Title: A Conserved Domain and Membrane Targeting of Nef from HIV and SIV Are Required for Association with a Cellular Serine Kinase Activity
Abstract: Among the primate lentiviruses (human immunodeficiency virus (HIV) −1, HIV-2, and simian immunodeficiency virus (SIV)), the nef gene is highly conserved and encodes a myristylated protein of ~27 kDa (HIV-1) or ~34 kDa (HIV-2, SIV). Previously, we found Nef expressed either as a CD8-Nef fusion protein or as a native protein in virally infected T cell lines associates with a cellular serine kinase. This kinase activity phosphorylated two proteins of 62 and 72 kDa that coimmunoprecipitate with Nef in in vitro kinase assays. Using transient expression, various Nef alleles and mutants have been analyzed for association with the cellular kinase activity. The ability of Nef to associate with the kinase activity is conserved among several alleles of HIV-1 as well as SIVmac239open and is observed in non-lymphoid cell lines of simian and murine origins. Two separate regions of HIV-1SF2 Nef are critical for the associated kinase activity. One domain overlaps with a central highly conserved region found in all primate lentivirus nef genes and has been provisionally mapped to amino acids 45-127. Because membrane localization of Nef is important for the associated cellular kinase activity, the second domain represents a membrane targeting signal. Moreover, point mutations within the central region that abrogate the Nef-associated kinase activity in HIV-1SF2 Nef have the same effect when introduced into SIVmac239open Nef. Among the primate lentiviruses (human immunodeficiency virus (HIV) −1, HIV-2, and simian immunodeficiency virus (SIV)), the nef gene is highly conserved and encodes a myristylated protein of ~27 kDa (HIV-1) or ~34 kDa (HIV-2, SIV). Previously, we found Nef expressed either as a CD8-Nef fusion protein or as a native protein in virally infected T cell lines associates with a cellular serine kinase. This kinase activity phosphorylated two proteins of 62 and 72 kDa that coimmunoprecipitate with Nef in in vitro kinase assays. Using transient expression, various Nef alleles and mutants have been analyzed for association with the cellular kinase activity. The ability of Nef to associate with the kinase activity is conserved among several alleles of HIV-1 as well as SIVmac239open and is observed in non-lymphoid cell lines of simian and murine origins. Two separate regions of HIV-1SF2 Nef are critical for the associated kinase activity. One domain overlaps with a central highly conserved region found in all primate lentivirus nef genes and has been provisionally mapped to amino acids 45-127. Because membrane localization of Nef is important for the associated cellular kinase activity, the second domain represents a membrane targeting signal. Moreover, point mutations within the central region that abrogate the Nef-associated kinase activity in HIV-1SF2 Nef have the same effect when introduced into SIVmac239open Nef. INTRODUCTIONThe nef gene was first identified as an open reading frame that overlaps with the 3′-long terminal repeat of the human immunodeficiency virus type 1 (HIV( 1The abbreviations used are: HIVhuman immunodeficiency virusSIVsimian immunodeficiency virus.) -1)1(1Allan J.S. Coligan J.E. Lee T.H. McLane M.F. Kanki P.J. Groopman J.E. Essex M. Science. 1985; 230: 810-813Crossref PubMed Scopus (156) Google Scholar). It is conserved among all primate lentiviruses, i.e. HIV-1, HIV-2, and simian immunodeficiency virus (SIV)(2Colombini S. Arya S.K. Reitz M.S. Jagodzinski L. Beaver B. Wong-Staal F. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 4813-4817Crossref PubMed Scopus (35) Google Scholar, 3Shibata R. Miura T. Hayami M. Ogawa K. Sakai H. Kiyomasu T. Ishimoto A. Adachi A. J. Virol. 