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Journal of Virology, April 2006, p. 3773-3780, Vol. 80, No. 8
0022-538X/06/$08.00+0     doi:10.1128/JVI.80.8.3773-3780.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Stable Association of Herpes Simplex Virus with Target Membranes Is Triggered by Low pH in the Presence of the gD Receptor, HVEM

J. Charles Whitbeck,^1,2* Yi Zuo,¹ Richard S. B. Milne,^1,2 Gary H. Cohen,¹ and Roselyn J. Eisenberg^1,2

School of Dental Medicine,¹ School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104²

Received 13 August 2005/ Accepted 31 January 2006

Top Abstract Introduction Materials and Methods Results Discussion References

 
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°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°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.

Top Abstract Introduction Materials and Methods Results Discussion References

 
A common feature of enveloped virus entry is that in the presence of an activation signal(s), one or more envelope proteins undergo structural rearrangements leading to membrane fusion (10). Activation signals differ among enveloped viruses but fall into two broad categories based upon the requirement of low pH in entry (7, 30). The fusion mechanism of pH-independent viruses, such as human immunodeficiency virus, paramyxoviruses, and coronaviruses, is activated by receptor binding at the plasma membrane. In contrast, the fusion of pH-dependent viruses, such as influenza, rhabdoviruses, and alphaviruses/flaviviruses, is triggered by mildly acidic pH within an endosome.

Herpes simplex virus (HSV) employs at least three different entry pathways. Early studies suggested that entry into Vero and Hep-2 cells occurred by direct fusion with the plasma membrane (41). More recently, two endocytic entry pathways have been described. Nicola et al. have demonstrated that in CHO K1 and HeLa cells (25) as well as in primary human keratinocytes (27), HSV is internalized into an endocytic compartment prior to pH-dependent virus entry. Interestingly, Milne et al. (22) described a second endocytic entry pathway that is pH independent and is employed in a different subset of cell types (our unpublished data). Thus, HSV is capable of utilizing both pH-independent and pH-dependent entry pathways.

Complicating the study of HSV entry is the existence of multiple viral entry glycoproteins as well as multiple cellular receptors for virus entry (32). Among the 11 or so viral envelope glycoproteins, 4 are essential for entry. These are gB, gD, gH, and gL. The cellular receptors include two cell adhesion molecules from the immunoglobulin superfamily (nectin-1 and nectin-2), a tumor necrosis factor receptor family member (HVEM), and certain moieties within heparan sulfate proteoglycans generated by a specific isoform of 3-O-sulfotransferase (33). For each of these receptors, gD is the viral ligand. The necessity of a gD receptor for HSV entry (as opposed to simple attachment or internalization) holds true regardless of whether entry is pH dependent or pH independent (22, 26). Although the fusion protein(s) of HSV has not yet been identified, the likely candidates appear to be gB and the gH/gL complex. These glycoproteins constitute the core fusion machinery of all herpesviruses studied to date, whereas gD homologues are present in a subset of alphaherpesviruses (32).

The association of virions with liposomes in response to receptor binding, low pH, or other conditions is viewed as evidence of the induction of conformational changes in the viral fusion protein(s), resulting in exposure of a hydrophobic fusion peptide (7). Liposome-binding assays have been used to define the conditions required for triggering membrane association by a number of viruses (3, 14, 34, 35, 40, 42, 43). In this study, we used a liposome-binding assay to examine the activation of HSV to a state capable of membrane association. In the presence of soluble HVEM (HVEMt), but not nectin-1t or nectin-2t, mildly acidic pH triggered association of HSV with liposomes at 37°C, suggesting that these conditions provide coactivation signals for membrane association. In the absence of membranes, these same conditions led to virus inactivation for both liposome association and host cell infection, suggesting that the conformational changes triggered by HVEM binding at mildly acidic pH may be irreversible. The effects of HVEM binding and exposure to mildly acidic pH on HSV in vitro may mimic the events that occur during virus entry into CHO K1 or HeLa cells.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Virus and cell culture. All cell lines were grown at 37°C in the presence of 5% CO₂. Vero cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco) supplemented with 5% fetal bovine serum (FBS) and antibiotics (100 U/ml penicillin G and 100 U/ml streptomycin sulfate; Gibco). B78H1 murine melanoma cells were also cultured in DMEM supplemented with 5% FBS and antibiotics. CHO K1 cells were cultured in Ham's F-12 medium (Gibco) supplemented with 10% FBS and antibiotics. HSV-1(KOS) as well as the ß-galactosidase reporter virus, HSV-1(KOS)tk12 (38), was propagated, and the titers of the virus were determined on Vero cells. Sucrose gradient-purified HSV (16) was used throughout the studies reported here.

