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Journal of Virology, March 2004, p. 2875-2883, Vol. 78, No. 6
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.6.2875-2883.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

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

Eduardo Mortola,¹ Rob Noad,¹ and Polly Roy^1,2*

Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, WC1E 7HT, United Kingdom,¹ Department of Geographic Medicine, University of Alabama at Birmingham, Birmingham, Alabama 35294²

Received 29 September 2003/ Accepted 1 December 2003

Top Abstract Introduction Materials and Methods Results Discussion References

 
Bluetongue virus (BTV) is transmitted by Culicoides sp. biting midges to livestock, causing severe hemorrhagic disease in sheep, but is asymptomatic in the insect host. Similarly, BTV causes rapid cell death in infected mammalian cells in culture, whereas infections of insect cells are long-term and unapparent, despite productive virus replication. To assess whether apoptosis plays any role in these two distinct cell responses, we have investigated apoptosis in cultured insect and mammalian cells. Three different mammalian cell lines and three different insect cell lines including Culicoides variipennis (KC) cells were infected with BTV serotype 10, and the key apoptosis indicators of cell morphology, chromosomal DNA fragmentation, and caspase-3 activation were monitored. BTV infection induced apoptosis with the activation of the transcription factor nuclear factor B (NF-B) in all three mammalian cell lines. In contrast, no evidence for apoptosis was detected in any of the three insect cell lines in response to BTV infection. Using inhibitors of endosomal acidification and UV-inactivated virus, we established that virus uncoating, but not productive virus replication, is necessary for BTV to trigger apoptosis in mammalian cells. Intracellular expression of the viral outer capsid proteins VP2 and VP5 or the two major nonstructural proteins NS1 and NS2 was not sufficient to trigger an apoptotic response. However, extracellular treatment with a combination of purified recombinant VP2 and VP5, but not with each protein used separately, resulted in an apoptotic response. Virus- and VP2-VP5-stimulated apoptotic responses were both inhibited by inhibitors of endosomal acidification. Thus, for BTV the viral outer capsid proteins alone are sufficient to trigger apoptosis.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Bluetongue virus (BTV), which is vectored to vertebrate species by biting midges (Culicoides spp.), causes a hemorrhagic disease of sheep and other ruminants. BTV is able to replicate in both insect and mammalian hosts; however, infection is asymptomatic in insects but causes a severe pathogenesis in sheep and other livestock (23, 24, 26). This is reflected in tissue culture, where insect cells show little or no cytopathic effect (CPE) in response to viral infection, despite productive virus replication, whereas mammalian cells show dramatic CPE. This type of differential host response is not limited to BTV but is shared by a number of other arboviruses (19). For Sindbis virus, the difference in host cell CPE following virus infection strongly correlates with the amount of apoptosis seen in the cells. Sindbis virus rapidly induces apoptotic cell death in vertebrate cell cultures, but infection of Aedes albopictus cells with the same virus results in very little detectable apoptosis, and in general infections are long-term and persistent (18, 19). Whether the correlation between CPE and apoptosis holds for other arboviruses is as yet unclear. Previous microscopic analysis of primary cultures of endothelial cells from sheep and bovine lungs infected with BTV suggests that apoptosis may contribute to the pathogenesis of bluetongue disease in the mammalian host (10, 11). However, neither the degree of apoptosis in insect cells infected with BTV nor the initiators and effectors of BTV-induced apoptosis in mammalian cells have been reported.

BTV is a nonenveloped icosahedral virus within the Reoviridae family. Attachment of BTV to mammalian cells is mediated by the outer capsid protein VP2 (15). The virus is then internalized by receptor-mediated endocytosis. During internalization, VP2 is believed to be degraded, and the partly denuded BTV virion causes destabilization of the vesicle membrane to allow the penetration of the newly uncoated core particles into the cytoplasm. The release of the BTV core has been shown to be dependent on acidic pH (12), and evidence is accumulating that the second outer capsid protein, VP5, has amphipathic alpha-helices in its structure that are involved in permeabilization of the endosomal membrane in a pH-dependent manner (17). In insect cells, in addition to the VP2-VP5-mediated entry pathway, the initial stages of BTV may also use an alternate mechanism based on the outer protein of the core, VP7 (27, 30).

