NIH researchers find that the SARS-CoV-2 Nsp15’s uridine cleavage can evade the immune response

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) uses a uridine-specific endoribonuclease, called nonstructural protein 15 (Nsp15), to evade the immune system. It works by cleaving viral RNA and preventing the activation of double-stranded RNA sensors. Although how Nsp15 recognizes its RNA target for cleavage remains unclear.

Research conducted by scientists at the National Institutes of Health in the USA shows that Nsp15 targets uridines (U). Following detection of U, Nsp15 can then further cleave a broad spectrum of RNA substrates.

The research team writes:

Overall, this work establishes SARS-CoV-2 Nsp15 as a largely non-specific endoribonuclease with recognition for a minimal consensus motif (N)(U)^(R>U>>C) (where N is any base and R is a purine). Our data show that Nsp15 acts in a distributive fashion to catalyze cleavage following uridines.”

Understanding the mechanism behind Nsp15’s cleavage targets in coronaviruses could help develop future therapeutics that prevent immune evasion.

The study “Characterization of SARS2 Nsp15 Nuclease Activity Reveals it’s Mad About U” is available as a preprint on the bioRxiv* server.

How they did it

The researchers used a combination of techniques including cryo-EM, molecular dynamic simulations, and in vitro RNA cleavage assays to evaluate Nsp15’s substrate preferences beyond U. They performed a cryo-EM reconstruction of Nsp15 that’s RNA bound through information collected in its pre- and post-cleavage states.

The team looked at how the nucleotide 5’ and 3’ of uridine affected cleavage to determine RNA specificity. They also looked at how Nsp15 cleavages viral RNA substrates such as polyuridine and the transcriptional regulatory sequence.

Uridines help guide Nsp15’s towards RNA binding and cleavage

Nsp15 did not have any other base binding sites for RNA binding and recognition, indicating detection of U is critical for proper RNA cleavage.

Several N-terminal domain residues from a nearby protomer interacted with the B₂ adenine in the cryo-EM model. Considering they are essential for oligomerization and nuclease activity, the results suggest the N-terminal domain could help with engaging the RNA in the active site.

Thus, our structure-based point mutations suggest Nsp15 could be inhibited by disrupting the EndoU/NTD interface at the edge of the active site, which should destabilize the hexamer and lead to inactive monomeric enzyme,” wrote the researchers.

N278 influences uridine preference

N278 was the key residue found for maintaining Nsp15’s uridine preference. The residue is identical in Nsp15’s found in SARS-CoV-2 and MERS, but it differed across other coronaviruses. N278 interacts with S294 to move hydrogen bonding to favor uridines. This was further confirmed with a variation of N278 showing reduced activity on cleaving uridine-containing substrates.

The authors note that other factors not studied in this research cannot be ruled out and that there may be a possibility of others modulating sequence specificity and Nsp15’s preference towards uridine. However, the results provide strong evidence towards the N278/S294 pair supporting a high preference for cleaving uridines.

Substrate specificity points toward purines 3’ following a cleaved uridine

While Nsp15 can cleave polyuridine in RNA, there is a stronger preference between Nsp15 and the purines 3’ of the cleaved uridine outside of PolyU tracts. The results suggest Nsp15 evolved to have a strong preference for targeting uridine rather than cleaving randomly.

This work reveals that similar to RNase A, Nsp15 is a broad-spectrum endoribonuclease primarily guided to its cleavage targets by recognition of a single uridine.”

*Important Notice

bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.

Written by

Jocelyn Solis-Moreira

Jocelyn Solis-Moreira graduated with a Bachelor's in Integrative Neuroscience, where she then pursued graduate research looking at the long-term effects of adolescent binge drinking on the brain's neurochemistry in adulthood.