Antibodies. Rabbit polyclonal antibodies raised against gB (R69 [9]), gC (R47 [9]), and gD (R7 [17]) have been described previously.

Preparation of recombinant soluble receptor molecules. The production and purification of soluble forms of HVEM [HVEM(200t)] (39), nectin-1 [HveC(346t)] (18), and nectin-2 [HveB(361t)] (38) using a baculovirus expression system have been described previously. For simplicity, these soluble receptors will be referred to here as HVEMt, nectin-1t, and nectin-2t, respectively.

Liposome preparation. Large unilamellar vesicles were prepared as described elsewhere (29, 31). Briefly, 4 mg (5.26 µmol) phosphatidylcholine (0.4 ml of 10 mg/ml in chloroform; Avanti Polar Lipids) was combined with 1 mg (2.59 µmol) cholesterol (20 µl of 50 mg/ml in chloroform; Avanti Polar Lipids) in a 5-ml borosilicate glass vial. The final lipid mixture thus contained a 2:1 molar ratio of phosphatidylcholine to cholesterol. To coat the glass surface, the lipid mixture was dried under a gentle stream of argon gas while the vial was rotated. The dried lipid mixture was then lyophilized for 1 h to ensure complete removal of chloroform. The dried lipid film was hydrated in 1 ml phosphate-buffered saline (PBS) (10 mM sodium phosphate [pH 7.3], 150 mM NaCl) at 50°C with frequent, vigorous vortexing to yield large multilamellar vesicles. These large multilamellar vesicles were converted to large unilamellar vesicles by 5 cycles of freezing (dry ice/methanol bath) and thawing (37°C water bath), followed by 21 passes through a 0.8-µm Nuclepore track-etched membrane (Whatman) using a Mini-Extruder (Avanti Polar Lipids).

Liposome flotation experiments. Liposome flotation experiments were adapted from previously described methods (6, 7, 31). HSV-1(KOS) (2 x 10⁷ PFU) was combined with soluble receptors (5 µM) and 50 µl of liposomes. The final reaction volume was adjusted to 100 µl with PBS. Receptor/virus/liposome reaction mixtures were incubated at 4°C or 37°C for 1 h. Reactions were then adjusted to 50% sucrose by the addition of 200 µl of 75% sucrose in PBS. The virus/receptor/liposome mixtures were then overlaid with 2 ml of 40% sucrose and 2 ml of 20% sucrose and centrifuged for 20 h in a Beckman SW50.1 rotor at 100,000 x g and 4°C. Six equal fractions (approximately 700 µl each) were collected starting from the top of the gradient and spotted onto nitrocellulose filters using a vacuum manifold (Schleicher and Schuell). Filters were then probed for HSV envelope glycoproteins using a mixture of polyclonal antibodies raised against purified gB (R69), gC (R47), and gD (R7). Dot blots were then incubated with horseradish peroxidase-conjugated goat anti-rabbit antibodies and visualized using enhanced chemiluminescence (Amersham). For flotation experiments involving pH changes, the basic protocol was the same as that described above, with the following exceptions. Receptor/virus/liposome mixtures were incubated at 37°C for 1 h prior to pH change. Samples were then shifted to 4°C or left at 37°C prior to pH change. The pH of each reaction was lowered by adding 25 µl of 200 mM sodium citrate buffer (pH-adjusted samples contained a final concentration of 40 mM sodium citrate and 8 mM sodium phosphate). Typically, the final sample pH was 0.3 units higher than the pH of the sodium citrate solution added due to the buffering effect of sodium phosphate (pH 7.3) already present in each sample (i.e., samples receiving 25 µl of 200 mM sodium citrate [pH 5.0] had a final pH of 5.3). The final pH is reported in each figure. Prior to being loaded onto gradients, the samples were neutralized (pH 7) by the addition of a predetermined volume of 2 M Tris (pH 9.0).

