The first immunoglobulin-like domain of HveC is sufficient to bind herpes simplex virus gD with full affinity, while the third domain is involved in oligomerization of HveC

Abstract

The human herpesvirus entry mediator C (HveC/PRR1) is a member of the immunoglobulin family used as a cellular receptor by the alphaherpesviruses herpes simplex virus (HSV), pseudorabies virus, and bovine herpesvirus type 1. We previously demonstrated direct binding of the purified HveC ectodomain to purified HSV type 1 (HSV-1) and HSV-2 glycoprotein D (gD). Here, using a baculovirus expression system, we constructed and purified truncated forms of the receptor containing one [HveC(143t)], two [HveC(245t)], or all three immunoglobulin-like domains [HveC(346t)] of the extracellular region. All three constructs were equally able to compete with HveC(346t) for gD binding. The variable domain bound to virions and blocked HSV infection as well as HveC(346t). Thus, all of the binding to the receptor occurs within the first immunoglobulin-like domain, or V-domain, of HveC. These data confirm and extend those of Cocchi et al. (F. Cocchi, M. Lopez, L. Menotti, M. Aoubala, P. Dubreuil, and G. Campadelli-Fiume, Proc. Natl. Acad. Sci. USA 95:15700, 1998). Using biosensor analysis, we measured the affinity of binding of gD from HSV strains KOS and rid1 to two forms of HveC. Soluble gDs from the KOS strain of HSV-1 had the same affinity for HveC(346t) and HveC(143t). The mutant gD(rid1t) had an increased affinity for HveC(346t) and HveC(143t) due to a faster rate of complex formation. Interestingly, we found that HveC(346t) was a tetramer in solution, whereas HveC(143t) and HveC(245t) formed dimers, suggesting a role for the third immunoglobulin-like domain of HveC in oligomerization. In addition, the stoichiometry between gD and HveC appeared to be influenced by the level of HveC oligomerization.

FIG. 1

(A) Schematic representation of HSV receptors. Full-length HveC is shown as a solid line with amino acids numbered from the initial methionine. The open box indicates the HveC natural signal peptide. (B) Schematic representation of gD constructs. gD from HSV-1 KOS is represented with amino acids numbered from the N terminus of the mature gD after cleavage of the gD signal peptide (shaded box). Disulfide bonds are indicated by dotted lines. The black circles represent putative N-linked carbohydrates. The hatched box represents the mellitin signal peptide used in the baculovirus constructs. Baculovirus-expressed proteins are truncated (t) at the indicated amino acid prior to the transmembrane region (TMR). H6, six-histidine tag added at the C terminus.

FIG. 2

Purified soluble receptors. Receptors purified from recombinant baculovirus-infected Sf9 cell supernatants were separated by SDS-PAGE. (A) Purified proteins were electrophoresed on a 12% polyacrylamide gel under reducing and denaturing conditions and then visualized by silver staining. The molecular mass markers are indicated in kilodaltons. (B) Endoglycosidase digestions. HveC(245t) (lanes 1 to 4) and HveC(143t) (lanes 5 to 8) were subjected to digestion with PNGase F (F) or endoglycosidase H (H) or were mock treated (−) prior to electrophoresis on a 16% acrylamide gel under reducing and denaturing conditions. After the Western blotting, proteins were detected with the anti-tetra-His MAb. (C) Native Western blot. Proteins were run under nondenaturing and nonreducing conditions on a 12% polyacrylamide gel and detected with MAb R1.302 (35).

FIG. 3

Binding of MAbs and gD to HveC MAbs by ELISA. Detection of immobilized truncated HveC proteins with anti-tetra-His Ig (Qiagen, Inc.) (A) or R1.302 Ig (Beckman Coulter) (B). Bound immunoglobulins were detected with horseradish peroxidase-conjugated anti-mouse IgG secondary antibody and substrate. (C) Plates coated with HveC truncated proteins were incubated with increasing concentrations of gD(306t) from HSV-1 strain KOS. Bound gD was detected with R7 antiserum, followed by peroxidase-conjugated secondary antibody and substrate. Absorbance was read at 405 nm.

