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Journal of Virology, May 2009, p. 4297-4307, Vol. 83, No. 9
0022-538X/09/$08.00+0     doi:10.1128/JVI.02321-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Protein X of Borna Disease Virus Inhibits Apoptosis and Promotes Viral Persistence in the Central Nervous Systems of Newborn-Infected Rats {triangledown}

Marion Poenisch,{dagger} Nils Burger,{ddagger} Peter Staeheli, Georg Bauer, and Urs Schneider*

Department of Virology, University of Freiburg, D-79104 Freiburg, Germany

Received 6 November 2008/ Accepted 31 January 2009


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ABSTRACT
 
Borna disease virus (BDV) is a neurotropic member of the order Mononegavirales with noncytolytic replication and obligatory persistence in cultured cells and animals. Here we show that the accessory protein X of BDV represents the first mitochondrion-localized protein of an RNA virus that inhibits rather than promotes apoptosis induction. Rat C6 astroglioma cells persistently infected with wild-type BDV were significantly more resistant to death receptor-dependent and -independent apoptotic stimuli than uninfected cells or cells infected with a BDV mutant expressing reduced amounts of X. Confocal microscopy demonstrated that X colocalizes with mitochondria and expression of X from plasmid DNA rendered human 293T and mouse L929 cells resistant to apoptosis induction. A recombinant virus encoding a mutant X protein unable to associate with mitochondria (BDV-XA6A7) failed to block apoptosis in C6 cells. Furthermore, Lewis rats neonatally infected with BDV-XA6A7 developed severe neurological symptoms and died around day 30 postinfection, whereas all animals infected with wild-type BDV remained healthy and became persistently infected. TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) staining revealed a significant increase in the number of apoptotic cells in the brain of BDV-XA6A7-infected animals, whereas the numbers of CD3+ T lymphocytes were comparable to those detected in animals infected with wild-type BDV. Our data thus indicate that inhibition of apoptosis by X promotes noncytolytic viral persistence and is required for the survival of cells in the central nervous system of BDV-infected animals.


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INTRODUCTION
 
Virus infection of a host triggers numerous cellular defense responses designed to inhibit efficient viral replication and to eliminate viruses from the organism. Apoptosis is a controlled and highly regulated cell death program, which is critical for the removal of virus-infected cells. A key regulatory event in most forms of apoptosis induction is the change of mitochondrial membrane permeabilization (MMP) resulting in the release of cytochrome c and other proapoptotic factors from the mitochondria into the cytoplasm and activation of the initiator caspase-9 (55). In most cases, this event represents the point of no return in the apoptotic process and thus is tightly regulated by pro- and antiapoptotic members of the Bcl-2 family of proteins (28). A change of the MMP can be induced by intrinsic factors such as oxidative stress, DNA damage, or viral infections. Similarly, extrinsic proapoptotic signals received on the cell surface through ligation of cellular death receptors such as Fas (CD95 and Apo-1) can lead to altered MMP through proteolytic activation of the proapoptotic BH3 (Bcl-2 homology domain 3)-only protein Bid by initiator caspase-8 (8).

Viruses have developed many ways to manipulate cellular defense mechanisms of infected cells for their own benefit. They selectively inhibit or induce apoptosis to increase viral propagation or evade efficient host immune responses. A broad range of viral proteins was identified that modulate the efficiency of apoptosis induction at different stages of the induction process. (4, 18, 34, 42). In particular, many DNA viruses encode proteins that modulate MMP by direct physical interaction with mitochondrial target molecules. The Bcl-2 homologues encoded by lymphotropic gammaherpesviruses (19, 20, 31), the E1B-19K protein of adenovirus (36, 49), vMIA of human cytomegalovirus (2), and the M11L protein of myxoma virus (12), among many others, are able to abrogate the activity of proapoptotic Bcl-2 members that act on mitochondria. RNA viruses from different families are similarly able to interfere with apoptosis at different stages of the process (9, 29, 30, 43). However, all mitochondrion-localized proteins of RNA viruses identified thus far stimulate rather than inhibit apoptosis. The nonstructural protein 3A of hepatitis C virus accumulates on mitochondria, rendering the cells sensitive to apoptosis (35). The matrix protein of Mokola virus, a low-pathogenicity lyssavirus, promotes apoptosis by targeting subunit 1 of the cytochrome c oxidase of the mitochondrial respiratory chain (14). The small PB1-F2 protein of influenza A virus and the viral protein R (Vpr) of human immunodeficiency virus type 1 promote apoptosis by interaction with the adenine nucleotide translocator and the voltage-dependent anion channel 1 components of the permeability transition pore (PTP) complex and significantly contribute to the pathogenic potential of these viruses (6, 25, 54).

Borna disease virus (BDV) is a nonsegmented negative-strand RNA virus (Mononegavirales) that, despite continuous gene expression, efficiently persists in cultured cells and in the central nervous system (CNS) of a broad range of vertebrates (10, 26). BDV persistence in cultured cells, including primary neuronal cultures, is not associated with any overt cytotoxicity. In vivo, the outcome of BDV infections largely depends on the age and immune status of the host (21, 22). Immunocompetent rats receiving BDV as adults almost invariably develop severe immune-mediated meningoencephalitis, resulting in massive neuronal destruction throughout the CNS. Adult athymic nude rats and newborn rats infected within the first 24 h of birth, in contrast, fail to mount an efficient cellular immune response and survive BDV infection, resulting in the establishment of life-long viral persistence and secretion of infectious virus in the urine (44). Persistence in newborn-infected rats is associated with distinct pathological changes in defined areas of the CNS. One of the affected areas is the dentate gyrus (DG) of the hippocampus, where the dentate granule cells (DGC) gradually undergo apoptosis during BDV infection (23). It was proposed that virus-induced endoplasmatic reticulum stress might play a critical role in the induction of apoptosis in DGC of newborn-infected rats (51). However, data obtained in BDV-infected organotypic hippocampal slice cultures suggests that apoptosis induction in DGC is a consequence of their failure to maintain synaptic contact to pyramidal cells of the CA3 region (33).

