Capturing intermediates and membrane remodeling in class III viral fusion

Enveloped viruses enter cells by fusing their envelopes to host cell membranes. Vesicular stomatitis virus (VSV) glycoprotein (G) is a prototype for class III fusion proteins. Although structures of the stable pre- and postfusion ectodomain of G are known, its fusogenic intermediates are insufficiently characterized. Here, we incubated VSV virions with late endosome-mimicking liposomes at pH 5.5 and used cryo–electron tomography (cryo-ET) to visualize stages of VSV’s membrane fusion pathway, capture refolding intermediates of G, and reconstruct a sequence of G conformational changes. We observe that the G trimer disassembles into monomers and parallel dimers that explore a broad conformational space. Extended intermediates engage target membranes and mediate fusion, resulting in viral uncoating and linearization of the ribonucleoprotein genome. These viral fusion intermediates provide mechanistic insights into class III viral fusion processes, opening avenues for future research and structure-based design of fusion inhibition-based antiviral therapeutics.