Attachment but Not Penetration of Bovine Herpesvirus 1 Is Necessary To Induce Apoptosis in Target Cells

Abstract

Bovine herpesvirus 1 (BHV-1) induces apoptotic cell death in bovine peripheral blood mononuclear cells and B-lymphoma cells. Using a BHV-1 glycoprotein H null mutant, we have demonstrated that although penetration of BHV-1 is not required, attachment of BHV-1 viral particles is essential for the induction of apoptosis.

Bovine herpesvirus 1 (BHV-1) is a member of the subfamily Alphaherpesvirinae (28). In addition to causing initial respiratory infections (36), BHV-1 can also predispose animals, presumably through immunosuppression (4, 8), to secondary bacterial infections which lead to severe pneumonia and death (36). There is increasing evidence that in a variety of acute viral infections, immunosuppression is directly associated with the induction of programmed death in cells involved in immunity (21, 26). Programmed cell death or apoptosis is an encoded suicide program which allows the elimination of cells that have been produced in excess, developed improperly, or sustained genetic damage (32). Apoptosis is characterized morphologically by cell shrinkage, apoptotic-body formation, and condensation of the chromatin (6, 20) and biochemically by fragmentation of DNA into oligonucleosomal DNA fragments (1, 35). BHV-1 can induce apoptosis in peripheral blood mononuclear cells (PBMC) (11, 16) and bovine B-lymphoma (BL-3) cells (10). The mechanism by which BHV-1 induces apoptotic cell death is not understood. However, since inactivated BHV-1 viral particles are still able to induce apoptosis in PBMC cultures, the mechanism of induction could involve either attachment, penetration, or decapsidation of BHV-1 (11). Several BHV-1 glycoproteins play an important role during the initial interactions of the viral particle with target cells. Glycoprotein B (gB) and gC have been shown to be involved in the attachment of BHV-1 to heparan sulfate proteoglycans on the cell surface (5, 18, 25, 33). In addition, gB and gD have been implicated in viral penetration (7, 17, 23, 29). A fourth glycoprotein, gH, which is highly conserved among members of the subfamily Alphaherpesvirinae (31), is also essential for entry of BHV-1 into target cells (22, 34). Deletion of the gene for gH in BHV-1 (22) causes a defect in viral penetration but not attachment. Therefore, to further characterize the mechanism by which BHV-1 induces apoptosis, we used a BHV-1 strain with the gene for gH deleted (22). This mutant virus offers the opportunity to test whether viral penetration is required for BHV-1 to induce apoptosis in BL-3 cells.

BL-3 cells (American Type Culture Collection CRL 8037) were cultured in Optimem medium (Gibco) containing 20% fetal calf serum (Gibco), 100-IU/ml penicillin (Gibco), and 100-μg/ml streptomycin (Gibco). As described by Meyer et al. (22), the BHV-1 gH null mutant was multiplied on gH-expressing Madin-Darby bovine kidney (MDBK) cells (American Type Culture Collection CCL22; multiplicity of infection [MOI] of 10) to generate a virus stock in which virions contain gH in the viral envelope but do not genetically encode gH (BHV-1 gH^−/+). A second virus stock in which virions do not contain gH (BHV-1 gH^−/−) and, consequently, are no longer infectious (22) was generated after multiplication of the BHV-1 gH null mutant on normal MDBK cells (MOI of 10). The parental wild-type (wt) strain used to construct the BHV-1 gH null mutant (22) is the BHV-1 LAM strain (kindly provided by J. T. van Oirschot, Lelystad, The Netherlands) and was propagated on MDBK cells. After multiplication of the viruses, the culture medium was clarified by centrifugation at 1,500 × g for 20 min at 4°C and the viruses were pelleted by ultracentrifugation at 26,000 × g for 2 h at 4°C. Viral pellets were then resuspended in Optimem medium containing 100-IU/ml penicillin and 100-μg/ml streptomycin and stored at −70°C until use.

