Project Summary/Abstract Herpes Simplex Virus 1 (HSV-1) enters and infects cells by fusing its viral envelope with target cell membranes. Membrane fusion is catalyzed by the HSV-1 fusogen, glycoprotein B (gB). Based on our knowledge of other viral fusogens, gB is thought to irreversibly refold from its unstable prefusion form to its low energy postfusion from through large conformational changes that merge the viral and target membranes. However, unlike most enveloped viruses which catalyze fusion using a single protein, the fusion pathway in HSV-1 is more complex and is unusual, requiring three additional glycoproteins, gD, gL, and gH. In the hypothesized fusion pathway, gD binds a receptor on the target cell and activates the gH/gL complex, which activates gB to refold. However, it is not known how gB is maintained in its metastable prefusion conformation prior to fusion triggering, nor how gH activates gB to cause fusion. Our prior work suggests the cytoplasmic regions of gB and gH regulate this process, with the gB cytoplasmic domain (CTD) stabilizing prefusion gB and the gH cytotail (CT) activating gB to refold. Elucidating the mechanisms by which these regions regulate gB fusogenic activity is essential for understanding how the viral glycoproteins accomplish membrane fusion during viral infection. The objective of this proposal is to determine how the gB CTD stabilizes the prefusion conformation prior to fusion triggering, and how the gH CT interacts with gB to cause fusion. In Aim 1, I will identify regions in the gB CTD that are responsible for stabilizing gB by mutagenesis and an unfolding assay and determine whether increased CTD stabilization decreases fusion. In Aim 2, I will identify residues involved in the gH-gB interaction by mutagenesis and a protein-protein interaction assay, and their effect on fusion via a cell-cell fusion assay. These experiments will illuminate how cytoplasmic regions of gB and gH interact and regulate gB activity in the HSV-1 fusion pathway. The proposal is significant because it investigates the unknown events between gH activation and gB refolding that currently limit our understanding of herpesvirus fusion. The proposal is innovative because it uses original hypotheses to investigate a novel mechanism of protein activity regulation in the unique herpesviral fusion pathway. Successful completion of the proposed research would advance the HSV-1 field by increasing our understanding of the different steps in HSV-1 fusion in molecular detail. In line with the mission of NIAID, this would be an important step towards better treating and preventing HSV-1 infection. This fellowship will provide outstanding research training on my path to becoming a physician-scientist.