The unfolded protein response components IRE1α and XBP1 promote human coronavirus infection

Abstract

The cellular processes that support human coronavirus replication and contribute to the pathogenesis of severe disease remain incompletely understood. Many viruses, including coronaviruses, cause endoplasmic reticulum (ER) stress during infection. IRE1α is a component of the cellular response to ER stress that initiates non-conventional splicing of XBP1 mRNA. Spliced XBP1 encodes a transcription factor that induces the expression of ER-related targets. Activation of the IRE1α–XBP1 pathway occurs in association with risk factors for severe human coronavirus infection. In this study, we found that the human coronaviruses HCoV-OC43 (human coronavirus OC43) and SARS-CoV-2 (severe acute respiratory syndrome coronavirus-2) both robustly activate the IRE1α–XBP1 branch of the unfolded protein response in cultured cells. Using IRE1α nuclease inhibitors and genetic knockdown of IRE1α and XBP1, we found that these host factors are required for optimal replication of both viruses. Our data suggest that IRE1α supports infection downstream of initial viral attachment and entry. In addition, we found that ER stress–inducing conditions are sufficient to enhance human coronavirus replication. Furthermore, we found markedly increased XBP1 in circulation in human patients with severe coronavirus disease 2019 (COVID-19). Together, these results demonstrate the importance of IRE1α and XBP1 for human coronavirus infection. IMPORTANCE There is a critical need to understand the cellular processes co-opted during human coronavirus replication, with an emphasis on identifying mechanisms underlying severe disease and potential therapeutic targets. Here, we demonstrate that the host proteins IRE1α and XBP1 are required for robust infection by the human coronaviruses, SARS-CoV-2 and HCoV-OC43. IRE1α and XBP1 participate in the cellular response to ER stress and are activated during conditions that predispose to severe COVID-19. We found enhanced viral replication with exogenous IRE1α activation, and evidence that this pathway is activated in humans during severe COVID-19. Together, these results demonstrate the importance of IRE1α and XBP1 for human coronavirus infection.

Human Betacoronavirus OC43 Interferes with the Integrated Stress Response Pathway in Infected Cells

“…Although the IRE1/XBP1s pathway has not been shown to upregulate GADD34, we tested whether IRE1 inhibition would perturb GADD34 expression in OC43-infected cells. As reported previously in another model [33], OC43 infection induced XBP1s in 293A cells (Figure 3E). The addition of specific IRE1 inhibitor 4µ8C inhibited XBP1s' induction triggered by OC43 infection or Tg treatment in a concentration-dependent manner (Figure 3E,F), but did not affect GADD34 induction.…”

“…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.…”

“…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-eIF2α in the pathway. Among the eIF2α 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 eIF2α at 24 hpi using Western blotting.…”

“…However, GADD34 can be induced by other transcription factors [ 37 , 38 , 39 ], and in addition to PERK, Tg and OC43 can activate other ER stress sensors. One ER stress sensor that is strongly activated by OC43 is inositol-requiring enzyme 1 (IRE1) [ 33 ]. IRE1 activation causes the non-canonical cytoplasmic splicing of mRNA-encoding transcription factor X-box binding protein 1 (XBP1), leading to the synthesis of XBP1s, an activator of transcription produced from spliced XBP1 mRNA [ 40 ].…”

Insights into the Activation of Unfolded Protein Response Mechanism during Coronavirus Infection

“…Indeed, the IRE1 overexpression in the Vero-infected cells induced the upregulation of cleaved caspase-9 as well as nFκB activation and TNF-a expression, which were ameliorated when the cells were treated with the antiviral agent Nelfinavir [97]. Although the findings from Oda et al in HCT-9, Calu-3, and Vero-3 indicated the activation of the UPR and IRE1a pathway, they contradict previous reports as they found in samples from patients with severe COVID-19 the overexpression of both activated IRE1a and XBP1s, thus indicating the RNase activity of IRE1 [98]. Nevertheless, all the reports indicate the significance of the IRE1a branch in SARS-CoV-2 infection.…”