Tsg101 and Alix Interact with Murine Leukemia Virus Gag and Cooperate with Nedd4 Ubiquitin Ligases during Budding
2005; Elsevier BV; Volume: 280; Issue: 29 Linguagem: Inglês
10.1074/jbc.m413735200
ISSN1083-351X
AutoresCarolina Segura-Morales, Christina Begon-Pescia, Christine Chatellard-Causse, Rémy Sadoul, Édouard Bertrand, Eugénia Basyuk,
Tópico(s)RNA regulation and disease
ResumoRetroviruses use endosomal machinery to bud out of infected cells, and various Gag proteins recruit this machinery by interacting with either of three cellular factors as follows: ubiquitin ligases of the Nedd4 family, Tsg101, or Alix/Aip1. Here we show that the murine leukemia virus Gag has the unique ability to interact with all three factors. Small interfering RNAs against Tsg101 or Alix and dominant-negative forms of Nedd4 can all reduce production of virus-like particles. However, inactivating the Nedd4-binding site abolishes budding, whereas disrupting Tsg101 or Alix binding has milder effects. Nedd4 ubiquitin ligases are therefore essential, and Tsg101 and Alix play auxiliary roles. Most interestingly, overexpression of Alix can stimulate the release of Gag, and this occurs independently of most Alix partners Tsg101, Cin85, Alg-2, and endophilins. In addition, Gag mutants that do not bind Tsg101 or Alix concentrate on late endosomes and become very sensitive to dominant-negative forms of Nedd4 that do not conjugate ubiquitin. This suggests that the direct interaction of Gag with Tsg101 and Alix favors budding from the plasma membrane and relieves a requirement for ubiquitination by Nedd4.1. Other Nedd4-dependent Gag proteins also contain binding sites for Tsg101 or Alix, suggesting that this could be a common feature of retroviruses. Retroviruses use endosomal machinery to bud out of infected cells, and various Gag proteins recruit this machinery by interacting with either of three cellular factors as follows: ubiquitin ligases of the Nedd4 family, Tsg101, or Alix/Aip1. Here we show that the murine leukemia virus Gag has the unique ability to interact with all three factors. Small interfering RNAs against Tsg101 or Alix and dominant-negative forms of Nedd4 can all reduce production of virus-like particles. However, inactivating the Nedd4-binding site abolishes budding, whereas disrupting Tsg101 or Alix binding has milder effects. Nedd4 ubiquitin ligases are therefore essential, and Tsg101 and Alix play auxiliary roles. Most interestingly, overexpression of Alix can stimulate the release of Gag, and this occurs independently of most Alix partners Tsg101, Cin85, Alg-2, and endophilins. In addition, Gag mutants that do not bind Tsg101 or Alix concentrate on late endosomes and become very sensitive to dominant-negative forms of Nedd4 that do not conjugate ubiquitin. This suggests that the direct interaction of Gag with Tsg101 and Alix favors budding from the plasma membrane and relieves a requirement for ubiquitination by Nedd4.1. Other Nedd4-dependent Gag proteins also contain binding sites for Tsg101 or Alix, suggesting that this could be a common feature of retroviruses. In the last years, our understanding of the mechanisms underlying retroviral budding has made enormous progress. It is now well appreciated that Gag proteins of many retroviruses hijack the budding machinery of multivesicular bodies (MVB), 1The abbreviations used are: MVB, multivesicular bodies; YFP, yellow fluorescent protein; HIV-1, human immunodeficiency virus, type 1; PBS, phosphate-buffered saline; CFP, cyan fluorescent protein; GFP, green fluorescent protein; GST, glutathione S-transferase; siRNA, small interfering RNA; VLPs, virus-like particles; MA, matrix; MLV, murine leukemia virus; RSV, Rous sarcoma virus; HTLV, human T-cell lymphotrophic virus; MPMV, Mason-Pfizer monkey virus. 1The abbreviations used are: MVB, multivesicular bodies; YFP, yellow fluorescent protein; HIV-1, human immunodeficiency virus, type 1; PBS, phosphate-buffered saline; CFP, cyan fluorescent protein; GFP, green fluorescent protein; GST, glutathione S-transferase; siRNA, small interfering RNA; VLPs, virus-like particles; MA, matrix; MLV, murine leukemia virus; RSV, Rous sarcoma virus; HTLV, human T-cell lymphotrophic virus; MPMV, Mason-Pfizer monkey virus.allowing the nascent viral particle to divert this machinery for its own use (1Garrus J. von Schwedler U. 