1990; 64: 742-747Crossref PubMed Google Scholar). The viral gene product is translated from multiply spliced transcripts and is expressed together with the regulatory proteins Tat and Rev early in the viral replicative cycle(4Feinberg M.B. Jarrett R.F. Aldovini A. Gallo R.C. Wong-Staal F. Cell. 1986; 46: 807-817Abstract Full Text PDF PubMed Scopus (418) Google Scholar, 5Robert-Guroff M. Popovic M. Gartner S. Markham P. Gallo R.C. Reitz M.S. J. Virol. 1990; 64: 3391-3398Crossref PubMed Google Scholar, 6Klotman M.E. Kim S. Buchbinder A. DeRossi A. Baltimore D. Wong-Staal F. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 5011-5015Crossref PubMed Scopus (159) Google Scholar, 7Munis J.R. Kornbluth R.S. Guatelli J.C. Richman D.D. J. Gen. Virol. 1992; 73: 1899-1901Crossref PubMed Scopus (32) Google Scholar).Nef encodes a myristylated, phosphorylated protein of approximately 27 kDa that forms homomeric oligomers and intramolecular disulfide bonds(1Allan J.S. Coligan J.E. Lee T.H. McLane M.F. Kanki P.J. Groopman J.E. Essex M. Science. 1985; 230: 810-813Crossref PubMed Scopus (156) Google Scholar, 8Guy B. Kieny M.P. Riviere Y. Le Peuch C. Dott K. Girard M. Montagnier L. Nature. 1987; 330: 266-269Crossref PubMed Scopus (402) Google Scholar, 9Guy B. Riviere Y. Dott K. Regnault A. Kieny M.P. Virology. 1990; 176: 413-425Crossref PubMed Scopus (89) Google Scholar, 10Kienzle N. Freund J. Kalbitzer H.R. Mueller-Lantzsch N. Eur. J. Biochem. 1993; 214: 451-457Crossref PubMed Scopus (29) Google Scholar, 11Zazopoulos E. Haseltine W.A. J. Virol. 1993; 67: 1676-1680Crossref PubMed Google Scholar, 12Bandres J.C. Luria S. Ratner L. Virology. 1994; 201: 157-161Crossref PubMed Scopus (22) Google Scholar, 13Yu G. Felsted R.L. Virology. 1992; 187: 46-55Crossref PubMed Scopus (88) Google Scholar, 14Zazopoulos E. Haseltine W.A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6634-6638Crossref PubMed Scopus (50) Google Scholar). Myristylation has been reported to be important for its function(13Yu G. Felsted R.L. Virology. 1992; 187: 46-55Crossref PubMed Scopus (88) Google Scholar, 14Zazopoulos E. Haseltine W.A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6634-6638Crossref PubMed Scopus (50) Google Scholar, 15Niederman T.M.J. Randall-Hastings Ratner L. Virology. 1993; 197: 420-425Crossref PubMed Scopus (69) Google Scholar, 16Chowers M.Y. Spina C.A. Kwoh T.J. Fitch N.J. Richman D.D. Guatelli J.C. J. Virol. 1994; 68: 2906-2914Crossref PubMed Google Scholar, 17Aiken C. Konner J. Landau N.R. Lenburg M.E. Trono D. Cell. 1994; 76: 853-864Abstract Full Text PDF PubMed Scopus (604) Google Scholar). In infected cells, Nef primarily localizes to the cytoplasm and intracellular membranes (18Franchini G. Robert-Guroff M. Ghrayeb J. Chang N.T. Wong-Staal F. Virology. 1986; 155: 593-599Crossref PubMed Scopus (118) Google Scholar) and preferentially associates with the cytoskeleton(15Niederman T.M.J. Randall-Hastings Ratner L. Virology. 1993; 197: 420-425Crossref PubMed Scopus (69) Google Scholar). However, it has also been reported to be present in the nucleus(19Kohleisen B. Neumann M. Herrmann R. Brack-Werner R. Krohn K.J. Ovod V. Ranki A. Erfle V. AIDS. 1992; 6: 1427-1436Crossref PubMed Scopus (56) Google Scholar, 20Murti K.G. Brown P.S. Ratner L. Garcia J.V. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11895-11899Crossref PubMed Scopus (36) Google Scholar).Initially, Nef was reported to have a negative effect on virus replication and transcription(12Bandres J.C. Luria S. Ratner L. Virology. 1994; 201: 157-161Crossref PubMed Scopus (22) Google Scholar, 13Yu G. Felsted R.L. Virology. 1992; 187: 46-55Crossref PubMed Scopus (88) Google Scholar, 21Terwilliger E.F. Sodroski J.G. Rosen C.A. Hazeltine W.A. J. Virol. 