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Cleavage of Stemloop RNA by SARS-CoV-2 Nsp15

ABSTRACT: Coronaviruses, such as SARS-CoV-2, use the endoribonuclease Nsp15 to evade the host immune system by cleaving double-stranded RNA (dsRNA) at uridine (U), thus regulating viral RNA levels. Although structural data shows that Nsp15 flips the target U out of the dsRNA helix to cleave it, the exact mechanism for this is unclear. Our study addresses this uncertainty by creating fluorinated dsRNA substrates to explore how a U's sequence context affects its tendency to flip and its susceptibility to Nsp15. Using a combination of nuclease assays, 19F NMR spectroscopy, mass spectrometry, and single-particle cryo-EM, we found that Nsp15 cleaves more effectively when U is unpaired and already flipped. Our results suggest that Nsp15's cleavage efficiency correlates with U's tendency to spontaneously flip, indicating that Nsp15's activity during infection may target bulged or accessible Us in the coronaviral genomic RNA, providing insights into Nsp15's role in immune evasion.

INSTRUMENT(S): Q Exactive Plus

ORGANISM(S): Sars-cov-2

SUBMITTER: Robin E. Stanley

PROVIDER: MSV000094614 | MassIVE | Wed Apr 24 15:27:00 BST 2024

REPOSITORIES: MassIVE

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