Avian Reoviruses Cause Apoptosis in Cultured Cells: Viral Uncoating, but Not Viral Gene Expression, Is Required for Apoptosis Induction

Cells, viruses, and reagents.

Primary cultures of CEF were prepared from 9- to 10-day-old chicken embryos and grown in medium 199 containing 10% tryptose phosphate broth and 7.5% calf serum. Mouse L fibroblasts, monkey kidney BSC-40 epithelial cells, and human epithelial HeLa cells were grown in monolayer cultures in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. Propagation and titering of vaccinia virus were performed on BSC-40 cells. The avian reoviruses used were provided by Laboratorios Intervet S.A. (Salamanca, Spain) and were grown on semiconfluent monolayers of CEF. Conditions for growing, radiolabeling, purifying, and titrating these viruses have been described previously (25, 45). Generation of UV-inactivated avian reovirions was performed as previously described for mammalian reovirus (64). Polyclonal antibodies against the avian reovirus nonstructural protein μNS were generated in rabbits with a gel-purified recombinant S1133 μNS expressed in insect cells as the antigen.

Cell culture reagents were from Life Technologies (Barcelona, Spain), and microscopy reagents were from Polysciences, Inc. (Warrington, Pa.). The ³⁵S cell-labeling mix was from Amersham Bioscience (Barcelona, Spain). All other reagents, enzymes, and inhibitors were from Sigma-Aldrich S.A. (Madrid, Spain).

Nuclear staining.

For nuclear staining, mock-infected or infected cells grown on glass cover slips were washed three times with cold phosphate-buffered saline (PBS) and fixed with methanol. Cover slips were kept at −20°C for 20 min, washed again with PBS, and incubated twice (5 min each) with PBS at room temperature. Nuclei were stained by incubation with Hoechst 33258 (50 ng/ml in PBS) for 10 min at room temperature in the darkness. Cells were then washed with PBS, dehydrated in 100% ethanol, and dried. Stained cells were mounted with Mowiol and visualized by fluorescence microscopy.

For the TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick-end labeling) assay, cells on cover slips were fixed with 4% paraformaldehyde and subsequently permeabilized and stained with an in situ cell death detection kit (TUNEL/fluorescein isothiocyanate [FITC] kit from Roche Applied Sciences, Barcelona, Spain) according to the manufacturer's instructions.

Virus infections, immunoprecipitation, and protein analysis.

Viral infections, virus purification, metabolic radiolabeling of cells with [³⁵S]methionine-cysteine, preparation of cytoplasmic extracts, immunoprecipitation, and electrophoretic analysis of radiolabeled proteins were performed as described previously (25, 44, 45).

Analysis of integrity of cellular DNA and rRNA.

For the detection of oligonucleosomal DNA fragments, a monolayer of either mock-infected or infected cells on a 100-mm plate was scraped off into the culture medium, and cells were collected by low-speed centrifugation. The cell pellet was resuspended in PBS and centrifuged again, and cells in the final pellet were resuspended (2 × 10⁷ cells/ml) and lysed in a buffer containing 10 mM Tris-HCl (pH 7.5), 10 mM EDTA, and 1% sodium dodecyl sulfate (SDS). Cell extracts were supplemented with 1 M NaCl, incubated for 12 h at 4°C, and centrifuged for 10 min at 10,000 × g and 4°C. The SDS concentration in the supernatant was adjusted to 0.5%, and the mixture was supplemented with 200 μg of proteinase K/ml and 50 μg of RNase A/ml and incubated for 1 h at 37°C. DNA was isolated by phenol-chloroform extraction, precipitation with 95% ethanol, and centrifugation. The DNA pellet was resuspended in TE buffer (10 mM Tris-HCl [pH 8.0], 1 mM EDTA) containing 50 μg of RNase A/ml, incubated for 15 min at 37°C, and subsequently resolved by electrophoresis in 1.5% agarose gels (4 μg/well).

