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 x 10(-6) M and gD2(306t) had a KD of 1.5 x 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(Delta290-299t)] exhibited a 100-fold enhancement in affinity for HveAt (KD = 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(Delta290-299t) 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.

FIG. 1

Schematic representation of truncated HSV gD produced by baculovirus-infected insect cells (33, 36). The protein has the honeybee melittin signal peptide (hatched box) in place of the wild-type gD signal for efficient translocation into the lumen of the endoplasmic reticulum, two extra amino acids (D and P) at the N terminus, and a histidine tag at the C terminus (33, 36, 44). The protein was truncated prior to the transmembrane region and has three N-CHO (balloons). The disulfide bonds are indicated by dashed lines. The functional regions of gD encompass the following amino acids: 27 to 43 for region I, 126 to 131 for region II, 225 to 246 for region III, and 277 to 300 for region IV (5).

FIG. 2

STEM. Individual molecules of protein were directly viewed, and molecular masses were calculated by comparison to a tobacco mosaic virus standard. The range of molecular masses for each protein was plotted on a histogram as percentages of the total numbers of particles counted. The average molecular masses are reported in Table 1.

FIG. 3

Example of raw sensorgram data. gD1 (Δ290-299t) at a concentration of 0.5 μM was injected over a surface containing 2,000 RU of HveAt (Fc1) in series with a blank surface (Fc2). At 120 s, the sample was replaced with buffer, and dissociation followed for 2 min. The change in refractive index (RI) represents a difference in solution composition between the buffer and the protein solution.

FIG. 4

Corrected sensorgram overlays for the gDt-HveAt interaction. (A) Repeat injections of gD1 (306t) at the indicated concentrations are represented by the small symbols. Data were collected at 5 Hz, but for clarity, one point per second is shown. The solid lines represent the best fits found by the global software analysis. The inset represents a plot of k_obs versus concentration, the negative slope of which is equal to k_on. The R² for the linear fit of the data was 0.999. (B) Repeat injections of gD1 (Δ290-299t) at the indicated concentrations. Data collection and analysis were the same as described for panel A. The R² for the linear fit of the data in the plot of k_obs versus concentration (inset) was 0.999.

FIG. 5

Equilibrium binding sensorgrams and Scatchard analysis of the binding of gD1 (306t) and gD1 (Δ290-299t) to immobilized HveAt. (A) Binding of gD1 (306t) to HveAt was monitored for 15 min for the 5, 2.5, 1.25, and 0.62 μM injections and for 20 min for the 0.31 and 0.15 μM injections. The arrows indicate the time points used for the Scatchard analysis. (B) Binding of gD1 (Δ290-299t) to HveAt was monitored for 10 min for all of the injections. The arrows indicates the time points used for the Scatchard analysis. (C and D) Scatchard analysis. C is the concentration of gDt that flowed across the surface. The negative slope of each line is equal to the association constant (the reciprocal is K_D). The R² for the linear fit of the data in panel C was 0.98. The R² for the linear fit of the data in panel D was 0.99.

FIG. 6

Corrected sensorgram overlays for the interaction between gD2 (306t) (A) and gD1 (QAAt) (B) in binding to immobilized HveAt. Sensorgrams are presented as described in the legend to Fig. 4.

FIG. 7

Binding of the cysteine variants to immobilized HveAt. (A) Binding of the gDt cysteine variants to HveAt by ELISA. An ELISA plate was coated with 200 nM HveAt in PBS, blocked, and incubated with various concentrations of gDt. Bound gDt was detected with antiserum R7 and then with peroxidase-conjugated secondary antibody and substrate. The data are averages of results from duplicate wells, and each experiment was repeated twice with similar results. Abs, absorbance. (B to D) Binding of gD1 (cys1,5), gD1 (cys2,6), and gD1 (cys3,4), respectively, to immobilized HveAt by SPR. Data are plotted and analyzed as described for Fig. 4.

FIG. 8

Binding of the functional region variants to immobilized HveAt. (A) Binding of the gDt functional region variants to HveAt by ELISA. ELISA was done as described in the legend to Fig. 7. Abs, absorbance. (B to D) Binding of gD1 (∇34t), gD1 (∇126t), and gD1 (∇243t), respectively, to immobilized HveAt by SPR. Data are plotted and analyzed as described for Fig. 4.