Faculty Collaboration Database

Coronaviruses lacking exoribonuclease activity are susceptible to lethal mutagenesis: evidence for proofreading and potential therapeutics. PLoS Pathog 2013 Aug;9(8):e1003565

Date

08/24/2013

Pubmed ID

23966862

Pubmed Central ID

PMC3744431

DOI

10.1371/journal.ppat.1003565

Scopus ID

2-s2.0-84883438898 (requires institutional sign-in at Scopus site)   314 Citations

Abstract

No therapeutics or vaccines currently exist for human coronaviruses (HCoVs). The Severe Acute Respiratory Syndrome-associated coronavirus (SARS-CoV) epidemic in 2002-2003, and the recent emergence of Middle East Respiratory Syndrome coronavirus (MERS-CoV) in April 2012, emphasize the high probability of future zoonotic HCoV emergence causing severe and lethal human disease. Additionally, the resistance of SARS-CoV to ribavirin (RBV) demonstrates the need to define new targets for inhibition of CoV replication. CoVs express a 3'-to-5' exoribonuclease in nonstructural protein 14 (nsp14-ExoN) that is required for high-fidelity replication and is conserved across the CoV family. All genetic and biochemical data support the hypothesis that nsp14-ExoN has an RNA proofreading function. Thus, we hypothesized that ExoN is responsible for CoV resistance to RNA mutagens. We demonstrate that while wild-type (ExoN+) CoVs were resistant to RBV and 5-fluorouracil (5-FU), CoVs lacking ExoN activity (ExoN-) were up to 300-fold more sensitive. While the primary antiviral activity of RBV against CoVs was not mutagenesis, ExoN- CoVs treated with 5-FU demonstrated both enhanced sensitivity during multi-cycle replication, as well as decreased specific infectivity, consistent with 5-FU functioning as a mutagen. Comparison of full-genome next-generation sequencing of 5-FU treated SARS-CoV populations revealed a 16-fold increase in the number of mutations within the ExoN- population as compared to ExoN+. Ninety percent of these mutations represented A:G and U:C transitions, consistent with 5-FU incorporation during RNA synthesis. Together our results constitute direct evidence that CoV ExoN activity provides a critical proofreading function during virus replication. Furthermore, these studies identify ExoN as the first viral protein distinct from the RdRp that determines the sensitivity of RNA viruses to mutagens. Finally, our results show the importance of ExoN as a target for inhibition, and suggest that small-molecule inhibitors of ExoN activity could be potential pan-CoV therapeutics in combination with RBV or RNA mutagens.

Author List

Smith EC, Blanc H, Surdel MC, Vignuzzi M, Denison MR

Author

Matthew C. Surdel PhD Assistant Professor in the Medicine department at Medical College of Wisconsin

MESH terms used to index this publication - Major topics in bold

Animals
Antiviral Agents
Apoptosis
Astrocytoma
Brain Neoplasms
Cell Proliferation
Coronavirus
Coronavirus Infections
Exoribonucleases
Genome, Viral
Humans
Mice
Mutagenesis
Mutagens
Mutation
RNA, Messenger
RNA, Viral
Real-Time Polymerase Chain Reaction
Reverse Transcriptase Polymerase Chain Reaction
Tumor Cells, Cultured
Viral Proteins
Virus Replication