Examination of the Kinetics of Herpes Simplex Virus Glycoprotein D Binding to the Herpesvirus Entry Mediator, Using Surface Plasmon Resonance

Abstract

Previously, we showed that truncated soluble forms of herpes simplex virus (HSV) glycoprotein D (gDt) bound directly to a truncated soluble form of the herpesvirus entry mediator (HveAt, formerly HVEMt), a cellular receptor for HSV. The purpose of the present study was to determine the affinity of gDt for HveAt by surface plasmon resonance and to compare and contrast the kinetics of an expanded panel of gDt variants in binding to HveAt in an effort to better understand the mechanism of receptor binding and virus entry. Both HveAt and gDt are dimers in solution and interact with a 2:1 stoichiometry. With HveAt, gD1(306t) (from the KOS strain of HSV-1) had a dissociation constant (KD ) of 3.2 × 10−6 M and gD2(306t) had a KD of 1.5 × 10−6 M. The interaction between gDt and HveAt fits a 1:1 Langmuir binding model, i.e., two dimers of HveAt may act as one binding unit to interact with one dimer of gDt as the second binding unit. A gD variant lacking all signals for N-linked oligosaccharides had an affinity for HveAt similar to that of gD1(306t). A variant lacking the bond from cysteine 1 to cysteine 5 had an affinity for HveAt that did not differ from that of the wild type. However, variants with double cysteine mutations that eliminated either of the other two disulfide bonds showed decreased affinity for HveAt. This result suggests that two of the three disulfide bonds of gD are important for receptor binding. Four nonfunctional gDt variants, each representing one functional domain of gD, were also studied. Mutations in functional regions I and II drastically decreased the affinity of gDt for HveAt. Surprisingly, a variant with an insertion in functional region III had a wild-type level of affinity for HveAt, suggesting that this domain may function in virus entry at a step other than receptor binding. A variant with a deletion in functional region IV [gD1(Δ290-299t)] exhibited a 100-fold enhancement in affinity for HveAt (KD = 3.3 × 10−8 M) due mainly to a 40-fold increase in its kinetic on rate. This agrees with the results of other studies showing the enhanced ability of gD1(Δ290-299t) to block infection. Interestingly, all the variants with decreased affinities for HveAt exhibited decreased kinetic on rates but only minor changes in their kinetic off rates. The results suggest that once the complex between gDt and HveAt forms, its stability is unaffected by a variety of changes in gD.

Antibody-Induced Conformational Changes in Herpes Simplex Virus Glycoprotein gD Reveal New Targets for Virus Neutralization

“…Biosensor analysis indicated that the increased affinity of gD2(306t) for HVEM and nectin-1 caused by MC2 or MC14 was due to an increase in the rate of association (k on ), with little change in dissociation (k off ) (data not shown). This pattern was identical to that seen for the kinetics of gD2(285t) binding to HVEM and nectin-1 (44,60). These experiments support the concept that binding of MC2 or MC14 to gD induces a more "open" conformation of the C-term, thereby enhancing the ability of gD to bind the receptor.…”

“…The anti-MYC antibody control reflects the normal binding capacity of each form of gD to receptors. As expected (44,60), gD2(285t) bound to HVEM and nectin-1 better than gD2(306t). Interestingly, MC2 enhanced the binding of gD2(306t) to HVEM almost as well as the C-terminal truncation [gD2(285t)] (Fig.…”

“…To what extent do MC2, MC5, and MC14 enhance gD binding to receptor? Previously, we found that the affinity of gD(306t) for both HVEM and nectin-1 is in the micromolar range whereas the affinity of gD(285t) for these receptors is enhanced significantly (approximately 50-fold) (44,60). This is explained by crystallographic data that showed that the C-terminal residues of the gD ectodomain lie across the core of gD and interact with the N terminus, thereby impeding receptor binding (16,30,60).…”

“…Map competition by surface plasmon resonance. Experiments were carried out on a BIACORE 3000 optical biosensor (Biacore AB) at 25°C as previously described (1,27,60). The running buffer for the experiments was HBS-EP (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.005% polysorbate 20).…”

Crystal Structure of Herpes Simplex Virus 2 gD Bound to Nectin-1 Reveals a Conserved Mode of Receptor Recognition

“…The molecule also serves as a functional receptor for both HSV-1 and HSV-2 (12) and is shown to play a more determinant role than other gD receptors (e.g., HVEM) in HSV-2 infection (28,29). With a truncated gD molecule (spanning residues 1 to 285), which according to previous reports (32)(33)(34) shows a much higher affinity for receptors than the longer gD form (spanning residues 1 to 306), the molecular basis of the binding between HSV-1 gD and nectin-1 has been successfully elucidated via costructures (35,36). It was revealed that the gD molecule contains a V-set immunoglobulin-like (IgVlike) core that is wrapped by large terminal extensions and utilizes the extensive N-and C-terminal extension elements to engage nectin-1 (35,36).…”