TITLE: Investigating Interferon Antagonists in Delaying Innate Immune Responses to SARS-CoV-2 PI: Susan C. Baker, PhD, Loyola University Chicago Stritch School of Medicine The goal of this proposal is to determine how viral interferon antagonists function in the replication and pathogenesis of coronaviruses, particularly during replication of Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Coronaviruses (CoVs) are a diverse family of positive-sense RNA viruses that include pathogenic strains infecting human and animal hosts. CoVs have repeatedly jumped from animal reservoirs into human circulation, causing severe disease and pandemics, as we are currently experiencing with SARS-CoV-2. Developing appropriate protective measures against emerging CoVs, including SARS-CoV- 2, will depend upon gaining an understanding of coronavirus-host interactions. We discovered that the endoribonuclease (EndoU), a highly conserved component of the CoV replicase complex, reduces dsRNA species recognized by host pattern recognition receptor MDA5, delaying the induction of interferon. We reported that viruses expressing an inactive form of EndoU replicate as efficiently as wild type virus in IFN non- responsive cells. Importantly, replication of EndoU mutant CoVs in interferon-responsive cells activate robust immune responses, which extinguishes virus replication and reduces pathogenesis in animals. Recently, we identified the target of EndoU activity to be poly-uridine containing negative sense RNA, which we term PUN RNA. This removal of the PUN RNA delays the generation of dsRNA species that are recognized by host pattern recognition receptor MDA5. We hypothesize that EndoU activity contributes to the delay in the innate immune response to SARS-CoV-2 replication. Here, we propose to investigate the mechanism of how EndoU acts in SARS-CoV-2, how EndoU associates with the replicase complex, and how PUN RNA contributes to activating MDA5. In Aim 1, we will evaluate EndoU and other IFN antagonists for their role as modulators of Type I and Type III IFN responses to SARS-CoV-2 infection in primary human airway cells and in enterocytes. We will use reverse genetics to generate viruses with inactive IFN antagonists and evaluate the effects of combining inactivation of EndoU with inactivating mutations of other viral protein IFN antagonists. In Aim 2 we will delineate and disrupt EndoU interactions within the coronavirus replicase complex. The results of these studies will guide strategies for disruption of EndoU from the CoV replicase complex, which would activate protective immune responses to CoV infections. In Aim 3, we will identify regions of poly-uridine negative-sense RNA, termed PUN RNA, required for recognition by EndoU and MDA5. These studies will provide new information on how PUN RNAs are recognized by EndoU and MDA5. Overall, these studies will define a new mechanism for how an endoribonuclease acts as a virulence factor. This new information ca...