The BDV genome codes for five structural proteins (N, P, M, G, and L) and one nonstructural protein, a short polypeptide of 87 amino acids, termed X (11, 45). X protein expression is tightly regulated by translational and transcriptional mechanisms (38, 41) and interacts with P (52), a cofactor of the RNA-dependent RNA polymerase L. X was shown to be an important regulator of viral RNA synthesis and polymerase complex assembly (38, 39). This regulatory function of X is pivotal for BDV replication, since recombinant viruses encoding an inactivated X gene or an X protein with a nonfunctional P-binding domain are not viable (40). In the present study, we identified an additional function of X in BDV infection. We show that X associates with mitochondria and efficiently prevents apoptosis induction in infected cells receiving death receptor-dependent and -independent proapoptotic stimuli. We further show that the mitochondrial localization of X reduces pathogenesis and is required for BDV persistence in newborn-infected rats.


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MATERIALS AND METHODS
 
Cells and transfection. Vero cells (African green monkey kidney), 293T cells (human embryonic kidney), C6 astroglioma cells, and mouse L929 cells were maintained in Dulbecco modified Eagle medium supplemented with 10% fetal calf serum (FCS). Cells were kept at 37°C in a 5% CO2 humidified atmosphere. Cells were transfected in 35-mm (six-well) dishes as described previously (47).

Induction of apoptosis. Peroxynitrite (PON) in alkaline solution was obtained from Calbiochem and was stored at –80°C. Before use, PON was diluted in ice-cold phosphate-buffered saline (PBS) and used immediately. For induction of apoptosis via PON, 293T cells (104 cells) were seeded into 48-well dishes 20 h after transfection, and apoptosis was induced by treatment with 50 µM PON for 6 h. Apoptotic cells were stained with 5 µg of DAPI (4',6'-diamidino-2-phenylindole)/ml. For induction of apoptosis via {alpha}-Fas antibody (Fas/CD95/Apo-1, clone DX2 from Sigma), C6 or L929-apo cells (1.2 x 104 cells) were seeded into a 96-well dish and maintained in Eagle minimal essential medium (MEM) supplemented with 10% FCS and 10 µg of {alpha}-Fas antibody/ml. To determine apoptosis, 5 µg of DAPI/ml was added to the cell culture medium 30 min before the percentage of apoptotic cells in a total of at least 200 cells was determined.

Glucose oxidase (GOX) was obtained from Sigma Aldrich and was stored as a stock solution of 6,000 U/ml at 4°C. Working dilutions were prepared in MEM containing 5% FCS before use. GOX (2 mU/ml) was added to 3 x 103 cells in a 96-well dish, containing 100 µl of MEM-5% FCS. Equal distribution of the cells is crucial for reproducible apoptosis induction by GOX. Apoptotic cells were determined at the indicated times as described above, based on the morphological criteria of chromatin condensation, fragmentation, and membrane blebbing.

Inhibition of apoptosis by caspase inhibitors. C6 cells were treated with 10 µg of {alpha}-Fas antibody/ml in the presence of either 50 µM caspase-3 and -7 inhibitor (Z-DEVD-FMK), 50 µM caspase-8 inhibitor (Z-IETD-FMK), or 50 µM caspase-9 inhibitor (Z-LEHD-FMK), all purchased from R&D Systems. Apoptotic cells were stained with 5 µg of DAPI/ml.

Recombinant viruses. BDV was recovered from cDNA as described previously (32, 38, 46, 48). Briefly, semiconfluent 293T cells in 35-mm (six-well) dishes were transfected with 4 µg of pBRPol II-HrBDV encoding the antigenome of BDV strain He80, 0.5 µg of pCA-N, 0.1 µg of pCA-L, and 0.1 µg of pCA-at0.8. At 72 h posttransfection, the 293T cells were seeded into 94-mm plates, together with 106 Vero cells. These cells were maintained in coculture for at least 15 days. Indirect immunofluorescence analysis was used to screen for the recovery of recombinant BDV. BDV-XA6A7 was generated in an identical manner using pBRPol II-HrBDV-XA6A7. The mutations were inserted into pBRPol II-HrBDV by mutagenesis PCR using the primers XA6A7(+) and XA6A7(–) (the primer sequences are available from the authors upon request). A fragment containing the mutated sequence was excised and subcloned into unmodified pBRPol II-HrBDV using unique restriction sites XhoI and AgeI. The correctness of the transferred sequence was verified.

Preparation of virus stocks. BDV stocks were prepared from persistently infected Vero cells as described previously (5). Stocks were dialyzed for 2 days against PBS and then titrated on Vero cells.

Infection of rats. Newborn Lewis rats (Charles River) were infected by intracerebral injection of 300 focus-forming units (FFU) of wild-type BDV or BDV-XA6A7 in a final volume of 20 µl. The animals were examined daily for neurological symptoms up to 60 days postinfection.

Histology and immunohistochemical analysis. Brain sectioning and immunohistochemistry were performed as described previously (13). Viral load in the brain was assessed by immunohistological staining of 5-µm paraffin-embedded brain sections with monoclonal antibody Bo18 directed against the nucleoprotein of BDV and counterstained with Mayer's hematoxylin. Immunohistochemistry for the detection of the CD3 epsilon chain was performed using rabbit polyclonal serum ab5690 (Abcam, Cambridge, United Kingdom) according to the supplier's recommendations. The protocol included a 10-min boiling step in 10 mM citrate buffer (pH 6.0) for the heat-mediated antigen retrieval before commencing with the immunohistochemical staining protocol.

TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) staining. Paraffin-embedded dehydrated brain sections were incubated with proteinase K (20 µg/ml in 10 mM Tris-HCl [pH 7.4]) for 30 min and washed two times with PBS. For inactivation of endogenous peroxidase, the sections were treated with 0.3% H2O2 for 13 min and washed with PBS for 5 min. The brain sections were covered with the terminal desoxynucleotidyl transferase for 60 min at 37°C according to the manufacturer's protocol (In Situ Cell Death Detection Kit, POD; Roche). The sections were washed three times, incubated with converter-POD for 30 min at 37°C, and stained with DAB (3,3'-diaminobenzidine tetrahydrochloride) for 10 s. The reaction was stopped by adding H2O, and the brain sections were counterstained with Mayer's hematoxylin.

Fluorescence microscopy. Cells were fixed for 10 min in 3% paraformaldehyde and permeabilized for 5 min in PBS containing 0.5% Triton X-100. X protein of transfected cells was detected as described previously by using a polyclonal rabbit anti-X antisera (38) and a goat anti-rabbit antibody carrying a Cy2 fluorophore. The X protein in BDV-infected cells were visualized by using a polyclonal rabbit anti-X antiserum and the TSA fluorescein system (Perkin-Elmer Life Science) according to the manufacturer's protocol.

Western blot analysis. Vero cells were lysed in radioimmunoprecipitation assay buffer (50 mM Tris [pH 8.0], 150 mM NaCl, 1% NP-40, 0.5% deoxycholate), and the protein content was determined by Bradford analysis (Bio-Rad). Proteins were separated by electrophoresis through 15% sodium dodecyl sulfate-polyacrylamide gels and blotted onto polyvinylidene difluoride membranes (Millipore). Bound proteins were detected using rabbit antisera against the BDV proteins X and N. As a loading control, the blot was stained with a mouse monoclonal antibody (Sigma) against actin. The visualized proteins were quantified with a Chemidoc system from Bio-Rad, and the X and N signals were normalized to the actin signal.


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RESULTS
 
Persistent BDV infection inhibits induction of apoptosis in rat C6 cells. Rat C6 astroglioma cells are highly susceptible to infection with BDV (16) and express Fas death receptors on their surface (Fig. 1A, top panel). Monoclonal antibody DX2 (Sigma) directed against human Fas/CD95/Apo-1 ({alpha}-Fas) was previously shown to induce apoptosis upon binding to human Fas (7). We found that antibody DX2 recognized endogenous Fas on C6 cells (Fig. 1A, lower panel) and stimulated apoptosis (Fig. 1B, top panels). To determine whether the presence of BDV interferes with the induction of apoptosis, we incubated noninfected (n.i.) and persistently infected C6 cells in medium supplemented with 10 µg of {alpha}-Fas antibody/ml. In independent experiments we observed that between 68 and 96 h after the addition of DX2 a significant fraction of n.i. C6 cells displayed the typical morphological changes associated with apoptosis, whereas the morphology of BDV-infected C6 cells appeared unchanged (Fig. 1B). At this time point 5 µg of DAPI/ml was added to the medium for 30 min before apoptotic cells were counted. DAPI associates with cellular DNA, which under these conditions results in faint blue staining in healthy cells and in bright blue staining in apoptotic cells due to the condensation of DNA (Fig. 1B). To determine the percentage of apoptotic cells, we analyzed at least 200 cells per well in duplicate experiments for apoptosis-associated morphological changes and intensity of the DAPI staining. In three independent experiments, we found a significant (P < 0.03 in a paired Wilcoxon test) almost fivefold reduction of apoptosis in BDV-infected C6 cultures (Fig. 1C). Apoptosis induction in n.i. C6 cells was strongly inhibited by a caspase-9 inhibitor (Z-LEHD-FMK) and to a lesser extent by inhibitors of caspase-3 and caspase-7 (Z-DEVD-FMK) and caspase-8 (Z-IETD-FMK), indicating a possible contribution of the mitochondrial pathway to apoptosis induction in these cells (Fig. 1D). None of the caspase inhibitors affected the residual apoptosis induction seen in BDV-infected C6 cells (Fig. 1D), suggesting that BDV-mediated inhibition of apoptosis was efficient. Caspase-9 activation in {alpha}-Fas-treated C6 cells was further confirmed by immunofluorescence microscopy using a caspase-9 inhibitor coupled to a fluorescent dye (FAM-LEHD-FMK, Fig. 1E).


