Bovine herpes virus type 1 induces apoptosis through Fas-dependent and mitochondria-controlled manner in Madin-Darby bovine kidney cells
- Xingang Xu†1,
- Kuan Zhang†1,
- Yong Huang1,
- Li Ding1,
- Guangda Chen1,
- Honglei Zhang1 and
- Dewen Tong1Email author
© Xu et al.; licensee BioMed Central Ltd. 2012
Received: 9 April 2012
Accepted: 13 September 2012
Published: 17 September 2012
Bovine herpesvirus type 1 (BHV-1) is an important pathogen in cattle that is responsible for substantial economic losses. Previous studies suggest that BHV-1 may induce apoptosis in Madin-Darby bovine kidney (MDBK) cells via a mechanism only involving caspases and p53. However, the mechanism for BHV-1-induced MDBK cell apoptosis still requires more research.
MDBK was used as a model to study the precise signaling pathways of apoptosis induced by BHV-1 infection.
BHV-1 infection activated a Fas/FasL-mediated apoptotic pathway, resulting in activation of caspase-8 and cleavage of Bid. In addition, BHV-1 infection down-regulated Bcl-2 and up-regulated Bax expression, thereby initiating the release of cytochrome c followed by caspase-9 activation. The combined activation of the extrinsic and intrinsic pathways resulted in activation of downstream effecter caspase-3 and poly ADP-ribose polymerase (PARP), leading to apoptosis. Furthermore, blocking apoptosis using caspase inhibitors improved BHV-1-infected MDBK cell viability to different extent. BHV-1 infection did not induce significant DNA fragmentation in MDBK cells pretreated with ammonium chloride (NH4Cl) or cells infected with UV-inactivated BHV-1. Blocking caspases activation increased BHV-1 replication.
BHV-1 induces apoptosis in MDBK cells through extrinsic and intrinsic pathways and there might be cross-talk between the two pathways. In addition, BHV-1 replication may be necessary for the induction of apoptosis in BHV-1-infected cells, and prolonged cell viability benefits BHV-1 replication.
KeywordsBHV-1 MDBK cells Apoptosis Caspase cascades Fas Mitochondria
Bovine herpes virus type 1 (BHV-1), an alphaherpesvirinae subfamily member, is an important pathogen in cattle that gives rise to substantial economic losses as a result of effects including reproductive failures, increased calf mortality, as well as enteric and respiratory disease. As a viral pathogen in cattle, BHV-1 causes severe respiratory infection, conjunctivitis, abortion, vulvovaginitis, balanopostitis, and systemic infection in neonate calves . Most of these problems are caused by increased susceptibility to secondary infection which correlates with BHV-1-induced immunosuppression [2, 3]. This immunosuppression may be partly due to apoptosis of infected lymphocytes because reduction of CD4+ T lymphocytes was detected in peripheral blood mononuclear cells (PBMCs) and lymph nodes during acute infection of BHV-1 and those CD4+ T lymphocytes undergo apoptosis .
Apoptosis is a major form of death caused by some types of virus infection. This process is characterized by detachment, plasma membrane blebbing, nuclear collapse and chromatin condensation. An important regulatory event in the apoptotic process is the activation of caspases, a family of cysteine proteases. Caspase cascades are involved in both intrinsic and extrinsic signal pathways which regulate the apoptotic process . The relationship between virus infection and apoptosis is bidirectional. On one hand, virus infected cells can be eliminated by apoptosis mediated by components in the innate and adaptive immune systems. On the other hand, viruses have evolved strategies to regulate apoptosis, either by blocking a specific step of apoptotic cascade within the host cells, and thus maximizing virus production, or by actively inducing apoptosis, which consequently facilitates the spreading of virus progeny .
A previous study found that BHV-1 could induce PBMC subpopulations (B lymphocytes, T lymphocytes and monocytes) to undergo apoptosis individually . Moreover, although penetration of BHV-1 is not required, attachment of BHV-1 viral particles is essential for the induction of apoptosis . Also, it has been proved that the apoptosis induced by BHV-1 infection in MDBK cells involves p53-dependent mechanism . To better understand the apoptotic process initiated by BHV-1 infection, in this work we further characterize caspases activation cascades during BHV-1 induced apoptosis in MDBK cells, focusing on the cell surface death receptor pathway and the mitochondria-initiated pathway. Our results demonstrate that BHV-1 infection appears to activate caspase-8 by Fas-dependent mechanism and to turn on caspase-9 by the mitochondria-dependent pathway. In addition, the two pathways could be associated through Bid.
