# Human Betacoronavirus OC43 Interferes with the Integrated Stress Response Pathway in Infected Cells
## 3 Results  
The Targeting Subunit of eIF2\(\alpha\) Holophophasatase GADD34 Is Strongly Upresulated in OC43-Infected Cells  
Our previously published analyses of eIF2\(\alpha\) phosphorylation in OC43-infected cells showed that at 24 and 48 h post-infection (hpi), OC43 causes a small but consistent increase in p-eIF2\(\alpha\) levels [29]. Therefore, to begin characterizing the effects of the virus on ISR, we wanted to test the activation status of host factors upstream and downstream of phospho-eIF2\(\alpha\) in the pathway. Among the eIF2\(\alpha\) kinases, we focused on PKR because of its primary function in recognition of viral dsRNA replication intermediates, and PERK, because coronaviruses are known to induce ER stress [33; 34]. We infected 293A cells with OC43 at a multiplicity of infection (MOI) = 1.0 and analyzed the phosphorylation statuses of PKR, PERK, and eIF2\(\alpha\) at 24 hpi using Western blotting. For positive controls, we used transfection with polyinosinic-polyctidylic acid (poly(I:C)) dsRNA mimic for PKR activation and treatment with the sarcoendoplasmic reticulum calcium ATPase (SERCA) inhibitor thapsigargin (Tg) for the induction of ER stress and the activation of PERK (Figure 1A,B). In both mock- and OC43-infected cells, poly(IC) transfection induced the activation of PKR (as measured by phosphorylation at Threonine-451) and strong eIF2\(\alpha\) phosphorylation, while no phospho-PKR signal could be detected in untransfected mock- or OC43-infected cells (Figure 1A). These results indicate that in our infection model, OC43 successfully blocks the detection of its dsRNA molecules by PKR. By contrast, virus infection caused the activation of PERK (as indicated by the appearance of an upstream shifted band corresponding to phosphorylated PERK), similar to the activation observed in mock-infected cells upon 1 h Tg treatment (Figure 1B, lanes 1-3). As expected, 4 h Tg treatment led to the induction of ATF4 and GADD34 and decreased phospho-eIF2\(\alpha\) in both mock- and OC43-infected cells (Figure 1B, lanes 5,6). However, even without Tg treatment, OC43 infection strongly induced GADD34 to levels exceeding those observed after 4 h Tg treatment in mock cells (Figure 1B, compare lanes 2 and 5). To test if GADD34 protein accumulation was caused by increased mRNA levels, we isolated total RNA from mock- and OC43-infected cells and performed a reverse transcription-quantitative polymerase chain reaction (RT-QPCR) analysis of the GADD34 transcript. This analysis showed that, similar to 4 h Tg treatment, OC43 infection caused the strong upregulation of GADD34 mRNA levels (Figure 1C). Control GAPDH transcript was not significantly affected by Tg treatment or infection (Figure 1D). To confirm minimal eIF2\(\alpha\) phosphorylation and strong GADD34 upregulation by OC43 infection in a different cell type, we used Vero cells. Similar to the infection of 293A cells, OC43 caused a small increase in eIF2\(\alpha\) phosphorylation in Vero cells and decreased the maximal levels of phospho-eIF\(\alpha\) upon Tg treatment (Figure 1E). The virus also caused the significant upregulation of GADD34 mRNA levels in Vero cells (Figure 1F,G), confirming that this phenotype is not specific to the 293A infection model. The Activation of the Integrated Stress Response Pathway Is Not Responsible for GADD34 Induction in OC43-Infected Cells  
Next, we wanted to determine whether ISR activation by OC43 was responsible for GADD34 upregulation and, consequently, the inhibition of As-induced eIF2\(\alpha\) phosphorylation. To block phospho-eIF2\(\alpha\)-mediated changes in translation initiation, we treated mock- and OC43-infected cells with ISR inhibitor (ISRIB, [35]). ISRIB does not affect eIF2\(\alpha\) phosphorylation, but blocks its effects on the regeneration of the initiation-competent  
Figure 1: OC43 strongly induces GADD34 expression. 293A cells were infected with OC43 at MOI = 1.0.