Modulation and characterization of the selective activation of the IRE1 stress sensor by SARS-CoV2

Infection with most enveloped viruses induces the ER stress response and an IFN response. However, unlike other coronaviruses, SARS-CoV-2 infection elicits an unorthodox response from the major stress sensor inositol reactive enzyme 1 (IRE1). It activates IRE1, as normally, by dimerization and phosphorylation, but fails to activate its RNase domain, and thus is not accompanied by the generation of the transcription factor X box binding protein (XBP1) nor by the RIDD activity on select transcripts, which control adaptive vs. apoptotic outcomes. To investigate this selective IRE1 activation further, I will complement a SARS-CoV-2 cell model lacking the endogenous IRE1 gene with functional fusions to a fluorescent protein tag or to the promiscuous peroxidase APEX2, infect it with SARS-CoV-2 and ask: 1) can the RNAse domain of IRE1 be activated with the help of chemical ligands?; 2) do the IRE1 dimers in these cells oligomerize into distinct structures that characterize the selective activation state? and 3) how is IRE1 interactome, in infected cells, different from a full activation state of the sensor? In Aim 1, I will determine if luteolin, or other small molecule modulators of IRE1, can restore RNase activity in SARS-CoV-2 infected cells and whether it induces IRE1-dependent caspase activation. In Aim 2, I will take advantage of live cell imaging of IRE1 to determine whether its cellular distribution reports on the selective activation state, characteristic of SARS-CoV-2 infection. In Aim 3, I will employ isolation, enrichment and determination of RNA and protein species in close proximity to IRE1 to establish whether its molecular interactome reports on such unique mode of action. Altogether, these orthogonal methods applied in biological relevant model systems will characterize, molecularly and throughout, the status of IRE1. In the future, this characterization will be used to screen variants of SARS-CoV-2 or the other enveloped viruses that induce the ER stress response differentially and define their effects on the proteome or transcriptome. The results may provide a totally novel strategy against SARS-CoV-2.