Bluetongue Virus Outer Capsid Proteins Are Sufficient To Trigger Apoptosis in Mammalian Cells

Cells and virus.

Three different mammalian cell lines were used in these experiments: HeLa (human cervical epithelial carcinoma), BSR (baby hamster kidney), and HEK 293T (human embryonic kidney). HeLa and BSR cells were maintained in Dulbecco's modified Eagle's medium (Gibco BRL, Gaithersburg, Md.), and 293T cells were maintained in minimal essential medium (MEM) (Gibco BRL) containing 10% fetal calf serum (FCS), 100 U of penicillin/ml, and 100 μg of streptomycin (Sigma-Aldrich Chemical Co., St. Louis, Mo.)/ml; these cells were incubated at 37°C. Also, three insect cell lines were used: KC (embryonic Culicoides variipennis), S2 (embryonic Drosophila melanogaster), and C6/36 (mosquito [A. albopictus]). KC and S2 cells were grown in Schneider's medium (Sigma), and C6/36 cells were grown in Leibovitz L-15 medium (Sigma), containing 10% FCS, 100 U of penicillin per ml, and 100 μg of streptomycin (Sigma)/ml; these cells were incubated at 28°C.

Spodoptera frugiperda (Sf9) cells were grown in Sf900-II medium (Invitrogen, Carlsbad, Calif.) by using standard techniques (9). Recombinant Autographica californica nuclear polyhedrosis virus (AcNPV) expressing glutathione S transferase-tagged VP5 or S-peptide-tagged VP2 was used to express viral outer capsid proteins as described previously (16, 17).

BTV serotype 10 (BTV-10), titered by a plaque assay at 2.5 × 10⁷ PFU/ml, was used for cell infection. Cell monolayers were washed with FCS-free growth medium and then incubated with viruses at the required multiplicity of infection (MOI). Virus adsorptions were carried out for 2 h, followed by incubation in growth medium supplemented with 5% FCS.

Electron microscopy.

For detection of morphological cell alterations characteristic of apoptosis, infected cells were examined by electron microscopy. Briefly, cells were harvested, pelleted by low-speed centrifugation, and fixed twice in 3% glutaraldehyde-0.2 M sodium cacodylate buffer (pH 7.4) and then in 1% osmium tetroxide in 0.2 M sodium cacodylate buffer. After fixation, the cells were washed in water, agar embedded, cut into smaller cubes, and dehydrated through a series of graded ethanol solutions. The cubes were embedded in epoxy resin, and ultrathin sections were cut and mounted onto copper grids. Sections were stained with uranyl acetate and Reynolds lead citrate solutions and then examined and photographed under a JEOL 1200EX transmission electron microscope.

DNA fragmentation.

For detection of chromosomal DNA fragmentation, approximately 2 × 10⁶ mock-infected or infected cells were resuspended and lysed in 0.5% Triton X-100-5 mM Tris-HCl (pH 7.4)-5 mM EDTA for 20 min on ice, and nuclei were removed by centrifugation at 10,000 × g for 15 min. Supernatants were treated with 50 μg of RNase A/ml for 1 h at 37°C, and DNAs were extracted with phenol-chloroform and precipitated with ethanol. Pellets were dissolved in Tris-EDTA (pH 7.5) and separated by electrophoresis in 2% agarose gels. For quantitation of relative apoptosis, a nucleosome enzyme-linked immunosorbent assay (ELISA) kit based on a nonisotopic assay for in vitro quantitation of free nucleosomes in apoptotic cells was used according to the manufacturer's instructions (Oncogene Research Products; Merck KGaA, Darmstadt, Germany).

Caspase-3 activation.

For the caspase-3 activity assay, mock-infected or infected cells (∼10⁶) were collected, rinsed twice with cold 150 mM phosphate-buffered saline (PBS), resuspended, and lysed in a solution containing 50 mM piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES)-KOH (pH 6.5), 2 mM EDTA, 0.1% 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS), 5 mM dithiothreitol (DTT), 20 μg of leupeptin/ml, 10 μg of pepstatin A/ml, 10 μg of aprotinin/ml, and 2 mM phenylmethylsulfonyl fluoride (PMSF). Cell pellets were subjected to three freeze-thaw cycles in dry ice-methanol, and then the lysate was centrifuged at 4°C for 30 min at 20,000 × g and the supernatant fraction was recovered. Cell extracts were analyzed by Western blotting, revealed with a monoclonal anti-caspase-3 antibody (eBioscience, San Diego, Calif.) which reacts with both the 32- and 17-kDa forms of caspase-3, and detected by an enhanced chemiluminescence procedure using high-performance chemiluminescence film (Hyperfilm ECL; Amersham). A positive control was performed with staurosporine-induced apoptosis.

