Using Split Luciferase Assay And Anti-Herpes Simplex Virus Glycoprotein Monoclonal Antibodies To Predict A Functional Binding Site Between Gd And Gh/Gl

Herpes simplex virus (HSV) entry and cell-cell fusion require glycoproteins gD, gH/gL, and gB. HSV entry begins with gD binding its receptor (nectin-1), which then activates gH/gL to enable the conversion of prefusion gB to its active form to promote membrane fusion. Virus-neutralizing monoclonal antibodies (Mabs) interfere with one or more of these steps, and the localization of their epitopes identifies functional sites on each protein. Utilizing this approach, we have identified the gH/gL binding face on gD and the corresponding gD binding site on gH/gL. Here, we used combinations of these Mabs to define the orientations of gD and gH/gL relative to each other. We reasoned that if two Mabs, one directed at gD and the other at gH/gL, block fusion more effectively than when either Mab was used alone (additive), then their epitopes would be spatially distanced, and the binding of one would not directly interfere with the binding of the other during fusion. However, if the two Mabs blocked fusion with an efficacy equal to or lesser than that when either Mab was used alone (indifferent), we propose that their epitopes would be in close proximity in the complex. Using a live-cell fusion assay, we found that some Mab pairings blocked the fusion with different mechanisms, while others had similar mechanisms of action. Grouping the different combinations of antibodies into indifferent and additive groups, we present a model for the orientation of gD vis-a-vis gH/gL in the complex.IMPORTANCE Virus entry and cell-cell fusion mediated by HSV require four essential glycoproteins, gD, gH/gL, gB, and a cellular 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. Thus, the epitopes of these Mabs overlap and point to critical, functional sites on their target proteins. Here, we combined gD and gH/gL antibodies to determine whether they work in an additive or a nonadditive (indifferent) fashion to block specific events in glycoprotein-driven cell-cell fusion. Identifying combinations of antibodies that have additive effects will help in the rational design of an effective therapeutic "polyclonal antibody" to treat HSV disease. In addition, identification of the exact contact regions between gD and gH/gL can inform the design of small molecules that would interfere with gD-gH/gL complex formation, thus preventing the virus from entering the host cell.