1 Department of Aquaculture Kochi University, Nankoku, Kochi 783-8502, Japan
2 Department of Medical Biology, Kochi Medical School, Japan
Correspondence
Syun-ichirou Oshima
S-Oshima{at}cc.kochi-u.ac.jp
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ABSTRACT |
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INTRODUCTION |
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Studies attempting to identify birnavirus receptors have been done mainly for infectious bursal disease virus (IBDV), a member of the genus Avibirnavirus. IBDV is the contagious agent of a highly infectious disease that affects young chickens (Dobos et al., 1979). Two different serotypes (1 and 2) of IBDV have been identified. All IBDV serotype 1 strains are well-known pathogens, have a selective tropism for pre-B lymphocytes within the bursa of Fabricius and cause severe immune suppression. Serotype 2 strains can infect chickens and turkeys but do not cause clinical signs of disease (McFerran et al., 1980
). In the case of cell-adapted IBDV serotypes 1 and 2, virus binding sites for proteins with molecular masses of 40 and 46 kDa have been shown in the plasma membrane of chicken embryo fibroblast (CEF) or chicken lymphoid cells (Nieper & Müller, 1996
). The host range of some virulent strains of IBDV serotype 1 is controlled mainly by the presence of a virus receptor composed of N-glycosylated proteins of B lymphocytes (Ogawa et al., 1998
). Ogawa et al. (1998)
suggested that studies would be needed to examine if the 40 and 46 kDa proteins reported by Nieper & Müller (1996)
are receptor molecules for the virulent IBDV serotype 1 strains because CEF cell-adapted serotype 1 IBDV became adapted in CEF cells by serial passage but, conversely, became attenuated in its natural host. Thus, the nature of IBDV receptors is not understood sufficiently.
MABV has been isolated from various marine fish, such as Japanese flounder Paralichthys olivaceus, red sea bream Pagrus major and amberjack S. dumerili, as well as shellfish and sea squirt (Kusuda et al., 1989, 1993
, 1994
; Suzuki et al., 1997
, 1998
). Such a wide host range by MABV suggests either that it may use characteristic common cellular receptors among different sea animals or that it can use multiple receptors. In contrast to IBDV, however, the nature of aquabirnavirus receptors, including those for IPNV and MABV, still remains unclear.
In the case of IPNV, virus penetration by peripheral vesicular compartments was observed morphologically in fish cell lines by electron microscopy (Couve et al., 1992; Granzow et al., 1997
). This evidence supports the idea that the early stages of aquabirnavirus infection are penetration by endocytosis through specific virus receptors located on the host cell surface.
This study aimed to find the MABV adsorption/penetration mechanisms using electron microscopy and also to find the MABV binding protein using virus overlay protein blotting assays (VOPBAs) in four fish cell lines: Chinook salmon embryo (CHSE-214) (Fryer et al., 1965) and red sea bream kidney (RSBK-2) (Kusuda & Kawarasaki, 1993
) cell lines, both of which are susceptible to MABV infection (Jung, 1998
), and fathead minnow caudal peduncle (FHM) (Gravell & Malsberger, 1965
) and epithelioma papulosum cyprini (EPC) (Fijan et al., 1983
) cell lines, which are both resistant to MABV infection (Jung, 1998
).
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METHODS |
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Virus purification.
Virus was propagated in CHSE-214 cells until a massive cytopathic effect occurred. After infection for 2 or 3 days, the supernatant was collected by centrifugation at 2000 g for 20 min and the virus was concentrated with polyethylene glycol. The remaining pellet was resuspended in TNE buffer (0·1 M Tris/HCl, pH 7·4, 0·1 M NaCl, 1 mM EDTA) and the virus was extracted with chloroform. The combined viruses from the supernatant and pellet were layered over 20 % sucrose in a 1·27, 1·30 and 1·40 g cm-3 CsCl gradient. After gradient ultracentrifugation (160 000 g for 90 min at 4 °C), the virus fraction was collected, dialysed against PBS overnight at 4 °C and stored at -80 °C until use.
Reactivity of serum against purified virus and lysates of virus-infected cells.
