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 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 got for HveAt by surface plasmon resonance and to compare 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 solution and interact with a 2:1 stoichiometry, With HveAt, gD1(306t) (from 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 dimers 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 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 go are important for receptor binding. Four nonfunctional got variants, each representing one functional domain of go, were also studied. Mutations in functional regions I and II drastically decreased the affinity of got 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 TV [gD1(Delta 290-299t)] exhibited a 100-fold enhancement in affinity for HveAt (K-D = 3.3 x 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(Delta 290-299t) to block infection. Interestingly, all the variants,vith 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 go.