1986; 60: 754-760Crossref PubMed Google Scholar, 22Luciw P.A. Cheng-Mayer C. Levy J.A. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 1434-1438Crossref PubMed Scopus (218) Google Scholar, 23Cheng-Mayer C. Iannello P. Shaw K. Luciw P.A. Levy J.A. Science. 1989; 246: 1629-1632Crossref PubMed Scopus (136) Google Scholar, 24Tsunetsugu-Yokota Y. Matsuda S. Maekawa M. Saito T. Takemori T. Takebe Y. Virology. 1992; 191: 960-963Crossref PubMed Scopus (11) Google Scholar, 25Ahmad N. Venkatesan S. Science. 1988; 241: 1481-1485Crossref PubMed Scopus (283) Google Scholar, 26Niederman T.M.J. Thielan B.J. Ratner L. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 1132-1186Crossref Scopus (189) Google Scholar, 27Maitra R.K. Ahmad N. Holland S.M. Venkatesan S. Virology. 1991; 182: 522-533Crossref PubMed Scopus (31) Google Scholar, 28Niederman T.M.J. Garcia J.V. Randall-Hastings W. Luria S. Ratner L. J. Virol. 1992; 66: 6213-6219Crossref PubMed Google Scholar, 29Niederman T.M.J. Randall-Hastings W. Luria S. Bandres J.C. Ratner L. Virology. 1993; 194: 338-344Crossref PubMed Scopus (45) Google Scholar). However, later studies demonstrated that Nef expression has no effect on viral replication in T-cell lines but may have a positive effect on virus replication in peripheral blood mononuclear cells (30de Ronde A. Klaver B. Keulen W. Smit L Goudsmit J. Virology. 1992; 188: 391-395Crossref PubMed Scopus (143) Google Scholar, 31Spina C.A. Kwoh T.J. Chowers M.Y. Guatelli J.C. Richman D.D. J. Exp. Med. 1994; 179: 115-123Crossref PubMed Scopus (361) Google Scholar, 32Miller M.D. Warmerdam M.T. Gaston I. Greene W.C. Feinberg M.B. J. Exp. Med. 1994; 179: 101-113Crossref PubMed Scopus (477) Google Scholar) or fetal thymic and liver implants in mice having severe combined immunodeficiency(33Jamieson B.D. Aldrovandi G.M. Planelles V. Jowett J.B. Gao L. Bloch L.M. Chen I.S. Zack J.A. J. Virol. 1994; 68: 3478-3485Crossref PubMed Google Scholar). Thus, the role of this protein on virus replication is controversial. This may be due, in part, to the use of various alleles of Nef and different cell culture systems. Nevertheless, studies in SIV-infected macaques have revealed that the preservation of a full-length Nef is necessary for the maintenance of high viral loads and for the progression of disease(34Kestler III, H.W. Ringler D.J. Mori K. Panicali D.L. Sehgal P.K. Daniel M.D. Desrosiers R.C. Cell. 1991; 65: 651-662Abstract Full Text PDF PubMed Scopus (1420) Google Scholar). Although the basis for this requirement is still not known, it may be related to a role of Nef in T-cell activation(28Niederman T.M.J. Garcia J.V. Randall-Hastings W. Luria S. Ratner L. J. Virol. 1992; 66: 6213-6219Crossref PubMed Google Scholar, 29Niederman T.M.J. Randall-Hastings W. Luria S. Bandres J.C. Ratner L. Virology. 1993; 194: 338-344Crossref PubMed Scopus (45) Google Scholar, 35Luria S. Chambers I. Berg P. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 5326-5330Crossref PubMed Scopus (162) Google Scholar, 36Skowronski J. Parks D. Mariani R. EMBO J. 1993; 12: 703-713Crossref PubMed Scopus (256) Google Scholar, 37De S.K. Marsh J.W. J. Biol. Chem. 1994; 269: 6656-6660Abstract Full Text PDF PubMed Google Scholar, 38Rhee S.S. Marsh J.W. J. Immunol. 1994; 152: 5128-5134PubMed Google Scholar, 39Rhee S.S. Marsh J.W. J. Virol. 1994; 68: 5156-5163Crossref PubMed Google Scholar, 40Baur A.S. Sawai E.T. Dazin P. Fantl W.J. Cheng-Mayer C. Peterlin B.M. Immunity. 1994; 1: 373-384Abstract Full Text PDF PubMed Scopus (278) Google Scholar).Indeed, several studies indicate that the expression of Nef affects a signal transduction pathway. Nef has been shown to down-regulate the expression of CD4 on T-lymphocytes in vitro(41Garcia J.V. Miller A.D. Nature. 