The phosphorylation of Gag at Ser487 promotes the incorporation of Vpr into HIV-1 virions. (A) Bimolecular fluorescence complementation (BiFC) of 293T cells expressing Gag-KGC and KGN-Vpr (upper panel). 293T cells were transfected with KGN-Vpr (WT or Q44E) and Gag-KGC or Gag (S487A)-KGC, were harvested and analyzed by flow cytometry to determine the BiFC signal at 48 hours post-transfection. Results represent the means of three independent experiments. The cells were harvested and subjected to western blot analysis. (B) The phosphorylation of Gag at Ser487 promotes interaction of Vpr with Gag. HA-tagged Vpr was co-transfected with Flag tagged Gag into 293T cells in presence of 5 μM aPKC inhibitor or DMSO, followed by pulldown with Flag-beads and immunoblotting with indicated antibodies. Bottom panel shows input for pulldown assay. (C) Differential effect of Gag Ser487 phosphorylation on Alix and Vpr incorporation into VLPs. 293T cells were transfected with GFP-tagged Gag (Gag-WT) or with the Ser487 point mutated Gag (Gag-S487A), with either HA-tagged Alix, or HA-tagged Vpr expression vectors. Cells and VLPs were collected at 24 hours post-transfection, and their protein extracts were resolved using SDS-PAGE and subjected to western blot analysis. (D) aPKC-mediated Gag phosphorylation at Ser487 facilitates Vpr incorporation into virions. 293T cells were transiently transfected with Gag-Pol, either Flag-tagged Vpr (WT) or Gln44 point mutated Vpr (Q44E), and with either V5-tagged aPKC (WT) or kinase-negative aPKC (Kn). Cell lysates and VLPs were collected 24 hours post-transfection and subjected to western blot analysis. (E) The phosphorylation of Gag-pol-Ser487 is required for Vpr incorporation into HIV-1 virions. 293T cells were transfected with Flag-tagged Vpr and either with Gag-Pol (WT) or with the Ser487 point mutated Gag-Pol (S487A). The transfected 293T cells were treated with DMSO or aPKC inhibitor for 24 hours. Cell lysates and VLPs were collected and subjected to western blot analysis.