Structural and Functional Characterization of SARS-CoV-2 RNA Processing Factors

National Institute Of Environmental Health Sciences

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Abstract

SARS-CoV-2 is the virus responsible for the current Covid-19 global pandemic which has infected millions worldwide. Nsp15 is a viral endoribonuclease found in all coronaviruses that processes viral RNA to prevent detection by the host immune system. Nsp15 is a promising anti-viral target, however how it cuts RNA is poorly understood. Through the combination of cryo-EM, mass-spectrometry, biochemistry, and molecular dynamics we determined how Nsp15 recognizes and cleaves both single and double stranded RNA substrates. Through structural-based mutagenesis we uncovered the role of critical Nsp15 residues in mediating RNA substrate specificity, cleavage, and oligomerization. By capturing a cryo-EM structure of Nsp15 bound to double stranded (ds) RNA we discovered that Nsp15 is able to process ds-RNA substrates through a unique base-flipping mechanism. We also analyzed the GISAID database to identify mutants of Nsp15 that have emerged during the pandemic. We selected a subset of these mutants and determined how they impact oligomerization and cleavae activity in vitro. Overall our work has defined the basic mechanisms of RNA recognition and cleavage by SARS-CoV-2 Nsp15.