Structure and dynamics of SARS-CoV-2 proofreading exoribonuclease ExoN

Abstract

High-fidelity replication of the large RNA genome of coronaviruses (CoVs) is mediated by a 3'-to-5' exoribonuclease (ExoN) in non-structural protein 14 (nsp14), which excises nucleotides including antiviral drugs mis-incorporated by the low-fidelity viral RNA-dependent RNA polymerase (RdRp) and has also been implicated in viral RNA recombination and resistance to innate immunity. Here we determined a 1.6-Å resolution crystal structure of SARS-CoV-2 ExoN in complex with its essential co-factor, nsp10. The structure shows a highly basic and concave surface flanking the active site, comprising several Lys residues of nsp14 and the N-terminal amino group of nsp10. Modeling suggests that this basic patch binds to the template strand of double-stranded RNA substrates to position the 3' end of the nascent strand in the ExoN active site, which is corroborated by mutational and computational analyses. Molecular dynamics simulations further show remarkable flexibility of multi-domain nsp14 and suggest that nsp10 stabilizes ExoN for substrate RNA-binding to support its exoribonuclease activity. Our high-resolution structure of the SARS-CoV-2 ExoN-nsp10 complex serves as a platform for future development of anti-coronaviral drugs or strategies to attenuate the viral virulence.

The Antiviral Effect of the Chemical Compounds Targeting DED/EDh Motifs of the Viral Proteins on Lymphocytic Choriomeningitis Virus and SARS-CoV-2

“…Accordingly, the ExoN activity of coronavirus nsp14 appears to be an important drug target to combat coronaviruses, including SARS-CoV-2 [ 14 ]. The structure of SARS-CoV-2 nsp14 has not yet been published in the protein data bank [ 15 ]; therefore, a predicted structure based on the SARS-CoV nsp14 was constructed ( Figure 4 A). Using this model, we calculated the stability of the DEEDh motif of SARS-CoV-2 nsp14 upon binding two chemical compounds, ATA and PV6R, in silico.…”

SARS-CoV-2 Nsp14 activates NF-κB signaling and induces IL-8 upregulation

“…Nsp14 is a conserved, multifunctional viral factor participating in synthesizing and modifying coronaviral sub-genomic (sg) RNAs (9). Nsp14 possesses a 3' to 5' exonuclease activity that excises mismatched base pairs during viral RNA replication (10)(11)(12), providing a proofreading function that increases the fidelity of viral RNA synthesis (13,14). Nsp14 also possesses RNA methyltransferase activity required for guanine-N7 methylation (15).…”

An atomistic model of the coronavirus replication-transcription complex as a hexamer assembled around nsp15

“…While a small degree of conformational flexibility of this protein has been observed in the available SARS-CoV x-ray crystal structures, a much larger conformational change has recently been demonstrated via MD simulations. [17] The conformational change we observe here is energetically unfavorable for isolated nsp14, but is compensated by binding to nsp15 and nsp16. The conformational change that nsp14 undergoes upon complexing with nsp15 and nsp16 enables its binding to nsp12.…”

“…[10][11][12][13] Unlike many other viral families, structures have been determined for all key proteins that are presumed to make up the coronavirus replication-transcription complex (RTC), several of which are shown in Figure S1. To date, there are x-ray crystal structures of coronavirus nsp13, [14,15] heterodimeric nsp14/nsp10, [6,16,17] heterodimeric nsp16/nsp10, [11,12,[18][19][20][21] hexameric nsp15, [22][23][24][25][26][27] dimeric nsp9, [28][29][30] and the N protein NTD bound to both dsRNA and the specific viral RNA oligo known as the transcription regulatory sequence (TRS), which is critical to the unusual template switching process that occurs during transcription. [31] Cryo-EM has been especially successful in illuminating the structure of the polymerase complex, made up of nsp12, nsp7, two subunits of nsp8, and up to two subunits of nsp13.…”

“…The available structures of SARS-CoV nsp14 suggest at least some limited flexibility between the two domains exists. [6,16] As they are linked by a 14-residue loop (residues 286-299), we considered the possibility that there may be greater flexibility between these two domains, an hypothesis supported by a recently published molecular dynamics (MD) study of SARS-CoV-2 nsp14 [17] and made more likely in the context of protein-protein perturbations. Thus, given our assessment that the ExoN could interact with the dsRNA on both the polymerase complex and the nsp15 hexamer, and that it could potentially adopt a range of conformations with respect to the MTase, we decided to handle the two domains separately, first optimizing the position of the ExoN (residues 1-285) and nsp10 on nsp15 and then docking the MTase (residues 300-526).…”