For reovirus, a member of the Reoviridae family that is not transmitted by insect vectors, apoptosis is triggered in mammalian cells through the activation of specific pathways that differ between cell and tissue types (3, 13). Moreover, different virus isolates also differ in their abilities to induce apoptosis, and apoptosis induction segregates with the genome segments that encode the outer capsid proteins (22, 28). This segregation appears to be linked to the abilities of variants of the outer capsid protein, 1, to bind different types of cell membrane receptors (1, 2, 4, 7). However, receptor binding alone is not sufficient to trigger apoptosis; virus disassembly in the endosome, but not subsequent steps in virus replication, is also required (8). Avian reoviruses differ from their mammalian counterparts in their lack of hemagglutination and their ability to cause cell fusion (20). However, it has recently been reported that, like mammalian reovirus, avian reovirus can trigger activation of apoptosis very early in infection subsequent to virion disassembly (20).

While apoptosis has previously been linked to the pathogenesis of BTV in sheep cells (10, 11), the response of the Culicoides insect host to BTV infection, the trigger for apoptosis in BTV-infected mammalian cells, and biochemical intermediates of this response in BTV infections have not been described previously. In this study, an experimental system which uses three different mammalian cell lines and three different insect cell lines including Culicoides (KC) cells to determine the induction of apoptosis by BTV, was developed. We report here that BTV infection triggers apoptosis in mammalian cells but not in insect cells and, as with orthoreovirus and avian reovirus, uncoating of BTV, but not BTV replication, is required to trigger apoptosis. Moreover, we demonstrate here that extracellular treatment with a combination of the viral outer capsid proteins, the cellular receptor binding protein VP2, and the cell penetration protein VP5 is sufficient to trigger apoptosis. However, neither VP2 nor VP5 on its own to is able to perform this function.

Top Abstract Introduction Materials and Methods Results Discussion References

 
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 x 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 x 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 x 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 x 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-B (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 x 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-B detection by SDS-PAGE and Western blot analysis with two secondary polyclonal antibodies, against NF-B p50 and NF-B p65 (Oncogene, San Diego, Calif.). The presence of NF-B subunits was detected by an enhanced chemiluminescence procedure using high-performance chemiluminescence film (Hyperfilm ECL; Amersham).

Top Abstract Introduction Materials and Methods Results Discussion References

 
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).

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FIG. 1. BTV-10 induces apoptosis in mammalian cells at an MOI of 1 PFU per cell. (A) Micrographs of mock-infected (a) or BTV-10-infected (b) HeLa cells showing morphological characteristics of apoptosis at 24 h p.i. (B) Electron micrographs of HeLa cells after mock infection (a) and 24 h post-BTV-10 infection (b). Note that BTV-infected cells show chromatin condensation, blebbing of the plasma membrane, cellular vacuolization, and shrinkage. (C) Pattern of chromosomal DNA extracted from three different mammalian cells at different times after BTV-10 infection. DNA ladders typical for apoptosis were detected at 36 and 48 h p.i. for all three cell lines.

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).

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FIG. 2. Time course of caspase-3 activation and expression of viral proteins in BTV-10-infected mammalian cells. (A) Cleavage of the inactive 32-kDa procaspase-3 into 17-kDa active caspase-3 in three different mammalian cell lines at 16 and 24 h after infection with BTV-10 at an MOI of 1. (B) Western blot analysis showing the reactivity of a polyclonal anti-BTV-10 antibody with different BTV proteins in lysates of the same three mammalian cell lines at 36 h p.i.

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).

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FIG. 3. BTV-10 does not cause apoptosis in insect cells. (A) Microscopic images of mock-infected (a) and BTV-10-infected (b) KC cells. None of the gross morphological features associated with apoptosis were noted in any of the insect cell lines tested. (B and C) Chromosomal DNA extracted from KC cells at the time points indicated (B) and from C6/36 and S2 cells at 5 days p.i. (C). No chromosomal DNA fragmentation was detected in BTV-infected insect cells even at 7 days p.i.

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FIG. 4. Caspase-3 detection and expression of viral proteins in BTV-10-infected insect cells. (A) Western blot analysis of cleavage of 32-kDa procaspase-3 into active 17-kDa caspase-3 over 72 h in BTV-infected insect cells. (B) Western blot analysis with a polyclonal anti-BTV-10 antibody showing the presence of different BTV proteins in lysates of insect cells at 36 h p.i.

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.

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FIG. 5. Effect of UV treatment on induction of apoptosis by BTV. HeLa cells were analyzed 36 h after mock infection or treatment with UV-inactivated BTV-10 (+UV-BTV). (A) Fragmentation of chromosomal DNA typical of apoptosis. (B) Detection of the active 17-kDa subunit of caspase-3.

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).

