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• W2000622245 abstract "The host cell MAP kinase ERK-2 incorporated within human immunodeficiency virus type 1 particles plays a critical role in virus infectivity by phosphorylating viral proteins. Recently, a fraction of the virus incorporated late (L) domain-containing p6gag protein, which has an essential function in the release of viral particles from the cell surface, was reported to be phosphorylated by an unknown virus-associated cellular protein kinase (Muller, B., Patschinsky, T., and Krausslich, H. G. (2002) J. Virol. 76, 1015–1024). The present study demonstrates the contribution of the MAP kinase ERK-2 in p6gag phosphorylation. According to mutational analysis, a single ERK-2-phosphorylated threonine residue, belonging to a highly conserved phosphorylation MAP kinase consensus site, was identified at position 23 within p6gag. Substitution by an alanine of the Thr23 phosphorylable residue within the pNL4.3 molecular clone was found to decrease viral release from various cell types. As observed from electron microscopy experiments, most virions produced from this molecular clone remained incompletely separated from the host cell membrane with an immature morphology and displayed a reduced infectivity in single round infection experiments. Analysis of protein processing by Western blotting experiments revealed an incomplete Pr55gag maturation and a reduction in the virion-associated reverse transcriptase proteins was observed that was not related to differences in intracellular viral protein expression. Altogether, these data suggest that phosphorylation of p6gag protein by virus-associated ERK-2 is involved in the budding stage of HIV-1 life cycle. The host cell MAP kinase ERK-2 incorporated within human immunodeficiency virus type 1 particles plays a critical role in virus infectivity by phosphorylating viral proteins. Recently, a fraction of the virus incorporated late (L) domain-containing p6gag protein, which has an essential function in the release of viral particles from the cell surface, was reported to be phosphorylated by an unknown virus-associated cellular protein kinase (Muller, B., Patschinsky, T., and Krausslich, H. G. (2002) J. Virol. 76, 1015–1024). The present study demonstrates the contribution of the MAP kinase ERK-2 in p6gag phosphorylation. According to mutational analysis, a single ERK-2-phosphorylated threonine residue, belonging to a highly conserved phosphorylation MAP kinase consensus site, was identified at position 23 within p6gag. Substitution by an alanine of the Thr23 phosphorylable residue within the pNL4.3 molecular clone was found to decrease viral release from various cell types. As observed from electron microscopy experiments, most virions produced from this molecular clone remained incompletely separated from the host cell membrane with an immature morphology and displayed a reduced infectivity in single round infection experiments. Analysis of protein processing by Western blotting experiments revealed an incomplete Pr55gag maturation and a reduction in the virion-associated reverse transcriptase proteins was observed that was not related to differences in intracellular viral protein expression. Altogether, these data suggest that phosphorylation of p6gag protein by virus-associated ERK-2 is involved in the budding stage of HIV-1 life cycle. As other intracellular parasites, the human immunodeficiency virus type 1 (HIV-1) 1The abbreviations used are: HIV-1, human immunodeficiency virus type 1; RT, reverse transcriptase; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; GST, glutathione S-transferase; PR, protease; WT, wild type; mAb, monoclonal antibody; siRNA, small interfering RNA; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; E2, ubiquitin carrier protein. 