Ebola virus disease is a global concern given its periodic occurrence, high lethality, and rapid spread, coupled with a lack of approved therapeutics and vaccines. New tools are needed to gain fundamental insight into the virus life cycle, particularly entry and fusion of the viral envelope with the host membrane, and to combat the spread of the disease. Some drugs that alter endo-lysosomal calcium can inhibit Ebola virus infection, hinting that calcium may play a role in Ebola virus entry. Calcium is known to mediate fusion of other enveloped viruses as well as synaptic vesicles (Chapter 1). Chapter 2 demonstrates that Zaire ebolavirus (EBOV) infection is increased in the presence of calcium and that increased infectivity is a result of calcium interacting with the EBOV fusion peptide. Calcium interacts with viral glycoprotein residues D522 and E540 to promote insertion of the fusion peptide into the host membrane. Notably, these residues are highly conserved across Filoviridae, suggesting that calcium-interfering drugs are important candidates in the search for antivirals against not only Ebola, but all filoviruses. Although receptor binding, low pH, removal of the glycan cap, and calcium have all been implicated in Ebola virus fusion, the exact trigger remains unknown. Techniques that enable the visualization of individual virions undergoing fusion can contribute substantially to clarifying the fusion trigger. Methods that track virions in live cells lend themselves to “top-down” experiments where individual cellular factors are knocked out either genetically or by drug treatment. Alternatively, biomimetic systems, such as those that monitor viral fusion with supported lipid bilayers, enable “bottom-up” approaches where factors are added in one by one. These approaches are discussed in detail in Chapter 3. Chapter 4 describes the construction of a biomimetic platform for investigating Ebolavirus fusion, including incorporation of the host receptor and control over exposure to potential fusion triggers. The ...