Recent insights into the structure and function of coronavirus ribonucleases

Abstract

Coronaviruses use approximately two‐thirds of their 30‐kb genomes to encode nonstructural proteins (nsps) with diverse functions that assist in viral replication and transcription, and evasion of the host immune response. The SARS‐CoV‐2 pandemic has led to renewed interest in the molecular mechanisms used by coronaviruses to infect cells and replicate. Among the 16 Nsps involved in replication and transcription, coronaviruses encode two ribonucleases that process the viral RNA—an exonuclease (Nsp14) and an endonuclease (Nsp15). In this review, we discuss recent structural and biochemical studies of these nucleases and the implications for drug discovery.

Innovation and renovation: FEBS Open Bio in 2023

“…Our third special issue of the year placed the limelight firmly on one specific virus: SARS‐CoV‐2. Guest editor Alexander Wlodawer commissioned three Review articles focussing on the structures of different proteins of SARS‐CoV‐2: Robin Stanley and co‐authors discussed the structure and function of ribonucleases [ 10 ]; Franck Martin and colleagues focussed on viral and cellular translation during SARS‐CoV‐2 infection, as mediated by NSP1 [ 11 ]; and finally, Xinquan Wang, Jiwan Ge and co‐authors discussed the evolution of and therapeutic targeting of the spike glycoprotein [ 12 ].…”

Substrate Specificity and Kinetics of RNA Hydrolysis by SARS-CoV-2 NSP10/14 Exonuclease

“…Scientists around the world have published enzymatic mechanisms and structures of viral proteins at an unprecedented rate. In particular, the kinetics and mechanism of fast RNA replication (300 nt/s) by the RNA-dependent-RNA polymerase (RdRp) and its inhibition by the nucleoside analog remdesivir have been established through a combination of kinetic and structural analyses. , The bifunctional NSP10/NSP14 exonuclease/methyltransferase complex has been implicated in genome proofreading , and RNA capping, and structural biologists have provided atomic structures of the exonuclease complex in isolation − as well as bound to the RdRp complex (NSP12/NSP7/NSP8) and the NSP13 helicase . However, important details regarding kinetics and specificity of the proofreading exonuclease are lacking.…”

“…11 However, there are contradictions in the literature as to whether a remdesivir incorporated into the primer strand of the RNA can be efficiently excised by the exonuclease complex 7,8,12 and whether the enzyme preferentially hydrolyzes single-stranded RNA over base-paired RNA or RNA with terminal mismatches. 3,4,7,8,10 To date, the ambiguity present in currently available studies on the exonuclease arises largely because initial experiments to characterize the exonuclease reaction have been performed under steady-state conditions with a single long time point, anywhere from 20 to 45 min, which does not provide a valid measure of substrate specificity. 13 Fixed time point assays famously underestimate enzyme discrimination because the amounts of product formed after long incubation times do not reflect the different rates of reaction for various substrates.…”

“…However, important details regarding kinetics and specificity of the proofreading exonuclease are lacking. Other groups have reported expression, purification, and preliminary activity assays for the SARS-CoV-2 exonuclease complex. ,− ,, From these studies, it is clear that the exonuclease activity of NSP14 (1) requires either Mg 2+ or Mn 2+ for catalysis ,,, (2) strongly prefers RNA bases over DNA bases, , and (3) is greatly stimulated by the addition of the noncatalytic NSP10 . However, there are contradictions in the literature as to whether a remdesivir incorporated into the primer strand of the RNA can be efficiently excised by the exonuclease complex ,, and whether the enzyme preferentially hydrolyzes single-stranded RNA over base-paired RNA or RNA with terminal mismatches. ,,,, …”

Structural investigations of proteins encoded by SARS‐CoV‐2

“…Robin Stanley kand her colleagues review the properties of two ribonucleases that process the viral RNA [ 1 ]. Nsp14 is an exonuclease, whereas Nsp15 is an endonuclease.…”