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 cleavages viral RNA. Through structural-based mutagenesis we uncovered the role of critical Nsp15 residues in mediating specificity, cleavage, and oligomerization. Surprisingly our cryo-EM structures and molecular dynamic simulations did not reveal a large RNA binding interface within the Nsp15 hexamer. Therefore, we turned to FRET and gel-based nuclease assays to determine how the sequence of the RNA influences cleavage by Nsp15. We discovered that Nsp15 has a strong preference for purines 3 of the cleaved uracil. We further probed Nsp15s ability to cleave RNA sequences from the SARS-CoV-2 viral genome in vitro and found that it efficiently cleaves 3 of uridines located within polyU tracks and the transcriptional regulatory sequence.