<p dir="ltr">Viruses are obligate, intracellular pathogens meaning they are dependent on a host to produce progeny virus. Generally, viruses contain genetic material that is encapsulated in a protein shell. This protein shell can be encased in a lipid layer with glycoproteins classifying them as enveloped viruses. Flaviviruses are enveloped, positive sense RNA viruses transmitted through the bites of infected arthropods. Flaviviruses cause a variety of symptoms including general malaise, encephalitis, meningitis, hemorrhagic fever, congenital defects, and death. Approximately 3 billion people are at risk of contracting a flavivirus infection each year and with limited vaccine options, the development of effective therapeutics is critical for global human health. This thesis focuses on characterizing the effects flavivirus membrane perturbation has on structure and infectivity. The rationale behind this work was to determine if the viral membrane can be leveraged as a target for viral inactivation and vaccine development.</p><p dir="ltr">Flavivirus structural studies have identified targets for the development of antivirals and vaccines as well as bottlenecks hindering successful therapeutic development. Chapter 1 situates and expands on the global burden, life cycle, and structure of flaviviruses. Additional information on vaccine availability and how flavivirus structure and antibodies guide vaccine development is provided with emphasis on the importance of understanding flavivirus structural dynamics for successful vaccines.</p><p dir="ltr">Chapter 2 introduces the thesis’s study, where the flavivirus lipid membrane was leveraged as an inactivation target for the development of new vaccine methodology. After learning that Tween 20, a non-ionic detergent, was capable of flavivirus inactivation, a combination of imaging and molecular tools were utilized to characterize the particles after Tween 20 treatment. Increasing temperatures to 37°C improved viral inactivation determined by plaque assay. After determining what concentrations of Tween 20 impaired infectivity, Kunjin Virus (KUNV) was used as a model virus to assess structural changes that occurred during Tween 20 incubation. Resulting particles lacked homogeneity in the size and morphology of the glycoprotein shell but consistently had a reduction of internal structural protein Capsid and similar quantity of viral RNA. Repeated study trails with the French Polynesian strain of Zika Virus (ZIKV) yielded similar results.</p><p dir="ltr">Further experimentation detailed in Chapter 3 describes attempts to expand current knowledge of flavivirus uncoating and assembly mechanisms by disrupting flavivirus structure and protein-RNA interactions. To expand on the results discussed in chapter 2, Tween 20 was used to test disruption of Capsid-RNA binding reactions in vitro as well as induce genome leakage. It was determined that Tween 20 was not directly interfering with the retention of RNA and loss of Capsid, but more likely a result from Tween 20 integrating into the viral membrane. Additionally, after Tween 20 treatments, the RNA remaining was exposed, so ribonuclease protection assays were utilized to see if there was a region of genome that was consistently protected after treatment. The incubation of KUNV with Tween 20 and ribonucleases XRN1, Exo T, and RNase A determined that there was not a genome region consistently protected. Chapter 3 also includes preliminary results of an intra-lab collaboration to develop styrene maleic lipid nanoparticles from purified KUNV as a tool to probe for glycoprotein-Capsid protein interactions.</p><p dir="ltr">In Chapter 4, further analysis of flavivirus asymmetric reconstructions were done with the ZIKV Dakar strain. Previously, flaviviruses have been observed to have imperfect glycoprotein shells which could expose the presumably completely covered lipid membrane. To further evaluate asymmetric features, purified ZIKV was incubated with antigen binding fragments (Fabs) to stabilize the virions for improved resolution. Mature ZIKV was incubated with potently neutralizing Fab ZV-117 and immature ZIKV with ZV-67. In the mature and immature asymmetric reconstructions, a disruption in the glycoprotein shell was observed suggesting that the viral membrane is exposed.</p><p><br></p>

<p>Human noroviruses (HuNoVs) are the leading cause of an acute form of non-bacterial gastroenteritis, where strains belonging to genogroup (G) II are dominant. Upon expression with the baculovirus culture system, virus-like particles (VLPs) of HuNoVs are expected to assemble into T = 3 icosahedral capsid particles resembling the structure of the infectious virion particles. However, some strains were found to assemble into either T = 1 or T = 4 capsids, or a combination of two different capsid forms. In this study, we showed that VLPs of the Virginia 1997 387 (VA387) GII.4 outbreak strain assembled into T = 1, T = 3, and T = 4 capsids upon expression in insect cell culture, the first case for a naturally occurring HuNoV strain to assemble into all three capsid states. TEM analysis revealed that T = 1 icosahedral particles were the most abundant in purified samples, which contrasts previous findings where either T = 3 or T = 4 were the most abundant. We resolved the cryo-EM structures of the T = 1 shell (S) domain, T = 3, and T = 4 particles to 2.24, 2.44, and 3.43 Å, respectively, making them the most resolved norovirus (NoV) structures to date. Single particle cryo-EM 3D analysis showed that the protruding (P) domain of T = 1 and T = 4 VLPs are highly dynamic. Additionally, we showed that T = 3 VLPs are resistant while T = 1 and T = 4 VLPs are sensitive to digestion in the presence of trypsin. This suggested that T = 1 and T = 4 capsids are less stable among the VLPs, which is consistent with the highly dynamic P domain inferred from our cryo-EM 3D analysis. During infection, HuNoVs travel through the gastrointestinal (GI) tract where they encounter a broad range of variable conditions that include pH, ionic strength, and host defenses (e.g., proteases). Our analyses suggest that virions are T = 3 particles as they can survive the GI tract upon exiting the host. We determined the first cryo-EM structure of T = 3 VLPs in complex with the known HuNoV host cell receptor, histo-blood group antigen (HBGA), to a resolution of 2.51 Å, demonstrating that NoV VLPs can serve as a platform in the structural characterization of small ligand molecules. Lastly, we identified a histidine residue retained in the S domain of all identified caliciviruses critical in the assembly of capsids. Our structures and their characterization will contribute to the development of therapeutic agents to combat noroviruses. </p>