Mechanistic insights into the inhibition of stress granule formation by a viral protein

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. However, the significance of SG modulation during virus infection is 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, I 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. I 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.Medicine, Faculty ofBiochemistry and Molecular Biology, Department ofGraduat

Targeting Nup358/RanBP2 by a viral protein disrupts stress granule formation.

Viruses have evolved mechanisms to modulate cellular pathways to facilitate infection. One such pathway is the formation of stress granules (SG), which are ribonucleoprotein complexes that assemble during translation inhibition following cellular stress. Inhibition of SG assembly has been observed under numerous virus infections across species, suggesting a conserved fundamental viral strategy. However, the significance of SG modulation during virus infection is not fully understood. The 1A protein encoded by the model dicistrovirus, Cricket paralysis virus (CrPV), is a multifunctional protein that can bind to and degrade Ago-2 in an E3 ubiquitin ligase-dependent manner to block the antiviral RNA interference pathway and inhibit SG formation. Moreover, the R146 residue of 1A is necessary for SG inhibition and CrPV infection in both Drosophila S2 cells and adult flies. Here, we uncoupled CrPV-1A's functions and provide insight into its underlying mechanism for SG inhibition. CrPV-1A mediated inhibition of SGs requires the E3 ubiquitin-ligase binding domain and the R146 residue, but not the Ago-2 binding domain. Wild-type but not mutant CrPV-1A R146A localizes to the nuclear membrane which correlates with nuclear enrichment of poly(A)+ RNA. Transcriptome changes in CrPV-infected cells are dependent on the R146 residue. Finally, Nup358/RanBP2 is targeted and degraded in CrPV-infected cells in an R146-dependent manner and the depletion of Nup358 blocks SG formation. We propose that CrPV utilizes a multiprong strategy whereby the CrPV-1A protein interferes with a nuclear event that contributes to SG inhibition in order to promote infection