Coronaviruses (CoVs) are positive-sense RNA viruses that can emerge from endemic reservoirs and infect zoonotically, causing significant morbidity and mortality. CoVs encode an endoribonuclease designated EndoU that facilitates evasion of host pattern recognition receptor MDA5, but the target of EndoU activity was not known. Here, we report that EndoU cleaves the 5′-polyuridines from negative-sense viral RNA, termed PUN RNA, which is the product of polyA-templated RNA synthesis. Using a virus containing an EndoU catalytic-inactive mutation, we detected a higher abundance of PUN RNA in the cytoplasm compared to wild-type−infected cells. Furthermore, we found that transfecting PUN RNA into cells stimulates a robust, MDA5-dependent interferon response, and that removal of the polyuridine extension on the RNA dampens the response. Overall, the results of this study reveal the PUN RNA to be a CoV MDA5-dependent pathogen-associated molecular pattern (PAMP). We also establish a mechanism for EndoU activity to cleave and limit the accumulation of this PAMP. Since EndoU activity is highly conserved in all CoVs, inhibiting this activity may serve as an approach for therapeutic interventions against existing and emerging CoV infections.
Results: EndoU Activity Reduces the Accumulation of an Epitope Recognized by an Anti-dsRNA Antibody in CoV-Infected Hepatocytes. Previously, we reported that EndoU activity delays the accumulation of an epitope recognized by the K1 antibody in the cytoplasm of IFNAR−/− bone marrow-derived macrophages (BMDMs) as measured by immunofluorescence (11). The K1 antibody was shown to recognize Cells carry sensors that are primed to detect invading viruses. To avoid being recognized, coronaviruses express factors that interfere with host immune sensing pathways. Previous studies revealed that a coronavirus endoribonuclease (EndoU) delays activation of the host sensor system, but the mechanism was not known. Here, we report that EndoU cleaves a viral polyuridine sequence that would otherwise activate host immune sensors. This information may be used in developing inhibitors that target EndoU activity and prevent diseases caused by coronaviruses.
Reference & Source Information: https://www.pnas.org/
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