1 Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan, Republic of China
2 Institute of Biomedical Sciences, Academia Sinica, No. 128, Sec. 2, Yen-Jiou-Yuan Rd, Taipei 11529, Taiwan, Republic of China
3 Department of Microbiology and Immunology, National Defense Medical Center, Taipei, Taiwan, Republic of China
Correspondence
Yi-Ling Lin
yll{at}ibms.sinica.edu.tw
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ABSTRACT |
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INTRODUCTION |
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Immediately after virus infection, robust host responses are initiated to limit virus replication, including specific immune reactions and intracellular defensive mechanisms. To cope with such powerful antiviral effects, many viruses have evolved different strategies to avoid host destruction until sufficient progeny have been produced (Roulston et al., 1999). Since apoptosis may serve as an intracellular defence mechanism to restrict virus production, especially at the early stage of infection, several viruses have evolved genes able to counteract the apoptotic cascade during infection. On the other hand, some viruses encode proteins that are able to induce apoptotic processes at the late stages of the virus life cycle, from which the viruses may conceivably benefit due to promotion of their spreading without causing noticeable immune responses. We have demonstrated that JEV infection induces severe cytopathic effects (CPEs) in different types of cultured cells and the replication of JEV appears to be essential for induction of apoptosis in those infected cells, albeit at a late time of infection (Liao et al., 1997
). The cross-talk between virus and host cell at an earlier stage of JEV infection remains largely elusive.
Oxidative stress has been implicated in the pathophysiology of many neurological diseases (Gilgun-Sherki et al., 2001; Maher & Schubert, 2000
). Excessive production of reactive oxygen species (ROS), free radical derivatives of molecular oxygen and hydrogen peroxide, may damage various intracellular macromolecules, which leads to oxidative stress often accompanied by loss of cell function, and apoptosis and/or necrosis (Nordberg & Arner, 2001
). In addition, ROS may trigger a variety of signalling pathways that involve transcriptional activation and protein phosphorylation (Kamata & Hirata, 1999
; Suzuki et al., 1997
). ROS are primarily generated by mitochondria, which possess an antioxidant system capable of neutralizing the damaging effects of ROS under normal conditions (Yu, 1994
). When excessive amounts of ROS result, nucleic acids, proteins and lipids are extensively modified by oxidation, thereby giving rise to mitochondrial dysfunction (Richter et al., 1995
). Of these, an increase in mitochondrial membrane permeabilization (MMP) may constitute a common event in cell death by both apoptosis and necrosis (Green & Reed, 1998
; Kroemer et al., 1998
; Kroemer & Reed, 2000
). Several studies have shown that viral infections can generate ROS and induce oxidative stress, such as in the case of human immunodeficiency virus (HIV) (Baruchel & Wainberg, 1992
; Israel & Gougerot-Pocidalo, 1997
), Sendai virus (Peterhans, 1979
), cytomegalovirus (CMV) (Speir et al., 1996
), influenza virus (Akaike et al., 1990
), hepatitis B virus (HBV) (Hagen et al., 1994
), dengue virus serotype 2 (Jan et al., 2000
), JEV (Raung et al., 2001
) and tobacco mosaic virus (Allan et al., 2001
). It has been shown that generation of ROS in the target cells in response to infection plays a role in virus replication and pathogenesis (Everett & McFadden, 2001a
, b
; Schwarz, 1996
).
