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Journal of Virology, October 2003, p. 10537-10547, Vol. 77, No. 19
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.19.10537-10547.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Glycoprotein of Nonpathogenic Rabies Viruses Is a Key Determinant of Human Cell Apoptosis

Christophe Préhaud,1 Stéphanie Lay,1 Bernhard Dietzschold,2 and Monique Lafon1*

Unité de Neuroimmunologie Virale, Département de Neuroscience, Institut Pasteur, Paris, France,1 Center for Neurology, Department of Microbiology and Immunology, Thomas Jefferson University, Philadelphia, Pennsylvania2

Received 7 February 2003/ Accepted 20 June 2003


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ABSTRACT
 
We showed that, unlike pathogenic rabies virus (RV) strain CVS, attenuated RV strain ERA triggers the caspase-dependent apoptosis of human cells. Furthermore, we observed that the induction of apoptosis is correlated with a particular virus antigen distribution: the overexpression of the viral G protein on the cell surface, with continuous localization on the cytoplasmic membrane, and large cytoplasmic inclusions of the N protein. To determine whether one of these two major RV proteins (G and N proteins) triggers apoptosis, we constructed transgenic Jurkat T-cell lines that drive tetracycline-inducible gene expression to produce the G and N proteins of ERA and CVS individually. The induction of ERA G protein (G-ERA) expression but not of ERA N protein expression resulted in apoptosis, and G-ERA was more efficient at triggering apoptosis than was CVS G protein. To test whether other viral proteins participated in the induction of apoptosis, human cells were infected with recombinant RV in which the G protein gene from the attenuated strain had been replaced by its virulent strain counterpart (CVS). Only RV containing the G protein from the nonpathogenic RV strain was able to trigger the apoptosis of human cells. Thus, the ability of RV strains to induce apoptosis is largely determined by the viral G protein.


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INTRODUCTION
 
Many viruses have developed strategies to manipulate the survival or the death of the cells that they infect (27). Different viruses use different mechanisms, and these mechanisms have been studied in detail (23). However, a large number of groups are trying to identify proteins that directly trigger apoptosis and to determine the precise roles of these proteins (8, 11, 14, 34, 50). As well as improving knowledge of virus pathogenicity and virulence, the identification of "viral death proteins" may have practical applications for human therapeutics. The induction of apoptosis by peptides or polypeptides might make it possible to eliminate undesirable cells, such as tumor, infected, and autoimmune cells, or to trigger the formation of apoptotic bodies, which are thought to be powerful immune response activators (1, 3, 32, 45, 49, 51).

Rabies virus (RV) is an enveloped, bullet-shaped virus belonging to the Rhabdoviridae family and the Lyssavirus genus. The viral particle consists of a membrane composed of host lipids and two viral proteins, the G and M proteins, surrounding a helical nucleocapsid (NC). The NC is composed of a viral negative-strand RNA molecule protected by the N protein, the P protein, and the RNA-dependent RNA polymerase, the L protein. RV proteins are not synthesized in equal amounts in infected cells and are not present at the same ratios in viral particles (13, 37). Indeed, the N, G, and M proteins are, in this order, the most prominent species in virions.

RV strain CVS is a highly neurotropic virus strain. In contrast, SAG-2, ERA, and SN-10-SAD are attenuated strains, probably because they have been subjected to several passages in nonneuronal cultures. They grow in vitro in lymphocytes (57). These disabled viruses are candidates for live vaccines. SAG-2, an SN-10-SAD mutant, has been successfully used in oral vaccination programs to eradicate rabies from wildlife reservoirs in western Europe (4, 5, 19, 38). The attenuated live RV vaccine strain, ERA, triggers apoptosis in the human lymphoblastoid T-cell line Jurkat and activates caspases 3, 8, and 9. RV-induced apoptosis is blocked by the overexpression of Bcl-2, suggesting that RV induces apoptosis via a mitochondrial pathway (58). The direct interaction of RV with receptors located on the cytoplasmic membrane is not sufficient to induce apoptosis (25, 57). Apoptosis requires that the infectious cycle be completed. Apoptosis is correlated with the detection of the G protein, suggesting that viral proteins, the G protein in particular, play a role in this process (15, 57). Moreover, Morimoto et al. (43) showed that the accumulation of the G protein, but not the N protein, is correlated with the induction of apoptosis in primary mouse neuron cultures. Nevertheless, differential NC distributions are observed in CVS- and ERA-infected Jurkat T cells (57, 58). Thus, the intrinsic properties of the N protein as an apoptosis inducer remain unclear.

The goal of this study was to identify the RV protein that causes death in two types of human cells, Jurkat T cells and neuroblastoma SK-N-SH cells. In a first instance, we decided to focus our investigations on the viral N and G proteins. First, we assessed whether apoptosis, which occurs in Jurkat T cells, could also take place in neuronal cells. Second, we analyzed the expression and the distribution of the G and N proteins in cells infected with apoptotic and nonapoptotic virus strains. Third, we constructed transgenic Jurkat T-cell lines that drive tetracycline-inducible gene expression to compare the abilities of the G and N proteins of nonapoptotic RV strain CVS and of proapoptotic RV strain ERA to trigger the apoptosis of Jurkat T cells. Finally, we used recombinant viruses obtained by reverse genetics to determine whether other proteins from strain ERA, such as the M, L, and P proteins, were inducers of apoptosis.

Our data indicated that the proapoptotic properties of the attenuated RV strain are dependent on the viral G protein in both human lymphoblastoid and human neuronal cell lines and suggest that the accumulation of the viral G protein at the cytoplasmic membrane is an important factor in the triggering of apoptosis.