We first investigated whether BHV-1 virions devoid of gH (BHV-1 gH^−/−) induce DNA fragmentation in BL-3 cells. For this purpose, BL-3 cells were mock infected or infected with BHV-1 gH^−/− and incubated for 48 h. As a control, the effects of wt BHV-1 and BHV-1 gH^−/+ used at an MOI of 10 were also investigated. After being harvested, the cells were further processed to detect the occurrence of DNA fragmentation by in situ DNA fragment labeling and by agarose gel electrophoresis as previously described (9). The percentages of cells undergoing DNA fragmentation were determined by flow cytometry using a Becton Dickinson fluorescence-activated cell sorter. Figure 1 shows that BHV-1 gH^−/− is able to induce DNA fragmentation at an even higher level (52%) than is wt BHV-1 (30.6%) or BHV-1 gH^−/+ (29.5%). The occurrence of DNA fragmentation was confirmed by agarose gel electrophoresis, by which the apoptosis-specific internucleosomal laddering was clearly observable in the DNA obtained from BL-3 cells incubated with wt BHV-1, BHV-1 gH^−/+, or BHV-1 gH^−/−, respectively (Fig. 2, lanes 2 to 4). Mock-infected cultures always showed background levels of DNA fragmentation (Fig. 1 and 2, lane 1). We also determined, by electron microscopy, the morphological characteristics of BL-3 cells incubated with BHV-1 gH^−/−. After 48 h of incubation, a significant proportion of BL-3 cells had membrane-bound apoptotic bodies and distinctive condensation of the chromatin (Fig. 3B). Comparable results were obtained with wt BHV-1 and BHV-1 gH^−/+ (data not shown). Together, these results demonstrate the accumulation of cells with biochemical and morphological characteristics of apoptosis in a BL-3 cell culture incubated with BHV-1 gH^−/−.

FIG. 1.

Percentages of cells with DNA fragmentation in cultures of BL-3 cells mock infected or infected with wt BHV-1, BHV-1 gH^−/+, or BHV-1 gH^−/−. Cells were incubated for 48 h. Each value represents the average ± the standard deviation of triplicate cultures.

FIG. 2.

Agarose gel electrophoresis of DNA extracted from BL-3 cells mock infected (lane 1) or infected with wt BHV-1 (lane 2), BHV-1 gH^−/+ (lane 3), or BHV-1 gH^−/− (lane 4). Cells were incubated for 48 h. One representative experiment out of three is shown.

FIG. 3.

Electron micrographs of BL-3 cells mock infected (A) or infected with BHV-1 gH^−/− (B). (B) BL-3 cells incubated for 24 h with condensed chromatin in several electron-dense masses. Bars, 1 μm.

To exclude the possibility that induction of apoptosis by BHV-1 gH^−/− was due to phenotypically or genotypically rescued virions, standard plaque assays were performed on normal and gH-expressing MDBK cells. BHV-1 gH^−/− was not able to induce plaque formation on normal MDBK cells, while few plaques developed on gH-expressing MDBK cells. We calculated that the amount of BHV-1 gH^−/− used to infect BL-3 cells still contained 0.001 PFU/BL-3 cell (MOI of 0.001). This residual infectivity of BHV-1 gH^−/− could not be responsible for the induction of DNA fragmentation in BL-3 cells. Indeed, the incubation of BL-3 cells with BHV-1 gH^−/+ at an MOI of 0.1 only induced 12.4% DNA fragmentation (Fig. 1), which is not significantly higher than the percentage obtained in mock-infected cultures (11.6%) (Fig. 1). The induction of apoptosis by BHV-1 gH^−/− in BL-3 cells was therefore not due to phenotypically or genotypically rescued virions present in this virus stock.

Virions devoid of gH (BHV-1 gH^−/−) are unable to enter MDBK cells (22). However, this observation has not been confirmed with other cell lines, including BL-3 cells. We therefore investigated whether BHV-1 gH^−/− is indeed unable to enter BL-3 cells. Since BHV-1 gH^−/− contains the Escherichia coli β-galactosidase gene under control of the mouse cytomegalovirus immediate-early gene promoter-enhancer in place of the gH gene (22), we quantified β-galactosidase activity to monitor virus entry into target cells. Indeed, production of β-galactosidase indicates that the virus has entered the cell, released its genome into the nucleus, and activated the constitutive promoter driving β-galactosidase expression (19). For this assay, MDBK or BL-3 cells were mock infected or infected with wt BHV-1 (MOI of 10), BHV-1 gH^−/+ (MOI of 10), or BHV-1 gH^−/− (MOI of 0.001) and incubated for 24 h. After being harvested, the cells were further processed to determine the percentage of cells expressing β-galactosidase as previously described (11). In cultures of BL-3 cells incubated with BHV-1 gH^−/+, 22.4% of the cells expressed β-galactosidase (Fig. 4D). In contrast, BL-3 cells which were mock infected (Fig. 4A) or infected with wt BHV-1 (Fig. 4B) or BHV-1 gH^−/− (Fig. 4C) yielded very low levels of β-galactosidase expression (0.5, 1.7, and 1.8%, respectively) (Fig. 4). Similarly, in cultures of MDBK cells incubated with BHV-1 gH^−/+, 78.1% of the cells expressed β-galactosidase while only 1.5, 2, and 3%, respectively, of those in cultures which were mock infected or infected with BHV-1 gH^−/− or wt BHV-1 did so. These observations are consistent with the results obtained by Meyer et al. (22) and demonstrate that BHV-1 gH^−/− is unable to penetrate BL-3 cells.