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FIG. 6. Identification of BTV proteins sufficient for the induction of apoptosis in mammalian cells. (A and B) Chromosomal DNA was extracted from 293T cells transfected with expression plasmids encoding the BTV proteins indicated. No DNA fragmentation (A) or activation of caspase-3 (B) was detected in any of the transfected cells. Note that cell transfection with a plasmid encoding VP5 protein resulted in slight cytotoxicity, with a smear of DNA attributable to cell necrosis, a characteristic feature of VP5 expression. VP2/5, mixture of VP2 and VP5. (C) Chromosomal DNA extracted from 293T cells treated with purified VP2 and/or VP5 protein. After treatment for 24 h, a fragmented ladder of chromosomal DNA typical of apoptosis was found only in cells treated with VP2/5. (D) Presence of the activated 17-kDa subunit of caspase-3 in 293T cells 24 h after treatment with VP2/5. (E) Quantitation of relative apoptosis by ELISA specific for free nucleosomes in 293T cells 24 h after BTV infection or VP2/VP5 treatment. Apoptosis in BTV-infected cells at 24 h p.i. was set at 100%. Other values are expressed relative to this standard on the basis of ELISA results. The percentage of apoptotic cells treated with a mixture of VP2 and VP5 was slightly lower than that for BTV-infected cells. Values are means from three independent experiments. Error bars, standard deviations.

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 x10⁶ 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.

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FIG. 7. Effects of endosomal acidification inhibitors on the induction of apoptosis by BTV-10 infection. (A) Chromosomal DNA extracted from HeLa cells 24 h after BTV-10 infection or 24 h after treatment with purified VP2 and VP5 proteins. Cells treated with CQ or AC 2 h before infection or addition of purified proteins failed to show chromosomal DNA fragmentation compared with mock-treated cells. (B) CQ- or AC-treated cells failed to show detectable activation of caspase-3 compared with mock-treated cells.

NF-B is activated in response to both BTV infection and VP2-VP5 treatment of cells. The activation of transcription factor NF-B 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-B, 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-B 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-B 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-B complex to the nuclei of cells and suggesting that most likely NF-B plays a role in the BTV apoptosis machinery.

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FIG. 8. Translocation of NF-B to the nucleus in HeLa cells infected with BTV-10 or treated with a mixture of purified VP2 and VP5 (VP2/5). Nuclear extracts were prepared from infected or treated cells at 12 h after infection or treatment. The major NF-B subunit, p65 (A), the minor subunit, p50 (B), and both subunits (C) were detected by Western blot analysis.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Previous studies have reported that apoptosis in response to BTV infection occurs in primary sheep endothelial cell cultures (10, 11). These studies have characterized apoptosis mainly in terms of the exclusion of propidium iodide from cells in fluorescence-activated cell sorter analysis. In this report we have investigated several different morphological and biochemical markers of apoptosis in both mammalian and insect cells. Consistent with the previous description of apoptosis in the arbovirus Sindbis virus (19), we find that the apoptotic responses of mammalian and insect cells to BTV infection in culture reflect the apparent pathology of the virus in insect and mammalian hosts. Thus, while we were able to detect clear signs of apoptosis in three different mammalian cell lines in response to BTV infection, there was no evidence of apoptosis in insect cell lines, including a cell line derived from C. variipennis, a natural vector for BTV. This was true even upon prolonged exposure of insect cells to virus (7 days), despite productive replication of virus and expression of viral proteins. Apoptosis in insect cells is well documented (5, 6), and thus, this finding suggests that the signaling pathway for the induction of apoptosis is not triggered by BTV infection of insect cells. It may be that BTV and other arboviruses that do not trigger disease in the insect host are genetically more fit, as they stand an increased chance of being transmitted to the mammalian host. Thus, there is a continual selection for viral variants that are asymptomatic in the insect host (or for insects that do not suffer from disease upon virus infection).

One of the characteristic differences between the replication cycles of BTV in mammalian versus insect hosts is the proportion of newly synthesized virus particles that remains cell associated. In mammalian cells, virions remain mainly associated with cellular components, and only a minority of particles (<10%) are found in the extracellular medium, presumably as a result of release from apoptosis and death of infected cells. In contrast, in insect cells, only 10% of the virus particles are recovered from the intracellular compartments, whereas 90% are purified from the supernatant and no CPE is observed (14). Therefore, based on our initial findings, it was reasonable to suppose that the accumulation of viral proteins and nascent viral particles could be responsible for triggering apoptosis in mammalian cells. In order to test this possibility, we inactivated the virus by UV treatment and used it to treat cells that showed an apoptotic response to viral infection. In this experiment, apoptosis was clearly triggered in response to treatment of cells with UV-inactivated virus. Thus, a productive viral replication cycle was not required to trigger apoptosis. We also found that at least four times as much UV-inactivated BTV as infectious BTV was required to elicit an apoptotic response. We would explain this result by suggesting that replication of BTV and release of BTV from infected cells trigger multiple rounds of infection and thus amplify the proapoptotic signals.

