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.

Structural biology of SARS-CoV-2: open the door for novel therapies

“…SARS-CoV-2 Nsp15 consists of 347 amino acids, including the N-terminal domain (ND, residues 1–64), the middle domain (residues 65–182), and the C-terminal EndoU domain (residues 207–347). 295 The overall structure of SARS-CoV-2 Nsp15 is a homo-hexamer with D3 symmetry 289 , 295 – 297 (Fig. 7c ).…”

“…Several conserved residues in Nsp15 (including His235, His250, Lys290, Thr341, Trp333, Tyr343, Ser294, Gly248, Lys345, Val295, and Gln245) are involved in substrate specificity, nuclease activity, and oligomerization of EndoU. 295 , 298 His235, His250, and Lys290 constitute the catalytic triad and utilize the general acid-base catalytic mechanism to complete the cleavage reaction. 289 , 295 – 297 The EndoU domain has conformational variability, and the substrate uridine-5′-Monophosphate (UMP) restrains this dynamic.…”

“… 295 , 298 His235, His250, and Lys290 constitute the catalytic triad and utilize the general acid-base catalytic mechanism to complete the cleavage reaction. 289 , 295 – 297 The EndoU domain has conformational variability, and the substrate uridine-5′-Monophosphate (UMP) restrains this dynamic. 295 Notably, allosteric regulation exists in the excision reaction of EndoU, 297 , 299 and the base binding sites are different between RNA in the pre- and post-cleavage states 297 (Fig.…”

“… 289 , 295 – 297 The EndoU domain has conformational variability, and the substrate uridine-5′-Monophosphate (UMP) restrains this dynamic. 295 Notably, allosteric regulation exists in the excision reaction of EndoU, 297 , 299 and the base binding sites are different between RNA in the pre- and post-cleavage states 297 (Fig. 7d, e ).…”

“… 295 , 300 In the Nsp15/AU-3′P complex (PDB 7N06), Trp333 forms π–π stacking interactions with uracil, but Ser294 no longer forms hydrogen bonds with uracil due to a conformational flip. 297 The ND domain also plays an indispensable role in the function of Nsp15, participating in protein oligomerization and RNA binding. 297 , 299 …”