Peptide inhibitors recognize prefusion viral fusion proteins with heterogeneous stoichiometry and rapid kinetics.

Peptide fusion inhibitors, an important class of antivirals, block viral entry by targeting fusion proteins required for membrane fusion. However, their interactions with intact trimeric fusion proteins remain elusive; direct observation of binding on virions or native-like trimers has been lacking. Here, we developed a single-molecule imaging platform to visualize peptide binding in real time. LP-98 bound HIV-1 Envelope (Env) trimers on virions and, unexpectedly, prefusion-stabilized soluble Env trimers, with higher affinity for virion-associated Env and, among soluble trimers, a mutant Env. RSV fusion-inhibiting T-118 and 4ca similarly engaged prefusion-stabilized fusion (F) trimers with rapid kinetics and high-nanomolar affinities. Binding to prefusion Env or F demonstrates that peptide inhibitors can act earlier than the canonical prehairpin-intermediate model suggests. Individual binding events revealed heterogeneous peptide-to-trimer stoichiometries, with single-peptide occupancy predominating, while stepwise and simultaneous events revealed multiple routes to higher occupancy. These findings expand the canonical model of peptide fusion inhibition and provide previously inaccessible mechanistic insights into how antiviral peptide fusion inhibitors act.