Structural and functional studies of the Flock House virus cell entry mechanism

When a virus infects a cell, its genome must cross the cellular membrane into the cytoplasm where viral replication takes place. Little is known about the cell entry mechanism used by nonenveloped viruses. Nonenveloped viruses lack a viral membrane and therefore must rely on a mechanism other than membrane fusion to deliver their genome into the cell. To study this mechanism we used Flock House virus, a nonenveloped virus with a single stranded positive sense RNA genome, as a model system. The mature Flock House virus capsid is composed of two structural proteins, beta and gamma. Much is known about Flock House virus capsid structure and stability from years of biochemical and structural analysis in vitro. The goal of the research described in this dissertation was to apply what is known about Flock House virus capsid structure and stability in vitro to understand the Flock House virus cell entry mechanism in vivo.; This dissertation is divided into two main topics of study. In the first set of studies, Flock House virus particles with a distinct conformation were identified and characterized. These "eluted" particles were isolated from cell-associated particles that fall away from cells during virus entry. The mature Flock House virus protein capsid is extremely stable to changes in the extracellular environment, including changes in redox state, pH, ionic strength, and temperature. However, once the virus adheres to a permissive cell the viral protein capsid undergoes conformational changes that lead to formation of eluted particles. The eluted particle conformation exhibits a significant change in stability that render eluted particles sensitive to environmental changes, changes that the mature Flock House virus capsid conformation is resistant to. These results led us to hypothesize that the eluted particle conformational changes are critical for the proper spatial and temporal delivery of the viral genome into the cell.; In the second set of studies we investigated the role of the Flock House virus capsid protein, gamma, during cell infection in vivo. The gamma capsid protein is membrane active in vitro, and a fraction of the 180 gamma proteins are lost from the viral capsid during formation of the eluted particle conformation. We hypothesize that gamma interacts with the cellular membrane and facilitates viral genome cell entry. We show that gamma interacts with cell membranes during viral infection and when supplied in trans, can restore the infectivity of gamma deficient virions. Our studies have led to the development of a new hypothesis for Flock House virus cell entry that furthers our knowledge of cellular entry by nonenveloped viruses.