Dissecting the IRE1a-related branch of the Unfolded Protein Response in SARS-CoV-2 infected cells

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiological agent of recent COVID-19 disease. SARS-CoV-2 belongs to the Coronaviridae family. Members of this family include enveloped, (+)-single-stranded RNA viruses with a broad host range and high pandemic potential. Coronaviruses are known to induce high amounts of viral glycoproteins and a massive morphological alteration of endoplasmic reticulum (ER), usually leading to ER stress and activation of unfolded protein response (UPR). However, conflicting results have been obtained regarding the role of UPR in SARS-CoV-2 infection. Indeed, the UPR is only marginally induced in early SARS-CoV-2 infection, which suggests a viral inhibitory mechanism. The UPR pathway is responsible for homeostatic control of protein synthesis and folding in the ER. One of the most important protein sensors of ER stress is inositol-requiring protein 1 (IRE1a). Upon activation, IRE1a RNase domain cleaves a 26nt-long intron from X-box binding protein 1 (XBP1) mRNA. This non-conventional cytosolic splicing is then completed by the activity of the tRNA ligase RTCB. The goal of this project is to investigate the molecular pathway leading to an attenuated UPR in SARS-CoV-2 infected cells. RNA sequencing of SARS-CoV-2 infected cells revealed absent or limited induction of IRE1a-related genes and XBP1 splicing. Transcriptomic data were confirmed in vitro: no significant induction of XBP1 splicing was observed in CoV-2-infected cells at an early (6hpi) and a late (24hpi) timepoint. Moreover, a time-course experiment performed using an inducible IRE1a-mNG reporter cell line showed that the virus is not affecting IRE1a phosphorylation and clustering but levels of XBP1 splicing are inhibited in the early phase of viral replication. Taken together, our data suggests a time-dependent viral inhibitory activity specifically targeting the splicing reaction. The ability of the virus to inhibit XBP1 splicing was then demonstrated by timely pharmacological induction of the reaction in infected cells. To identify potential viral inhibitors, overexpression of single CoV-2 proteins was performed in sterile conditions and splicing of XBP1 mRNA was checked. However, no protein was observed to directly interfere with the splicing reaction. Then, we investigated the role of host protein RTCB as viral target. RTCB was shown to localise in proximity of viral replication sites in infected cells. Moreover, infection of an RTCB knock-down cell line led to reduction of viral replication, suggesting a pro-viral role of the RNA ligase. In conclusion, SARS-CoV-2 infection has been observed to induce an early inhibition of IRE1a signalling, by specifically targeting the XBP1 splicing event. The RTCB protein, which is involved in the molecular mechanism of splicing, was shown to be a host factor supporting viral replication. Further investigations will provide a better understanding of molecular events regulating the role of RTCB in SARS-CoV-2 infection.