UBC Theses and Dissertations
Multiple viral protein genome linked proteins in dicistrovirus infection Warsaba, Reid
Viral protein genome-linked (VPg) protein plays an essential role in protein-primed replication of positive-sense single-stranded RNA viruses. VPg is covalently linked to the 5’ end of the viral RNA genome via a phosphodiester bond typically at a conserved amino acid. Whereas most viruses have a single VPg, some viruses encode multiple VPgs that are proposed to have redundant yet undefined roles in viral replication. Here, we use the dicistrovirus, Cricket paralysis virus (CrPV), which encodes four non-identical copies of VPg, as a model to characterize the role of VPg copies in infection. Dicistroviruses encode two main open reading frames (ORFs) that are driven by distinct internal ribosome entry sites (IRESs). We systematically generated single and combinatorial deletions and mutations of VPg1-4 within the CrPV infectious clone and monitored viral yield in Drosophila S2 cells. Deletion of one to three VPg copies progressively decreased viral yield and delayed viral replication, suggesting a threshold number of VPgs for productive infection. Mass spectrometry analysis of CrPV VPg-linked RNAs revealed viral RNA linkage to either a serine or threonine in VPg, from which mutations in all VPgs attenuated infection. Mutating serine 4 in a single VPg abolished viral infection, indicating a dominant-negative effect. I hypothesized that the multiple VPgs in dicistroviruses compensate for the relatively weaker translation of the non-structural proteins compared to the structural proteins was tested using minigenome reporter constructs. Viral minigenome reporters that monitor dicistrovirus 5’ untranslated region (UTR) and IRES translation revealed a relationship between VPg copy number and the ratio of IGR IRES:5’ UTR IRES translation. We uncover a novel viral strategy whereby VPg copies in dicistrovirus genomes compensate for the relative IRES translation efficiencies to promote virus infection. We also performed a bioinformatic analysis of VPgs from the family Dicistroviridae and find many novel dicistroviruses containing repeated VPgs, up to eight copies and find that the VPg type but not the number correlates with the RdRp evolution of Dicistroviruses.
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