Cells were seeded in 35 mm2 tissue culture dishes (Falcon) and incubated overnight at 20 °C. After washing briefly with PBS, cells were infected with virus at an m.o.i. of 0·01 p.f.u. per cell. Virus- and mock-infected cells were harvested after infection for 20 h and lysed in 1x sample buffer (62·5 mM Tris/HCl, 2 % SDS, 10 % glycerol, 0·005 % bromophenol blue, 5 % 2-mercaptoethanol, pH 6·8). Purified virus proteins and cell lysates were separated by SDS-PAGE on a reducing 14 % polyacrylamide gel and were transferred electrophoretically onto a nitrocellulose membrane for Western blot analysis. The membrane was blocked overnight at 4 °C in PBS containing 1 % skimmed milk and was incubated with anti-MABV Y-6 virion rabbit serum at a dilution of 1 : 100 in PBS containing 1 % skimmed milk. The membrane was then washed three times with PBS and incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG at a dilution of 1 : 2500 in PBS containing 1 % skimmed milk. Bound antibodies were detected with Konica Immunostaining HRP-1000 (Konica).
Indirect immunofluorescence.
Cells were seeded in 8-well chamber slides (Falcon) and incubated overnight at 20 °C. After washing briefly with PBS, cells were infected with virus at an m.o.i. of 50 p.f.u. per cell. After adsorption for 30 min, cells were washed three times with PBS to remove unbound virus. The cells were then fixed in cold acetone and blocked with PBS containing 5 % skimmed milk for 1 h at room temperature. Anti-MABV Y-6 virion rabbit serum at a dilution of 1 : 100 in PBS was added to the cells, which were then incubated for a further 30 min at room temperature. FITC-conjugated goat anti-rabbit IgG at a dilution of 1 : 100 in PBS was added to the cells prior to incubation for 30 min at room temperature, followed by several washings. Cells were counterstained with CYTO 64 RED Fluorescent Nucleic Acid (Funakoshi) for 10 min at room temperature and observed by fluorescence microscopy. Mock-infected cells were prepared as a control for nonspecific binding using the same procedure as described for virus-adsorbed cells.
Immunoelectron microscopy.
Immunomarking was done directly with monolayer cells using a preembedding technique. Cells were seeded in 2-well chamber slides (Falcon) and incubated overnight at 20 °C. After washing briefly with PBS, cells were infected with virus at an m.o.i. of 50 p.f.u. per cell. After adsorption for 30 min, cells were washed three times with PBS to remove unbound virus. Cells were then fixed in 2 % formaldehyde and 0·1 % glutaraldehyde and blocked with PBS containing 5 % skimmed milk for 1 h at room temperature. Anti-MABV Y-6 virion rabbit serum at a dilution of 1 : 100 in PBS was added to the cells and they were then incubated for 30 min at room temperature. Gold (10 nm)-conjugated goat anti-rabbit IgG at a dilution of 1 : 100 in PBS was added to the cells, which were then incubated for 30 min at room temperature, followed by several washings. Cells were fixed in 1·5 % glutaraldehyde for 10 min to strengthen antibody binding. After washing with PBS, the cells were processed by a conventional technique for electron microscopy (H-7100 Hitachi).
Transmission electron microscopy.
Cells were seeded in 2-well chamber slides (Falcon) overnight at 20 °C. After washing briefly with PBS, cells were infected with virus at an m.o.i. of 50 p.f.u. per cell. After adsorption for 0, 10, 20, 30, 40, 50 and 60 min, the cells were fixed in 1·5 % glutaraldehyde for 10 min. After washing with PBS, the cells were post-fixed with 1 % OsO4, dehydrated with a graded ethanol series and embedded in epoxy resin. Ultrathin sections were prepared using an ultramicrotome. Sections were placed on grids and then stained with uranyl acetate and lead citrate. Stained grids were observed by electron microscopy. The numbers of pits beneath the virions per cell after adsorption for 20 min and vesicles containing the virions per cell after adsorption for 30, 40, 50 or 60 min were counted. Each sample was counted in triplicate and each time more than 100 cells were counted for statistical analysis using one-way analysis of variance (ANOVA) followed by Tukey's test to find the statistically significant difference (P<0·01).
Cellular protein preparations.
Cells were seeded in 75 cm2 tissue culture flasks (Falcon) overnight at 20 °C. After washing with PBS, scraped cells were resuspended in lysis buffer (250 mM sucrose, 20 mM HEPES, pH 7·4, 1 mM EDTA, 5 µg aprotinin ml-1, 3 µg leupeptin ml-1, 1 % Triton-X 100). After incubation for 10 min at room temperature, the suspension was sonicated and ultracentrifuged at 120 000 g for 1 h at 4 °C. The supernatant was collected and dialysed against 0·01 M Tris/HCl (pH 7·5) overnight at 4 °C. The protein concentration of this preparation was measured using the Bradford assay with BSA as a standard.
VOPBAs.