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MATERIALS AND METHODS
 
Cells and viruses. Jurkat rtTA cells (31) were cultivated in RPMI 1640 medium containing 2 mM L-glutamine, 100 U of penicillin/ml, 100 µg of streptomycin/ml, 10% tetracycline-free fetal bovine serum, and 2.5 µg of puromycin/ml. Cells were cultured at 37°C in a 5% CO2-95% air atmosphere and split twice a week. The human neuroblastoma cell line SK-N-SH (HTB11; American Type Culture Collection [ATCC]) was propagated in Dulbecco modified Eagle medium containing 2 mM L-glutamine, 1 mM Na pyruvate, 10% fetal calf serum, 2% Na bicarbonate, 100 U of penicillin/ml, and 100 µg of streptomycin/ml at 37°C and treated with trypsin twice a week. The nonpathogenic RV laboratory strain ERA and the pathogenic RV laboratory strain CVS (ATCC vr332 and ATCC vr959, respectively) were propagated as previously described (57). SN-10 is a nonpathogenic virus strain derived from SAD B19 (53). The recombinant virus R-N2c was used to introduce the entire coding region of the G protein gene from CVS-N2c, a neuroinvasive strain, into the SN-10 genome (42, 43). SN-10 and R-N2c were propagated in mouse neuroblastoma cell line N2a.

Abs, MAbs, and reagents. Fluorescein isothiocyanate (FITC)-conjugated affinity-purified F(ab')2 fragments of anti-mouse goat immunoglobulin G (IgG) and 5-(4,6-dichlorotriazinyl)aminofluorescein-conjugated and Cy3-conjugated streptavidin were purchased from Jackson ImmunoResearch (distributed by Immunotech/Beckman Coulter, Orsay, France). FITC-conjugated NC-specific rabbit antibodies (Abs) were obtained from Sanofi Diagnostics (Marnes la Coquette, France). Phycoerythrin (PE)-conjugated streptavidin was obtained from Dako (Glostrup, Denmark). Biotinylated anti-RV G protein monoclonal antibody (MAb) 8-2 (41) was produced in the laboratory. A mixture of three anti-N protein MAbs and anti-G protein MAb 15-13 were gifts from N. Minamoto (University of Gifu, Gifu, Japan) (35). An Ab specific for a linear RV G protein epitope (6-15-C4) was a gift from A. D. M. E. Osterhaus (Institute of Virology, Erasmus Medical Center, Rotterdam, The Netherlands) (9). The control consisted of an irrelevant FITC-conjugated antitrinitrophenol (TNP) IgG1 antibody (BD-Pharmingen, Le Pont de Claix, France). A CaspaTag caspase 8 (leucylglutamylthreonylaspartic [LETD]) activity kit and Hoechst 33342 were obtained from Intergen (distributed by Quantum Biogen, Illkirch, France). Cellfix was supplied by Becton-Dickinson Biosciences (Le Pont de Claix, France). Fluoromount-G was purchased from Cliniscience. Doxycycline and tetracycline-free fetal bovine serum were obtained from Ozyme (St. Quentin-en-Yvelines, France). Puromycin and hexadimethrine bromide (Polybrene) were purchased from Sigma Aldrich (St. Quentin Fallavier, France). Hygromycin B was obtained from Roche Diagnostics (Meylan, France). Streptomycin, penicillin, and G-418 were purchased from Gibco BRL (Cergy Pontoise, France). Trevigen Apoptotic Cell System annexin V-FITC was obtained from R&D Systems Europe (Abingdon, United Kingdom).

Inducible transgenic cell lines. We used the Rev-Tet System, developed by Clontech Laboratories, to construct transgenic Jurkat T-cell lines that drive tetracycline-inducible gene expression. All experiments were carried out according to the manufacturer's instructions (Rev-Tet System user manual PT 3223-1). Briefly, the open reading frames corresponding to the genes for the G protein of ERA (G-ERA) (GenBank accession number AF406693), the G protein of CVS (G-CVS) (AF406694), the N protein of ERA (N-ERA) (AF406695), and the N protein of CVS (N-CVS) (AF406696) were amplified by PCR and inserted into pRevTRE that had been cut with BamHI and HpaI and dephosphorylated. Recombinant Moloney murine leukemia viruses (retroviruses) carrying the RV genes were isolated after transfection of RetroPack PT67 cells with the resulting plasmids. Hygromycin-resistant clones were subsequently identified. These viruses were used to infect Jurkat rtTA cells (31), and clones resistant to hygromycin and puromycin were selected. RV protein expression was monitored by flow cytometry analysis after 18 h in the presence of 1 µg of doxycycline/ml.

Infection of cell cultures. Monolayers of SK-N-SH cells in 12-well plates (105 cells) were infected with ERA, CVS, SN-10, and the recombinant virus R-N2c (multiplicity of infection [MOI], 3) or mock infected for 1 h at 37°C. The virus inoculum was then replaced by 1 ml of culture medium, and the cells were incubated for 48 h at 37°C.

Jurkat rtTA cells in 12-well plates were infected with ERA, CVS, and SN-10 (MOI, 3) and with R-N2c (MOI, 40) or mock infected. Cells were incubated for 48 h at 37°C in a 5% CO2-95% air atmosphere for further analysis.

