1 Departments of Microbiology, University of Ulsan College of Medicine, Asan Medical Center, Songpa, PO Box 145, Seoul, Korea
2 Departments of Anatomy and Cell Biology, University of Ulsan College of Medicine, Asan Medical Center, Songpa, PO Box 145, Seoul, Korea
3 Departments of Pathology, University of Ulsan College of Medicine, Asan Medical Center, Songpa, PO Box 145, Seoul, Korea
Correspondence
Heuiran Lee
heuiran{at}amc.seoul.kr
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ABSTRACT |
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These authors contributed equally to this work.
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INTRODUCTION |
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We previously found that CVB infection of permissive Vero cells led to productive virus replication, different types of cell death and eventual lysis of the infected cells, facilitating the dissemination of progeny virus (Ahn et al., 2003). In addition, we observed that CVB4 induced rapid death of rat primary neuronal cells, indicating the occurrence of apoptotic alterations by 24 h post-infection (p.i.) (Joo et al., 2002
). Other studies have also suggested that neuronal cell damage resulting from viral infection can involve apoptosis (Griffin & Hardwick, 1999
; White, 1996
). Moreover, apoptotic cell death has been shown to be important in the pathogenesis of human viral diseases (Allsopp & Fazakerley, 2000
; Allsopp et al., 1998
). For example, infected neuronal cells show a number of morphological and biochemical changes indicative of apoptosis, including compaction of chromatin, oligonucleosomal DNA degradation and fragmentation into membrane-bound bodies (Schwartzman & Cidlowski, 1993
). Caspases, a family of cysteine-dependent aspartate-directed proteases, catalyse the proteolysis of substrates involved in the cell death pathway (Cryns & Yuan, 1998
; Earnshaw et al., 1999
).
The detailed nature of CVB-induced cytopathogenesis and the preferential association of certain CVB serotypes with neural disease and disease severity are still poorly understood. To gain insight into the mechanism of cytopathic effects (CPE) resulting from the infection of neuronal cells with CVB, we investigated various cellular alterations resulting from the infection of primary mouse cortical neuronal cells with each of the six CVB serotypes. In addition to determining differential virus replication and the morphological and biochemical alterations resulting from infection, we assayed the effects of the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (Z-VAD.fmk) and the macromolecule synthesis inhibitor actinomycin D (Act D) on infected cells.
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METHODS |
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CVB serotypes CVB1 (VR-687), CBV2 (VR-29), CBV3 (VR-30), CVB4 (VR-184), CBV5 (VR-1036) and CBV6 (VR-1037) were purchased from the ATCC and propagated and titrated by conventional methods in Vero cells as described previously (Minor, 1996). For one-step virus growth, the neuronal cells were infected at an m.o.i. of 1 for 1 h. The virus inocula were thoroughly washed several times and the media and cells were harvested simultaneously. Production of progeny virus was estimated by plaque assay in Vero cells.
Z-VAD.fmk (Enzyme System Products) was maintained as a stock solution of 50 mM in DMSO and added to the cells at a final concentration of 100 µM. Act D, maintained as a stock solution of 10 mg ml1 in DMSO, was added to the cells at a final concentration of 0·1 µg ml1.
Low molecular mass DNA fragmentation analysis.
Degradation of oligonucleosomal DNA from the nucleus and cytoplasm was analysed as described previously (Saeki et al., 1997) with minor modifications. Briefly, cells in a 10 cm dish were harvested and treated with 350 µl lysis buffer (10 mM Tris/HCl, pH 8·0, 0·6 % SDS, 0·1 % EDTA) for 10 min at room temperature. To each lysate, 21 µl 5 M NaCl was added and the samples were incubated at 4 °C for at least 8 h and centrifuged at 15 000 r.p.m. at 4 °C for 20 min. Each supernatant was treated with 10 µg heat-inactivated RNase A ml1 at 45 °C for 90 min followed by 200 µg proteinase K ml1 at 55 °C for 60 min. Nuclear DNA was recovered by phenol extraction and ethanol precipitation. One-third of each DNA sample was used on a 1·5 % agarose gel in TAE solution and the DNA fragmentation pattern was examined by ethidium bromide staining.
