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• W1963510765 abstract "INI1/hSNF5/BAF47/SMARCB1 is an HIV-1 integrase (IN)-binding protein that modulates viral replication in multiple ways. A minimal IN-binding domain of INI1, S6 (amino acids 183–294), transdominantly inhibits late events, and down-modulation of INI1 stimulates early events of HIV-1 replication. INI1 both stimulates and inhibits in vitro integration depending on IN concentration. To gain further insight into its role in HIV-1 replication, we purified and biochemically characterized INI1. We found that INI1 forms multimeric structures. Deletion analysis indicated that the Rpt1 and Rpt2 motifs form the minimal multimerization domain. We isolated mutants of INI1 that are defective for multimerization using a reverse yeast two-hybrid system. Our results revealed that INI1 residues involved in multimerization overlap with IN-binding and nuclear export domains and are required for nuclear retention and co-localization with IN. Multimerization-defective mutants are also defective for mediating the transdominant effect of INI1-S6-(183–294). Furthermore, we found that INI1 is a minor groove DNA-binding protein. Although IN binding and multimerization are required for INI1-mediated inhibition, the acceptor DNA binding property of INI1 may be required for stimulation of in vitro strand transfer activities of IN. Binding of INI1 to IN results in the formation of presumably inactive high molecular weight IN-INI1 complexes, and the multimerization-defective mutant was unable to form these complexes. These results indicate that the multimerization and IN binding properties of INI1 are necessary for its ability to both inhibit integration and influence assembly and particle production, providing insights into the mechanism of INI1-mediated effects in HIV-1 replication. INI1/hSNF5/BAF47/SMARCB1 is an HIV-1 integrase (IN)-binding protein that modulates viral replication in multiple ways. A minimal IN-binding domain of INI1, S6 (amino acids 183–294), transdominantly inhibits late events, and down-modulation of INI1 stimulates early events of HIV-1 replication. INI1 both stimulates and inhibits in vitro integration depending on IN concentration. To gain further insight into its role in HIV-1 replication, we purified and biochemically characterized INI1. We found that INI1 forms multimeric structures. Deletion analysis indicated that the Rpt1 and Rpt2 motifs form the minimal multimerization domain. We isolated mutants of INI1 that are defective for multimerization using a reverse yeast two-hybrid system. Our results revealed that INI1 residues involved in multimerization overlap with IN-binding and nuclear export domains and are required for nuclear retention and co-localization with IN. Multimerization-defective mutants are also defective for mediating the transdominant effect of INI1-S6-(183–294). Furthermore, we found that INI1 is a minor groove DNA-binding protein. Although IN binding and multimerization are required for INI1-mediated inhibition, the acceptor DNA binding property of INI1 may be required for stimulation of in vitro strand transfer activities of IN. Binding of INI1 to IN results in the formation of presumably inactive high molecular weight IN-INI1 complexes, and the multimerization-defective mutant was unable to form these complexes. These results indicate that the multimerization and IN binding properties of INI1 are necessary for its ability to both inhibit integration and influence assembly and particle production, providing insights into the mechanism of INI1-mediated effects in HIV-1 replication. HIV-1 3The abbreviations used are: HIV-1human immunodeficiency virus, type 1INHIV-1-encoded integraseNi-NTAnickel-nitrilotriacetic acidEMSAelectrophoretic mobility shift assayLTRlong terminal repeatLEDGFlens epithelium-derived growth factorBSAbovine serum albuminHAhemagglutininNESnuclear export signalDAPI4′,6-diamidino-2-phenylindoleONPGo-nitrophenyl-β-d-galactopyranosideGFPgreen fluorescent proteinGSTglutathione S-transferaseHAPhydroxylapatite. replication is a dynamic process that is modulated by the interaction of several host cellular proteins (1Sorin M. Kalpana G.V. Curr. HIV Res. 2006; 4: 117-130Crossref PubMed Scopus (28) Google Scholar). A genome-wide siRNA-mediated knockdown indicated that hundreds of host factors are involved in the stimulation or inhibition of HIV-1 replication (2Brass A.L. Dykxhoorn D.M. Benita Y. Yan N. Engelman A. Xavier R.J. Lieberman J. Elledge S.J. Science. 