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

Brian D Carey,Shih-Chao Lin,Bibha Dahal,Kevin J Sokoloski,Caitlin W Lehman,Kylene Kehn-Hall,Nicole Bracci,Ivan Akhrymuk,Sarah Finstuen-Magro,Chelsea L Pinkham,Mark T Heise

doi:10.1371/journal.ppat.1008282

Abstract

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 PKCδ, but not other PKC isoforms and siRNA knockdown of PKCδ caused a decrease in viral replication. Furthermore, knockdown of PKCδ 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 PKCδ mediated capsid phosphorylation regulates viral RNA binding and assembly, significantly impacting viral pathogenesis.

Highlights

Epidemics or epizootics caused by mosquito-borne Venezuelan equine encephalitis virus (VEEV) have resulted in devastating outbreaks of VEE involving equine and human cases in Columbia, Venezuela, and Trinidad since the 1930s [1]
Venezuelan equine encephalitis virus causes flu-like symptoms that can lead to severe encephalitic disease and sometimes death
We have discovered that PKCδ modulates VEEV capsid phosphorylation and that phosphorylation of VEEV capsid is important for viral RNA binding, assembly, and pathogenesis

Summary

Introduction

Epidemics or epizootics caused by mosquito-borne Venezuelan equine encephalitis virus (VEEV) have resulted in devastating outbreaks of VEE involving equine and human cases in Columbia, Venezuela, and Trinidad since the 1930s [1]. The virus causes flulike symptoms including myalgia, fever, fatigue, nausea, and pharyngitis in humans. In up to 14%, of cases, severe neurological complications can occur due to encephalitis, including seizures, confusion, blurred vision and coma. The genome is approximately 11.4kb and is positive sense single stranded RNA encoding two open reading frames. Four non-structural proteins (nsP1-4) are encoded by the first reading frame which begins at the 5’ end of the genome. The second open reading frame is controlled by a 26S promoter on the negative strand and encodes for the structural proteins including capsid, E2, 6K, and E1. A small E3 protein is formed during post-translational modification of the precursor E2 protein [6]

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