The Vaccinia virus chondroitin sulfate binding protein drives host membrane curvature to facilitate fusion

Abstract

Virus binding serves to define virus tropism and species specificity1. Virus binding proteins are classically considered as facilitators of cell surface attachment prior to receptor engagement and virus internalization. For efficient entry vaccinia virus (VACV) - the prototypic poxvirus - relies on four binding proteins and an eleven-protein entry fusion complex (EFC)2. We recently demonstrated that VACV binding and fusion proteins are organized into distinct functional domains, with localization of EFC proteins to virion tips directly influencing membrane fusion activity3. However, the relationship between virus binding protein distribution, virion binding orientation and subsequent membrane fusion remain unexplored. Here, we show that virus binding proteins guide side-on virion binding and promote curvature of the host membrane towards EFC-containing virion tips to facilitate virus fusion. Using a cell-derived membrane-bleb model system together with super-resolution and electron microscopy we found that side-bound VACV virions induce membrane invagination in the presence of low pH. Repression or deletion of individual binding proteins revealed that three of four contribute to binding orientation, amongst which the chondroitin sulphate binding protein, D8, is required for host membrane bending. Consistent with low-pH dependent macropinocytic entry of vaccinia virus4,5, loss of D8 prevents virion-associated macropinosome membrane bending, disrupts fusion pore formation and infection kinetics. Our results extend the role of viral binding proteins from mere attachment factors to active participants in successful viral membrane fusion and further illustrate the influence of virus protein architecture on successful infection.