Browsing by Author "Dahal, Bibha"

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    PERK Is Critical for Alphavirus Nonstructural Protein Translation
    Dahal, Bibha; Lehman, Caitlin W.; Akhrymuk, Ivan V.; Bracci, Nicole R.; Panny, Lauren; Barrera, Michael D.; Bhalla, Nishank; Jacobs, Jonathan L.; Dinman, Jonathan D.; Kehn-Hall, Kylene (MDPI, 2021-05-12)
    Venezuelan equine encephalitis virus (VEEV) is an alphavirus that causes encephalitis. Previous work indicated that VEEV infection induced early growth response 1 (EGR1) expression, leading to cell death via the protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK) arm of the unfolded protein response (UPR) pathway. Loss of PERK prevented EGR1 induction and decreased VEEV-induced death. The results presented within show that loss of PERK in human primary astrocytes dramatically reduced VEEV and eastern equine encephalitis virus (EEEV) infectious titers by 4–5 log10. Loss of PERK also suppressed VEEV replication in primary human pericytes and human umbilical vein endothelial cells, but it had no impact on VEEV replication in transformed U87MG and 293T cells. A significant reduction in VEEV RNA levels was observed as early as 3 h post-infection, but viral entry assays indicated that the loss of PERK minimally impacted VEEV entry. In contrast, the loss of PERK resulted in a dramatic reduction in viral nonstructural protein translation and negative-strand viral RNA production. The loss of PERK also reduced the production of Rift Valley fever virus and Zika virus infectious titers. These data indicate that PERK is an essential factor for the translation of alphavirus nonstructural proteins and impacts multiple RNA viruses, making it an exciting target for antiviral development.
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    Protein Kinase C subtype delta interacts with Venezuelan equine encephalitis virus capsid protein and regulates viral RNA binding through modulation of capsid phosphorylation
    Carey, Brian D.; Akhrymuk, Ivan V.; Dahal, Bibha; Pinkham, Chelsea L.; Bracci, Nicole R.; Finstuen-Magro, Sarah; Lin, Shih-Chao; Lehman, Caitlin W.; Sokoloski, Kevin J.; Kehn-Hall, Kylene (PLoS, 2020-03-01)
    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.