Abstract
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.
Highlights
Coronaviruses (CoVs) are positive-sense RNA viruses that can emerge from endemic reservoirs and infect zoonotically, causing significant morbidity and mortality
We show that CoV EndoU activity limits the abundance and length of the polyuridine extension on 5′-polyU-containing, negative-sense (PUN) RNAs for both the beta-CoV mouse hepatitis virus strain A59 (MHV-A59) and the alpha-CoV PEDV
We propose a mechanism for EndoU, which is to cleave polyU sequences from PUN RNAs, limiting the formation of a pathogen-associated molecular pattern (PAMP) and impeding the ability of MDA5 to activate the innate immune response to infection
Summary
Coronaviruses (CoVs) are positive-sense RNA viruses that can emerge from endemic reservoirs and infect zoonotically, causing significant morbidity and mortality. More recent findings revealed that EndoU catalytic mutant (EndoUmut) viruses replicate as well as wild-type virus in IFN-nonresponsive cells, but are severely impaired for replication in IFN-responsive macrophages [10, 11] These recent results revealed that EndoU activity is important for limiting the sensing of viral RNA by host dsRNA sensors such as MDA5, PKR, and OAS/RNaseL. The negative-sense RNAs function as templates for synthesis of positive-sense genomic RNA and sgRNA [1, 2] This replication strategy can generate long double-stranded RNA (dsRNA) intermediates [3], that may act as pathogen-associated molecular patterns (PAMPs) recognized by cytoplasmic pattern recognition receptors (PRRs) [4, 5]. Initial studies revealed that EndoU colocalizes with the viral replication complex
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