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• W2148176502 abstract "Transport of the viral genome into the nucleus required phosphorylation of components in the preintegration complex by virion-associated host cellular kinases. In this study, we showed that ERK-2/MAPK is associated with simian immunodeficiency virus (SIV) virions and regulated the nuclear transport of Vpx and virus replication in non-proliferating target cells by phosphorylating Vpx. Suppression of the virion-associated ERK-2 activity by MAPK pathway inhibitors impaired both Vpx nuclear import and viral infectivity without affecting virus particle maturation and release. In addition, mutation analysis indicated that the inactivation of Vpx phosphorylation precluded nuclear import and reduced virus replication in macrophage cultures, even when functional integrase and Gag matrix proteins implicated in viral preintegration complex nuclear import are present. In this study, we also showed that co-localization of Vpx with Gag precursor in the cytoplasm is a prerequisite for Vpx incorporation into virus particles. Substitution of hydrophobic Leu-74 and Ile-75 with serines in the helical domain abrogated Vpx nuclear import, and its incorporation into virus particles, despite its localization in the cytoplasm, suggested that the structural integrity of helical domains is critical for Vpx functions. Taken together, these studies demonstrated that the host cell MAPK signal transduction pathway regulated an early step in SIV infection. Transport of the viral genome into the nucleus required phosphorylation of components in the preintegration complex by virion-associated host cellular kinases. In this study, we showed that ERK-2/MAPK is associated with simian immunodeficiency virus (SIV) virions and regulated the nuclear transport of Vpx and virus replication in non-proliferating target cells by phosphorylating Vpx. Suppression of the virion-associated ERK-2 activity by MAPK pathway inhibitors impaired both Vpx nuclear import and viral infectivity without affecting virus particle maturation and release. In addition, mutation analysis indicated that the inactivation of Vpx phosphorylation precluded nuclear import and reduced virus replication in macrophage cultures, even when functional integrase and Gag matrix proteins implicated in viral preintegration complex nuclear import are present. In this study, we also showed that co-localization of Vpx with Gag precursor in the cytoplasm is a prerequisite for Vpx incorporation into virus particles. Substitution of hydrophobic Leu-74 and Ile-75 with serines in the helical domain abrogated Vpx nuclear import, and its incorporation into virus particles, despite its localization in the cytoplasm, suggested that the structural integrity of helical domains is critical for Vpx functions. Taken together, these studies demonstrated that the host cell MAPK signal transduction pathway regulated an early step in SIV infection. A critical step in the process of retrovirus infection is the transfer of viral DNA into the nucleus of the infected cells (1Cullen B.R. Cell. 1998; 93: 685-692Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar, 2Trono D. Nat. Med. 1998; 4: 1368-1369Crossref PubMed Scopus (19) Google Scholar, 3Emerman M. Curr. Biol. 1996; 6: 1096-1103Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). Lentiviruses like HIV 1The abbreviations used are: HIV, human immunodeficiency virus; MAPK, mitogen activated protein kinase; ERK, extra cellular signal-regulated kinase; SIV, simian immunodeficiency virus; HIV-1, HIV type 1; HIV-2, HIV type 2; PIC, preintegration complex; NE, nuclear envelope; NLS, nuclear localization signal; MA, matrix; PMA, phorbol myristate acetate; GST, glutathione S-transferase; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; PBMC, peripheral blood mononuclear cell; Vif, virion infectivity factor./SIV are capable of infecting non-dividing cells such as terminally differentiated macrophages and memory T-cells, which are important for viral dissemination and persistence (4Hirsch V.M. Sharkey M.E. Byrum R. Elkins W.R. Hahn B.H. Lifson J.D. Stevenson M. Nat. Med. 1998; 4: 1401-1408Crossref PubMed Scopus (154) Google Scholar). In contrast, prototypic retroviruses do not replicate efficiently in non-dividing cells (5Lewis P.F. Emerman M. J. Virol. 