BHV-1 infection induced apoptosis in MDBK cells
Consistent with the CPE resulting from BHV-1 infection, mock-infected and BHV-1-infected MDBK cells were examined for characteristic signs of apoptosis using fluorescent microscope, agarose gel electrophoresis and flow cytometry. The BHV-1-infected cells showed typical apoptotic features with chromatin condensation and nuclear fragmentation to different extent after AO/EB staining (Figure 1B and C, lower panel). As shown in Figure 1D, DNA ladders could be detected as early as 24 h p.i., as MDBK cells were infected with BHV-1 at 2 MOI. Flow cytometry was used to measure the percentage of apoptotic cells. Increased rates of apoptotic cells were detected in BHV-1-infected MDBK cells. The percentage of apoptotic cells increased from 4.1% at 24 h p.i. to 70.5% at 60 h p.i. (Figure 1E). In contrast, mock-infected MDBK cells at corresponding times did not show obvious apoptosis. The above results provided biochemical evidence that the cell viability reduction and morphological changes observed in the BHV-1-infected MDBK cells are due to the induction of apoptosis.
BHV-1 infection provoked activation of caspase-8, 9 and 3
To further determine the contribution of caspase-8, -9, -3 in apoptosis, we examined the cell viability of BHV-1-infected cells pre-treated with caspase-8, -9 and −3 specific inhibitors z-IETD-FMK, z-LEHD-FMK and Z-DEVD-FMK, respectively. The results show that caspases-8, -9 and −3 specific inhibitors significantly prevent cell death significantly (Figure 2B). To determine the effect of two initiator caspase-8 and −9 in caspase-3 activation, we detected the activity of caspase-3 in the cells pretreated with caspase-8, caspase-9, and both of that inhibitors. The activity of caspase-3 was decreased in cells in the presence of caspase-8 inhibitor or caspase-9 inhibitor. Combining both of the two inhibitors inhibited the activity of caspase-3 more significantly (Figure 2C). Taken together, these results suggest that both extrinsic and intrinsic pathways are involved in caspase-3 activation and BHV-1-induced apoptosis.
Fas/FasL-mediated apoptotic pathway are involved in BHV-1- induced apoptosis
It is known that activated caspase-8 is able to cleave full-length Bid to tBid, which is important in crosstalk between the death-receptor pathway and the mitochondrial pathway in some cell types . Bid activation results in the destruction of mitochondria integrity and cytochrome c release to cytosol, which further facilitates caspase-9 activation. To test this possibility in BHV-1-induced apoptosis, we determined whether Bid was cleaved upon BHV-1 infection and whether blocking caspase-8 activity could affect Bid cleavage and caspase-9 activation. Western blot analysis revealed that tBid was detected in BHV-1-infected cells but not in mock-infected cells. tBid clearly appeared at 24 h p.i. and maintained a high level in subsequent hours (Figure 3B). Following incubation with z-IETD-FMK, a specific inhibitor of caspase-8, tBid was not detected in BHV-1-infected cells (Figure 3C). However, the activation of caspase-9 was partly blocked in BHV-1-infected cells (Figure 3D). These results suggest that apoptosis signals from the FasL were transmitted to caspase-8 which in turn cleaves Bid and is followed by caspase-9 activation, and that other apoptosis signals also contribute to the activation of caspase-9.
BHV-1 infection regulates the expression of the Bcl-2 family of proteins and promotes the release of cytochrome c from mitochondria
Interaction between virus replication and cell apoptosis
To further examine whether apoptosis induction by BHV-1 requires virus replication, we used UV treatment to abrogate replication of BHV-1. After BHV-1 was subjected to UV treatment, no viral progeny was detected. Consistently, induced apoptosis disappeared in cells infected with UV-inactivitated BHV-1. The capacity of BHV-1 to induce apoptosis was investigated. When BHV-1 was subjected to UV treatment, a drastic reduction in viral progeny titer was observed (Figure 5D). In cells infected with UV-inactivated BHV-1, apoptosis induction was suppressed when compared to UV-untreated BHV-1 (Figure 5E). Taken together, our results suggests that viral replication is needed for apoptosis induction in BHV-1-infected cells.