Virus inactivation. For virus inactivation, 40-µl samples containing 1 x 10⁶ PFU of HSV, 5 µM HVEMt, and 5 µM bovine serum albumin were prepared in PBS. Samples were then incubated at either 4°C or 37°C for 1 h. Each sample then received 10 µl of PBS (control samples at pH 7.3) or 10 µl of 200 mM sodium citrate (of various pH levels), and incubation was continued for an additional hour. Samples were then shifted to ice and quickly diluted 10-fold by adding 450 µl of Vero cell growth medium. Serial 10-fold dilutions of this virus were prepared and plated on Vero cells. Following 1 h of adsorption at 37°C, cell monolayers were overlaid with Vero cell growth medium containing 1% carboxymethylcellulose. Infected monolayers were incubated at 37°C for 2 days to allow plaque formation, fixed in 2.5% formaldehyde (in PBS), incubated with rabbit polyclonal antibodies against gB, gC, and gD, followed by horseradish peroxidase-conjugated protein A (Amersham), and visualized via staining with a solution of 0.1 mg/ml 4-chloro-1-naphthol and 0.1% sodium peroxide in PBS (36). Plaques were then counted, and the number in each experimental sample was plotted as a percentage of plaques present in the control sample.

HSV entry into receptor-deficient cells triggered by soluble receptors. Gradient-purified HSV-1(KOS)tk12 was diluted in DMEM, 5% fetal calf serum, and 30 mM HEPES (pH 7.0). Virus was added to 5 x 10⁴ B78H1 or CHO K1 cells in 96-well plates (10⁵ PFU/well) and incubated at 4°C for 90 min to allow virus attachment. During this time, the cell cultures were centrifuged (spinoculated) at 250 x g (11). Following spinoculation, the virus inoculum was removed and various concentrations of soluble receptors diluted in PBS (50 µl per well) were added to triplicate wells of the plate. The plates were incubated for an additional 60 min at 4°C in the presence of the soluble receptors. Finally, warm (37°C) medium (150 µl per well) was added (without removing the soluble receptors), and the cultures were incubated at 37°C for 8 h. Cells were then lysed by adding a one-fourth volume (50 µl per well) of PBS and 2.5% NP-40. ß-Galactosidase activity in the lysates was determined as described previously (38). Similar levels of ß-galactosidase activity were induced in the absence of added soluble receptors on both CHO-K1 and B78H1 cells.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Association of HSV with liposomes is triggered by soluble HVEMt at mildly acidic pH. Our working hypothesis is that the requirements for HSV association with a host cell membrane during virus entry can be studied in vitro using purified virions, soluble gD receptors, and liposomes as target membranes. The soluble receptors HVEMt and nectin-1t bind to gD (18, 39) as well as to purified HSV (24), whereas nectin-2t fails to bind gD of wild-type HSV strains (38) and was therefore used as a negative control. In the first experiment (Fig. 1A to F), duplicate samples of the virus were mixed with HVEMt, nectin-1t, or nectin-2t in the presence of liposomes at 37°C in PBS for 1 h. The pH of each sample was then either maintained at 7.3 or lowered to 5.0 to mimic late endosomal pH (15), and the mixtures were incubated for an additional hour at 37°C. Following incubation, the virus/receptor or virus/receptor/liposome mixtures were layered beneath a 20 to 40% discontinuous sucrose gradient and centrifuged. Fractions were collected, spotted onto nitrocellulose, and probed with a mixture of polyclonal antibodies against gB, gC, and gD. Under these conditions, liposomes moved to the top of the gradient along with any associated virions; any virions not associated with liposomes remained at the bottom of the gradient. At pH 7.3, virions were detected only in the bottom fractions (Fig. 1A, C, and E), indicating that no liposome association had occurred in the presence of any of the receptors. In contrast, when the virus was incubated at 37°C and pH 5.0, HSV associated with liposomes in the presence of HVEMt (Fig. 1B). However, neither nectin-2t (Fig. 1D) nor nectin-1t (Fig. 1F) triggered liposome association of the virus under these conditions. To confirm the requirement of liposomes for HSV flotation in a sucrose gradient, we incubated parallel samples of the virus with HVEMt at 37°C and adjusted the pH to 5.0 either in the absence (Fig. 1G) or in the presence (Fig. 1H) of liposomes. Virus was detected only in the upper fractions of the sucrose gradient in the presence of liposomes. This showed that the density of HSV particles was not altered under these conditions such that liposome-independent flotation could occur. Based on these results, we focused our attention on HVEMt in subsequent experiments.

