Single Particle Kinetic And Electron Cryo-Microscopy Studies Of Flavivirus Membrane Fusion

Flaviviruses (a family that includes West Nile, Dengue, Tick-borne encephalitis and Zika viruses) use the envelope protein E, to promote membrane fusion during viral entry by undergoing a low-pH triggered conformational reorganization. We have examined the mechanism of WNV fusion and sought insight for intermediates during the conformational transition by following hemifusion of WNV virus-like particles in a single particle format, with small molecule inhibitors and using single particle electron cryo-microscopy (Cryo-EM). At the level of individual E subunits, trimer formation and membrane engagement of three fold clustered fusion loops is a kinetic bottleneck. Simulation of the kinetics indicates hemifusion requires at least two adjacent trimers, and that the availability of competent monomers within the contact zone between virus and target membrane is limiting. We present an initial Cryo-EM reconstruction of the full length post-fusion trimer and discuss how these visualization of the transmembrane regions, completed by further kinetic studies can describe principles of flavivirus entry generally applicable to mechanisms of membrane fusion.