The Coronavirus Helicase and Exoribonuclease in Viral Replication and Fitness

Abstract

Coronavirus replication is directed by viral proteins that function as critical regulators of infection. The significant public health and agricultural burden of coronavirus diseases emphasizes the need to identify conserved determinants of replication. These studies are necessary in order to support surveillance efforts of circulating and emerging coronaviruses and develop targets for antiviral drugs. In this dissertation research, I investigate the role of the coronavirus helicase and exoribonuclease in virus replication and fitness. The enzymatic activities of both proteins have been established in vitro and have been shown to be important for efficient viral replication, but the contribution of discrete amino acid residues and protein domains was not well-studied. This work used structure-guided mutagenesis, passaging experiments, and amino acid alignments to generate mutant viruses for study in combination with in vitro experiments testing mutant protein function. This parallel approach leveraged viral genetics and biochemistry to define key protein residues across multiple systems. This work identifies a novel determinant of resistance to the approved antiviral remdesivir in the helicase, demonstrates that the helicase zinc binding domain is sensitive to mutagenesis, characterizes a novel regulator of replication kinetics in the exoribonuclease, and presents evidence toward a new model for the spatial-temporal regulation of the coronavirus multi-protein replication transcription complex during early and late infection.