Western blot analysis of viral proteins.

Mock-infected or virus-exposed cells were collected and washed in PBS, and the pellets were dissolved and boiled in loading buffer containing 10% glycerol, 1% sodium dodecyl sulfate (SDS), and 10 mM DTT. Samples were then resolved by SDS-12% polyacrylamide gel electrophoresis in Tris-glycine buffer, transferred to nitrocellulose membranes by a semidry electroblotter, and probed with the appropriate antibodies as described by Sambrook and Russell (25).

Transfection of cells.

Full-length NS1, NS2, VP2, and VP5 genes from BTV-10 were PCR amplified and ligated into a pCAG-GS vector (The CABRI Consortium) carrying the Pol II promoter. The orientation of the plasmids was examined by restriction enzyme analysis, and the authenticity of each construct was confirmed by automated DNA sequencing. Plasmids were transfected into Escherichia coli and purified by using commercial affinity columns (Qiagen, Valencia, Calif.). Monolayer cultures of 293T cells were grown in MEM supplemented with 10% FCS. At 70% of cell confluency, the plasmids were transfected by using Lipofectamine reagent (Invitrogen) in Optimem I medium (Gibco BRL) according to the manufacturer's instructions. After 3 h, the mixture was replaced by normal MEM.

Expression and purification of recombinant VP2 and VP5.

Recombinant VP2 and VP5 proteins were expressed and purified as described previously (16, 17) Purified VP5 and VP2 proteins were quantified by a bicinchoninic acid protein assay reagent kit (Pierce, Rockford, Ill.).

For treatment of cells with purified proteins, confluent cells were rinsed in MEM, and then 5 μg of purified VP5 and/or 10 μg of VP2 was added to the cells. Protein adsorptions were carried out for 2 h, followed by incubation in MEM supplemented with 5% FCS.

Apoptosis inhibitor treatments and UV inactivation of BTV.

Chloroquine (CQ) and ammonium chloride (AC) (both from Sigma) were dissolved in PBS at a stock concentration of 1 mM and syringe filtered (pore size, 0.2 μm). Confluent cultures of HeLa cells were rinsed with MEM and then exposed to 50, 100, or 200 μM CQ or to 1, 5, or 10 μM AC in MEM for 2 h prior to, simultaneously with, or 1 h after BTV infection. In parallel, duplicate cells were treated with proteins as described above in the presence of inhibitors at the same concentrations. Cell lysates were then processed for viral replication by plaque assay and detection of apoptosis.

BTV-10 was exposed to UV light (400 μJ/cm²) for 30 min. The effectiveness of this treatment at inactivating the virus was confirmed by plaque assay. Confluent cultures of HeLa cells were infected with the UV-inactivated virus as previously described by using the intact virus.

Detection of NF-κB (p50 and p65).

Confluent cultures of HeLa cells were either infected with BTV-10 or treated with a VP2-VP5 protein mixture. After incubation for 12 h, nuclear extracts were prepared by washing the cells in PBS and incubating at 4°C for 20 min in 10 mM HEPES (pH 7.9) containing 10 mM KCl, 1.5 mM MgCl₂, 0.5 mM DTT, 0.5 mM PMSF, 20 μg of leupeptin/ml, 10 μg of pepstatin A/ml, and 10 μg of aprotinin/ml. Each sample was then lysed by addition of Nonidet P-40 to a final concentration of 0.5% (wt/vol), followed by centrifugation at 10,000 × g for 15 min. Each pellet was subsequently resuspended in 20 mM HEPES (pH 7.9) buffer containing 0.5 M NaCl, 1.5 mM MgCl₂, 0.2 mM EDTA, 25% glycerol, 0.5 mM DTT, 0.5 mM PMSF, 20 μg of leupeptin/ml, 10 μg of pepstatin/ml, and 10 μg of aprotinin/ml at 4°C for 3 h. Samples were centrifuged, and the supernatant was used for NF-κB detection by SDS-PAGE and Western blot analysis with two secondary polyclonal antibodies, against NF-κB p50 and NF-κB p65 (Oncogene, San Diego, Calif.). The presence of NF-κB subunits was detected by an enhanced chemiluminescence procedure using high-performance chemiluminescence film (Hyperfilm ECL; Amersham).

