EXAMINATION OF THE KINETICS OF HERPES-SIMPLEX VIRUS GLYCOPROTEIN-D BINDING TO THE HERPESVIRUS ENTRY MEDIATOR, USING SURFACE-PLASMON RESONANCE

Previously, we showed that truncated soluble forms of herpes simplex v irus (HSV) glycoprotein D (gDt) bound directly to a truncated soluble form of the herpesvirus entry mediator (HveAt, formerly HVEMt), a cell ular receptor for HSV. The purpose of the present study was to determi ne the affinity of got for HveAt by surface plasmon resonance and to c ompare and contrast the kinetics of an expanded panel of got variants in binding to HveAt in an effort to better understand the mechanism of receptor binding and virus entry. Both HveAt and got are dimers in so lution and interact with a 2:1 stoichiometry, With HveAt, gD1(306t) (f rom the KOS strain of HSV-1) had a dissociation constant (K-D) of 3.2 x 10(-6) M and gD2(306t) had a K-D of 1.5 x 10(-6) M. The interaction between got and HveAt fits a 1:1 Langmuir binding model, i.e., two dim ers of HveAt may act as one binding unit to interact with one dimer of got as the second binding unit. A go 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 h ad an affinity for HveAt that did not differ from that of the wild typ e. However, variants with double cysteine mutations that eliminated ei ther of the other two disulfide bonds showed decreased affinity for Hv eAt, This result suggests that two of the three disulfide bonds of go are important for receptor binding. Four nonfunctional got variants, e ach representing one functional domain of go, were also studied. Mutat ions in functional regions I and II drastically decreased the affinity of got for HveAt. Surprisingly, a variant with an insertion in functi onal region III had a wild-type level of affinity for HveAt, suggestin g that this domain may function in virus entry at a step other than re ceptor binding. A variant with a deletion in functional region TV [gD1 (Delta 290-299t)] exhibited a 100-fold enhancement in affinity for Hve At (K-D = 3.3 x 10(-8) M) due mainly to a 40-fold increase in its kine tic on rate. This agrees with the results of other studies showing the enhanced ability of gD1(Delta 290-299t) to block infection. Interesti ngly, all the variants,vith decreased affinities for HveAt exhibited d ecreased 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 go.