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• W2059708939 abstract "HIV-1 integrase (IN) orchestrates the integration of the reverse transcribed viral cDNA into the host cell genome and participates also in other steps of HIV-1 replication. Cellular and viral factors assist IN in performing its multiple functions, and post-translational modifications contribute to modulate its activities. Here, we show that HIV-1 IN is modified by SUMO proteins and that phylogenetically conserved SUMOylation consensus motifs represent major SUMO acceptor sites. Viruses harboring SUMOylation site IN mutants displayed a replication defect that was mapped during the early stages of infection, before integration but after reverse transcription. Because SUMOylation-defective IN mutants retained WT catalytic activity, we hypothesize that SUMOylation might regulate the affinity of IN for co-factors, contributing to efficient HIV-1 replication. HIV-1 integrase (IN) orchestrates the integration of the reverse transcribed viral cDNA into the host cell genome and participates also in other steps of HIV-1 replication. Cellular and viral factors assist IN in performing its multiple functions, and post-translational modifications contribute to modulate its activities. Here, we show that HIV-1 IN is modified by SUMO proteins and that phylogenetically conserved SUMOylation consensus motifs represent major SUMO acceptor sites. Viruses harboring SUMOylation site IN mutants displayed a replication defect that was mapped during the early stages of infection, before integration but after reverse transcription. Because SUMOylation-defective IN mutants retained WT catalytic activity, we hypothesize that SUMOylation might regulate the affinity of IN for co-factors, contributing to efficient HIV-1 replication. HIV-1 IN 6The abbreviations used are: INintegrasePICpreintegration complexSUMOsmall ubiquitin-like modifierCAcapsidVSVgglycoprotein G of vesicular stomatitis virus. is a 288-amino acid protein consisting of three functionally independent domains. The N-terminal domain harbors a highly conserved HHCC zinc binding motif that contributes to IN multimerization and enzymatic activities. The central core domain contains the catalytic DDE motif that is conserved in all retroviral and retrotransposon INs and in certain bacterial transposases. The C-terminal domain is the least conserved among retroviral IN and binds DNA nonspecifically (for reviews, see Refs. 1Craigie R. J. Biol. Chem. 2001; 276: 23213-23216Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar and 2Esposito D. Craigie R. Adv. Virus. Res. 1999; 52: 319-333Crossref PubMed Google Scholar). The best characterized activity of HIV-1 IN is the catalysis of integration, which is crucial for HIV-1 replication (3Vandegraaff N. Engelman A. Expert Rev. Mol. Med. 2007; 9: 1-19Crossref PubMed Scopus (54) Google Scholar). This reaction can be reproduced in vitro in the presence of recombinant IN alone and synthetic DNA species mimicking the viral LTR ends and an acceptor substrate (4Craigie R. Fujiwara T. Bushman F. Cell. 1990; 62: 829-837Abstract Full Text PDF PubMed Scopus (331) Google Scholar, 5Katz R.A. Merkel G. Kulkosky J. Leis J. Skalka A.M. Cell. 1990; 63: 87-95Abstract Full Text PDF PubMed Scopus (297) Google Scholar). However, other components of the preintegration complex (PIC) contribute to the specificity and efficiency of integration in vivo (6Carteau S. Gorelick R.J. Bushman F.D. J. Virol. 