期刊
出版社
NATL ACAD SCIENCES
DOI: 10.1073/pnas.2106379119
关键词
SARS-CoV-2; exoribonuclease; proofreading; molecular dynamics simulations; crystal structure
资金
- US NIH [National Institute of General Medical Sciences (NIGMS)] [R35-GM118047, R01-GM132826]
- US NIH [National Cancer Institute] [P01-CA234228]
- NSF Rapid Response Research [MCB-2032054]
- RCSA Research Corp.
- University of California San Diego Moores Cancer Center 2020 SARS-COV-2 seed grant
- US NIH [NIGMS P30 GM124165]
- NIH Office of Research Infrastructure Programs High-End Instrumentation Grant [S10 RR029205]
- DOE Office of Science [DE-AC02-06CH11357]
- NSF [OAC-1818253]
This study reveals the structure of the ExoN protein in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which plays a crucial role in the accurate replication of the virus's RNA genome. The structure demonstrates how ExoN binds to its cofactor nsp10 and interacts with RNA substrates. The findings have implications for the development of antiviral drugs and strategies to mitigate the virulence of coronaviruses.
High-fidelity replication of the large RNA genome of coronaviruses (CoVs) is mediated by a 3'-to-5' exoribonuclease (ExoN) in nonstructural protein 14 (nsp14), which excises nucleotides including antiviral drugs misincorporated 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-angstrom resolution crystal structure of severe acute respiratory syndrome CoV 2 (SARS-CoV-2) ExoN in complex with its essential cofactor, 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. We also show that the ExoN activity can rescue a stalled RNA primer poisoned with sofosbuvir and allow RdRp to continue its extension in the presence of the chain-terminating drug, biochemically recapitulating proofreading in SARS-CoV-2 replication. Molecular dynamics simulations further show remarkable flexibility of multidomain nsp14 and suggest that nsp10 stabilizes ExoN for substrate RNA binding to support its exonuclease activity. Our high-resolution structure of the SARS-CoV-2 ExoN-nsp10 complex serves as a platform for future development of anticoronaviral drugs or strategies to attenuate the viral virulence.
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