Figure 1
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  		FIG. 1. Persistent BDV infection inhibits mitochondrial activation of apoptosis in rat C6 cells. (A) Fas expression on rat C6 astroglioma cells. Rat C6 cells (5 x 105) were fixed and incubated in 100 µl of PBS containing 1 µg of rabbit polyclonal antiserum AAP-221 (StressGen, Victoria, British Columbia, Canada) recognizing residues 29 to 44 of mouse Fas (upper panel) or 1 µg of mouse monoclonal antibody DX2 (Sigma) directed against residues 1 to 173 of human Fas (lower panel). Bound antibodies were detected by incubation in 100 µl of PBS containing a 1 to 100 dilution of a Cy3-coupled goat anti-rabbit serum (upper panel) or a Cy3-coupled goat anti-mouse serum (lower panel). The thin lines represent the isotype controls. (B) Detection of DNA condensation by staining of C6 cells with DAPI. n.i. and persistently infected C6 cells (BDV) in a 96-well culture plate were either mock treated or incubated for 68 to 96 h in medium containing 10 µg of {alpha}-Fas antibody (human {alpha}-Fas/CD95/Apo-1 monoclonal, clone DX2 [Sigma])/ml. Accumulation of DAPI in the nucleus indicates DNA condensation and apoptosis. (C) Quantification of the experiment shown in panel B. The graph shows a representative result of three independent duplicate experiments. A significant reduction in apoptotic cells (P < 0.03 calculated by a paired Wilcoxon test) in BDV-infected cells was observed. The standard deviations are indicated. (D) Inhibition of apoptosis induction by the addition of specific caspase inhibitors. Apoptosis was induced as described above in the presence of either 50 µM caspase-3 and -7 inhibitor (Z-DEVD-FMK), 50 µM caspase-8 inhibitor (Z-IETD-FMK), or 50 µM caspase-9 inhibitor (Z-LEHD-FMK). DAPI-stained condensed nuclei from two independent duplicate experiments were counted. The standard deviations are indicated. (E) Detection of activated caspase-9 in n.i. and BDV-infected C6 cells. C6 cells in a 96-well culture plate were incubated with {alpha}-Fas antibody as described before. Cleaved caspase-9 was stained by the addition of carboxyfluorescein FLICA (named for fluorochrome inhibitor of caspases, immunochemistry) (Immunochemistry Technologies, Bloomington, MN) according to the manufacturer's protocol. Phase-contrast microscopy combined with DAPI staining demonstrates the presence of similar numbers of cells. (F) Fas expression on BDV-infected rat C6 astroglioma cells. Cells were stained with either a rabbit preimmune serum (–) or with the rabbit antiserum AAP-221 (+) directed against residues 29 to 44 of mouse Fas. Shown are the average mean fluorescence values of three independent experiments. The standard deviations are indicated by error bars. (G) Death receptor-independent induction of apoptosis by GOX. n.i. and BDV-infected C6 cells (104) in 96-well plates were incubated in medium containing 2 mU of GOX/ml in the presence or absence of 50 µM caspase-9 inhibitor for 1.5 and 2 h, respectively, before 5 µg of DAPI was added to the medium. After 30 min, the percentage of apoptotic cells in at least 200 cells was determined. The graphs represent the average number of apoptotic cells found in two independent duplicate experiments. The standard deviations are indicated.

Subsequent analysis of Fas expression on C6 cells persistently infected with various recombinant BDV revealed a slight reduction of Fas receptor on the surface of these cell lines (Fig. 1F). To analyze whether reduced Fas expression was responsible for the block in apoptosis induction in C6 cells infected with wild-type BDV, we induced apoptosis through a death receptor-independent pathway. n.i. and BDV-infected C6 cells were incubated in medium containing 2 mU of GOX/ml (Fig. 1G), which generates hydroxyl radicals by spontaneous Fenton chemistry and leads to the activation of the mitochondrial pathway of apoptosis induction. GOX-mediated oxidative stress provides a very strong proapoptotic signal that induces strong apoptosis within the first few hours after the addition of GOX and massive necrotic cell death at later time points (3, 24). To reduce the possible impact of necrosis on the outcome of our experiment, we determined the percentage of apoptotic C6 cells at 2 and 2.5 h after the addition of GOX. In two independent duplicate experiments we detected a fourfold (at 2 h) and a sevenfold (at 2.5 h) reduction in the number of apoptotic cells in BDV-infected versus n.i. C6 cultures (Fig. 1G). The addition of a specific caspase-9 inhibitor reduced apoptosis in n.i. C6 cells (Fig. 1G), whereas a caspase-8 inhibitor failed to protect C6 cells from apoptosis (data not shown). Taken together, these results indicated that persistent BDV infection actively blocks apoptosis in C6 cells through inhibition of the mitochondrial induction pathway.

Accessory viral protein X associates with mitochondria and blocks apoptosis. Since many viral and cellular proteins regulate apoptosis by direct association with target molecules on mitochondria we tested whether one of the abundantly expressed BDV proteins N (nucleoprotein), P (phosphoprotein), or X colocalizes with a mitochondrial marker. Vero cells were cotransfected with BDV expression vector pCA-N, pCA-P, or pCA-X (47) and with plasmid pDsRed2-mito (Clontech) encoding a humanized red fluorescence protein targeted to mitochondria. Confocal microscopy demonstrated almost complete localization of X with the mitochondrial marker protein (Fig. 2A, top panels), whereas N and P showed no mitochondrial localization (data not shown). In BDV-infected cells, X associates with the viral polymerase complex through binding to P (52) and is predominantly found in the nucleus (Fig. 2A, lower panels). To test whether in infected cells a fraction of X interacts with mitochondria, we transfected pDsRed2-mito into persistently infected Vero cells. To enhance the X signal in the cytosol of BDV-infected cells, we used the TSA fluorescein amplification system. Our analysis showed that the cytosolic fraction of X almost completely localized to mitochondria (Fig. 2A, lower panels). We further analyzed the colocalization of X with mitochondria in C6 and human oligodendroglioma (oligo) cells, another cell line that is highly susceptible to BDV infection. X staining in the cytoplasm of C6 cells was too diffuse to convincingly assess colocalization with the mitochondrial marker (Fig. 2B, top panels). In oligonucleotide cells, we detected abundant cytoplasmic colocalization of X with mitochondria (Fig. 2B, bottom panels), indicating that X colocalization with mitochondria is not restricted to Vero cells.


