The substitution of residues in this domain causes a decrease in the Vpr incorporation levels compared with full length HIV 1 Gag protein, indicating that this conserved region is essential for this process. HIV 1 Vpr is a non structural full read protein that is incorpo rated into the viral particles and possesses several charac teristic features that are known to play important roles in HIV 1 replication and disease progression. Vpr mediates multiple functions, including the nuclear import of the HIV 1 pre integration complex, G2 cell cycle arrest, the transactivation of both viral replication and host genes, and the induction of apoptosis. Vpr interacts with the LXXLF binding domain of Gag p6 and is thereby pack aged into the virus particles.
Virion incorporated Vpr is known to positively regulate the infection of non dividing cells and enhance virus production in macrophages and in resting T cells. However, it remains elusive whether and how Vpr incorporation is indeed regulated. Furthermore, although p6 has been shown to be post translationally modified by phosphorylation, it is unknown whether Inhibitors,Modulators,Libraries this phosphorylation Inhibitors,Modulators,Libraries event has any functional relevance to Vpr incorporation and HIV 1 infectivity. In our current study, we utilized an in vitro high throughput protein protein interaction assay using full length HIV 1 Gag and host protein kinases synthesized by the wheat germ cell free protein production system in an attempt to identify the kinase that directs the phosphorylation Inhibitors,Modulators,Libraries of Gag p6 to promote virus replication.
We here report that atypical protein kinase C is a functional interactor of HIV 1 Gag and facilitates viral infectivity by promoting the incorporation of Vpr into virions. Inhibitors,Modulators,Libraries We provide evidence that Gag Ser487 is phosphorylated by aPKC, and that this phosphory lation is essential for p6 Vpr interactions and the re sultant Vpr incorporation within viral particles. Using computer assisted structural modeling, we further ex plore the biological significance of the phosphorylation of Gag p6 Ser487 by aPKC for the physiological inter action between Gag and Vpr. Our current study sheds new light on the molecular link between Gag phospho rylation and viral infectivity through the incorporation of Vpr into virions. Results aPKC binds and phosphorylates HIV 1 Gag Our initial goal was to identify host kinases that phos phorylate the HIV 1 Gag protein.
Because Gag phospho rylation is important for its functional role, we focused on human protein kinases as potential Gag regulators. We synthesized more than 287 full length protein kinases using Inhibitors,Modulators,Libraries a wheat germ cell free protein production system, and screened them for their association with Gag with the amplified selleck bio luminescent proximity homogenous assay. In this method, the extent of the protein protein interaction was measured by assaying the luminescence intensity. Full length Gag and human protein kinases were synthesized using a wheat germ cell free system and subjected to an AlphaScreen assessment.