Light, fluorescence and transmission electron microscopy.
Cytopathic effects were examined by light microscopy (LM) and the pattern of nuclear condensation was examined by fluorescence microscopy (FM). Cells in 48-well plates were incubated for 30 min with the membrane-permeable fluorescent dye Hoechst 33342 (Molecular Probe; maintained as a stock solution of 10 mg ml1 in dH2O), at a final concentration of 5 µg ml1.
For transmission electron microscopy (TEM), neuronal cells were cultured on glass plates pre-coated with poly-L-lysine and fixed overnight in 4 % glutaraldehyde at 4 °C. The cells were washed three times with 0·2 M cacodylate buffer (pH 7·2), post-fixed with 2 % osmium tetroxide for 1 h at room temperature and again washed three times in cacodylate buffer. The cells were stained en bloc for 1 h at room temperature with 0·5 % uranyl acetate, dehydrated through a graded ethanol/acetone series and embedded in Mollenhauer's (1964) Epon-Araldite epoxy mixture No. 1 at 70 °C for 2 days. Ultrathin sections were prepared using a Sorvall MT5000 microtome and collected on 150-mesh copper grids. Sections were stained with 1 % uranyl acetate and/or lead citrate and photographed in a JEOL 100CX transmission electron microscope.
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RESULTS |
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DISCUSSION |
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Virus-induced apoptosis, however, may have detrimental effects on the host. For example, in susceptible cells, poliovirus (PV)-induced apoptotic effects may be responsible for virus pathogenesis, which is directly associated with viral load (Couderc et al., 2002; Girard et al., 1999
). Using cells from transgenic mice expressing the PV receptor, it has been shown that, following PV treatment, virus replication and apoptotic cell death are co-localized in both primary neuronal cells and neurons of spinal cords from paralysed mice.
Since both TEM and Hoechst staining of CVB-infected cells in the presence of Z-VAD.fmk showed substantial inhibition of DNA condensation and nuclear fragmentation, we hypothesized that caspases may be involved in CVB-induced apoptosis in primary cortical neuronal cells. When cells were treated with the caspase inhibitor Z-VAD.fmk, we observed no inhibition of virus production or cell death (data not shown). Previous studies suggested that Z-VAD.fmk treatment also did not inhibit cytopathic changes in CVB-infected Vero cells or Hela cells, although caspase activation and cleavage of substrates were substantially inhibited (Ahn et al., 2003; Carthy et al., 1998
). Using PV-infected Hela cells, it was shown that 100 µM Z-VAD.fmk affected apoptosis, but had no effect on virus growth or cellular pathological changes, suggesting that these cells undergo two types of cell death (Agol et al., 1998
, 2000
). The findings presented here suggest that interference with the caspase-dependent apoptotic phenotype using Z-VAD.fmk did not completely prohibit the cytotoxic effects of CVB on infected neuronal cells and are in good agreement with previous results.
In contrast to Z-VAD.fmk, Act D treatment dramatically prevented both cytopathic effects and virus replication. Act D has been widely utilized as an RNA synthesis inhibitor, which acts by incorporating into double-stranded DNA via deoxyguanosine residues. Act D can also be incorporated into double-stranded RNA, such as yeast rRNA (D'Arcy, 1983). In this regard, the findings presented here suggest that novel host RNA synthesis is essential for active virus production and that productive virus infection might be necessary to induce CPE.