1991; 350: 508-511Crossref PubMed Scopus (648) Google Scholar, 42Garcia J.V. Alfano J. Miller A.D. J. Virol. 1993; 67: 1511-1516Crossref PubMed Google Scholar, 43Inoue M. Koga Y. Djordjijevic D. Fukuma T. Reddy E.P. Yokoyama M.M. Sagawa K. Int. Immunol. 1993; 5: 1067-1073Crossref PubMed Scopus (12) Google Scholar, 44Mariani R. Skowronski J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5549-5553Crossref PubMed Scopus (153) Google Scholar). This effect of Nef is mediated by an endocytotic mechanism (17Aiken C. Konner J. Landau N.R. Lenburg M.E. Trono D. Cell. 1994; 76: 853-864Abstract Full Text PDF PubMed Scopus (604) Google Scholar, 39Rhee S.S. Marsh J.W. J. Virol. 1994; 68: 5156-5163Crossref PubMed Google Scholar, 45Sanfridson A. Cullen B.R. Doyle C. J. Biol. Chem. 1994; 269: 3917-3920Abstract Full Text PDF PubMed Google Scholar) that involves the targeting of CD4 for lysosomal degradation(17Aiken C. Konner J. Landau N.R. Lenburg M.E. Trono D. Cell. 1994; 76: 853-864Abstract Full Text PDF PubMed Scopus (604) Google Scholar, 39Rhee S.S. Marsh J.W. J. Virol. 1994; 68: 5156-5163Crossref PubMed Google Scholar, 45Sanfridson A. Cullen B.R. Doyle C. J. Biol. Chem. 1994; 269: 3917-3920Abstract Full Text PDF PubMed Google Scholar). Furthermore, we recently reported that the expression of a CD8-Nef fusion protein in T-cells leads to inhibition or activation of early T cell signaling events depending on its localization within the cell (40). When expressed at the inner surface of the plasma membrane, the activation markers CD69 and CD25 were induced, and the cells died by apoptosis. Cells that survived contained truncated Nefs.To elucidate the pathway by which Nef functions, attempts have been made to identify cellular proteins that complex with Nef. Harris and Coates (46Harris M. Coates K. J. Gen. Virol. 1993; 74: 1581-1589Crossref PubMed Scopus (44) Google Scholar) reported that baculovirus-expressed glutathione S-transferase-Nef fusion proteins associate with cellular proteins of various sizes which depend on their subcellular location. We demonstrated that Nef expressed either as a CD8-Nef fusion protein or native Nef itself specifically interacted with a serine kinase in human T-lymphocytes(47Sawai E.T. Baur A. Struble H. Peterlin B.M. Levy J.A. Cheng-Mayer C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1539-1543Crossref PubMed Scopus (209) Google Scholar). This kinase was found to phosphorylate proteins of 62 and 72 kDa that coimmunoprecipitated with Nef.In the present study, the regions of Nef that are important for these interactions with the kinase activity were investigated. Using a transient expression assay, we determined that various alleles of Nef are capable of associating with the kinase, and the kinase is present in non-lymphoid cells from several different species. We found that stability of the molecule affects the association of Nef with the cellular kinase activity. Moreover, two different regions of Nef are critical for the association with this activity; the first has been mapped to a domain that overlaps a centrally located, highly conserved portion of the molecule, and the second represents a membrane targeting signal.MATERIALS AND METHODSCells and AntibodiesThe Jurkat T-cell lines that constitutively express the CD8/HIV-1SF2 Nef fusion protein (J.CN) and HUT 78 cells chronically infected with HIV-1SF2 (E-line) were cultured as described previously(47Sawai E.T. Baur A. Struble H. Peterlin B.M. Levy J.A. Cheng-Mayer C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1539-1543Crossref PubMed Scopus (209) Google Scholar). COS-7 cells were grown in Dulbecco's minimal essential medium containing 10% fetal bovine serum, 1% glutamine, and 