2008; 319: 921-926Crossref PubMed Scopus (1183) Google Scholar). Understanding the interplay between the host proteins and the HIV-1 viral proteins is essential to fully comprehend the dynamic relationship between the virus and the host. human immunodeficiency virus, type 1 HIV-1-encoded integrase nickel-nitrilotriacetic acid electrophoretic mobility shift assay long terminal repeat lens epithelium-derived growth factor bovine serum albumin hemagglutinin nuclear export signal 4′,6-diamidino-2-phenylindole o-nitrophenyl-β-d-galactopyranoside green fluorescent protein glutathione S-transferase hydroxylapatite. INI1/hSNF5/BAF47/SMARCB1 is a core component of the SWI/SNF chromatin-remodeling complex. It interacts directly with the HIV-1-encoded integrase (IN) required for the integration of the viral DNA into the host chromosome (3Kalpana G.V. Marmon S. Wang W. Crabtree G.R. Goff S.P. Science. 1994; 266: 2002-2006Crossref PubMed Scopus (460) Google Scholar, 4Van Maele B. Busschots K. Vandekerckhove L. Christ F. Debyser Z. Trends Biochem. Sci. 2006; 31: 98-105Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar). IN mediates the insertion of viral cDNA into host chromosomal DNA by sequential steps of 3′ processing and strand transfer (or joining) (4Van Maele B. Busschots K. Vandekerckhove L. Christ F. Debyser Z. Trends Biochem. Sci. 2006; 31: 98-105Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar, 5Engelman A. Cherepanov P. PLoS Pathog. 2008; 4: e1000046Crossref PubMed Scopus (191) Google Scholar). INI1 binds directly to HIV-1 IN in vitro and in vivo and modulates several steps of HIV-1 replication (3Kalpana G.V. Marmon S. Wang W. Crabtree G.R. Goff S.P. Science. 1994; 266: 2002-2006Crossref PubMed Scopus (460) Google Scholar, 6Sorin M. Yung E. Wu X. Kalpana G.V. Retrovirology. 2006; 3: 56Crossref PubMed Scopus (30) Google Scholar, 7Maroun M. Delelis O. Coadou G. Bader T. Ségéral E. Mbemba G. Petit C. Sonigo P. Rain J.C. Mouscadet J.F. Benarous R. Emiliani S. J. Biol. Chem. 2006; 281: 22736-22743Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 8Yung 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). The ectopically expressed minimal IN-binding domain of INI1 transdominantly and potently inhibits HIV-1 assembly and particle production (8Yung 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). The inhibitory effect is dependent on IN-INI1 interaction and is abrogated when an IN mutant defective for interaction with INI1 is used (8Yung 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, particle production is minimal in cells lacking INI1, and reintroduction of INI1 into these cells can partially correct the defect (6Sorin M. Yung E. Wu X. Kalpana G.V. Retrovirology. 2006; 3: 56Crossref PubMed Scopus (30) Google Scholar). These results indicate that INI1 is required for HIV-1 late events. Additional studies have indicated that INI1 is selectively incorporated into HIV-1 but not other retroviral and lentiviral particles (9Yung E. Sorin M. Wang E.J. Perumal S. Ott D. Kalpana G.V. J. Virol. 2004; 78: 2222-2231Crossref PubMed Scopus (63) Google Scholar). Virally encapsidated INI1 is required for post-entry early events of HIV-1 replication prior to integration (6Sorin M. Yung E. Wu X. Kalpana G.V. Retrovirology. 2006; 3: 56Crossref PubMed Scopus (30) Google Scholar). These studies indicate that producer cell-associated as well as virion-associated INI1 is required for HIV-1 replication. Contrary to these proviral functions of INI1, siRNA-mediated knockdown studies indicate that INI1 in the target cells inhibits early events of HIV-1 replication (7Maroun M. Delelis O. Coadou G. Bader T. Ségéral E. Mbemba G. Petit C. Sonigo P. Rain J.C. Mouscadet J.F. Benarous R. Emiliani S. J. Biol. Chem. 2006; 281: 22736-22743Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). These studies indicate that whereas INI1 in the target cells may act as an antiviral host protein, HIV-1 may subvert the INI1 antiviral effect, and HIV-1 may utilize this host factor for late events in the producer cells and for early preintegration events in the target cells. Interestingly, in an earlier study, we demonstrated that partially purified INI1 both inhibits and stimulates in vitro integration in a manner dependent on IN concentration (3Kalpana G.V. Marmon S. Wang W. Crabtree G.R. Goff S.P. Science. 