Structural basis for membrane anchoring and fusion regulation of the Herpes Simplex Virus fusogen gB

Viral fusogens merge viral and cell membranes during cell penetration. Their ectodomains drive fusion by undergoing large-scale refolding, but little is known about the functionally important regions located within or near the membrane. Here, we report the crystal structure of the full-length glycoprotein B, the fusogen from Herpes Simplex Virus, complemented by electron spin resonance measurements. The membrane-proximal (MPR), transmembrane (TMD), and cytoplasmic (CTD) domains form a uniquely folded trimeric pedestal beneath the ectodomain, which balances dynamic flexibility with extensive, stabilizing membrane interactions. Hyperfusogenic mutations within the CTD destabilize it, targeting trimeric interfaces, structural motifs, and membrane-interacting elements. Thus, we propose that the CTD trimer observed in the structure stabilizes gB in its prefusion state despite being appended to the postfusion ectodomain. Our data suggest a model for how this dynamic, membrane-dependent “clamp” controls the fusogenic refolding of gB.