1999; 73: 6670-6679Crossref PubMed Google Scholar, 7Hindmarsh P. Leis J. Adv. Virus. Res. 1999; 52: 397-410Crossref PubMed Google Scholar, 8Bowerman B. Brown P.O. Bishop J.M. Varmus H.E. Genes Dev. 1989; 3: 469-478Crossref PubMed Scopus (284) Google Scholar). Independently of its enzymatic activity, HIV-1 IN plays additional roles during the viral life cycle that are still ill defined. Indeed, many catalytically active IN mutants have pleiotropic effects impairing reverse transcription and/or uncoating (9Hehl E.A. Joshi P. Kalpana G.V. Prasad V.R. J. Virol. 2004; 78: 5056-5067Crossref PubMed Scopus (95) Google Scholar, 10Tasara T. Maga G. Hottiger M.O. Hübscher U. FEBS Lett. 2001; 507: 39-44Crossref PubMed Scopus (60) Google Scholar, 11Tsurutani N. Kubo M. Maeda Y. Ohashi T. Yamamoto N. Kannagi M. Masuda T. J. Virol. 2000; 74: 4795-4806Crossref PubMed Scopus (113) Google Scholar, 12Wu X. Liu H. Xiao H. Conway J.A. Hehl E. Kalpana G.V. Prasad V. Kappes J.C. J. Virol. 1999; 73: 2126-2135Crossref PubMed Google Scholar, 13Zhu K. Dobard C. Chow S.A. J. Virol. 2004; 78: 5045-5055Crossref PubMed Scopus (135) Google Scholar, 14Leavitt A.D. Robles G. Alesandro N. Varmus H.E. J. Virol. 1996; 70: 721-728Crossref PubMed Google Scholar, 15Engelman A. Englund G. Orenstein J.M. Martin M.A. Craigie R. J. Virol. 1995; 69: 2729-2736Crossref PubMed Scopus (0) Google Scholar, 16Shin C.G. Taddeo B. Haseltine W.A. Farnet C.M. J. Virol. 1994; 68: 1633-1642Crossref PubMed Google Scholar, 17Briones M.S. Dobard C.W. Chow S.A. J. Virol. 2010; 84: 5181-5190Crossref PubMed Scopus (40) Google Scholar), PIC nuclear import (18Gallay P. Hope T. Chin D. Trono D. Proc. Natl. Acad. Sci. U.S.A. 1997; 94: 9825-9830Crossref PubMed Scopus (420) Google Scholar, 19Ikeda T. Nishitsuji H. Zhou X. Nara N. Ohashi T. Kannagi M. Masuda T. J. Virol. 2004; 78: 11563-11573Crossref PubMed Scopus (53) Google Scholar), and virion protein composition and/or morphology (16Shin C.G. Taddeo B. Haseltine W.A. Farnet C.M. J. Virol. 1994; 68: 1633-1642Crossref PubMed Google Scholar, 20Bukovsky A. Göttlinger H. J. Virol. 1996; 70: 6820-6825Crossref PubMed Google Scholar, 21Quillent C. Borman A.M. Paulous S. Dauguet C. Clavel F. Virology. 1996; 219: 29-36Crossref PubMed Scopus (64) Google Scholar). integrase preintegration complex small ubiquitin-like modifier capsid glycoprotein G of vesicular stomatitis virus. Post-translational modifications contribute to the regulation of IN activities. HIV-1 IN interacts with and is acetylated by both histone acetyltransferases p300 and GCN5 on C-terminal lysine residues (22Cereseto A. Manganaro L. Gutierrez M.I. Terreni M. Fittipaldi A. Lusic M. Marcello A. Giacca M. EMBO J. 2005; 24: 3070-3081Crossref PubMed Scopus (145) Google Scholar, 23Terreni M. Valentini P. Liverani V. Gutierrez M.I. Di Primio C. Di Fenza A. Tozzini V. Allouch A. Albanese A. Giacca M. Cereseto A. Retrovirology. 2010; 7: 18Crossref PubMed Scopus (62) Google Scholar). Acetylation increases IN affinity for the viral cDNA, enhances its strand transfer activity in vitro, and might regulate the interaction between IN and cellular factors (24Allouch A. Cereseto A. Amino Acids. 2009; (in press)PubMed Google Scholar). However, the role of this modification during HIV-1 replication is still controversial (25Topper M. Luo Y. Zhadina M. Mohammed K. Smith L. Muesing M.A. J. Virol. 2007; 81: 3012-3017Crossref PubMed Scopus (45) Google Scholar). HIV-1 IN is also ubiquitinated and subsequently degraded by the proteasome (26Mousnier A. Kubat N. Massias-Simon A. Ségéral E. Rain J.C. Benarous R. Emiliani S. Dargemont C. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 13615-13620Crossref PubMed Scopus (76) Google Scholar, 27Mulder L.C. Muesing M.A. J. Biol. Chem. 2000; 275: 29749-29753Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 28Devroe E. Engelman A. Silver P.A. J. Cell Sci. 2003; 116: 4401-4408Crossref PubMed Scopus (56) Google Scholar). In the viral context, IN degradation seems to occur after integration and to be required for correct gap repair (29Emiliani S. Mousnier A. Busschots K. Maroun M. Van Maele B. Tempé 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 (210) Google Scholar, 30Yoder K.E. Bushman F.D. J. Virol. 