1994; 266: 2002-2006Crossref PubMed Scopus (460) Google Scholar). Although INI1 stimulates in vitro strand transfer reactions at low IN concentrations, it inhibits the reaction at high concentrations (3Kalpana G.V. Marmon S. Wang W. Crabtree G.R. Goff S.P. Science. 1994; 266: 2002-2006Crossref PubMed Scopus (460) Google Scholar). Further structure-function analysis of INI1 is required to understand this complex and dual role of INI1 during HIV-1 replication. INI1 gene is also a tumor suppressor that is biallelically deleted in aggressive pediatric cancers known as rhabdoid tumors (10Versteege I. Sévenet N. Lange J. Rousseau-Merck M.F. Ambros P. Handgretinger R. Aurias A. Delattre O. Nature. 1998; 394: 203-206Crossref PubMed Scopus (1233) Google Scholar). INI1 mutations have been found in other soft tissue cancers (11Hulsebos T.J. Plomp A.S. Wolterman R.A. Robanus-Maandag E.C. Baas F. Wesseling P. Am. J. Hum. Genet. 2007; 80: 805-810Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar, 12Modena P. Lualdi E. Facchinetti F. Galli L. Teixeira M.R. Pilotti S. Sozzi G. Cancer Res. 2005; 65: 4012-4019Crossref PubMed Scopus (275) Google Scholar, 13Sévenet N. Sheridan E. Amram D. Schneider P. Handgretinger R. Delattre O. Am. J. Hum. Genet. 1999; 65: 1342-1348Abstract Full Text Full Text PDF PubMed Scopus (351) Google Scholar). The mechanism of INI1-mediated tumor suppression is not fully understood. INI1 protein has two highly conserved domains that are imperfect direct repeats (termed Rpt1 and Rpt2) of each other and a third conserved coiled coil domain (termed homology region 3 or HR3) at the C terminus. The Rpt1 and Rpt2 domains appear to be involved in protein-protein interaction with various cellular and viral proteins (3Kalpana G.V. Marmon S. Wang W. Crabtree G.R. Goff S.P. Science. 1994; 266: 2002-2006Crossref PubMed Scopus (460) Google Scholar, 14Rozenblatt-Rosen O. Rozovskaia T. Burakov D. Sedkov Y. Tillib S. Blechman J. Nakamura T. Croce C.M. Mazo A. Canaani E. Proc. Natl. Acad. Sci. U.S.A. 1998; 95: 4152-4157Crossref PubMed Scopus (213) Google Scholar, 15Wu D.Y. Kalpana G.V. Goff S.P. Schubach W.H. J. Virol. 1996; 70: 6020-6028Crossref PubMed Google Scholar, 16Lee D. Sohn H. Kalpana G.V. Choe J. Nature. 1999; 399: 487-491Crossref PubMed Scopus (1004) Google Scholar, 17Cheng S.W. Davies K.P. Yung E. Beltran R.J. Yu J. Kalpana G.V. Nat. Genet. 1999; 22: 102-105Crossref PubMed Scopus (318) Google Scholar, 18Morozov A. Yung E. Kalpana G.V. Proc. Natl. Acad. Sci. U.S.A. 1998; 95: 1120-1125Crossref PubMed Scopus (82) Google Scholar). Additionally, the Rpt2 domain harbors a masked nuclear export signal, and the C-terminal domain is involved in inhibiting the nuclear export of the protein in the steady state. INI1 exhibits nonspecific DNA binding activity (18Morozov A. Yung E. Kalpana G.V. Proc. Natl. Acad. Sci. U.S.A. 1998; 95: 1120-1125Crossref PubMed Scopus (82) Google Scholar). The cancer-associated mutations occur throughout the open reading frame of the INI1 gene, suggesting that mutation in any one of the INI1 domains may inactivate the protein and that multiple domains are required for its function (19Schmitz U. Mueller W. Weber M. Sévenet N. Delattre O. von Deimling A. Br. J. Cancer. 2001; 84: 199-201Crossref PubMed Scopus (107) Google Scholar, 20Sévenet N. Lellouch-Tubiana A. Schofield D. Hoang-Xuan K. Gessler M. Birnbaum D. Jeanpierre C. Jouvet A. Delattre O. Hum. Mol. Genet. 1999; 8: 2359-2368Crossref PubMed Scopus (268) Google Scholar, 21Yuge M. Nagai H. Uchida T. Murate T. Hayashi Y. Hotta T. Saito H. Kinoshita T. Cancer Genet. Cytogenet. 