# Human Betacoronavirus OC43 Interferes with the Integrated Stress Response Pathway in Infected Cells
## 4 Discussion  
In this work, we examined some of the key aspects of the ISR pathway in 293A cells infected with human common cold virus OC43 (Figure 6). Previously, we used the same infection model to demonstrate that OC43 infection proceeds efficiently in these cells and does not trigger strong eIF2\(\alpha\) phosphorylation or SG formation [29]. Most importantly, OC43-infected cells become less responsive to As, a strong ISR inducer that triggers robust eIF2\(\alpha\) phosphorylation and SG formation in mock- but not in OC43-infected cells. Due to its potency in triggering eIF2\(\alpha\) phosphorylation and translation arrest, As is often used in research labs to induce ISR-dependent SG formation [4]. We probed the response to As in infected cells for the same reason; however, the level of oxidative stress induced by 50 \(\upmu\)M As is not physiological and only serves as a probe to reveal potential eIF2\(\alpha\) phosphorylation  
Figure 5: GADD34 is not responsible for the inhibition of eIF2\(\alpha\) phosphorylation in OC43-infected cells. GADD34 expression was silenced in 293A cells using specific siRNAs (siGADD34#1 and siGADD34#2) or cells were transfected with non-targeting control siRNA (siNT). (**A**,**B**) Western blot analysis of untreated or As-treated mock- or OC43 infected cells performed at 24 hpi. (**C**,**D**) Relative level of eIF2\(\alpha\) phosphorylation was quantified from Western blot images represented in (**A**,**B**). (**E**) Levels of GADD34 transcript were determined by RT-QPCR. (**F**) Immunofluorescence staining of OC43-infected cells pre-treated with the indicated siRNAs. Infected cells at 12, 16, and 20 hpi were visualised using the antibody to OC43 nucleoprotein (OC43, green). Nuclei were stained with Hoechst dye (blue). (**G**) Total RNA was isolated from infected cells at the indicated times post-infection and levels of OC43 genomic RNA were determined by RT-QPCR. Values were normalized to 18S. (**H**) The production of infectious virions was measured in cell supernatants. For all plots, each data point represents an independent biological replicate. Error bars = standard deviation. Two-way ANOVA and Tukey multiple comparisons tests were performed to determine statistical significance (****, \(p\)-value \(<\) 0.0001; ****, \(p\)-value \(<\) 0.001; \(p\)-value \(<\) 0.01; \(p\)-value \(<\) 0.05; ns, non-significant). inhibition. Since coronavirus mRNAs are sensitive to ISR-induced translation arrest [28], they must evolve mechanisms to suppress ISR to ensure robust viral protein synthesis and replication. To characterize the upstream activation of eIF2\(\alpha\) kinases by OC43, we focused on PKR and PERK, the two kinases that are often activated by virus infections [9]. For example, both kinases are activated by severe acute respiratory syndrome coronavirus and chicken gammacoronavirus infectious bronchitis virus [41; 42], and PERK is shown to be activated by a number of different coronaviruses and can be involved in virus-induced cell death and inflammation [28; 34; 43; 44]. In our infection model, we did not detect PKR activation by OC43. Moreover, we did not observe the inhibition of PKR activation by poly(IC) transfection in OC43-infected cells compared to mock. This suggests that OC43 is efficient at avoiding the detection of its dsRNA molecules by PKR by shielding them inside the double membrane replication transcription compartments and/or by the action of the Nsp15 protein [45], and does not actively block PKR activation. By contrast, OC43 infection caused a detectable activation of PERK, consistent with its ability to trigger ER stress in infected cells [33; 46; 47]. Interestingly, PERK activation was previously shown to either inhibit (transmissible gastroenteritis virus, [28]) or promote (porcine epidemic diarrhea virus, [44]) coronavirus replication. In our study, we demonstrate that PERK is an antiviral factor for OC43, as the silencing of PERK expression in 293A cells enhanced viral protein accumulation and infectious virion release.