Figure 2
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  		FIG. 2. BDV-X colocalizes with mitochondria and possesses antiapoptotic activity. (A) Confocal microscopy demonstrates colocalization of BDV-X with a mitochondrial marker. Approximately 105 n.i. Vero cells (top panel) on coverslips were transfected with 1 µg of the BDV-X expression plasmid pCA-X (47) and 0.1 µg of pDsRed2-mito encoding a humanized red fluorescence protein fused to the MTS from subunit VIII of human cytochrome c oxidase (Clontech). Vero cells persistently infected with BDV (lower panel) received 0.1 µg of pDsRed2-mito only. At 20 h after transfection, the cells were fixed, and localization of X in pCA-X-transfected cells was visualized by using a polyclonal rabbit {alpha}-X serum (38) and a goat {alpha}-rabbit antibody carrying a Cy2 fluorophore (green). In BDV-infected cells X was detected by using the rabbit {alpha}-X serum and the TSA fluorescein system (green) to enhance the signal. The merge indicates colocalization of X with the mitochondrial marker (yellow). (B) Colocalization of BDV-X with mitochondrial marker DsRed2-mito in rat C6 astroglioma and human oligo cells transfected with expression vector pCA-X. The sites of potential colocalization in C6 cells (empty arrowheads) and highly defined cytoplasmic structures indicative of colocalization in oligonucleotide cells (solid arrowheads) are marked. Transfection of cells and immunofluorescence analysis was performed as described in the legend to Fig. 2A. (C) Antiapoptotic activity of BDV-X in transfected 293T cells. 293T cells in a six-well plate were transfected with 1 µg of an empty vector (pCA-{phi}), a BDV-P expressing plasmid (pCA-P), or pCA-X. At 20 h after transfection, the cells were seeded into a 48-well plate, and apoptosis was induced by treatment with 50 µM PON for 6 h or cells were left untreated as a control. Cells were stained with DAPI, and the percentage of apoptotic cells was determined. The graph represents the average of two independent duplicate experiments. The standard deviations are indicated. (D) Antiapoptotic activity of BDV-X in transfected L929-apo cells stably expressing human Fas. L929-apo cells were cotransfected with 0.1 µg of a GFP-expressing vector (pCA-eGFP) and 1 µg of either pCA-zxzxswslzxzxO, pCA-P, or pCA-X. The transfected cells were either treated for 68 to 96 h with 10 µg of {alpha}-Fas/ml or were left untreated. GFP-positive cells were analyzed for apoptosis induction in two independent duplicate experiments. The standard deviations are indicated.

We next tested whether X alone is capable of conferring resistance to death receptor-dependent and -independent proapoptotic stimuli. We transfected human embryonic kidney 293T cells with either an empty vector (pCA-{phi}) or with pCA-P and pCA-X, respectively (Fig. 2C). At 20 h after transfection the 293T cells received medium containing 50 µM PON, which stimulates apoptosis in a manner similar to stimulation by GOX but appeared less toxic for 293T cells than GOX in our hands. Apoptosis induction was monitored continuously and quantified 6 h after the addition of PON by analysis of morphological changes and intensity of DAPI staining in at least 200 cells in duplicate experiments. Evaluation of two independent experiments showed a roughly fourfold reduction in the number of apoptotic cells in 293T cells receiving pCA-X compared to cells receiving pCA-{phi} or pCA-P (Fig. 2C). We further analyzed the effect of transfected X on death receptor-dependent apoptosis induction in mouse L929 cells stably expressing human Fas (Fig. 2D). Cells were transfected with a 1:10 mixture of pCA-eGFP and either pCA-{phi}, pCA-P, or pCA-X. Apoptosis was induced by the addition of 10 µg {alpha}-Fas/ml 20 h after transfection and was evaluated only in green fluorescent protein (GFP)-positive cells to account for the low transfection efficacy (~10%) observed in L929 cells. The presence of BDV-X reduced apoptosis induction almost to background level in two independent experiments (Fig. 2D).

We next evaluated the antiapoptotic function of X in BDV-infected cells. Since we previously showed that a recombinant BDV lacking a functional X gene is not viable (40), we analyzed apoptosis inhibition mediated by a recombinant virus encoding an inactive X gene in the normal position but expressing reduced amounts of X from an X gene inserted into an additional transcription unit located near the 5' end of the BDV genome (BDV-LRD-{Delta}X-X5') (40). As a consequence of the attenuating effect of these mutations, we only succeeded to recover this virus by inserting two replication-enhancing mutations into the viral polymerase protein (L) (BDV-LRD) (1). Western blot analysis demonstrated that the X protein level in BDV-LRD-{Delta}X-X5'-infected C6 cells was ~3-fold lower than in BDV-LRD-infected cells, whereas N protein levels were normal (Fig. 3A). BDV-LRD-infected and BDV-LRD-{Delta}X-X5'-infected C6 cells expressed similar levels of Fas on their surfaces (Fig. 1F). However, BDV-LRD-infected C6 cells were highly resistant to apoptotic stimuli (Fig. 3B and 3C), whereas BDV-LRD-{Delta}X-X5' lost the ability to efficiently block Fas-mediated apoptosis (Fig. 3B) and displayed an intermediate sensitivity to apoptosis induced by GOX (Fig. 3C).


Figure 3
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  		FIG. 3. Reduced X expression levels enhance sensitivity of BDV-infected C6 cells to apoptotic stimuli. (A) Western blot analysis of 5 µg of total protein derived from C6 cells that were either n.i. or persistently infected with either BDV-LRD or BDV-LRD-{Delta}X-X5'. Viral proteins were detected using rabbit antisera directed against BDV-N and BDV-X, respectively. Staining with antiserum specific for actin served as loading control. Actin, N, and X signals were quantified on a Chemidoc system from Bio-Rad, and the N and X signals were normalized to the actin signal. (B) Apoptosis induction in n.i. and C6 cells persistently infected with the indicated viruses by the addition of 10 µg of {alpha}-Fas/ml to the medium as previously described. The graph shows the average number of apoptotic cells found in three independent duplicate experiments. The standard deviations are indicated. (C) GOX-mediated apoptosis induction in C6 cells persistently infected with BDV-LRD or BDV-LRD-{Delta}X-X5'. The experiment was performed exactly as described in the legend to Fig. 2C. The graph shows the average number of apoptotic cells found in two independent duplicate experiments. The standard deviations are indicated.