2000; 74: 11191-11200Crossref PubMed Scopus (178) Google Scholar) and viral gene expression (26Mousnier A. Kubat N. Massias-Simon A. Ségéral E. Rain J.C. Benarous R. Emiliani S. Dargemont C. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 13615-13620Crossref PubMed Scopus (76) Google Scholar). Recently, phosphorylation of HIV-1 IN by cellular JNK has also been proposed to modulate its stability and to be necessary for efficient integration (31Manganaro L. Lusic M. Gutierrez M.I. Cereseto A. Del Sal G. Giacca M. Nat. Med. 2010; 16: 329-333Crossref PubMed Scopus (89) Google Scholar). SUMOylation consists of the covalent attachment of small ubiquitin-like modifier (SUMO) peptides to a Lys residue within the consensus motif (ΨKX(E/D), where Ψ is a large hydrophobic residue) of a substrate protein. SUMO conjugation is mediated by SUMO-specific E1-activating, E2-conjugating, and E3-ligating enzymes and is reversed by SUMO-specific proteases (reviewed in Ref. 32Geiss-Friedlander R. Melchior F. Nat. Rev. Mol. Cell Biol. 2007; 8: 947-956Crossref PubMed Scopus (1382) Google Scholar). In mammals, three major forms of SUMO proteins are expressed. SUMO-1 has ∼45% amino acid sequence homology to SUMO-2 and SUMO-3, which are 96% identical to each other. SUMO modification is implicated in numerous cellular processes, including signal transduction, protein stability and localization, transcriptional regulation, chromatin structure, and genome stability (32Geiss-Friedlander R. Melchior F. Nat. Rev. Mol. Cell Biol. 2007; 8: 947-956Crossref PubMed Scopus (1382) Google Scholar). It is also well established that viruses interfere with and/or hijack the cellular SUMOylation machinery to replicate (for reviews, see Refs. 33Boggio R. Chiocca S. Curr. Opin. Microbiol. 2006; 9: 430-436Crossref PubMed Scopus (82) Google Scholar and 34Wilson V.G. Rangasamy D. Virus Res. 2001; 81: 17-27Crossref PubMed Scopus (40) Google Scholar). Interactions between murine leukemia virus capsid (CA) protein and components of the SUMOylation pathway are required for proper execution of the early steps of replication after reverse transcription but before integration (35Yueh A. Leung J. Bhattacharyya S. Perrone L.A. de los Santos K. Pu S.Y. Goff S.P. J. Virol. 2006; 80: 342-352Crossref PubMed Scopus (45) Google Scholar). SUMOylation events have also been implicated in the early phase of HIV-1 infection. Indeed, SUMO-2 and RanBP2 (Ran-binding protein 2), a SUMO E3 ligase, were identified in genome-wide screens for cell factors that promote HIV-1 reverse transcription and PIC nuclear import, respectively (36König R. Zhou Y. Elleder D. Diamond T.L. Bonamy G.M. Irelan J.T. Chiang C.Y. Tu B.P. De Jesus P.D. Lilley C.E. Seidel S. Opaluch A.M. Caldwell J.S. Weitzman M.D. Kuhen K.L. Bandyopadhyay S. Ideker T. Orth A.P. Miraglia L.J. Bushman F.D. Young J.A. Chanda S.K. Cell. 2008; 135: 49-60Abstract Full Text Full Text PDF PubMed Scopus (800) Google Scholar, 37Brass 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 (1189) Google Scholar). Interaction of HIV-1 or Mason-Pfitzer monkey virus Gag proteins with SUMO-1 and the E2-conjugating enzyme Ubc9 during the late phases of replication have also been reported and are probably involved in the production of fully infectious virions (38Gurer C. Berthoux L. Luban J. J. Virol. 2005; 79: 910-917Crossref PubMed Scopus (71) Google Scholar, 39Martinez N.W. Xue X. Berro R.G. Kreitzer G. Resh M.D. J. Virol. 