1994; 68: 510-516Crossref PubMed Google Scholar). Although the mechanisms that underlie this restriction are not fully understood, inefficient nuclear transport of viral DNA appears to be one of them. HIV/SIV possess determinants that ensure effective nuclear import of viral DNA to the nucleus in non-dividing cells by exploiting cellular pathways (6Pancio H.A. Vander Heyden N. Ratner L. J. Virol. 2000; 74: 6162-6167Crossref PubMed Scopus (62) Google Scholar, 7Henderson L.E. Sowder R.C. Copeland TD. Benveniste R.E. Oroszlan S. Science. 1988; 241: 199-202Crossref PubMed Scopus (103) Google Scholar, 8Kappes J.C. Morrow C.D. Lee S.W. Jameson B.A. Kent S.B.H. Hood L.E. Shaw G.M. Hahn B.H. J. Virol. 1989; 62: 3501-3505Crossref Google Scholar, 9Wu X. Conway J.A. Kim J. Kappes J.C. J. Virol. 1994; 68: 6161-6169Crossref PubMed Google Scholar, 10Fletcher T.M. Brichacek B. Sharova N. Newman M.A. Stivahtis G. Sharp P.M. Emerman M. Hahn B.H. Stevenson M. EMBO J. 1996; 15: 6155-6165Crossref PubMed Scopus (205) Google Scholar, 11Accola M.A. Bukovsky A.A. Jones M.S. Go ̈ttlinger H.G. J. Virol. 1999; 73: 9992-9999Crossref PubMed Google Scholar). After entry of the virus into the cell, the genomic HIV/SIV RNA is reverse-transcribed into linear double-stranded DNA, which remains associated with a nucleoprotein complex called the preintegration complex (PIC) (12Bukrinsky M.I. Haggerty S. Dempsey M.P. Sharova N. Adzhubel A. Spitz L. Lewis P. Goldfarb D. Emerman M. Stevenson M. Nature. 1993; 365: 666-669Crossref PubMed Scopus (741) Google Scholar, 13Gallay P. Swingler S. Aiken C. Trono D. Cell. 1995; 80: 379-381Abstract Full Text PDF PubMed Scopus (294) Google Scholar, 14Heinzinger N.K. Bukinsky 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 (757) Google Scholar, 15Zennou V. Petit C. Guetard D. Nerhbass U. Montagnier L. Charneau P. Cell. 2000; 14: 173-185Abstract Full Text Full Text PDF Scopus (713) Google Scholar). The viral PICs are then imported into the nucleus through the nuclear envelope via an active mechanism within 4-6 h after infection (3Emerman M. Curr. Biol. 1996; 6: 1096-1103Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). One cis-acting element, central DNA flap (15Zennou V. Petit C. Guetard D. Nerhbass U. Montagnier L. Charneau P. Cell. 2000; 14: 173-185Abstract Full Text Full Text PDF Scopus (713) Google Scholar), and at least three different proteins, namely Integrase (16Gallay P. Hope T. Chin D. Trono D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9825-9830Crossref PubMed Scopus (420) Google Scholar), Gag matrix (MA) (13Gallay P. Swingler S. Aiken C. Trono D. Cell. 1995; 80: 379-381Abstract Full Text PDF PubMed Scopus (294) Google Scholar, 16Gallay P. Hope T. Chin D. Trono D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9825-9830Crossref PubMed Scopus (420) Google Scholar), and Vpr (14Heinzinger N.K. Bukinsky 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 (757) Google Scholar), have been identified as possible mediators of the nuclear import of the HIV-1 PIC; however, the roles of GagMA protein and central DNA flap in this process are still controversial (17Fouchier R.A. Meyer B.E. Simon J.H. Fischer U. Malim M.H. EMBO J. 1997; 16: 4531-4539Crossref PubMed Scopus (302) Google Scholar, 18Dvorin J.D. Bell P. Maul G.G. Yamashita M. Emerman M. Malim M.H. J. Virol. 2002; 76: 12087-12096Crossref PubMed Scopus (144) Google Scholar, 19Limon A. Nakajima N. Lu R. Ghory H.Z. Engelman A. J. Virol. 2002; 76: 12078-12086Crossref PubMed Scopus (88) Google Scholar). Based on its karyophilic properties, Vpr protein has been implicated in this process (20Lu Y.L. Spearman P. Ratner L. J. Virol. 