To obtain structural based information on Gag phospho rylation on Ser487 and how it affects the interaction of Gag with Alix or Vpr, we conducted computer assisted molecular modeling of the Gag p6 domain inhibitor Pazopanib coupled with Inhibitors,Modulators,Libraries peptides derived from either Alix or Vpr. The models con structed in this study included unphosphorylated and phosphorylated Gag p6, and its SerAla substituted mutant on Ser487. Mo lecular modeling calculations with thermodynamically op timized three dimensional structures Inhibitors,Modulators,Libraries 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 Alix was not affected by the phosphorylation or SerAla substitution of Gag Ser487.

On the other hands, the binding of Gag p6 with Vpr was facilitated since the phosphorylation of Ser487 can create another hydrogen Inhibitors,Modulators,Libraries bond between Gag p6 and Vpr. The Ser487 was predicted to form no hydro gen bonds with Vpr in non phosphorylated state, whereas the phosphorylated Ser487 could form the hydrogen bond with Gln44 of Vpr. Consequently, binding energy calculated with Molecular Operating Environment was signifi cantly increased by phosphorylation of Ser487 only for the Gag p6 Vpr complex. These data suggest that the phosphorylation of Gag p6 on Ser487 could indeed affect the binding affinity of Gag p6 with Vpr but not Alix. Based on our structural modeling results, we next asked whether the phosphorylation of Gag at Ser487 has any effect on the interaction between Vpr and Gag.

We have selected Bimolecular Fluorescence Complementa tion system to quantify the Vpr Gag interaction in live cells as previously reported. Plasmids encoding C terminally KGC tagged Gag and N terminally KGN tagged Vpr were transfected and evaluated for BiFC Inhibitors,Modulators,Libraries signal by flow cytometry. Flow cytometry analysis revealed that the interaction Inhibitors,Modulators,Libraries of Vpr with Gag Ser487Ala mutant was reduced as com pared with wild type Gag. To further assess whether the phosphorylation of Gag at Ser487 provides another hydrogen bond with Vpr Gln44 to facilitate Gag Vpr interaction, we constructed Vpr Q44E mutant for BiFC analysis. Results demonstrated that Vpr Q44E mu tant exhibited weker interactions to Gag and Gag S487A as compared with wild type Vpr.

We further found that aPKC inhibitor Erlotinib CAS suppressed the interaction bet ween Gag Flag and HA Vpr in imunoprecipitation ana lysis. The phosphorylation of Gag at Ser487 affects Vpr incorporation into virions and viral infectivity We next examined whether the phosphorylation of Gag at Ser487 has any effects on the incorporation of Vpr into HIV 1 virus like particles. As shown in Figure 4B, we found no distinct changes in the incorporation of Alix into VLPs regardless of a SerAla substitution at Gag Ser487 in 293T cells.