1The abbreviations used are: HIV-1, human immunodeficiency virus type 1; RT, reverse transcriptase; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; GST, glutathione S-transferase; PR, protease; WT, wild type; mAb, monoclonal antibody; siRNA, small interfering RNA; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; E2, ubiquitin carrier protein. is necessarily dependent upon cell factors for replication. Participation of host cell components is specially required for any of the many HIV-1 gag-encoded functions. Indeed, a variety of Pr55gag-interacting factors have been identified including actin (1Rey O. Canon J. Krogstad P. Virology. 1996; 220: 530-534Google Scholar, 2Liu B. Dai R. Tian C.J. Dawson L. Gorelick R. Yu X.F. J. Virol. 1999; 73: 2901-2908Google Scholar, 3Ott D.E. Coren L.V. Johnson D.G. Kane B.P. Sowder 2nd, R.C. Kim Y.D. Fisher R.J. Zhou X.Z. Lu K.P. Henderson L.E. Virology. 2000; 266: 42-51Google Scholar, 4Wilk T. Gowen B. Fuller S.D. J. Virol. 1999; 73: 1931-1940Google Scholar), calmodulin (5Radding W. Williams J.P. McKenna M.A. Tummala R. Hunter E. Tytler E.M. McDonald J.M. AIDS Res. Hum. Retroviruses. 2000; 16: 1519-1525Google Scholar), the motor protein KIF-4 (6Tang Y. Winkler U. Freed E.O. Torrey T.A. Kim W. Li H. Goff S.P. Morse 3rd, H.C. J. Virol. 1999; 73: 10508-10513Google Scholar), the nuclear transporter karyopherin α (7Gallay P. Stitt V. Mundy C. Oettinger M. Trono D. J. Virol. 1996; 70: 1027-1032Google Scholar, 8Agostini I. Popov S. Li J. Dubrovsky L. Hao T. Bukrinsky M. Exp. Cell Res. 2000; 259: 398-403Google Scholar), the human nuclear shuttling protein VAN (9Gupta K. Ott D. Hope T.J. Siliciano R.F. Boeke J.D. J. Virol. 2000; 74: 11811-11824Google Scholar), the translation elongation factor 1-α (10Cimarelli A. Luban J. J. Virol. 1999; 73: 5388-5401Google Scholar), the translation initiation factor 2 (11Wilson S.A. Sieiro-Vazquez C. Edwards N.J. Iourin O. Byles E.D. Kotsopoulou E. Adamson C.S. Kingsman S.M. Kingsman A.J. Martin-Rendon E. Biochem. J. 1999; 342: 97-103Google Scholar), the HO3 hystidyl-tRNA synthase (12Lama J. Trono D. J. Virol. 1998; 72: 1671-1676Google Scholar), the heat shock protein 70 (13Gurer C. Cimarelli A. Luban J. J. Virol. 2002; 76: 4666-4670Google Scholar), and a number of polycomb group proteins (14Peytavi R. Hong S.S. Gay B. d'Angeac A.D. Selig L. Benichou S. Benarous R. Boulanger P. J. Biol. Chem. 1999; 274: 1635-1645Google Scholar). As a consequence, a number of these host cell proteins has been found to be incorporated within HIV-1 virions in addition to virus-encoded components (15Ott D.E. Rev. Med. Virol. 2002; 12: 359-374Google Scholar, 16Tremblay M.J. Fortin J.F. Cantin R. Immunol. Today. 1998; 19: 346-351Google Scholar). A specific implication of host cell factors was reported to occur during the late budding stage. Indeed, the Pr55gag precursor protein is transported to the budding site by an unknown mechanism that most likely utilizes host cell factors and associates with the plasma membrane through cotranslational modifications by the cellular myristyl-S-transferase (17Bryant M. Ratner L. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 523-527Google Scholar, 18Gottlinger H.G. Sodroski J.G. Haseltine W.A. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 5781-5785Google Scholar). The accumulation of the Pr55gag protein at the plasma membrane then allows its multimerization and is followed by the projection outward of the cell of a spherical budding particle. Recently, the recruitment at the budding site of class E Vps proteins including AIP1/ALIX (19Strack B. Calistri A. Craig S. Popova E. Gottlinger H.G. Cell. 2003; 114: 689-699Google Scholar, 20von Schwedler U.K. Stuchell M. Muller B. Ward D.M. Chung H.Y. Morita E. Wang H.E. Davis T. He G.P. Cimbora D.M. Scott A. Krausslich H.G. Kaplan J. Morham S.G. Sundquist W.I. Cell. 2003; 114: 701-713Google Scholar) and the ubiquitin-conjugating enzyme homologue Tsg101 (21Demirov D.G. Ono A. Orenstein J.M. Freed E.O. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 955-960Google Scholar, 22Garrus J.E. von Schwedler U.K. Pornillos O.W. Morham S.G. Zavitz K.H. Wang H.E. Wettstein D.A. Stray K.M. Cote M. Rich R.L. Myszka D.G. Sundquist W.I. Cell. 2001; 107: 55-65Google Scholar, 23Martin-Serrano J. Zang T. Bieniasz P.D. Nat. Med. 2001; 7: 1313-1319Google Scholar, 24VerPlank L. Bouamr F. LaGrassa T.J. Agresta B. Kikonyogo A. Leis J. Carter C.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 7724-7729Google Scholar) through interactions with the Pr55gag polyprotein, was reported to be required for the complete release of the viral particle. Interactions with Tsg101, mediated via the PTAP motif identified as the L-domain contained within the p6gag region, were demonstrated to be enhanced by ubiquitination of the Pr55gag precursor, suggesting a possible interaction with unknown ubiquitin-conjugating enzymes (22Garrus J.E. von Schwedler U.K. Pornillos O.W. Morham S.G. Zavitz K.H. Wang H.E. Wettstein D.A. Stray K.M. Cote M. Rich R.L. Myszka D.G. Sundquist W.I. Cell. 2001; 107: 55-65Google Scholar, 25Ott D.E. Coren L.V. Copeland T.D. Kane B.P. Johnson D.G. Sowder 2nd, R.C. Yoshinaka Y. Oroszlan S. Arthur L.O. Henderson L.E. J. Virol. 1998; 72: 2962-2968Google Scholar). Soon after the viral particle pinches off and is released in the extracellular compartment, the viral protease becomes activated and the Pr55gag polyprotein is cleaved into MAp17gag, CAp24gag, NCp7gag, and p6gag proteins and p1, p2 spacer peptides. Most of these mature products have also been shown to interact with cellular proteins. As an example, interactions occurring with CAp24gag have been documented as the mechanism directing incorporation into virions of cyclophilin A (26Franke E.K. Luban J. Virology. 1996; 222: 279-282Google Scholar, 27Thali M. Bukovsky A. Kondo E. Rosenwirth B. Walsh C.T. Sodroski J. Göttlinger H.G. Nature. 1994; 372: 363-365Google Scholar), a host cell compound that acts as an uncoating factor and participates to the initial uptake of HIV-1 by target cells (28Sherry B. Zybarth G. Alfano M. Dubrovsky L. Mitchell R. Rich D. Ulrich P. Bucala R. Cerami A. Bukrinsky M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1758-1763Google Scholar, 29Saphire A.C. Bobardt M.D. Gallay P.A. EMBO J. 1999; 18: 6771-6785Google Scholar). In addition, a number of these Pr55gag processing products was also suggested to interact with host cell protein kinases as they were found to be phosphorylated. Phosphorylation of the viral MAp17gag by host cell kinase(s) was found to occur at various steps of the HIV-1 replicative cycle. Indeed, the C-terminal phosphorylation of MAp17gag protein during and immediately after virus production was proposed to facilitate the dissociation of the viral matrix protein from the cellular membrane (30Gallay P. Swingler S. Aiken C. Trono D. Cell. 1995; 80: 379-388Google Scholar). Additional phosphorylation of MAp17gag by the MAP kinase (MAPK) ERK-2 was proposed to promote membrane dissociation of the reverse transcription complex from the cell membrane at the site of entry, allowing its nuclear translocation (31Bukrinskaya A.G. Ghorpade A. Heinzinger N.K. Smithgall T.E. Lewis R.E. Stevenson M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 367-371Google Scholar, 32Jacque J.M. Mann A. Enslen H. Sharova N. Brichacek B. Davis R.J. Stevenson M. EMBO J. 1998; 17: 2607-2618Google Scholar). For CAp24gag, three distinct phosphorylation sites on serine residues were identified (33Cartier C. Sivard P. Tranchat C. Decimo D. Desgranges C. Boyer V. J. Biol. Chem. 1999; 274: 19434-19440Google Scholar). As mutations at these sites were observed to block HIV-1 replicative cycle at an early reverse transcription step, CAp24gag phosphorylation was proposed to contribute to the early post-entry step by promoting the dissociation of the viral core. Recently, we observed that CAp24gag interacts with and is phosphorylated by the PKA-Cα subunit that we found to be incorporated within HIV-1 particles (34Cartier C. Hemonnot B. Gay B. Bardy M. Sanchiz C. Devaux C. Briant L. J. Biol. Chem. 2003; 278: 35211-35219Google Scholar). More recently, the proline-rich peptide of 52 amino acids termed p6gag, derived from the C terminus of the Pr55gag precursor, was reported to be the predominant phosphoprotein of HIV-1 and the contribution of one or several virus-associated kinase(s) was hypothesized (35Muller B. Patschinsky T. Krausslich H.G. J. Virol. 