Immunostaining. For the detection of RV NC and its main constituent, the N protein, which is not expressed on the cell surface, and for the detection of total intracytoplasmic RV G proteins, transgenic or infected Jurkat rtTA cells and SK-N-SH cells were fixed with 3% paraformaldehyde (PFA) for 20 min at 4°C. Cells were then incubated for 1 h at 37°C with a mixture of three anti-N protein MAbs, a biotinylated anti-RV G protein MAb, or an FITC-conjugated Ab diluted in permeabilization buffer (1% heat-inactivated fetal calf serum, 0.1% [wt/vol] sodium azide, and 0.1% [wt/vol] saponin in phosphate-buffered saline). Cells were further incubated with FITC-conjugated affinity-purified F(ab')2 fragments of anti-mouse goat IgG or PE-conjugated streptavidin. For the surface expression of the G protein, PFA treatment was omitted, and cells were incubated for 1 h at 37°C with the biotinylated anti-RV G protein MAb diluted in culture medium (Jurkat rtTA or SK-N-SH cells) and then incubated with PE-conjugated or FITC-conjugated streptavidin. For flow cytometry analysis, cells were washed several times in staining buffer and fixed in Cellfix (1/10 in staining buffer [phosphate-buffered saline, 1% fetal calf serum, and 0.1% sodium azide, pH 7.6]) (directly for Jurkat rtTA cells and after scraping for SK-N-SH cells). Viral proteins were detected in a cell population (104 cells) that was gated to exclude dead cells and cell debris. Viral proteins in cell cultures were measured by determining the frequency of events with FITC or PE signals higher than a given threshold.

Assessment of viability by light-scattering analysis. Morphological changes were assessed by side and forward light-scattering flow cytometry and used to identify apoptotic Jurkat T cells as previously described (28).

Detection of nuclear DNA fragmentation by Hoechst staining. The nuclear morphology of normal and apoptotic cells was assayed by staining with Hoechst 33342 (1 µg/ml) and visualized with a Zeiss UV microscope. The percentage of cells with fragmented nuclei in 20 microscope fields (magnification, x63) was determined with offline software (Metamorph software imaging system; Universal Imaging Corporation, Downingtown, Pa.).

Detection of nuclear DNA fragmentation by the TUNEL method. DNA fragmentation was assessed by using the terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) technique as previously described (21) with minor modifications (57). Slides were observed with a Zeiss UV microscope. Results are expressed as the number of cells with fluorescent nuclei per 500 or 5,000 cells per field (magnification, x63).

Detection of PS exposure. For detection of phosphatidylserine (PS) exposure, SK-N-SH cells adhering to a 5-cm2 surface were washed with phosphate-buffered saline before being incubated for 15 min in the dark with 25 ng of Annexin V-FITC at room temperature. Cells were washed, fixed in 3% PFA, and analyzed by confocal microscopy.

Detection of caspase activation. Activated caspases were detected by use of FITC-conjugated FAM-LETD-FMK, a caspase inhibitor analog that enters cells and irreversibly binds to activated caspase 8 and with a reduced efficiency to caspases 1, 6, 9, and 10. Live cells (3 x 105 in 300 µl) were incubated with 10 µl of FITC-conjugated FAM-LETD-FMK for 1 h at 37°C in a 5% CO2 atmosphere in the dark. After two washes, cells were resuspended in 500 µl of staining buffer-50 µl of Cellfix before being analyzed by flow cytometry. To exclude nonspecific binding of the staining reagents to dead or necrotic cells, flow cytometry analyses were gated on a viable population (104 cells). Caspase activation following infection or the production of different proteins was determined by measuring the number of events with FITC signals higher than 3 log units.

Confocal microscopy. Confocal microscopy was performed with a Zeiss LSM 510 confocal microscope equipped with a helium-neon laser (1 mV). Cells were analyzed at 488 nm with a narrow-band filter centered on 535 nm for FITC signal detection. For each observation, the results for 12 optical xy sections taken at 0.5-µm intervals were recorded. Data were analyzed with Image Browser version 2.8. Images were treated with Adobe Photoshop 5.5.


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RESULTS
 
RV strain ERA triggers apoptosis in infected human SK-N-SH cells. SK-N-SH cells were infected with RV ERA or CVS, and the apoptosis of the infected cells was monitored by costaining with annexin V or Hoechst 33342 and Abs specific for virus antigens. Only cell monolayers in which more than 70% of the cells were infected were included in the subsequent analysis, such that apoptosis was monitored mostly in infected cells.

The flux of PS from the inner to the outer leaflet of the plasma membrane is an early marker of apoptosis that can be detected by annexin V staining (18, 54). ERA-infected SK-N-SH cells were stained with an RV G protein-specific Ab (Fig. 1, section I, panel A) and with annexin V (Fig. 1, section I, panel B). Costaining (Fig. 1, section I, panel C) revealed that infected cells expressing the G protein (red) on the cytoplasmic membrane contained PS on the outer leaflet (green). Annexin V-positive cells were not labeled by an irrelevant Ab (anti-TNP-FITC; data not shown) or by propidium iodide (Fig. 1, section I, panel D), a result that can be taken as evidence that PS exposure occurs only on viable cells which have not completely lost their membrane integrity. No annexin V staining was observed in CVS-infected SK-N-SH cells (data not shown), suggesting that CVS-infected cells do not encounter apoptosis.



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  		FIG. 1. ERA-infected SK-N-SH cells undergo cell death. (I) Confocal microscopy analysis of PS exposed on the outside of the cytoplasmic membrane of ERA-infected SK-N-SH cells (A) Detection of viral G protein on the cytoplasmic membrane of ERA-infected SK-N-SH cells (red). (B) Detection of PS with FITC-annexin V (green). (C) Annexin V and viral G protein labeling are not strictly colocalized. (D and E) Detection of nuclei after propidium iodide staining on infected cells not treated (D) or treated with PFA (E). These images are representative of 69 observations. (II) Nuclear fragmentation in ERA-infected SK-N-SH cells (magnification, x40). Nonapoptotic strain CVS-infected (panels labeled 1) and proapoptotic strain ERA-infected (panels labeled 2) SK-N-SH cells were stained with Hoechst 33342 (blue) and anti-NC Ab (green). Fragmented nuclei, which are characteristic of apoptosis, were observed in ERA- but not in CVS-infected SK-N-SH cells. These images are representative of 70 observations.

