# Human Betacoronavirus OC43 Interferes with the Integrated Stress Response Pathway in Infected Cells
## 3 Results
(**A**) Western blot analysis of lysates from mock- or OC43-infected cells collected at 24 h post-infection (hpi) with or without poly(I:C) transfection at 22 hpi (2 h poly(I:C) treatment). (**B**) Western blot analysis of lysates from mock- or OC43-infected cells collected at 24 hpi with or without Tg treatment for 1 or 4 h prior to harvesting. (**C**,**D**) Total RNA was isolated from mock- and infected cells treated as indicated and levels of GADD34 (**C**) and GAPDH (**D**) transcripts were determined by RT-QPCR. (**E**–**G**) Vero cells were infected with OC43 at MOI = 1.0. (**E**) Western blot analysis of lysates from mock- or OC43-infected Vero cells collected at 24 hpi with or without Tg treatment for 1 h prior to harvesting. (**F**,**G**) Total RNA was isolated from mock- and levels of GADD34 (**F**) and ACTB (**G**) transcripts were determined by RT-QPCR. On all plots, values were normalized to 18S and expressed as fold changes relative to mock untreated cells. Each data point represents an independent biological replicate (N \(\geq\) 3). Error bars = standard deviation. Two-way ANOVA and [PERSON]’s multiple comparisons tests were performed in C and D, and Student’s _t_-Test in (**F**,**G**) to determine statistical significance (*****==, _p_-value \(<\) 0.0001; **==, _p_-value \(<\) 0.001; **==, _p_-value \(<\) 0.01; *, _p_-value \(<\) 0.01; *, _p_-value \(<\) 0.05; _ns, non-significant). GTP-bound form of eIF2 and prevents translation arrest [36]. In our infection model, ISRIB treatment did not alter the inhibition of As-induced SG formation by OC43, while it significantly decreased As-induced SG formation in mock-infected cells, as expected (Figure 2A,B). It also did not affect OC43-mediated GADD34 induction (Figure 2C), the inhibition of As-induced eIF2\(\alpha\) phosphorylation by OC43 (Figure 2D,E), or virus replication (Figure 2F). To test whether the ISR-dependent ATF4 and GADD34 induction pathway is intact in 293A cells, we verified the effects of ISRIB on Tg-induced ISR in this model. As expected, the Tg-mediated induction of ATF4 and GADD34 was blocked by ISRIB (Figure 2G-I). Taken together, these results show that neither GADD34 induction nor the inhibition of eIF2\(\alpha\) phosphorylation by OC43 are due to ISR activation by the virus. Figure 2: Integrated stress response inhibitor does not prevent GADD34 upregulation or the inhibition of eIF2\(\alpha\) phosphorylation by OC43. The 293A cells were infected with OC43 at MOI = 1.0 and treated  
with ISRIB at 1 hpi or left untreated. At 23 hpi, p-eIF2\(\alpha\) phosphorylation and SG formation was induced by the sodium arsenite treatment (+As). (**A**) SG formation in infected and mock-infected cells was analyzed using immunofluorescence staining for SG marker TIAR (magenta). Infected cells were identified by staining for nucleoprotein (N(OC43), teal). (**B**) SG formation was quantified from immunofluorescence staining images represented in (**A**). (**C**) Total RNA was isolated from mock- and infected cells treated as indicated and levels of GADD34 transcripts were determined by RT-QPCR. Values were normalized to 18S and expressed as fold changes relative to mock untreated cells. (**D**) Western blot analysis of lysates from mock- or OC43-infected cells treated as indicated. (**E**) Relative level of eIF2\(\alpha\) phosphorylation was quantified from Western blot images represented in (**D**). (**F**) Production of infectious virions in untreated and ISRIB-treated cells was measured in cell supernatants at 24 hpi. (**G-I**) ISRIB blocks Tg-induced ATF4 and GADD34 expression in 293A cells. Uninfected cells were treated with Tg with or without the addition of ISRIB as indicated. (**G**,**H**) Levels of GADD34 (**G**) and GAPDH (**H**) transcripts were determined by RT-QPCR. Values were normalized to 18S and expressed as fold changes relative to mock untreated cells. (**I**) Western blot analysis of lysates from cells treated as indicated. For all plots, each data point represents an independent biological replicate (N \(\geq\) 3).