Nuclear factor kappa B (NF-B) was one of the first transcription factors shown to be regulated by ROS and is often considered to be a primary sensor of oxidative stress in cells (Kamata & Hirata, 1999
; Sen & Packer, 1996
; Suzuki et al., 1997
). The activation of NF-
B occurs via the phosphorylation and degradation of an inhibitory protein, I
B-
, thereby releasing NF-
B from the cytoplasm and allowing its translocation into the nucleus. NF-
B binds to nuclear
B elements of target genes and activates transcription of genes mediating cell growth, differentiation, inflammation, oncogenesis, pro- and anti-apoptotic reactions, etc. (Baeuerle & Baltimore, 1996
; Barkett & Gilmore, 1999
; Hatada et al., 2000
; Karin & Lin, 2002
; Mogensen & Paludan, 2001
). In addition, NF-
B activation is also a hallmark of most infections (Barkett & Gilmore, 1999
; Mogensen & Paludan, 2001
). At the early steps of viral infections, such as for herpes simplex virus (HSV), CMV, EpsteinBarr virus (EBV), HIV (Mogensen & Paludan, 2001
) and reovirus (Barton et al., 2001
), NF-
B can be activated by the interaction between viral envelope glycoproteins and cellular receptors. This NF-
B activation can either increase virus replication because the viruses have NF-
B-binding sites in their promoter, to enhance viral pathogenicity, or to block or promote apoptosis. Some viruses may block apoptosis by NF-
B activation in infected cells (Barkett & Gilmore, 1999
); in contrast, certain viruses may induce apoptosis through NF-
B activation, such as in infection by sindbis virus (Lin, K. I. et al., 1995
), dengue virus (Marianneau et al., 1997
) and reovirus (Barton et al., 2001
).
To study the effect of JEV replication on host cells, we used ultraviolet-inactivated JEV (UV-JEV) as the control; unexpectedly, we found that the virions of replication-defective JEV could cause a unique cell death of the target cells. This killing event occurred only in actively growing cells and appeared to be cell-type-dependent, i.e. only the vigorously growing neuronal cells were vulnerable to assault by UV-JEV. Both ROS generation and NF-B activation were observed in UV-JEV-treated mouse neuroblastoma N18 cells. Blocking either ROS generation or NF-
B activation could readily suppress the cell death induced by UV-JEV. The possible mechanisms and the significance of such JEV-replication-independent cytotoxicity are discussed in the present study.
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METHODS |
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Purification and UV inactivation of JEV.
For virus purification, JEV supernatant was clarified at 8000 r.p.m. for 30 min, then centrifuged through a 20 % sucrose cushion at 27 000 r.p.m. for 3·5 h at 4 °C. The pellet was resuspended in RPMI 1640 medium and the viral titre [plaque-forming units per millilitre (p.f.u. ml-1)] was determined by a plaque assay on BHK-21 cells as described previously (Chen et al., 1996b). JEV inactivation was carried out with a Stratalinker 2400 (Stratagene) using short-wavelength UV radiation (UVC, 254 nm) at a distance of 5 cm for 30 min on ice. Virus inactivation was verified by plaque assay, indicating its infectious titre had been reduced by more than 105-fold, and immunofluorescence assay using anti-JEV antibodies (Chen et al., 1996a
), showing no positive staining (data not shown).
Lactate dehydrogenase (LDH) assay.
Cell viability was assessed by the release of the cytoplasmic enzyme LDH using a commercial kit (Cytotoxicity Detection Kit; Boehringer Mannheim) according to the manufacturer's instructions. Percentage of LDH release was calculated as [(experimental LDH-medium control)/(total cellular LDH-medium control)]x100. Percentage of cytotoxicity was determined by using the formula [(experimental LDH-medium control)/(UV-JEV LDH-medium control)]x100.
Measurement of NF-B activity by luciferase reporter assay.
N18 cells were stably transfected with the reporter plasmid pNF-B-Luc (Stratagene), which carried the luciferase gene downstream of five NF-
B-binding sites. At various experimental time points, cells were harvested, lysed and the luciferase activity [counts per second (c.p.s.)] was determined using a Luciferase Assay System kit purchased from Promega.
Measurement of intracellular ROS by 2',7'-dichlorofluorescin (DCF) fluorescence.
This was done using a fluorescent probe, 2',7'-dichlorofluorescin diacetate (DCFH-DA), purchased from Molecular Probes. Briefly, DCFH-DA diffuses through the cell membrane and is enzymically hydrolysed by intracellular esterases to the highly fluorescent compound DCF. N18 cells were loaded with 2 µM DCFH-DA for 30 min, and washed with PBS twice before the treatment. The fluorescence intensity was monitored on a spectrofluorometer (Fluoroskan Ascent; Labsystems) using 485 nm excitation and 538 nm emission.
Terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) assay.
Apoptosis-induced DNA strand breaks were end-labelled with dUTP by terminal deoxynucleotidyl transferase (TdT) using the In situ Cell Death Detection kit (Boehringer Mannheim) according to the manufacturer's instructions. The labelled cells were observed under a Leica fluorescence microscope.
Hoechst 33258 staining of cells.
Cell monolayers were fixed with 70 % ethanol at 4 °C for 1 h, then washed three times with PBS before treatment with Hoechst 33258 (Molecular Probes) for 15 min at room temperature. Cells were then washed three times with PBS and observed under a Leica fluorescence microscope.
Detection of the mitochondrial membrane potential (m).
The m was determined by using a DePsipher kit (Trevigen); this kit uses a unique cationic dye (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide) to indicate the loss of the mitochondrial potential. Healthy cells will appear red following aggregation of the DePsipher dye within the mitochondria; in cells with disrupted mitochondrial potential, the dye remains in the cytoplasm as a green fluorescent monomeric form.
Electron microscopy.
Cells were collected as pellets, washed with RPMI 1640 medium and fixed with freshly prepared 2·5 % glutaraldehyde in RPMI 1640 medium for 90 min at room temperature. After fixation, the pellets were washed three times in RPMI 1640 medium, post-fixed with 1 % osmium in distilled water for 90 min and then dehydrated in a graded series of ethanol. The dehydrated pellets were embedded in LR white acrylic resin. Sections were double-stained by floating on a fresh 50 % aqueous saturated solution of uranyl acetate, rinsed twice with distilled water and stained immediately with lead citrate. Thin-sections were examined under a JEM 1200 EX electron microscope.
Immunofluorescence and confocal laser scanning microscopy.
Cells were transfected with plasmid pEYFP-Mito (Clontech), which encodes a fusion protein of enhanced yellow fluorescent protein (EYFP) with the targeting sequence from subunit VIII of cytochrome c oxidase. Transfected cells were untreated (control) or treated with UV-JEV (m.o.i. 30) for 5 h. The cells were rinsed twice with PBS and fixed with 2 % formaldehyde for 30 min followed by permeabilization with 0·5 % Triton X-100 for 10 min. Subsequently, staining for cytochrome c was performed with a mAb against cytochrome c (Pharmingen clone 6H2.B4) and probed with a Cy3-conjugated secondary antibody (Jackson ImmunoReseach). Images were collected using a ZEISS LSM5 Pascal confocal laser scanning microscope (Carl Zeiss, Oberkchen, Germany) equipped with an argon laser attached to a Zeiss Axiovert-100 M microscope with a LD-Alhroplan 40x oil-immersion objective lens. EYFP was measured using 488 nm excitation and 505 nm emission, and Cy3 was measured using 543 nm excitation and 590 nm emission.
Western immunoblot analysis.
Cells were lysed in SDS sample buffer (62·5 mM Tris/HCl, pH 6·8, 2 % SDS, 10 % (v/v) glycerol, 50 mM DTT, 0·1 % bromophenol blue) containing a cocktail of protease inhibitors. Proteins were separated by SDS-PAGE and then transferred to a nitrocellulose membrane (Hybond-C Super; Amersham). Non-specific-antibody-binding sites were blocked with 5 % skim milk in TBS-T (25 mM Tris, 0·8 % NaCl, 2·68 mM KCl, pH 7·4, with 0·1 % Tween 20) and membranes were reacted with the primary antibody. Blots were then treated with a horseradish-peroxidase-conjugated secondary antibody (Amersham) and developed using the ECL system (Amersham).
