Flipped over U: structural basis for dsRNA cleavage by the SARS-CoV-2 endoribonuclease

Abstract

Coronaviruses generate double-stranded (ds) RNA intermediates during viral replication that can activate host immune sensors. To evade activation of the host pattern recognition receptor MDA5, coronaviruses employ Nsp15, which is a uridine-specific endoribonuclease. Nsp15 is proposed to associate with the coronavirus replication-transcription complex within double-membrane vesicles to cleave these dsRNA intermediates. How Nsp15 recognizes and processes dsRNA is poorly understood because previous structural studies of Nsp15 have been limited to small single-stranded (ss) RNA substrates. Here we present cryo-EM structures of SARS-CoV-2 Nsp15 bound to a 52nt dsRNA. We observed that the Nsp15 hexamer forms a platform for engaging dsRNA across multiple protomers. The structures, along with site-directed mutagenesis and RNA cleavage assays revealed critical insight into dsRNA recognition and processing. To process dsRNA Nsp15 utilizes a base-flipping mechanism to properly orient the uridine within the active site for cleavage. Our findings show that Nsp15 is a distinctive endoribonuclease that can cleave both ss- and dsRNA effectively.

Figure 1.

DsRNA binds Nsp15 through interactions with two platforms in addition to the EndoU active site. (A) Selected 2D classifications of Nsp15 bound to dsRNA. DsRNA, indicated by arrows is visible extending away from the complex. Some classes suggest binding of more than one dsRNA substrate; however these were not the predominant views. (B) Side view of the complex in ribbon (left), EM density (middle), and local resolution (right) views.

Figure 2.

Nsp15 can cleave both ss- and dsRNA. (A) DsRNA interacts with three Nsp15 protomers, across both trimers. P1 and P6 form platforms that support RNA cleavage by P3. (B) Close-up of the active site (P3). Critical residues are shown in stick format. Uridine flips out to interact with S294 and N278, which provides optimal positioning for the catalytic triad. The 3′ base is stabilized by W333. (C) Cryo-EM density for the RNA engaged in the active site. (D) Time-course cleavage reaction for the dsRNA as well as each strand alone. Nsp15 cleaves ssRNA more quickly than dsRNA, and prefers different positions depending on that context. OH: alkaline hydrolysis of dsRNA. ssRNA(for) is the Cy5 labeled strand (red); ssRNA(rev) is the FI-labeled strand (blue).

Figure 3.

Comparison of WT Nsp15 activity on ss- and ds-RNA. (A) Time course reactions with WT Nsp15 were performed on double-labeled ssRNA ± the unlabeled complementary strand. Cleavage products are labeled to the right of the gel, with the color corresponding to which product (Cy5 or FI) it is. (B) RNA cleaved was quantified by disappearance of the uncleaved RNA band, normalized to the 0 time point for that reaction. The average and standard deviation for at least three independent reactions are graphed.

Figure 4.

Combined dsRNA surface mutant disrupts dsRNA cleavage. (A) Panels depicting the mutated residues at the P1, P3, and P6 interfaces (left to right). RNA cryo-EM density is shown as a transparent surface, with the modeled RNA shown as ribbons and slabs. (B) Nsp15 with mutations in residues near the dsRNA binding interface (Q19A, K111A, T113A, D129A, N137A, H243A, S136A and E340A) was tested for cleavage activity with ss- and dsRNA. A representative time-course cleavage gel from three technical replicates is shown.

Figure 5.

EndoU domain mutations in Nsp15 affect dsRNA cleavage. (A) Zoomed in depiction of the active site mutants, focused on W333. (B) Time course reactions for Nsp15 active site mutants were performed with double-labeled ssRNA ± the unlabeled complementary strand. A representative time course is shown for Nsp15 W333A. Cleavage products are labeled to the right of the gel, with the color corresponding to which product (Cy5 or FI) it is. (C, D) RNA cleaved was quantified by disappearance of the uncleaved RNA band, normalized to the 0 time point for that reaction. The average and standard deviation for at least three independent reactions are graphed.

Figure 6.

Model comparing ss- and dsRNA cleavage by Nsp15. Nsp15 cleaves ssRNA mainly through engagement of the U in the active site, with limited binding by upstream or downstream nucleotides. ssRNA is readily accessible to the Nsp15 active site. Nsp15 cleaves dsRNA by multiple interaction sites, spanning three protomers and both trimers that compose the hexamer. The U base must flip out of the base-paired helix to be cleaved; distortion in the RNA is stabilized through π-stacking with W333. Figure made in Biorender.