ABSTRACT
Ras-GTPase-activating protein SH3-domain-binding proteins (G3BP) are multifunctional RNA-binding proteins, pivotal in the initiation of stress granules (SGs). SARS-CoV-2 nucleocapsid (N) protein exhibits strong binding affinity for G3BP and inhibition of SG formation. However, pro-viral role(s) of the G3BP-N interaction have remained unclear. Here, we have comprehensively examined the importance of G3BP for SARS-CoV-2 infection both in vitro and in vivo. Using reverse genetics, we constructed a viral mutant, SARS-CoV-2 RATA, which exhibits stronger and more persistent SG response in infected cells. We also show that in SARS-CoV-2 infected cells, G3BP-N complexes are targeted to the pore complex of double membrane vesicles (DMV) from which nascent viral RNA emerges. Furthermore, through interaction with 40S ribosomal subunits, G3BP-N complexes promote highly localized translation of viral mRNAs at the viral factories and thus facilitate viral gene expression and replication. This work provides a mechanistic understanding of the roles of G3BP in SARS-CoV-2 infection.