2008; 82: 9937-9950Crossref PubMed Scopus (68) Google Scholar, 40Jaber T. Bohl C.R. Lewis G.L. Wood C. West Jr., J.T. Weldon Jr., R.A. J. Virol. 2009; 83: 10448-10459Crossref PubMed Scopus (24) Google Scholar, 41Weldon Jr., R.A. Sarkar P. Brown S.M. Weldon S.K. Virology. 2003; 314: 62-73Crossref PubMed Scopus (21) Google Scholar). Here, we show that HIV-1 IN is SUMOylated and that three Lys residues, which are found within conserved consensus motifs, represent the major SUMO acceptor sites. In the viral context, mutation of SUMO acceptor residues in IN led to reduced infectivity and slower replication kinetics. Biogenesis, release, and reverse transcription steps of mutant HIV-1 particles were not affected. However, cells infected with viruses harboring SUMOylation-defective IN mutants showed a significant decrease in integration events compared with HIV-1WT-infected cells. Because SUMOylation-site IN mutants retained WT catalytic activity, we inferred that modification by SUMO might participate in the modulation of the HIV-1 IN interaction network by regulating its affinity for co-factors, which are required for the efficient execution of early events of HIV-1 replication. HeLa and 293T cells were grown in DMEM (Invitrogen). CEM-GFP cells (AIDS Reagent Program), a human T cell line harboring the GFP reporter gene under the control of HIV-1 LTR (42Gervaix A. West D. Leoni L.M. Richman D.D. Wong-Staal F. Corbeil J. Proc. Natl. Acad. Sci. U.S.A. 1997; 94: 4653-4658Crossref PubMed Scopus (208) Google Scholar), were grown in RPMI (Invitrogen). Media were supplemented with 10% fetal calf serum (Invitrogen), 100 units/ml penicillin, and 100 μg/ml streptomycin. Antibodies used were as follows: mouse anti-IN (IN-2), mouse anti-His, rabbit anti-SUMO-1 (FL-101) (Santa Cruz Biotechnology, Inc. (Santa Cruz, CA)), rabbit anti-IN 756 and 757 (AIDS Reagent Program), rabbit anti-SUMO-2/3 (Zymed Laboratories Inc.), mouse anti-CA (Hybridolab), mouse anti-FLAG M2 (Sigma-Aldrich), mouse anti-HA 12CA5 (Roche Applied Science), and rabbit anti-acetyl-lysine (Abcam). psPAX2, pWPI, and pMD2.G (a gift from D. Trono); pNL4–3EnvFsGFP (a gift from D. Gabuzda), which contains a complete HIV-1 provirus with an env-inactivating mutation and enhanced GFP in the place of Nef (43He J. Chen Y. Farzan M. Choe H. Ohagen A. Gartner S. Busciglio J. Yang X. Hofmann W. Newman W. Mackay C.R. Sodroski J. Gabuzda D. Nature. 1997; 385: 645-649Crossref PubMed Scopus (823) Google Scholar); and INWT-FLAG, which encodes a codon-optimized IN gene harboring an ATG initiation codon and a C-terminal FLAG tag (44Cherepanov P. Pluymers W. Claeys A. Proost P. De Clercq E. Debyser Z. FASEB J. 2000; 14: 1389-1399PubMed Google Scholar), have been described previously. The cDNAs encoding WT IN, SUMO-1, -2, and -3, were amplified by PCR from IN-FLAG and YFP-SUMO-1, -2, and -3 (45Ayaydin F. Dasso M. Mol. Biol. Cell. 2004; 15: 5208-5218Crossref PubMed Scopus (161) Google Scholar) and were subcloned in frame with an N-terminal His6 tag into the pcDNA3.1(−) vector (Invitrogen), yielding His-INWT and His-SUMO-1, -2, and -3. The cDNA encoding the C-terminal region of LEDGF/p75 (amino acids 325–530) was amplified by PCR from WT and D366N mutant HA-LEDGF/p75 (46Cherepanov P. Sun Z.Y. Rahman S. Maertens G. Wagner G. Engelman A. Nat. Struct. Mol. Biol. 2005; 12: 526-532Crossref PubMed Scopus (205) Google Scholar). To produce IN mutants, changes were introduced by PCR into the IN sequence of the suitable plasmid using the Expand Long Templates PCR System (Roche Applied Science). The entire recombinant IN coding fragment was confirmed by sequencing and swapped for the corresponding WT fragment