Dissecting the role of the coronavirus proofreading exoribonuclease in RNA recombination

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.