Multiple Sites on Glycoprotein H (gH) Functionally Interact with the gB Fusion Protein to Promote Fusion during Herpes Simplex Virus (HSV) Entry

Enveloped virus entry requires fusion of the viral envelope with a host cell membrane. Herpes simplex virus 1 (HSV-1) entry is mediated by a set of glycoproteins that interact to trigger the viral fusion protein glycoprotein B (gB). In the current model, receptor-binding by gD signals a gH/gL heterodimer to trigger a refolding event in gB that fuses the membranes. To explore functional interactions between gB and gH/gL, we used a bacterial artificial chromosome (BAC) to generate two HSV-1 mutants that show a small plaque phenotype due to changes in gB. We passaged the viruses to select for restoration of plaque size and analyzed second-site mutations that arose in gH. HSV-1 gB was replaced either by gB from saimiriine herpesvirus 1 (SaHV-1) or by a mutant form of HSV-1 gB with three alanine substitutions in domain V (gB3A). To shift the selective pressure away from gB, the gB3A virus was passaged in cells expressing gB3A. Sequencing of passaged viruses identified two interesting mutations in gH, including gH-H789Y in domain IV and gH-S830N in the cytoplasmic tail (CT). Characterization of these gH mutations indicated they are responsible for the enhanced plaque size. Rather than being globally hyperfusogenic, both gH mutations partially rescued function of the specific gB version present during their selection. These sites may represent functional interaction sites on gH/gL for gB. gH-H789 may alter the positioning of a membrane-proximal flap in the gH ectodomain, whereas gH-S830 may contribute to an interaction between the gB and gH CTs.IMPORTANCE Enveloped viruses enter cells by fusing their envelope with the host cell membrane. Herpes simplex virus 1 (HSV-1) entry requires the coordinated interaction of several viral glycoproteins, including gH/gL and gB. gH/gL and gB are essential for virus replication and both proteins are targets of neutralizing antibodies. gB fuses the membranes after being activated by gH/gL, but the details of how gH/gL activates gB are not known. This study examined the gH/gL-gB interaction using HSV-1 mutants that displayed reduced virus entry due to changes in gB. The mutant viruses were grown over time to select for additional mutations that could partially restore entry. Two mutations in gH (H789Y and S830N) were identified. The positions of the mutations in gH/gL may represent sites that contribute to gB activation during virus entry. Enveloped viruses enter cells by fusing their envelope with the host cell membrane. Herpes simplex virus 1 (HSV-1) entry requires the coordinated interaction of several viral glycoproteins, including gH/gL and gB. gH/gL and gB are essential for virus replication and both proteins are targets of neutralizing antibodies. gB fuses the membranes after being activated by gH/gL, but the details of how gH/gL activates gB are not known.