DNA fragmentation is a hallmark of intermediate-stage apoptosis resulting from the caspase-dependent activation of endonucleases that degrade chromatin into discrete fragments (61). Thus, we assessed DNA fragmentation in ERA- or CVS-infected SK-N-SH cells by costaining with Hoechst 33342 and NC-specific Abs (Fig. 1, section II). The nuclei of ERA-infected cells were clearly fragmented (Fig. 1, section II, panel 2), whereas those of CVS-infected cells were not (Fig. 1, section II, panel 1). These data indicate that ERA- but not CVS-infected cells undergo apoptosis.

We then quantified the extent of apoptosis in ERA-infected neuroblastoma populations and compared the results to those for ERA-infected Jurkat rtTA cells. We measured the apoptosis of ERA-and CVS-infected Jurkat rtTA cells and SK-N-SH cells by means of DNA fragmentation (TUNEL and Hoechst 33342 staining) and caspase activation (Fig. 2). In addition, for lymphoblastoid cells, apoptosis was quantified by light-scattering analysis (28) in order to determine the extent of cells with morphological changes. At similar levels of infection, ERA induced the apoptosis of the human lymphoblastoid cell line Jurkat rtTA (Fig. 2A and B) and of the neuroblastoma cell line SK-N-SH (Fig. 2C and D), whereas CVS did not (Fig. 2). Interestingly, caspases were activated in ERA-infected SK-N-SH cells, in agreement with the presence of the caspase 8 protein in these cells (46), a feature that is not shared by all neuroblastoma cells (2, 55, 56). Altogether, these data confirm that ERA induces apoptosis, whereas CVS does not. The similarities between the infections caused by the two RV strains in the two cell types indicate that the proapoptotic property of strain ERA does not depend on a specific cell environment.



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  		FIG. 2. ERA induces the apoptosis of Jurkat rtTA and SK-N-SH cells. (A) Apoptosis in 2-day-old cultures of Jurkat rtTA cells infected with ERA or CVS, as measured by determining the percentage of R2 cells in the cell population (black bars) or by determining the percentage of cells with fragmented nuclei stained by Hoechst 33342 (white bars; 500 cells counted) or by the TUNEL technique (stippled bars; 500 cells counted). This experiment is representative of five experiments (means and standard deviations [SD]). (B) Detection of infection (black bars) and caspase activation (white bars) of Jurkat rtTA cells infected with either ERA or CVS. Results are the means and SD of three experiments. (C) Induction of apoptosis in 2-day-old cultures of SK-N-SH cells infected with ERA or CVS, as measured by determining the percentage of cells with fragmented nuclei stained by Hoechst 33342 (white bars; 500 cells counted) or by the TUNEL technique (stippled bars; 500 cells counted). This experiment is representative of two experiments. (D) Detection of infection (black bars) and caspase activation (white bars) of SK-N-SH cells infected with either ERA or CVS. Results are the means and SD of three experiments.

The RV G protein and the NC accumulate to different levels in cells infected with proapoptotic and nonapoptotic virus strains. SK-N-SH cells were infected with RV ERA and CVS, and we determined the overall distribution of the NC by immunofluorescence. Typical NC staining was observed in both types of infected cells (Fig. 1, section II). However, ERA-infected cells exhibited many large inclusions of NC, whereas CVS-infected SK-N-SH cells contained both large and small inclusions together with a more diffuse pattern of staining throughout the cytoplasm.

We then investigated the behavior of the RV G protein. The G protein is synthesized in the cytoplasm of infected cells and trafficked through the endoplasmic reticulum and the Golgi apparatus before being anchored at the cytoplasmic membrane of virus-infected cells. SK-N-SH cells were infected with ERA and CVS, and the level of infection, the amount of G protein inside the cells, and the amount of cytoplasmic membrane-associated G protein were checked by flow cytometry (Fig. 3E). Interestingly, we found that the amount of cytoplasmic membrane-bound G protein relative to the amount of G protein inside the cells was higher in ERA- than in CVS-infected neuroblastoma cells. The G protein/N protein ratio shows that this difference was not due to a difference in the levels of infection (Fig. 3F). To try to explain this difference, we carried out G-protein-specific confocal immunofluorescence studies with living ERA- and CVS-infected SK-N-SH cells and with three different G-protein-specific MAbs and an irrelevant MAb. RV-infected neuroblastoma cells were not stained by the irrelevant antibody, showing that the infected cells had not lost their membrane integrity (data not shown). However, intense labeling was detected with the three G-protein-specific MAbs (Fig. 3A to D; data are shown for two of them). The distributions of cytoplasmic membrane-bound G protein were very different in ERA- and CVS-infected cells. In ERA-infected cells, the G protein was observed in localized areas of the cytoplasmic membrane, where it formed a fairly large ribbon-like protein structure. On the contrary, in CVS-infected cells, the G protein was distributed in the cytoplasmic membrane as bright patches. As already illustrated in Fig. 1, the ERA-infected cells subsequently underwent apoptosis, whereas the CVS infected cells did not. Similar data were obtained with Jurkat rtTA cells (data not shown).



