Cryo-EM Structures of the SARS-CoV-2 Endoribonuclease Nsp15

Abstract

New therapeutics are urgently needed to inhibit SARS-CoV-2, the virus responsible for the on-going Covid-19 pandemic. Nsp15, a uridine-specific endoribonuclease found in all coronaviruses, processes viral RNA to evade detection by RNA-activated host defense systems, making it a promising drug target. Previous work with SARS-CoV-1 established that Nsp15 is active as a hexamer, yet how Nsp15 recognizes and processes viral RNA remains unknown. Here we report a series of cryo-EM reconstructions of SARS-CoV-2 Nsp15. The UTP-bound cryo-EM reconstruction at 3.36 Å resolution provides molecular details into how critical residues within the Nsp15 active site recognize uridine and facilitate catalysis of the phosphodiester bond, whereas the apo-states reveal active site conformational heterogeneity. We further demonstrate the specificity and mechanism of nuclease activity by analyzing Nsp15 products using mass spectrometry. Collectively, these findings advance understanding of how Nsp15 processes viral RNA and provide a structural framework for the development of new therapeutics.

Fig.1. Nsp15 is a central regulator of SARS-CoV-2 RNA processing.

Upon viral entry, the single-strand SARS-CoV-2 positive-sense genomic RNA (+gRNA; yellow) is released into the cytoplasm and translated by host ribosomes to generate viral polyproteins pp1a and pp1ab. Subsequent proteolytic cleavage of the polyproteins results in a variety of non-structural proteins (Nsp) essential for diverse viral functions. Transcription of the positive-sense genomic RNA produces a negative-sense genomic RNA (−gRNA; green) intermediate. The negative-sense strand is a template for reverse transcription (RT) generating a series of positive-sense subgenomic RNAs (+sgRNA; brown), which are translated into diverse structural proteins. In addition, the negative-sense strand is also the template for viral replication. The non-structural protein Nsp15 (orange) is a poly-(U) specific endonuclease that cleaves 3′ to uridines within the viral genomic RNA. Nsp15 cleavage sites (blue arrowheads) have been mapped all along the positive-sense genomic RNA and 5′-end of the negative-sense genomic RNA. Nsp15 viral RNA processing plays an important role for evading detection by the host innate immune response.

Fig. 2.. Architecture of hexameric SARS-CoV-2 Nsp15.

(A) Domain organization of Nsp15. The numbering corresponds to the amino acid residues at the domain boundaries. The Nsp15 N-terminal domain (ND) is shown in orange, the middle domain (MD) is shown in red, and the poly-U specific endonuclease domain (endoU) is shown in beige. (B) Orthogonal views of the cryo-EM map reconstruction of UTP-bound Nsp15 (left) and its corresponding model shown as a cartoon (right). Each Nsp15 protomer is colored as green (P1), purple (P2), coral (P3), tan (P4), gray (P5), and blue (P6). (C) Model of Nsp15 protomer shown as a cartoon and colored as seen in panel A. The position of the Nsp15 endoU active site is highlighted with a black box and the modeled nucleotide is shown as sticks (cyan). N and C mark the N- and C-termini, respectively.

Fig. 3.. 5′-UMP coordination by the Nsp15 endoU active site.

(A) Amino acid sequence alignment of endoU active site residues from Nsp15 homologs. Secondary structure motifs observed in the nucleotide-bound Nsp15 cryo-EM structure are shown above with their corresponding amino acid residue boundaries. (B) Nucleotide-bound Nsp15 cryo-EM map reconstruction with protomers colored as seen in Fig. 2B. Excess UTP was added to the sample resulting in additional density within all six endoU active sites. The nucleotide density is colored in cyan and the black box demarcates the endoU active site. (C) Nsp15 coordination of 5′-UMP ligand. Due to the poor density of the UTP β- and γ-phosphates, 5′-UMP was modeled into the active site. Cartoon model of the 5′-UMP-bound Nsp15 endoU active site where 5′-UMP (cyan) and individual residues H235, H250, K290, S294, and Y343 are shown as sticks (top). Model of uracil base discrimination shown as sticks (bottom). Black dotted lines represent potential hydrogen bonds (2.1–3.0 Å). (D) RNA cleavage activity of Nsp15 variants (2.5 nM) incubated with FRET RNA substrate (0.8 μM) over time. RNA cleavage was quantified from three technical replicates. The mean and standard deviation are plotted and p values of wt-Nsp15 compared to H235A (blue) and H250A (red) are reported from two-tailed Student’s t tests.

Fig. 4.. Extracted ion chromatograms and MS spectra of Nsp15 RNA cleavage products.

(A) Extracted ion chromatograms of the 3′-TAMRA labeled AA-TAMRA RNA cleavage product. 5′-HO-AA-TAMRA is readily observed in the presence of Nsp15 (+Nsp15) as a singly charged ion at m/z 1463.42, but is undetectable in the absence of enzyme (−Nsp15). (B) MS spectrum confirms the identity of 5′-HO-AA-TAMRA and does not detect the presence of alternative 5′-cleavage products. (C) Extracted ion chromatograms of the 5′-fluorescein labeled (FI) FI-AAAU cleavage product. FI-AAAU is only detected in the presence of Nsp15. (D) MS spectrum confirms the identity of FI-AAAU. The 3′-product is observed primarily as a doubly charged ion at m/z 914.14, which corresponds to a 2′3′-cP terminated moiety. A doubly charged ion at m/z 923.14 is also present and corresponds to a 3′-P terminated species. MSMS spectra of m/z 1463.14 and m/z 914.14 unambiguously confirm the identity of these ions (Fig. S5) (E) Extracted ion chromatograms of the 5′-FI-AAAU-2′3′-cP cleavage product (top) and the 5′-FI-AAAU-3′-P cleavage product (bottom) set to the same scale to demonstrate that under the conditions employed, the majority of the cleavage product is terminated by a cyclic phosphate. The graphical representation of the areas under the curve of the extracted ion chromatograms show that approximately 80% of the cleavage product is the cyclic phosphate (assuming similar ionization efficiencies of the two species). (F) Superimposition of SARS-CoV-2 Nsp15 (beige) and Bos taurus RNase A (PDB ID: 1o0n; magenta) active site residues bound to 3′-UMP (cyan) shown as sticks.

Fig. 5.. Conformational heterogeneity of the Nsp15 endoU domain.

(A) 3D variability analysis (30) of cryo-EM reconstructions of Nsp15 apo-states. Frame 1 (gray) and frame 20 (blue) of a 20-frame set describing the motion by the first eigenvector of the cryo-EM reconstructions. Black outlines demarcate the unique views of the endoU domains and dotted arrows demarcate regions of conformational heterogeneity. (B) Dynamical cross-correlation matrix (DCCM) analysis of a 5′-UMP-bound protomer and its 5′-UMP-free form from hexamer systems. Both inter- and intra-domain positive correlations are significantly enhanced due to 5′-UMP-binding. The correlations were calculated from the equally spaced 100-configurations extracted from the last 500 ns of each simulation. Black boxes highlight the positive correlated motions between the endoU domains within the hexameric assembly (box a) and the endoU domain relative to the N-terminal half of the protomer (box b).