The 2013–2016 Ebola virus (EBOV) disease epidemic was orders of magnitude larger than any previous EBOV outbreak. Preliminary data indicate that GP-A82V, an EBOV glycoprotein mutant that came to dominate the outbreak, increases infectivity in human cells. To elucidate the mechanism by which GP-A82V increases infectivity, and to clarify its significance for Ebola virus replication and transmission, we have assembled a team that leverages NIAID resources at the IRF-Fort Detrick and the Genomic Center for Infectious Diseases at The Broad Institute. Aim 1 will be to investigate the mechanism by which GP-A82V increases virion fusogenicity. Computer modeling suggests that GP-A82V destabilizes glycoprotein conformation. Mutations engineered based on the models will be tested for effects on infectivity, the reorganization of critical interactions as determined by molecular dynamics simulations, conformational equilibrium as determined by smFRET, novel assays for GP fusion, Cryo-EM of GP trimers, and crystal complexes with the NPC1 C-loop. Aim 2 will be to assess the effect of GP-A82V in the context of the EBOV Makona variant on infectivity in human cells in vitro and in humanized mice. We will generate a reverse genetic system for the ancestral EBOV Makona lineage and test the effect of GP-A82V on this background. Replication of WT and GP-A82V will be compared in U20S cells, in human dendritic cells, and in a novel humanized mouse model where the effect of GP-A82V on virus sequence adaptation to specific tissue compartments will be assessed. From these experiments we expect to clarify the significance of GP-A82V for viral replication and transmission, taking into account the genetic background of the EBOV and the species-specific effects of GP-A82V. Aim 3 will be to examine the effect of GP-A82V on neutralizing antibodies. Preliminary data indicate that GP-A82V is relatively resistant to neutralization by particular antibodies. Using a panel of monoclonal antibodies targeting different parts of GP, we will determine whether neutralization resistance is a general property of GP-A82V, or if this trait is specific to antibodies targeting particular regions of GP. If differential neutralization is observed with particular antibodies, the effect of these on viral titer will be tested in the humanized mouse model. We will also determine whether GP-A82V alters neutralization sensitivity to convalescent sera from Guineans infected early or later in the outbreak, and from individuals treated at Emory University. From these studies we hope to determine whether the antibody response to EBOV was different depending on whether a person was infected with virus bearing GP-A82 or GP-A82V. If differences in neutralization titer correlate with virus genotype it would contribute to understanding the factors that determine survival in an infected individual or the efficiency of transmission to people who come into contact with infected body fluids. Finally, these studies will provide valuable experimental tools that will inform our studies on GP structure and function.