2002; 76: 1015-1024Google Scholar). Having demonstrated that the cellular MAPK ERK-2 is specifically incorporated within HIV-1 viral particles (36Cartier C. Deckert M. Grangeasse C. Trauger R. Jensen F. Bernard A. Cozzone A. Desgranges C. Boyer V. J. Virol. 1997; 71: 4832-4837Google Scholar), it remained to investigate its role in p6gag phosphorylation. The present study was thus designed to define the precise contribution of ERK-2 in the phosphorylation of the p6gag viral protein, to determine critical residues phosphorylated within p6gag and to elucidate the involvement of p6gag phosphorylation in the HIV-1 life cycle. As observed from in vitro phosphorylation assays, the p6gag protein and its p15gag precursor were found to be phosphorylated by ERK-2 at the level of a single threonine residue (Thr23) located within a conserved MAPK canonical consensus sequence. By analyzing point mutation of the p6gag protein within a pNL4.3 molecular clone, we observed that substitution of the ERK-2 phosphorylable residue within p6gag leads to the production of immature viruses unable to separate from the host cell membrane. A faint number of particles released from the producing cells was isolated that were found to display altered morphology and size. Such particles were characterized by incomplete maturation of Pr55gag precursors accompanied by modifications in the incorporation of reverse transcriptase subunits. As defined from single round infectivity assays performed in the MAGI indicator cell line, Thr23 mutated virions displayed a reduced infectivity as compared with wild type viruses. Altogether, our data indicate that phosphorylation of a unique site of the p6gag domain by ERK-2 plays a critical role in the late stage of the HIV-1 life cycle, by contributing to the regulation of viral assembly and/or release. Cell Culture and Transfections—Human embryonic kidney 293T cells and MAGI cells (37Luukkonen B.G. Fenyo E.M. Schwartz S. Virology. 1995; 206: 854-865Google Scholar) were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum (Invitrogen), 100 units/ml penicillin, 100 μg/ml streptomycin, 2 mm glutamine. In transfection experiments, cells (3 × 105) were plated in 6-well culture plates and incubated in the presence of viral DNA diluted in ExGen 500 transfection reagent (Euromedex) at a ratio of 6 units/μg of DNA. Western Blot Analysis—Proteins were separated on a 14% ProSieve® 50 polyacrylamide gel (FMC), then transferred to polyvinylidene difluoride membrane (Immobilon P, Millipore) and revealed by immunoblotting using an anti-CAp24gag mouse monoclonal antibody (mAb) (ICN), an anti-RT rabbit polyclonal serum (kindly provided by J. L. Darlix, ENS, Lyon, France), or an anti-p6gag polyclonal serum obtained after immunization of rabbits with GST-p6gag fusion protein. Anti-MAp17gag mAb was obtained from Fitzgerald Industries International, Inc., anti-ERK-2 from Santa Cruz Biotechnology, Inc., and anti-GST from Amersham Biosciences. Secondary antibodies conjugated to horseradish peroxidase were revealed by enhanced chemiluminescent detection. Virion Production and Purification—Viral stocks were obtained by transfection of the pNL4.3 HIV-1 molecular clone into 293T cells. Briefly, culture supernatants collected 40 h after transfection were cleared from cellular debris by low-speed centrifugation and filtered on 0.45-μm pore size membranes (Millipore). Virions were sedimented by ultracentrifugation through 20% sucrose cushions, at 25,000 rpm for 2 h 30 min at 4 °C (SW28 rotor), resuspended in 200 μl of phosphate-buffered saline and layered onto the top of an Optiprep velocity gradient (6–20% (w/v) iodixanol; Abcys