The Role of Low PH and Disulfide Shuffling in the Entry and Fusion of Semliki Forest Virus and Sindbis Virus

Semliki Forest virus (SFV), an enveloped alphavirus, infects cells via a membrane fusion reaction that is induced by the low pH in endocytic vesicles. The role of low pH in the entry of the alphavirus Sindbis virus (SIN) is unclear, and an alternative fusion mechanism involving receptor-induced disulfide bond rearrangements at neutral pH has been proposed. The entry properties of SFV and SIN were here compared in parallel using treatment with the weak base NH4Cl or the vacuolar ATPase inhibitors bafilomycin A-1 or concanamycin to neutralize endosome pH. Three membrane impermeant thiol modifying reagents, 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB), p-chloromercuriphenylsulfonic acid (pCMBS), and monobromotrimethylammoniobimane (Thiolyte MQ), were used to inhibit thiol-disulfide exchange reactions. Primary infection by both SFV and SIN was inhibited by neutralization of endosome pH using NH4Cl, bafilomycin, or concanamycin. The concentration of NH4Cl or bafilomycin required for inhibition correlated with the pH dependence of membrane fusion for SFV, SIN, and a pH-shift mutant of SFV. SFV and SIN infection were partially inhibited by the thiol blocker DTNB, but not by pCMBS or Thiolyte MQ. Our data suggest that acidic endosomal pH induces the fusion activity of both SFV and SIN during virus infection.