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  		FIG. 3. Distributions of viral G proteins differ in ERA- and CVS-infected SK-N-SH cells. (A to D) Confocal microscopy analysis of cytoplasmic membrane-anchored G protein in ERA-infected (A and B) and CVS-infected (C and D) SK-N-SH cells. G protein staining is shown for two MAbs: 6-15-C4 and 8.2. G-ERA formed a large ribbon-like structure. G-CVS was localized on the membrane as patches. (E and F) Quantitative cytofluorimetry. (E) NC antigens that accumulated in the cytoplasm (black bars) were detected in PFA-treated ERA- or CVS-infected SK-N-SH cells. G protein, which is trafficked through the endoplasmic reticulum and Golgi apparatus before being exported to the plasma membrane, was measured with an anti-G protein MAb on live cells to detect membrane-bound G protein (mbG; gray bars) or on PFA-treated cells to detect internal G protein (intG; white bars). Error bars indicate standard deviations. (F) mbG/intG (stippled bars), mbG/NC (black bars), or intG/NC (white bars) ratio. Proapoptotic strain ERA had a much higher mbG/intG ratio than nonapoptotic strain CVS. Thus, mbG accumulates on the surface of ERA-infected cells but not on that of CVS-infected cells.

It is noteworthy that the morphology of ERA-infected SK-N-SH cells changed following infection. Indeed, they lost their neuronal shape and became rounder (Fig. 1, section II, panel 2). This change did not occur in CVS-infected cells.

To summarize, SK-N-SH cells infected with proapoptotic strains exhibited patterns of NC and G protein expression different from those of cells infected with nonapoptotic strains (Fig. 1 and 3). Furthermore, the pro- and antiapoptotic phenotypes were characterized by different distributions of the RV G protein in the cytoplasmic membrane and morphological modifications.

Doxycycline-inducible expression of RV N and G proteins in Jurkat rtTA cells. To examine the consequences of the overproduction of the RV G protein or the RV N protein on apoptosis in a nonviral context, we constructed Jurkat T-cell lines that produced the RV proteins following induction. Jurkat T cells were selected for production of the reverse tetracycline transactivator protein rtTA (puromycin resistant) (31) and then infected with recombinant Moloney murine leukemia viruses (retroviruses) carrying the N or G protein gene of RV ERA and CVS as well as the neomycin resistance gene. Two transgenic cell lines that were resistant to hygromycin and puromycin were obtained after infection with the two types of recombinant virus. After doxycycline treatment, RV protein production in PFA-treated cells was monitored by flow cytometry (Fig. 4). After 18 h in the presence of doxycycline, N-ERA (Fig. 4A) was present in 57.4% of the live cells, N-CVS was present in 64.3% (Fig. 4B), G-ERA was present in 34.4% (Fig. 4C), and G-CVS was present in 19.6% (Fig. 4D). These data are representative of four independent experiments. Thus, the production of the RV G and N proteins can be induced in this system. These results show that the percentages of cells expressing the G protein from both RV strains were slightly lower than those expressing the N protein and that the production of G-ERA was more efficient than that of G-CVS.



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  		FIG. 4. Inducible expression of RV proteins in Jurkat T cells. The expression of virus proteins in transgenic Jurkat rtTA cells (JrtTA) containing the gene encoding N-ERA (A), N-CVS (B), G-ERA (C), or G-CVS (D) was analyzed after 18 h in the presence of doxycycline by flow cytometry with a MAb specific for the G or N protein (bold lines). Thin lines show Ab reactivity with cells that did not express the protein (cells without doxycycline). The numbers show the percentages of doxycycline-treated cells that expressed a fluorescence signal that was higher than fluorescence channel 5. These graphs are representative of four experiments.

The induction of G-ERA production triggers the apoptosis of Jurkat rtTA cells. We then tested whether the production of the RV G and N proteins could induce apoptosis in this system. Three different methods were used (Fig. 5A to C). First, the induction of apoptosis was measured by flow cytometry by comparing side and forward light scattering in G-ERA-transgenic Jurkat rtTA cells treated with doxycycline (Fig. 5A, right panel) or not treated (Fig. 5A, left panel). The number of dying Jurkat rtTA cells (R2 population) increased when G-ERA was produced. The difference between the levels of apoptosis in the presence and absence of doxycycline was 31.4% (39.7 - 8.3%). Second, we assessed apoptosis by monitoring nuclear fragmentation. Typical nuclear fragmentation (Fig. 5B, right panel, arrows) was observed in doxycycline-treated Jurkat rtTA cells expressing G-ERA (28.2%) but rarely in doxycycline-treated control Jurkat rtTA cells (10%) (Fig. 5B, left panel). Finally, we monitored the ability of G-ERA to activate caspases. The detection of cells expressing activated caspases in Jurkat rtTA cells (Fig. 5C, left panel) and in Jurkat rtTA cells expressing G-ERA (Fig. 5C, right panel) either treated with doxycycline or not treated indicates that G-ERA expression triggers the specific activation of caspases in 19% of the population (after subtraction of the value for doxycycline-treated Jurkat rtTA cells). Thus, the production of G-ERA triggers caspase-dependent apoptosis, leading to the fragmentation of cell nuclei. As commonly observed, the percentages of apoptosis measured by the different methods were not the same, but they all correlated (5, 52).



