Structural and Functional Characterization of the SARS-CoV-2 Endoribonuclease Nsp15

National Institute Of Environmental Health Sciences

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Abstract

The first two-thirds of the SARS-CoV2 viral genome encode for a group of proteins known as the non-structural proteins (NSPs), which play various roles in supporting the viral life cycle. Many of the NSPs are promising anti-viral drug targets, underscoring the need to understand their structure and function. Nsp15 (also known as EndoU) is a viral endoribonuclease that is found in all coronaviruses and most nidoviruses, that cleaves viral RNA 3’ of uridines. Loss of Nsp15 nuclease activity has been shown to cause an accumulation of double stranded (ds) RNA leading to the activation of the host dsRNA sensor MDA5 and triggers an interferon response. While there is an established connection between Nsp15 and evasion of the host immune response how Nsp15 selects and cleaves its viral RNA targets is still not well defined. Over the past several years we have explored the structure and cleavage activity of Nsp15 through the combination of cryo-EM, mass-spectrometry, biochemistry, and molecular dynamics. Our work has helped to establish the structural basis for uracil specificity and define the mechanism of cleavage through an RNase-A like reaction. We have also established that Nsp15 can cleave both single stranded (ss) and ds-RNA substrates. In order for Nsp15 to accommodate dsRNA within its active site the RNA must under-go a structural rearrangement. A cryo-EM structure of Nsp15 bound to dsRNA revealed that Nsp15 stabilizes a uridine in a flipped-out conformation, but it has remained unclear if Nsp15 plays a direct or passive role in base-flipping. To answer this question, we performed in vitro cleavage assays and NMR spectroscopy with fluorine labeled substrates. Collectively these assays support that Nsp15 more efficiently cleaves uridines which are unpaired. We explored different sequence contexts and discovered that uridines that are more likely to be in a flipped-out conformation are more efficiently cleaved by Nsp15. These results suggest that Nsp15 has a strong preference for cleaving more accessible uridines within the viral RNA.