Receptor-dependent herpes simplex virus (HSV)-induced cell-cell fusion requires glycoproteins gD, gH/gL, and gB. Our current model posits that during fusion, receptor-activated conformational changes in gD activate gH/gL, which subsequently triggers the transformation of the prefusion form of gB into a fusogenic state. To examine the role of each glycoprotein in receptor-dependent cell-cell fusion, we took advantage of our discovery that fusion by wild-type herpes simplex virus 2 (HSV-2) glycoproteins occurs twice as fast as that achieved by HSV-1 glycoproteins. By sequentially swapping each glycoprotein between the two serotypes, we established that fusion speed was governed by gH/gL, with gH being the main contributor. While the mutant forms of gB fuse at distinct rates that are dictated by their molecular structure, these restrictions can be overcome by gH/gL of HSV-2 (gH ₂ /gL ₂ ), thereby enhancing their activity. We also found that deregulated forms of gD of HSV-1 (gD ₁ ) and gH ₂ /gL ₂ can alter the fusogenic potential of gB, promoting cell fusion in the absence of a cellular receptor, and that deregulated forms of gB can drive the fusion machinery to even higher levels. Low pH enhanced fusion by affecting the structure of both gB and gH/gL mutants. Together, our data highlight the complexity of the fusion machinery, the impact of the activation state of each glycoprotein on the fusion process, and the critical role of gH/gL in regulating HSV-induced fusion. IMPORTANCE Cell-cell fusion mediated by HSV glycoproteins requires gD, gH/gL, gB, and a gD receptor. Here, we show that fusion by wild-type HSV-2 glycoproteins occurs twice as fast as that achieved by HSV-1 glycoproteins. By sequentially swapping each glycoprotein between the two serotypes, we found that the fusion process was controlled by gH/gL. Restrictions imposed on the gB structure by mutations could be overcome by gH ₂ /gL ₂ , enhancing the activity of the mutants. Under low-pH conditions or when using deregulated forms of gD ₁ and gH ₂ /gL ₂ , the fusogenic potential of gB could only be increased in the absence of receptor, underlining the exquisite regulation that occurs in the presence of receptor. Our data highlight the complexity of the fusion machinery, the impact of the activation state of each glycoprotein on the fusion process, and the critical role of gH/gL in regulating HSV-induced fusion.

Virus entry and cell-cell fusion mediated by HSV require four essential glycoproteins, gD, gH/gL, gB, and a gD receptor. Virus-neutralizing antibodies directed against any of these proteins bind to residues within key functional sites and interfere with essential steps in the fusion pathway.

Virus entry and cell-cell fusion mediated by HSV require gD, gH/gL, gB, and a gD receptor. Neutralizing antibodies directed against any of these proteins bind to residues within key functional sites and interfere with an essential step in the fusion pathway. Thus, the epitopes of these MAbs identify critical, functional sites on their target proteins. Unlike many anti-gD MAbs, which block binding of gD to a cellular receptor, two, MC2 and MC5, block a separate, downstream step in the fusion pathway which is presumed to be the activation of the modulator of fusion, gH/gL. By combining epitope mapping of a panel of gD mutants with fusion and virus entry assays, we have identified residues that are critical in the binding and function of these two MAbs. This new information helps to define the site of the presumptive interaction of gD with gH/gL, of which we have limited knowledge.

Herpes simplex virus (HSV) requires four essential virion glycoproteins—gD, gH, gL, and gB—for virus entry and cell fusion. To initiate fusion, the receptor binding protein gD interacts with one of two major cell receptors, HVEM or nectin-1. Once gD binds to a receptor, fusion is carried out by the gH/gL heterodimer and gB. A comparison of free and receptor-bound gD crystal structures revealed that receptor binding domains are located within residues in the N-terminus and core of gD. Problematically, the C-terminus lies across and occludes these binding sites. Consequentially, the C-terminus must relocate to allow for both receptor binding and the subsequent gD interaction with the regulatory complex gH/gL. We previously constructed a disulfide bonded (K190C/A277C) protein that locked the C-terminus to the gD core. Importantly, this mutant protein bound receptor but failed to trigger fusion, effectively separating receptor binding and gH/gL interaction. Here, we show that “unlocking” gD by reducing the disulfide bond restored not only gH/gL interaction but fusion activity as well, confirming the importance of C-terminal movement in triggering the fusion cascade. We characterize these changes, showing that the C-terminus region exposed by unlocking is: (1) a gH/gL binding site; (2) contains epitopes for a group (competition community) of monoclonal antibodies (Mabs) that block gH/gL binding to gD and cell–cell fusion. Here, we generated 14 mutations within the gD C-terminus to identify residues important for the interaction with gH/gL and the key conformational changes involved in fusion. As one example, we found that gD L268N was antigenically correct in that it bound most Mabs but was impaired in fusion, exhibited compromised binding of MC14 (a Mab that blocks both gD–gH/gL interaction and fusion), and failed to bind truncated gH/gL, all events that are associated with the inhibition of C-terminus movement. We conclude that, within the C-terminus, residue 268 is essential for gH/gL binding and induction of conformational changes and serves as a flexible inflection point in the critical movement of the gD C-terminus.

Herpes simplex viruses (HSV-1 and HSV-2), which can be transmitted both orally and sexually, cause lifelong morbidity and in some cases, meningitis and encephalitis. While both the passive transfer of neutralizing antibodies and placental transfer of anti-HSV monoclonal antibodies (Mabs) have shown therapeutic promise in animal models, clinical trials have yet to identify approved immunotherapeutics for herpes infection. Here, we present strategies for the generation of recombinant bispecific antibodies (BsAbs) that target different domains of glycoprotein D (gD), crucial for HSV entry, that have the potential to outperform the effect of individual Mabs to curb herpes infection. Specifically, we selected three pairs of Mabs from our extensive panel for BsAb design and production based on their binding site and ability to block virus entry. Actual binding of BsAbs to gD and epitope availability on gD after BsAb binding were characterized using surface plasmon resonance (SPR) and inhibition by IgG Fab fragments generated from selected Mabs. While one BsAb exhibited an additive effect similar to that observed using a combination of the Mabs utilized for its generation, two showed antagonistic effects, suggesting that the simultaneous engagement of two epitopes or selective binding to one affected their activity against HSV. One BsAb (DL11/1D3) targeting the binding site for both nectin-1 and HVEM receptors demonstrated synergistic inhibitory activity against HSV, outperforming the effect of the individual antibodies. Recombinant DL11/1D3 antibody variants, in which the size of one or both paratopes was decreased to single chains (scFv-Fc), highlighted differences in potency depending on antibody size and format. We propose that BsAbs to individual glycoproteins offer a potential avenue for herpes therapeutics, but their design, mechanism of action, antibody format, and epitope engagement require careful consideration of structure for optimal efficacy.

Herpesviridae comprise a large family of enveloped DNA viruses with a unifying ability to establish a latent infection in their host