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  		FIG. 5. Only G-ERA triggers apoptosis in Jurkat rtTA cells. (A) Induction of apoptosis was measured by flow cytometry analysis comparing side and forward light scattering in Jurkat rtTA (JrtTA) cells containing the gene encoding G-ERA (JrtTA-G-ERA) and treated with doxycycline (+Dox) or not treated (-Dox). The numbers represent the percentages of R2 cells that were apoptotic. (B) Typical nuclear fragmentation, detected by Hoechst staining (arrows in right panel), was observed in doxycycline-treated JrtTA-G-ERA cells. (C) Comparison of percentages of cells expressing activated caspases in JrtTA cells and JrtTA-G-ERA cells either treated with doxycycline (bold lines) or not treated (broken lines). (D) Comparison of induction of apoptosis (R2 cells, Hoechst 33342 staining, and caspase activation) in control cells (JrtTA cells) and JrtTA cells expressing various proteins. These different measurements of apoptosis were obtained simultaneously. The data are representative of three independent experiments. Boldface highlights G-ERA data. (E) Correlation between the induction of apoptosis (measurement of R2 cells) in JrtTA-G-CVS cells (squares) and JrtTA-G-ERA cells (diamonds) and the production of the viral G protein. R values are regression coefficients.

We used the same methods to study apoptosis in three other transgenic Jurkat rtTA cell lines expressing G-CVS, N-ERA, or N-CVS. The production of G-ERA led to the highest level of apoptosis (Fig. 5D). The absence of apoptosis in Jurkat rtTA cells expressing G-CVS, N-CVS, or N-ERA could not have resulted from a lower level of expression, since even at a high level of G-CVS expression (Fig. 5E) or at a high level of N-ERA or N-CVS expression (Fig. 4 and 5), apoptosis did not occur. These data strongly suggest that G-ERA is proapoptotic, whereas N-ERA and G-CVS are not.

Is the viral G protein the only major apoptosis inducer? A recombinant virus was used to determine whether other RV proteins, namely, the N, P, M, and L proteins, can play major roles in apoptosis induction. Recombinant virus R-N2c was constructed from parental virus SN-10, a nonpathogenic virus that is closely related to ERA. In R-N2c, the G protein gene of strain SN-10 is replaced by the G protein gene of strain CVS-N2c (Fig. 6A) (42). R-N2c is transcribed and replicates as efficiently as SN-10, as shown by its rate of virus production and by the amount of N protein mRNA (42).



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  		FIG. 6. The G protein is the only RV protein to induce apoptosis in Jurkat rtTA and SK-N-SH cells. (A) Schematic representation of recombinant virus R-N2c. The G protein gene of RV SN-10 (stippling) was replaced by the G protein gene of RV CVS-N2c (hatching) (as described by Morimoto et al. [42]). (B) Comparison of apoptosis in 2-day-old cultures of Jurkat rtTA and SK-N-SH cells infected with SN-10 or recombinant R-N2c. Apoptosis was measured in Jurkat rtTA cells by the following methods. (Panel 1) Determination of the percentage of R2 cells in the cell population (black bars) or the percentage of cells with fragmented nuclei stained by Hoechst 33342 (white bars; 500 cells counted) or by the TUNEL technique (stippled bars; 500 cells counted). This experiment is representative of five experiments (means and standard deviations [SD]). (Panel 2) Detection of infection (black bars) and caspase activation (white bars). Apoptosis was measured for SK-N-SH cells. (Panel 3) Determination of the percentage of cells with fragmented nuclei stained by Hoechst 33342 (white bars; 500 cells counted) or by the TUNEL technique (stippled bars; 500 cells counted). Panel 3, like panel 1, represents five experiments. (Panel 4) Measurement of infection (black bars) and caspase activation (white bars) by techniques described in the legend to Fig. 2. Results in panels 2 and 4 are the means and SD of three experiments.

Infection of Jurkat rtTA and SK-N-SH cells with nonpathogenic strain SN-10 triggered apoptosis, as determined by the percentage of cells in the R2 population, the percentage of cells with fragmented nuclei, and the percentage of cells harboring activated caspases (Fig. 6B). In contrast, R-N2c did not induce apoptosis. The absence of apoptosis in R-N2c-infected cultures was associated with an absence of caspase activation (Fig. 6B). The absence of apoptosis in R-N2c-infected cells was not associated with a lower level of infection, as 70 to 85% of Jurkat rtTA and SK-N-SH cells were infected by R-N2c and SN-10 in these experiments.

Altogether, these data indicate that only the removal of the G protein gene from the proapoptotic RV strain is sufficient to inhibit the proapoptotic property of this RV strain. Furthermore, the N, P, M, and L proteins of the nonpathogenic viral strains (i.e., ERA and SN-10) on their own cannot counteract this loss of apoptosis. Altogether, these data show that the G protein is a key element in apoptosis induction. Whether the G protein acts on its own or in combination with other viral and/or cellular proteins remains to be elucidated.

The NC and G protein distributions are driven by the origin of the G protein gene. As described above, the G protein and NC of proapoptotic RV strains have distributions in infected cells different from those of nonapoptotic RV strains. Furthermore, large amounts of NC accumulate in ERA- and CVS-infected SK-N-SH cells without the induction of apoptosis, and the pro- and antiapoptotic phenotypes are characterized by different distributions of the RV G protein in the cytoplasmic membrane. These data raised the question of whether transferring the G protein gene from a nonapoptotic RV strain to a proapoptotic RV strain would affect the RV antigen distributions in SK-N-SH cells. Thus, neuroblastoma cells were infected with either SN-10 or R-N2c, and the accumulation of NC or the G protein was monitored. Interestingly, the distributions of NC (Fig. 7A) and the G protein (data not shown) in SN-10 and R-N2c were identical to those observed following ERA and CVS infections, respectively (Fig. 1, section II, and Fig. 3). As already shown for ERA and CVS, the amount of G protein associated with the cytoplasmic membrane was much greater than the amount of G protein detected internally for proapoptotic strain SN-10 (Fig. 7B, panel 1). This was not the case for recombinant virus R-N2c. As we observed for ERA-infected cells, ribbon-like membrane-bound G protein fluorescence (Fig. 3A and B) was detected for SN-10-infected SK-N-SH cells. On the contrary, patchy membrane-bound G protein fluorescence (Fig. 3C and D) was identified for R-N2c-infected cells, as shown previously for CVS infection. These data show that the transfer of the G protein gene from a nonapoptotic RV strain genome to a proapoptotic RV strain genome is sufficient to confer a nonapoptotic phenotype, as illustrated by the distributions of NC and the G protein. It is noteworthy that the levels of NC and G protein expression in R-N2c were lower than those in SN-10 (Fig. 7B, panel 1). The same data were observed for CVS and ERA infections (Fig. 3E). Whether G-CVS is able to regulate viral or cellular gene expression, as has been shown for vesicular stomatitis virus (VSV) G protein, remains to be elucidated (39).