into the appropriate recipient vector. Bacterially expressed His-tagged full-length IN, N-terminal (INΔN, encoding amino acids 50–288) or C-terminal (INΔC, encoding amino acids 1–213) truncation forms (47Carayon K. Leh H. Henry E. Simon F. Mouscadet J.F. Deprez E. Nucleic Acids Res. 2010; 38: 3692-3708Crossref PubMed Scopus (25) Google Scholar), and IN3KR mutant were purified as described previously (48Leh 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). Next, recombinant His-tagged full-length or mutant IN proteins (200 nm) were used to perform an in vitro assay with the SUMOylation kit (BIOMOL) according to the manufacturer's instructions. 293T cells (3 × 106) were seeded into 10-cm dishes and transfected 24 h later using a calcium phosphate precipitation technique with plasmids encoding FLAG-tagged WT or mutant IN proteins and vectors expressing Ubc9 and His-SUMO-1, -2, or -3 or an appropriate empty vector. After 40 h, cells were lysed under denaturing conditions in buffer A (6 m guanidium HCl, 0.1 m Na2HPO4/NaH2PO4, 10 mm imidazole, pH 8.0) and sonicated (10 cycles, 40-s pulse, 15-s pause with the BioruptorTM (Diagenode)). Cell lysates were incubated with nickel-NTA-agarose beads (Qiagen) (3 h, room temperature) and next extensively washed with decreasing amounts of guanidium HCl. Bound proteins were eluted by boiling in Laemmli buffer with 200 mm imidazole and resolved by SDS-PAGE. Tagged proteins were probed for by Western blot. For indirect immunostaining, HeLa cells were grown on glass coverslips and transfected with Polyfect reagent (Qiagen) according to the manufacturer's instructions. After 48 h, cells were fixed with phosphate-buffered saline (PBS), 4% paraformaldehyde (10 min, 4 °C), permeabilized with ice-cold methanol (5 min, 4 °C), and incubated with primary antibodies overnight at 4 °C, followed by corresponding secondary antibodies conjugated to Alexa-Fluor488 (Jackson ImmunoResearch Laboratories, Inc.). Images were acquired on a laser-scanning confocal microscope (LSM510 Meta; Carl Zeiss) equipped with an Axiovert 200 M inverted microscope, using a Plan Apo 63/1.4 numerical aperture oil immersion objective. For fractionation experiments, 293T cells expressing FLAG-tagged WT, 3KR, or 3EQ IN were lysed in buffer C (10 mm Tris-Cl, pH 7.4, 0.15 m NaCl, 1% CHAPS, EDTA-free complete protease inhibitors (Roche Applied Science)) (30 min, 4 °C). Supernatant (cytosol) and pellet (nuclei) were separated by centrifugation (top speed, 5 min, 4 °C). Nuclear content was extracted in buffer N (buffer C with 0.85 m NaCl final) (30 min, 4 °C). For IN stability studies, cycloheximide (100 μg/ml; Sigma) or MG132 (5 μm; Calbiochem) were added to the culture medium 24 h after transfection. Next, cells were lysed in buffer N. Total protein content was measured with a Bradford assay (Sigma). Proteins (25 μg/lane) were resolved by SDS-PAGE and detected by Western blot. Single-round viruses were produced by co-transfection of 293T cells using a standard calcium phosphate precipitation technique with a plasmid encoding WT or mutant HIV-1-packaging DNA (psPAX2) and the genomic transfer vector encoding GFP (pWPI) or the pNL4–3EnvFsGFP vector and an expression vector for the glycoprotein G of vesicular stomatitis virus (VSVg) (pMD2.G). Replication-competent viruses were produced by transfecting the pNL4-3 plasmid that encodes a complete HIV-1 infectious provirus. Supernatants were collected 40 h post-transfection, clarified by low speed centrifugation, filtered through 0.45-μm pore size filters, and treated with 10 units/ml Turbo DNase (Ambion) (1 h, room temperature). Viral particles were concentrated by ultracentrifugation (24,000 rpm, 1 h 30 min, 4 °C) using a SW32 rotor (Beckman) on a 20% sucrose cushion. All viral stocks were normalized for the p24CA antigen content, as determined by ELISA (Zeptometrix) and used to infect target cells (6 × 104 293T or 1 × 106 CEM-GFP cells). After 48 h, the percentage of GFP-expressing cells was measured by flow cytometry on a FACSCalibur flow cytometer with CellQuest software (BD Biosciences). Viral proteins associated with virions or with infected cells were analyzed by Western blot with anti-CA and anti-IN antibodies. For quantification of virion-associated CA and IN proteins, secondary antibodies coupled with IRDye near infrared dyes (IRDye800CW and IRDye680LT, Science Tec) were used. Proteins were visualized on an Odyssey infrared imager and quantified with Odyssey software (LI-COR Biosciences). Total genomic DNA was extracted using a blood and body fluid kit (Qiagen) from 293T cells (5 × 105) infected with single-round viruses. Full-length reverse transcripts, integrated HIV-1 DNA, and 2-LTR circles were quantified using a previously described protocol (49Brussel A. Sonigo P. J. Virol. 2004; 78: 11263-11271Crossref PubMed Scopus (67) Google Scholar). Parallel infections with heat-inactivated HIV-1WT viruses were performed to control for residual levels of plasmid DNA that may have resisted DNase treatment. Viral RNA was extracted with the RNeasy Mini kit (Qiagen) and amplified with the HIV-1 real time RT-PCR kit (BioEvolution). Real-time PCR and RT-PCR were performed on a Lightcycler 1.0 (Roche Applied Science). Viral stocks generated by co-transfecting 293T cells with pNLX.Luc(R-Env−), pRL2P-Vpr-IN WT or mutant, and pNLXE7 were used to infect Jurkat cells (2 × 106 cells/ml, 5 × 105 reverse transcriptase cpm), as described (50Lu R. Limón A. Ghory H.Z. Engelman A. J. Virol. 2005; 79: 2493-2505Crossref PubMed Scopus (78) Google Scholar). Cells were harvested 48 h after infection and lysed in passive lysis buffer (Promega). Frozen and thawed lysates were clarified by centrifugation (18,730 × g, 15 min, 4 °C), and supernatants were analyzed for luciferase activity in duplicate using the Promega luciferase assay system, an EG&G Berthold Microplate LB 96V luminometer, and a Microlite 1 flat bottom microtiter plate (Thermo Labsystems). Luciferase activity was normalized to the protein concentration as determined by the Bio-Rad protein assay kit (Bio-Rad) and corrected for background levels from lysates of cells infected with Env-negative controls. 293T cells were co-transfected with plasmids encoding FLAG-tagged WT or 3KR or 3EQ IN and WT or D366N HA-LEDGF/p75Cter. The immunoprecipitation assay was performed as described (46Cherepanov P. Sun Z.Y. Rahman S. Maertens G. Wagner G. Engelman A. Nat. Struct. Mol. Biol. 2005; 12: 526-532Crossref PubMed Scopus (205) Google Scholar). Briefly, precleared cell extracts were incubated with HA.11 affinity matrix (Covance) (3 h, 4 °C). Following extensive washing, bound proteins were eluted in Laemmli buffer. Cell extracts and immunoprecipitates were analyzed by Western blot. Pairwise comparison between groups was performed using Student's t test. p < 0.05 was set as a threshold for statistical significance. By analyzing HIV-1 IN sequences, we identified three Lys residues at positions 46, 136, and 244 (HXB2 numbering scheme) within canonical SUMOylation consensus motifs, which represent potential sites for modification by SUMO (SUMOplot; SUMO sp 2.0 (51Xue Y. Zhou F. Fu C. Xu Y. Yao X. Nucleic Acids Res. 2006; 34: W254-W257Crossref PubMed Scopus (160) Google Scholar)). SUMO consensus motifs harboring Lys46 and Lys244 are conserved in HIV-2, SIVcpz, and SIVmac (Fig. 1A and supplemental Fig. S1), whereas the SUMO consensus harboring