Herpes simplex virus types 1 and 2 differ in their interaction with heparan sulfate

Abstract

Cell surface heparan sulfate (HS) serves as an initial receptor for many different viruses, including herpes simplex virus types 1 and 2 (HSV-1 and 2, respectively). Glycoproteins C and B (gC and gB) are the major components of the viral envelope that mediate binding to HS. In this study, purified gB and gC homologous proteins as well as purified HSV-1 and HSV-2 virions were compared for the ability to bind isolated HS receptor molecules. HSV-1 gC and HSV-2 gC bound comparable amounts of HS. Similarly, HSV-1 gB and its HSV-2 counterpart showed no difference in the HS-binding capabilities. Despite the similar HS-binding potentials of gB and gC homologs, HSV-1 virions bound more HS than HSV-2 particles. Purified gC and gB proteins differed with respect to sensitivity of their interaction with HS to increased concentrations of sodium chloride in the order gB-2 > gB-1 > gC-1 > gC-2. The corresponding pattern for binding of whole HSV virions to cells in the presence of increased ionic strength of the medium was HSV-2 gC-neg1 > HSV-1 gC(-)39 > HSV-1 KOS 321 > HSV-2 333. These results relate the HS-binding activities of individual glycoproteins with the cell-binding abilities of whole virus particles. In addition, these data suggest a greater contribution of electrostatic forces for binding of gB proteins and gC-negative mutants compared with binding of gC homologs and wild-type HSV strains. Binding of wild-type HSV-2 virions was the least sensitive to increased ionic strength of the medium, suggesting that the less extensive binding of HS molecules by HSV-2 than by HSV-1 can be compensated for by a relatively weak contribution of electrostatic forces to the binding. Furthermore, gB and gC homologs exhibited different patterns of sensitivity of binding to cells to inhibition with selectively N-, 2-O-, and 6-O-desulfated heparin compounds. The O-sulfate groups of heparin were found to be more important for interaction with gB-1 than gB-2. These results indicate that HSV-1 and HSV-2 differ in their interaction with HS.

Figures

FIG. 1

Electrophoretic analysis of affinity-purified gB-1, gB-2, gC-1, and gC-2. Glycoproteins were subjected to electrophoresis under reducing conditions on a 10% acrylamide tris-glycine precast gel and stained with a colloidal Coomassie blue kit.

FIG. 2

(A) HS-binding capabilities of purified gB-1, gB-2, gC-1, and gC-2. 35S-labeled HS from GMK AH1 cells was incubated for 2 h at room temperature with purified viral proteins. Bound HS was trapped on nitrocellulose filters. Values shown are averages of four individual determinations from two separate experiments. Data for gC-2 (HEp-2) and the rest of the proteins were obtained in experiments done at different times. (B) Binding of purified gB-1, gB-2, gC-1, and gC-2 to HS in the presence of increasing concentrations of sodium chloride. Purified proteins were incubated with 35S-labeled HS from GMK AH1 in phosphate buffer supplemented with specific concentrations of sodium chloride. The rest of the procedure was as described for panel A. Values shown are averages of two individual determinations from two separate experiments.

FIG. 3

HSV binding to cells in the presence of increasing concentrations of sodium chloride. Purified radiolabeled (A) or nonlabeled (B) HSV-1 KOS 321, HSV-1 gC−39, HSV-2 333, and HSV-2 gC-neg1 were incubated with GMK AH1 cells for 20 min at 4°C in the presence specific concentrations of sodium chloride. Experiments were carried out at 4°C (cold room). Sodium chloride solutions were left on cells for a maximum of 40 min. For further details, see Materials and Methods. Values shown are averages of four individual determinations from two separate experiments.

FIG. 4

Carbohydrate modification and HS binding by gC-1 and gC-2. (A) Electrophoretic analysis of mock (M)-, sialidase (S)-, sialidase and O-glycanase (SO)-, and endoglycosidase F/N (N)-treated gC-1 and gC-2. Sizes are indicated in kilodaltons. (B) Binding of HS by sialidase and/or glycosidase-treated gC-1 and gC-2. Results are expressed as percent bound HS relative to mock-treated controls. For further details, see the legend to Fig. 2A.

FIG. 5

Infection of heparinase-treated cells by HSV wild-type and gC-negative mutant strains. GMK AH1 cells in six-well plates cells were pretreated with the indicated number of heparinase Sigma units (1 IU corresponds to approximately 600 Sigma units) and then approximately 200 PFU of KOS 321, gC−39, or 333 or gC-neg1 was added. Values shown for HSV-2 are means of six replicates from three separate experiments. For HSV-1, the results represent means of two replicates from a single experiment.

FIG. 6

Sensitivities of HSV-1, HSV-2, and purified gC and gB homologs to selectively desulfated heparin compounds. (A) HSV-1 and HSV-2 were preincubated for 10 min at either 4 or 37°C in the presence of fivefold-increasing concentrations of N-, 2-O-, or 6-O-desulfated samples of heparin. Mixtures were transferred to GMK AH1 cells and held for 1 h at either 4 or 37°C. The number of PFU is expressed as a percentage of the average number of plaques formed in the absence of competitor. Two to three separate experiments were carried out in duplicate for each sample. (B) Purified gB-1, gB-2, gC-1, and gC-2 were preincubated for 10 min at 4°C in the presence of 10-fold-increasing concentrations of modified heparin samples. The mixtures were transferred to GMK AH1 cells in 96-well plates and left for attachment for 1 h at 4°C. Bound glycoproteins were detected by an ELISA-based procedure. Results are means of six replicates from two separate experiments.