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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).

To examine the importance of temperature, HSV was combined with liposomes in the presence of soluble HVEMt as described before and then incubated at 4°C or 37°C for 1 h. Two samples were incubated at each temperature; one was adjusted to pH 6.5 and the other to pH 5.0 by the addition of 200 mM sodium citrate. Regardless of pH, virus samples maintained at 4°C did not associate with liposomes (Fig. 2A and B). Additionally, virus incubated with HVEMt at 37°C and pH 6.5 did not associate with liposomes (Fig. 2C). However, as described before, HSV incubated with HVEMt and liposomes at 37°C and pH 5.0 associated with the liposomes (Fig. 2D). Thus, we conclude that in addition to being receptor and pH dependent, the association of the virus with liposomes is also temperature dependent. This experiment also demonstrated that it was pH and not buffer composition alone (citrate versus phosphate buffer) that affected virus association with liposomes.

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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.

The pH optimum for HVEMt-triggered liposome association occurs below 5.6. To define the pH range for liposome association, we incubated virions with liposomes in the presence of HVEMt at various pH levels between 5.0 and 6.5 (Fig. 3). For this experiment, we separated liposome flotation gradients into three fractions instead of six fractions as shown elsewhere. This allowed us to focus on major rather than more subtle differences in liposome association between individual samples. Similar levels of liposome association were observed at pH 5.3 and 5.0 (Fig. 3E and F). In contrast, little or no liposome association occurred at pH 6.5, 6.2, and 5.9 (Fig. 3A, B, and C). Some evidence of liposome association was observed in the sample incubated at pH 5.6 (Fig. 3D). These data indicate that the highest levels of virus-liposome association occur below pH 5.6.

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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.

Membranes must be present during low-pH incubation for virion association to occur. Thus far, we found that HSV associated with liposomes when the virus, HVEMt, and liposomes were coincubated at 37°C at mildly acidic pH. Since some viruses, such as influenza (34) and rabies (12), rapidly lose the ability to associate with membranes following exposure to mildly acidic pH, we wondered whether the association of HSV with membranes could still occur if liposomes were added after the pH of the virus-HVEMt mixture was lowered. Three samples containing HSV and HVEMt were incubated at 37°C. In the first (Fig. 4A), liposomes were added to the virus/receptor mixture at the start of the incubation as shown in Fig. 1 to 3. In the second (Fig. 4B), the virus and receptor were incubated for 1 h at 37°C at pH 7.3; then liposomes were added, and the pH was immediately adjusted to 5.0. The mixture was then incubated for an additional hour. In both cases, a substantial portion of the virus was found in the upper fractions of the gradient, indicative of liposome association. However, when liposomes were added 30 min after the pH change (Fig. 4C), none of the virus was found at the top of the gradient, indicating no association with liposomes. This experiment showed that liposomes must be present at the same time that the pH is lowered in order for virus association to occur.