BTV induces apoptosis in mammalian cells.

To characterize the induction of apoptosis by BTV in mammalian cells, BSR, HeLa, and 293T cells were infected with BTV-10 at an MOI of 1 PFU per cell and were analyzed for morphological hallmarks of apoptosis over a time course of 48 h. Microscopic examination and electron micrographs of infected HeLa cells at 24 h postinfection (p.i.) showed the nuclear condensation, blebbing of the plasma membrane, cellular vacuolization, and shrinkage which are characteristic features of apoptosis (Fig. 1A and B).

To confirm that the morphological alterations observed in the infected mammalian cells were due to apoptosis, nuclear DNA was extracted from infected cells as described in Materials and Methods and resolved in 2% agarose gels. The presence of a fragmented chromosomal DNA ladder typical of apoptosis was detectable after 36 and 48 h p.i., but no DNA fragmentation was found in mock-infected cells (Fig. 1C).

In order to obtain further insight into the mechanism of BTV-induced apoptosis, since caspases have been shown to play a central role in programmed cell death, we examined the activation of caspase-3, a key agent of apoptosis, by Western blot analysis using a specific anti-caspase-3 antibody (Fig. 2A). Cellular extracts obtained at 16 and 24 h after infection were analyzed. The active 17-kDa fragment was clearly visible in the infected cells, while the 32-kDa pro-caspase-3 was observed only in mock infected cells. BTV-10 infection of mammalian cells was verified by Western blot analysis using an anti-BTV-10 polyvalent serum (Fig. 2B).

Together these data provide evidence that in vitro infection of three different mammalian cell lines with BTV-10 results in apoptosis at 24 to 48 h p.i.

BTV does not cause apoptosis in insect cells.

To investigate if BTV-10 infection induces an apoptotic response in Culicodes cells similar to that seen in mammalian cells, KC cells were infected with BTV-10 at an MOI of 1, 10, or 20 PFU per cell and examined daily for induction of apoptosis. None of the gross morphological features associated with apoptosis were noted (Fig. 3A). Furthermore, there was no detectable chromosomal DNA fragmentation in infected KC cells over a time course of 7 days (Fig. 3B), and no caspase-3 activation was detected (Fig. 4A). The normal cell characteristics exhibited in these experiments were independent of the input MOI between 1 and 20 (data not shown). Despite these observations, we found that, in agreement with previous reports (21, 29, 30), KC cells are fully susceptible to BTV infection (Fig. 4B). To test whether the lack of apoptotic response was specific to KC cells, Drosophila S2 and A. albopictus C6/36 cells were also infected in the same manner and then analyzed in detail for DNA fragmentation (Fig. 3C) and activation of caspase-3 (Fig. 4A) after BTV-10 infection. Neither of these two cell lines displayed any characteristics that could be associated with apoptotic events, despite efficient production of viral proteins (Fig. 4B).

Induction of apoptosis by BTV in mammalian cells does not require virus replication.

To determine if virus replication is necessary for BTV-10-induced apoptosis, the effect of UV treatment on the capacity of the virus to induce apoptosis was investigated. Complete inactivation of BTV by UV treatment was confirmed by a plaque assay prior to addition to mammalian cells at an effective MOI of 4. UV treatment had little effect on the ability of virus infection to induce DNA fragmentation and caspase-3 activation (Fig. 5A and B, respectively). The data suggest that the full virus replication cycle is not required for apoptosis. However, four times as many input virus particles were required for induction of apoptosis by UV-inactivated BTV as by the replicating virus.

Identification of BTV proteins responsible for inducing apoptosis in mammalian cells.