For analysis of the integrity of rRNAs, cell pellets were resuspended (3.3 × 10⁷ cells/ml) in a buffer containing 10 mM Tris-HCl (pH 8.6), 0.14 M NaCl, 1.5 mM MgCl₂, 0.5% Nonidet P-40, and 1 mM dithiothreitol, and cells were lysed by incubation in this buffer for 5 min at 4°C. After centrifugation for 90 s at 10,000 × g and 4°C, the supernatant was supplemented with an equal volume of 0.2 M Tris-HCl (pH 8.0), 25 mM EDTA, 0.3 M NaCl, 0.2% SDS, and 100 μg of proteinase K/ml and incubated for 30 min at 37°C. Cytoplasmic RNA was isolated by phenol and phenol-chloroform (24:1) extractions, followed by ethanol precipitation. After centrifugation, the RNA pellet was resuspended in water, incubated in RNA sample buffer (50% formamide, 2.2 M formaldehyde, and 40 μg of ethidium bromide per ml) for 5 min at 60°C and resolved in 1% agarose-0.22 M formaldehyde gels (4 μg/well).

Histone-associated DNA fragments were detected by immunoassay with the Cell Death Detection enzyme-linked immunosorbent assay (ELISA) kit from Roche Applied Sciences, following the manufacturer's instructions.

Flow cytometric analysis.

At the end of the infection, adherent and floating cells were collected and centrifuged at 500 × g for 10 min. Pelleted cells were then resuspended in PBS, centrifuged, and resuspended again in a minimum volume of PBS. Cells were then permeabilized by the dropwise addition of 80% ice-cold ethanol to a final concentration of 70% and incubated overnight at 4°C. Cells were centrifuged, resuspended in PBS at a final concentration of 10⁶ cells/ml, and subsequently incubated for 30 min at 37°C with 50 μg of propidium iodide/ml and 100 μg of RNase A/ml (Sigma). Aliquots of 10⁶ cells were subjected to flow cytometric analysis with a FACSCalibur flow cytometer equipped with an argon-ion laser (488 nm), and the data were acquired and analyzed with CELLQuest software (Becton Dickinson).

To detect translocation of phosphatidylserine from the inner to the outer leaflet of the plasma membrane, adherent and floating cells were collected and assayed with Vybrant apoptosis assay kit #3 (Molecular Probes, Eugene, Oreg.), following the manufacturer's instructions.

Avian reovirus-infected CEF undergo apoptosis.

The infection of monolayers of CEF with avian reovirus S1133 causes a strong cytopathic effect. Thus, whereas mock-infected cells show the typical fibroblast-like morphology, most of the avian reovirus-infected cells manifest shrinkage, rounding, and detachment from the plate. Furthermore, infected cells, but not their uninfected counterparts, suffer nucleus damage, with chromatin condensation and margination (Fig. 1A). Since the morphology of avian reovirus-infected cells is very similar to that reported for apoptotic cells, we next performed different assays to assess the apoptotic state of avian reovirus-infected cells. For this, CEF monolayers were infected with 20 PFU of avian reovirus S1133/cell, and, at 24 h postinfection, infected cells were examined for different biochemical hallmarks of apoptosis.

Internucleosomal DNA fragmentation into an oligonucleosome-length DNA ladder (Fig. 1B) and rRNA degradation (Fig. 1C) were evident in avian reovirus-infected CEF but not in either uninfected cells or vaccinia virus-infected cells. Apoptosis in avian reovirus-infected cells could also be observed by ELISA detection of cytoplasmic histone-associated DNA fragments (Fig. 1D) and by in situ visualization at the single-cell level by the TUNEL assay (Fig. 1E). These results clearly demonstrate that S1133-infected CEF undergo apoptosis.

We next quantified the extent of apoptosis in infected cells by flow cytometry, with propidium iodide and fluorescein-conjugated annexin V as fluorochromes. DNA staining with propidium iodide showed that while only 1% of the uninfected cells displayed a sub-G₀/G₁ DNA content, this fraction was increased up to 60% in avian reovirus-infected cells (Fig. 2A). A similar increase was also detected by cytometric analysis of the redistribution of phosphatidylserine to the outer layer of cell membranes with fluorescein-conjugated annexin V (Fig. 2B).

Apoptosis is a virus-induced event that is triggered at an early infection stage. In order to demonstrate the connection between viral infection and induction of apoptosis, we next compared the effects of time and multiplicity of infection on both apoptosis induction and viral growth. When CEF monolayers were infected with a viral multiplicity of 20 PFU/cell, oligonucleosomal DNA laddering could be detected as soon as 8 h postinfection, and the intensity of the ladder bands increased with time of infection, reaching a plateau at 16 h postinfection (Fig. 3A). Similar results were obtained when the extent of apoptosis was quantified by flow cytometric analysis of propidium iodide-stained cells; in this case, an increase in the percentage of sub-G₀/G₁ cells was already evident at 4 h postinfection, and the fraction of low-DNA-containing cells increased almost linearly until it reached a plateau at 16 h postinfection (Fig. 3B). In contrast, the production of infectious virus progeny increased almost linearly from 8 to 24 h postinfection, and no increase in infectious progeny virus production was observed at 4 h postinfection (Fig. 3C).