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  		FIG. 7. NC and G protein distributions in R-N2c-infected SK-N-SH cells are identical to those of the CVS phenotype.(A) SK-N-SH cells were infected with SN-10 or R-N2c. At 48 h later, cells were fixed in PFA,and the NC distribution was determined by staining with an FITC-conjugated anti-NC Ab. The images are representative of 80 observations (magnification, x0). NC staining was distributed in large patches in SN-10-infected cells. In R-N2c-infected cells, patches and diffuse staining were observed throughout the cytoplasm. (B) Quantitative flow cytometry. (Panel) NC antigens that accumulated in the cytoplasm were detected in PFA-treated SN-10- or R-N2c-infected SK-N-SH cells (black bars). Membrane expression of the G protein was measured with an anti-G protein Ab on live cells (mbG; gray bars), and internal G protein expression was detected on PFA-treated cells (intG; white bars). Error bars indicate standard deviations. (Panel) mbG/intG (stippled bars), mbG/NC (black bars), or intG/NC (white bars) ratio. Proapoptotic strain SN-10 was characterized by a much higher mbG/intG ratio than nonapoptotic strain R-N2c.


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DISCUSSION
 
In this report, using both inducible expression models with Jurkat rtTA cells and infecting human neuronal and lymphoblastoid cells with a recombinant virus, we showed that the viral G protein encoded by nonpathogenic RV strains is a major apoptosis inducer in human neuroblastoma and lymphoblastoid cells. In addition, a comparison of the surface expression of the G protein in pathogenic and nonpathogenic RV strains indicated that the accumulation of the nonpathogenic G protein at the cytoplasmic membrane could be important for the induction of apoptosis.

The finding that the G protein from a nonpathogenic strain of RV can induce apoptosis is fully consistent with a study by Thoulouze et al. (57) showing that Jurkat T cells undergo apoptosis as the viral G protein accumulates. It is also in agreement with the results obtained by Morimoto et al. (43) using mouse primary cortical neuronal cell cultures. This study revealed that the proapoptotic property of RV strains is correlated with the capacity of the RV G protein to accumulate in neuronal cells. Furthermore, it is in accordance with a study by Faber et al. (15) showing that mutant recombinant RVs encoding two copies of the G protein were stronger inducers of apoptosis than the wild-type virus encoding one copy. Finally, our results are in agreement with in vivo experiments with mouse brains which showed that the number of TUNEL-positive cells is correlated with the level of G protein expression (26).

The induction of the expression of the N protein provided evidence that the N proteins from the nonpathogenic and pathogenic RV strains cannot trigger apoptosis. We also showed that no viral protein other than the G protein contributes as a key effector to the proapoptotic property of nonpathogenic RV strain SN-10 because no apoptosis occurred when the G protein gene of SN-10 was replaced by the G protein gene of nonapoptotic RV strain CVS. Nevertheless, whether G-CVS is able to modulate the expression of nonidentified virus or cellular genes which cooperate with G-ERA to trigger apoptosis remains to be investigated.

Some viral glycoproteins have been proposed to be inducers of apoptosis. Most of these glycoproteins are envelope proteins that play a role in the entry and egress of virus particles into and out of host cells. The induction of apoptosis affects the ability of some of these glycoproteins to disrupt receptor signaling. In particular, reovirus sigma1 is able to induce apoptosis after binding to the cell surface (60). Human immunodeficiency virus gp120 and gp41 induce apoptosis by interacting with CD4 and CxCR4, whereas the Sindbis virus E2 protein targets an early step in the replication process that occurs shortly after entry. Similarly, the entry of vaccinia virus or herpes simplex virus type 1 can induce apoptosis (20, 48). For RV, the direct interaction of virus particles with the membrane is not sufficient to induce apoptosis, as cells treated with inactivated noninfectious virus particles do not undergo apoptosis (25, 57). This observation, together with the delay between RV infection and the onset of apoptosis, indicates that it is unlikely that the entry of the virus is the critical factor for triggering apoptosis. RV-mediated apoptosis requires the infectious cycle to be launched, suggesting that newly produced viral G protein plays a role in this process.