Consistent with the fact that expression of functional viral proteins is not required to trigger apoptosis; the intracellular expression of viral proteins (VP2, VP5, NS1, and NS2) had no proapoptotic effect. However, intracellular coexpression of the outer capsid proteins VP2 and VP5 in this way is a poor mimic for the events involved in viral entry, because during virus entry it is the extracellular side of the plasma membrane that is first exposed to these proteins. To test whether treatment of cells with purified VP2 or VP5 was sufficient to trigger apoptosis, we expressed and purified each protein by using the baculovirus expression system, and we treated cells with each protein individually and with the two proteins in combination. Surprisingly, while neither VP2 nor VP5 alone was able to trigger apoptosis, when a combination of these proteins was applied to the cells, a dramatic apoptotic response was noted (Fig. 6C to E).

Reports of apoptosis for orthoreovirus and avian reovirus have shown that receptor binding is not sufficient to trigger apoptosis, but those downstream events dependent on acidification of the endosome and thus on viral uncoating are also necessary (8). In a similar way, we have found that CQ and AC, two inhibitors of endosomal acidification, dramatically inhibit both apoptosis and virus replication. Interestingly, while BTV VP2 has been shown to be involved in virus attachment, (16) it has been found that the N terminus of VP5 contains amphipathic alpha-helices that are capable of membrane permeabilization and thus may play a role in the release of the viral core from the endosomal vesicle (17). We would hypothesize that a complex of VP2 and VP5 is internalized in cells treated exogenously with purified protein and that it is the combination of the receptor binding activity of VP2 and the membrane permeabilization activity of VP5 that is responsible for triggering apoptosis. Consistent with this hypothesis, apoptosis in response to extracellular treatment with VP2-VP5 is inhibited by prior treatment of the cells with either CQ or AC. Thus, activation of VP5 by acidification of endosomal vesicles is critical to the induction of apoptosis.

In reovirus infections, activation of the transcription factor NF-B is necessary for virus-induced apoptosis (9). We have also found that either BTV infection or treatment of cells with purified VP2-VP5 is sufficient to trigger translocation of NF-B to the nucleus within 12 h p.i. Whether, as in reovirus infections, the activation of this transcription factor is necessary for BTV-induced apoptosis will be the subject of future research. However, the requirement for both receptor binding and subsequent endosomal acidification for the induction of apoptosis by BTV reveals that the process of apoptosis for this virus is surprisingly similar to that of orthoreovirus and avian reovirus. This is highly intriguing, since there are significant differences in host range and mode of transmission between these viruses. It may be that the common structural features of the multilayered capsids of members of the Reoviridae predispose the uncoating virion to trigger certain cellular responses to virus invasion. What is also quite remarkable for BTV is that the same virion structure that triggers a dramatic apoptotic response in mammalian cells causes no detectable apoptosis in insect cells. This may be evidence for the coevolution of the insect vector with the virus or simply an indication that a virus that does not cause disease in the insect is more likely to be passed on to the next stage in the virus infection cycle.

Apoptosis plays an important role in BTV-induced tissue injury in vivo, and it has been demonstrated that the level of cellular apoptosis correlates with viral pathogenicity (10). Although the exact cellular pathways involved in BTV-induced apoptosis and pathogenesis are still not fully understood, the results obtained from our in vitro studies suggest that when apoptosis is induced by BTV infection in vivo, the target cells eventually die, thus releasing a complete new generation of virions.

In this study we provide evidence for apoptosis induction following BTV infection and we show that the BTV outer capsid proteins are specifically involved in this induction. Although several aspects of BTV-induced apoptosis remain unclear and studies will be needed to gain insights into the nature of the pathways involved, the first steps in the process, interaction of BTV capsid proteins with the cell surface, are presented here. Our study adds an important new dimension to BTV-host interactions and provides the basis for a study of the role of apoptosis, and apoptosis inhibitors, in viral pathogenesis.

 
This work is supported partly by grants from the BBSRC and by the Wellcome Trust of the United Kingdom.

 
* Corresponding author. Mailing address: Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel St., London WC1E 7HT, United Kingdom. Phone: 44 (0) 20 7927 2324. Fax: 44 (0) 20 7927 2839. E-mail: polly.roy{at}lshtm.ac.uk.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Journal of Virology, March 2004, p. 2875-2883, Vol. 78, No. 6
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.6.2875-2883.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

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