CHARACTERIZING THE MODULATION OF STRESS GRANULE RESPONSES BY CORONAVIRUSES

Abstract

Coronaviruses (CoVs) are a family of viruses that circulate in human populations causing mild common colds as well as severe and fatal respiratory disease. Stress granules (SGs) are cytoplasmic condensates that often form as part of the cellular antiviral response. Despite growing interest in SGs and other biological condensates, the role of SG formation during CoV infection is poorly understood. In this work, I analyzed SG formation during infection of the human common cold CoV OC43. Little SG formation was observed in infected cells and OC43 inhibited SG formation induced by exogenous stress (e.g. sodium arsenite or silvestrol treatment). Furthermore, OC43 inhibited phosphorylation of eukaryotic initiation factor 2α (eIF2α) – the hallmark of translation inhibition induced by integrated stress response (ISR). Inhibition of eIF2α phosphorylation by OC43 was unaffected by treatment with the integrated stress response inhibitor (ISRIB) or knockdown of GADD34, suggesting that OC43 can limit eIF2α phosphorylation independently from activation of the ISR or upregulation of GADD34. Further, GADD34 knockdown decreased OC43 replication. Upon ectopic overexpression, the nucleocapsid (N) and non-structural protein 1 (Nsp1) from both OC43 and the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) inhibited SG formation. Furthermore, SARS-CoV2 Nsp1 expression resulted in depletion of G3BP1 protein – the master regulator of SG formation. This phenotype was dependent on Nsp1-mediated depletion of cytoplasmic mRNAs, which was also associated with nuclear retention of TIAR, another SG-nucleating protein. To test the role of G3BP1 in CoV replication, we infected cells overexpressing EGFP-tagged G3BP1 with OC43 and observed a significant decrease in infection compared to parental or control cells expressing EGFP. The antiviral role of G3BP1 and the existence of multiple SG suppression mechanisms that are conserved between the common cold OC43 and the pandemic SARS-CoV2 suggests that SG formation may represent an important antiviral host defense that CoVs target to ensure efficient replication.