Second-site revertants of a Semliki Forest virus fusion-block mutation reveal the dynamics of a class II membrane fusion protein

The alphavirus Semliki Forest virus (SFV) infects cells through low-pH-induced membrane fusion mediated by the El protein, a class 11 virus membrane fusion protein. During fusion, El inserts into target membranes via its hydrophobic fusion loop and refolds to form a stable El homotrimer. Mutation of a highly conserved histidine (the H230A mutation) within a loop adjacent to the fusion loop was previously shown to block SFV fusion and infection, although the mutant El protein still inserts into target membranes and forms a homotrimer. Here we report on second-site mutations in El that rescue the H230A mutant. These mutations were located in a cluster within the hinge region, at the membrane-interacting tip, and within the groove where the El stem is believed to pack. Together the revertants reveal specific and interconnected aspects of the fusion protein refolding reaction.