Identification and characterization of E266Q mutation in Nsp15 endoribonuclease from SARS-CoV-2 Epsilon variant

SARS-CoV-2 is responsible for the ongoing global pandemic of COVID-19. Nsp15, an endoribonuclease from SARS-CoV-2 plays active roles in immune evasion and hence emerged as a drug target for COVID-19. Here we report the identification and characterization of a high frequency mutation in Nsp15 from the SARS-CoV-2 variant, Epsilon. First detected in California, USA in July 2020, Epsilon exhibited increased transmissibility compared to other SARS-CoV-2 variants circulating at the time. We performed multiple sequence alignment of 126 genomes of epsilon and identified four non-synonymous amino acid changes in Nsp15 (V66L, A81V, D183N and E266Q). We created these four single mutants to study their impact on Nsp15 catalysis. We also created a catalytically inactive mutant (H234A) for comparative analysis. Initial protein expressions revealed, E266Q which had a mutation rate of 4.76% exhibited very low expression levels compared to wild type (WT) whereas the inactive H234A exhibited very higher expression levels. This observation was reiterated by large-scale protein purification as well. We hypothesize that being an endoribonuclease, Nsp15 might be cleaving its own mRNA during recombinant expression, thus the activity levels of E266Q, WT, and H234A directly correlate to their corresponding expression levels. We performed a preliminary activity assay which demonstrated catalytic efficiency of E266Q is equal to or even slightly higher than WT. Enzyme kinetics experiments are underway to confirm this phenomenon. We also designed a new mutation E266A to further explore the effect of the E266 residue in Nsp15 activity. Currently, we are pursuing comparative structural and functional studies on E266Q, E266A, WT, and H234A to understand how E266Q mutation renders increased catalytic activity to Nsp15 and influences Epsilon's disease transmissibility.