Protein Kinase C subtype δ interacts with Venezuelan equine encephalitis virus capsid protein and regulates viral RNA binding through modulation of capsid phosphorylation.

Protein phosphorylation plays an important role during the life cycle of many viruses. Venezuelan equine encephalitis virus (VEEV) capsid protein has recently been shown to be phosphorylated at four residues. Here those studies are extended to determine the kinase responsible for phosphorylation and the importance of capsid phosphorylation during the viral life cycle. Phosphorylation site prediction software suggests that Protein Kinase C (PKC) is responsible for phosphorylation of VEEV capsid. VEEV capsid co-immunoprecipitated with PKC delta, but not other PKC isoforms and siRNA knockdown of PKC delta caused a decrease in viral replication. Furthermore, knockdown of PKC delta by siRNA decreased capsid phosphorylation. A virus with capsid phosphorylation sites mutated to alanine (VEEV CPD) displayed a lower genomic copy to pfu ratio than the parental virus; suggesting more efficient viral assembly and more infectious particles being released. RNA:capsid binding was significantly increased in the mutant virus, confirming these results. Finally, VEEV CPD is attenuated in a mouse model of infection, with mice showing increased survival and decreased clinical signs as compared to mice infected with the parental virus. Collectively our data support a model in which PKC delta mediated capsid phosphorylation regulates viral RNA binding and assembly, significantly impacting viral pathogenesis. Author summary Venezuelan equine encephalitis virus causes flu-like symptoms that can lead to severe encephalitic disease and sometimes death. Understanding the molecular mechanisms of host-virus interactions can aid in the design of intervention strategies in a field that is severely lacking FDA-approved therapeutics and vaccines. The significance of our research is identifying the mechanism behind VEEV capsid phosphorylation and unveiling its importance during infection and disease development. We have discovered that PKC delta modulates VEEV capsid phosphorylation and that phosphorylation of VEEV capsid is important for viral RNA binding, assembly, and pathogenesis. Thus, capsid phosphorylation events could represent a potential target in the development of vaccine design strategies.