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The Zinc Fingers of HIV Nucleocapsid Protein NCp7 Direct Interactions with the Viral Regulatory Protein Vpr

1997; Elsevier BV; Volume: 272; Issue: 49 Linguagem: Inglês

10.1074/jbc.272.49.30753

ISSN

1083-351X

Autores

Hugues de Rocquigny, Patrice Petitjean, Valérie Tanchou, Didier Décimo, Laurent Drouot, Thierry Delaunay, Jean‐Luc Darlix, Bernárd P. Roques,

Tópico(s)

RNA Interference and Gene Delivery

Resumo

The 96-amino acid protein Vpr functions as a regulator of cellular processes involved in human immunodeficiency virus, type 1 (HIV-1) life cycle, in particular by interrupting cells division in the G2 phase. Incorporation of Vpr in the virion was reported to be mediated by the C-terminal domain of the Pr55Gag polyprotein precursor, which includes NCp7, a protein involved in the genomic RNA encapsidation and p6, a protein required for particle budding. To precisely define the Gag and Vpr sequences involved in this protein-protein interaction, NCp7, p6, and Vpr as well as a series of derived peptides were synthesized using Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry. Binding assays were carried out by Far Western experiments and by competition studies using (52–96)Vpr immobilized onto agarose beads. The results show that interaction between NCp7 and Vpr occurs in vitroby a recognition mechanism requiring the zinc fingers of NCp7 and the last 16 amino acids of Vpr. Moreover, NCp10, the equivalent of NCp7 in Moloney murine leukemia virus but not polysine inhibits Vpr-NCp7 complexation. Interestingly enough, Vpr was found to interact with Gag, NCp15, and NCp7 but not with mature p6 in vitro.In vivo mutations in NCp7 zinc fingers in an HIV-1 molecular clone led to viruses with important defects in Vpr encapsidation. Together, these results suggest that NCp7 cooperates with p6 to induce Vpr encapsidation in HIV-1 mature particles. The NCp7-Vpr complex could also be important for interaction of Vpr with cellular proteins involved in cell division. The 96-amino acid protein Vpr functions as a regulator of cellular processes involved in human immunodeficiency virus, type 1 (HIV-1) life cycle, in particular by interrupting cells division in the G2 phase. Incorporation of Vpr in the virion was reported to be mediated by the C-terminal domain of the Pr55Gag polyprotein precursor, which includes NCp7, a protein involved in the genomic RNA encapsidation and p6, a protein required for particle budding. To precisely define the Gag and Vpr sequences involved in this protein-protein interaction, NCp7, p6, and Vpr as well as a series of derived peptides were synthesized using Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry. Binding assays were carried out by Far Western experiments and by competition studies using (52–96)Vpr immobilized onto agarose beads. The results show that interaction between NCp7 and Vpr occurs in vitroby a recognition mechanism requiring the zinc fingers of NCp7 and the last 16 amino acids of Vpr. Moreover, NCp10, the equivalent of NCp7 in Moloney murine leukemia virus but not polysine inhibits Vpr-NCp7 complexation. Interestingly enough, Vpr was found to interact with Gag, NCp15, and NCp7 but not with mature p6 in vitro.In vivo mutations in NCp7 zinc fingers in an HIV-1 molecular clone led to viruses with important defects in Vpr encapsidation. Together, these results suggest that NCp7 cooperates with p6 to induce Vpr encapsidation in HIV-1 mature particles. The NCp7-Vpr complex could also be important for interaction of Vpr with cellular proteins involved in cell division. The protein Vpr, which contains 96 amino acids (see Fig.1 A) and could form oligomers, was reported to enhance virus replication, particularly by inducing arrest in cell cycle in the G2 phase (1Zhao L.J. Wang L. Mukherjee S. Narayan O. J. Biol. Chem. 1994; 269: 32131-32137Abstract Full Text PDF PubMed Google Scholar, 2He J. Choe S. Walker R. Di Marzio P. Morgan D.O. Landau N.R. J. Virol. 