Project Summary Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is a recently emergent, currently pandemic virus and etiological agent of Coronavirus Induced Disease-19 (COVID-19). Despite a flood of scientific investigation, critical gaps remain in our understanding of the basic cellular processes that facilitate replication of coronaviruses, including SARS-CoV-2, and contribute to the pathogenesis of severe disease. Our preliminary data demonstrate that IRE1α, a component of the cellular response to endoplasmic reticulum (ER) stress, is required for SARS-CoV-2 replication and inflammatory cytokine responses. However, the stage(s) of the viral life cycle and downstream cellular pathways that mediate these effects remain completely unknown. ER stress and IRE1α activation are well-associated with conditions such as obesity, diabetes, hypertension, and aging, all of which are risk factors for severe manifestations of COVID-19. We hypothesize that comorbidity-associated ER stress primes both exuberant viral replication and pathogenic inflammatory cytokine production via IRE1α. This project leverages our unique ability to test this hypothesis using cell culture infection models, as well as specimens from patients with COVID-19. IRE1α inhibitors are under evaluation for treatment of non-infectious human diseases, and we propose that this project will provide preclinical evidence for the novel application of these drugs to treat infections with SARS-CoV-2 and potentially other human coronaviruses. The experiments outlined in this proposal will determine the molecular mechanism(s) by which IRE1α supports SARS-CoV-2 infection. IRE1α is a nuclease which initiates nonconventional splicing of XBP1 mRNA, which encodes a pleiotropic transcription factor. IRE1α also targets other specific RNAs leading to their degradation. We will determine whether the requirement for IRE1α is XBP1-dependent or -independent and dissect the downstream cellular processes that facilitate SARS-CoV-2 replication and inflammatory cytokine responses (Aim 1). We will systematically identify stage(s) of the SARS-CoV-2 life cycle that require IRE1α (Aim 2). We predict that IRE1α most likely supports biogenesis of ER-derived viral replication platforms, and will focus experiments on this hypothesis. We predict that IRE1α represents a target for dual anti-viral and anti- inflammatory therapy and will test this in mouse models of SARS-CoV-2 infection (Aim 3). Finally, we will determine whether IRE1α activation occurs during human infection and ER stress is a prognostic marker for severe COVID-19. Together, the results of this project will reveal basic cellular processes occurring during coronavirus infection and host factors critical for the pathogenesis of COVID-19. .