Chemical modulation of the unfolded protein response reveals an antiviral role for the PERK pathway in human coronavirus 229E infection

Broad spectrum antivirals are critical to respond rapidly to the threat posed by newly emerging RNA viruses. One potential candidate is the natural compound thapsigargin (Tg). Tg potently induces endoplasmic reticulum (ER) stress and activates the unfolded protein response (UPR). Recent studies have demonstrated that Tg has robust antiviral activity against several human coronaviruses (CoVs), including SARS-CoV-2, although the specific antiviral mechanism(s) have remained unclear. Here, we aimed to characterize the role of the UPR in the antiviral activity of Tg against HCoV-229E, a model common cold CoV. Consistent with previous findings, we show that a short 30-minute priming of A549 cells with Tg potently inhibits HCoV-229E infection. Time-of-addition assays showed that Tg is most effective when added up to 8 hours post-infection. Furthermore, Tg inhibits the accumulation of double-stranded RNA in infected cells, suggesting that Tg inhibits early stages of viral RNA replication. Using selective UPR pathway inhibitors to narrow down the role of these pathways in mediating the antiviral effect of Tg, we show that the inhibition of IRE1 or ATF6 does not impair the ability of Tg to inhibit HCoV-229E infection. The use of stable knockdown A549 cells in which IRE1, PERK, or ATF6 expression was silenced further revealed that the antiviral activity of Tg is not dependent on the expression of any of the three UPR sensors individually. However, HCoV-229E replication is inhibited in A549-shIRE1 cells, or in cells treated with the IRE1 inhibitor (KIRA6), suggesting that IRE1 activation may play a pro-viral role during HCoV-229E infection. Selective UPR pathway activators were used to further probe down the role of each pathway during HCoV-229E infection. Selective activation of the PERK pathway, but not IRE1 or ATF6 pathways, inhibits HCoV-229E infection. Lastly, to more broadly test the antiviral role of PERK against CoV RNA replication, we used BHK-21 cells that stably express a SARS-CoV-2 replicon. We show that selective PERK activation robustly inhibits SARS-CoV-2 replication, comparable to Tg. Overall, these findings provide insight into the antiviral mechanism(s) of Tg against CoV infection and demonstrate that modulation of the UPR may be exploited as an antiviral strategy.