SA). Centrifugation was run for 3 h at 26,000 × g at 4 °C. Virions were recovered from virus-containing fractions by additional ultracentrifugation at 95,000 rpm for 5 min at 4 °C (TLA100.2 rotor) and lysed. Additional preparation of a highly purified virus (HIV strain HZ321, from clade A) obtained by multiple density gradient purification from infected HUT78 supernatant (38Trauger R.J. Ferre F. Daigle A.E. Jensen F.C. Moss R.B. Mueller S.H. Richieri S.P. Slade H.B. Carlo D.J. J. Infect. Dis. 1994; 169: 1256-1264Google Scholar) was kindly provided by R. Trauger (Immune Response Corp., Carlsbad, CA). GST Fusion Proteins, Site-directed Mutagenesis, and Plasmid Production—p6gag and p15gag sequences were amplified by PCR experiments from the pNL4.3 proviral DNA with 5′-GCGTGGATCCCAGAGCAGAC, 5′-CGGGAATTCTCATTGTGAC and 5′-GCGGATCCCAAAGAAAGACTGTTAAG, 5′-CCGAATTCTTATTGTGACGAGGGGTC oligonucleotide primer pairs, respectively, and cloned in-frame into the pGEX 5X.1 expression vector (Amersham Biosciences). Vectors encoding GST-p6gag mutants, where codons encoding each serine/threonine residue were replaced by sequences encoding an alanine, and the pNL4.3T23A plasmid were obtained by site-directed mutagenesis using the Gene Editor mutagenesis kit (Promega) and primers are listed in Table I. All mutant sequences were confirmed by sequencing. GST-NCp7gag, GST-MAp17gag, and GST-CAp24gag expression vectors were kindly provided by J. Darlix (ENS, Lyon) and vector encoding GST-Elk was a gift of D. A. Brenner (Department of Pharmacology, University of North Carolina, Chapel Hill).Table ISerine/threonine residues in the p6gag sequence encoded by the GST-p6gag plasmid or the pNL4.3T23A molecular clone were replaced by an alanine by site-directed mutagenesis using the Gene Editor mutagenesis kit (Promega) and the corresponding primersPrimerResidue mutatedOligonucleotide sequencePosition in pNL4.3 sequenceS3ASer35′-GCTCTGGTCTGGCCTGGGATCCACGC2126-2151T8AThr85′-CTGGTGGGGCTGCTGGCTCTGGTCTG2142-2167S14ASer145′-CCAAACCTGAAGGCCTCTTCTGGTG2162-2186T21AThr215′-GAGGGAGTTGTTGCCTCTTCCCCAAAG2181-2207T22AThr225′-CTGAGAGGGAGTTGCTGTCTCTTCC2187-2211T23AThr235′-CTGAGAGGGAGCTGTTGTCTCTTCC2187-2211S25ASer255′-CTCCTGCTTCTGAGCGGGAGTTGTTG2198-2220S40ASer405′-AAGAGTGATCTGAGGGCAGCTAAAGG2242-2267S43ASer435′-GCTGCCAAAGAGTGCTCTGAGGGAAG2249-2274S47ASer475′-GACGAGGGGTCGGCGCCAAAGAGTG2261-2285S50ASer505′-TTATTGTGACGCGGGGTCGCTGCCAAAG2265-2292S51ASer515′-TTATTGTGCCGAGGGGTCGCTGCCAAAG.2126-2151pNL4.3T23AThr235′-CTTCTGAGAGGGAGCTGTTGTCTCTT2189-2214 Open table in a new tab In Vitro Phosphorylation Assays—Viral lysate phosphorylation assays were performed in the presence of 2 μCi of [γ-32P]ATP and kinase buffer containing 50 mm Hepes (pH 7.5), 5 mm MnCl2 at 30 °C for 30 min. When phosphorylation of peptides or GST-fused proteins was assayed, 20 ng of recombinant activated ERK-2 (Stratagene) (39Zheng C.F. Guan K.L. J. Biol. Chem. 1993; 268: 11435-11439Google Scholar) was added to the reaction mixture. Phosphorylated products were separated by SDS-PAGE and detected by autoradiography. Two-dimensional Gel Electrophoresis—Purified cell-free virions were analyzed by two-dimensional gel electrophoresis as follows. Virions were solubilized in buffer containing 8 m urea, 2 m thiourea, 4% CHAPS, 65 mm dithioerythritol, 40 mm Tris, and protease inhibitor for 2 h on a wheel. Viral lysates were clarified by centrifugation for 30 min at 20,000 × g. Each sample was added to a rehydratation solution containing 8 m urea, 2 m thiourea, 4% CHAPS, 65 mm dithioerythritol, bromphenol blue, and 1% Pharmalytes pH 3–10 solution (Amersham Biosciences). Immobiline DryStrips, 18 cm, covering a pH range of 3–10 were allowed to rehydrate in the above solution for 4 h without current and then for 12 h under a 30-V current under low viscosity paraffin oil. Isoelectrofocusing was then performed according to the following voltage/time profile: 500 V for 1 h, 1,000 V for 1 h, linearly increasing the gradient to 8,000 V for 2 