Infections with several alphaherpesviruses induce apoptotic cell death in various cell types. Anatid herpesvirus type 1 (AHV-1) infection induces apoptosis in duck embryo fibroblast cells . Herpes simplex virus type 2 (HSV-2) activates the apoptotic process in T cells . For BHV-1, it was reported that the attachment of BHV-1 virions to cells induces apoptotic cell death in PBMC cells  and bovine BL-3 (B lymphoma cells) . However, the apoptotic signal pathways in BHV-1-infected cells are not well understood. The stimultaneous activation of caspase-8 and caspase-9 suggests that BHV-1 can trigger the death-receptor pathway and the mitochondrial pathway separately and in parallel. It will be interesting to reveal the upstream signals that trigger these two pathways following BHV-1 infection.
In many cases caspase are required to propagate the signal to commit suicide [17, 18]. Many viruses trigger the activation of these caspase cascades, which in turn are responsible for apoptosis induction in infected cells . In BHV-1-infected MDBK cells, the activation of caspase-8, 9 and 3 and cleavage of PARP were observed. Furthermore, incubation of specific caspase inhibitors significantly enhanced the cell viability. These results are consistent with a previous study which showed that PARP is cleaved during BHV-1 infection  and suggests that BHV-1-induced apoptosis involves caspase-8 and caspase-9 activation.
Several viruses have been shown to readily activate the caspase-8-associated apoptotic pathway . In the case of BEFV, the induced apoptosis involves increasing the expression of Fas/FasL and activation of caspase-8 . On the other hand, although caspase-8 is often activated through the death-receptor pathway in many systems, the Sendai virus can active apical caspase-8 without involvement of the upstream death receptors during the apoptotic process . Our research provided evidence that BHV-1 induces apoptosis in MDBK cells, involving caspase-8-dependent pathways. This suggests that an extrinsic pathway was involved in the BHV-1-induced apoptosis. The increasing expression of Fas and FasL indicates that caspase-8 activation in BHV-1-infected MDBK cells can be mediated by Fas/FasL signal payhway. However, just how Fas expression is regulated by BHV-1 remains unclear, and further studies are under way to elucidate the mechanism of Fas regulation in BHV-1-infected MDBK cells. Caspase-8 has been shown to activate a mitochondria pathway following the cleavage of Bid, a pro-apoptotic member of Bcl-2 family [22, 23]. Cleavage of Bid in BHV-1-infected cells was consistent with such a mechanism. We also propose that the release of cytochrome c from mitochondria and increasing expression of Apaf-1 are critical steps in the BHV-1-initiated apoptosis. These results are consistent with current theory that tBid transfers to the surface of the mitochondria causing cytochrome c release . Thereafter, cytochrome c helps Apaf-1 to recruit and activate caspase-9 preprotein, which in turn activates caspase-3 . Our research provides evidence that BHV-1 infection induced apoptosis occurs through a mitochondria pathway. There are also some other types of virus which induce apoptosis through mitochondria pathway, such as the equine arteritis virus  and the dengue virus . Suppressing the activation of caspase-8 and caspase-9 separately degrades the activation of caspase-3 but did not stop it totally, indicating that both the extrinsic and intrinsic pathways contribute to activation of caspase-3 and cleavage of PARP progressively.
Mitochondria-mediated apoptosis is strictly controlled by Bcl-2 family members, which include a number of pro-apoptotic and anti-apoptotic proteins to regulate apoptosis by changing relative levels . In some cases, some viruses either encode proteins homologous to Bcl-2 to inhibit apoptosis or influence Bcl-2 expression to establish a persistent infection. Many viruses inducing apoptosis by modulating expression of Bcl-2 family proteins have been reported . Our results show that the BHV-1 infection up-regulated Bax, down-regulated Bcl-2 expression, and induced the release of cytochrome c, resulting in MDBK cells apoptosis. These results are consistent with the observation in other herpes viruses [28, 29], which suggest that modulation of Bcl-2 members is a key step for herpes viruses induction of CPE and eventually apoptosis.
In most cases of virus-induced apoptosis, apoptosis is a result of crosstalk between extrinsic and intrinsic pathways [19, 30]. In this pathway, activated caspase-8 cleaves Bid to tBid which, in turn, translocates to mitochondria and initiates the release of cytochrome c into the cytosol activating caspase-9. Our results also provide evidences for the potential cross-talk between extrinsic and intrinsic pathways in BHV-1-induced apoptosis. Blocking caspase-8 activity using z-IETD-FMK, a specific inhibitor of caspase-8, does not completely abrogate caspase-9 activation. This suggests that caspase-9 is activated not only by caspase-8 activation but also by other upstream apoptotic signals. In previous study, P53 appeared to be involved in the BHV-1-induced apoptosis in MDBK cells because the p53 level and promoter activity increased after infection .
