During cell entry of flaviviruses, low endosomal pH triggers the rearrangement of the viral surface glycoproteins to a fusion-active state that allows the release of the infectious RNA into the cytoplasm. In this work, West Nile virus was complexed with Fab fragments of the neutralizing mAb E16 and was subsequently exposed to low pH, trapping the virions in a pre-fusion intermediate state. The structure of the complex was studied by cryo-electron microscopy and provides the first structural glimpse of a flavivirus fusion intermediate near physiological conditions. A radial expansion of the outer protein layer of the virion was observed compared to the structure at pH 8. The resulting ∼60 Å-wide shell of low density between lipid bilayer and outer protein layer is likely traversed by the stem region of the E glycoprotein. By using antibody fragments, we have captured a structural intermediate of a virus that likely occurs during cell entry. The trapping of structural transition states by antibody fragments will be applicable for other processes in the flavivirus life cycle and delineating other cellular events that involve conformational rearrangements.
West Nile Virus (WNV) and other related viruses such as dengue virus enter their host cell by a process that involves fusion between the viral membrane and the membrane of cellular vesicles (endosomes) resulting in the release of the viral genome into the cytoplasm of the cell. This fusion event is initiated by low pH in the endosomes. Little is known regarding structural changes within the viral particle that render the viral surface proteins capable of fusion. In the present study, we used antibody fragments as a tool to trap virions in a pre-fusion intermediate state and examined these particles by cryo-electron microscopy. We showed that low pH triggered a radial displacement of the virion's external protein layer. The surface proteins moved away from the viral membrane, a shift made possible by the outward extension of a small alpha-helical region of the surface protein. The process gives the proteins greater sideways freedom for their reorganization into the fusion-active state. Our results provide a first structural glimpse into the low pH-induced conformational rearrangement of the flavivirus particle that occurs prior to fusion of viral and endosomal membranes. Such dissection of the fusion process highlights targets for the development of antiviral strategies.