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A. 2001; 98: 7724-7729Crossref PubMed Scopus (504) Google Scholar). The first are the ubiquitin ligases of the Nedd4 family, which are involved in the ubiquitination of membrane-associated proteins to initiate their sorting toward internal MVB vesicles (10Raiborg C. Rusten T. Stenmark H. Curr. Opin. Cell Biol. 2003; 15: 446-455Crossref PubMed Scopus (405) Google Scholar, 14Marmor M. Yarden Y. Oncogene. 2004; 23: 2057-2070Crossref PubMed Scopus (320) Google Scholar, 33Staub O. Gautschi I. Ishikawa T. Breitschopf K. Ciechanover A. Schild L. Rotin D. EMBO J. 1997; 16: 6325-6336Crossref PubMed Scopus (597) Google Scholar). Nedd4 proteins bind short PPXY motifs on Gag through conserved WW domains, and they have been shown to interact with MLV, RSV, HTLV, and MPMV Gag (19Strack B. Calistri A. Accola M. Palu G. Gottlinger H. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 13063-13068Crossref PubMed Scopus (280) Google Scholar, 21Blot V. Perugi F. Gay B. Prevost M. Briant L. Tangy F. Abriel H. Staub O. Dokhelar M. Pique C. J. Cell Sci. 2004; 117: 2357-2367Crossref PubMed Scopus (127) Google Scholar, 22Bouamr F. Melillo J. Wang M. Nagashima K. de Los Santos M. Rein A. Goff S. J. Virol. 2003; 77: 11882-11895Crossref PubMed Scopus (114) Google Scholar, 23Freed E. J. Virol. 2002; 76: 4679-4687Crossref PubMed Scopus (375) Google Scholar, 24Garnier L. Wills J. Verderame M. Sudol M. Nature. 1996; 381: 744-745Crossref PubMed Scopus (102) Google Scholar, 25Gottwein E. Bodem J. Muller B. Schmechel A. Zentgraf H. Krausslich H. J. Virol. 2003; 77: 9474-9485Crossref PubMed Scopus (109) Google Scholar, 26Harty R. Brown M. Wang G. Huibregtse J. Hayes F. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 13871-13876Crossref PubMed Scopus (377) Google Scholar, 27Heidecker G. Lloyd P. Fox K. Nagashima K. Derse D. J. Virol. 2004; 78: 6636-6648Crossref PubMed Scopus (69) Google Scholar, 28Kikonyogo A. Bouamr F. Vana M. Xiang Y. Aiyar A. Carter C. Leis J. Proc. Natl. Acad. Sci. U. S. 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U. S. A. 2001; 98: 7724-7729Crossref PubMed Scopus (504) Google Scholar). In these cases, binding occurs through P(T/S)AP motifs present in Gag, which mimic the natural interaction of Tsg101 with its cellular partners (38Pornillos O. Higginson D. Stray K. Fisher R. Garrus J. Payne M. He G. Wang H. Morham S. Sundquist W. J. Cell Biol. 2003; 162: 425-434Crossref PubMed Scopus (206) Google Scholar). Tsg101 also binds ubiquitin through a UEV domain, and ubiquitination of Gag increases its affinity for Tsg101 (1Garrus J. von Schwedler U. Pornillos O. Morham S. Zavitz K. Wang H. Wettstein D. Stray K. Cote M. Rich R. Myszka D. Sundquist W. Cell. 2001; 107: 55-65Abstract Full Text Full Text PDF PubMed Scopus (1152) Google Scholar, 38Pornillos O. Higginson D. Stray K. Fisher R. Garrus J. Payne M. He G. Wang H. Morham S. Sundquist W. J. Cell Biol. 2003; 162: 425-434Crossref PubMed Scopus (206) Google Scholar). Finally, and more recently, Alix has also been shown to be involved directly in HIV-1 and EIAV budding (3von Schwedler U. Stuchell M. Muller B. Ward D. Chung H. Morita E. Wang H. Davis T. He G. Cimbora D. Scott A. Krausslich H. Kaplan J. Morham S. Sundquist W. Cell. 2003; 114: 701-713Abstract Full Text Full Text PDF PubMed Scopus (692) Google Scholar, 4Strack B. Calistri A. Craig S. Popova E. Gottlinger H. Cell. 2003; 114: 686-699Abstract Full Text Full Text PDF Scopus (672) Google Scholar, 29Martin-Serrano J. Yarovoy A. Perez-Caballero D. Bieniasz P. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 12414-12419Crossref PubMed Scopus (331) Google Scholar). Alix is the homolog of yeast Bro1, an essential protein of the MVB pathway (16Odorizzi G. Katzmann D. Babst M. Audhya A. Emr S. J. Cell Sci. 2003; 116: 1893-1903Crossref PubMed Scopus (173) Google Scholar). In mammals, it has been shown to interact with both Tsg101 and CHMP4, a core component of ESCRT-III (3von Schwedler U. Stuchell M. Muller B. Ward D. Chung H. Morita E. Wang H. Davis T. He G. Cimbora D. Scott A. Krausslich H. Kaplan J. Morham S. Sundquist W. Cell. 2003; 114: 701-713Abstract Full Text Full Text PDF PubMed Scopus (692) Google Scholar, 4Strack B. Calistri A. Craig S. Popova E. Gottlinger H. Cell. 2003; 114: 686-699Abstract Full Text Full Text PDF Scopus (672) Google Scholar, 29Martin-Serrano J. Yarovoy A. Perez-Caballero D. Bieniasz P. