To further investigate whether the phosphorylation of HIV 1 Gag at Ser487 is mediated by endogenous aPKC activity, we employed a myristoylated PKC�� pseudosub strate peptide as an aPKC inhibitor. This PKC�� pseu dosubstrate peptide mimics the substrate binding site in PKC�� and PKC��, and suppresses the activity of endogenous PKC�� and PKC��. HIV 1 Gag Pol e pression plasmids were transfected into 293T cells with or without aPKC inhibitor treatment. Immunoblot analysis revealed that the aPKC inhibitor suppressed Gag phosphorylation at Ser487. Subsequent titration analysis demonstrated a dose dependent inhibitory effect of the PKC�� pseudosubstrate peptide by showing an 74. 9% and 70. 4% decrease in Gag phosphorylation at 2 uM and 5 uM doses, respectively.

Note that at these concentrations the aPKC inhibitor did not affect the e pression levels of endogenous aPKC as well as a house keeping protein Vinculin. Fur thermore, cell viability was not prominently affected by aPKC inhibitor when cells were assessed by trypan blue e clusion. Conventional PKC, Akt, CDK and PI3 kinases have been reported previously to affect HIV 1 replication through their phosphory lation of HIV 1 or of host proteins. We thus also investigated using specific inhibitors whether these kinases could mediate the phosphorylation of HIV 1 Gag at Ser487. Our results show that neither PKC nor PKCB specific pseudosubstrates affect Gag phospho rylation at Ser487. Similarly, neither Akt inhibitor, the CDK inhibitor roscovitine nor the PI3K inhibitor wortmannin blocked Gag phosphorylation at Ser487.

Taken together, these observations indicate that aPKC specifically phosphorylates HIV 1 Gag at Ser487 both in vitro and in vivo. The phosphorylation of Gag Ser487 facilitates the interaction between Gag and Vpr HIV 1 Gag p6 contains a late domain consisting of three protein binding motifs, PTAP, LYP nL and C terminal Vpr. Ser487 is located in the Ali binding motif and is also adjacent to the Vpr binding motif spanning amino acids 488 492. To obtain structural based information on Gag phospho rylation on Ser487 and how it affects the interaction of Gag with Ali or Vpr, we conducted computer assisted molecular modeling of the Gag p6 domain coupled with peptides derived from either Ali or Vpr. The models con structed in this study included unphosphorylated and phosphorylated Carfilzomib Gag p6, and its Ser Ala substituted mutant on Ser487.

Mo lecular modeling calculations with thermodynamically op timized three dimensional structures showed less than 1 of positional shifts of C atoms of Gag p6 by phosphory lation, suggesting no obvious difference in the basic struc ture of Gag p6 irrespective of the phosphorylation status. Furthermore, binding interface between Gag p6 and Ali was not affected by the phosphorylation or Ser Ala substitution of Gag Ser487.