Using Split Luciferase Assay and anti-HSV 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 pre-fusion gB to its active form to promote membrane fusion. Virus-neutralizing monoclonal antibodies (Mabs) interfere with one or more of these steps and 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 orientation 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 were used alone (additive), then their epitopes would be spatially distanced and binding of one would not directly interfere with binding of the other during fusion. However, if the two Mabs blocked fusion with equal or lesser efficacy that when either were 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 other had a 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-à-vis gH/gL in the complex.