1995; 69: 6705-6711Crossref PubMed Scopus (0) Google Scholar, 3Jowett J.B.M. Planelles V. Poon B. Shah N.P. Chen M.L. Chen I.S.Y. J. Virol. 1995; 69: 6304-6313Crossref PubMed Google Scholar, 4Rogel M.E. Wu L.I. Emerman M. J. Virol. 1995; 69: 882-888Crossref PubMed Google Scholar, 5Emerman M. Curr. Biol. 1996; 6: 1096-1103Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 6Gras-Masse H. Ameisen J.C. Boutillon C. Gesquière J.C. Vian S. Neyrinck J.L. Drobecq H. Capron A. Tartar A. Int. J. Pep. Protein Res. 1990; 36: 219-226Crossref PubMed Scopus (16) Google Scholar). Moreover, Vpr was shown to participate with the matrix protein MA in the nuclear transport of the preintegration complex (7Heinzinger N.K. Bukrinsky M.I. Haggerty S.A. Ragland A.M. Kewalramani V. Lee M.-A. Gendelman H.E. Ratner L. Stevenson M. Emerman M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7311-7315Crossref PubMed Scopus (768) Google Scholar). Vpr is present in virions, and its encapsidation has been reported to be dependent on the presence of NCp15, the C-terminal part of the polyprotein Gag (8Cohen E.A. Dehni G. Sodroski J.G. Haseltine W.A. J. Virol. 1990; 64: 3097-3099Crossref PubMed Google Scholar, 9Lu Y.-L. Spearman P. Ratner L. J. Virol. 1993; 67: 6542-6550Crossref PubMed Google Scholar, 10Paxton W. Connor R.I. Landau N.R. J. Virol. 1993; 67: 7229-7237Crossref PubMed Google Scholar, 11Lavallée C. Yao X.J. Ladha A. Göttlinger H.G. Haseltine W.A. Cohen E.A. J. Virol. 1994; 68: 1926-1934Crossref PubMed Google Scholar, 12Kondo E. Mammano F. Cohen E.A. Göttlinger H.G. J. Virol. 1995; 69: 2759-2764Crossref PubMed Google Scholar, 13Lu Y.-L. Bennett R.B. Wills J.W. Gorelick R. Ratner L. J. Virol. 1995; 69: 6873-6879Crossref PubMed Google Scholar, 14Kondo E. Göttlinger H.G. J. Virol. 1996; 70: 159-164Crossref PubMed Google Scholar). In the virions, NCp15 is cleaved by the viral protease to form NCp7 and p6 (15Di Marzo Veronese F. Rahman R. Copland T.D. Orozlan S. Gallo R.C. Sarngadharan M.G. AIDS Res. Hum. Retroviruses. 1987; 3: 253-264Crossref PubMed Scopus (91) Google Scholar). NCp7 is a small basic protein of 72 amino acids (see Fig.1 A) characterized by the presence of two spatially close zinc fingers of the CX 2CX 4HX 4C type (16Morellet N. Jullian N. de Rocquigny H. Maigret B. Darlix J.L. Roques B.P. EMBO J. 1992; 11: 3059-3065Crossref PubMed Scopus (219) Google Scholar, 17Morellet N. de Rocquigny H. Mély Y. Jullian N. Déméné H. Ottmann M. Gérard D. Darlix J.L. Fournié-Zaluski M.C. Roques B.P. J. Mol. Biol. 1994; 235: 287-301Crossref PubMed Scopus (138) Google Scholar), NCp7 exhibits nucleic acid binding and annealing activities in vitro and is required for proviral DNA synthesis and virion formation in vivo (reviewed in Ref.18Darlix J.L. Lapadat-Tapolsky M. de Rocquigny H. Roques B.P. J. Mol. Biol. 1995; 254: 523-537Crossref PubMed Scopus (381) Google Scholar). The function of p6 remains unclear, although mutations in the sequence of this protein cause alterations in a late step of viral budding (19Göttlinger H.G. Dorfman T. Sodroski J.E. Haseltine W.A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3195-3199Crossref PubMed Scopus (546) Google Scholar). Vpr was recently reported to interact with NCp7 in vitro (20Li M.