K136 is found in about one-third of HIV-1 strains (52Kuiken C.L. Foley B. Freed E. Hahn B. Korber P.A. Marx F. McCutchan J.W. Wolinksy S. HIV Sequence Compendium 2002. Theoretical Biology and Biophysics Group, Los Alamos National Laboratory, Los Alamos, NM2002Crossref Google Scholar). The extent of conservation of these motifs prompted us to assess whether HIV-1 IN is post-translationally modified by SUMO proteins. With this aim, we performed an in vitro SUMOylation assay. Slow migrating bands reactive to an antibody against IN were observed when purified recombinant IN bearing an N-terminal His6 tag was incubated with components of the SUMOylation machinery in the presence but not in the absence of ATP (Fig. 1, B and C, lanes 1 and 2). The appearance of several high molecular weight species indicates the addition of multiple SUMO moieties to IN. Of note, similar levels of modification but slightly different patterns of conjugation by SUMO-1 or SUMO-2/3 were observed when IN was incubated in the presence of each SUMO protein separately (Fig. 1B, lanes 3–5). Because putative SUMO attachment sites localize to each IN functional domain, we examined the modification of IN mutants in which the N-terminal or the C-terminal region was deleted. SUMOylated species were observed when INΔC was subject to the in vitro SUMOylation reaction (Fig. 1C), whereas modified forms of INΔN could not be detected, probably due to technical limits (absence of the epitope or low affinity of the antibodies used) (data not shown). To confirm that sites identified in silico are SUMOylated, we simultaneously replaced Lys residues at positions 46, 136, and 244 by Arg, an amino acid with similar positive charge that cannot be modified by SUMO, into the His-IN plasmid. The resulting triple IN mutant (IN3KR) was then tested in the in vitro SUMOylation assay. Under these settings, conjugation of SUMO to IN3KR was dramatically decreased compared with INWT (Fig. 1D). Altogether, these results show that HIV-1 IN is modified with similar efficiency, but with slightly different specificity, by the three SUMO paralogues on candidate consensus sites in vitro. Because we found that HIV-1 IN is covalently modified by SUMO proteins in vitro, we next assessed SUMO conjugation in a cellular context. With this aim, 293T cells were co-transfected with a plasmid encoding HIV-1 IN bearing a C-terminal FLAG tag (INWT-FLAG) and expression vectors for Ubc9 and for N-terminal His-tagged SUMO-1, -2 or -3 or the appropriate control empty vector. Forty hours after transfection, purification by immobilized metal ion affinity chromatography in highly denaturing conditions was performed to ensure that only molecules covalently linked to IN were recovered and that the activity of SUMO proteases was blocked. Although IN expression levels were comparable in all samples (Fig. 2A, bottom, lanes 1–4), modified species reactive to an anti-IN antibody and migrating at the size expected for IN conjugated to one (∼36 + 12 = 48 kDa) or several SUMO moieties, were observed when SUMO-1, -2, or -3 was expressed but not in the control (Fig. 2A, top, lanes 1–4). Because Lys-to-Arg changes at positions 46, 136, and 244 lead to a drastic reduction of IN SUMOylation in vitro, the same mutations were introduced into the INWT-FLAG plasmid. Candidate SUMOylation sites were disrupted either individually or in various combinations, and modified IN derivatives were analyzed as described above. Single and double mutant IN proteins displayed SUMOylation profiles analogous to INWT (data not shown) (supplemental Fig. S2). As expected, the enrichment of modified forms of IN3KR, which was expressed at levels similar to INWT, was considerably