SARS-CoV-2 Nonstructural Proteins 3 and 4 tune the Unfolded Protein Response.

Coronaviruses (CoV), including SARS-CoV-2, modulate host proteostasis pathways during infection through activation of stress-responsive signaling pathways such as the Unfolded Protein Response (UPR). The UPR regulates protein translation, increases protein folding capacity and enhances endoplasmic reticulum (ER) biogenesis to alleviate ER stress caused by accumulation of misfolded proteins. CoVs depend on host machinery to generate large amounts of viral protein and manipulate ER-derived membranes to form double-membrane vesicles (DMVs), which serve as replication sites, making the UPR a key host pathway for CoVs to hijack. Despite the importance of CoV nonstructural proteins (nsps) in mediating replication, little is known about the role of nsps in modulating the UPR. We characterized the impact of SARS-CoV-2 nsp4, which is a key driver of DMV formation, on the UPR using quantitative proteomics. We find nsp4 preferentially activates the ATF6 and PERK branches of the UPR. Previously, we found an N-terminal truncation of nsp3 (nsp3.1) can suppress pharmacological activation of the ATF6 pathway. To determine how nsp3.1 and nsp4 might tune the UPR in concert, both proteins were co-expressed demonstrating that nsp3.1 does not suppress nsp4-mediated ATF6 activation but does suppress PERK activation. A meta-analysis of SARS-CoV-2 infection proteomics data reveals a time-dependent activation of PERK protein markers early in infection, which subsequently fades. This temporal regulation suggests a role for nsps tuning the PERK pathway to attenuate host translation beneficial for viral replication while avoiding later apoptotic signaling caused by chronic PERK activation. This work furthers our understanding of CoV-host proteostasis interactions and identifies potential areas to target for anti-viral therapies.