Herpes simplex virus type 2 glycoprotein G is targeted by the sulfated oligo- and polysaccharide inhibitors of virus attachment to cells

Abstract

Variants of herpes simplex virus type 2 (HSV-2) generated by virus passage in GMK-AH1 cells in the presence of the sulfated oligosaccharide PI-88 were analyzed. Many of these variants were substantially resistant to PI-88 in their initial infection of cells and/or their cell-to-cell spread. The major alteration detected in all variants resistant to PI-88 in the initial infection of cells was a frameshift mutation(s) in the glycoprotein G (gG) gene that resulted in the lack of protein expression. Molecular transfer of the altered gG gene into the wild-type background confirmed that the gG-deficient recombinants were resistant to PI-88. In addition to PI-88, all gG-deficient variants of HSV-2 were resistant to the sulfated polysaccharide heparin. The gG-deficient virions were capable of attaching to cells, and this activity was relatively resistant to PI-88. In addition to having a drug-resistant phenotype, the gG-deficient variants were inefficiently released from infected cells. Purified gG bound to heparin and showed the cell-binding activity which was inhibited by PI-88. Many PI-88 variants produced syncytia in cultured cells and contained alterations in gB, including the syncytium-inducing L792P amino acid substitution. Although this phenotype can enhance the lateral spread of HSV in cells, it conferred no virus resistance to PI-88. Some PI-88 variants also contained occasional alterations in gC, gD, gE, gK, and UL24. In conclusion, we found that glycoprotein gG, a mucin-like component of the HSV-2 envelope, was targeted by sulfated oligo- and polysaccharides. This is a novel finding that suggests the involvement of HSV-2 gG in interactions with sulfated polysaccharides, including cell surface glycosaminoglycans.

FIG. 1.

Effect of PI-88 or heparin on infectivity of PI-88 escape variants of HSV-2. Approximately 100 to 200 PFU of a specific AC variant (A and B) or an A or C variant (C and D) were mixed with PI-88 (0.16 to 100 μg/ml) or heparin (0.16 to 100 μg/ml) and incubated for 15 min at room temperature prior to being added to and incubated with GMK AH1 cells for 2 h at 37°C. The cells were then washed and overlaid with methylcellulose solution without an inhibitor. The results are expressed as percentages of the number of viral plaques of the PI-88/heparin-treated virus relative to that of the mock-treated controls. The values shown are the means of duplicate determinations from two separate experiments.

FIG. 2.

Effect of PI-88 or heparin on infectivity of gG-deficient mutants of HSV-2. Approximately 200 PFU of gG-deficient recombinants 333+AC9gG and 333+AC6gG or gG-proficient strains 333 and 333(gG⁺) were mixed with 0.16 to 100 μg/ml of PI-88 (A) or heparin (B) and incubated for 15 min at room temperature prior to being added to and incubated with GMK AH1 cells for 2 h at 37°C. The cells were then washed and overlaid with methylcellulose solution without an inhibitor. The results are expressed as percentages of the number of viral plaques of the PI-88/heparin-treated virus relative to that of the mock-treated controls. The values shown are the means of duplicate determinations from two separate experiments.

FIG. 3.

Infection of GMK AH1 cells with the gG-deficient mutant of HSV-2. The cells were infected with the gG-deficient recombinant virus 333+AC6gG (A and C) or the gG-proficient 333(gG⁺) strain (B and D) at a multiplicity of infection of 0.001. The cells were washed and maintained in Eagle's medium without methylcellulose or human gamma globulin. The cells were immunostained with the monoclonal anti-HSV gB antibody B11D8 at 24 h (A and B) or 48 h (C and D) after infection.

FIG. 4.

Production of infectious virus in GMK AH1 cells infected with gG-deficient mutants of HSV-2. The cells were infected with the gG-deficient recombinant viruses 333+AC6gG and 333+AC9gG or the gG-proficient 333(gG⁺) strain at a MOI of 0.001 or 1. At specific times after infection, the production of extracellular (E) or cell-associated (C) virus was assessed by titration in GMK AH1 cells. Two separate experiments were carried out for each virus. The values shown were derived from the first experiment.

FIG. 5.

Effect of PI-88 on attachment of gG-negative mutants of HSV-2 to cells. (A) Purified radiolabeled virions of gG-deficient mutant viruses 333+AC6gG, 333+AC9gG, or AC9 and gG-proficient strains 333 and 333(gG⁺) were mixed with PI-88 (100 μg/ml) and incubated for 10 min at 4°C prior to being added to and incubated with GMK AH1 cells for 90 min at 4°C. The results are expressed as percentages of the counts per minute (cpm) of viruses attaching to cells in the presence of an inhibitor relative to that for the mock-treated controls. (B) Purified radiolabeled virions of gG-deficient and gG-proficient viruses were incubated with GMK AH1 cells for 90 min at 4°C. The results are expressed as percentages of the counts per minute of viruses binding to cells relative to that for the added virus. The values shown are the means of the results of two (333 and AC9) or five (333gG⁺, 333+AC6gG, and 333+AC9gG) separate experiments.

FIG. 6.

Binding of purified gG of HSV-2 to immobilized heparin. Purified gG was run through columns loaded with Sephadex G10-heparin or Sephadex G10 alone. The beads were eluted in a stepwise manner with increasing concentrations of NaCl. The input (A), flow-through (B), wash-out (not shown), and pooled fractions of protein eluted with 0.15 to 0.3 M (C), 0.4 to 0.6 M (D), or 0.7 to 2 M NaCl (E) were concentrated and electrophoresed under reducing conditions on a 10% acrylamide NuPAGE Bis-Tris precast gel. The electroblotted proteins were immunostained with the monoclonal anti-HSV-2 gG antibody O1C5. +, with heparin; −, without heparin.

FIG. 7.

Binding of purified gG of HSV-2 to cells. Purified proteins at specific concentrations were incubated with GMK AH1 (A) or HaCaT (C) cells for 1 h at 4°C. Alternately, purified proteins (4 μg) were incubated with PI-88 for 15 min prior to being added to and during a 1-h period of protein adsorption to GMK AH1 (B) or HaCaT (D) cells at 4°C. The bound glycoproteins were detected by an enzyme-linked immunosorbent assay-based procedure. The amount of bound protein is expressed as an absorbance value (A and C) or as a percentage of cell-bound protein detected in the PI-88-treated sample relative to that in the mock-treated controls (B and D). The values shown are the means of duplicate determinations from two separate experiments. O.D.450, optical density at 450 nm.