Re-visiting the functional Relevance of the highly conserved Serine 40 Residue within HIV-1 p6(Gag).

BACKGROUND: HIV-1 formation is driven by the viral structural polyprotein Gag, which assembles at the plasma membrane into a hexagonal lattice. The C-terminal p6(Gag) domain harbors short peptide motifs, called late domains, which recruit the cellular endosomal sorting complex required for transport and promote HIV-1 abscission from the plasma membrane. Similar to late domain containing proteins of other viruses, HIV-1 p6 is phosphorylated at multiple residues, including a highly conserved serine at position 40. Previously published studies showed that an S40F exchange in p6(Gag) severely affected virus infectivity, while we had reported that mutation of all phosphorylatable residues in p6(Gag) had only minor effects. FINDINGS: We introduced mutations into p6(Gag) without affecting the overlapping pol reading frame by using an HIV-1 derivative where gag and pol are genetically uncoupled. HIV-1 derivatives with a conservative S40N or a non-conservative S40F exchange were produced. The S40F substitution severely affected virus maturation and infectivity as reported before, while the S40N exchange caused no functional defects and the variant was fully infectious in T-cell lines and primary T-cells. CONCLUSIONS: An HIV-1 variant carrying a conservative S40N exchange in p6(Gag) is fully functional in tissue culture demonstrating that neither S40 nor its phosphorylation are required for HIV-1 release and maturation. The phenotype of the S40F mutation appears to be caused by the bulky hydrophobic residue introduced into a flexible region.

Comprehensive mutational analysis reveals p6Gag phosphorylation to be dispensable for HIV-1 morphogenesis and replication.

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.