-S. Garcia-Asua G. Bhattacharyya U. Mascagni P. Austen B.M. Roberts M.M. Biochem. Biophys. Res. Commun. 1996; 218: 352-355Crossref PubMed Scopus (28) Google Scholar) in agreement with experiments in which Vpr was co-precipitated with Gag products from infected cells (11Lavallée C. Yao X.J. Ladha A. Göttlinger H.G. Haseltine W.A. Cohen E.A. J. Virol. 1994; 68: 1926-1934Crossref PubMed Google Scholar) and with results showing that virus mutants with truncation of the C-terminal domain of Gag were unable to export Vpr from the cell (9Lu Y.-L. Spearman P. Ratner L. J. Virol. 1993; 67: 6542-6550Crossref PubMed Google Scholar, 10Paxton W. Connor R.I. Landau N.R. J. Virol. 1993; 67: 7229-7237Crossref PubMed Google Scholar). Various HIV-1 1The abbreviations used are: HIV-1, human immunodeficiency virus, type 1; MoMuLV, Moloney murine leukemia virus; MW, molecular weight; Fmoc,N-(9-fluorenyl)methoxycarbonyl; HPLC, high pressure liquid chromatography; PAGE, polyacrylamide gel electrophoresis. gene manipulations such as selective deletion of p6 or co-transfection of genes encoding Vpr and heterogenous Gag polyproteins supported a critical role for p6 in Vpr incorporation (12Kondo E. Mammano F. Cohen E.A. Göttlinger H.G. J. Virol. 1995; 69: 2759-2764Crossref PubMed Google Scholar). The leucine-rich motif LXSLFG of p6 was suggested to be critical for virion association of Vpr (13Lu Y.-L. Bennett R.B. Wills J.W. Gorelick R. Ratner L. J. Virol. 1995; 69: 6873-6879Crossref PubMed Google Scholar, 14Kondo E. Göttlinger H.G. J. Virol. 1996; 70: 159-164Crossref PubMed Google Scholar). However, all the chimeric HIV-1 Gag polyproteins leading to Vpr incorporation contained NCp12 from avian leukosis sarcoma virus or NCp10 from MoMuLV in place of the native NCp7, suggesting that the presence of a nucleocapsid protein preceding the p6 sequence could play a role in incorporation of Vpr in virions. This hypothesis is supported by the observed replacement of a NC protein by another nucleocapsid protein in some steps of the retroviral life cycle (21Berkowitz R.D. Ohagen A. Höglund S. Goff S.P. J. Virol. 1995; 69: 6445-6456Crossref PubMed Google Scholar, 22Zhang Y. Barklis E. J. Virol. 1995; 69: 5716-5722Crossref PubMed Google Scholar) and by the similarities in the tridimensional structure of NCp7 and NCp10 (16Morellet N. Jullian N. de Rocquigny H. Maigret B. Darlix J.L. Roques B.P. EMBO J. 1992; 11: 3059-3065Crossref PubMed Scopus (219) Google Scholar,23Déméné H. Jullian N. Morellet N. de Rocquigny H. Cornille F. Maigret B. Roques B.P. J. Biomol. NMR. 1994; 4: 153-170Crossref PubMed Scopus (40) Google Scholar). Moreover, the sequence of Vpr necessary for its incorporation into virions remains to be elucitated because the C-terminal domain was suggested to be involved (10Paxton W. Connor R.I. Landau N.R. J. Virol. 1993; 67: 7229-7237Crossref PubMed Google Scholar), whereas other groups have proposed the involvement of the N-terminal sequence (24Yao X.-J. Subbramanian R.A. Rougeau N. Boisvert F. Bergeron D. Cohen E.A. J. Virol. 1995; 69: 7032-7044Crossref PubMed Google Scholar, 25Mahalingam S. Khan S.A. Murali R. Jabbar M.A. Monken C.E. Collman R.G. Srinivasan A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3794-3798Crossref PubMed Scopus (69) Google Scholar). With the aim to characterize more precisely, the interaction of Vpr with the maturation products of NCp15, various in vitrotests were performed using the first chemically synthesized active Vpr.In vitro interactions of this protein with synthetic NCp7, p6, and cellular extracts containing Gag and NCp15 were measured by various methods. In these