# Human Betacoronavirus OC43 Interferes with the Integrated Stress Response Pathway in Infected Cells
## 3 Results
Error bars = standard deviation. Two-way ANOVA and Tukey multiple comparison tests or an unpaired _t_-Test (panel F only) were performed to determine statistical significance (***, _p_-value! 0.0001; ***, _p_-value! 0.001; **, _p_-value! 0.01; ns, non-significant). The principal upstream transcription factor induced upon eIF2\(\alpha\) phosphorylation by PERK is ATF4. To confirm that ATF4 is not responsible for the transcriptional upregulation of GADD34 in OC43-infected cells, we silenced its expression using two lentivirus-driven shRNA constructs. As a non-targeting control, we used shRNA against IL2, the cytokine that is not expressed in epithelial cells. Both shRNA constructs were effective in decreasing Tg-induced ATF4 expression to nearly undetectable levels in uninfected cells, as well as OC43-induced ATF4 expression (Figure 3A). At the same time, neither shRNA affected the induction of GADD34 protein by OC43 infection or Tg treatment (Figure 3A). ATF4 silencing also did not affect OC43 protein accumulation, virus replication, or GADD34 mRNA induction (Figure 3B-D). In a classical ISR pathway, ATF4 induces the CHOP transcription factor, which then induces GADD34. However, GADD34 can be induced by other transcription factors [37; 38; 39], and in addition to PERK, Tg and OC43 can activate other ER stress sensors. One ER stress sensor that is strongly activated by OC43 is inositol-requiring enzyme 1 (IRE1) [33]. IRE1 activation causes the non-canonical cytoplasmic splicing of mRNA-encoding transcription factor X-box binding protein 1 (XBP1), leading to the synthesis of XBP1s, an activator of transcription produced from spliced XBP1 mRNA [40]. Although the IRE1/XBP1s pathway has not been shown to upregulate GADD34, we tested whether IRE1 inhibition would perturb GADD34 expression in OC43-infected cells. As reported previously in another model [33], OC43 infection induced XBP1s in 293A cells (Figure 3E). The addition of specific IRE1 inhibitor 4\(\mu\)8C inhibited XBP1s' induction triggered by OC43 infection or Tg treatment in a concentration-dependent manner (Figure 3E,F), but did not affect GADD34 induction. The PERK Arm of the Unfolded Protein Response Negatively Affects OC43 Protein Synthesis and Replication  
Having determined that OC43 infection activates PERK, we wanted to determine what role PERK plays in virus replication. First, we silenced PERK expression in 293A cells by transduction with the lentiviral-vector-encoding CRISPR/Cas9 system with guide RNA targeting PERK (gRNA-PERK). After 2 days, transduced cells expressed measurably lower PERK levels compared to parental untransduced cells (Figure 4A). When transduced cells were infected with OC43, they accumulated more viral proteins and GADD34 compared to parental cells (Figure 4A, lanes 3,4). Consistent with PERK being an upstream regulator of ATF4 expression, when transduced cells were treated with Tg, they induced lower levels of ATF4 protein compared to parental cells (Figure 4A, lanes 5,6). A similar effect was also observed in OC43-infected cells (Figure 4A, lanes 7,8). This suggests that PERK is not required for GADD34 upregulation by OC43 and that PERK-mediated ISR may have an antiviral role. Indeed, compared to parental cells, gRNA-PERK transduced cells generated significantly higher infectious virus titers (Figure 4B). Figure 3: The upregulation of GADD34 expression by OC43 is ATF4-independent. (**A**–**D**) Cells transduced with lentivirususes encoding shRNAs against ATF4 (shATF4-1 and shATF4-2) or control IL-2 shRNA (shLL2) were infected with OC43 at multiplicity of infection (MOI) = 1.0 for 24 h or were treated with Tg for 4 h. (**A**) Western blot analysis of lysates from cells treated as indicated. (**B**) Production of infectious virions was measured in cell supernatants at 24 hpi. (**C**) Total RNA was isolated from infected cells and levels of OC43 genomic RNA were determined by RT-QPCR. Values were normalized to 18S and expressed as fold changes relative to shLL2 cells.