Key residues in SARS-CoV-2 NSP3 hyper variable region are necessary to modulate early stress granule activity.

Antagonism of the host responses that limits viral replication is critical to the success of infection. Recently, we identified that the hypervariable region (HVR) of SARS-CoV-2 NSP3 binds to FXR1 and disrupts stress granule formation during the early stages of infection. Despite variation across the rest of the HVR, a 20-amino acid region, highly conserved in the Sarbecovirus family, is required for NSP3-FXR1 binding, but the critical residues remained unresolved. In this study, we explore the individual residues in NSP3 driving FXR1 binding and determine their impact on viral replication, pathogenesis, and stress granule formation. Our results indicate that the tyrosine at position 138 (Y138) and a phenylalanine at position 145 (F145) are required for FXR1 binding and affinity. Using reverse genetics, we showed mutating NSP3 Y138A/F145A (YF mutant) reduced viral replication in vitro and in vivo. Importantly, we demonstrate that attenuation is not due to differential type I interferon responses, but rather loss of stress granule control by the NSP3 mutant as compared to WT. Together, our findings demonstrate the importance of Y138 and F145 within the NSP3-HVR in regulating stress granule formation at the early times post infection.