1993; 67: 6542-6550Crossref PubMed Google Scholar, 21Yao X.J. Subbramanian R.A. Rougeau N. Boisvert F. Bergeron D. Cohen E.A. J. Virol. 1995; 69: 7032-7044Crossref PubMed Google Scholar, 22Mahalingam S. Khan S.A. Jabbar M.A. Monken C.E. Collman R.G. Srinivasan A. Virology. 1995; 207: 297-302Crossref PubMed Scopus (83) Google Scholar, 23Vodicka M.A. Koepp D.M. Silver P.A. Emerman M. Genes Dev. 1998; 12: 175-185Crossref PubMed Scopus (296) Google Scholar, 24Jenkins Y. McEntee M. Weis K. Greene W.C. J. Cell Biol. 1998; 143: 875-885Crossref PubMed Scopus (177) Google Scholar, 25Fouchier R.A. Meyer B.E. Simon J.H. Fischer U. Albright A.V. Gonzalez-Scarano F. Malim M.H. J. Virol. 1998; 72: 6004-6013Crossref PubMed Google Scholar, 26de Noronha C.M. Sherman M.P. Lin H.W. Cavrois M.V. Moir R.D. Goldman R.D. Greene W.C. Science. 2001; 294: 1105-1108Crossref PubMed Scopus (226) Google Scholar). Viruses in the HIV-2/SIVsm/SIVmac lineage contain a vpr gene as well as an evolutionarily related vpx gene (27Hahn B.H. Shaw G.M. De Cock K.M. Sharp P.M. Science. 2000; 287: 607-614Crossref PubMed Scopus (893) Google Scholar). A recent report (10Fletcher T.M. Brichacek B. Sharova N. Newman M.A. Stivahtis G. Sharp P.M. Emerman M. Hahn B.H. Stevenson M. EMBO J. 1996; 15: 6155-6165Crossref PubMed Scopus (205) Google Scholar) has demonstrated that SIVsm Vpr and Vpx proteins have distinct and non-complementary functions. Vpr induces cell cycle arrest at the G2 stage (13Gallay P. Swingler S. Aiken C. Trono D. Cell. 1995; 80: 379-381Abstract Full Text PDF PubMed Scopus (294) Google Scholar, 14Heinzinger N.K. Bukinsky 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 (757) Google Scholar, 15Zennou V. Petit C. Guetard D. Nerhbass U. Montagnier L. Charneau P. Cell. 2000; 14: 173-185Abstract Full Text Full Text PDF Scopus (713) Google Scholar, 16Gallay P. Hope T. Chin D. Trono D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9825-9830Crossref PubMed Scopus (420) Google Scholar, 17Fouchier R.A. Meyer B.E. Simon J.H. Fischer U. Malim M.H. EMBO J. 1997; 16: 4531-4539Crossref PubMed Scopus (302) Google Scholar, 18Dvorin J.D. Bell P. Maul G.G. Yamashita M. Emerman M. Malim M.H. J. Virol. 2002; 76: 12087-12096Crossref PubMed Scopus (144) Google Scholar, 19Limon A. Nakajima N. Lu R. Ghory H.Z. Engelman A. J. Virol. 2002; 76: 12078-12086Crossref PubMed Scopus (88) Google Scholar, 20Lu Y.L. Spearman P. Ratner L. J. Virol. 1993; 67: 6542-6550Crossref PubMed Google Scholar, 21Yao X.J. Subbramanian R.A. Rougeau N. Boisvert F. Bergeron D. Cohen E.A. J. Virol. 1995; 69: 7032-7044Crossref PubMed Google Scholar, 22Mahalingam S. Khan S.A. Jabbar M.A. Monken C.E. Collman R.G. Srinivasan A. Virology. 1995; 207: 297-302Crossref PubMed Scopus (83) Google Scholar, 23Vodicka M.A. Koepp D.M. Silver P.A. Emerman M. Genes Dev. 1998; 12: 175-185Crossref PubMed Scopus (296) Google Scholar, 24Jenkins Y. McEntee M. Weis K. Greene W.C. J. Cell Biol. 1998; 143: 875-885Crossref PubMed Scopus (177) Google Scholar, 25Fouchier R.A. Meyer B.E. Simon J.H. Fischer U. Albright A.V. Gonzalez-Scarano F. Malim M.H. J. Virol. 1998; 72: 6004-6013Crossref PubMed Google Scholar, 26de Noronha C.M. Sherman M.P. Lin H.W. Cavrois M.V. Moir R.D. Goldman R.D. Greene W.C. Science. 2001; 294: 1105-1108Crossref PubMed Scopus (226) Google Scholar, 27Hahn B.H. Shaw G.M. De Cock K.M. Sharp P.M. Science. 2000; 287: 607-614Crossref PubMed Scopus (893) Google Scholar, 28Rogel M.E. Wu L.I. Emerman M. J. Virol. 1995; 69: 882-888Crossref PubMed Google Scholar), whereas Vpx is mainly involved in the nuclear import of the viral PIC (9Wu X. Conway J.A. Kim J. Kappes J.C. J. Virol. 1994; 68: 6161-6169Crossref PubMed Google Scholar, 10Fletcher T.M. Brichacek B. Sharova N. Newman M.A. Stivahtis G. Sharp P.M. Emerman M. Hahn B.H. Stevenson M. EMBO J. 1996; 15: 6155-6165Crossref PubMed Scopus (205) Google Scholar). Vpx is an 18-kDa, 112-amino acid protein, which is highly conserved among all the divergent isolates of HIV-2 and SIVsm (7Henderson L.E. Sowder R.C. Copeland TD. Benveniste R.E. Oroszlan S. Science. 