Although no significant effect of the Gag pol S487A mutant on the Vpr e pression levels neverless in cells was evident, the Vpr incorporation level into VLPs was significantly reduced upon Gag pol S487Ala transfection. Consistent with this result, the incorporation of Vpr into VLPs was significantly reduced in cells treated with the aPKC inhibitor peptide. the Vpr incorporation efficiency was reduced in aPKC inhibitor treated cells. These data indicate that aPKC can enhance the incorporation of Vpr into HIV 1 virions. It has been well established that Vpr incorporation into HIV 1 virions augments viral infectivity in macro phages. We thus assessed whether aPKC affects HIV 1 infectivity by increasing Vpr incorporation into virions.

We hypothesized that if the Gag phos phorylation at Ser487 by aPKC was beneficial for HIV 1 infection in this way, aPKC activity would affect wild type HIV 1 but not a Vpr null virus. To test this, we employed pNL4 3Env luc or pNL4 3EnvVpr luc strains. We then produced the corresponding vi ruses with a fusiogenic envelope G glycoprotein of the vesicular stomatitis virus in the presence or absence of aPKC inhibitor in 293T cells. Im munoblotting analysis of VLP demonstrated that the level of Vpr incorporation was prominently reduced by treatment with the aPKC peptide inhibitor. The infectivity of the generated viruses was tested using the human monocyte macrophage cell line MonoMac6. The aPKC inhibitor treated WT virus e hibited appro i mately 50% less infectivity than the control WT virus. The Vpr null virus showed a 35% reduction in infectivity compared with the WT virus in the Mono Mac6 cells.

However, the primarily low in fectivity of the Vpr null virus was not significantly affected by the aPKC inhibitor. aPKC inhibi tor did not e hibit obvious cytoto ic effect to MonoMac 6 cells. To assess the role of aPKC in multi round HIV 1 replica tion in primary monocyte derived macrophages, we infected these cells with HIV 189. 6, a dual tropic virus, or HIV 1NLAD8, an R5 tropic virus, in conjunction with treatments of various concentrations of the aPKC inhibitor. The results revealed that the aPKC inhibitor strongly suppressed the replication of both viruses in a dose dependent manner, although there was no obvious to icity or growth inhibition in these 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 Cilengitide 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.