Mutagenesis of the E1 Domain of Sindbis Virus Glycoprotein Alters its pH-Dependent Fusion

Abstract

Sindbis virus pathogenesis can be affected by mutations at residues 75 and 237, individually or in combination, of the E1 domain of the viral glycoprotein. To understand the changes in fusion properties driven by these mutations, we constructed three new fusogen molecules (FMs) derived from the Sindbis virus glycoprotein (G75D, S237A and G75DS237A), and investigated their fusion activity using lentiviral nanovectors. The co-expression of FMs and antibodies on the viral particles, were shown to mediate viral transduction on targeted cells. We found that the G75D mutation enhances the transduction efficiency by broadening the pH range required for fusion. Conversely, the S237A mutation impairs infectivity; however, adding the G75D site mutation to this molecule (G75DS237A) was able to partially restore infectivity. We further demonstrate that G75D can initiate virus fusion in both the early and the late endosomes, while S237A requires that the virus be functionally transported to the late endosomes for fusion to occur. This study sheds light on the mechanism by which mutations in FMs can affect viral fusion and transduction, and explores the use of our FM-bound lentiviral nanovector platform as tool to elucidate the mechanisms of viral membrane fusion.