Doctor of Philosophy, Case Western Reserve University, 2021, Molecular Virology

Extracellular vesicles (EVs) are lipid-bilayer enclosed, cell-derived nanoparticles released constitutively by most cells. EVs carry proteins and nucleic acids from their cells of origin, facilitating intercellular communication through a variety of different pathways. These include signaling through cellular receptors, triggering immune responses, and delivering bioactive cargos after EV internalization. Viruses share many characteristics with EVs, including size, structure, and biogenesis pathways. Virally infected cells continue to release EVs, resulting in the production of virus-induced EVs that carry viral elements like proteins or RNAs. Viral fusion proteins – viral envelope glycoproteins that mediate virus/cell membrane fusion – are often incorporated by virus-induced EVs. In this work, we investigated the effect that viral fusion protein incorporation has on EV communication and functionality. First, we investigated the spike protein of the current pandemic virus SARS-CoV-2. Interestingly, we found that EVs released from spike expressing cells did incorporate this type I viral fusion protein. These spike(+) EVs displayed the spike protein in a conformation available for antibody binding, allowing the EVs to serve as decoy targets for neutralizing antibodies. This held true with both convalescent patient serum and a commercial neutralizing antibody, with the effect of reducing virus neutralization and increasing infection. The second component of this work concerned the development of EV/cell membrane fusion assays to monitor EV cargo delivery. This EV communication pathway is particularly important in disease, where EVs are thought to be capable of influencing cells through membrane fusion and delivery of bioactive cargos to cell cytosol. We successfully developed two EV fusion assays, based on delivery of the proteins beta-lactamase and Cre-recombinase. In the third and final component of this work, we utilized these fusion assays to investigate the in (open full item for complete abstract)

Committee: John Tilton M.D. (Advisor); Alan Levine Ph.D. (Committee Chair); Alex Huang M.D./Ph.D. (Committee Member); Scott Sieg Ph.D. (Committee Member) Subjects: Biomedical Research; Molecular Biology; Virology

Master of Science in Biomedical Sciences (MSBS), University of Toledo, 2008, College of Graduate Studies

Vesicular stomatitis virus (VSV) is an RNA virus commonly used for the study of RNA virus evolution. Like other RNA viruses, VSV has a high mutation rate, averaging one mutation per genome per round of replication. This high mutation rate leads to a genetically heterogeneous population; each produced virus will have at least one new mutation. This collection of mutants is termed a quasispecies. Bottlenecking of a population results in the fixation of random mutations, which are more likely to be deleterious than those that are fixed through selection. Previous studies have found bottlenecked virus strains with adaptability defects that are unable to adapt and gain fitness normally. In the set of experiments presented here, I explored the quasispecies of these adaptability-deficient strains. Plaque sizes were measured for each strain as a surrogate of fitness values, and then fitness values for at least 200 clones of each strain were measured. Results suggested a lack of robustness in adaptability-deficient strains, as these strains produced a greater amount of deleterious mutants. In addition, these strains were less able to generate beneficial mutations. A lack of robustness would explain data collected both previously and in these experiments. This could have implications in vaccine design and treatment of RNA virus diseases.

Committee: Isabel Novella PhD (Committee Chair); Dorothea Sawicki PhD (Committee Member); Robert Blumenthal PhD (Committee Member) Subjects: Genetics; Molecular Biology; Virology

Master of Science in Biomedical Sciences (MSBS), University of Toledo, 2008, College of Graduate Studies

RNA viruses, such as HIV, influenza, and hepatitis viruses, are major sources ofhuman infection and disease. Although vesicular stomatitis virus (VSV) is not a major human pathogen, it serves as an excellent model for RNA virus evolution. In this work, I used VSV to test two predictions of ecological theory that are relevant to speciation. The first prediction is that viruses replicating in homogeneous environments will become specialists as a result of fitness tradeoffs or costs due to differences in fitness landscapes, while viruses that replicate in heterogeneous environments will become generalists. Results show one example of fitness trade-off and two examples of costs associated with fitness landscapes. In contrast to previous works and predictions from ecological theory, results did not show frequent fitness trade-offs. The second prediction is that phenotypic variance in generalist populations will be higher than phenotypic variance in specialist populations. Once again, the prediction was incorrect, and there was no correlation between the history or behavior of a population and it level of variation. However, populations adapting under high-MOI conditions did result in a substantial increase in variance, probably due to the ability of complementation to preserve variation.

Committee: Isabel Novella PhD (Committee Chair); R. Mark Wooten PhD (Committee Member); Nancy H. Collins PhD (Committee Member) Subjects: Virology