In order to determine which BTV protein(s) is responsible for inducing apoptosis in mammalian cells, four major BTV proteins were selected: two outer capsid proteins, VP2 and VP5, and the two nonstructural proteins accumulating most abundantly in BTV-infected cells, NS1 and NS2. The role of each protein in apoptosis when expressed intracellularly was examined by using 293T cells. Plasmids encoding each of the full-length individual proteins NS1, NS2, VP2, and VP5 expressed by the hybrid CAG promoter were constructed as described in Materials and Methods. Each purified plasmid was transfected into the cells, and protein expression was allowed to proceed for 36 h. Under these conditions, all proteins were expressed, but no signs of apoptosis were detected in the cells when plasmids were individually transfected (Fig. 6A and B). Since VP2 and VP5 are responsible for virus entry into the cells, in order to examine their possible synergistic effect, plasmids expressing these two proteins were also cotransfected. As with the individual proteins, no sign of apoptosis could be detected (Fig. 6A and B), although cells transfected with the VP5 plasmid showed slight cytotoxicity (cell necrosis), as reported in a previous study (17).

Based on the premise that the binding of cell receptors might be the trigger for apoptosis, an alternate experimental study was undertaken. Uninfected 293T cells were treated extracellularly with purified recombinant VP2 and VP5. To obtain sufficient purified VP2 and VP5, recombinant baculoviruses that expressed VP2 tagged with the S-peptide and VP5 protein tagged with glutathione S-transferase were used. Both tags were completely removed before recombinant proteins were used in the experimental system. Since VP5 has been shown to be toxic for the cell in high concentrations, 5 μg of purified VP5 was used to treat approximately 8 ×10⁶ cells in a 75-cm² flask. For the same number of cells, 10 μg of VP2 was used. No signs of apoptosis were found when the cells were treated with either VP2 or VP5 independently. However, when VP2 and VP5 were added together to the cells, microscopic examination revealed hallmarks of cellular apoptosis at 24 h posttreatment, as well as a typical pattern of DNA fragmentation and the presence of the activated 17-kDa subunit of caspase-3. Quantitation of apoptosis by ELISA revealed that cells treated with VP2-VP5 had 82% apoptosis relative to that of BTV-infected cell controls (Fig. 6C to E).

Acidification of the endosome is necessary for triggering of apoptosis in response to BTV infection.

To investigate if acid-dependent viral uncoating is required for BTV to trigger apoptosis, we studied the effects of two inhibitors of endosomal acidification, CQ and AC, on the induction of apoptosis in HeLa cells. The cells were exposed to different concentrations of the drugs at different times as described in Materials and Methods; then they were either infected with BTV-10 or treated with purified VP2 and VP5 proteins together and harvested 24 h later. CQ at a 200 μM concentration or AC at 10 μM, added 2 h before infection, protected the cells from apoptosis induced by BTV-10 or purified VP2 and VP5 proteins. Fragmentation of chromosomal DNA was not detectable, and both inhibitors were able to delay the activation of caspase-3 (Fig. 7A and B, respectively). The yield of the virus was determined after CQ or AC addition, and virus replication was 2 to 3 log₁₀ units lower than that of the control (data not shown). Both inhibitors of endosomal acidification were able to block effective induction of apoptosis by BTV and by VP2-VP5 treatment of cells, as well as significantly reducing the replication of the virus.

NF-κB is activated in response to both BTV infection and VP2-VP5 treatment of cells.

The activation of transcription factor NF-κB in response to BTV and VP2-VP5 was studied in order to further characterize the activation of apoptosis in mammalian cells in this system. Activation of NF-κB, a heterodimer composed of proteins p50 and p65, results in its translocation from the cytoplasm to the nucleus, where it potentiates the cellular apoptotic response. HeLa cells were infected with BTV-10 or treated with a purified VP2-VP5 mixture as described above. When the nuclear extracts from harvested cells were analyzed by Western blotting and chemoluminescence using anti-p50 and anti-p65 antibodies, clear signals of NF-κB activation in HeLa cells by BTV were visible. Similar positive signals were also detected in cells treated with the purified VP2-VP5 mixture. Both p65, the major NF-κB subunit, and p50 were detected in the nuclei of the cells 12 h after BTV infection or VP2-VP5 treatment (Fig. 8), demonstrating the translocation of the NF-κB complex to the nuclei of cells and suggesting that most likely NF-κB plays a role in the BTV apoptosis machinery.