Kinetic analysis of RNA cleavage and substrate specificity of coronavirus Nsp15 endoribonuclease

Nidhi Kalia, Kimberly Snell, Michael E. Harris

University of Florida

SARS-CoV-2, the virus responsible for the COVID-19 pandemic, relies on various nonstructural proteins (NSPs) for its replication and pathogenicity. Among these, Nsp15 stands out as a key player. This uridine-specific viral endonuclease is highly conserved among vertebrate infecting viruses of the nidovirales order and plays a critical role in the virus's strategy to evade the host immune response and enhance its replication. Understanding the functional properties and interactions of Nsp15 is essential for unraveling the intricate mechanisms of SARS-CoV-2 and developing potential therapeutic interventions to mitigate its impact on global health. Previous literature states that Nsp15 requires millimolar concentrations of divalent metal ions to perform its function, but our experiments indicate that divalent ions are dispensable for catalysis. We used single- and multiple-turnover kinetics on model substrates to demonstrate that Nsp15 is not a metalloenzyme. The pH-rate profiles in the presence and absence of Mn²⁺ reveal active-site ionizable groups. This data provides evidence for conventional acid–base catalysis and substrate-dependent divalent ion activation. Our kinetic data also demonstrates that Mn²⁺ activates catalysis by stabilizing alternative enzyme states with faster rates of substrate cleavage on the enzyme. Our ongoing studies focus on the determinants of sequence and structure specificity with initial evidence that proves that Nsp15 has a preference for bulge and single-stranded substrates over double-stranded substrates. These results shed new light on Nsp15 catalytic mechanism and substrate specificity and provide more information on defining biological substrates for Nsp15.