1988; 241: 199-202Crossref PubMed Scopus (103) Google Scholar, 8Kappes J.C. Morrow C.D. Lee S.W. Jameson B.A. Kent S.B.H. Hood L.E. Shaw G.M. Hahn B.H. J. Virol. 1989; 62: 3501-3505Crossref Google Scholar). Vpx mutant SIVsm is significantly reduced in its ability to replicate in non-dividing target cells such as macaque macrophages (6Pancio H.A. Vander Heyden N. Ratner L. J. Virol. 2000; 74: 6162-6167Crossref PubMed Scopus (62) Google Scholar, 10Fletcher T.M. Brichacek B. Sharova N. Newman M.A. Stivahtis G. Sharp P.M. Emerman M. Hahn B.H. Stevenson M. EMBO J. 1996; 15: 6155-6165Crossref PubMed Scopus (205) Google Scholar, 29Rajendra Kumar P. Singhal P.K. Vinod S.S. Mahalingam S. J. Mol. Biol. 2003; 331: 1141-1156Crossref PubMed Scopus (22) Google Scholar). Vpx is also essential for efficient in vivo dissemination and spread of SIVsm following mucosal and intravenous infection of macaques (4Hirsch V.M. Sharkey M.E. Byrum R. Elkins W.R. Hahn B.H. Lifson J.D. Stevenson M. Nat. Med. 1998; 4: 1401-1408Crossref PubMed Scopus (154) Google Scholar). Within viral particles, Vpx seems to be localized within the viral core (30Kewalramani V.N. Emerman M. Virology. 1996; 1: 159-168Crossref Scopus (45) Google Scholar). Vpx is packaged efficiently in the progeny virions formed in the absence of the pol and env gene products and is independent of viral RNA encapsidation (6Pancio H.A. Vander Heyden N. Ratner L. J. Virol. 2000; 74: 6162-6167Crossref PubMed Scopus (62) Google Scholar, 8Kappes J.C. Morrow C.D. Lee S.W. Jameson B.A. Kent S.B.H. Hood L.E. Shaw G.M. Hahn B.H. J. Virol. 1989; 62: 3501-3505Crossref Google Scholar, 14Heinzinger N.K. Bukinsky 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 (757) Google Scholar). These results indicate that expression of the HIV-2/SIV Gag precursor (Pr55Gag) is sufficient to mediate the incorporation of Vpx into virions. It is likely that the mechanism by which Vpx enters the assembling virion also dictates its association with the viral PICs. Recent studies (7Henderson L.E. Sowder R.C. Copeland TD. Benveniste R.E. Oroszlan S. Science. 1988; 241: 199-202Crossref PubMed Scopus (103) Google Scholar, 9Wu X. Conway J.A. Kim J. Kappes J.C. J. Virol. 1994; 68: 6161-6169Crossref PubMed Google Scholar, 11Accola M.A. Bukovsky A.A. Jones M.S. Go ̈ttlinger H.G. J. Virol. 1999; 73: 9992-9999Crossref PubMed Google Scholar, 29Rajendra Kumar P. Singhal P.K. Vinod S.S. Mahalingam S. J. Mol. Biol. 2003; 331: 1141-1156Crossref PubMed Scopus (22) Google Scholar, 31Kappes J.C. Parkin J.S. Conway J.A. Kim J. Brouillette C.G. Shaw G.M. Hahn B.H Virology. 1993; 193: 222-233Crossref PubMed Scopus (54) Google Scholar, 32Mahalingam S. Van Tine B. Santiago M.L. Gao F. Shaw G.M. Hahn B.H. J. Virol. 2001; 75: 362-374Crossref PubMed Scopus (47) Google Scholar) have shown that the packaging of Vpx into viral particles depends on the C-terminal p6 domain of the Gag polyprotein. As the Vpx protein is incorporated into the virion it becomes available during early replication events, immediately following entry of the new virion into a target cell even before de novo viral protein synthesis could start. Based on such late expression during virus production and early availability during initial infection, it has been proposed that Vpx is involved in the early stages of viral life cycle, particularly in the efficient import of viral genome into the nuclear compartment of non-proliferating target cells. The domain(s) and/or the amino acid(s) required for various functions of Vpx and its relevance to the optimal virus replication in non-dividing target cells have not been reported so far. Also, the mechanism by which Vpx incorporates into the virus particles and mediates the nuclear import of HIV-2/SIVsm PICs remains unknown. Mutations within the nuclear targeting domain abrogate nuclear import function of Vpx and attenuate replication of HIV-2 and SIV in macrophages (6Pancio H.A. Vander Heyden N. Ratner L. J. Virol. 