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 12414-12419Crossref PubMed Scopus (331) Google Scholar), and it binds Gag proteins through LYPX1–3L motifs (3von Schwedler U. Stuchell M. Muller B. Ward D. Chung H. Morita E. Wang H. Davis T. He G. Cimbora D. Scott A. Krausslich H. Kaplan J. Morham S. Sundquist W. Cell. 2003; 114: 701-713Abstract Full Text Full Text PDF PubMed Scopus (692) Google Scholar, 4Strack B. Calistri A. Craig S. Popova E. Gottlinger H. Cell. 2003; 114: 686-699Abstract Full Text Full Text PDF Scopus (672) Google Scholar, 29Martin-Serrano J. Yarovoy A. Perez-Caballero D. Bieniasz P. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 12414-12419Crossref PubMed Scopus (331) Google Scholar). Gag proteins can also recruit cellular factors involved in endosomal function but not directly linked to the MVB pathway. For instance, MLV Gag has been shown to interact with endophilins (39Wang M. Kim W. Gao G. Torrey T. Morse H.I. De Camilli P. Goff S. J. Biol. 2003; 3: 4Crossref PubMed Scopus (1) Google Scholar). HIV-1 Gag also interacts with the clathrin adaptor protein complex 3 (40Dong X. Li H. Derdowski A. Ding L. Burnett A. Chen X. Peters T. Dermody T. Woodruff E. Wang J. Spearman P. Cell. 2005; 120: 663-674Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar), and this has been shown to control its intracellular routing. It is not clear why the viral Gag proteins have selected Nedd4, Tsg101, and Alix among all other proteins of the MVB pathway. Even more puzzling, viral Gag proteins often contain several interacting motifs, and in different viruses, their respective importance can vary. For instance, HIV-1 Gag binds Tsg101 and Alix, but Tsg101 is essential, whereas Alix plays only an auxiliary role (1Garrus J. von Schwedler U. Pornillos O. Morham S. Zavitz K. Wang H. Wettstein D. Stray K. Cote M. Rich R. Myszka D. Sundquist W. Cell. 2001; 107: 55-65Abstract Full Text Full Text PDF PubMed Scopus (1152) Google Scholar, 3von Schwedler U. Stuchell M. Muller B. Ward D. Chung H. Morita E. Wang H. Davis T. He G. Cimbora D. Scott A. Krausslich H. Kaplan J. Morham S. Sundquist W. Cell. 2003; 114: 701-713Abstract Full Text Full Text PDF PubMed Scopus (692) Google Scholar, 4Strack B. Calistri A. Craig S. Popova E. Gottlinger H. Cell. 2003; 114: 686-699Abstract Full Text Full Text PDF Scopus (672) Google Scholar, 29Martin-Serrano J. Yarovoy A. Perez-Caballero D. Bieniasz P. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 12414-12419Crossref PubMed Scopus (331) Google Scholar). In contrast, Alix binds to EIAV Gag and is required for its budding (4Strack B. Calistri A. Craig S. Popova E. Gottlinger H. Cell. 2003; 114: 686-699Abstract Full Text Full Text PDF Scopus (672) Google Scholar, 29Martin-Serrano J. Yarovoy A. Perez-Caballero D. Bieniasz P. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 12414-12419Crossref PubMed Scopus (331) Google Scholar), whereas Tsg101 binds to MPMV Gag but is not essential (25Gottwein E. Bodem J. Muller B. Schmechel A. Zentgraf H. Krausslich H. J. Virol. 2003; 77: 9474-9485Crossref PubMed Scopus (109) Google Scholar). In this work, we have analyzed the budding partners of MLV Gag, and we have found that, most surprisingly, it interacts with Nedd4, Tsg101, and Alix. This is the first Gag protein shown to interact with all three factors, and it gave us a unique opportunity to analyze their respective role in budding. Plasmids, Mutagenesis, and Two-hybrid Assays—Moloney MLV Gag was cloned in the donor vector of the Gateway cloning system (Invitrogen), and mutagenesis was performed on this plasmid with the quickstrand mutagenesis kit (Stratagene). RSV Gag (a gift of Dr. J. L. Darlix, GenBank™ accession number M37980) was also cloned in the Gateway system. Wild-type and mutant Gag cDNAs were then cloned into two-hybrid plasmids (pACT-II and pAS2ΔΔ) and into mammalian