FIG. 4.

Expression of β-galactosidase activity in BL-3 cells mock infected (A) or infected with wt BHV-1 (B), BHV-1 gH^−/+ (D), or BHV-1 gH^−/− (C). Cells were incubated for 24 h. One representative experiment out of three is shown.

Since attachment of BHV-1 is mediated through interactions of viral glycoproteins with heparinlike moieties (15, 25), we investigated the effect of heparin (Sigma), a well-known inhibitor of BHV-1 attachment (15, 25), on the ability of BHV-1 gH^−/− to induce apoptosis in BL-3 cells. For this purpose, BL-3 cells were mock infected or infected with wt BHV-1 (MOI of 10), BHV-1 gH^−/+ (MOI of 10), or BHV-1 gH^−/− (MOI of 0.001) with or without simultaneous addition of heparin at concentrations of 10, 100, and 1,000 IU/ml. After incubation for 4 h at 37°C, the cells were washed, resuspended in fresh medium, and further cultivated for 44 h. The cells were then harvested, and the occurrence of DNA fragmentation was detected by in situ DNA fragment labeling. In the absence of heparin, we observed 19% DNA fragmentation in cultures incubated with BHV-1 gH^−/− (Fig. 5). In contrast, cultures containing heparin at 10, 100, and 1,000 IU/ml showed lower levels of DNA fragmentation (4.7, 3.8, and 1.5%, respectively) (Fig. 5). Comparable results were obtained with wt BHV-1 and BHV-1 gH^−/+ (data not shown). Furthermore, heparin only slightly reduced the background level of apoptosis in a mock infected culture (Fig. 5), indicating that the effect of heparin on the ability of BHV-1 to induce apoptosis is a specific phenomenon. Altogether, these data provide strong evidence for the involvement of viral attachment in the induction of apoptosis by BHV-1.

FIG. 5.

Effect of heparin on the ability of BHV-1 gH^−/− to induce apoptosis in BL-3 cells. Cells were incubated for 48 h. Each value represents the average ± the standard deviation of triplicate cultures.

The observation that attachment, but not penetration, of BHV-1 is necessary to induce apoptosis in target cells provides important information about the mechanism by which BHV-1 induces apoptosis. Engagement of a cellular receptor by a BHV-1 envelope protein(s) during the attachment process could be responsible for induction of the apoptotic process in target cells. Similarly, in human immunodeficiency virus-infected patients, interaction of the soluble human immunodeficiency virus envelope protein gp120 with CD4 molecules is thought to induce a defective signal transduction that leads to apoptosis of the T-helper population (3, 13, 14, 30). In this context, to identify the BHV-1 envelope protein(s) which could be involved in the activation of the apoptotic process, we have already tested BHV-1 mutants with the gC, gI, gE, or gG gene deleted (kindly provided by F. A. M. Rijsewijk, Lelystad, The Netherlands) (27). These BHV-1 mutants are still able to induce apoptosis in PBMC and BL-3 cells (11a). Therefore, it seems most likely that glycoproteins gC, gE, gI, and gG are not involved in the induction of the apoptotic process. Among the other BHV-1 glycoproteins, gD could be a potential candidate for the induction of apoptosis. It has been shown that gD of herpes simplex virus type 1, another member of the subfamily Alphaherpesvirinae (28), interacts with a member of the tumor necrosis factor-nerve growth factor receptor family (24). These receptors are implicated in a variety of cellular functions, including proliferation, differentiation, and apoptosis (2). In addition, the expression of BHV-1 gD in bovine cells has been shown to be toxic (7, 17). On the basis of these observations, we are currently investigating the possible involvement of gD in BHV-1-induced apoptosis.

The involvement of a BHV-1 envelope protein(s) in the induction of apoptosis opens a new area of investigation. Following BHV-1 infection, the high susceptibility of infected animals to secondary bacterial infection has been shown to be associated with immunosuppression (4, 8). Even a modified live BHV-1 vaccine decreases the immune response against an antigen administrated simultaneously to animals (12). We previously reported that BHV-1 is able to induce apoptosis in T lymphocytes, B lymphocytes, and monocytes, which play an important role in the immune system (10). Subtle amino acid substitutions in a specific BHV-1 envelope protein(s) may eliminate the ability of BHV-1 to induce apoptosis and, presumably, the immunosuppression. This strategy could have important applications in the development of new, more effective, and safer vaccines and also help us to better understand the pathogenesis of BHV-1.