Mitochondrial localization of X is essential for blocking apoptosis. We next determined whether the mitochondrial localization of X is essential for the antiapoptotic activity. The X protein shares no detectable homology with any other cellular or viral protein and does not contain a typical mitochondrion-targeting signal (MTS). The only secondary structure that was consistently predicted by various algorithms was a rather short {alpha}-helical domain stretching from amino acid residue 6 to residue 16 (Fig. 4A). The predicted {alpha}-helix is part of an unusual importin-{alpha} binding motif (residues 6 to 19) (53) and harbors the domain responsible for binding to BDV-P (residues 8 to 15) (52). We determined whether di-alanine mutations inserted into the predicted {alpha}-helical domain (XA6A7, XA8A9, and XA14A15 [Fig. 4A]) or slightly downstream (XA18A19 [Fig. 4A]) interfere with mitochondrial localization of X. Confocal microscopy on transfected Vero cells showed that XA6A7 and XA14A15 were no longer localized to mitochondria (Fig. 4B), whereas mutant XA8A9 and XA18A19 associated with mitochondria such as unmodified X (Fig. 4B and Fig. 2A), indicating that the positive charges of the arginine residues (Fig. 4A) are critical for the mitochondrial localization of X. Fusion of GFP to either the N or the C terminus of full-length X interfered with the mitochondrial localization of the protein (data not shown), a finding that prevented a more precise mapping of the mitochondrion-targeting motif of X by standard experimental approaches.


Figure 4
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  		FIG. 4. Mitochondrial targeting of X is essential for inhibiting apoptosis. (A) Mutational analysis of the N-terminal 19 amino acids of X harboring a predicted {alpha} helix (framed). Residues mutated to alanine in four di-alanine X mutants are indicated in blue, and the residues responsible for binding to P are underlined. (B) Analysis of the subcellular localization of the di-alanine X mutants in Vero cells transfected with the corresponding pCA expression constructs. Transfection and immunofluorescence analysis were performed as described in Fig. 2. (C) Western blot analysis of 5 µg of total protein derived from Vero cells that were either n.i. or persistently infected with either wild-type BDV or BDV-XA6A7. Viral proteins were detected using rabbit antisera directed against BDV-N and BDV-X, respectively. Staining with antiserum specific for actin served as loading control. Actin, N, and X signals were quantified on a Chemidoc system from Bio-Rad, and the N and X signals were normalized to the actin signal. (D) Apoptosis induction in n.i. and C6 cells persistently infected with the indicated viruses by addition of 10 µg/ml of {alpha}-Fas to the medium as previously described. The graph shows the average number of apoptotic cells found in three independent duplicate experiments. Standard deviations are indicated.

Residues 6 and 7 of the X protein are outside the P binding domain (52) essential for BDV multiplication (40). A virus encoding a mutant XA6A7 protein instead of wild-type X (BDV-XA6A7) was successfully recovered from copy DNA and used to infect C6 cells. Fas levels on BDV-XA6A7-infected C6 cells were comparable to those found on n.i. cells (Fig. 1F) and Western blot analysis demonstrated that wild-type BDV and BDV-XA6A7 expressed almost identical amounts of X (Fig. 4C). C6 cells persistently infected with BDV-XA6A7 were highly sensitive to Fas-mediated apoptosis (Fig. 4D), showing that mitochondrial localization of X was essential for blocking apoptosis.

Antiapoptotic activity of X reduces viral pathogenesis in newborn-infected Lewis rats. Lewis rats infected with BDV as neonates usually do not develop neurological symptoms and survive infection due to the immaturity of the immune system at their first encounter with the virus (23). To test whether the nonpathogenic BDV infection of newborn rats requires efficient suppression of apoptosis in brain cells, we inoculated neonates intracerebrally with 300 FFU of either wild-type BDV or BDV-XA6A7 within the first 24 h of birth. As expected, all animals receiving wild-type BDV (Fig. 5A, n = 10) remained healthy for the entire observation period of 60 days. In contrast, all animals inoculated with BDV-XA6A7 (Fig. 5A, n = 5) started to show first neurological symptoms at around day 26 postinfection. The severity of the symptoms rapidly increased thereafter until the animals developed hind leg ataxia, paralysis, and epileptic seizures and became moribund on day 28 or 29. At the same time point we sacrificed control animals that received either no virus (n = 1) or wild-type BDV (n = 3). Immunohistochemistry using monoclonal antibody Bo18 directed against BDV-N (13) and hematoxylin-eosin staining showed that BDV-infected and BDV-XA6A7-infected animals were similarly well infected and identified an comparable amount of immune cell infiltration into the brain (Fig. 5B).


Figure 5
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  		FIG. 5. Ablation of mitochondrial targeting of X promotes lethality in newborn-infected rats. (A) Survival of newborn Lewis rats inoculated intracerebrally with 300 FFU of either wild-type BDV or BDV-XA6A7. The animals were checked daily for the onset of clinical symptoms for up to 60 days postinfection. Severely diseased animals were killed, and the brains were recovered and paraffin embedded for subsequent analysis. (B) Infection status of BDV-infected and BDV-XA6A7-infected animals at the time of sacrifice. The brains of n.i. and infected animals were analyzed by immunohistochemistry using monoclonal antibody Bo-18 directed against the BDV-N protein. Shown are low-magnification pictures of the entire brain section and high magnifications of selected areas. Dark brown staining is indicative of the presence of viral antigen. Hematoxylin-eosin staining (H&E) was performed to analyze immune cell infiltration into the brains of infected animals.

