Defining the determinants and outcomes of coronavirus recombination

Abstract

Coronaviruses (CoVs) are a genetically diverse group of positive-sense single-stranded RNA viruses containing the largest known genome of any RNA virus, >30 kb, and responsible for the current COVID-19 pandemic (SARS-CoV-2). CoV RNA-RNA recombination is essential for CoV sub-genomic mRNA synthesis and defective viral genome generation (DVG), and is proposed to be critical for novel CoV genome generation, evolution, and repair of mistakes during RNA synthesis. Work in this dissertation shows that multiple betacoronaviruses including murine hepatitis virus (MHV), MERS-CoV, and SARS-CoV-2, generate extensive and diverse recombination products during replication in vitro. Further, analysis of an attenuated CoV with engineered loss of the nsp14 3’-to-5’ exoribonuclease (nsp14-ExoN) is shows that nsp14-ExoN is required for normal CoV recombination and determines recombination junction site selection during replication and transcription. MHV can adapt to replication defects resulting from genetic inactivation of nsp14-ExoN activity, and that adaptation is associated with increased recombination that equals or exceeds WT levels. And finally, these studies show that alteration of recombination and RNA synthesis by the antiviral nucleoside analog β-D-N4-hydroxycytidine (NHC), the parent molecule of the clinically relevant therapeutic molnupiravir (MOV, EIDD-2801, MK-4482), may represent an additional mechanism of inhibition and a potential generalizable principle of nucleoside analogs. Work in this dissertation defines the extent and patterns of CoV recombination and its association and requirement for virus fitness. The reported results also identify a novel recombination function for nsp14-ExoN in addition to demonstrated roles in fidelity, resistance to nucleoside analogs, antagonism of innate immunity, and in vivo virulence. Taken together with the effect of NHC treatment on CoV recombination and RNA synthesis, these results increasingly emphasize the importance of CoV recombination as a conserved and vulnerable target for inhibition and attenuation.