# Dissecting the role of the coronavirus proofreading exoribonuclease in RNA recombination

> **NIH NIH F31** · VANDERBILT UNIVERSITY · 2021 · $11,105

## Abstract

PROJECT SUMMARY
Coronaviruses (CoVs) are a family of positive-sense RNA viruses that cause respiratory illnesses in humans
ranging from the common cold to severe and lethal disease. The epidemic of severe acute respiratory syndrome
coronavirus (SARS-CoV) in 2002-2004 and emergence of Middle East respiratory syndrome coronavirus
(MERS-CoV) since 2012 emphasize the capacity of CoVs to emerge by strain recombination and cause new
zoonotic infections with pandemic potential. Despite the high mortality rates of these infections, no therapeutics
or vaccines against any CoVs are currently available. Coronaviruses, whose genomes are several times larger
than other positive-strand RNA viruses, encode a proofreading exoribonuclease in nonstructural protein 14
(nsp14-ExoN) that is distinct from the RNA-dependent RNA polymerase (nsp12-RdRp). We have demonstrated
that catalytic inactivation of nsp14-ExoN (ExoN(-)) decreases replication fidelity, resulting in increased mutation
frequency, increased sensitivity to RNA mutagens, decreased replication, decreased fitness, and stable
attenuation in vivo. Recent findings in other RNA viruses have linked replication fidelity and recombination, and
our preliminary studies support scientific premise that replication fidelity contributes to recombination by
demonstrating that ExoN(-) mutants of the model coronavirus, murine hepatitis virus (MHV) have impaired
recombination. The goals of this proposal are to define the role of nsp14-ExoN in CoV recombination and test
whether the structural and amino acid determinants of ExoN fidelity regulation are linked to recombination. In
Specific Aim 1 we will compare WT and ExoN(-) MHV for changes in RNA recombination by next-generation
sequencing and in vitro infection assays. In Specific Aim 2 we will use structure-directed mutagenesis to
determine the impact of nsp14-ExoN structural elements and amino acid residues on MHV recombination,
replication fidelity, and fitness. Together, these studies will define the nsp14-ExoN determinants of CoV
recombination and explore the potential role of nsp14-ExoN in new strain emergence. This research also will
inform the development of next-generation sequencing approaches to investigate CoV recombination and RNA
synthesis. Finally, these studies will utilize recombinant RNA as tools to better understand CoV replication
fidelity, fitness, and viral emergence and evolution.

## Key facts

- **NIH application ID:** 10268982
- **Project number:** 5F31AI147560-02
- **Recipient organization:** VANDERBILT UNIVERSITY
- **Principal Investigator:** Jennifer Gribble
- **Activity code:** F31 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $11,105
- **Award type:** 5
- **Project period:** 2020-09-01 → 2021-12-26

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10268982

## Citation

> US National Institutes of Health, RePORTER application 10268982, Dissecting the role of the coronavirus proofreading exoribonuclease in RNA recombination (5F31AI147560-02). Retrieved via AI Analytics 2026-05-26 from https://api.ai-analytics.org/grant/nih/10268982. Licensed CC0.

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