To analyze whether preferential infiltration of T-lymphocytes into the brains of BDV-XA6A7-infected animals contributed to the unexpected occurrence of disease, we probed sagittal brain sections with a polyclonal rabbit antiserum directed against the CD3-epsilon chain (Abcam). Brain sections derived from the n.i. control animal did not contain detectable CD3+ cells (Fig. 6A). In contrast, CD3+ cells were detected in all brain regions of BDV-infected and BDV-XA6A7-infected animals, including the cerebellum and brain stem, but were predominantly located in the cortex and in the hippocampus (Fig. 6A). Quantification of the CD3+ cells in the hippocampus on at least three consecutive brain sections from each animal revealed some variation between individual animals, but no obvious difference in the number of CD3+ cells in BDV-infected and BDV-XA6A7-infected animals sacrificed 28 or 29 days after infection (Fig. 6B). In addition, we analyzed brain sections of animals infected with wild-type BDV that were maintained for 60 days and sacrificed without overt signs of disease (Fig. 5A). Remarkably, the numbers of CD3+ cells detected in the hippocampus of these animals were comparable to those found in animals sacrificed around day 30 (Fig. 6B). These findings suggest that the presence of CD3+ cells was not responsible for the mortality of BDV-XA6A7-infected animals.


Figure 6
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  		FIG. 6. Infiltration of CD3+ T lymphocytes into the brain of BDV-infected and BDV-XA6A7-infected animals. (A) Exemplary pictures of the hippocampus and the cortex of the indicated animals (numbers correspond to those in Fig. 6B). (B) Quantification of CD3+ cell infiltration into the brain of neonatally infected rats. Sagittal brain sections were probed with a rabbit polyclonal antiserum (ab5690; Abcam) recognizing the CD3 epsilon chain. The animals were sacrificed when moribund (BDV-XA6A7) or 30 and 60 days postinfection (BDV), respectively. CD3+ cell infiltration was quantified by counting CD3+ cells in the hippocampus (HC) on at least three consecutive brain sections from each animal.

TUNEL staining on paraffin-embedded brain sections showed that all animals infected with BDV-XA6A7 had a strongly increased number of apoptotic cells in all brain regions as shown, for example, in Fig. 7A for the DG, the CA1 region of the hippocampus, and a region of the cortex. Quantification of apoptotic cells in the hippocampus revealed a highly significant, >10-fold-increased number (P < 0.003, calculated by using the Student t test on the basis of logarithmic values) of apoptotic cells in BDV-XA6A7-infected animals (Fig. 7B), suggesting that enhanced apoptosis was primarily responsible for the unexpected deaths of these animals.


Figure 7
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  		FIG. 7. Ablation of mitochondrial targeting of X stimulates apoptosis in the brain of newborn-infected rats. (A) Detection of apoptotic cells in various brain regions. Apoptotic cells were detected by TUNEL staining using the in situ cell death detection system from Roche. Representative pictures of the DG, the CA1 region of the hippocampus and a region of the cortex are shown. (B) Quantification of TUNEL-positive cells in the hippocampus (HC) of n.i. (n = 1), BDV-infected (n = 3), and BDV-XA6A7-infected (n = 4) animals, respectively. The number of TUNEL-positive cells was determined on at least three consecutive brain sections for each animal. The statistical significance was determined by using the Student t test on the basis of logarithmic values.


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DISCUSSION
 
RNA viruses need to continuously produce significant amounts of viral RNA in order to maintain infection. Viral genomic RNA and doubled-stranded RNA intermediates generated during viral RNA synthesis usually provide important danger signals that efficiently trigger cellular innate immunity and apoptosis (17, 27, 50). It is therefore highly remarkable that BDV replication does not cause any overt cytotoxicity and that the virus readily establishes persistent infections. The lack of detectable cell loss during infection suggested that BDV is able to interfere with the induction of apoptosis, but the mechanisms by which the virus is able to do so had, to date, remained unknown. We demonstrate here that the accessory protein X of BDV is targeted to mitochondria and confers resistance to caspase-dependent apoptosis through inhibition of the mitochondrial activation pathway. We further show that the antiapoptotic activity of X reduces pathogenesis in persistently infected animals. To the best of our knowledge, the present study provides the first description of a mitochondrion-localized protein of an RNA virus that significantly inhibits apoptosis in cultured cells and infected animals.

Several viral proteins that block apoptosis contain Bcl-2-like MTSs that mediate their insertion into the outer mitochondrial membrane. These targeting signals are usually located close to the C terminus of the proteins and are composed of positively charged anchor residues upstream and downstream of putative membrane spanning domains consisting of 18 to 24 residues (12). The X protein clearly lacks a Bcl-2-like MTS, indicating that X most likely does not interfere with the activity of proapoptotic members of the Bcl-2 family of proteins. The only secondary structure in the X protein that was consistently predicted by various algorithms is a short amphipathic helix located close to the N terminus (Fig. 4A) that is followed by a stretch of positively charged residues farther downstream. This structure showed some similarity to mitochondrial targeting and sorting signals that mediate import of cellular proteins into the inner membrane or the intermembrane space (37). Furthermore, the length and the amino acid composition of the predicted helical domain strongly resembles those of the amphipathic helices in the C-terminal part of PB1-F2 of influenza A virus and Vpr of human immunodeficiency virus type 1 that are responsible for the mitochondrial targeting of these proteins (15, 25). In support of the mitochondrial targeting function of the helical domain, we found that the mutation of the positively charged arginine residues in position 6 (XA6A7) and positions 14 and 15 (XA14A15) resulted in the complete loss of mitochondrial localization of X, while mutation of the uncharged residues 8 and 9 (XA8A9) and of residues 18 and 19 located outside the helical domain (XA18A19) did not affect X localization (Fig. 4B). These findings demonstrate that the positively charged residues were essential for the mitochondrial targeting of X. In addition, mutant proteins XA6A7, XA8A9, and XA14A15 showed enhanced nuclear localization compared to wild-type X and XA18A19 (Fig. 2A, top panels, and Fig. 4B), which is in accordance with the previously reported function of the helical domain in the nuclear import of X (53). The fact that the cytoplasmic fraction of XA8A9 remained nevertheless strongly associated with mitochondria, whereas XA6A7 and XA14A15 were no longer targeted to mitochondria (Fig. 4B), suggest that nuclear import and mitochondrial targeting of X are two independent functions of the helical domain with different structural requirements. A third function of the small helical domain of X is the direct interaction with BDV-P (52), which is critical for the regulation of viral RNA synthesis (39) and essential for BDV multiplication (40). It is unclear at present how the small helical domain is able to sustain three elementary functions for the virus at the time. Our observation that a slight (threefold) reduction of X expression resulted in the complete loss of protection against apoptosis induction (Fig. 3B) indicates that the tight translational and transcriptional regulation of X expression (38, 41) is critical for the maintenance of the various X functions and might be a prerequisite for viral replication and the establishment of BDV persistence.