Several epidemiological studies have shown that, in patients with neural disease, the most frequently observed CVB serotypes are CVB3, -4 and -5, with CVB1, -2 and -6 observed less frequently (Draganescu et al., 1980; Rotbart, 1995b
; Rotbart & Romero, 1995
; Xie & Xiang, 2000
). Our results, showing that all six CVB serotypes can actively grow in mouse primary neuronal cells, seem to be at odds with these clinical findings. The clinical results may simply reflect the incidence rate of each serotype in the population. It may also indicate, however, that cytopathogenesis ex vivo cannot fully explain CVB pathogenicity in vivo. Our results with CVB2, which did not show any obvious cytotoxicity, even during active virus production, may indicate that productive virus replication is not sufficient for CPE and that an unidentified viral or cellular factor is needed to trigger cell toxicity.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Agol, V. I., Belov, G. A., Bienz, K., Egger, D., Kolesnikova, M. S., Romanova, L. I., Sladkova, L. V. & Tolskaya, E. A. (2000). Competing death programs in poliovirus-infected cells: commitment switch in the middle of the infectious cycle. J Virol 74, 55345541.
Ahn, J. J. C., Seo, I., Kim, Y. K., Kim, D., Hong, H. N. & Lee, H. (2003). Characteristics of apoptotic cell death induced by coxsackievirus B in Vero cells. Intervirology 46, 245251.[CrossRef][Medline]
Allsopp, T. E. & Fazakerley, J. K. (2000). Altruistic cell suicide and the specialized case of the virus-infected nervous system. Trends Neurosci 23, 284290.[CrossRef][Medline]
Allsopp, T. E., Scallan, M. F., Williams, A. & Fazakerley, J. K. (1998). Virus infection induces neuronal apoptosis: a comparison with trophic factor withdrawal. Cell Death Differ 5, 5059.[CrossRef][Medline]
Carthy, C. M., Granville, D. J., Watson, K. A., Anderson, D. R., Wilson, J. E., Yang, D., Hunt, D. W. & McManus, B. M. (1998). Caspase activation and specific cleavage of substrates after coxsackievirus B3-induced cytopathic effect in HeLa cells. J Virol 72, 76697675.
Couderc, T., Guivel-Benhassine, F., Calaora, V., Gosselin, A. S. & Blondel, B. (2002). An ex vivo murine model to study poliovirus-induced apoptosis in nerve cells. J Gen Virol 83, 19251930.
Cryns, V. & Yuan, J. (1998). Proteases to die for. Genes Dev 12, 15511570.
D'Arcy, P. F. (1983). Reactions and interactions in handling anticancer drugs. Drug Intell Clin Pharm 17, 532538.[Medline]
Draganescu, N., Nereantiu, F. & Girjabu, E. (1980). Coxsackie B2 virus isolation from a case of postnatal meningoencephalitis. Virologie 31, 912.[Medline]
Earnshaw, W. C., Martins, L. M. & Kaufmann, S. H. (1999). Mammalian caspases: structure, activation, substrates, and functions during apoptosis. Annu Rev Biochem 68, 383424.[CrossRef][Medline]
Gadaleta, P., Vacotto, M. & Coulombie, F. (2002). Vesicular stomatitis virus induces apoptosis at early stages in the viral cycle and does not depend on virus replication. Virus Res 86, 8792.[CrossRef][Medline]
Girard, S., Couderc, T., Destombes, J., Thiesson, D., Delpeyroux, F. & Blondel, B. (1999). Poliovirus induces apoptosis in the mouse central nervous system. J Virol 73, 60666072.
Griffin, D. E. & Hardwick, J. M. (1999). Perspective: virus infections and the death of neurons. Trends Microbiol 7, 155160.[CrossRef][Medline]
Hay, S. & Kannourakis, G. (2002). A time to kill: viral manipulation of the cell death program. J Gen Virol 83, 15471564.
Johnson, R. T. (1998a). Historical background. In Viral Infections of the Nervous System, pp. 310. Edited by R. T. Johnson. Philadelphia: LippincottRaven.
Johnson, R. T. (1998b). Meningigis, encephalitis, and poliomyelitis. In Viral Infections of the Nervous System, pp. 87132. Edited by R. T. Johnson. Philadelphia: LippincottRaven.