2000; 74: 6162-6167Crossref PubMed Scopus (62) Google Scholar, 29Rajendra Kumar P. Singhal P.K. Vinod S.S. Mahalingam S. J. Mol. Biol. 2003; 331: 1141-1156Crossref PubMed Scopus (22) Google Scholar, 33Belshan M. Ratner L. Virology. 2003; 311: 7-15Crossref PubMed Scopus (28) Google Scholar). Phosphorylation plays a critical role in the nuclear localization signal (NLS)-mediated nuclear transport, cell cycle progression, and gene expression (34Fridell R.A. Truant R. Thorne L. Benson R.E. Cullen B.R. J. Cell Sci. 1997; 110: 1325-1331Crossref PubMed Google Scholar, 35Jans D.A. Hubner S. Physiol. Rev. 1996; 76: 651-685Crossref PubMed Scopus (389) Google Scholar, 36Peterson R.T. Schreiber S.L. Curr. Biol. 1999; 9: R521-R524Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 37Schakney S.E. Shankey T.V. Cytometry. 1999; 35: 97-116Crossref PubMed Scopus (49) Google Scholar). Phosphorylation-regulated NLSs were found to control nuclear transport in eukaryotic cells from yeast and plants to higher mammals (35Jans D.A. Hubner S. Physiol. Rev. 1996; 76: 651-685Crossref PubMed Scopus (389) Google Scholar). For example, the archetypal NLS-containing simian virus 40 large T-antigen is regulated by the CcN motif. This motif comprises the T-antigen NLS (N), the phosphorylation site (C) of casein kinase II, 13 amino acids N-terminal to the NLS modulating the rate of nuclear import, and a cyclin-dependent kinase site (c) adjacent to the NLS regulating the maximal level of nuclear accumulation. Phosphorylation has been shown to regulate the progression of cell cycle and gene expression by changing the nuclear localization of various proteins, as well as their association with transcriptional activation factors (36Peterson R.T. Schreiber S.L. Curr. Biol. 1999; 9: R521-R524Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 37Schakney S.E. Shankey T.V. Cytometry. 1999; 35: 97-116Crossref PubMed Scopus (49) Google Scholar). Recent studies demonstrated that serine-threonine kinases of the host cell are incorporated within HIV-1 particles (38Camaur D. Gallay P. Swingler S. Trono D. J. Virol. 1997; 71: 6834-6841Crossref PubMed Google Scholar, 39Gallay P. Swingler S. Song J. Bushman F. Trono D. Cell. 1995; 83: 569-576Abstract Full Text PDF PubMed Scopus (315) Google Scholar, 40Luo T. Downing J.R. Garcia J.V. J. Virol. 1997; 71: 2535-2539Crossref PubMed Google Scholar, 41Paul M. Jabbar M.A. Virology. 1997; 232: 207-216Crossref PubMed Scopus (66) Google Scholar, 42Jacque J.M. Mann A. Enslen H. Sharova N. Brichacek B. Davis R.J. Stevenson M. EMBO J. 1998; 17: 2607-2618Crossref PubMed Scopus (148) Google Scholar) and regulate early steps in the viral life cycle (38Camaur D. Gallay P. Swingler S. Trono D. J. Virol. 1997; 71: 6834-6841Crossref PubMed Google Scholar, 39Gallay P. Swingler S. Song J. Bushman F. Trono D. Cell. 1995; 83: 569-576Abstract Full Text PDF PubMed Scopus (315) Google Scholar, 40Luo T. Downing J.R. Garcia J.V. J. Virol. 1997; 71: 2535-2539Crossref PubMed Google Scholar, 41Paul M. Jabbar M.A. Virology. 1997; 232: 207-216Crossref PubMed Scopus (66) Google Scholar). Phosphorylation of HIV-1 Gagp17MA and Gagp6 by the virion-associated ERK-2/MAPK is shown to be essential for its association with the viral PIC and also for the release of the virus particle from the infected cells (42Jacque J.M. Mann A. Enslen H. Sharova N. Brichacek B. Davis R.J. Stevenson M. EMBO J. 1998; 17: 2607-2618Crossref PubMed Scopus (148) Google Scholar, 43Hemonnot B. Cartier C. Gay B. Rebuffat S. Bardy M. Devaux C. Boyer V. Briant L. J. Biol. Chem. 2004; 279: 32426-32434Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). By having demonstrated that Vpx is the critical determinant for HIV-2/SIV replication in non-dividing cells, the present study was designed to understand the contribution of the Vpx nuclear transport and phosphorylation in the HIV-2/SIV life cycle. In this study, we show for the first time that ERK-2/MAPK is packaged into the SIVsm(PBj1.9) virions and that it phosphorylates Vpx and regulates SIV replication. Furthermore, we demonstrate that the phosphorylation was directly implicated in the Vpx nuclear import and subsequent virus infection but not on viral maturation, assembly, and Vpx packaging into virus particles. These results reveal a new level of regulation of HIV-2 and SIV infection. Construction of Vpx Expression Vectors—Mutational analyses were performed by using the infectious molecular clone SIVsm(PBj1.9) (44Dewhurst S. Embretson J.E. Anderson D.C. Mullins J.I. Fultz P.N. Nature. 