Characterization of SARS2 Nsp15 Nuclease Activity Reveals it’s Mad About U

Abstract

Nsp15 is a uridine specific endoribonuclease that coronaviruses employ to cleave viral RNA and evade host immune defense systems. Previous structures of Nsp15 from across Coronaviridae revealed that Nsp15 assembles into a homo-hexamer and has a conserved active site similar to RNase A. Beyond a preference for cleaving RNA 3′ of uridines, it is unknown if Nsp15 has any additional substrate preferences. Here we used cryo-EM to capture structures of Nsp15 bound to RNA in pre- and post-cleavage states. The structures along with molecular dynamics and biochemical assays revealed critical residues involved in substrate specificity, nuclease activity, and oligomerization. Moreover, we determined how the sequence of the RNA substrate dictates cleavage and found that outside of polyU tracts, Nsp15 has a strong preference for purines 3′ of the cleaved uridine. This work advances our understanding of how Nsp15 recognizes and processes viral RNA and will aid in the development of new anti-viral therapeutics.

Biochemical Characterization of Emerging SARS-CoV-2 Nsp15 Endoribonuclease Variants

“…Mutations with a high number of isolates are an easy selection criterion; our selection of K13, T34, T115, and R207 agree with bioinformatics analysis of main mutated residues across the SARS-CoV-2 proteome published in early 2021 [18]. Knowledge of molecular mechanisms is also important; this led us to select N-terminal residue and active site residue mutations to further test the established roles of these amino acids [29,35,36,42]. We recently determined a structure of Nsp15 bound to dsRNA [43]; of the Nsp15 mutants we characterized here, the only non-active site residues in the area of the dsRNA are D133 (~5 Å) and V128 (~9 Å).…”

“…We selected several prominent mutations to study from these 53 non-synonymous variants: K13N (1716), K13R (1339), T34I (9900), A93S (1081), T115A (1810), V128F (4457), D133Y (1982), L163F (2072), P206S (5849), R207S (6174), D220Y (10323), H235Y (3630) and K260R (4938). In addition to these variants, others were chosen based on previous biochemical data [28, 29, 35, 36]. These variants were G18E (304), G18R (123), K290N (396), and W333C (304).…”

“…We assessed the impact of mutations on K13, G18, and T34I (Figure 3A) from the NTD of Nsp15, which has been shown to be critical for oligomerization [29, 37, 42]. In particular, K13 sits at the interface between neighboring protomers, and participates in water-mediated interactions with RNA ([29], Supplemental Figure 2). Given the importance of the NTD in oligomerization, we compared the amount of hexamer and monomer from each purified variant.…”

“…Molecular studies of Nsp15 have revealed important details regarding how it processes RNA. SARS-CoV-2 Nsp15 is a hexamer formed from a dimer of trimers; monomeric Nsp15 is inactive [28][29][30][31]. Each protomer consists of an N-terminal (NTD), middle (MD), and catalytic endoribonuclease (EndoU) domain.…”

Nsp1 proteins of human coronaviruses HCoV-OC43 and SARS-CoV2 inhibit stress granule formation

“…Nsp15 is a nuclease that is conserved in coronaviruses. It preferentially cleaves polyuridine RNA sequences and inhibits accumulation and detection of viral dsRNA in infected cells (36). Another viral protein that was shown to affect SG formation is Nsp1, a host shutoff factor encoded by the first N-terminal portion of ORF1ab of betacoronaviruses (37).…”