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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.

Soluble HVEM induces a pH-dependent loss of HSV infectivity. We hypothesize that incubation of the virus with HVEMt at 37°C and pH 5.0 in some way induces a transient membrane-reactive state. Perhaps due to the exposure of a hydrophobic domain within one or more envelope glycoproteins, virus in this membrane-reactive state may be unstable in an aqueous environment and may quickly lose its ability to associate with membranes. To test this hypothesis, we incubated HSV with HVEMt at either 4°C or 37°C for 1 h. Some samples were then adjusted to a lower pH with sodium citrate, and the incubation was continued for an additional hour. Two control samples lacking HVEMt were included, with one incubated at pH 7.3 and the other at pH 5.0, both at 37°C. A third control was incubated in the absence of a receptor at pH 5.0 and 4°C. To determine the amount of infectious virus remaining, the titers of virus in each sample were determined on Vero cells (Fig. 5). Residual infectivity was calculated as the ratio of the experimental samples to the control virus sample lacking a receptor and incubated at pH 7.3. The two other control samples (held at pH 5.0) retained 90% infectivity. Samples incubated at 37°C with HVEMt at pH 6.5 or 6.2 retained greater than 80% infectivity. Samples incubated with HVEMt at more acidic pH values lost infectivity in a pH-dependent manner. At pH 5.3 and 5.0, virus samples retained only 16% and 6% infectivity, respectively (84% and 94% inactivation). Thus, the temperature, receptor, and pH requirements for virus inactivation are strikingly similar to those previously observed for virus-liposome association.

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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).

Nicola et al. (25) reported inactivation of HSV at mildly acidic pH in the absence of any gD receptor. Although we cannot explain this discrepancy, there were many differences between the two protocols used for inactivation. Nicola et al. treated virions at various pH levels in cell culture medium (containing 10% fetal bovine serum and buffered with 44 mM sodium bicarbonate and 1 mM sodium phosphate) supplemented with HEPES, MES [2-(N-morpholino)ethanesulfonic acid], and succinate (5 mM each) and then neutralized with 0.05 N NaOH. Our samples were treated in a solution of 120 mM NaCl buffered with 40 mM sodium citrate and 8 mM sodium phosphate and were neutralized by a 10-fold dilution in culture medium. Perhaps the presence of 10% fetal bovine serum provided a factor capable of effecting HSV inactivation at low pH.

Inactivation by low pH in the presence of HVEMt occurs rapidly. To determine the kinetics of virus inactivation by HVEMt at low pH, we measured the extent of inactivation over time (Fig. 6). Here, virus samples were combined with HVEMt and incubated at either 4°C or 37°C at neutral pH. After 1 h, the pH was adjusted to 5.0, and the virus-receptor mixtures were incubated at 4°C or 37°C for an additional hour. Control samples were maintained at pH 7.3 and 37°C. At various times, the titers of virus in the aliquots were determined to quantitate the amount of infectious virus remaining. Prior to the pH change, infectivity remained intact at both 4°C and 37°C. However, following the shift to pH 5.0, virus incubated at 37°C was rapidly inactivated. Within 5 min, the sample had lost almost 80% infectivity and by 15 min, infectivity dropped to less than 10% of that of the control (virus incubated with a receptor in PBS at 4°C). Samples held at 4°C were not inactivated following the pH shift. Thus, changes that lead to virus inactivation are rapidly induced by a combination of low pH and HVEMt and a temperature of 37°C.

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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.

