The SARS-CoV-2 pandemic (COVID-19) threatens the entire world?s health, economy and social stability and is likely to continue for the foreseeable future. The capacity of this virus to cause protean manifestations and resist public health control demonstrates its profound evolutionary and adaptive capacity. We have studied the experimental evolution and determinants of fidelity and adaptation of CoVs for more than 20 years. The parent grant (R01 AI108197) for this proposed supplement defines the determinants of CoV replicase proteins in virus fidelity and pathogenesis, and is specifically directed toward understanding the role of the unique CoV exoribonuclease encoded in nonstructural protein 14 (nsp14-ExoN). Using SARS-CoV, MERS- CoV and MHV, we have shown that nsp14-ExoN mediates RNA proofreading and is responsible for: i) CoV high fidelity replication; ii) resistance to nucleoside analog inhibitors; iii) virus fitness; iv) evasion of host immunity; and v) virulence in vivo. Engineered mutants of MHV and SARS-CoV lacking ExoN (ExoN(-)) are impaired in all of the above functions and thus define ExoN as an exceptionally conserved and vulnerable virus encoded target for inhibition and attenuation. In this administrative supplement, we propose in vitro and in vivo studies of SARS- CoV-2 nsp14-ExoN, with the long-term goal define its role in virus replication and as a target for inhibitors and attenuation. We will rescue SARS-CoV-2 mutants of nsp14-ExoN and define their impact on replication and disease.
In Aim 1, we will introduce mutations into SARS-CoV-2CoV-2 nsp14-ExoN that are known in SARS- CoV, MERS-CoV, and/or MHV to abolish proofreading, alter nucleoside analog sensitivity, impact virus replication and fitness, or decrease virulence. Recovered viruses will be tested for these phenotypes.
In Aim 2, we will select replication-competent ExoN mutants with defined phenotypes for testing in highly relevant human airway epithelial (HAE) cultures and in a mouse model for SARS-CoV-2 replication and disease. The long- standing and highly productive collaboration between the Denison and Baric labs has already resulted in development of SARS-CoV-2 reverse genetics, initial analysis SARS-CoV-2 recombination, and potential animal models, all of which, in combination with our established bioinformatics pipelines, will allow rapid progress on the proposed supplement aims and will provide data for longer-term detailed studies of the role of ExoN and the viral polymerase. The significance and urgency of these studies is high, as they will rapidly result in identification of nsp14-ExoN targets for small molecule inhibitors and multiple pathways to stable and universal attenuation of SARS-CoV-2 and future zoonotic CoVs.

Public Health Relevance

SARS-CoV-2 is a devastating zoonotic pandemic CoV. Studies in this proposal will generate SARS- CoV2 with changes in the proofreading exoribonuclease that impair virus fitness and virulence. The outcomes will establish nsp14-ExoN as a target for our long-term goals of development of novel countermeasures including small molecule inhibitors and virus attenuation strategies.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Project (R01)
Project #
Application #
Study Section
Program Officer
Stemmy, Erik J
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Vanderbilt University Medical Center
United States
Zip Code
Graham, Rachel L; Deming, Damon J; Deming, Meagan E et al. (2018) Evaluation of a recombination-resistant coronavirus as a broadly applicable, rapidly implementable vaccine platform. Commun Biol 1:179
Menachery, Vineet D; Gralinski, Lisa E; Mitchell, Hugh D et al. (2018) Combination Attenuation Offers Strategy for Live Attenuated Coronavirus Vaccines. J Virol 92:
Athmer, Jeremiah; Fehr, Anthony R; Grunewald, Matthew E et al. (2018) Selective Packaging in Murine Coronavirus Promotes Virulence by Limiting Type I Interferon Responses. MBio 9:
Nakagawa, Keisuke; Narayanan, Krishna; Wada, Masami et al. (2018) The Endonucleolytic RNA Cleavage Function of nsp1 of Middle East Respiratory Syndrome Coronavirus Promotes the Production of Infectious Virus Particles in Specific Human Cell Lines. J Virol 92:
Case, James Brett; Li, Yize; Elliott, Ruth et al. (2018) Murine Hepatitis Virus nsp14 Exoribonuclease Activity Is Required for Resistance to Innate Immunity. J Virol 92:
Agostini, Maria L; Andres, Erica L; Sims, Amy C et al. (2018) Coronavirus Susceptibility to the Antiviral Remdesivir (GS-5734) Is Mediated by the Viral Polymerase and the Proofreading Exoribonuclease. MBio 9:
Sheahan, Timothy P; Sims, Amy C; Graham, Rachel L et al. (2017) Broad-spectrum antiviral GS-5734 inhibits both epidemic and zoonotic coronaviruses. Sci Transl Med 9:
Athmer, Jeremiah; Fehr, Anthony R; Grunewald, Matthew et al. (2017) In Situ Tagged nsp15 Reveals Interactions with Coronavirus Replication/Transcription Complex-Associated Proteins. MBio 8:
Graepel, Kevin W; Lu, Xiaotao; Case, James Brett et al. (2017) Proofreading-Deficient Coronaviruses Adapt for Increased Fitness over Long-Term Passage without Reversion of Exoribonuclease-Inactivating Mutations. MBio 8:
Case, James Brett; Ashbrook, Alison W; Dermody, Terence S et al. (2016) Mutagenesis of S-Adenosyl-l-Methionine-Binding Residues in Coronavirus nsp14 N7-Methyltransferase Demonstrates Differing Requirements for Genome Translation and Resistance to Innate Immunity. J Virol 90:7248-7256

Showing the most recent 10 out of 23 publications