Herpesviruses are a broad family of viruses that can cause lifelong infections in humans. Three envelope-bound glycoproteins are conserved across all herpesviruses: glycoproteins B, H, and L, which are necessary for membrane fusion and viral entry. Upon host cell attachment and triggering, glycoprotein B (gB), the fusogen, undergoes a dramatic refolding event, transitioning from a metastable prefusion conformation to an energetically favorable postfusion form. This conformational change overcomes energetic barriers to fusion of host and viral membranes, allowing for viral entry and infection. gB is a trimeric, single-pass transmembrane protein. Membrane-interacting regions of gB stabilize the prefusion conformation and regulate membrane fusion, infection, and viral spread. However, how gB interacts with the membrane is unclear because all known gB structures either lack membrane-interacting regions or the lipid environment of the native membrane. Here, we have developed and optimized protocols for the extraction of gB using synthetic polymer nanodiscs, which preserves native membrane-protein interactions. We demonstrate that the use of polymer nanodiscs enables the efficient capture of purified, recombinant gB in its metastable prefusion conformation. This method will enable the determination of the gB structure in its native membrane environment, thereby clarifying its regulatory mechanism, which is conserved across herpesviruses. Additionally, this work supports the hypothesis that membrane interactions stabilize the prefusion conformation of gB. Purified native prefusion gB can be used to assess fusion triggers, identify conformational intermediates, and analyze the lipid environment of herpesviral glycoproteins. Overall, this work furthers our understanding of the viral protein dynamics involved during infection.
Cieslewicz et al. (Sun,) studied this question.