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RESULTS |
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DISCUSSION |
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Binding of UV-inactivated viruses to target cells has been shown to activate various cellular genes. For example, UV-inactivated virions of Human cytomegalovirus (HCMV) on human lung fibroblasts (Carlquist et al., 1999) and respiratory syncytial virus on mouse alveolar macrophages (Stadnyk et al., 1997
) could stimulate the expression of interleukin-6 as efficiently as their infectious counterparts. In addition, UV-inactivated, non-replicating HSV-1 appears to trigger early and transient synthesis of alpha/beta interferon in mouse regional lymph nodes when delivered in the dermis of the ear (Riffault et al., 2000
). Also, the binding of either purified live or UV-inactivated HCMV to the cell surface can quickly upregulate the expression of the cellular transcription factors Sp1 and NF-
B (Yurochko et al., 1997
). In this study, we also found that the virions of UV-inactivated, non-replicating JEV were able to activate the NF-
B pathway in a ROS-dependent manner (Fig. 7
). Whether UV-JEV is also capable of triggering certain cellular events such as those triggered by the viruses described above remains to be determined. Our preliminary results indicated that UV-JEV was not able to induce beta interferon production in a cultured cell system, probably due to a failure of UV-JEV to activate interferon regulatory factor-3 phosphorylation (data not shown). The potential activation of other cytokines by UV-JEV remains elusive. Several flaviviruses have been shown to activate the NF-
B signalling cascade (Jan et al., 2000
; Kesson & King, 2001
; Liao et al., 2001
; Marianneau et al., 1997
), which probably plays a crucial role in the life cycle of flaviviruses. Both infectious JEV and UV-JEV activated NF-
B (Fig. 7A
); however, infectious JEV did not induce early cell death whereas UV-JEV did, suggesting that distinct downstream NF-
B-dependent genes might be activated by JEV and UV-JEV. However, which NF-
B-regulated gene(s) in the target cells is/are responsible for UV-JEV cytotoxicity remains to be determined.
UV-JEV-induced cellular death occurs quickly after the virus encounters the receptors on the cell surface, much faster than the way infectious JEV can kill its infected targets (Liao et al., 1997). On the other hand, co-infection with infectious JEV was found to attenuate UV-JEV cytotoxicity on neuronal cells (Fig. 3
). Competition between live and killed JEV for the cell-surface receptors that transmit the death signal could be a possible mechanism for this phenomenon. Alternatively, this observation suggests that replicating JEV may trigger not only cell-death but also cell-survival signals, as similar observations have been reported for other viruses (Roulston et al., 1999
; Tschopp et al., 1998
). Since non-replicating UV-JEV (Fig. 5
) could induce stronger oxidative stress in neuronal cells than infectious JEV, co-infection of JEV (Fig. 3
) may create a reducing state in infected cells to suppress UV-JEV cytotoxicity. In fact, it has been shown that influenza virus infection can robustly enhance expression of metallothioneins, which appeared to maintain the intracellular redox balance (Ghoshal et al., 2001
). It would be of interest to know whether JEV replication can suppress UV-JEV-induced CPEs by modulation of the intracellular redox pathways.
Although cell proliferation and cell death are two seemingly opposing processes, accumulating evidence now indicates that the two are closely linked under normal circumstances (Evan & Littlewood, 1998). The previous reports of impaired generation of ROS (Pani et al., 2000
) and lower activation of the stress-activated protein kinases (Lallemand et al., 1998
) in confluent cells strongly indicate a link between redox changes and cell growth. Our data (Fig. 2
), which show that only actively growing cells are vulnerable to killing by UV-JEV-triggered ROS, might also reflect a link between redox status and cell growth. The reason why only the neuronal and not other cell types were killed by UV-JEV (Fig. 2
) might reflect the fact that the oxygen metabolism rate in brain cells is much higher than that of average tissues (Maher & Schubert, 2000
).
Overall, in this study, we show that UV-JEV can kill actively growing neuronal cells by triggering a ROS-dependent, partly NF-B-mediated pathway. Although it involves mitochondrial injury, this killing is a cytochrome-c- and caspase-independent cell-death process. Taken together, our results seem to suggest that the interaction between the JEV virion and its cell-surface receptor probably triggers a killing process as a host defence mechanism to restrict virus replication, whereas replicating JEV can somewhat modify the cellular environment to overcome the cytotoxicity induced by this receptor engagement and make it suitable for timely productive replication before the target cells inevitably undergo apoptosis through various mechanisms.
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ACKNOWLEDGEMENTS |
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Received 9 July 2003;
accepted 7 October 2003.