# Human Betacoronavirus OC43 Interferes with the Integrated Stress Response Pathway in Infected Cells
## 1 Introduction
S is a receptor binding protein that binds sialic acid on the cell surface [21] and mediates viral entry [14]. Once the virus enters a host cell, its large 30 kb positive sense capped and polyadeninated RNA genome associates with host translation machinery to initiate the synthesis of viral non-structural proteins (Nsps) involved in genome replication, subgenomic mRNA production, and the inhibition of host antiviral responses [14]. The replication of the viral genome in the cytoplasm can generate double-stranded RNA (dsRNA) intermediates which may activate cytoplasmic sensors, including retinoic acid-inducible gene I (RIG-I), melanoma differentiation-associated gene 5 (MDA5), and dsRNA-activated protein kinase (PKR) [7; 22]. However, in CoV-infected cells, dsRNA is largely shielded inside double membrane replication transcription compartments that exclude and limit the activation of cytosolic sensors [14; 23]. These double membrane compartments are derived from the endoplasmic reticulum (ER) of the host cell by the concerted action of viral Nsp3, Nsp4, and Nsp6 proteins [24; 25]. Newly generated viral sub-genomic mRNAs that encode structural proteins are released from these compartments through specific crown-shaped pores and then associate with either free or ER-associated ribosomes in the cytoplasm to be translated [26]. Genomic RNA and sub-genomic mRNAs of coronaviruses are capped and polyadeninated and therefore resemble host mRNAs in their translation initiation mechanism, which is sensitive to the disruption of the cap-directed assembly of the translation preinitiation complex or eIF2\(\alpha\) phosphorylation [27; 28]. Therefore, coronaviruses must limit ISR to ensure robust protein synthesis and replication. Our previous work revealed that OC43 infection actively inhibits the phosphorylation of eIF2\(\alpha\)[29]. In our cell culture infection model, very little eIF2\(\alpha\) phosphorylation was detected in infected cells, and when they were treated with a potent inducer of eIF2\(\alpha\) phosphorylation sodium arsenite (As), they had significantly reduced levels of p-elF2\(\alpha\) compared to mock-infected cells [29]. As treatment induces oxidative stress and activates HRI kinase [4]. This usually causes very strong eIF2\(\alpha\) phosphorylation and the formation of stress granules (SGs), cytoplasmic condensates of polysome-free messenger ribonucleoprotein complexes that accumulate following the inhibition of translation initiation [9]. OC43 infection also inhibits the formation of As-induced stress granules (SGs) [29]. In the present work, we aimed to further characterize the modulation of ISR by OC43. We demonstrate that the OC43 infection of human embryonic kidney 293A cells strongly induces GADD34, the negative regulator of ISR. Although OC43 infection causes ER stress and the activation of PERK, which negatively affects viral protein synthesis and replication, PERK-mediated ATF4 production is not responsible for GADD34 upregulation in infected cells. Remarkably, we also show that OC43-induced GADD34 protein does not function in mediating increased eIF2\(\alpha\) dephosphorylation.