Browsing by Author "Ykema, Matthew Ryan"

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    Discovery and Characterization of Lipid Disruption Activity in the Human Astrovirus Capsid
    (2022-12-02) Ykema, Matthew Ryan; McNew, James; Tao, Yizhi J
    Astroviruses (AstVs) are non-enveloped, positive single stranded RNA viruses that cause a wide range of inflammatory diseases in mammalian and avian hosts. The T=3 viral capsid is unique in its ability to infect host cells is a process driven by host proteases. Intercellular protease cleavages allow for viral egress from a cell, while extracellular cleavages allow for the virus to enter a new host cell to initiate infection. High resolution models of the capsid core indicate a large, exposed region enriched with protease cleavage sites. During maturation, the capsid goes through significant structural changes including the loss of many surface spikes. However, there is still a lack of structural information surrounding the immature VP90 capsid assembly, along with the matured VP70 and VP34/27/25 assemblies. I have expressed the VP90 protein and correlated the maturation of the viral protein to a previously uncharacterized function, disruption of lipid membranes in vitro. The original goal of this project was to model the stages of the capsid maturation cycle at high resolution. I was able to purify the VP90 state of the virus through expression and purification in a bacterial system, however full capsid assemblies were unable to be obtained. These purified VP90 proteins were used as a template for functional assays. It has been previously proposed that the AstV capsid can function as a toxic protein that causes an increased cellular membrane flux and disruption, but this function has not been observed in other conditions. I developed an assay to use artificially generated liposomes to test the lipid-disruption activity of AstV protein in vitro. I was the first to observe that the AstV VP90 protein has high levels of lipid-disruption activity only after maturation by trypsin treatment. This liposome assay was used to test many AstV proteins and lipid conditions. I have concluded that the lipid-disruption activity originates from the cleavage of trypsin sites from amino acids 299 to 313 in the AstV Outer Core domain, and the activity is dependent on the types of lipids present in the liposomes. Further understanding of the viral capsid structure and maturation process can contribute to characterizing the toxic properties of the AstV capsid, gastric therapeutics, and viral engineering applications.