Entry of enveloped viruses relies on insertion of hydrophobic residues of the viral fusion protein into the host cell membrane. However, the intermediate conformations during fusion remain unknown. Here, we address the fusion mechanism of Rift Valley fever virus. We determine the crystal structure of the Gn glycoprotein and fit it with the Gc fusion protein into cryo-electron microscopy reconstructions of the virion. Our analysis reveals how the Gn shields the hydrophobic fusion loops of the Gc, preventing premature fusion. Electron cryotomography of virions interacting with membranes under acidic conditions reveals how the fusogenic Gc is activated upon removal of the Gn shield. Repositioning of the Gn allows extension of Gc and insertion of fusion loops in the outer leaflet of the target membrane. These data show early structural transitions that enveloped viruses undergo during host cell entry and indicate that analogous shielding mechanisms are utilized across diverse virus families.

An emergent viral pathogen termed severe fever with thrombocytopenia syndrome virus (SFTSV) is responsible for thousands of clinical cases and associated fatalities in China, Japan, and South Korea. Akin to other phleboviruses, SFTSV relies on a viral glycoprotein, Gc, to catalyze the merger of endosomal host and viral membranes during cell entry. Here, we describe the postfusion structure of SFTSV Gc, revealing that the molecular transformations the phleboviral Gc undergoes upon host cell entry are conserved with otherwise unrelated alpha-and flaviviruses. By comparison of SFTSV Gc with that of the prefusion structure of the related Rift Valley fever virus, we show that these changes involve refolding of the protein into a trimeric state. Reverse genetics and rescue of site-directed histidine mutants enabled localization of histidines likely to be important for triggering this pH-dependent process. These data provide structural and functional evidence that the mechanism of phlebovirushost cell fusion is conserved among genetically and pathophysiologically distinct viral pathogens.emerging virus | phlebovirus | viral membrane fusion | bunyavirus | structure

Many phleboviruses (family Bunyaviridae) are emerging as medically important viruses. These viruses enter target cells by endocytosis and low pH-dependent membrane fusion in late endosomes. However, the necessary and sufficient factors for fusion have not been fully characterized. We have studied the minimal fusion requirements of a prototypic phlebovirus, Uukuniemi virus, in an in vitro virus-liposome assay. We show that efficient lipid mixing between viral and liposome membranes requires close to physiological temperatures and phospholipids with negatively charged headgroups, such as the late endosomal phospholipid bis(monoacylglycero)phosphate. We further demonstrate that bis(monoacylglycero)phosphate increases Uukuniemi virus fusion beyond the lipid mixing stage. By using electron cryotomography of viral particles in the presence or absence of liposomes, we observed that the conformation of phlebovirus glycoprotein capsomers changes from the native conformation toward a more elongated conformation at a fusion permissive pH. Our results suggest a rationale for phlebovirus entry in late endosomes.