2000; 122: 37-42Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). To gain further insight into the mechanism of its action, we purified INI1 protein to homogeneity and characterized it biochemically. Here we report, for the first time, that INI1 forms dimeric and higher order multimeric structures. We have characterized the multimerization domain of INI1 and found that multimerization and IN binding activities of INI1 are required for inhibition of in vitro integration. Furthermore, we found that the multimerization, IN binding, and nuclear export properties of INI1 are important for transdominant effects. In addition, we found that INI1 possesses a minor groove DNA binding activity and that the nonspecific acceptor DNA binding activity of INI1 may be required for stimulation of in vitro integration. Finally, we found that multimerization of the full-length protein is necessary for its ability to be retained in the nucleus and to co-localize with HIV-1 IN in the nucleus. Thus, our studies provide novel insights into the mechanism by which INI1 regulates HIV-1 replication. The plasmids pGEX-INI1, pGEX-IN, pSH2-INI1, and pGADNotINI1 and deletion fragments pGADNotINI1-(183–294), pSH2IN, pBABEpuro-INI1, pCGNINI1, and pQE32-INI1 have been described previously (18Morozov A. Yung E. Kalpana G.V. Proc. Natl. Acad. Sci. U.S.A. 1998; 95: 1120-1125Crossref PubMed Scopus (82) Google Scholar). Details of the construction of pQE32-INI1-(141–304) and generation of the random mutagenesis library of pGADNotINI1-(183–294) are as described in the supplemental material. CFP-INI1, DD5, and DD6 constructs were generated by PCR amplification and subcloning of the subsequent fragments at the EcoRI-BamHI sites of pECFP vector (Clontech). His6-INI1 and His6-INI1-(183–294) were purified to homogeneity as described in the supplemental material. His6-LEDGF was purified as reported (27Vandegraaff N. Devroe E. Turlure F. Silver P.A. Engelman A. Virology. 2006; 346: 415-426Crossref PubMed Scopus (88) Google Scholar) except that His6-LEDGF was not digested with PreScission protease, and two purification steps, viz. Ni-NTA and Mono-S Sepharose, were used. The partially purified protein was dialyzed against IN dialysis buffer (3Kalpana G.V. Marmon S. Wang W. Crabtree G.R. Goff S.P. Science. 1994; 266: 2002-2006Crossref PubMed Scopus (460) Google Scholar). C-terminal His-tagged IN was purified as described (3Kalpana G.V. Marmon S. Wang W. Crabtree G.R. Goff S.P. Science. 1994; 266: 2002-2006Crossref PubMed Scopus (460) Google Scholar). These tests were performed as described, and details are provided in the supplemental “Materials and Methods” (3Kalpana G.V. Marmon S. Wang W. Crabtree G.R. Goff S.P. Science. 1994; 266: 2002-2006Crossref PubMed Scopus (460) Google Scholar). The details of the methods used are provided in the supplemental “Materials and Methods.” Briefly, Mono-Q Sepharose eluate of His-INI1 was separated on a 15–35% glycerol gradient at 40,000 rpm (SW41 Ti rotor) for 24 h. Fractions (400–450 μl) were collected and subjected to trichloroacetic acid precipitation. The precipitated proteins were subjected to Western blot analysis using an anti-His monoclonal antibody as probe. To compare the oligomeric status of wild type and mutant INI1 proteins, the protein was incubated in 500 μl of the buffer for 3 h on ice and then subjected to glycerol gradient centrifugation as described above. Gel filtration chromatography was performed in 20 mm Tris-HCl (pH 7.5), 100 mm NaCl, 1 mm dithiothreitol, with 200 μg of His-INI1-(183–294) eluate from the Mono-Q Sepharose column. Fractions (500 μl) were collected and subjected to Western blot analysis using anti-His antibody as probe. The joining assays were performed with 32P-labeled 3′ preprocessed duplex DNA (U5.5 and U5.4) as donor DNA and pcDNA as target DNA as described previously (3Kalpana G.V. Marmon S. Wang W. Crabtree G.R. Goff S.P. Science. 