1% penicillin-streptomycin. The hybridomas, 51.1 and MH-SVM26, which produce monoclonal antibodies directed against the human CD8 molecule or HIV-1 gp41, respectively, were obtained from the American Type Culture Collection and cultured in HY media containing 20% fetal bovine serum. Hybridoma supernatants containing CD8-specific monoclonal antibody (α-CD8) or gp41-specific monoclonal antibody (α-gp41) were collected and either partially purified by ammonium sulfate precipitation or used directly for immunoprecipitation analyses. The rabbit anti-HIV-1SF2Nef antibody (α-Nef) was provided by Chiron Corporation (Emeryville, CA).Plasmid Construction and TransfectionTo express native Nef in COS-7 cells, the complete nef gene of HIV-1SF2 was inserted into the pRc/CMV expression vector (Invitrogen) generating the plasmid pCMV/SF2Nef. In addition, the complete nef gene of HIV-1SF2 and the pathogenic SIVmac239open strain were fused to the extracellular and transmembrane domains of the human CD8 molecule thereby generating a hybrid CD8-SF13Nef or CD8-SIVmac239openNef fusion that was subsequently cloned into the pRc/CMV plasmid as described previously(40Baur A.S. Sawai E.T. Dazin P. Fantl W.J. Cheng-Mayer C. Peterlin B.M. Immunity. 1994; 1: 373-384Abstract Full Text PDF PubMed Scopus (278) Google Scholar). These plasmids were designated pCMV/CD8-SF13Nef and pCMV/CD8-SIVmac239openNef, respectively.Site-specific mutations, carboxyl-terminal truncations, and amino-terminal deletions in Nef were introduced into the pCMV/CD8-SF2Nef, pCMV/CD8-SIVmac239openNef, or the pCMV/SF2Nef expression vectors using the single-stranded oligonucleotide-directed mutagenesis strategy (Bio-Rad). The amino-terminal deletion mutants of Nef were fused in-frame to the extracellular and transmembrane domains of CD8. All of the mutations were confirmed by DNA sequencing and by the presence of introduced restriction endonuclease cleavage sites.Chimeric CD8-Nef plasmids that reciprocally exchanged the centrally located conserved region of HIV-1SF2 Nef for the homologous region of SIVmac239open Nef were constructed as follows. Unique KpnI and EcoRV sites, which did not alter the amino acid sequence of the Nef protein, were engineered into the SIVmac239open plasmid by site-directed mutagenesis. The KpnI-EcoRV fragments from the HIV-1SF2 and SIVmac239open Nefs were subsequently exchanged. The domain substitution results in the replacement of amino acids 78-139 of HIV-1SF2 Nef for amino acids 107-167 of SIVmac239open Nef and vice versa.Plasmid DNA (15 μg) was transfected into ~5 ´ 105 COS-7 cells by calcium-phosphate precipitation. Twenty-four h after transfection, the cultures were split in half and propagated for an additional 24 h before harvesting for protein labeling and kinase analyses.Metabolic Labeling, Immunoprecipitation, in Vitro Kinase Assay, and Pulse-chase AnalysisThe procedures for metabolic labeling, cellular extraction, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, and the in vitro kinase assay were performed as described previously(47Sawai E.T. Baur A. Struble H. Peterlin B.M. Levy J.A. Cheng-Mayer C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1539-1543Crossref PubMed Scopus (209) Google Scholar). For pulse-chase analyses, transfected COS-7 cells were starved with methionine- and cysteine-free RPMI 1640 media for 30 min and pulse labeled for 15 min with labeling medium containing 500 μCi/ml 35S-Translabel (ICN) at 37°C. After washing the monolayers twice with Dulbecco's phosphate-buffered saline, 10 ml of Dulbecco's modified Eagle's medium containing 10% fetal bovine serum was added to the cells, except for those representing the zero time point which was extracted immediately. After chase times of 30 min, 1, 2 and 