conditions, Vpr was shown to interact with NCp7 but not with p6. The zinc finger domains of NCp7 and the C-terminal part of Vpr are involved in the complex whose formation is strongly reduced by mutation of the Trp residue on the NCp7 distal zinc finger. NCp10 from MoMuLV, which share common structural features with NCp7 but not with unrelated basic proteins was also found able to recognize Vpr. These results were confirmed and extended by means of site-directed mutagenesis done in the nucleocapsid domain of the molecular clone pNL4.3, suggesting that NCp7 and p6 could cooperate to encapsidate Vpr. Moreover, the NCp7-Vpr complex could play a role in the functions of both proteins during virus replication as recently shown for the activation of the phosphatase protein phosphatase 2A involved in cell division (26Lim T.H.Y. de Rocquigny H. Zhao L.J. Cayla X. Roques B.P. Ozon R. FEBS Lett. 1997; 401: 197-201Crossref PubMed Scopus (48) Google Scholar). NCp7, p6, Vpr, (1Zhao L.J. Wang L. Mukherjee S. Narayan O. J. Biol. Chem. 1994; 269: 32131-32137Abstract Full Text PDF PubMed Google Scholar, 2He J. Choe S. Walker R. Di Marzio P. Morgan D.O. Landau N.R. J. Virol. 1995; 69: 6705-6711Crossref PubMed Scopus (0) Google Scholar, 3Jowett J.B.M. Planelles V. Poon B. Shah N.P. Chen M.L. Chen I.S.Y. J. Virol. 1995; 69: 6304-6313Crossref PubMed Google Scholar, 4Rogel M.E. Wu L.I. Emerman M. J. Virol. 1995; 69: 882-888Crossref PubMed Google Scholar, 5Emerman M. Curr. Biol. 1996; 6: 1096-1103Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 6Gras-Masse H. Ameisen J.C. 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A. 1992; 89: 6472-6476Crossref PubMed Scopus (281) Google Scholar). A biotinyl group was added on the N-terminal residue of peptides when necessary. Amino acids chains were protected byt-butyloxycarbonyl or trityl groups and Nα by a Fmoc. The hydroxymethylphenoxy-methyl-polystyrene resin was used, and amino acids were incorporated using N,N′-dicyclohexyl carbodiimide/hydroxybenzotriazole as a coupling reagent. At the end of the synthesis, the peptidyl resin was treated for 2 h with trifluoroacetic acid in the presence of scavengers to obtain the crude peptide. Purification was performed by reverse phase HPLC with acetonitril-water gradients. The purity of the peptides already synthesized was checked by electrospray mass spectroscopy. NCp7, Cys23-NCp7 and NCp fragments (13–30; 34–51; 12–53), Acr37(12–53)NCp7, and NCp10 were lyophilized with 1.5 equivalents of ZnCl2 per zinc finger. Details on the synthesis of p6, Vpr, and derivatives will be given elsewhere. 2H. de Rocquigny, P. Petitjean, T. Delaunay, A. Caneparo and B. P. Roques, manuscript in preparation. Electrospray mass spectroscopy was used to confirm the purity of the peptides: NCp7 (MWth = 8388.75; MWcal = 8388.2), p6 (MWth = 5807.34; MWcal = 5806), Vpr (MWth = 11394.9; MWcal = 11392.6), (1Zhao L.J. Wang L. Mukherjee S. Narayan O. J. Biol. Chem. 1994; 269: 32131-32137Abstract Full Text PDF PubMed Google Scholar, 2He J. Choe S. Walker R. Di Marzio P. Morgan D.O. Landau N.R. J. Virol. 1995; 69: 6705-6711Crossref PubMed Scopus (0) Google Scholar, 3Jowett J.B.M. Planelles V. Poon B. Shah N.P. Chen M.L. Chen I.S.Y. J. Virol. 1995; 69: 6304-6313Crossref PubMed Google Scholar, 4Rogel M.E. Wu L.I. Emerman M. J. Virol. 1995; 69: 882-888Crossref PubMed Google Scholar, 5Emerman M. Curr. Biol. 1996; 6: 1096-1103Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 6Gras-Masse H. Ameisen J.C. Boutillon C. Gesquière J.C. Vian S. Neyrinck J.L. Drobecq H. Capron A. Tartar A. Int. J. Pep. 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