h, and a final phase of 8,000 V for 40,000 V/h up to a total amount of 50,000 V/h. After the first dimensional run, the individual strip was equilibrated, and then put on the top of the second dimensional gel (15%) with the Hofer Dalt System (Amersham Biosciences) at 150 V/10 gels. Proteins were then transferred onto polyvinylidene difluoride, hybridized with anti-p6gag serum and horseradish peroxidase-conjugated secondary antibodies, and revealed with ECL reagents. Viral Infectivity Assays—MAGI indicator cells, which stably express the β-galactosidase reporter gene cloned downstream of the HIV-1 LTR promoter, were platted at 8 × 104 cells per well, in 24-well plates and exposed to HIV-1 stock solutions normalized according to the CAp24gag antigen content by enzyme-linked immunosorbent assay (Beckman-Coulter) or to RT activity determined as previously reported (40Briant L. Benkirane M. Girard M. Hirn M. Iosef C. Devaux C. J. Virol. 1996; 70: 5213-5220Google Scholar). Forty-eight hours post-infection, viral infectivity was monitored by quantification of o-nitrophenyl β-d-galactopyranoside hydrolysis from the cell lysates as previously described (41Briant L. Robert-Hebmann V. Sivan V. Brunet A. Pouyssegur J. Devaux C. J. Immunol. 1998; 160: 1875-1885Google Scholar). β-Galactosidase activity evaluated by measuring absorbance at 410 nm was normalized according to total protein content in the cell lysate. Electron Microscopy Analysis—Transfected 293T cells were processed for thin-layer electron microscopy as follows: 40 h post-transfection, cells were fixed in situ with 2.5% glutaraldehyde in cacodylate buffer (pH 7.4) for 60 min at 4 °C. Cells were then post-fixed with 2% osmium tetroxide, washed in cacodylate buffer containing 0.5% tanic acid, and embedded in epon (Embed-812, Electron Microscopy Sciences Inc.). Cell-free virions sedimented by ultracentrifugation for 10 min at 95,000 × g were fixed for 2 h at 4 °C in 2.5% glutaraldehyde in cacodylate buffer (pH 7.2). After extensive washes in 0.1 m Sorensen phosphate buffer (pH 7.2), viruses were included in a fibrin clot as described by Charret and Fauré-Fremiet (42Charret R. Fauré-Fremiet E. J. Microscopie. 1967; 6: 1063-1066Google Scholar). Viruses were then post-fixed with 2% osmium tetroxide and 0.5% tannic acid, dehydrated, and embedded in epon. Sections were counterstained with uranyl acetate and lead citrate and examined with an Hitachi H7100 transmission electron microscope. ERK-2 Knock-out Experiments—ERK-2 knock-out was performed by transfection of specific siRNA using the MAPK1(ERK-2) siRNA/siAb assay kit (Dharmacon RNA Technologies). Briefly, 100 pmol of pooled siRNA duplexes specific for the erk-2 sequence or control siRNA duplexes were transfected into 293T cells using the OligofectAMINE transfection reagent at a ratio of 1 μl for 20 pmol of siRNA. Inhibition of ERK-2 expression was assayed by immunoblotting of the total cell extracts using anti-ERK-2 mAbs. p6gag Protein and Its p15gag Precursor Are Phosphorylated by ERK-2 Virus-associated Kinase—The p6gag protein was recently shown to be phosphorylated in vivo and the possible contribution of virus-associated kinase(s) in these phosphorylation events was evoked (35Muller B. Patschinsky T. Krausslich H.G. J. Virol. 2002; 76: 1015-1024Google Scholar). To investigate such hypothesis, a lysate of highly purified NL4.3 virions was incubated in the presence of [γ-32P]ATP and appropriate kinase buffer. Phosphorylated proteins were separated by SDS-PAGE and revealed by autoradiography. As shown in Fig. 1A, several phosphoproteins evidenced and their nature were investigated by probing the membrane with antibodies raised against HIV-1 proteins. In these experimental conditions, phosphorylated products at 55 and 6 kDa were revealed by using anti-p6gag serum. This observation indicates that the p6gag protein and its Pr55gag precursor are both phosphorylated by host cell protein kinase(s) incorporated within HIV-1 particles. The