Exploring regulatory functions and enzymatic activities in the nidovirus replicase

Members of the order Nidovirales (Coronaviridae, Arteriviridae, and Roniviridae) employ genomes with mRNA polarity (plus-strand) and encode one of the most complex RNA virus replicases currently known. This replicase is expressed from the viral genome by translation of two large 5__-proximal ORFs into two polyproteins, which are processed by virus-encoded proteases in 13-16 individual nonstructural proteins (nsps). The nsps direct the formation of an RNA-synthesizing complex that mediates viral genome replication, as well as the synthesis of a 3__-coterminal nested set of subgenomic (sg) mRNAs, from which the viral genes located downstream of the replicase gene are expressed. Arteriviruses and coronaviruses presumably employ a unique mechanism of discontinuous minus-strand extension to generate subgenome-length templates for sg mRNA synthesis. This thesis focused on the functional characterization of two replicase subunits and their roles in coupling different processes in the replicative cycle of equine arteritis virus (EAV), the arterivirus prototype. The biological importance of a conserved domain unique to nidoviruses (NendoU), mapping to arterivirus nsp11 and proposed to function as an endoribonuclease, was addressed. We demonstrated the recombinant nsp11 exhibits pyrimidine-specific endoribonuclease activity, and showed the critical importance of the NendoU domain for EAV RNA synthesis. In addition, we identified a multidomain replicase subunit, EAV nsp1, as a key coordinator of EAV genome replication, sg mRNA synthesis, and virus production. Our results reveal that the relative abundance of EAV mRNAs is tightly controlled by nsp1 and is critical for efficient production of new virus particles. The protein was implicated in modulating the accumulation of full-length and subgenome-length minus-strand templates for EAV mRNA synthesis. A protocol for purification of soluble recombinant nsp1, which can be used in future research on the molecular mechanisms of nsp1 function, is described.