1990; 345: 636-640Crossref PubMed Scopus (161) Google Scholar). The quick-change site-directed mutagenesis kit (Stratagene) was used to introduce mutations into the PBj1.9 vpx gene (subcloned as an internal SpeI-ClaI DNA fragment). Mutagenized vpx genes were PCR-amplified and inserted into the mammalian expression vector pCDNA3 (Invitrogen) and also were reinserted into the PBj1.9 proviral vector using a series of subcloning steps. None of the introduced nucleotide substitutions resulted in amino acid changes in overlapping the virion infectivity factor (Vif) open reading frame. All introduced mutations were confirmed by DNA sequence analysis. Cell Culture, Transfection, and Infection—293T, COS-7, Vero, and HeLa cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with penicillin (100 units/ml), streptomycin (100 μg/ml), and 10% fetal bovine serum (FBS). CEMx174 and Jurkat cells were maintained in RPMI 1640 supplemented with 2 mm l-glutamine, penicillin (100 units/ml), streptomycin (100 μg/ml), and 10% FBS. Macaque PBMCs were obtained from heparin-treated whole blood using lymphocyte separation medium (Organon Teknika). Macrophages were purified from unstimulated macaque PBMCs as described elsewhere (32Mahalingam S. Van Tine B. Santiago M.L. Gao F. Shaw G.M. Hahn B.H. J. Virol. 2001; 75: 362-374Crossref PubMed Scopus (47) Google Scholar). Briefly, 3 × 106 macaque PBMCs were placed in 12-well tissue culture plates in macrophage medium containing 10% autologous macaque serum and conditioned medium to supply growth factors. Non-adherent cells were removed after 30-60 min of incubation at 37 °C, followed by extensive washing with phosphate-buffered saline. Cells were allowed to differentiate in macrophage medium for 10-12 days prior to virus infection. To examine the effects of MAPK inhibitor on virus infectivity, T-cells were stimulated with PMA, and the infectious virus particles were produced in the presence or absence of the MAPK inhibitor hypericin and were used for infection in macaque macrophages. For the generation of virus stocks, 293T cells were transfected with wild type and vpx mutant SIVsm(PBj1.9) proviral DNAs (10 μg) using the Effectene transfection kit (Qiagen). Cell culture supernatants were collected 48 h after transfection and analyzed for core antigen (p27Gag) content using an SIV core antigen assay (Coulter). CEMx174 cells were then infected with supernatants containing 10 ng of p27Gag and were incubated overnight at 37 °C in 5% CO2. Terminally differentiated macaque macrophages were infected with virion preparations containing 10 ng of p27Gag in 12-well plates overnight at 37 °C in 5% CO2. Infected cells were washed extensively to remove residual virus and incubated at 37 °C. Culture supernatants were collected after every 3 days and were frozen at -70 °C for p27Gag determination at the conclusion of the experiments. Metabolic Labeling and Immunoprecipitation—The infection-transfection protocol for the vaccinia virus expression system was as described elsewhere (29Rajendra Kumar P. Singhal P.K. Vinod S.S. Mahalingam S. J. Mol. Biol. 2003; 331: 1141-1156Crossref PubMed Scopus (22) Google Scholar). Briefly, Vero cells were grown to 90% confluence on 60-mm-diameter plates, infected for 1 h at 37 °C with vTF7-3, a vaccinia virus expressing T7 RNA polymerase (45Fuerst T.R. Earl P.L. Moss B. Mol. Cell. Biol. 1987; 7: 2538-2544Crossref PubMed Scopus (334) Google Scholar), at a multiplicity of infection of 10, and then transfected with wild type or relevant vpx mutant