The host cell MAP kinase ERK-2 regulates viral assembly and release by phosphorylating the p6gag protein of HIV-1.
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 p6(gag) 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 p6(gag) 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 p6(gag). Substitution by an alanine of the Thr(23) 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 Pr55(gag) 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 p6(gag) protein by virus-associated ERK-2 is involved in the budding stage of HIV-1 life cycle.
Hemonnot
Bénédicte
B
Laboratoire Infections Rétrovirales et Signalisation Cellulaire, Centre National pour la Recherche Scientifique, UMR 5121-Université Montpellier 1, Institut de Biologie, 4 Boulevard Henri IV, CS89508, 34960 Montpellier cedex 2, France.
Cartier
Christine
C
Gay
Bernard
B
Rebuffat
Sandra
S
Bardy
Martine
M
Devaux
Christian
C
Boyer
Véronique
V
Briant
Laurence
L
D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D015153:Blotting, Western; D002460:Cell Line; D004252:DNA Mutational Analysis; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D015683:Gene Products, gag / Q000737:chemistry / Q000378:metabolism*; D005982:Glutathione Transferase / Q000378:metabolism; D015497:HIV-1 / Q000378:metabolism; D006801:Humans; D008854:Microscopy, Electron; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D034741:RNA, Small Interfering / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013912:Threonine / Q000737:chemistry; D014162:Transfection; D014771:Virion / Q000378:metabolism; D014780:Viruses / Q000378:metabolism; D054301:gag Gene Products, Human Immunodeficiency Virus
D015683:Gene Products, gag; D034741:RNA, Small Interfering; D054301:gag Gene Products, Human Immunodeficiency Virus; C083002:p6 gag protein, Human immunodeficiency virus 1; D013912:Threonine; D005982:Glutathione Transferase; D019950:Mitogen-Activated Protein Kinase 1; D000409:Alanine