Studies on fusion peptide mutants of Semliki Forest virus
Shome, Swati Ghosh
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Semliki Forest Virus (SFV) is an enveloped RNA virus that infects cells by a low pH dependent membrane fusion reaction catalyzed by the virus spike protein. The SFV spike protein has 3 subunits, El, E2 and E3. The El subunit contains a highly hydrophobic and conserved region, called the fusion peptide domain, that is thought to interact with the target membrane during fusion. Mutagenesis of the fusion peptide domain of the spike protein identified position 91 of the El subunit as being important for virus budding and fusion.;The first objective of this thesis was to further analyze the effect of mutations at position 91 and 83 using the SFV infectious clone.;Fluorescent lipid dye mixing assays were established to detect hemifusion, an intermediate in membrane fusion. Using this assay we showed that G91D was blocked in both hemifusion and complete fusion upon low pH treatment.;These studies demonstrate that mutations in the fusion peptide domain of the spike protein can affect virus assembly and virus fusion at a number of different stages. However not a glycine residues in the fusion peptide domain have similar function, since a G83D mutation at position 83 does not affect virus infectivity and causes only a slight shift in the pH threshold of fusion while a G91D mutation is completely blocked in fusion.;The second aim of this research addressed whether there are other regions of the spike protein that can compensate for inhibitory mutations in the fusion peptide domain. For this we selected for revertants of lethal mutations in the fusion peptide domain.;Naturally occurring infectious revertants of G91D and G91P were selected. Two infectious revertants of Delta91 were isolated, both of which regained a glycine at position 91. Thus all revertants of mutations at position 91 regained a glycine at that position even under conditions that greatly favored second site revertants. This result demonstrates that it is crucial for the viability of SFV to have a glycine at position 91. (Abstract shortened by UMI.).