1994; 266: 2002-2006Crossref PubMed Scopus (460) Google Scholar) using the indicated amounts of proteins except that ∼7 ng of radiolabeled substrate and 100 ng of target DNA were used. Salt concentrations were varied between 10 and 150 mm NaCl. For agarose-based gel retardation assay about 8 and 16 pmol of hydroxylapatite eluate of the wild type INI1 or mutant INI1 were incubated with 200 ng of pcDNA in buffer containing 20 mm HEPES-KOH (pH 6.8), 100 mm NaCl, 0.1 mm EDTA, 1 mm dithiothreitol, 10% glycerol, 3 mm MnCl2, and protease inhibitors at 30 °C for 1 h. Protein-DNA complexes were resolved by 1% agarose gel electrophoresis and stained with ethidium bromide. For EMSA ∼7 ng of radiolabeled 3′ preprocessed duplex DNA (U5.5 and U5.4) was incubated with 5 pmol of hydroxylapatite eluate of wild type INI1 in strand transfer reaction buffer and protease inhibitors at 37 °C for 10 min. 10× cold viral LTR DNA was added to the reaction mixture. Minor groove inhibitors were added to the reaction mixture as indicated. About 10 μl of reaction mixture was run on a 6% native polyacrylamide gel made in Tris borate-EDTA, and the gel was run at 10 mA for 2 h. The gel was dried and subjected to autoradiography. These were carried out as described previously, and the details are provided in the supplemental material (8Yung 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, 23Craig E. Zhang Z.K. Davies K.P. Kalpana G.V. EMBO J. 2002; 21: 31-42Crossref PubMed Scopus (82) Google Scholar). To understand the structure-function relationships of INI1, we biochemically purified bacterially expressed His-tagged INI1 (His-INI1) through three consecutive chromatography steps, viz. Ni-NTA-agarose, hydroxylapatite, and Mono-Q Sepharose columns (see supplemental “Materials and Methods”). After elution through the Mono-Q Sepharose column, INI1 was purified to near homogeneity (>95%; Fig. 1A). To test whether recombinant INI1 is an active protein, we carried out in vitro strand transfer reactions using different concentrations of purified INI1 and different IN:INI1 molar ratios. Previous reports had indicated that INI1 is able to activate and inhibit in vitro strand transfer activity of HIV-1 IN depending on the IN concentration and IN:INI1 molar ratios (3Kalpana G.V. Marmon S. Wang W. Crabtree G.R. Goff S.P. Science. 1994; 266: 2002-2006Crossref PubMed Scopus (460) Google Scholar). We employed the hydroxylapatite eluate for carrying out IN reactions because of the poor yield after the Mono-Q column purification step. INI1 stimulated joining of the substrate to the target DNA in a dose-dependent manner at low concentrations (0.25 pmol) of IN (Fig. 1B, compare lane 2 with lanes 5 and 8, indicated by arrows) but inhibited strand transfer reaction at higher concentration of IN (1.0 pmol) (Fig. 1B, compare lanes 4 and 7 with lane 10, indicated by arrowheads). At or near the physiological salt concentration (100, 125, and 150 mm) of NaCl, we found that 0.5 pmol of INI1 stimulated integrase activity at lower integrase input (0.25 pmol) and a lower INI1:IN ratio (2:1) but inhibited integrase activity at higher integrase input (0.5 pmol) and a higher INI1:IN ratio (4:1) (Fig. 1C). At physiological salt concentrations, a higher amount of INI1 was required to obtain robust inhibition of integrase activity. Similar results were obtained with another IN-binding protein, LEDGF. We found that purified full-length LEDGF stimulated IN strand transfer activity at a low IN concentration and an IN:LEDGF ratio of 1:4 and inhibited the reaction at a high IN concentration and an IN:LEDGF ratio of 1:2 (Fig. 1D, lanes 1–12). Furthermore, the addition of 5.3 μm IN inhibitor (S-1360) resulted in inhibition of integrase strand transfer activities under these conditions (Fig. 1D, lanes 13–15). During the course of these studies, we observed that INI1 forms a higher order structure and has a tendency to aggregate at high concentration. This and additional observations using in vitro and in vivo binding studies suggested that INI1 is a multimer, as detailed below. To determine whether INI1 exists as a multimer in solution, purified INI1 eluted from the Mono-Q Sepharose column was subjected to glycerol gradient centrifugation. Purified marker proteins were run as a control to estimate the apparent molecular weight relative to the fractions. At low concentrations (<5 nm), purified INI1 fractionated in a single peak after BSA (67 kDa) in glycerol gradients (Fig. 1E). However, at higher protein concentrations (>100 nm), INI1 migrated in