Biochemical and genetic analysis of alphavirus entry and membrane fusion

Abstract

In order to initiate infection, the membrane of the alphaviruses, Semliki Forest virus (SFV) and Sindbis virus (SIN), must undergo fusion with a cellular membrane to deliver the RNA genome into the cell cytoplasm. Fusion is mediated by the viral spike glycoprotein, which consists of 3 subunits, E1, E2, and E3. SFV membrane fusion occurs within the endosome via low pH triggered conformational changes of the spike. SIN virus fusion at the plasma membrane, potentiated by disulfhydryl exchange reactions has also been proposed. E3 remains associated with the SFV virus particle, but not with the SIN particle perhaps influencing the mechanism and site of membrane fusion.;The first objective of this thesis research was to compare the early entry mechanisms of SFV and SIN, evaluating the contributions of disulfide-exchange and low pH as fusion triggering mechanisms. The effects of membrane impermeant sulfhydryl blockers, and endosomal acidification inhibitors on SFV and SIN entry was evaluated during virus infection. The endosomal acidification inhibitors dramatically inhibited both SFV and SIN infection whereas, the sulfhydryl inhibitors only showed slight inhibition. In addition, SIN was found to display a lower pH threshold for virus-cell fusion than SFV. These results indicate that both SFV and SIN require low pH to trigger fusion within the endosome.;The spike glycoprotein undergoes ordered conformational changes upon low pH exposure. The second aim of the thesis research was to characterize the biochemical and genetic defect of a previously isolated SFV pH conditional mutant, fus-1. Extensive biochemical analysis of the fus-1 mutant showed that dissociation of the fus-1 E1-E2 dimer required a more acidic pH of 5.4, as did the subsequent E1 and E2 conformational changes. The structural protein genes of fus-1 have been subcloned into an SFV infectious clone. The phenotype of the subclone was found to be indistinguishable from fus-1. Comprehensive sequencing studies identified a point mutation which changed the fus-1 E2 Thr{dollar}\sb{lcub}12{rcub}{dollar} to Ile. Defining the fus-1 mutation has identified a novel region in the E2 subunit involved in controlling the pH dependence of SFV fusion.