Soluble receptors trigger HSV entry into receptor-deficient cells. We reasoned that if soluble HVEM is capable of providing the same activation signal to HSV virions that the full-length receptor provides in cells, then it might enhance virus entry into receptor-deficient CHO K1 or B78H1 cells. It has been shown that HSV enters CHO K1 cells via a pH-dependent endocytic pathway. Although internalization into these cells is not dependent upon a receptor, the ability of the internalized virus to initiate productive infection is receptor dependent (26). In contrast, HSV enters B78H1 cells via a pH-independent, endocytic pathway (22). In these experiments, HSV(KOS)tk12, a ß-galactosidase reporter virus, was added to CHO K1 and B78H1 cells for 90 min at 4°C to allow virus attachment via heparan sulfate. HVEMt (or nectin-2t as a negative control) was then added to the cells, and the cultures were shifted to 37°C. At 8 h postinfection, the cells were lysed with NP-40. Virus entry was monitored by measuring ß-galactosidase activity in cell extracts (Fig. 7). Enhanced entry into CHO K1 cells was first evident in the presence of 40 nM HVEMt, in which entry reached levels approximately three times background levels. Entry into CHO K1 cells increased further at higher HVEMt concentrations, reaching 15.5 times background levels in the presence of 5 µM HVEMt. Although HVEMt also triggered entry into B78H1 cells, it did so with much lower efficiency. In this case, HVEMt increased entry less than threefold over background levels at the highest concentration tested (5 µM). HSV entry into B78H1 cells was enhanced approximately 10-fold in the presence of 5 µM nectin-1t (data not shown), indicating that these cells were susceptible to soluble receptor-mediated infection. As expected, soluble nectin-2 failed to trigger entry into either CHO K1 or B78H1 cells. These data are consistent with the notion that HVEMt provides a trigger rather than simply an attachment point for virus entry into HSV-resistant cells. However, the fact that HVEMt was much less efficient in promoting HSV entry into B78H1 cells suggests that HVEMt alone is insufficient to trigger entry and that additional cell-specific factors (such as trafficking internalized virions through a low-pH environment) are also required.

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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 ß-galactosidase activity was measured as an indication of virus entry. Results are reported as increases (n-fold) in ß-galactosidase activity over entry in the absence of added soluble receptor. Data shown represent the means of triplicate samples.

Top Abstract Introduction Materials and Methods Results Discussion References

 
HVEM and nectin-1 are two structurally different cellular receptors for HSV. Expression of either protein on receptor-deficient cells is sufficient to allow HSV entry (33). We previously showed that the ectodomains of HVEM and nectin-1 were able to bind HSV virion gD (24), and we asked whether a gD receptor alone might be sufficient to trigger HSV association with a target membrane. To address this question, we combined soluble receptors with purified virions in the presence of liposomes. At neutral pH, little or no association of virions with liposomes occurred at the physiological temperature of 37°C, suggesting that receptor binding alone was insufficient to trigger such an association. We hypothesized that an additional trigger or triggers, provided by the host cell, must also be necessary in order for membrane interaction to occur. Recently, Nicola et al. (25) showed that HSV entered CHO K1 and HeLa cells via a pH-dependent endosomal pathway, suggesting the possibility that the mildly acidic pH within an endosome could provide a "coactivation signal" for HSV along with a functional viral receptor. With this in mind, we repeated the prior experiment but shifted some of the samples to pH 5.0 following receptor binding to mimic the pH of a late endosome (15). Under these conditions, we observed HSV association with liposomes in the presence of HVEMt and at 37°C. Further studies showed that the highest levels of liposome association occurred at pH 5.3 and 5.0. Interestingly, nectin-1t failed to induce detectable levels of virus association with liposomes under any of the conditions tested. Perhaps nectin-1t induces a membrane-reactive state in HSV that is in some way different from that induced by HVEMt and that was not detectable in this assay. Alternatively, additional cellular factors may be required in conjunction with nectin-1t binding to trigger HSV association with membranes. These possibilities will be addressed in future studies.

