PROJECT SUMMARY/ABSTRACT Herpes Simplex virus type 1 (HSV-1) requires four glycoproteins for cell entry and membrane fusion – gB, gD, gH, and gL – in addition to a cellular receptor. A thorough knowledge of membrane fusion mechanisms is essential for understanding how herpesviruses penetrate cells and finding ways to inhibit this process. Due to the complexity of this process, many fundamental questions endure despite decades of research. Moreover, traditional entry assays rely on downstream reporters and do not measure fusion directly. Studies of cell-cell fusion of uninfected receptor-bearing cells expressing the four essential HSV entry glycoproteins have yielded much of the current mechanistic knowledge of membrane fusion, including the prevalent regulatory cascade model. However, the cell-cell fusion system, while informative, fails to capture the proper context of virus-cell fusion. Accurate, systematic dissection of the HSV-mediated membrane fusion mechanism thus requires an experimentally tractable system that enables direct visualization and kinetic measurements of the viral fusion. The goal of this exploratory proposal is to reconstitute HSV-1 fusion in vitro and to characterize it at a single- virion level by imaging fusion of individual virions with fluid, supported lipid bilayers using total internal reflection microscopy. This approach will be used to visualize different stages in fusion, measure their kinetic parameters, identify kinetic intermediates, and correlate them with structural rearrangements in gB. In addition to HSV-1, the proposed studies will employ Vesicular Stomatitis Virus (VSV) virions lacking the native fusogen G and pseudotyped with HSV-1 entry glycoproteins gB, gD, gH, and gL (VSVDG-BHLD). The use of this simplified, “bare-bones” system will benefit this work by excluding the potential effects of the 12 other envelope proteins. In-vitro reconstitution of fusion and its characterization at a single particle level will address the lingering questions in HSV-mediated membrane fusion mechanism, with the ultimate goal of reconstructing the HSV-1-mediated fusion pathway more fully.