Joo, C. H., Kim, Y. K., Lee, H., Hong, H., Yoon, S. Y. & Kim, D. (2002). Coxsackievirus B4-induced neuronal apoptosis in rat cortical cultures. Neurosci Lett 326, 175178.[CrossRef][Medline]
Koyama, A. H., Fukumori, T., Fujita, M., Irie, H. & Adachi, A. (2000). Physiological significance of apoptosis in animal virus infection. Microbes Infect 2, 11111117.[CrossRef][Medline]
Kurokawa, M., Koyama, A. H., Yasuoka, S. & Adachi, A. (1999). Influenza virus overcomes apoptosis by rapid multiplication. Int J Mol Med 3, 527530.[Medline]
Minor, P. D. (1996). Growth, assay and purification of picornaviruses. In Virology: a Practical Approach, pp. 2042. Edited by B. W. J. Mahy. Washington, DC: IRL.
Mosmann, T. (1983). Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 65, 5563.[CrossRef][Medline]
Muir, P. (1993). Enteroviruses and heart disease. Br J Biomed Sci 50, 258271.[Medline]
Nigrovic, L. E. (2001). What's new with enteroviral infections? Curr Opin Pediatr 13, 8994.[CrossRef][Medline]
Roivainen, M. (1999). Enteroviruses and myocardial infarction. Am Heart J 138, S479483.[Medline]
Roivainen, M., Rasilainen, S., Ylipaasto, P., Nissinen, R., Ustinov, J., Bouwens, L., Eizirik, D. L., Hovi, T. & Otonkoski, T. (2000). Mechanisms of coxsackievirus-induced damage to human pancreatic beta-cells. J Clin Endocrinol Metab 85, 432440.
Rotbart, H. A. (1995a). Enteroviral infections of the central nervous system. Clin Infect Dis 20, 971981.[Medline]
Rotbart, H. A. (1995b). Meningitis and encephalitis. In Human Enterovirus Infections, pp. 271290. Edited by H. Rotbart. Washington, DC: American Society for Microbiology.
Rotbart, H. A. & Romero, J. R. (1995). Laboratory diagnosis of enteroviral infections. In Human Enterovirus Infections, pp. 401408. Edited by H. Rotbart. Washington, DC: American Society for Microbiology.
Roulston, A., Marcellus, R. C. & Branton, P. E. (1999). Viruses and apoptosis. Annu Rev Microbiol 53, 577628.[CrossRef][Medline]
Rueckert, R. R. (1996). Picornaviridae: the viruses and their replication. In Fields Virology, 3rd edn, pp. 609654. Edited by B. N. Fields, D. M. Knipe & P. M. Howley. Philadelphia: LippincottRaven.
Saeki, K., Yuo, A., Kato, M., Miyazono, K., Yazaki, Y. & Takaku, F. (1997). Cell density-dependent apoptosis in HL-60 cells, which is mediated by an unknown soluble factor, is inhibited by transforming growth factor beta1 and overexpression of Bcl-2. J Biol Chem 272, 2000320010.
Schwartzman, R. A. & Cidlowski, J. A. (1993). Apoptosis: the biochemistry and molecular biology of programmed cell death. Endocr Rev 14, 133151.[Medline]
Slee, E. A., Zhu, H., Chow, S. C., MacFarlane, M., Nicholson, D. W. & Cohen, G. M. (1996). Benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethylketone (Z-VAD.FMK) inhibits apoptosis by blocking the processing of CPP32. Biochem J 315, 2124.[Medline]
White, E. (1996). Life, death, and the pursuit of apoptosis. Genes Dev 10, 115.[CrossRef][Medline]
Xie, R. & Xiang, X. (2000). Detection of viral aetiology in cerebral spinal fluid samples from 580 clinical cases. Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi 14, 373375.[Medline]
Zaoutis, T. & Klein, J. D. (1998). Enterovirus infections. Pediatr Rev 19, 183191.
Received 30 September 2003;
accepted 17 February 2004.