expression vectors with and without C-terminal YFP tags. Mouse Alix was also cloned into pACT-II, and two-hybrid vectors for human Tsg101 and rat Nedd4 were gifts from Dr. W. Sundquist and Dr. D. Rotin, respectively (1Garrus J. von Schwedler U. Pornillos O. Morham S. Zavitz K. Wang H. Wettstein D. Stray K. Cote M. Rich R. Myszka D. Sundquist W. Cell. 2001; 107: 55-65Abstract Full Text Full Text PDF PubMed Scopus (1152) Google Scholar, 41Staub O. Dho S. Henry P. Correa J. Ishikawa T. McGlade J. Rotin D. EMBO J. 1996; 15: 2371-2380Crossref PubMed Scopus (735) Google Scholar). The dominant-negative Vps4 mutant was a gift of Dr. Woodman and Dr. Ohsumi (42Yoshimori T. Yamagata F. Yamamoto A. Mizushima N. Kabeya Y. Nara A. Miwako I. Ohashi M. Ohsumi M. Ohsumi Y. Biol. Cell. 2000; 11: 747-763Google Scholar, 43Bishop N. Woodman P. Mol. Biol. Cell. 2000; 11: 227-239Crossref PubMed Scopus (213) Google Scholar). For the Cter-PSAP mutant, the sequence GGLPPSAPSLP was introduced in-frame at the C terminus of the MLV Gag triple mutant that binds neither Tsg101, Alix, nor Nedd4. Mouse Alix had a FLAG tag (44Chatellard-Causse C. Blot B. Cristina N. Torch S. Missotten M. Sadoul R. J. Biol. Chem. 2002; 277: 29108-29115Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). AlixΔPGY was deleted for amino acids 802–813; Alix ΔTsg101 was deleted for amino acids 717–720; AlixΔCin85 was deleted for amino acids 739–745; and AlixΔPP14 was deleted for amino acids 748–761. Wild-type Nedd4.2, as well as wild-type and mutant Nedd4.1, were gifts of O. Staub (21Blot V. Perugi F. Gay B. Prevost M. Briant L. Tangy F. Abriel H. Staub O. Dokhelar M. Pique C. J. Cell Sci. 2004; 117: 2357-2367Crossref PubMed Scopus (127) Google Scholar). The dominant-negative form of Nedd4.1 was the full-length protein with cysteine 867 replaced by serine (21Blot V. Perugi F. Gay B. Prevost M. Briant L. Tangy F. Abriel H. Staub O. Dokhelar M. Pique C. J. Cell Sci. 2004; 117: 2357-2367Crossref PubMed Scopus (127) Google Scholar). Wild-type Nedd4.1 and Nedd4.2 were re-cloned at the C terminus of YFP. GST-Gag, GST-MAp12, and GST-Alix were generated by cloning corresponding cDNAs at the C terminus of GST, in the pDest27 plasmid (Invitrogen). CFP-TiVamp was cloned with the Gateway technology and was expressed under the control of mouse ribosomal L30 promoter. For the two-hybrid assays, plasmids were introduced into the appropriate haploid strains (CG929 or YL455), which were then crossed. Diploids were plated on double or triple selectable media (minus Leu, Trp, or minus Leu, Trp, His), and growth was assessed 3 days later. Analyses of Virus-like Particles (VLPs), Formation and Release— 293T cells cultured in standard conditions were transfected with the calcium-phosphate procedure (10 μg of plasmids for a 60-mm plate), as described previously (45Basyuk E. Galli T. Mougel M. Blanchard J. Sitbon M. Bertrand E. Dev. Cell. 2003; 5: 161-174Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). After 15 h, cell culture supernatants were collected and replaced by fresh media. After an additional 24 h, supernatants were collected, and cells were resuspended in Laemmli buffer. Supernatants were filtered on 0.45-μm membranes, and VLPs were then centrifuged for 2 h at 20,000 × g. VLP production was determined by Western blotting with an anti-capsid monoclonal antibody (45Basyuk E. Galli T. Mougel M. Blanchard J. Sitbon M. Bertrand E. Dev. Cell. 2003; 5: 161-174Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). Western blots were revealed by ECL and quantified either by densitometry scanning of the films or by the direct measurement of light with a CCD camera (GeneGnome, Ozyme). When the signals were too different to be quantified on the same membrane, dilutions of the strongest sample were prepared and reloaded with the other samples on a single gel. The signals of these samples were then compared with the dilution series. Gag signals were first quantified in cellular extracts, and normalized amounts of both cellular and VLPs extracts were then reloaded on novel Western blots and re-quantified as above. Cell Imaging—293T cells were plated on glass coverslips and co
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