In-vitro reconstitution of Herpes Simplex Virus 1 fusion identifies low pH as a fusion co-trigger

Membrane fusion mediated by Herpes Simplex virus 1 (HSV-1) is a complex, multi-protein process that is receptor-triggered and can occur both at the cell surface and in endosomes. To deconvolute this complexity, we reconstituted HSV-1 fusion with synthetic lipid vesicles in vitro. Using this simplified, controllable system, we discovered that HSV-1 fusion required not only a cognate host receptor but also low pH. On the target membrane side, efficient fusion required cholesterol, negatively charged lipids found in the endosomal membranes, and an optimal balance of lipid order and disorder. On the virion side, the four HSV-1 entry glycoproteins gB, gD, gH, and gL were sufficient for fusion. We propose that low pH is a biologically relevant co-trigger for HSV-1 fusion. The dependence of fusion on low pH and endosomal lipids could explain why HSV-1 enters most cell types by endocytosis. We hypothesize that under neutral pH conditions, other, yet undefined, cellular factors may serve as fusion co-triggers. The in-vitro fusion system established here can be employed to systematically investigate HSV-1-mediated membrane fusion. IMPORTANCEHerpes simplex virus 1 (HSV-1) causes life-long, incurable infections and diseases ranging from mucocutaneous lesions to fatal encephalitis. Fusion of viral and host membranes is a critical step in HSV-1 infection of target cells that requires multiple factors on both the viral and host sides. Due to this complexity, many fundamental questions remain unanswered, such as the viral and host factors that are necessary and sufficient for HSV-1-mediated membrane fusion and the nature of the fusion trigger. Here, we developed a simplified in-vitro fusion assay to examine the fusion requirements and identified low pH as a co-trigger for virus-mediated fusion in vitro. We hypothesize that low pH has a critical role in cell entry and, potentially, pathogenesis.