Expression of SARS-CoV‑2 Nonstructural Proteins 3 and 4 Can Tune the Unfolded Protein Response in Cell Culture

Coronaviruses
(CoV), including SARS-CoV-2, modulate host proteostasis
through the activation of stress-responsive signaling pathways such
as the Unfolded Protein Response (UPR), which remedies misfolded protein
accumulation by attenuating translation and increasing protein folding
capacity. While CoV nonstructural proteins (nsps) are essential for
infection, little is known about the role of nsps in modulating the
UPR. We characterized the impact of overexpression of SARS-CoV-2 nsp4,
a key driver of replication, on the UPR in cell culture using quantitative
proteomics to sensitively detect pathway-wide upregulation of effector
proteins. We find that nsp4 preferentially activates the ATF6 and
PERK branches of the UPR. Previously, we found that an N-terminal
truncation of nsp3 (nsp3.1) can suppress pharmacological ATF6 activation.
To determine how nsp3.1 and nsp4 tune the UPR, their coexpression
demonstrated that nsp3.1 suppresses nsp4-mediated PERK, but not ATF6
activation. Reanalysis of SARS-CoV-2 infection proteomics data revealed
time-dependent activation of PERK targets early in infection, which
subsequently fades. This temporal regulation suggests a role for nsp3
and nsp4 in tuning the PERK pathway to attenuate host translation
beneficial for viral replication while avoiding later apoptotic signaling
caused by chronic activation. This work furthers our understanding
of CoV-host proteostasis interactions and highlights the power of
proteomic methods for systems-level analysis of the UPR.