multiple peaks (Fig. 1F). The first peak of INI1 was observed just after the 79-kDa marker, indicating that it is a dimer. Furthermore, other peaks of INI1 migrated with approximate molecular masses indicative of tetramers and octamers and higher order forms (Fig. 1F). Taken together, these studies demonstrated that recombinant INI1 is a dimer in solution at low concentrations but forms higher order structures at high concentrations. To further confirm that INI1 self-associates, we carried out other in vitro and in vivo interaction assays. A GST pulldown assay was carried out using GST-INI1 and His6-INI1 expressed in bacteria. The bacterial lysates expressing His6-INI1 were treated with DNase I, and the binding reaction was carried out in the presence of ethidium bromide to avoid DNA-mediated association (22Lai J.S. Herr W. Proc. Natl. Acad. Sci. U.S.A. 1992; 89: 6958-6962Crossref PubMed Scopus (398) Google Scholar). In this assay, although the control GST bound to beads failed to pull down His-INI1, GST-INI1 was able to interact robustly with His-INI1, confirming that INI1 interacts with itself in vitro (Fig. 1G). To investigate whether INI1 self-associates in vivo, 293T cells were transfected with HA-tagged INI1 (HA-INI1) or FLAG-tagged INI1 (FL-INI1) alone or together. The lysates were pretreated with micrococcal nuclease to avoid scoring for a DNA-mediated association. FLAG-INI1 was able to coimmunoprecipitate HA-INI1 (Fig. 1H, lane 4) but not when expressed alone (Fig. 1H, lane 2). Taken together these results demonstrate that INI1 self-associates both in vitro and in vivo. To determine which domain of INI1 is involved in self-association, a deletion analysis was carried out using the yeast two-hybrid assay. A series of truncation mutants of INI1 as a fusion to GAL4AD (Fig. 2A) were tested for their ability to interact with full-length INI1 fused to the LexADBD (Fig. 2B). GAL4AD-INI1 efficiently interacted with LexADBD-INI1 in yeast (Fig. 2B). Mutants with deletion in the N terminus of INI1 that contained the Rpt1, Rpt2, and HR3 domains retained their ability to interact with full-length INI1, whereas the N-terminal fragment INI1-(1–130) failed to interact with LexADBD-INI1 (Fig. 2B). Deletion fragments INI1-(1–245) and INI1-(262–385), containing either the Rpt1 or Rpt2 motif, respectively, retained their ability to bind LexADBD-INI1 (Fig. 2B) although to a lesser extent (10 and 14%, respectively). Truncation of the C-terminal coiled coil domain of INI1 did not affect the ability of INI1-(1–294) to bind LexADBD-INI1 robustly (Fig. 2B). Interestingly, the mutant INI1-(1–294), which retained both the Rpt and the N-terminal domains, exhibited robust activity. These results suggested that although the N-terminal fragment itself is not sufficient for self-association, it may be required for proper folding of INI1 that allows efficient self-association. The fact that INI1-(141–304) retained its ability to bind LexADBD-INI1 (Fig. 2B) demonstrates that the Rpt1 and Rpt2 motifs form the minimal multimerization domain. To determine whether the minimal multimerization domain of INI1 can self-associate in solution, deletion fragment S7 (amino acids 141–304) was expressed in bacteria and purified through three chromatographic columns, viz. Ni-NTA, hydroxylapatite, and Mono-Q Sepharose, respectively. The purified INI1-(141–304) polypeptide migrated in SDS-PAGE as two bands (Fig. 2C, left panel). Our analysis indicated that the faster migrating polypeptide is neither a degradation product of INI1-(141–304) nor a bacterial contaminant because: (i) antibodies against both the N-terminal His epitope tag and the C terminus of INI1 recognized the two polypeptides in the purified INI1-(141–304) preparation (Fig. 2C, middle and right panels), and (ii) mass spectrometric analysis revealed a single polypeptide species in purified preparations (data not shown). We found that the faster migrating polypeptide was an artifact of detergent solubilization, as it was absent when the protein was purified in the absence of IGEPAL, and the single band stained positively with INI1 antibody (Fig. 2D and data not shown). These results indicated that we were able