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. This number far exceeds the number of glycoproteins utilized by most other enveloped viruses complicating mechanistic studies of HSV-1-mediated entry and fusion. We now know that in HSV-1, as in other herpesviruses, the receptor-binding, regulatory, and fusogenic functions are distributed among several glycoproteins. gB is the conserved fusogen that, by analogy with other viral fusogens, is thought to facilitate membrane merger by undergoing large-scale refolding that brings the viral and cellular membranes together. However, gB is unusual in requiring several additional proteins for function. Therefore, understanding how gB works to mediate fusion during cell entry requires direct measurements of its fusogenic activity. Recently, single-particle tracking (SPT) has emerged as a powerful tool for quantitative studies of fusion of individual virions with fluid, supported lipid bilayers using total internal reflection microscopy. The SPT approach enables direct visualization and kinetic measurements of the viral fusion pathway and has been successfully used with both pH-triggered and receptor-triggered fusogens. The goal of this exploratory proposal is to develop single-particle imaging of HSV-1 fusion with supported lipid bilayers to visualize different stages in fusion, measure their kinetic parameters, identify kinetic intermediates, and correlate them with structural rearrangements in gB. Given that the SPT approach has not yet been applied to viruses that utilize >1 glycoprotein and may be challenging to apply to HSV-1 that has 15 envelope proteins, Aim 1 will utilize Vesicular Stomatitis Virus (VSV) virions lacking the native fusogen G and pseudotyped with HSV-1 entry glycoproteins gB, gD, gH, and gL (VSVDG-BHLD), which retain key characteristics of HSV-1 entry.
In Aim 2, the SPT approach will be extended to HSV-1, a more complicated yet more biologically relevant system. The scientific premise of the proposed work is that harnessing the power of single-particle imaging of virion fusion provides a unique opportunity to address the lingering questions in HSV-mediated membrane fusion mechanism, with the ultimate goal of reconstructing the HSV-1-mediated fusion pathway more fully.
Herpes simplex viruses type 1 and 2 (HSV-1 and HSV-2) infect their human hosts for life, causing cold sores, genital herpes, blindness, encephalitis, and life-threatening conditions in the immunocompromised individuals and newborns. No cure or vaccine is currently available. HSV-mediated membrane fusion is a prerequisite for infection, and the detailed knowledge of the mechanism is necessary for designing anti-herpesvirus therapeutics to combat both viral infections.