Comprehensive Mutational Analysis Reveals p6 ^Gag Phosphorylation To Be Dispensable for HIV-1 Morphogenesis and Replication

Abstract

bThe 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.

The Nucleocapsid Domain of Gag Is Dispensable for Actin Incorporation into HIV-1 and for Association of Viral Budding Sites with Cortical F-Actin

“…3B; see also Movie S2 in the supplemental material), while distinct individual assemblies remained in the case of the wild-type constructs throughout the observation period (see Movie S1 in the supplemental material). Pronounced coalescence of individual buds was also not observed in our previous live-cell analyses of HIV-1 assembly (24,38,42). The formation of large fluorescent Gag patches was in agreement with extended electron-dense regions resembling the Gag lattice and found adjacent to or in direct connection with distinct virus budding sites upon cET of U-373 MG cells transduced with AdGag(LZ) (Fig.…”

“…3B; see also Movies S1 and S2 in the supplemental material). Kinetic analysis of the early exponential phase of Gag.eGFP accumulation at individual assembly sites confirmed that the assembly rate for the uncoupled construct (k ϭ 0.0057 Ϯ 0.0035 s Ϫ1 ) was similar to that previously determined for wildtype HIV-1 (0.0043 Ϯ 0.00054 s Ϫ1 ) (24). Analysis of cells expressing fluorescent wild-type Gag or Gag(LZ) revealed the appearance of very similar punctae following exponential kinetics in an early phase of expression in both cases.…”

“…Replacing the NC domain of Gag with LZ in the context of the viral genome (rather than in a vector expressing only Gag) would destroy the Gag-Pol translational frameshift signal and alter the overlapping pol reading frame, which could affect assembly kinetics as well. We therefore made use of pCHIVunc eGFP (24), a variant of pCHIV eGFP in which the gag and pol reading frames are genetically uncoupled. This allows alteration of the 3= region of gag without affecting frameshifting efficiency or the pol open reading frame (ORF) (24).…”

“…We therefore made use of pCHIVunc eGFP (24), a variant of pCHIV eGFP in which the gag and pol reading frames are genetically uncoupled. This allows alteration of the 3= region of gag without affecting frameshifting efficiency or the pol open reading frame (ORF) (24). Replacement of NC by LZ in this context yielded plasmids pCHIVunc(LZ) and pCHIVunc(LZ) eGFP , respectively.…”

“…Plasmids pCHIVunc(LZ) and pCHIVunc (LZ) eGFP are based on the non-replication-competent plasmid pCHIV (23). A synthetic BstEII-XmaI gene fragment encoding the GCN4 leucine zipper sequence (Geneart AG, Regensburg, Germany) was used to replace the corresponding BstEII-XmaI fragment (encoding the NC region) of the infectious proviral construct pNL43(unc) (24). From this, non-replication-competent derivatives were generated by cloning an SwaI fragment from pNL4-3unc(LZ) into pCHIV or pCHIV eGFP .…”