Alphavirus entry and exit: Mechanisms of Semliki Forest virus budding
Lu, Yanping Emily
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Semliki Forest virus (SFV) and Sindbis virus (SIN), are two enveloped alphaviruses that are used extensively to study the mechanisms of membrane fusion and virus assembly. SFV and SIN enter the cells via endocytic pathway and low pH-mediated membrane fusion, and bud from the plasma membrane in a reaction requiring both the viral spike glycoproteins and the nucleocapsid.;The major part of my thesis focused on the establishment of a quantitative biochemical assay to study SFV budding, and its use to characterize the molecular requirements and mechanisms of SFV budding. This assay was based on cell-surface biotinylation of newly synthesized virus spike proteins and retrieval of biotinylated virions using strepavidin-conjugated magnetic particles. Budding of biotin-tagged SFV was continuous for at least two hours, independent of microfilaments and microtubules, strongly temperature dependent, and relatively independent of continuing exocytic transport. Studies of cell surface spike proteins at early times of infection showed that these spikes did not efficiently bud into virus particles and were rapidly degraded. In contrast, at later times of infection, spike protein degradation was markedly reduced and efficient budding was then observed, suggesting that efficient virus budding was dependent on a critical spike protein pool during the infection. The previously described cholesterol requirement in SFV exit was shown to be due to a block in the budding from the cell surface, and correlated with the continued degradation of spike proteins at all times of virus infection in sterol-deficient cells. In addition, the cholesterol requirement for virus budding could be modulated by a single point mutation in the spike protein. A model has been proposed on the mechanisms of SFV budding and cholesterol requirement.;Another aspect of my thesis focused on the cholesterol requirement for SIN infection and exit. I have shown that SIN, a distantly related alphavirus to SFV, is also strongly dependent on cellular cholesterol for the growth, primary infection, fusion, and exit, suggesting a general role of cholesterol in alphavirus entry and exit. Mutagenesis studies demonstrated that, similar to SFV, SIN cholesterol requirement can be modulated by sequences within the viral spike proteins.