PacificCan announces over $11.1 million in funding for two projects led by Dr. Leonard Foster and Dr. Natalie Strynadka

Jibin Sadasivan

Title: When proteins go viral: Investigation on how a viral protein impairs stress granule formation  

Abstract: Stress granules (SG) are ribonucleoprotein aggregates that accumulate during cellular stress when translation is limited. Inhibition of SG assembly has been observed under virus infection across species, suggesting a conserved fundamental viral strategy. How this occurs and why this would benefit virus infection are not fully understood. The 1A protein encoded by the model dicistrovirus, cricket paralysis virus (CrPV), is a multifunctional viral protein that can inhibit SG formation and bind to and degrade Argonaute-2 (Ago-2) in an E3 ubiquitin ligase-dependent manner to block the antiviral RNA interference pathway. Moreover, the R146 residue at the C terminus of 1A is necessary for virus infection in Drosophila S2 cells and flies. Here, we uncouple CrPV-1A’s functions and provide insights into its underlying mechanism for SG inhibition. CrPV-1A’s ability to inhibit SG formation does not require the Ago-2 binding domain but does require the E3 ubiquitin ligase binding domain. Overexpression and infection studies in Drosophila and human cells showed that wild-type CrPV-1A but not mutant R146A CrPV-1A localizes to the nuclear membrane, which correlates with nuclear enrichment of poly(A)+ RNA. Transcriptome analysis demonstrated that a single R146A mutation dramatically dampens host transcriptome changes in CrPV-infected cells. Finally, inhibition of SG formation by CrPV-1A requires Ranbp2/Nup358 in an R146-dependent manner. Wse propose that CrPV utilizes a multifaceted strategy for productive virus infection whereby the CrPV-1A protein interferes with a nuclear event that contributes to the suppression of SG assembly.

Reid Warsaba

Title: Multiple Viral Protein Genome Linked Proteins in Dicistrovirus Infection 

Abstract: 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. Using 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 Dicistrovirus VPgs and find many novel Dicistroviruses with repeated VPgs, up to eight copies and find that the VPg type but not the number correlates with the RdRp evolution of Dicistroviruses. 

Monday, October 03, 2022 at 2:30 pm – 3:30 pm at LSC 3 and Zoom

Hosted by: Dr. Eric Jan