Stable association of herpes simplex virus with target membranes is triggered by low pH in the presence of the gD receptor, HVEM

Abstract

Using a liposome-binding assay, we investigated the requirements for activation of herpes simplex virus (HSV) into a state capable of membrane interaction. Virions were mixed with liposomes along with the ectodomain of one of three gD receptors (HVEMt, nectin-1t, or nectin-2t) and incubated under different pH and temperature conditions. Virions failed to associate with liposomes in the presence of nectin-1 or nectin-2 at any temperature or pH tested. In contrast, HVEMt triggered association of HSV with liposomes at pH 5.3 or 5.0 when incubated at 37 degrees C, suggesting that HVEM binding and mildly acidic pH at a physiological temperature provide coactivation signals, allowing virus association with membranes. Virions incubated with HVEMt at 37 degrees C without liposomes rapidly lost infectivity upon exposure to pH 5.0, suggesting that these conditions lead to irreversible virus inactivation in the absence of target membranes. Consistent with the idea that soluble receptor molecules provide a trigger for HSV entry, HVEMt promoted virus entry into receptor-deficient CHO K1 cells. However, in B78H1 cells, HVEMt promoted virus entry with markedly lower efficiency. Interestingly, HSV entry into receptor-bearing CHO K1 cells has been shown to proceed via a pH-dependent manner, whereas HSV entry into receptor-bearing B78H1 cells is pH independent. Based on these observations, we propose that the changes triggered by HVEM and mildly acidic pH that allow liposome association are similar or identical to changes that occur during pH-dependent HSV entry.

FIG. 1.

HSV association with liposomes in the presence of soluble receptors at neutral or low pH. Gradient-purified HSV was incubated with the indicated soluble receptors in the presence (A to F, H) or absence (G) of liposomes for 60 min at 37°C in PBS (pH 7.3). Samples B, D, F, G, and H were then adjusted to pH 5.0 by the addition of sodium citrate. The pH values of samples A, C, and E were maintained at 7.3. All samples were incubated for an additional 60 min at 37°C. The samples were then adjusted to 50% sucrose, overlaid with 40% and 20% sucrose, and centrifuged. Six equal fractions were collected, spotted onto nitrocellulose, and probed with a mixture of antibodies against gB, gC, and gD. Samples A through F were from the same experiment, and samples G and H were from a separate experiment. (A) HSV plus HVEMt, pH 7.3. (B) HSV plus HVEMt, pH 5.0. (C) HSV plus nectin-2t, pH 7.3. (D) HSV plus nectin-2t, pH 5.0. (E) HSV plus nectin-1t, pH 7.3. (F) HSV plus nectin-1t, pH 5.0. (G) HSV plus HVEMt, pH 5.0, without liposomes. (H) HSV plus HVEMt, pH 5.0, with liposomes (same conditions as those for sample B).

FIG. 2.

Effect of temperature on HSV association with liposomes triggered by HVEMt at low pH. Virions were incubated with liposomes in the presence of HVEMt for 60 min at 4°C or 37°C. The pH was then adjusted to 6.5 or 5.0 by the addition of sodium citrate, and the mixtures were incubated for an additional 60 min at either 4°C or 37°C. The samples were then adjusted to 50% sucrose, overlaid with 40% and 20% sucrose, and centrifuged. Six equal fractions were collected, spotted onto nitrocellulose, and probed using antibodies to gB, gC, and gD. (A) HSV plus HVEMt, pH 6.5, 4°C. (B) HSV plus HVEMt, pH 5.0, 4°C. (C) HSV plus HVEMt, pH 6.5, 37°C. (D) HSV plus HVEMt, pH 5.0, 37°C.

FIG. 3.

Effect of pH on HSV association with liposomes in the presence of HVEMt. Gradient-purified HSV was incubated with liposomes in the presence of HVEMt for 60 min at 37°C. The pH of each reaction was then adjusted to the indicated level with sodium citrate, and the mixtures were incubated for an additional 60 min at 37°C. The samples were then adjusted to 50% sucrose, overlaid with 40% and 20% sucrose, and centrifuged. Three equal fractions were collected starting from the top of the tube (B, bottom; M, middle; and T, top), spotted onto nitrocellulose, and probed using polyclonal antibodies against the HSV envelope glycoproteins gB, gC, and gD. (A) pH 6.5. (B) pH 6.2. (C) pH 5.9. (D) pH 5.6. (E) pH 5.3. (F) pH 5.0.

FIG. 4.

Effect of liposome addition at various times relative to the pH shift. Virions were incubated with HVEMt for 60 min at 37°C prior to shifting the sample pH to 5.0. After pH shift, the samples were held at 37°C for an additional 60 min. The samples were then adjusted to 50% sucrose, overlaid with 40% and 20% sucrose, and centrifuged. Six equal fractions were collected, spotted onto nitrocellulose, and probed with antibodies against gB, gC, and gD. (A) Liposomes were added at the start of the experiment. (B) Liposomes were added just prior to the pH shift. (C) Liposomes were added 30 min after the pH shift.

FIG. 5.

Inactivation of HSV by HVEMt/low-pH treatment. HSV-1(KOS) (10⁶ PFU) was incubated either alone or with HVEMt for 1 h at 4°C or 37°C. Each sample was adjusted to the pH values shown, and incubation was continued for 1 h. The titers of virus for each sample were determined on Vero cells. Data represent the means of duplicate samples and are plotted as percentages of plaques in control samples (HSV incubated at 37°C without HVEMt and without pH change). Hatched bars indicate samples that were incubated in the absence of HVEMt; gray bars indicate samples to which soluble HVEMt was added. The pH of each sample is indicated below each bar. The temperature of incubation for each sample is also shown (4°C or 37°C).

FIG. 6.

Time course of HSV inactivation by HVEMt/low pH. Individual samples containing 10⁶ PFU of HSV-1(KOS) were incubated with HVEMt at either 4°C or 37°C in PBS. The pH values of some of the samples were shifted to 5.0. At various time points, pairs of samples were quickly chilled on ice and then serially diluted in growth medium for titration on Vero cells. The titers of virus for three pairs of samples were determined prior to shifting the sample pH to 5.0, while the titers of virus for five pairs of samples were determined at various times following the pH shift. Time zero represents the point at which the sample pH was shifted. The titers of virus for data shown at 0 min were determined prior to the pH shift; data at this time represent the controls at each temperature. Data represent the means of duplicate samples and are plotted as percentages of plaques in the sample incubated briefly with HVEMt at 4°C without pH shift (−60 min). White squares indicate samples incubated at 4°C; white circles indicate samples incubated at 37°C.

FIG. 7.

Soluble receptor-mediated entry of HSV into B78H1 and CHO K1 cells. HSV-1(KOS)tk12 was added to B78H1 or CHO K1 cells in 96-well plates. The plates were centrifuged for 90 min at 4°C, the virus inoculum was replaced with fivefold dilutions of the indicated soluble receptors, and the plates were incubated for an additional 60 min at 4°C. Warm complete medium was added, and the cultures were incubated for 8 h at 37°C. Cells were then lysed, and β-galactosidase activity was measured as an indication of virus entry. Results are reported as increases (n-fold) in β-galactosidase activity over entry in the absence of added soluble receptor. Data shown represent the means of triplicate samples.