Species-Specific gB Ectodomain Interactions and Cytoplasmic Domain Stability Regulate Herpes Simplex Virus Fusion

Entry of herpesviruses into cells requires coordinated action of multiple viral glycoproteins, including gH/gL and gB which comprise the core fusion machinery conserved in herpesviruses. The gH/gL heterodimer activates the gB fusion protein, triggering its refolding from a prefusion to a postfusion form to drive membrane merger. The cytoplasmic tail domain (CTD) of gB is proposed to act as an inhibitory clamp that stabilizes the prefusion state, with interactions between gH and gB CTDs destabilizing this clamp. We previously found that herpes simplex virus 1 (HSV-1) and saimiriine herpesvirus 1 (SaHV-1) gB homologs are functionally interchangeable but mediate reduced fusion when coexpressed with heterotypic gH/gL. To map the regions of gB responsible for species-specific interactions, we generated HSV-1/SaHV-1 gB chimeras by swapping ectodomain, membrane-proximal region (MPR), transmembrane domain (TMD), and CTD segments. Our results show that homotypic CTD interactions alone are insufficient to trigger fusion, suggesting that gH/gL contacts the ectodomain of gB. We show that the HSV-1 gB CTD is hyperfusogenic relative to the SaHV-1 CTD, whereas the HSV-1 MPR is hypofusogenic relative to the SaHV-1 MPR. Together, these findings suggest that functional interaction between gH/gL-gB occur on both sides of the membrane and that gB maintains a balance between promotion and restraint of fusion through coordinated contributions of its domains.