Viral infection requires stable binding of viral fusion proteins to host membranes, which contain hundreds of lipid species. The mechanisms by which fusion proteins utilize specific host lipids to drive virus–host membrane fusion remains elusive. We conducted molecular simulations of classes I, II, and III fusion proteins interacting with membranes of diverse lipid compositions. Free energy calculations reveal that class I fusion proteins generally exhibit stronger membrane binding compared to classes II and III — a trend consistent across 74 fusion proteins from 13 viral families as suggested by sequence analysis. Class II fusion proteins utilize a lipid binding pocket formed by fusion protein monomers, stabilizing the initial binding of monomers to the host membrane prior to assembling into fusogenic trimers. In contrast, class III fusion proteins form a lipid binding pocket at the monomer–monomer interface through a unique fusion loop crossover. The distinct lipid binding modes correlate with the differing maturation pathways of classes II and III proteins. Binding affinity was predominantly controlled by cholesterol and gangliosides as well as via local enrichment of polyunsaturated lipids, thereby locally enhancing membrane disorder. Our study reveals energetics and atomic details underlying lipid recognition and reorganization by different viral fusion protein classes, offering insights into their specialized membrane fusion pathways.

Mpox is a zoonotic disease endemic to Central and West Africa. Since 2022, two human-adapted monkeypox virus (MPXV) strains have caused large outbreaks outside these regions. Tecovirimat is the most widely used drug to treat mpox. It blocks viral egress by targeting the viral phospholipase F13; however, the structural details are unknown, and mutations in the F13 gene can result in resistance against tecovirimat, raising public health concerns. Here we report the structure of an F13 homodimer using X-ray crystallography, both alone (2.1 Å) and in complex with tecovirimat (2.6 Å). Combined with molecular dynamics simulations and dimerization assays, we show that tecovirimat acts as a molecular glue that promotes dimerization of the phospholipase. Tecovirimat resistance mutations identified in clinical MPXV isolates map to the F13 dimer interface and prevent drug-induced dimerization in solution and in cells. These findings explain how tecovirimat works, allow for better monitoring of resistant MPXV strains and pave the way for developing more potent and resilient therapeutics.