FIG. 4

Competition ELISA. Plates were coated with HveC(346t) and incubated with a constant amount of purified gD, together with increasing concentrations of HveC truncated receptors as competitors. Panels show 1 μM gD-1(306t) from HSV-1 KOS (A), 0.1 μM gD-1(rid1t) (B), and 1 μM gD-2(306t) from HSV-2 strain 333 (C). gD bound to the immobilized receptor was detected with R7 antiserum. Relative binding is indicated as percentage of gD binding in the absence of soluble receptor.

FIG. 5

Analysis of gD binding to HveC in real time. HveC(346t) (A, C, and E) or HveC(143t) (B, D, and F) were immobilized on a CM5 biosensor chip to 1,600 and 300 RU, respectively, in a Biacore X instrument. Various concentrations of gD(306t) (A and B), gD(285t) (C and D), and gD(rid1t) (E and F) were flowed over the chip for 2 min (association) and then replaced by buffer for another 2 min (dissociation). Sensorgrams of corrected data are represented after the subtraction of signal from the control flow cell. Data points were collected at 5 Hz but, for clarity, only one every 25 points is represented here by a symbol. The solid line shows the best fit obtained after global fitting with the BIAevaluation 3.0 software (5).

FIG. 6

Blocking of HSV infection with soluble HveC truncations. HSV-1 KOS tk12 was preincubated with variable concentrations of soluble HveC truncations or BSA prior to addition to M3A (A), IMR5 (B), or SY5Y (C) cells. Cells were lysed at 5.5 h postinfection, and the β-galactosidase activity was measured. A value of 100% of entry corresponds to the β-galactosidase activity induced after infection with HSV-1 KOS tk12 at a similar multiplicity of infection (0.5 to 1 PFU/cell) in the absence of soluble inhibitor.

FIG. 7

Binding of soluble HveC to HSV particles. Purified HveC(346t) or HveC(143t) (100 μg) were incubated with 10⁷ PFU of purified HSV-1 KOS for 90 min at 4°C. Viral particles were then sedimented through a discontinuous sucrose gradient. The virus-containing fraction was collected, concentrated, and analyzed by Western blot for detection of HveC and virus. Receptors were detected with anti-tetra-His antibody, and VP5 capsid protein was detected with NC-1 polyclonal rabbit serum simultaneously.

FIG. 8

Gel filtration chromatography of HveC(245t) and HveC(143t) alone or in a complex with gD(285t). Elution profiles of HveC(245t) and HveC(143t) loaded at 33 and 66 μM respectively, on a Superdex 200 column are shown in panels A and B as solid lines. An elution profile of gD(285t) (26 μM) is shown as a dotted line in panels A and B. Elution profiles of gD(285t)-HveC(245t) complex (C and E) or gD(285t)-HveC(143t) complex (D and F) at the given ratios are shown. Molecular sizes were determined by calibrating the column with standards of proteins ranging from 13.7 to 669 kDa. The shaded area in panel F indicates the fraction used for quantification of HveC(143t) and gD(285t) in Fig. 9.

FIG. 9

Quantitation of gD(285t) and HveC(143t) in the complex. Two aliquots of a fraction containing the gD(285t)-HveC(143t) complex separated by gel filtration (Fig. 8F) were loaded onto a 16% polyacrylamide gel (lanes 5 and 6). Standards of known amounts of HveC(143t) and gD(285t) were loaded in lanes 1 to 4 and lanes 7 to 10, respectively, with amounts of proteins as indicated under the gel. After the silver staining of the gel, the intensity of the protein bands was measured by densitometry. The amount of each protein in the complex is indicated and is based on the comparison with the standards.