Druggability and Amino Acid Variability of the Catalytic Site of SARS-CoV-2 Nsp15 Across Coronaviruses and SARS-CoV-2 Samples – Post 26

Today’s post focuses on another protein encoded by many coronaviruses, the non-structural protein 15 (nsp15), an endoribonuclease. Nsp15 is a nidoviral RNA uridylate-specific endoribonuclease (NendoU) with C-terminal catalytic activity. This protein belongs to the EndoU family, and they all carry an RNA endonuclease activity to produce 2’-3’ cyclic phosphodiester and 5’-hydroxyl termini. (Kim et al. 2020)

SARS-CoV-2 nsp15 has 88% sequence identity (ID) with SARS-CoV-1 nsp15, 50% with the Middle East Respiratory Syndrome coronavirus (MERS‐CoV) nsp15, and 43% with human coronavirus (H‐CoV‐229E) nsp15. (Kim et al. 2020)

Mutant nsp15 of mouse hepatitis virus strain A59 (MHV-A59), a model coronavirus that expressed an unstable catalysis-deficient nsp15, led to an early protective immune response by inducing type I interferon and causing early apoptotic cell death. The inactive mutant form of nsp15 led to the activation of the host dsRNA sensors associated with an array of immune responses. Nsp15 loss of activity also shortened the symptoms of disease acute hepatitis in mice, highlighting its role in coronavirus pathogenesis. (Deng et al. 2017)

The SARS-CoV-2 nsp15 monomer structure consists of an N-terminal oligomerization domain, a middle-domain, and a C-terminal NendoU catalytic domain. (Figure 1, Zenodo report) The functionally active form of SARS-CoV-2 nsp15 is a hexamer similar to the SARS- and MERS-CoV nsp15s. (Kim et al. 2020)

Considering the importance of nsp15 for viral evasion from the immune system, we analyzed the nsp15 catalytic site’s druggability using the SARS-CoV-2 nsp15 crystal structure (PDB: 6wlc). We assessed this site’s amino acid variability across members of Alphacoronavirus and Betacoronavirus genera and SARS-CoV-2 samples from COVID-19 patients.

The druggability assessment was done using SiteMap (Schrodinger, NY), and based on the druggability score of the nsp15 catalytic site (Dscore: 0.573), this site is likely not druggable. (Figure 2, Zenodo report)

Using ICM PocketFinder and the nsp15 structure (PDB: 6zsl), we identified 14 amino acid sidechains lining its catalytic site, which is occupied by uridine 5’-monophosphate. (Figure 3, Zenodo report)

To evaluate the nsp15 catalytic site’s amino acid conservation across coronaviruses, a multiple sequence alignment was made using sequences of nsp15 protein from Alpha- and Betacoronaviruses genera.

This analysis is part of a bigger picture survey of viral proteins from Alpha- and Betacoronaviruses to identify the most conserved druggable sites.

Looking across twenty-seven nsp15 sequences belonging to Alpha- and Betacoronaviruses genera, its catalytic site has 57% sequence identity and 78% sequence conservation. (Figure 4, Zenodo report) The amino acid variability of this site is also mapped to the SARS-CoV-2 nsp15 crystal structure. (Figure 5, Zenodo report)

Nicola De Maio, our collaborator from Nick Goldman’s lab at the European Bioinformatics Institute, looked at more than 15000 SARS-CoV-2 genome samples from COVID-19 patients and identified all mutations at the nsp15 catalytic site. He identified 7 non-synonymous variants at this site. (Table 1, Zenodo report) The SARS-CoV-2 variants of this site are also mapped to the SARS-CoV-2 nsp15 crystal structure. (Figure 6, Zenodo report)

The uridine 5’-monophosphate binding pocket of nsp15 is shallow and is predicted to be undruggable. The six key catalytic site residues remain conserved in SARS-CoV-1, SARS-CoV-2, and MERS-CoV proteins: His235, His250, Lys290, Thr341, Tyr343, and Ser294. (Kim et al.2020) Beyond these three viral species, the catalytic site of nsp15 has 57% sequence identity across several entries of the Alpha- and Betacoronavirus genera and has 78% amino acid conservation.  Although some evidence suggests the importance of nsp15 for coronavirus evasion from the host immune response, the shallowness of the catalytic site and its relatively low sequence identity do not encourage targeting this site using a broad-spectrum anti-coronavirus drug. (Deng et al. 2017)

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