Mechanism and key RNA determinants of SARS-CoV-2 Nsp1-induced endonucleolytic cleavage of mRNA.

SARS-CoV-2 nonstructural protein 1 (Nsp1) binds to 40S ribosomal subunits and induces host protein synthesis shut off by inhibiting translation initiation and triggering endonucleolytic cleavage of cellular mRNAs. Irrespective of the mode of initiation, Nsp1-mediated cleavage is induced by the cooperative action of the N-terminal domain of Nsp1, the RRM domain of eIF3g and 40S subunits. Using in vitro reconstitution, we determined that cleavage occurs by transesterification following intramolecular nucleophilic attack of the 2'OH of the ribose on the adjacent phosphodiester bond yielding 5'OH and 2',3'-cyclic phosphate termini. Cleavage requires a guanosine ~10-22 nucleotides from the 5' end of mRNA, occurs within a narrow window upstream of this G, is most efficient between nucleotides at positions -6/-7 and -7/-8 relative to G, and shows a preference for Pu at positions -7 or -8 which provides the 2'OH for the nucleophilic attack. Zero-length UV cross-linking of Nsp1 to nucleotides at positions -1 and -2 suggests that the critical guanosine may be recognized by Nsp1. Resistance to Nsp1-mediated cleavage of SARSCoV-2 mRNA was ensured both by the relatively long distance between its G23G24 and the 5'end and by the preceding oligoPy stretch lacking purines at positions -7 or -8 upstream of G23G24.