4 h, the respective cells were washed twice with Dulbecco's phosphate-buffered saline and extracted. Immunoprecipitations were done as described above.RESULTSKinase Association Is a Highly Conserved Property of Nef and Can Be Detected in Several Cell TypesPreviously, using a CD8-Nef fusion protein or native Nef, we identified a serine kinase activity that interacts with HIV-1SF2 Nef expressed in human T-cell lines (47). This kinase was responsible for the phosphorylation of two proteins of 62 and 72 kDa that specifically coimmunoprecipitate with native Nef and CD8-Nef fusion proteins. We also reported that HIV-1SF2 Nef truncated at amino acid 95 (CD8-SF2Nef (1-94)) no longer associated with the kinase activity, as indicated by the absence of the 62 or 72 kDa phosphorylated proteins in Nef immunoprecipitates. In order to develop a rapid assay for screening different alleles and mutants of Nef for the associated kinase activity, transient expression of Nef in simian COS-7 cells was conducted (Fig. 1A). By metabolic labeling, substantial expression of CD8-SF2Nef, CD8-antisense Nef, CD8-SF2Nef (1-94), CD8-SIVmac239open Nef, and CD8-SF13 Nef could be detected in transfected COS-7 cells (Fig. 1A, lanes 2-6).In vitro kinase assays performed on immunoprecipitates from unlabeled, transfected cell extracts demonstrated that besides CD8-SF2Nef, CD8-SF13 Nef, and CD8-SIVmac239open Nef also interacted with this kinase (Fig. 1A, lanes 9, 11, and 12). The 62-kDa protein was specifically phosphorylated in these immunoprecipitates but not in immunoprecipitates containing CD8-antisense Nef (anti) or a truncated Nef (SF2Nef(1Allan J.S. Coligan J.E. Lee T.H. McLane M.F. Kanki P.J. Groopman J.E. Essex M. Science. 1985; 230: 810-813Crossref PubMed Scopus (156) Google Scholar, 2Colombini S. Arya S.K. Reitz M.S. Jagodzinski L. Beaver B. Wong-Staal F. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 4813-4817Crossref PubMed Scopus (35) Google Scholar, 3Shibata R. Miura T. Hayami M. Ogawa K. Sakai H. Kiyomasu T. Ishimoto A. Adachi A. J. Virol. 1990; 64: 742-747Crossref PubMed Google Scholar, 4Feinberg M.B. Jarrett R.F. Aldovini A. Gallo R.C. Wong-Staal F. Cell. 1986; 46: 807-817Abstract Full Text PDF PubMed Scopus (418) Google Scholar, 5Robert-Guroff M. Popovic M. Gartner S. Markham P. Gallo R.C. Reitz M.S. J. Virol. 1990; 64: 3391-3398Crossref PubMed Google Scholar, 6Klotman M.E. Kim S. Buchbinder A. DeRossi A. Baltimore D. Wong-Staal F. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 5011-5015Crossref PubMed Scopus (159) Google Scholar, 7Munis J.R. Kornbluth R.S. Guatelli J.C. Richman D.D. J. Gen. Virol. 1992; 73: 1899-1901Crossref PubMed Scopus (32) Google Scholar, 8Guy B. Kieny M.P. Riviere Y. Le Peuch C. Dott K. Girard M. Montagnier L. Nature. 1987; 330: 266-269Crossref PubMed Scopus (402) Google Scholar, 9Guy B. Riviere Y. Dott K. Regnault A. Kieny M.P. Virology. 1990; 176: 413-425Crossref PubMed Scopus (89) Google Scholar, 10Kienzle N. Freund J. Kalbitzer H.R. Mueller-Lantzsch N. Eur. J. Biochem. 1993; 214: 451-457Crossref PubMed Scopus (29) Google Scholar, 11Zazopoulos E. Haseltine W.A. J. Virol. 1993; 67: 1676-1680Crossref PubMed Google Scholar, 12Bandres J.C. Luria S. Ratner L. Virology. 1994; 201: 157-161Crossref PubMed Scopus (22) Google Scholar, 13Yu G. Felsted R.L. Virology. 1992; 187: 46-55Crossref PubMed Scopus (88) Google Scholar, 14Zazopoulos E. Haseltine W.A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6634-6638Crossref PubMed Scopus (50) Google Scholar, 15Niederman T.M.J. Randall-Hastings Ratner L. Virology. 1993; 197: 420-425Crossref PubMed Scopus (69) Google Scholar, 16Chowers M.Y. Spina C.A. Kwoh T.J. Fitch N.J. Richman D.D. Guatelli J.C. J. Virol. 