Next we examined the dependence of apoptosis and viral growth on the multiplicity of infection. When cells were infected with a multiplicity as low as 3 PFU/cell, DNA laddering was detected as early as 12 h postinfection, and its intensity increased with increasing multiplicity of infection (Fig. 3D). A linear increase in the extent of apoptosis with inocula from 1 to 30 PFU/cell was also observed by flow cytometric analysis of propidium iodide-stained cells (Fig. 3E). On the contrary, no significant change in the production of infectious progeny virus was observed in the inoculum multiplicity range between 1 and 30 PFU/cell (Fig. 3F), suggesting that the number of viral particles but not their replicative capacity is important for avian reovirus to elicit apoptosis. Furthermore, these results, and the fact that DNA laddering was not detected in either uninfected cells or vaccinia virus-infected cells, strongly suggest that avian reovirus infection is the direct trigger of apoptosis in infected cells.

Apoptosis induction is not unique to the S1133 strain of avian reovirus or to primary cultures of CEF cells. Different cells from a single host individual respond differently to a given viral infection (19, 27, 28, 39, 52). Equally, different virus strains may cause different cytopathic effects in a given cell type (31, 49, 62, 64). To assess whether the avian reovirus is able to induce apoptosis in cells other than CEF, internucleosomal DNA cleavage was analyzed in mammalian BSC-40, L, and HeLa cells infected with avian reovirus S1133. The virus was able to replicate (Fig. 4A) and to cause apoptosis (Fig. 4B) in all three S1133-infected mammalian cells examined.

Next, we investigated the potential of three different avian reovirus isolates to induce apoptosis. DNA laddering was evident in CEF infected with each of the three viruses (Fig. 4C). Similar results were obtained with BSC40, HeLa, and L cells (data not shown).

Cell death activation in infected cells occurs in the absence of viral gene expression. In order to determine whether viral gene expression is required for apoptosis induction, the effect of UV treatment on the capacity of avian reovirions to induce apoptosis was first investigated. When purified avian reovirions were subjected to a 30-min UV treatment, the capacity of the virus to synthesize viral proteins was greatly reduced, as demonstrated by metabolic labeling with [³⁵S]methionine and by immunoblot analysis of intracellular levels of the nonstructural μNS protein (Fig. 5A and B, compare lanes 2 and 3). This treatment also caused a drastic reduction in the intracellular production of infectious virus particles (Fig. 5D, compare lanes 2 and 6). In contrast, UV treatment did not affect the apoptosis-inducing ability of the virus, as revealed by DNA fragmentation (Fig. 5C) and flow cytometric analysis (Fig. 5E, compare lanes 2 and 6). Furthermore, similar kinetics of apoptosis induction were obtained by infection of CEF with avian reovirions, whether before or after UV irradiation (data not shown). These results strongly suggest that viral mRNA synthesis and subsequent steps in viral replication are not needed for apoptosis induction in avian reovirus-infected cells.

However, since the possibility still existed that gene expression by a small subset of reovirions not inactivated by the UV treatment could be the trigger for apoptosis induction, control experiments were subsequently performed. First, cells were infected with samples of purified reovirions that had been UV irradiated for various times. A comparative analysis of both virus production and the extent of apoptosis revealed that while the UV treatment caused a progressive and drastic reduction in viral replication (Fig. 5D, compare lanes 2 to 6), it did not diminish the extent of apoptosis (Fig. 5E, compare lanes 2 to 6). Furthermore, when infection was performed with two virus samples of similar infectivity (Fig. 5D, compare lanes 6 and 7) but markedly different reovirion content (40,000 versus 2 reovirions/cell), apoptosis was only triggered by the sample containing the large number of viral particles (Fig. 5E, compare lanes 6 and 7). These results demonstrate that the residual infectivity remaining in the sample of UV-treated virus is not sufficient to trigger apoptosis and suggest that a threshold number of input virus particles is required for apoptosis induction by avian reovirus. Taken together, our data strongly suggest that intracellular viral gene expression is dispensable for apoptosis induction in avian reovirus-infected cells.