contribution of host cell contaminating constituents was excluded as similar data were obtained using immunogen grade preparations of HIV strain HZ321 as a substrate (38Trauger R.J. Ferre F. Daigle A.E. Jensen F.C. Moss R.B. Mueller S.H. Richieri S.P. Slade H.B. Carlo D.J. J. Infect. Dis. 1994; 169: 1256-1264Google Scholar) (data not shown). In addition, 24- and 17-kDa phosphorylated proteins were recognized, respectively, with anti-CAp24gag and anti-MAp17gag mAbs, specific for processed forms of these proteins. This observation corroborates previous literature data indicating that MAp17gag and CAp24gag were phosphorylated by virus-associated kinases (32Jacque J.M. Mann A. Enslen H. Sharova N. Brichacek B. Davis R.J. Stevenson M. EMBO J. 1998; 17: 2607-2618Google Scholar, 34Cartier C. Hemonnot B. Gay B. Bardy M. Sanchiz C. Devaux C. Briant L. J. Biol. Chem. 2003; 278: 35211-35219Google Scholar). In agreement with our published observations (36Cartier C. Deckert M. Grangeasse C. Trauger R. Jensen F. Bernard A. Cozzone A. Desgranges C. Boyer V. J. Virol. 1997; 71: 4832-4837Google Scholar), an additional 42-kDa phosphorylated protein, which we previously identified as the host cell ERK-2 protein kinase incorporated within HIV-1 particles, was also observed from these phosphorylation experiments. We next investigated the contribution of ERK-2 in p6gag phosphorylation. GST fusion proteins containing the sequences encoding the p6gag protein or its p15gag precursor (see Fig. 1C, lower panel for its composition) were generated and used in an in vitro phosphorylation experiment in the presence of recombinant activated ERK-2 protein kinase (i.e. dually phosphorylated on threonine 183 and tyrosine 185 residues). As shown in Fig. 1B, the GST-p15gag precursor and GST-p6gag proteins were found to be phosphorylated. Interestingly, a stronger phosphorylation signal was detected with GST-p15gag compared with GST-p6gag. In contrast, no phosphorylation was observed using GST-NCp7gag or GST-CAp24gag as a substrate. As previously reported by others (32Jacque J.M. Mann A. Enslen H. Sharova N. Brichacek B. Davis R.J. Stevenson M. EMBO J. 1998; 17: 2607-2618Google Scholar), ERK-2-dependent phosphorylation of GST-MAp17gag was observed in our experimental conditions. GST alone and GST-Elk, a natural substrate of ERK-2, are shown as negative and positive controls, respectively. The amount of GST fusion proteins loaded in each lane was ascertained by Western blotting of the membrane with anti-GST mAb. Altogether, our results indicate that the p6gag protein packaged into virions is phosphorylated and suggest the contribution of ERK-2 virus-associated protein kinase as demonstrated by in vitro experiments. These phosphorylations are susceptible to occur before the complete processing of p6gag as suggested by the observation of phosphorylated forms of Pr55gag and p15gag precursors. A Single ERK-2 Consensus Sequence within the p6gag Protein Is Targeted by ERK-2—To define which amino acid residue(s) of p6gag is/are phosphorylated by the ERK-2 recombinant protein, the presence of the ERK-2 consensus motives was scanned in the total p6gag sequence. A single canonical ERK-2 motif represented by the (Ser/Thr)-Pro minimal sequence was identified at position 23 of the p6gag protein encoded by the pNL4.3 molecular clone. According to the compilation of HIV-1 sequences listed in the NIAID data base (43Kuiken C.L. Foley B. Hahn B. Korber B. McCutchan F. Marx P.A. Mellors J.W. Mullins J.I. Sodroski J. Wolinksy S. Biology Theoretical Group Biophysics Human Retroviruses and AIDS (1999). A Compilation and Analysis of Nucleic Acid and Amino Acid Sequences. Los Alamos National Laboratory, Los Alamos, NM1999Google Scholar), this motif was found to be conserved in most HIV-1 consensus sequences with a duplication observed in the consensus from clades M, F, and H (see Table II). In some cases (clades A and G), the Thr-Pro motif was absent but an equivalent Ser-Pro motif was found