constructs. Ten to 12 h after transfection, the cells were labeled for 5 h with 1.5 ml of phosphate-free DMEM containing 1.0 mCi of 32Pi (Bhabha Atomic Research Center), 1% dialyzed FBS, or Met/Cys-free DMEM containing 150 μCi of [35S]methionine (Bhabha Atomic Research Center). The labeled cells were lysed with radioimmunoprecipitation buffer without SDS (1% (v/v) Triton X-100, 0.5% (w/v) deoxycholate, and 0.2 mm phenylmethylsulfonyl fluoride in phosphate-buffered saline, with 0.2 mm Na2VO4 added to 32Pi-labeled samples). Then 3 μl of anti-Vpx antiserum was added, and the mixture was incubated for 2 h at 4 °C. Twenty microliters of immobilized protein-A beads was added, and the mixture was incubated for 90 min at 4 °C with gentle rotation. Immunoprecipitates were analyzed by SDS-12% PAGE after extensive washing in buffer containing high salt and bovine serum albumin. The gels were dried and exposed to a XAR-5 film (Eastman Kodak Co.) at -80 °C. Western Blot Analysis—COS-7 cells in 60-mm-diameter dishes were infected with vTF7-3 at a multiplicity of infection of 10 for 1 h in 5% CO2 at 37 °C and transfected with 5 μg of various Vpx expression plasmids using Lipofectin (Invitrogen). Transfected cells were lysed with lysis buffer (20 mm Tris-HCl (pH 7.4), 150 mm NaCl, 1 mm EGTA, 1% Triton X-100, 2.5 mm sodium pyrophosphate, 1 mm β-glycerophosphate, 1 mm sodium orthovanadate, 1 mm phenylmethylsulfonyl fluoride, 1 μg/ml leupeptin), solubilized in loading buffer (62.5 mm Tris-HCl (pH 6.8), 0.2% SDS, 5% 2-mercaptoethanol, 10% glycerol), and separated on a SDS-12% PAGE. Following electrophoresis, proteins were transferred to a Hybond-P membrane (Amersham Biosciences) and probed with the monoclonal Vpx antibody (1:1000). Protein-bound antibodies were probed with horseradish peroxidase-conjugated specific secondary antibodies (at a 1:2000 dilution) and developed using the enhanced chemiluminescence-plus detection system (Amersham Biosciences). For determining the ability of Vpx packaging into virus particles, virions from the culture supernatants (293T cells were transfected with Vpx wild type and mutant proviral clones) were concentrated by ultracentrifugation (125,000 × g, 2 h) through a 20% sucrose cushion, and viral pellets were separated on a SDS-12% PAGE. Following electrophoresis, Western blot was performed as described above using monoclonal anti-Vpx and anti-Gag antibodies. Isolation of Nuclear and Cytoplasmic Fractions—Vpx expression plasmids were transfected into Vero cells using Lipofectin as described above. Transfected cells were labeled with 32Pi for 5 h, and the cytoplasmic and nuclear extracts were prepared by resuspending the cell pellets with hypotonic buffer (10 mm HEPES (pH 7.9), 10 mm KCl, 1 mm EDTA, 1 mm EGTA, and 1.5 mm dithiothreitol) and incubated on ice for 45 min. Nuclear and cytoplasmic fractions were isolated by performing centrifugation of cell lysates at 12,500 rpm for 1 min after adding 0.1% Nonidet P-40 and were followed by immunoprecipitation using monoclonal Vpx antibody as described above. Fluorescence Microscopy—Vero cells in Chamber culture slides (Nunc) were infected with vTF7-3 and transfected with Vpx expression plasmids using Lipofectin as described above. Ten to 12 h after transfection, the cells were fixed with 3% paraformaldehyde and probed with the monoclonal anti-Vpx antibody (1:250) for 90 min at 37 °C. Alexa Fluor 488-conjugated affinity-purified goat anti-mouse IgG (Molecular Probes) was used as a secondary antibody to visualize the subcellular localization of Vpx proteins. Vpx and Gag co-localization in the Vpx and Gag expression vectors co-transfected cells were determined by probing the cells with monoclonal anti-Vpx antibody (1:250) and polyclonal FLAG antibody (1:500), which recognizes the FLAG-Gag fusion protein. Alexa Fluor 488-conjugated affinity-purified goat anti-mouse IgG and Alexa Fluor 594-conjugated affinity-purified goat anti-rabbit IgG were used as secondary antibodies to visualize the subcellular