Species-specific gB ectodomain interactions and cytoplasmic domain stability regulate herpes simplex virus fusion

ABSTRACT 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 the 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 a functional interaction between the gH/gL-gB ectodomains contributes to fusion and that gB maintains a balance between promotion and restraint of fusion through coordinated contributions of its domains. IMPORTANCE Herpes simplex virus type 1 (HSV-1) entry requires the coordinated interaction of gD, gH/gL, and gB. Both gH/gL and gB are conserved herpesvirus proteins that are required for viral replication and are key targets of neutralizing antibodies. Despite their importance, how these proteins interact to mediate herpesvirus entry into cells remains poorly understood. In this study, we examined gB function by creating chimeras that swapped distinct domains between HSV-1 and saimiriine herpesvirus 1 (SaHV-1) homologs. Using these chimeras, we demonstrate that a species-specific interaction occurs in the gB ectodomain. Additionally, we found that the HSV-1 cytoplasmic tail domain (CTD) is hyperfusogenic compared to SaHV-1, suggesting that different gB domains can compensate for one another to balance fusion. This study provides new insight into how gB is regulated to mediate virus entry at the right time and place.