Regulation of coronavirus nsp15 cleavage specificity by RNA structure

Abstract

SARS-CoV-2, the etiologic agent of the COVID-19 pandemic, has had a significantimpact on global public health. However, SARS-CoV-2 is only one of multiple pathogenic human coronaviruses (CoVs) to have emerged since the turn of the century. CoVs encode for several nonstructural proteins (nsps) that are essential for viral replication and pathogenesis. Among them is nsp15, a uridine-specific viral endonuclease that is important in evading the host immune response and promoting viral replication. Despite the established endonuclease function of nsp15, other determinants of its cleavage specificity have only recently begun to be investigated. In this study we investigate the role of RNA secondary structure in SARS-CoV-2 nsp15 endonuclease activity. We identified regions of differing predicted RNA secondary structure across the SARS-CoV-2 genome. Using a series of in vitro endonuclease assays, we observed that thermodynamically stable RNA structures were protected from nsp15 cleavage relative to RNAs lacking stable structure. We leveraged the s2m RNA from the SARS-CoV-1 3’UTR as a model RNA structure for our studies as it adopts a well-defined structure with several uridines, two of which are unpaired and thus highly probable targets for nsp15 cleavage. We found that SARS-CoV-2 nsp15 specifically cleaves s2m at the unpaired uridine within the pentaloop of the RNA. Further investigation revealed that the position of this uridine also impacted nsp15 cleavage efficiency suggesting that positioning within the pentaloop is necessary for optimal presentation of the scissile uridine and alignment within the nsp15 catalytic pocket. Our findings indicate that RNA secondary structure is an important determinant of nsp15 cleavage and provides insight into the molecular mechanisms of RNA recognition by nsp15. Understanding the broader implications of nsp15 activity will provide further insight not only into CoV biology but also into drug development against nsp15.