A Stable Prefusion Intermediate of the Alphavirus Fusion Protein Reveals Critical Features of Class II Membrane Fusion

Alphaviruses infect cells via a low-pH-triggered membrane fusion reaction mediated by the class II virus fusion protein E1, an elongated molecule with three extramembrane domains (DI-III). E1 drives fusion by inserting its fusion peptide loop into the target membrane and refolding to a hairpin-like trimer in which DIII moves toward the target membrane and packs against the central trimer. Three-dimensional structures provide static pictures of prefusion and postfusion E1 but do not explain this transition. Using truncated forms of E1, we reconstituted a low-pH-dependent intermediate composed of trimers of DI/II. Unexpectedly, DI/II trimers were stable in the absence of DIII. Once formed at a low pH, DI/II trimers efficiently and specifically bound recombinant DIII through a pH-independent reaction. Even in the absence of DIII, DI/II trimers interacted to form hexagonal lattices and to cause membrane deformation and tubulation. These studies identify a prefusion intermediate in class II membrane fusion.