SINDBIS VIRUS MEMBRANE-FUSION IS MEDIATED BY REDUCTION OF GLYCOPROTEIN DISULFIDE BRIDGES AT THE CELL-SURFACE

We have examined the role of thiol-disulfide exchange reactions during the penetration of cells by Sindbis virus. The protein-protein associ ations that form the rigid icosahedral lattice of the Sindbis virus en velope have been shown to be stabilized by disulfide bridges, and redu ction of these critical disulfide bridges during cell penetration may be the mechanism by which the rigid protein lattice is disrupted prior to fusion (R. Anthony and D. T. Brown, J. Virol. 65:1187-1194, 1991; R. Anthony, A. Paredes, and D. T. Brown, Virology 190:330-336, 1992). Reduction of disulfide bridges occurs at near neutral pHs via thiol-di sulfide exchange reactions, and these reactions can be blocked by cova lent modification of the thiol involved. In this study, the effects of the reducing agent 2-mercaptoethanol on Sindbis virus-mediated cell-c ell fusion from without and the effects of the membrane-impermeable th iol-alkylating reagent 5,5'-dithiobis(2-nitrobenzoic acid) on Sindbis virus penetration were determined. The presence of exogenous reducing agent was found to induce fusion from without under conditions unfavor able to both typical Sindbis virus-mediated fusion from without and cy steine-mediated thiol-disulfide exchange reactions. In addition, the t hiol-alkylating reagent was found to inhibit Sindbis virus entry when present during infection. These results are consistent with a model fo r Sindbis virus entry in which reduction of critical disulfide bridges at the cell surface disrupts the rigid protein-protein associations o f the envelope, allowing membrane fusion and release of the viral geno me into the cell.