to purify the deletion fragment to apparent homogeneity. To investigate the multimeric form of INI1-(141–304) we subjected the purified protein to gel filtration chromatography using a Superdex 200 HR 10/30 column. INI1-(141–304) eluted from the column soon after ovalbumin (43 kDa) and before chymotrypsinogen A (25 kDa), demonstrating that it is a dimer (∼42 kDa) (Fig. 2E). Western blot analysis of fractions eluted from the gel filtration column using an antibody against the His epitope tag confirmed that the eluted protein was INI1-(141–304) (Fig. 2E). To investigate the functional significance of multimerization of INI1 in HIV-1 replication, we isolated a panel of mutants of the minimal multimerization domain of INI1, S6 (amino acids 183–294), using a reverse yeast two-hybrid system. A random mutation library of INI1-S6-(183–294) fused to GAL4AD (8Yung 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) was screened against LexADB-INI1 to isolate a panel of multimerization-defective mutants with one or two amino acid substitutions (Table 1). These mutations were in either the Rpt1 or the Rpt2 motif and were mostly hydrophobic (Fig. 3A). These results suggested that hydrophobic interactions are likely to be important for INI1 self-association. Furthermore, among the residues mutated, amino acids Phe-204, Trp-206, Phe-233, Ile-237, Ile-263, Ile-264, and Ile-268 were partially or fully conserved phylogenetically (data not shown). In addition, several clusters of mutations were observed in Rpt1, and mutations in the Rpt2 motif overlapped with the nuclear export signal (NES) of INI1 (Fig. 3A). Previously we had identified mutants of INI1-(183–294) that are defective for interaction with IN (8Yung 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). Interestingly, we found that several residues of INI1-(183–294) necessary for IN binding were distinct from those required for INI1 multimerization (Fig. 3A and Table 2). Although IN binding-defective mutants were more frequent in the Rpt1 domain, multimerization-defective mutants were found in both the Rpt1 and Rpt2 regions.TABLE 1Interaction of dimerization-defective S6 with INI1 and INS6cloneMutation in Rpt1Mutation in Rpt2Interaction with INI1aPercentage of β-galactosidase activity representing the interaction of wild type S6 protein with wild type INI1 or IN.Interaction with INaPercentage of β-galactosidase activity representing the interaction of wild type S6 protein with wild type INI1 or IN.%%S6100100DD1F233L33<1DD2F204S33<1DD3V234A,I237T27<1DD4I195T,W206R28<1DD5I263T<510DD6I264T,I268T<110a Percentage of β-galactosidase activity representing the interaction of wild type S6 protein with wild type INI1 or IN. Open table in a new tab TABLE 2Interaction of IN-binding mutants of S6 with INI1 and INS6cloneMutationin Rpt1Mutationin Rpt2Interactionwith INaPercentage of β-galactosidase activity representing the interaction of wild type S6 protein with wild type INI1 or IN.Interactionwith INI1aPercentage of β-galactosidase activity representing the interaction of wild type S6 protein with wild type INI1 or IN.%%S6100100E3D225G2655E4T214A<293E5V185AI264T<110E6E183G,D191G<181a Percentage of β-galactosidase activity representing the interaction of wild type S6 protein with wild type INI1 or IN. Open table in a new tab To further investigate the relationship between IN binding and multimerization, we analyzed two panels of mutants of S6 INI-(183–294) for their ability to bind to both INI1 and IN, using a yeast two-hybrid system scoring for β-galactosidase activity (Fig. 3, Table 1). The first panel consisted of mutants isolated as defective for binding to INI1 using the reverse two-hybrid system. The second panel included mutants of S6 INI1-(183–294) that were previously isolated as defective for IN binding (8Yung 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). We found that the IN binding activity of S6 was stronger than its ability to bind t" @default.
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• W1963510765 title "Multimerization and DNA Binding Properties of INI1/hSNF5 and Its Functional Significance" @default.
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