localization of Vpx and Gag proteins, respectively. The cells were mounted in mounting medium (Vector Laboratories) containing 4,6-diamidino-2-phenylindole to stain the nuclei. Samples were viewed with an upright Nikon E800 microscope and photographed with a DXM1200 camera using Image Pro-plus software (Media Cybernetics Inc.). Confocal laser microscopy was performed on a Zeiss LSM510 META microscope. Construction, Expression, and Purification of His-Vpx, GST-Gag p17MA, and GST-c-Jun Fusion Proteins—Vpx from SIVsm(PBj1.9) was amplified by PCR using the forward (NdeI) primer 5′-GGTCGTCATATGATGTCAGATCCCAGGGAGAG-3′ and the reverse (BamHI) primer 5′-GATTAGGGATCCTTATGCTAGTCCTGGAGGGGG-3′. The PCR fragments were then cloned into pET16b vector at the NdeI and BamHI sites. JNK phosphorylation domain of c-Jun (forward primer BamHI, 5′-ATCGGGGATCCGATGACTGCAAAGATGGAA-3′, and reverse primer XhoI, 5′-CTAGGGCTCGAGTCAGGGGCACAGGAACTGGGT-3′) and SIVsm(PBj1.9) Gagp17MA (forward primer EcoRI, 5′-GATCCGAATTCTATGGGCGCGAGAAAC-3′, and reverse primer XhoI, 5′-GATCAGCTCGAGTTAGTAATTTCCTCCTTTGCCACT-3′) were amplified and cloned into pET41b vector as GST fusion. All the plasmids were transformed into the BL-21 DE3 strain of Escherichia coli and induced w" @default.
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• W2148176502 date "2005-03-01" @default.
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• W2148176502 title "Phosphorylation by MAPK Regulates Simian Immunodeficiency Virus Vpx Protein Nuclear Import and Virus Infectivity" @default.
• W2148176502 cites W1557691749 @default.
• W2148176502 cites W1573821584 @default.
• W2148176502 cites W1587940136 @default.
• W2148176502 cites W1683590180 @default.
• W2148176502 cites W1764504318 @default.
• W2148176502 cites W1807747199 @default.
• W2148176502 cites W1850756080 @default.
• W2148176502 cites W1942481415 @default.
• W2148176502 cites W1951130496 @default.
• W2148176502 cites W1961313548 @default.
• W2148176502 cites W1981864966 @default.
• W2148176502 cites W1982537658 @default.
• W2148176502 cites W1984077320 @default.
• W2148176502 cites W1984621096 @default.
• W2148176502 cites W1992807773 @default.
• W2148176502 cites W2000622245 @default.
• W2148176502 cites W2001243226 @default.
• W2148176502 cites W2002419494 @default.
• W2148176502 cites W2005298510 @default.
• W2148176502 cites W2006409246 @default.
• W2148176502 cites W2018558293 @default.
• W2148176502 cites W2024569169 @default.
• W2148176502 cites W2030483266 @default.
• W2148176502 cites W2031295164 @default.
• W2148176502 cites W2034173252 @default.
• W2148176502 cites W2035658978 @default.
• W2148176502 cites W2047028501 @default.
• W2148176502 cites W2050598058 @default.
• W2148176502 cites W2052406246 @default.
• W2148176502 cites W2060371587 @default.
• W2148176502 cites W2077455094 @default.
• W2148176502 cites W2084218665 @default.
• W2148176502 cites W2086691821 @default.
• W2148176502 cites W2088851522 @default.
• W2148176502 cites W2097744772 @default.
• W2148176502 cites W2111566256 @default.
• W2148176502 cites W2112156721 @default.
• W2148176502 cites W2120964363 @default.
• W2148176502 cites W2122140378 @default.
• W2148176502 cites W2122266844 @default.
• W2148176502 cites W2125299662 @default.
• W2148176502 cites W2125453887 @default.
• W2148176502 cites W2130493263 @default.
• W2148176502 cites W2146847077 @default.
• W2148176502 cites W2151557410 @default.
• W2148176502 cites W2153689167 @default.
• W2148176502 cites W2160368136 @default.
• W2148176502 cites W2160798495 @default.
• W2148176502 cites W2161635545 @default.
• W2148176502 cites W2162651796 @default.
• W2148176502 cites W2165895151 @default.
• W2148176502 cites W2165977771 @default.
• W2148176502 cites W2166090806 @default.
• W2148176502 cites W2169601134 @default.
• W2148176502 cites W2170434135 @default.
• W2148176502 cites W231371051 @default.
• W2148176502 cites W2415523443 @default.
• W2148176502 cites W4240204112 @default.
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