immediately beside, suggesting that a strong selection pressure leads to the conservation of an ERK-2 consensus at this site. ERK-2-dependent phosphorylation of the Thr23 residue was then analyzed in an in vitro phosphorylation assay using synthetic peptides mimicking either wild type (WT) p6gag spanning residues 12–39, or an equivalent peptide with an alanine replacing the threonine residue at position 23 (T23A) (Fig. 2A) incubated in the presence of recombinant activated ERK-2. As shown in Fig. 2B, phosphorylation of the WT peptide was evidenced from 0.3 to 15 nmol of peptide. In contrast, no phosphorylation signal was observed with equivalent amounts of T23A mutated peptide. These data indicate that residue Thr23 accounts for ERK-2-dependent phosphorylation of a p6gag peptide spanning residues 12 to 39. The existence of additional ERK-2 phosphorylation site(s) in p6gag was next analyzed. A systematic mutational analysis was performed to modify each serine or threonine residue within the p6gag sequence into an alanine residue. WT or mutated p6gag proteins were expressed in-frame with the GST sequence and used in an in vitro kinase experiment. As shown in Fig. 2C, p6gag protein was found to be phosphorylated when incubated with catalytically active ERK-2. ERK-2-dependent phosphorylation of p6gag was totally abolished only when the Thr23 residue was mutated into an alanine. No modification of GST-p6gag phosphorylation was observed when other serine or threonine residues within p6gag were substituted. Thus, the threonine residue at position 23 within p6gag appears to be the unique residue of this protein targeted by recombinant activated ERK-2 in vitro.Table IIConsequences of p6gagvariability on Pol sequence Open table in a new tab Fig. 2In vitro phosphorylation analysis of WT and mutated p6gag peptides and GST-fused p6gag protein.A, amino a" @default.
• W2000622245 created "2016-06-24" @default.
• W2000622245 creator A5032462021 @default.
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• W2000622245 title "The Host Cell MAP Kinase ERK-2 Regulates Viral Assembly and Release by Phosphorylating the p6 Protein of HIV-1" @default.
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• W2000622245 cites W1630981685 @default.
• W2000622245 cites W1813266103 @default.
• W2000622245 cites W1932084946 @default.
• W2000622245 cites W1968671288 @default.
• W2000622245 cites W1978002317 @default.
• W2000622245 cites W1978141733 @default.
• W2000622245 cites W1983108566 @default.
• W2000622245 cites W1984077320 @default.
• W2000622245 cites W1985610189 @default.
• W2000622245 cites W1992009621 @default.
• W2000622245 cites W1993877052 @default.
• W2000622245 cites W1994806672 @default.
• W2000622245 cites W2014212935 @default.
• W2000622245 cites W2023420720 @default.
• W2000622245 cites W2026659052 @default.
• W2000622245 cites W2028156315 @default.
• W2000622245 cites W2033320393 @default.
• W2000622245 cites W2038585810 @default.
• W2000622245 cites W2041344650 @default.
• W2000622245 cites W2044832767 @default.
• W2000622245 cites W2045185169 @default.
• W2000622245 cites W2048954075 @default.
• W2000622245 cites W2050779639 @default.
• W2000622245 cites W2053437843 @default.
• W2000622245 cites W2056119728 @default.
• W2000622245 cites W2057442177 @default.
• W2000622245 cites W2059841928 @default.
• W2000622245 cites W2060952336 @default.
• W2000622245 cites W2062710574 @default.
• W2000622245 cites W2076811542 @default.
• W2000622245 cites W2076877843 @default.
• W2000622245 cites W2077455094 @default.
• W2000622245 cites W2078248506 @default.
• W2000622245 cites W2084607977 @default.
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• W2000622245 cites W2115542461 @default.
• W2000622245 cites W2118233250 @default.
• W2000622245 cites W2119977018 @default.
• W2000622245 cites W2122673361 @default.
• W2000622245 cites W2123911636 @default.
• W2000622245 cites W2125453887 @default.
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