Several viruses lose infectivity upon triggering with a receptor or mildly acidic pH in the absence of cells. Two examples include influenza virus, which is inactivated by exposure to pH levels between 4.9 and 5.3 (28), and the coronavirus murine hepatitis virus (43), which is inactivated following incubation with a soluble form of its receptor (CECAM-1) at 37°C. In each case, inactivation has been directly correlated with irreversible pH- or receptor-induced conformational changes in the envelope glycoproteins. We found that HSV failed to associate with liposomes if the virus was first incubated with HVEMt and then shifted to pH 5 prior to liposome addition. Moreover, virus that had been incubated with HVEMt at mildly acidic pH and at 37°C rapidly lost the ability to infect cells. Based on these observations, we speculate that perhaps some sort of irreversible change(s) in one or more virion components (likely gB and/or gH/gL) is induced under these conditions. In the context of CHO K1 cells, we believe that these changes take place within the endosome, leading to membrane fusion and virus entry into its replicative cycle. In contrast, activation of the fusion machinery in the absence of a target membrane renders the virus inactive, perhaps due to trapping some portion of the fusion machinery in a nonfunctional state. In an attempt to detect possible conformational changes, we examined the structures of gB, gD, and gH in inactivated versus mock-inactivated virions using monoclonal antibodies (MAbs) as well as a panel of proteolytic enzymes. Using these methods, we have not yet seen evidence of any conformational changes. However, it is possible that none of our MAbs bind to regions of gB, gD, or gH that undergo these putative changes. We also examined gB, gD, and gH via "native" Western blotting (5) following incubation at various pH levels. Interestingly, gB incubated at pH levels above 5.6 migrated primarily as an oligomer, whereas gB incubated at pH levels below 5.6 migrated as a monomer (our unpublished results). This shift of gB from oligomer to monomer at lower pH was confirmed by a loss of reactivity with the oligomer-specific MAb DL16 (2). Based on this observation, we speculate that pH-dependent alterations in gB along with receptor engagement by gD provide necessary cues for pH-dependent HSV entry.

Virions treated with HVEMt at mildly acidic pH were inactivated at 37°C, but not at 4°C, just as liposome association was observed only at 37°C. This suggests that elevated temperature is necessary to allow the postulated changes on the surface of the virion to occur, leading to membrane association. These observations are consistent with the lack of HSV entry into cells at 4°C and suggest that the block to entry at low temperatures is not due solely to an effect on the host cell membrane but likely reflects a need for thermal energy in order to allow certain critical changes in the virus particle to occur.

To further examine the biological activity of soluble HVEM, we tested its ability to promote HSV entry into receptor-deficient CHO K1 and B78H1 cells. We reasoned that if HVEMt truly mimicked the effects of full-length, membrane-bound HVEM in its ability to provide a partial trigger for membrane (liposome) association, then it might also mediate virus entry into receptor-deficient cells. Interestingly, we observed a considerable difference in the potency of HVEMt in promoting virus entry into two different cell types. Whereas HVEMt promoted entry into CHO K1 cells at concentrations as low as 40 nM, a much higher HVEMt concentration (5 µM) was required to achieve an appreciable effect on virus entry into B78H1 cells. Since HSV entry into CHO K1 cells reportedly occurs through a pH-dependent, endocytic pathway (25), we suspect that in these cells, low endosomal pH provides the necessary coactivation signal (along with HVEMt) for virus entry. In contrast, B78H1 cells, in which HSV entry occurs via a pH-independent, endocytic pathway (22), do not provide an appropriate coactivation signal for HVEMt-mediated virus entry.

Several reports (1, 23, 31) have presented evidence that avian leukosis virus A (ALV-A) requires both receptor (Tva) binding and mildly acidic pH in order for membrane fusion and virus entry to occur (although the pH requirement for ALV-A fusion has been challenged by Earp et al. [8]). The authors of those studies have characterized receptor binding by ALV-A as "priming" virions to respond to the secondary stimulus of low endosomal pH. Our data suggest that HSV may be a second example of an enveloped virus that can be triggered to associate with membranes by the combined effects of receptor (HVEM) binding and low pH. The triggering mechanisms of ALV-A and HSV stand in contrast to those of the influenza virus, in which receptor (sialic acid) binding is required to direct the virion to an endocytic compartment and for target membrane attachment, but it is pH rather than the receptor that is required to induce the conformational changes in hemagglutinin that lead to membrane fusion (4, 20, 21, 28, 37).

