Post-receptor binding conformational changes that occur in gD and lead to the interaction with gHgL

Atanasiu, Doina, Cairns, Tina M, Saw, Wan Ting, Cohen, Gary H
University of Pennsylvania School of Dental Medicine, Department of Basic and Translational Sciences


Glycoprotein D (gD) is the receptor binding protein of herpes simplex virus (HSV) and binds to at least two distinct protein receptors, herpesvirus entry mediator (HVEM) and nectin-1. A comparison of free and receptor bound gD crystal structures shows that while both receptor binding regions are found within the first 234 amino acids, the C terminus of the gD ectodomain normally occludes the receptor binding sites. Introduction of a di-sulfide bond (K190C/A277C) that locked the C-terminus to the core allowed gD to bind receptor, but could not trigger fusion, effectively separating the two functions in gD: receptor binding and gH/gL interaction.


SIngle point mutations were generated using site-directed mutagenesis. Cell-based ELISA (CELISA): B78 cells were transfected with wt or mutant gDs for 24h. After fixation, cell were incubated with the indicated Mabs for 1h followed by secondary anti-mouse Ab (1h) and ABTS substrate. SIgnal at 405nm was registered with a BioTek plate reader. Split luciferase fusion assay: B78 cells transfected with gB, gD, gH, gL and split luciferase RLuc1-7 were co-cultured with B78-nectin cells transfected with RLuc8-11 in the presence of luciferase substrate EnduRen. Luciferase levels (as measure of fusion function) were determined with a BioTek plate reader. Surface plasmon resonance: Purified wt and gD mutant proteins were affixed to a CM5 chip via anti-His antibody. All injections were performed using HBS-EP. After each experiment, the chip surface was regenerated to baseline values with Na2CO3 (pH 10).


Here, we show that removal of the di-sulfide bond lock by treatment with reducing agents allowed for gain of fusion function confirming the importance of movement of the C-terminus in triggering the fusion cascade. Using a structure-function approach, we generated a series of point mutations in this region (262-282) with the purpose of identifying residues important for the function of gD. One of these mutants, L268N was not only impaired for function in a fusion assay, but the mutation also played a major role in the binding of a Mab (MC14) known to block the physical interaction between gD and gH/gL. Biosensor experiments with soluble purified protein showed that although the L268N mutant protein was antigenically correct, it was unable to physically bind gH/gL as measured by SPR. Further analysis showed that this was primarily due to a lack of movement of the C-terminus.


We conclude that residue 268 serves as an inflection point in the C-terminus of gD and can reduce flexibility of region 262-282 which is essential for gD function and interaction with gH/gL.