1994; 68: 2906-2914Crossref PubMed Google Scholar, 17Aiken C. Konner J. Landau N.R. Lenburg M.E. Trono D. Cell. 1994; 76: 853-864Abstract Full Text PDF PubMed Scopus (604) Google Scholar, 18Franchini G. Robert-Guroff M. Ghrayeb J. Chang N.T. Wong-Staal F. Virology. 1986; 155: 593-599Crossref PubMed Scopus (118) Google Scholar, 19Kohleisen B. Neumann M. Herrmann R. Brack-Werner R. Krohn K.J. Ovod V. Ranki A. Erfle V. AIDS. 1992; 6: 1427-1436Crossref PubMed Scopus (56) Google Scholar, 20Murti K.G. Brown P.S. Ratner L. Garcia J.V. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11895-11899Crossref PubMed Scopus (36) Google Scholar, 21Terwilliger E.F. Sodroski J.G. Rosen C.A. Hazeltine W.A. J. Virol. 1986; 60: 754-760Crossref PubMed Google Scholar, 22Luciw P.A. Cheng-Mayer C. Levy J.A. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 1434-1438Crossref PubMed Scopus (218) Google Scholar, 23Cheng-Mayer C. Iannello P. Shaw K. Luciw P.A. Levy J.A. Science. 1989; 246: 1629-1632Crossref PubMed Scopus (136) Google Scholar, 24Tsunetsugu-Yokota Y. Matsuda S. Maekawa M. Saito T. Takemori T. Takebe Y. Virology. 1992; 191: 960-963Crossref PubMed Scopus (11) Google Scholar, 25Ahmad N. Venkatesan S. Science. 1988; 241: 1481-1485Crossref PubMed Scopus (283) Google Scholar, 26Niederman T.M.J. Thielan B.J. Ratner L. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 1132-1186Crossref Scopus (189) Google Scholar, 27Maitra R.K. Ahmad N. Holland S.M. Venkatesan S. Virology. 1991; 182: 522-533Crossref PubMed Scopus (31) Google Scholar, 28Niederman T.M.J. Garcia J.V. Randall-Hastings W. Luria S. Ratner L. J. Virol. 1992; 66: 6213-6219Crossref PubMed Google Scholar, 29Niederman T.M.J. Randall-Hastings W. Luria S. Bandres J.C. Ratner L. Virology. 1993; 194: 338-344Crossref PubMed Scopus (45) Google Scholar, 30de Ronde A. Klaver B. Keulen W. Smit L Goudsmit J. Virology. 1992; 188: 391-395Crossref PubMed Scopus (143) Google Scholar, 31Spina C.A. Kwoh T.J. Chowers M.Y. Guatelli J.C. Richman D.D. J. Exp. Med. 1994; 179: 115-123Crossref PubMed Scopus (361) Google Scholar, 32Miller M.D. Warmerdam M.T. Gaston I. Greene W.C. Feinberg M.B. J. Exp. Med. 1994; 179: 101-113Crossref PubMed Scopus (477) Google Scholar, 33Jamieson B.D. Aldrovandi G.M. Planelles V. Jowett J.B. Gao L. Bloch L.M. Chen I.S. Zack J.A. J. Virol. 1994; 68: 3478-3485Crossref PubMed Google Scholar, 34Kestler III, H.W. Ringler D.J. Mori K. Panicali D.L. Sehgal P.K. Daniel M.D. Desrosiers R.C. Cell. 1991; 65: 651-662Abstract Full Text PDF PubMed Scopus (1420) Google Scholar, 35Luria S. Chambers I. Berg P. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 5326-5330Crossref PubMed Scopus (162) Google Scholar, 36Skowronski J. Parks D. Mariani R. EMBO J. 1993; 12: 703-713Crossref PubMed Scopus (256) Google Scholar, 37De S.K. Marsh J.W. J. Biol. Chem. 1994; 269: 6656-6660Abstract Full Text PDF PubMed Google Scholar, 38Rhee S.S. Marsh J.W. J. Immunol. 1994; 152: 5128-5134PubMed Google Scholar, 39Rhee S.S. Marsh J.W. J. Virol. 1994; 68: 5156-5163Crossref PubMed Google Scholar, 40Baur A.S. Sawai E.T. Dazin P. Fantl W.J. Cheng-Mayer C. Peterlin B.M. Immunity. 1994; 1: 373-384Abstract Full Text PDF PubMed Scopus (278) Google Scholar, 41Garcia J.V. Miller A.D. Nature. 1991; 350: 508-511Crossref PubMed Scopus (648) Google Scholar, 42Garcia J.V. Alfano J. Miller A.D. J. Virol. 1993; 67: 1511-1516Crossref PubMed Google Scholar, 43Inoue M. Koga Y. Djordjijevic D. Fukuma T. Reddy E.P. Yokoyama M.M. Sagawa K. Int. Immunol. 1993; 5: 1067-1073Crossref PubMed Scopus (12) Google Scholar, 44Mariani R. Skowronski J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5549-5553Crossref PubMed Scopus (153) Google Scholar, 45Sanfridson A. Cullen B.R. Doyle C. J. Biol. Chem. 1994; 269: 3917-3920Abstract Full Text PDF PubMed Google Scholar, 46Harris M. Coates K. J. Gen. Virol. 