title: Comprehensive mutational analysis reveals p6Gag phosphorylation to be dispensable for HIV-1 morphogenesis and replication.
abstract: The structural polyprotein Gag of human immunodeficiency virus type 1 (HIV-1) is necessary and sufficient for formation of virus-like particles. Its C-terminal p6 domain harbors short peptide motifs that facilitate virus release from the plasma membrane and mediate incorporation of the viral Vpr protein. p6 has been shown to be the major viral phosphoprotein in HIV-1-infected cells and virions, but the sites and functional relevance of p6 phosphorylation are not clear. Here, we identified phosphorylation of several serine and threonine residues in p6 in purified virus preparations using mass spectrometry. Mutation of individual candidate phosphoacceptor residues had no detectable effect on virus assembly, release, and infectivity, however, suggesting that phosphorylation of single residues may not be functionally relevant. Therefore, a comprehensive mutational analysis was conducted changing all potentially phosphorylatable amino acids in p6, except for a threonine that is part of an essential peptide motif. To avoid confounding changes in the overlapping pol reading frame, mutagenesis was performed in a provirus with genetically uncoupled gag and pol reading frames. An HIV-1 derivative carrying 12 amino acid changes in its p6 region, abolishing all but one potential phosphoacceptor site, showed no impairment of Gag assembly and virus release and displayed only very subtle deficiencies in viral infectivity in T-cell lines and primary lymphocytes. All mutations were stable over 2 weeks of culture in primary cells. Based on these findings, we conclude that phosphorylation of p6 is dispensable for HIV-1 assembly, release, and infectivity in tissue culture.
authors.affiliation: Department of Infectious Diseases, Virology, University of Heidelberg, Heidelberg, Germany.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Radestock
authors.forename: Ivonne
authors.initials: I
authors.lastname: Morales
authors.forename: Sheikh Abdul
authors.initials: SA
authors.lastname: Rahman
authors.forename: Sonja
authors.initials: S
authors.lastname: Radau
authors.forename: Bärbel
authors.initials: B
authors.lastname: Glass
authors.forename: René Peiman
authors.initials: RP
authors.lastname: Zahedi
authors.forename: Barbara
authors.initials: B
authors.lastname: Müller
authors.forename: Hans-Georg
authors.initials: HG
authors.lastname: Kräusslich
mesh_terms: D019943:Amino Acid Substitution; D015497:HIV-1 / Q000502:physiology*; D006367:HeLa Cells; D006801:Humans; D013058:Mass Spectrometry; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D012694:Serine / Q000378:metabolism; D013912:Threonine / Q000378:metabolism; D019065:Virus Assembly; D057074:Virus Release; D014779:Virus Replication; D054301:gag Gene Products, Human Immunodeficiency Virus / Q000737:chemistry / Q000378:metabolism*
chemical_list: D054301:gag Gene Products, Human Immunodeficiency Virus; C083002:p6 gag protein, Human immunodeficiency virus 1; D013912:Threonine; D012694:Serine