To confirm this suggestion, we next investigated the effect of ribavirin on apoptosis induction in avian reovirus-infected cells. Ribavirin is a guanosine nucleoside analog that has been reported to inhibit mammalian reovirus transcription and replication without affecting the expression of cellular genes or the capacity of mammalian reovirus to induce apoptosis (12, 53). Similar results showing that ribavirin likewise inhibits avian reovirus gene expression have been reported recently (7).

In order to determine the appropriate ribavirin concentration to be used in our experiments and to test its toxicity for CEF, we performed initial trials with uninfected cells. The presence of ribavirin in the culture medium of uninfected cells at concentrations up to 400 μM did not promote apoptosis (Fig. 6C and D, lanes 1 to 3) and did not affect either the viability of the cells for at least 3 days (data not shown) or their protein synthesis ability, as analyzed 16 h after addition of the inhibitor (Fig. 6A, lanes 1 to 3). In the experiments with infected cells, we found that whereas ribavirin did not prevent intracellular uncoating of parental reovirions (Fig. 7C, lane 4), it strongly and specifically inhibited viral but not cellular protein synthesis in a dose-dependent manner, as demonstrated by SDS-PAGE analysis of radiolabeled extracts from infected cells before and after immunoprecipitation with antireovirus antiserum (Fig. 6A and B, lanes 4 to 6).

Interestingly, while viral protein synthesis was completely abolished in cells treated with 400 μM ribavirin, the ability of avian reovirus to trigger apoptosis was not affected by ribavirin treatment (Fig. 6C and D, compare lanes 4 with lanes 6). Taken together, these results indicate that viral transcription and subsequent steps in viral replication are not required for apoptosis induction in avian reovirus-infected cells.

Intracellular viral uncoating is required for apoptosis induction in avian reovirus-infected cells. To identify the particular stage of the virus life cycle at which apoptosis is triggered in avian reovirus-infected cells and to assess whether apoptosis is induced by the virus from within or from without the host cell, we next investigated whether viral uncoating is required for apoptosis induction in S1133-infected CEF. Avian reovirus uncoating, which takes place within intracellular vacuoles, is largely dependent on the endosome-mediated proteolytic processing of the major outer capsid protein μB/μBC into two specific products, δ and δ′ (17) (Fig. 7C). This processing and virus replication are both blocked by specific inhibitors of the activity of the vacuolar proton-ATPase, such as bafilomycin A1 (our unpublished results), and by different lysosomotropic agents (17) (Fig. 7C), indicating both that avian reovirus penetrates cells via the receptor-mediated endocytosis route and that endosome acidification is necessary for viral uncoating and viral replication.

To assess the dependence of apoptosis on viral uncoating, we first tested the activity of several viral inhibitors in order to select those that do not trigger apoptosis in uninfected cells but block intracellular viral uncoating in infected cells (10, 43). Inhibitors such as bafilomycin A1 and concanamycin were discarded because of their proapoptotic activity in uninfected CEF (data not shown). Ammonium chloride and chloroquine, two weakly basic amines that act as lysosomotropic agents by neutralizing vacuolar acidification (56), were selected because they effectively blocked virus uncoating in S1133-infected CEF (Fig. 7C) without provoking apoptosis in uninfected cells (Fig. 7D and E, compare lane 1 with lanes 2 and 3) or affecting the synthesis of cellular proteins in uninfected or infected cells (Fig. 7A, compare lane 1 with lanes 2, 3, 5, and 6). However, when added to cells at the onset of the infection, the two inhibitors were very effective in blocking (i) viral protein synthesis (Fig. 7A and B, compare lane 4 with lanes 5 and 6); (ii) the intracellular uncoating of reovirions (Fig. 7C, compare lane 1 with lanes 2 and 3); (iii) viral replication (17) (our unpublished data); and (iv) the ability of the avian reovirus to induce apoptosis (Fig. 7D and E, compare lane 4 with lanes 5 and 6). Furthermore, the ability of the inhibitors to block these processes was lost when they were added to cells at 3 h postinfection (lanes 7 and 8 in Fig. 7A, B, D, and E; lanes 5 and 6 in Fig. 7C), suggesting that they block an early stage of the viral replication cycle. Similar results were obtained when avian reovirus-infected cells were cultivated in the presence of E64, a protease inhibitor that has been shown to specifically block the uncoating of mammalian reovirus in infected cells (2, 3) (data not shown). Taken together, these results demonstrate that virus uncoating is necessary for apoptosis induction in avian reovirus-infected cells.