Experiments with specific caspase inhibitors (Fig. 1D) and the interference of persistent BDV infection with apoptosis induction by reactive oxygen species (Fig. 1G and 3C) indicated that BDV efficiently prevents mitochondrial activation of apoptosis. Reactive oxygen species are known to damage the integrity of the mitochondrial membrane by increasing the permeability of the PTP (3). The overall similarity of X, PB1-F2, and Vpr and, in particular, of the helical domains responsible for the mitochondrial localization of these proteins implies that X might similarly target components of the PTP complex, thereby inhibiting rather than promoting a change in membrane permeability. The identification of the cellular target molecule(s) that mediate colocalization of X with mitochondria and a detailed analysis of X-mediated inhibition of apoptosis could therefore provide new insights into viral and cellular mechanisms to manipulate mitochondrial activation of apoptosis.

C6 cells persistently infected with BDV-XA6A7 and BDV-LRD-{Delta}X-X5' did not undergo apoptosis to a significant degree unless we provided a proapoptotic stimulus (Fig. 3B and 4D). These observations indicated that BDV infection of cultured cells provides only a weak stimulus for apoptosis and raised the possibility that apoptosis induction is only of minor importance for BDV-associated pathogenesis. To test the relevance of virus-induced apoptosis in vivo, we inoculated newborn Lewis rats intracerebrally with either BDV or BDV-XA6A7 and analyzed viral spread and pathogenesis. As expected, the neonates all survived the infection with wild-type BDV without overt neurological symptoms and became persistently infected (Fig. 5). Remarkably, all animals infected with BDV-XA6A7 developed severe symptoms and died within 29 days postinfection (Fig. 5A). One of the few known pathological consequences of persistent BDV infection in newborn-infected rats is the degeneration of DGC in the hippocampus (22). Analysis of the brains of BDV-XA6A7-infected animals showed a strong increase of apoptosis in the DG but, interestingly, also in the CA1 region of the hippocampus and in the cortex, two regions that are usually not affected by detectable cell death in newborn rats infected with wild-type BDV (Fig. 7A). The localization of the TUNEL-positive cells in the DG and the CA1 further suggests that the majority of cells undergoing apoptosis in these areas are neurons. However, additional experimentation will be required to determine whether other cell types such as microglia similarly undergo apoptosis and whether apoptosis induction in these animals is a direct consequence of BDV-infection or can be induced by bystander effects as well.

It is unclear at present what immediately caused the death of the BDV-XA6A7-infected animals. Phenotypically, the neurological symptoms resembled those induced by BDV infection in adult rats, suggesting that activation of the immune system contributed to the onset of disease. However, we did not find a significant difference in the number of CD3+ T lymphocytes in the brains of BDV-infected and BDV-XA6A7-infected animals (Fig. 6). Although we favor the hypothesis that the disease resulted from direct virus-triggered loss of neurons, at present we cannot exclude indirect immune cell-triggered pathogenesis. The possibility remains that differences in the composition of the infiltrating T-cell population or their differential activation contribute to the strong pathogenesis associated with BDV-XA6A7 infection.

In any case, our data suggest that inhibition of apoptosis is important for the prevention of pathogenesis and the establishment of persistence in newborn-infected rats. This may be of particular relevance for the maintenance and spread of BDV during natural infections. It was shown that newborn-infected rats start to secrete infectious virus in the urine only approximately 3 months after infection, and it has been proposed that intranasal infection with virus from the urine of persistently infected rodent vectors is the major mode of BDV transmission in the wild (44). These considerations suggest that the antiapoptotic activity of X is required to ensure the survival of persistently infected animals for a sufficiently long period of time to permit successful transmission of the virus.


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ACKNOWLEDGMENTS
 
We thank Rosita Frank for excellent technical support and Peter Aichele for help with the fluorescence-activated cell sorting analysis. We also thank Andreas Ackermann, Nadja Hoefs, Daniel Mayer, and Sandra Wille for critically reading the manuscript.

This study was supported by the Deutsche Forschungsgemeinschaft (grant SCHN 765/1-5).


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Virology, University of Freiburg, Hermann-Herder-Strasse 11, D-79104 Freiburg, Germany. Phone: 49-761-203-6222. Fax: 49-761-203-5053. E-mail: urs.schneider{at}uniklinik-freiburg.de Back

{triangledown} Published ahead of print on 11 February 2009. Back

{dagger} Present address: Department of Molecular Virology, University of Heidelberg, D-69120 Heidelberg, Germany. Back

{ddagger} Present address: Clinical Cancer Research Center, University Hospital Basel, CH-4031 Basel, Switzerland. Back


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Journal of Virology, May 2009, p. 4297-4307, Vol. 83, No. 9
0022-538X/09/$08.00+0     doi:10.1128/JVI.02321-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Poenisch, M., Wille, S., Schneider, U., Staeheli, P. (2009). Second-site mutations in Borna disease virus overexpressing viral accessory protein X. J. Gen. Virol. 90: 1932-1936 [Abstract] [Full Text]  

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