Kinetic Modeling of West Nile Virus Fusion Indicates an Off-pathway State

West Nile virus (WNV) is a prominent mosquito-borne flavivirus that causes febrile illness in humans. To infect host cells, WNV virions first bind to plasma membrane receptors, then initiate membrane fusion following endocytosis. The viral transmembrane E protein, triggered by endosomal pH, catalyzes fusion while undergoing a dimer-to-trimer transition. Previously, single-particle WNV fusion data was interrogated with a stochastic cellular automaton simulation, which modeled the E proteins during the fusion process. The results supported a linear fusion mechanism, with E protein trimerization being rate-limiting. Here, we present corrections to the previous simulation, and apply them to the WNV fusion data. We observe that a linear mechanism is no longer sufficient to fit the data. Instead, an off-pathway state is necessary; these results are corroborated by chemical kinetics modeling. When compared with a similar Zika virus fusion model, this suggests that off-pathway fusion mechanisms may characterize flaviviruses more broadly.