The results revealed that the aPKC inhibitor may strongly suppressed the replication of both viruses in a dose dependent manner, although there was no obvious toxicity or growth inhibition in these Inhibitors,Modulators,Libraries cells. Taken together, these results indicate that the phosphorylation of Gag by aPKC regulates Vpr incor poration and HIV 1 replication in macrophages. Discussion We here demonstrate that aPKC is a crucial regulator of HIV 1 infection via the phosphorylation of Gag p6 which enhances the incorporation of Vpr into virions. Our cur rent data strongly suggest that Ser487 is the specific phos phorylation site on HIV 1 Gag for aPKC and is crucial for the Gag p6 Vpr interaction that leads to Vpr incor poration into viral particles. Furthermore, our current data demonstrate that an aPKC inhibitor prominently inhibits HIV 1 replication in primary human macrophages.

Hence, the phosphorylation Inhibitors,Modulators,Libraries of Gag by aPKC may well be an important mechanism through which HIV 1 efficiently in fects macrophages and by which an excessive accumula tion of the cytotoxic Vpr protein in the host infected cells is prevented. The Gag p6 domain has been identified as the pre dominant site of phosphorylation in HIV 1 particles. Ser487 is a highly conserved residue in this p6 domain among various HIV 1 strains, suggesting that the phosphorylation of this residue is of fundamental functional importance. Votteler et al. have demonstrated that a HIV 1 Gag mutant with a deleted PTAP region and a phenylalanine substitution shows aberrant core formation and reduced viral infectivity in TZM b1 cells.

More recently, steady state affinity analysis using a surface plasmon resonance sensorgram has revealed that the phosphorylated form of Inhibitors,Modulators,Libraries p6 at Ser487 has a stable binding affinity Inhibitors,Modulators,Libraries for cyto plasmic membranes. These reports have therefore revealed that Gag Ser487 is a highly conserved phosphor ylation site of likely crucial importance for HIV 1 infec tion. On the other hand, Radestock et al. recently reported in tissue culture experiments that the phosphorylation of Gag p6 including Ser487 is dispensable for HIV 1 infecti vity. These authors showed that asparagine substitutions at five serine residues within the C terminus of Gag p6 produced no im pairment of Gag assembly or virus release and caused only very subtle deficiencies in viral infectivity in T cell lines and in primary lymphocytes.

These discrepancies may be due to different experimental approaches using different Gag substitution mutants as well as different cell types. In contrast, our present approach is distinct from these earlier studies as we initially attempted to identify the kinases responsible for Gag p6 phosphorylation and Inhibitors,Modulators,Libraries then explore their role in HIV 1 replication. Our current results clearly demonstrate that sellckchem aPKC phosphorylates Gag p6 and regulates the interaction of Gag with Vpr for the incorporation of Vpr into virus particles.