The non-structural protein 15 (NSP15) locus in Lineage A β-coronaviruses has two important functions during replication. The encoded endoribonuclease is conserved among coronaviruses. The function of the nsp15 protein is still not fully understood, but recent evidence suggests it may be involved in both replication and inhibiting viral sensing of double stranded RNA. In Lineage A β-coronaviruses, the RNA locus contains an inserted packaging signal (P/S). The P/S is essential for selectively packaging viral genomic RNA. While the P/S is required for selective packaging, it is not required for nsp15 protein function or viral replication. Utilizing this region, I studied the interactions of nsp15 protein during infection. Additionally, I studied the effect of selective packaging on virulence. Coronaviruses encode 16 nonstructural proteins in two open reading frames. These proteins are responsible for forming the replication/ transcription complex (RTC) and creating an environment conducive to viral replication. The RTC is an intricate complex of viral and host proteins with a largely unknown composition. While almost all nsps studied to date localize to sites of replication, the interactions between these proteins are not fully understood. In Chapter II, I describe studies of the interactions and localization of Nsp15 by creating an in situ hemagglutinin epitope tag. I found that mouse hepatitis virus nsp15 could tolerate an in situ tag when placed into the P/S (MHVNsp15-HA). MHVNsp15-HA had wild-type like replication in vitro. Nsp15 was localized to sites of replication throughout infection, with no localization to sites of assembly. Finally, nsp15 interacted with the RNA dependent RNA polymerase and putative primase during infection. These data demonstrate that nsp15 is a member of the RTC. During coronavirus replication two species of viral RNA are present, genomic RNA (gRNA) and sub-genomic RNA (sgRNA). These RNAs are co-terminal on both their 5’ and 3’ ends, containing the leader sequence and 3’ UTR/ polyA respectively. Even with these similarities, coronaviruses are adept at selectively packaging gRNA over sgRNA. This selective packaging is determined by the P/S, a 95 base pair stem-loop structure in the nsp15 locus. This RNA motif is sufficient for packaging of nonviral RNAs and has been shown to interact with the M protein from MHV. Moreover, when this RNA motif is deleted from MHV, (MHVPS-) selective packaging is lost during infection as sgRNAs become a large percentage of packaged viral RNA. In chapter IV I determined the effect of selective packaging on pathogenicity in vivo. Immunocompetent mice infected with MHVPS- had significantly better outcomes compared to MHV wild-type (MHVWT) infected mice. Peak viral loads were decreased in MHVPS- compared to MHVWT. Strikingly I found MHVPS- infected bone marrow derived macrophages had significant increases in type-I interferons (IFNs) and pathogenesis of MHVPS- was restored in mice deficient in IFN signaling. These data indicate that the P/S of MHV is an uncharacterized MHV virulence factor, which acts by preventing an increased IFN response during infection. In MHV, the nsp15 locus is translated into a functional protein and contains functional cis acting RNA elements both of which play a role in MHV replication. This work provides understanding of nsp15 localization and interactions which educate our understanding of the function of this conserved endoribonuclease. Additionally, this work demonstrates a unique function for the P/S not previously described. This work informs future studies of nsp15 protein function and the function of selective packaging during coronavirus infection.

Time-resolved cryo-EM has already provided ground-breaking discoveries in various fields, including structural biology, biochemistry, and drug development. Compared to traditional structural biology methods where mostly stabilized conformations are reconstructed, the main advantage of time-resolved cryo-EM is its ability to capture dynamic processes in biological samples at near-atomic resolution, which allows for studying biological structures as they change and interact in real-time. In this project, I focused on the expression and purification of the individual proteins of two dynamic molecular complexes – Staphylococcus aureus (S. aureus) DNA gyrase and Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) replication-transcription complex – and attempted to assemble them into their functional forms for cryo-EM imaging.  Both of these complexes are interesting drug targets as they play an essential role in nucleic acid replication. The function of DNA gyrase is to modulate DNA supercoiling, facilitate DNA replication, and resolve intertwined DNA molecules. The replication-transcription complex of SARS-CoV-2 comprises, among other proteins, the RNA-dependent RNA polymerase, which, together with non-structural proteins 7 and 8, is responsible for the replication of the viral genome. There are still many questions about the underlying mechanisms of these key processes, and time-resolved cryo-EM studies will provide valuable information to advance our understanding of them. Here I present expression and purification protocols for S. aureus DNA gyrase subunits A and B and SARS-CoV-2 non-structural proteins 7, 8 and 12. DNA gyrase subunits A and B were expressed in Escherichia coli (E. coli) and purified in several steps, including affinity chromatography (His-Trap), ion exchange chromatography (IEX) and size exclusion chromatography (SEC). Despite many challenges with gyrase A precipitation, I obtained enough of both subunits for the intended cryo-EM. Different strategies to assemble them into a functional tetramer were tested but did not result in the expected outcome. The gained knowledge about the behaviour of the subunits in solution will serve as a basis for further optimization of the protocols before the assembly of the complex can be attempted again. Non-structural proteins 7 and 8 were expressed in E. coli as a polyprotein and successfully purified using His-Trap and SEC. I obtained a great amount of the polyprotein and established a protocol for its cleavage. Nsp12 was expressed using the baculovirus-insect cell expression system. The immunofluorescence assay data showed that the tested lipofection protocol works, and nsp12 is being produced in sufficient quantities. This result provides a solid base for further experiments to establish a purification method and assemble the nsp12-nsp7-nsp8 complex for cryo-EM imaging.