For some types of viruses, viral replication is required for the induction of apoptosis during infection with these viruses. BHV-1 needs to penetrate MDBK cells in order to trigger cell death, but not to induce host cell intrinsic-recognition mechanism . Pan caspase inhibiter can prevent BHV-1-induced apoptosis and enhance the virus yield , in which down-stream caspase (caspase-3) leads a main role. In the meantime, caspase inhibitors increase cells’ viability, and the effect of caspase-3 inhibitor is more significant. We hypothesize that it was because caspase inhibitors prolonged the survival of MDBK cells that more virus was assembled in cells. In other words, apoptosis has a negative impact on the virus life cycle. However, the inhibition of caspases does not affect Canine Coronavirus Type II (CCoV-II) replication in canine fibro sarcoma cells (A-72 cells) . Treatment of MDBK cells with NH4Cl or infection with UV-inactivated BHV-1 was seen to abrogate virus apoptosis induction, suggesting that BHV-1-induced apoptosis in MDBK cells depends on viral replication. Although apoptosis occurred in MDBK cells after BHV-1 infection, UV-inactivated virus did not efficiently induce apoptosis. This finding is in contrast to those from previous studied using PBMC or activated CD4+ T cells. In general, lymphoid cells are prone to apoptosis, suggesting that inactivated virus can induce apoptosis in cells which easily undergo apoptosis. It is clear that BHV-1 induces cell death in a cell-type-dependent fashion, and this is probably because novel virus-host interactions are important for BHV-1-initiated apoptosis in different cell types. For example, a novel member of the tumor necrosis factor (TNF) NGF receptor family (HVEM)  mediates HSV-1 entry into activated T cells. Conversely, entry of HSV-1 into epithelial or other non-lymphoid cells is mediated by an unrelated membrane glycoprotein which resembles the poliovirus receptor .
In summary, our results demonstrate that BHV-1 infection activities caspase-8 through a death receptor pathway by regulating Fas/FasL expression, followed by the involvement of a mitochondria-mediate pathway, which leads to the release of cytochrome c and activation of caspases-9 and −3. Caspases are involved in controlling virus release by inducing infected cells apoptosis. In addition, BHV-1 replication may be necessary for the induction of apoptosis in BHV-1-infected cells, but in the mean time, prolonged cell viability benefits BHV-1 replication.
Antibodies, cells and virus
Monoclonal antibodies against caspase-8, caspase-9, caspase-3, PARP, Fas, FasL, Bid, Bcl-2, Bax, cytochrome c, Apaf-1, Cox4, β-actin were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Horseradish peroxidase (HRP)-conjugated secondary antibody was purchased from Pierce (Pierce, Rockford, IL, US). Madin Darby bovine kidney (MDBK) cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco BRL, Gaithersburg, MD, US) supplemented with 10% heat-inactivated new born bovine serum (Gibco BRL, Gaithersburg, MD, US), 100 IU of penicillin and 100 μg of streptomycin per ml, at 37°C in a 5% CO2 atmosphere incubator. The BHV-1 Shaanxi strain, which was isolated and characterized in 2009, was kindly provided by Prof. Jing-Yu Wang, College of Veterinary Medicine, Northwest A&F University . Virus titers were determined by 50% tissue culture infective doses (TCID50) as described previously .
Cell viability determination
Cell viability was determined by MTT assay as described previously . Briefly, 5,000 cells were seeded in 96-well plate chambers and then cells were infected with BHV-1 at different MOI (50, 10, 2, 0.4, 0.008, 0.016, and 0.0032 MOI). At every 12 hours post infection (h p.i.), cells were incubated with 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT, 5 mg/ml) for further 4 h. Then the medium was replaced with DMSO (Dimethyl sulfoxide) to solubilize the formazan crystals. 10 minutes later, absorbance at 570 nm was measured with a microplate reader (BioTek Instruments, Inc., Winooski, US). Appropriate controls were performed incubating cells with DMEM, but without BHV-1. Data are presented as a percentage of the control, and results are the mean ± SD of three independent experiments performed in duplicate.
