# Human Betacoronavirus OC43 Interferes with the Integrated Stress Response Pathway in Infected Cells
## 4 Discussion
Interestingly, despite PERK activation, eIF2\(\alpha\) phosphorylation levels in OC43 remained low, confirming that it is inhibited in virus-infected cells. In our infection model, we demonstrate the robust induction of the downstream ISR effector GADD34 by OC43. GADD34 is a multifunctional protein involved in cellular stress responses. Its primary role in ISR is the recovery of protein synthesis after stress by forming a complex with PP1 to dephosphorylate eIF2\(\alpha\)[13]. Beyond its involvement in the ISR, GADD34 plays a role in the modulation of cell death and cell cycle regulation, contributing to growth arrest under certain stress conditions [48; 49; 50]. The specific functions of GADD34 are context-dependent and vary based on the nature of the stress stimuli and cellular context [1; 48; 50]. Given robust GADD34 induction by OC43 infection, the inhibition of eIF2\(\alpha\) phosphorylation by OC43 could be explained by GADD34-mediated recruitment of PP1 and increased eIF2\(\alpha\) dephosphorylation rates. However, we were unable to alter the inhibition of As-induced eIF2\(\alpha\) phosphorylation in OC43-infected cells by silencing  
Figure 6: A schematic diagram representing the main findings of this work. Question marks indicate the main unanswered questions that should be addressed in future studies: How does OC43 inhibit HRI? What is the mechanism of GADD34 upregulation by OC43? What is the function of GADD34 in OC43-infected cells? GADD34. In mock-infected cells, Tg induced lower levels of GADD34 compared to those induced by OC43 infection, and this induction resulted in the apparent dephosphorylation of eIF2\(\alpha\) at 4 h post-treatment compared to its peak at 1 h post-treatment. These results indicate that most of the GADD34 induced by the virus is diverted to perform other functions, but that the ISR negative feedback loop is intact in 293A cells. The induction of GADD34 by Tg followed eIF2\(\alpha\) phosphorylation by PERK and the production of the ATF4 transcription factor that normally functions upstream of GADD34 induction in the ISR pathway. Our analyses showed that ATF4 is not required for GADD34 induction by ER stress in 293A cells, because GADD34 protein accumulation in response to Tg or OC43 infection was unaffected by ATF4 silencing. In addition to ATF4, GADD34 can be induced by transcription factors CHOP and ATF3 [1], as well as the interferon regulatory factor 3 (IRF3) [37]. Both CHOP and ATF3 are induced by ATF4; however, other stimuli can upregulate ATF3 [51; 52]. IRF3 is the main transcription factor activated by cellular sensors of viral nucleic acids and an important driver of type I and type III interferon expression [53]. OC43 was shown to activate IRF3 and induce interferon response genes in lung fibroblast cell line MRC5 [54], but in another study OC43 was demonstrated to be very efficient at avoiding type I and type III interferon induction in primary human bronchial epithelial cells [55]. Future studies will determine if ATF3, IRF3, or other transcription factors implicated in GADD34 induction [38; 39] are responsible for the upregulation of GADD34 in OC43-infected cells. To summarize, our work revealed that OC43 infection activates the PERK arm of the ISR that suppresses viral protein synthesis and replication. At the same time, OC43 robustly induces GADD34, a multifunctional protein normally involved in eIF2\(\alpha\) dephosphorylation. However, GADD34 silencing had no effect on the viral inhibition of As-induced eIF2\(\alpha\) phosphorylation or virus replication. For our infection model, we used 293A cells, a kidney-derived cell line that can be productively infected by OC43 [29]. The 293A cells are a clone of the human embryonic kidney 293 cell line that contains a stably integrated copy of the adenovirus E1 gene that encodes the E1a and E1b proteins. These viral proteins can potentially skew the cellular responses to OC43 infection, as they are shown to attenuate interferon induction [56; 57].