Cellular cholesterol abundance regulates potassium accumulation within endosomes and is an important determinant in Bunyavirus entry

The Bunyavirales order of segmented negative sense RNA viruses includes over 500 isolates that infect insects, animals, and plants, and are often associated with severe and fatal disease in humans. To multiply and cause disease, bunyaviruses must transport their genomes from outside the cell into the cytosol, achieved by transit through the endocytic network. We have previously shown that the model bunyaviruses Bunyamwera virus (BUNV) and Hazara virus (HAZV) exploit the changing potassium concentration ([K+]) of maturing endosomes to release their genomes at the appropriate endosomal location. K+ was identified as a biochemical cue to activate the viral fusion machinery, promoting fusion between viral and cellular membranes, consequently permitting genome release. In this study, we further define the biochemical prerequisites for BUNV and HAZV entry and their K+ dependence. We report four major findings: (1) BUNV and HAZV require cellular cholesterol during virus infection; (2) cholesterol is required during BUNV endosomal escape; (3) cholesterol depletion from host cells impairs their ability to accumulate K+ in maturing endosomes, revealing new insights into endosomal K+ homeostasis; (4) ‘priming’ BUNV virions with K+ prior to infection alleviates BUNV cholesterol requirement, revealing the mechanism of cholesterol dependence. Taken together, we provide a new model in which cholesterol abundance influences K+ endosomal homeostasis and consequently the efficiency of bunyavirus infection. The ability to inhibit bunyaviruses with existing cholesterol lowering drugs offers new options for future anti-bunyavirus interventions for pathogenic family members.