Herpes simplex virus (HSV) remains a major pathogen worldwide causing lifelong latent infections in humans. It is responsible for significant disease, ranging from cold sores and genital infections to blindness and fatal encephalitis. Herpesviral entry is a complex process involving multiple viral and cellular factors. The long-term goal of this project is to understand the molecular mechanisms that govern HSV entry into host cells. HSV particles are enveloped in a host cell-derived lipid bilayer membrane. To accomplish entry, viruses must surmount the barrier posed by the host membrane. Thus, the HSV entry process is intimately associated with membrane interactions. The roles of lipid components of virus and cell membranes in HSV entry are greatly understudied relative to membrane-associated proteins. Sphingomyelin (SM) is the most common sphingolipid component of biological membranes and is a signature constituent of lipid rafts. The specific role of SM in herpesviral entry is not well-understood. Based on our published and preliminary data, two complementary specific aims are proposed. A battery of assays will be employed to assess the importance of SM for specific steps in the entry process including cell attachment, endocytic uptake, intracellular vesicular transport, and membrane fusion. In Specific Aim # 1, we will elucidate the function of HSV envelope sphingomyelin in viral entry. Experiments in Specific Aim #2 follow from preliminary data indicating the selective importance of host cell SM in HSV entry by an endocytosis pathway. The precise step(s) in the entry cascade that require cellular SM will be defined. A combination of molecular, biochemical, and cell biological approaches will be used to fill key gaps in our understanding of HSV entry.