diminished regardless of the SUMO protein expressed (Fig. 2A, compare lanes 5–8 with lanes 1–4). We also generated an IN mutant in which the Glu residues at positions 48, 138, and 246 were substituted by Gln, yielding the IN3EQ mutant. Indeed, the acidic amino acid (Glu/Asp) at +2 of the SUMOylation consensus motifs is indispensable for SUMO conjugation to Lys (53Rodriguez M.S. Dargemont C. Hay R.T. J. Biol. Chem. 2001; 276: 12654-12659Abstract Full Text Full Text PDF PubMed Scopus (614) Google Scholar). In agreement with the results obtained with IN3KR, the SUMO-modified species of IN3EQ were much less abundant compared with that of INWT (Fig. 2A, compare lanes 9–12 with lanes 1–4). To evaluate whether disruption of IN SUMOylation motifs affected other post-translational modifications targeting Lys residues, FLAG-tagged WT or mutant IN proteins were expressed together with His-tagged ubiquitin in 293T cells, and purification in denaturing conditions was performed. Both IN3KR and IN3EQ displayed ubiquitination profiles similar to INWT, demonstrating that Lys-to-Arg or Glu-to-Gln changes specifically impaired SUMOylation but not ubiquitination (Fig. 2B). Finally, we analyzed conjugation of endogenous SUMO proteins to ectopically expressed IN. Thus, we generated plasmids encoding WT or 3KR IN with an N-terminal His6 tag that were used to transfect 293T cells, followed by affinity purification in denaturing conditions. Post-translational derivatives of IN enriched on nickel-NTA beads were detected with an anti-SUMO-2/3 antibody in the presence, but not in the absence, of INWT expression (Fig. 2C, top, lane 2). Under the same settings, weak but specific bands were also detected with an antibody against SUMO-1 (Fig. 2C, middle, lane 2). However, although IN3KR was expressed at levels similar to INWT (Fig. 2C, bottom, compare lanes 2 and 3), corresponding SUMO-1- and SUMO-2/3-modified forms were significantly reduced (Fig. 2C, top and middle). Analysis of acetylation and ubiquitination of WT and 3KR IN under the same settings showed that both proteins were modified to a similar extent (Fig. 2D) (data not shown). Altogether, these results confirm that HIV-1 IN is modified by the SUMO paralogues in vivo and that conserved Lys residues at positions 46, 136, and 244 are the principal sites of SUMOylation. Moreover, these data indicate that candidate SUMOylation sites are not targeted by ubiquitination or acetylation. SUMOylation has been shown to regulate numerous cellular processes, including protein localization and stability (32Geiss-Friedlander R. Melchior F. Nat. Rev. Mol. Cell Biol. 2007; 8: 947-956Crossref PubMed Scopus (1382) Google Scholar). First, the involvement of SUMO conjugation in HIV-1 IN subcellular distribution was studied by expressing FLAG-tagged WT or mutant IN proteins in HeLa cells, followed by immunofluorescence and confocal microscopy analysis. As already established, INWT was enriched in the nucleus of transfected cells (Fig. 3A). Likewise, IN3KR and IN3EQ concentrated in the nucleus (Fig. 3A). A similar distribution was also observed upon analysis of the steady state nucleocytoplasmic partitioning of FLAG-tagged INWT, IN3KR, or IN3EQ expressed in 293T cells by cell fractionation followed by Western blot with an anti-IN antibody. The proper separation of the nuclear and the cytoplasmic fraction was confirmed by immunoblotting with antibodies against the cytoplasmic protein LDH or the nuclear histone H2B, showing no detectable cross-contamination (Fig. 3B). Second, to investigate the involvement of SUMOylation on IN stability, we studied the half-life of INWT, IN3KR, and IN3EQ" @default.
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