In this report, we showed that the quantities of the G protein expressed on the cell surface of neuronal cells differed between apoptotic and nonapoptotic viruses. The G protein of the proapoptotic strain of RV accumulated on the surface of infected neuronal cells. This is an intrinsic property of the nonpathogenic strain and is not controlled by the rest of the genome, as a recombinant RV in which the nonpathogenic G protein gene had been replaced by a pathogenic G protein gene did not overexpress the G protein on the cell surface. The production of the viral gp120-gp41 complex envelope proteins of human immunodeficiency virus triggers apoptosis after interaction with CD4 receptors in the cell membrane (33). In the absence of virus particles, the formation of syncytia by infected cells expressing CD4 also induces apoptosis, involving the p53 pathway (10). The syncytia are characterized by an accumulation of gp120 on the membrane, suggesting that the accumulation of the viral G protein on the surface of cells is a critical factor in triggering apoptosis. Apoptosis induced by the binding of gp120 to CD4 requires the presence of the cytoplasmic region of CD4, indicating the need for signal transduction events (44). It is not yet known whether the accumulation of the RV G protein on the cell surface results from an interaction with some molecules at the cytoplasmic membrane. How the accumulation of a G protein in the plasma membrane can trigger apoptosis deserves further investigation. However, it is noteworthy that the RV G protein formed large ribbon-like structures in the cytoplasmic membranes of the proapoptotic RV strain and patches in the cytoplasmic membranes of the nonapoptotic RV strain. These distributions of a viral protein resemble what can be visualized for other cellular or viral proteins that are associated with membrane microdomains or lipid rafts (22). Indeed, it was recently demonstrated that the G protein of VSV (the prototype of the rhabdoviruses) is organized into membrane microdomains (7). Modifications of highly ordered lipid rafts have been shown to be associated with apoptosis (10). In neuronal cells, lipid rafts are associated with complex biological phenomena, including signal transduction, intracellular trafficking, neuronal cell adhesion, axon guidance, and synaptic transmission (59). These processes might be of major importance for the completion of the life cycle of a neurotropic virus in its host. The question of whether RV G proteins are associated with membrane microdomains is currently being investigated.

Why does the association between the viral G protein and the cytoplasmic membrane differe in ERA-infected cells and CVS-infected cells? One possibility is that the G protein of the proapoptotic RV strain is degraded more slowly than that of the nonapoptotic RV strain. The results of pulse-chase experiments and the N protein/G protein ratio (Fig. 3F and Fig. 7B, panel 2) are in agreement with this hypothesis (43). If this hypothesis is true, larger amounts of glycosylated G proteins should migrate toward the membrane in proapoptotic RV strains.

Morphological changes were observed in SK-N-SH cells infected with proapoptotic RV strains. One of the major characteristics of VSV infection is the rounding of infected cells (6). This rounding effect is mediated by the M protein (12, 30) and is linked to the induction of apoptosis (29). It involves the disruption of all three types of cytoskeletal elements (actin, vimentin, and tubulin) (36, 40). Similarly, for RV, proapoptotic strains induced morphological cell reshaping, such as rounding. Whether the induction of RV-mediated apoptosis is responsible for rounding is still questionable, since the role of caspase 3 in VSV M protein-mediated apoptosis induction is not related to the triggering of cell rounding (24). Nevertheless, it suggests that the cytoskeleton is disturbed in cells infected with the proapoptotic RV strain. Interestingly, the overall distributions of RV NC in infected SK-N-SH cells differ between strains showing rounding and those not showing rounding. A similar result was obtained by Morimoto et al. (43) using mouse primary neurons infected with pro- and nonapoptotic RV strains. Furthermore, the transfer of the G protein gene from the nonapoptotic strain genome to the proapoptotic strain genome is sufficient to change the NC distribution whatever the origin of the N, P, L, and M protein genes. Whether the RV G protein affects cytoskeleton organization directly or through an apoptosis signaling pathway remains to be determined.

An excess of G-ERA was observed in the plasma membrane. The induction of apoptosis exposed PS molecules on the outside of the plasma membrane, where they reacted with annexin V. This finding was confirmed by the exposure of PS in cells expressing G-ERA and in cells infected with ERA or SN-10 (43; this study). A loss of membrane asymmetry resulting in PS exposure is an important feature for the phagocytosis of apoptotic cells (16, 17). After engulfing apoptotic bodies, dendritic cells can cross-prime specific protective immune responses (1, 3, 45). The cytosolic expression of cytochrome c by a recombinant RV provides better antiviral immunity (higher viral antibody titers and better vaccine protection) than nonengineered RV alone (47). Similar finding were observed when the G protein was overexpressed in recombinant viruses (15). Nonpathogenic strains of RV are used as live vaccines to immunize wildlife and are extremely efficient. Due to the capacity of the viral G protein to induce apoptosis, thus generating cells that are engulfed by appropriate antigen-presenting cells, the proapoptotic property of the viral G protein could be responsible, at least in part, for the unique ability of attenuated RV strains to induce such efficient protective immunity.


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ACKNOWLEDGMENTS
 
Christophe Préhaud and Stéphanie Lay contributed equally to this work.

This work was supported by grants from the Institut Pasteur. S.L. is supported by a Ministry of Research and Technology fellowship.

We are grateful to N. Minamoto for the generous gift of RV G and N protein MAbs and to A. D. M. E. Osterhaus for the gift of MAb 6-15-C4. We are indebted to F. Colbere Garapin (Institut Pasteur, Paris, France) for the gift of SK-N-SH human neuroblastoma cells and J. Hiscott (McGill University, Montreal, Quebec, Canada) and A. Israel (Institut Pasteur, Paris, France) for the gift of the Jurkat rtTA cell line. We acknowledge the excellent technical assistance of M. Lafage for cell culture and virus production and E. Perret and P. Roux for confocal microscopy.


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FOOTNOTES
 
* Corresponding author. Mailing address: Institut Pasteur, Unité de Neuroimmunologie Virale, 25 Rue du Dr. Roux, 75724 Paris Cedex 15, France. Phone: 33-1-45-68-87-52. Fax: 33-1-40-61-33-12. E-mail: mlafon{at}pasteur.fr. Back


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Journal of Virology, October 2003, p. 10537-10547, Vol. 77, No. 19
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.19.10537-10547.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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