Betacoronaviruses Differentially Activate the Integrated Stress Response to Optimize Viral Replication in Lung Derived Cell Lines

Abstract

The betacoronavirus genus contains five of the seven human viruses, making it a particularly critical area of research to prepare for future viral emergence. We utilized three human betacoronaviruses, one from each subgenus-HCoV-OC43 (embecovirus), SARS-CoV-2 (sarbecovirus) and MERS-CoV (merbecovirus)- to study betacoronavirus interaction with the PKR-like ER kinase (PERK) pathway of the integrated stress response (ISR)/unfolded protein response (UPR). The PERK pathway becomes activated by an abundance of unfolded proteins within the endoplasmic reticulum (ER), leading to phosphorylation of eIF2 and translational attenuation in lung derived cell lines. We demonstrate that MERS-CoV, HCoV-OC43, and SARS-CoV-2 all activate PERK and induce responses downstream of p-eIF2, while only SARS-CoV-2 induces detectable p-eIF2 during infection. Using a small molecule inhibitor of eIF2 dephosphorylation, we provide evidence that MERS-CoV and HCoV-OC43 maximize replication through p-eIF2 dephosphorylation. Interestingly, genetic ablation of GADD34 expression, an inducible phosphatase 1 (PP1)-interacting partner targeting eIF2 for dephosphorylation, did not significantly alter HCoV-OC43 or SARS-CoV-2 replication, while siRNA knockdown of the constitutive PP1 partner, CReP, dramatically reduced HCoV-OC43 replication. Combining growth arrest and DNA damage-inducible protein (GADD34) knockout with peripheral ER membrane-targeted protein (CReP) knockdown had the maximum impact on HCoV-OC43 replication, while SARS-CoV-2 replication was unaffected. Overall, we conclude that eIF2 dephosphorylation is critical for efficient protein production and replication during MERS-CoV and HCoV-OC43 infection. SARS-CoV-2, however, appears to be insensitive to p-eIF2 and, during infection, may even downregulate dephosphorylation to limit host translation. IMPORTANCELethal human betacoronaviruses have emerged three times in the last two decades, causing two epidemics and a pandemic. Here, we demonstrate differences in how these viruses interact with cellular translational control mechanisms. Utilizing inhibitory compounds and genetic ablation, we demonstrate that MERS-CoV and HCoV-OC43 benefit from keeping p-eIF2 levels low to maintain high rates of virus translation while SARS-CoV-2 tolerates high levels of p-eIF2. We utilized a PP1:GADD34/CReP inhibitor, GADD34 KO cells, and CReP-targeting siRNA to investigate the therapeutic potential of these pathways. While ineffective for SARS-CoV-2, we found that HCoV-OC43 seems to primarily utilize CReP to limit p-eIF2a accumulation. This work highlights the need to consider differences amongst these viruses, which may inform the development of host-directed pan-coronavirus therapeutics.