Nicola et al. (25) reported inactivation of HSV between pH 5 and 6 in the absence of a gD receptor, suggesting that irreversible conformational changes had been triggered in one or more virion glycoproteins at these pH levels. In the current study, we observed some inactivation at pH 5 alone (12% loss of infectivity) but a much greater loss of infectivity (94%) when pH 5 treatment followed incubation of virions with soluble HVEM. Although there were numerous differences between the inactivation protocol used in this study and the one used by Nicola et al. (25), perhaps the most critical difference is that their virus samples were incubated at low pH in the presence of 10% fetal bovine serum. It seems possible that some component(s) of this complex mixture of proteins, etc., could have provided a necessary cofactor for pH-dependent virus inactivation. In spite of this, the requirement of receptor binding in addition to mildly acidic pH for triggering any conformational changes leading to fusion activation is consistent with our observation that virus entry into CHO K1 cells is enhanced by soluble HVEM. Since CHO K1 cells internalize the virus in a receptor-independent manner, one would not expect soluble HVEM to enhance HSV entry if pH alone were sufficient for fusion activation.

In summary, our data suggest that irreversible alterations to the HSV virion occur as a consequence of HVEMt binding at a pH of 5.3 or lower at 37°C. Virions triggered under these conditions become capable of stable membrane interaction as indicated by the ability of the virus to associate with liposomes. Virions triggered in the absence of membranes rapidly lose their ability to associate with membranes or to infect cells, suggesting that virions progress through a transient membrane-reactive state, followed by a more stable membrane-inactive state. Interestingly, these triggering conditions are met in vivo in CHO K1 or HeLa cells, in which the ability of the entering virus to initiate productive infection requires both a functional gD receptor and the mildly acidic pH environment of an endosome. Curiously, nectin-1t failed to trigger HSV association with liposomes under the conditions used here. One possible explanation for this result is that for nectin-1, other factors are required in order for fusion activation to occur. The fact that HSV productively infects cells (including CHO K1) expressing nectin-1 (13) suggests that any additional factors required for fusion are provided by the host cell. Recently, Kwon et al. (19) showed that a soluble form of nectin-1 promoted HSV entry into CHO K1 cells and, to a lesser extent, into J1.1-2 cells. Based on these observations, those authors have proposed that the binding of nectin-1 to virion gD is sufficient to activate the viral fusion machinery. While we agree that receptor binding by gD is necessary for fusion activation, our findings and those of others (22, 25, 26) are consistent with a model in which the binding of virion gD to a cell-surface receptor primes the viral entry machinery to respond to a secondary or "coactivation" signal. In the case of HVEM, the mildly acidic pH within an endosome may provide such a signal in cells such as CHO K1. For other cell types and other gD receptors, additional cellular factors are likely involved. It is noteworthy that our findings point to a mechanistic difference in how HSV employs these two receptors. However, we have observed that the virus incubated with HVEM at low pH and 37°C is inactivated for infection of several different cell types in which HSV exploits different entry pathways (i.e., pH-independent/nonendocytic, pH-independent/endocytic, and pH-dependent/endocytic pathways). This suggests that if there are multiple mechanisms for HSV fusion activation, then these pathways will ultimately converge at some common point.

 
This work was supported by Public Health Service grant AI-18289 from the National Institute of Allergy and Infectious Diseases, grant NS-36731 from the National Institute of Neurological Disorders and Stroke, and grant AI-056045 from the National Institute of Allergy and Infectious Diseases.

 
* Corresponding author. Mailing address: 4010 Locust Street, Levy Building, Room 212, School of Dental Medicine, University of Pennsylvania, Philadelphia, PA 19104. Phone: (215) 898-6553. Fax: (215) 898-8385. E-mail: whitbeck{at}biochem.dental.upenn.edu.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Journal of Virology, April 2006, p. 3773-3780, Vol. 80, No. 8
0022-538X/06/$08.00+0     doi:10.1128/JVI.80.8.3773-3780.2006
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