 siRNA. This amount remained higher at 24 and 48 h postinfection in cells where SNF5/Ini1 was inhibited (Fig. 2B). As expected, total HIV cDNA was barely detectable when infected cells were treated with a reverse transcriptase inhibitor, indicating that the quantitative PCR quantified de novo synthesized HIV cDNA. Interestingly, a 3- to 4-fold increase in 2-LTR circle forms was observed at 24 h postinfection in cells knocked down for SNF5/Ini1 (Fig. 2C). In addition, a 3- to 4-fold increase in the amount of integrated proviral DNA was also observed when SNF5/Ini1 expression was inhibited (Fig. 2D). Similar results were obtained when cells were transfected with a different so siRNA (SNF5.1) and infected at a lower m.o.i. (data not shown). Thus, these data indicate that the increase in HIV-1 replication observed after inhibition of SNF5/Ini1 expression correlates with an increase in the number of integrated copies as well as 2-LTR circular forms. Identification of Integrase Mutants Defective for SNF5/Ini1 Interaction—To further characterize the role of SNF5/Ini1
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