MDBK cells were grown on slides in 6-well plates and were infected with BHV-1 at 2 MOI. At indicated h p.i. cells were stained with acridine orange (AO, 200 μg /ml) and ethidium bromide (EB, 200 μg /ml) and then washed with phosphate buffer saline (PBS) to remove background staining. After that, slides were covered with cover slips and observed under a fluorescence microscope (Nikon Inc, Japan). The normal cells and early apoptotic cells can be stained by AO to appear bright green fluorescence, while the late apoptotic cells can be stained by EB to appear orange fluorescence.
DNA fragmentation assay
Mock-infected cells or BHV-1-infected cells were harvested, washed and incubated with lysis buffer (20 mM EDTA, 100 mM Tris, pH 8.0, 0.8% SDS) at room temperature for 1 h. After centrifugation for 10 min at 12000 × g, the supernatants were collected and treated with Rnase A (500 μg/ml) for 1 h at 37°C, followed by digestion with proteinase K (500 μg/ml) for 2 h at 55°C. The DNA was extracted using the phenol/chloroform/isoamylol (25:24:1), precipitated with ethanol, dissolved in TE buffer (10 mM Tris, pH 8.0, 1 mM EDTA), and subjected to 2.0% agarose gel electrophoresis for DNA fragmentation analysis.
DNA content assay
Flow cytometric analysis of apoptosis was performed by analyzing the reduced fluorescence of the DNA binding dye propidium iodide (PI) in the apoptotic nuclei as previously described . Briefly, MDBK cells were seeded in 6-well plastic tissue culture plates and mock infected or infected with BHV-1. Then cells were collected and washed twice with PBS. Following this, cells were fixed and permeabilized by suspension in 70% cold ethanol and incubated overnight at 4°C. Cell pellet was resuspended at room temperature in hypotonic solution consisting of 0.1% sodium citrate pH 6.5, 1% propidium iodide (PI) staining. Cells were analyzed after at least 1 h of incubation at 4 ~ 8°C in the dark with a flow cytometry (Beckman Coulter EPICS ALTRA, Orlando, US). At least 10,000 events were acquired for each sample.
Measurement of caspases activity
Caspases activities were measured by Caspases (caspase-3 and 9) Colorimetric Assay Kit (BioVision, Inc., Mountain View, California, US), according to manufacturer’s recommendations. Briefly, the cells pellet harvested by centrifugation was incubated for 30 min in chilled cell lysis buffer on ice. The lysate was centrifuged at 4°C and the supernatants were used for caspases activity assay. The supernatants protein content was estimated using BCA Protein Assay Reagent (Pierce, Rockford, IL, US). A total of 200 μg of proteins were incubated for 4 h at 37°C with 200 μM colorimetric substrate. The protease activity was determined by spectrophotometric detection. Absorbance at 405 nm was measured with a microplate spectrophotometer (BioTek Instruments, Inc., Winooski, US).
Western blot analysis
Cells were harvested and treated with ice-cold RIPA (Radio Immunoprecipitation Assay) lysis buffer with 1 mM phenylmethyl sulfonylfluoride (PMSF). Isolation of mitochondrial and cytosolic proteins was performed using the Mitochondria/cytosol Fractionation Kit (Pierce, Rockford, IL, US). Equivalent amounts of proteins were loaded and electrophoresed on 12% SDS-PAGE and transferred to PVDF membranes (Millipore Corp, Atlanta, GA, US). The membranes were blocked with 5% nonfat dry milk and then incubated with indicated primary antibodies over night at 4°C, followed by HRP-conjugated secondary antibodies. The signal was detected by ECL reagent (Pierce, Rockford, IL, US).
Quantitative real-time PCR (qRT-PCR) analysis
Sequences of bovine primer pairs used for qRT-PCR
Forward primer (5′–3′)
Reverse primer (5′–3′)
All data were means ± SD from three independent experiments in triplicate. Results were analyzed by Student’s t-test. A p-value less than 0.05 was considered to be statistically significant.
Dr. De-Wen Tong, professor of College of Veterinary Medicine, Northwest A&F University, Vice Dean of College of Veterinary Medicine, Northwest A&F University. Dr. Xin-Gang Xu and Dr. Yong Huang are assosiate professors of College of Veterinary Medicine, Northwest A&F University. Kuan Zhan , Li Ding , Guangda Chen and Honglei Zhang, graduate students of College of Veterinary Medicine, Northwest A&F University.
We thank Professor Jingyu Wang for providing us with BHV-1 strain, which was essential for this study. This work was supported by grants from the basic research and operating expenses of Northwest A&F University (Grant No. QN2012017 and No. Z109021119) and the international science and technology cooperation fund (Grant No. A213021202).
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