UCLA

Diseases caused by pathogenic microbes impose a substantial economic and public health burden on the world. Advancements in cryogenic electron microscopy (cryoEM) have revolutionized our ability to describe the atomic structures driving microbial pathogenesis, while eliminating artefacts introduced by classical structure methods. CryoEM improves both the biological context of these structures and broadens the scope of structural investigations to include large and dynamic complexes, where subtle differences can yield insights critical to combating these microbes. I began my thesis research by leveraging recent advancements in cryoEM image processing to address gaps in our understanding of the assembly and replication of double stranded RNA (dsRNA) viruses. We began with the study of a dsRNA virus with minimal genomic complexity, which lacks the capacity for intercellular infection. The resulting 3.6 Å structure of the viral capsid informs us on the essential features required for dsRNA virus replication. We next turned to a more complex dsRNA virus, Aquareovirus (ARV), which poses a serious threat to aquaculture. By resolving the asymmetric structure of ARV capsid core—an intermediate state of the replication process—to 3.3 Å, we could use the subtle differences between the complete and core particles to suggest a mechanism that explains the previously observed phenomenon of transcriptional inhibition in the complete particle. Our findings in both viruses deepen our understanding of viral replication in dsRNA viruses. Following this, we used cryoEM structures of the human cytomegalovirus (HCMV) capsid to guide the rational design of mutants targeting the protein-protein interface between the HCMV specific tegument protein, pp150, and the capsid. This structure guided mutagenesis allowed us to identify the potential mechanism of pp150 nuclear import and discover an attenuating mutation that slowed viral replication. Interestingly, this reduced replication rate did not compromise virion formation, suggesting such a mutation in clinical strains, may slow replication sufficiently for use as a vaccine strain, without affecting the virus’s antigenic profile. Lastly, we applied cryoEM to cytoskeletal elements from the common genitourinary parasite T. vaginalis (Tv). As Tv pathogenicity relies upon the multifunctional, motile flagella, we aim to characterize the flagellar cytoskeleton, namely the doublet microtubules of the axoneme. In doing so, we identified the minimally complex arrangement of microtubule inner proteins (MIPs) that facilitate motility through the viscous environment of the host genitourinary tract. Furthermore, we identified a Novel MIP Tv-specific protein as a potential drug target. This work demonstrates the versatility of cryoEM to resolve new high-resolution structures, identify novel drug targets, and guide the rational design of mutants to probe protein-protein interactions and inform on the mechanisms of pathogenesis.