1993; 74: 1581-1589Crossref PubMed Scopus (44) Google Scholar, 47Sawai E.T. Baur A. Struble H. Peterlin B.M. Levy J.A. Cheng-Mayer C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1539-1543Crossref PubMed Scopus (209) Google Scholar, 48Ratner L. Starchich B. Josephs S.F. Hahn B.H. Reddy E.P. Livak K.J. Petteway Jr., S.R. Pearson M.L. Haseltine W.A. Arya S.K. Wong-Staal F. Nucleic Acids Res. 1985; 13: 8219-8229Crossref PubMed Scopus (36) Google Scholar, 49Gurgo C. Guo H.-G. Franchini G. Aldovini A. Collati E. Farrell K. Wong-Staal F. Gallo R.C. Reitz Jr., M.S. Virology. 1988; 164: 531-536Crossref PubMed Scopus (98) Google Scholar, 50Altschul S.F. Gish W. Miller W. Myers E.W. Lipman D.J. J. Mol. Biol. 1990; 215: 403-410Crossref PubMed Scopus (69088) Google Scholar, 51Meyers G. Berzofsky J.A. Korber B. Smith R.F. Pavlakis G.N. Human Retroviruses and AIDS: A Compilation and Analysis of Nucleic Acid and Amino Acid Sequences. Los Alamos National Laboratory, Los Alamos, New Mexico1993Google Scholar, 52Eisenberg D. Schwarz E. Komaromy M. Wall R. J. Mol. Biol. 1984; 179: 125-142Crossref PubMed Scopus (1683) Google Scholar, 53Freund J. Kellner R. Houthaeve T. Kalbitzer H.R. Eur. J. Biochem. 1994; 221: 811-819Crossref PubMed Scopus (42) Google Scholar, 54Rogers S. Wells R. Rechesteiner M. Science. 1986; 234: 364-368Crossref PubMed Scopus (1943) Google Scholar, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94)) (Fig. 1A, lanes 8 and 10). Several other phosphorylated bands are observed in lanes 10 and 11. These were not observed reproducibly and, thus, were considered to be nonspecific (for example compare Fig. 2A, lane 10 and Fig. 1A, lane 10). The phosphorylated 62-kDa protein from T-cell lines and COS-7 cells was similar by partial V8-protease mapping (data not shown). Since three different alleles of Nef were capable of interacting with the kinase, it is likely that this property of Nef is conserved. Moreover, because the kinase activity was found in cultures of human T-cell lines (Jurkat, HUT-78)(47), simian fibroblasts (COS-7), and murine fibroblasts (NIH3T3; data not shown) that express Nef, the kinase itself is conserved. However, the phosphorylation of the 72-kDa substrate was not observed in the latter two cell types (COS-7 and NIH3T3) (Fig. 1B). Thus, phosphorylation of the 72-kDa protein may be T-cell-specific.Figure 2:The central conserved region of Nef is important for association with the cellular kinase activity. A, COS-7 cells transiently expressing CD8-SF2 Nef (SF2,lanes 1, 2, 8, and 9), CD8-SF2(1-94) (1-94,lanes 3 and 10), CD8-SF2(1-127) (1-127,lanes 4 and 11), CD8-SF2(45-210) (45-210,lanes 5 and 12), and CD8-SF2(70-210) (70-210,lanes 6 and 13) were extracted with (lanes 1-6) or without (lanes 8-13) metabolic labeling as in Fig. 1. Immunoprecipitations were performed either with an anti-gp41 monoclonal antibody (lanes 1 and 8) or a CD8-specific monoclonal antibody (lanes 2-6 and 9-13). An in vitro kinase assay was performed on the immunoprecipitates from unlabeled extracts (lanes 8-13). B, truncation of Nef at amino acid 127 results in an unstable protein. Pulse-chase analysis was performed using cells transiently expressing either CD8-SF2 Nef (lanes 5-8) or CD8-SF2(1-127) (lanes 1-4). Metabolically labeled cells were extracted at 0, 0.5, 1.0, and 2 h after the initial 15-min pulse. Immunoprecipitations were performed using a monoclonal antibody to CD8. The increased mobilities of CD8-SF2 Nef and CD8-SF2(1-127) during the chase period are probably due to the glycosylation of the CD8 portion of the molecule. The positions of CD8-SF2 Nef and CD8-SF2(1-127) are indicated on the right.View Large Image Figure ViewerDownload Hi-res