Cellular proteins (25 µg) were separated by SDS-PAGE in a reducing 5 % polyacrylamide gel and were transferred electrophoretically onto a nitrocellulose membrane for VOPBA analysis. The membrane was incubated overnight at 4 °C in PBS containing 1 % skimmed milk to block unbound sites. The membrane was incubated with purified virus particles for 2 h at room temperature. Virus binding was detected with anti-MABV Y-6 virion rabbit serum at a dilution of 1 : 100 in PBS containing 1 % skimmed milk and HRP-conjugated goat anti-rabbit IgG at a dilution of 1 : 2500 in PBS containing 1 % skimmed milk. After each reaction, the membrane was washed twice with Tween/PBS and then finally in PBS. Colour was developed using Konica Immunostaining HRP-1000. To control for nonspecific binding, PBS containing 1 % skimmed milk was added instead with purified virus.
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RESULTS |
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DISCUSSION |
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Many viruses can gain entry to the cell through the endosomal pathway (Sieczkarski & Whittaker, 2002). IPNV penetration occurs through receptor-mediated endocytosis in CHSE-214 cells (Couve et al., 1992
; Granzow et al., 1997
). Our investigations of the early stages of MABV infection by electron microscopy showed that virus penetration by vesicular peripheral compartments was observed morphologically in CHSE-214 cells, similar to IPNV. This finding indicates that the adsorption/penetration mechanisms by aquabirnaviruses are very similar to that found for many enveloped or other groups of naked viruses. In the case of the endosomal pathway, the low pH within endosomes can be important to trigger the release of the viral genome from the endosome into the cytosol. However, the acidic pH of endosomes is not required in IPNV replication in CHSE-214 cells (Espinoza & Kuznar, 1997
). The intact virions that were observed within large vesicles in CHSE-214 cells seemed to undergo structural degradation or transformation. This morphological change may correspond to an uncoating step, followed by the release of the viral genome from the endosome into the cytosol. This finding is the first morphological evidence to suggest that the uncoating process occurs, at least in part, within endosomes. Furthermore, it is necessary to clarify whether MABV undergoes the uncoating process completely within the endosome or are factors other than an acidic pH necessary, such as the activation of a protease for structural degradation of virions, to release the viral genome from the endosome into the cytosol.
We have shown also that the virions were within large vesicles in the cytoplasm in other cell lines, such as RSBK-2 and FHM cells, by electron microscopy but, in EPC cells, the small or large vesicles containing the virions were not observed until adsorption for 60 min and these remained bound to the cell surface. This indicates that MABV can bind to the cell surface of EPC cells but fails to enter the cytoplasm. Taken together, this suggests that the replication cycle of MABV infection may be restricted after virus adsorption in EPC cells. Interestingly, virions penetrated the cytoplasm in resistant FHM cells in a manner similar to susceptible CHSE-214 and RSBK-2 cells. Therefore, in contrast to EPC cells, the replication cycle of MABV infection appears to be restricted after virus penetration in FHM cells.
In VOPBA analysis, virions bound to a protein of around 250 kDa from CHSE-214, RSBK-2, FHM and EPC cells. Because cellular proteins prepared in this study probably included both membrane and cytosolic fractions, it is unclear whether this 250 kDa protein exists in the plasma membranes of four cell lines. However, virions could bind to the cell surfaces of four cell lines, suggesting that MABV may use a common macromolecule in these susceptible and resistant cell surfaces. Therefore, this 250 kDa protein may be a major MABV receptor in the plasma membrane. In general, virus penetration through the endocytotic pathway begins with the adsorption of the virus to its receptor in the plasma membrane of the host cell. In resistant EPC cells, virus adsorption was observed by electron microscopy but no virus was observed to enter the cytoplasm. The fact has become increasingly clear that virus infection is usually efficient in at least two steps (adsorption and penetration), presumably requiring at least two different cell surface macromolecules (a primary receptor and a so-called co-receptor). The example of such early stages in infection is human immunodeficiency virus type 1 (HIV-1), which uses the cell surface CD4 antigen as a site of adsorption followed by the use of one of the chemokine receptors for virus penetration (Berger et al., 1999). Similar to HIV-1, the co-receptor is very important for efficient penetration by adenoviruses and herpesviruses (Montgomery et al., 1996
; Bergelson et al., 1997
; Geraghty et al., 1998
; Laquerre et al., 1998
). MABV infection seemed to be restricted after the virus adsorption step in only EPC cells. If this 250 kDa protein is an MABV receptor, which exists commonly in four cell lines, this protein may be necessary as a specific receptor to bind the virus but it is not sufficient to cause the virus to penetrate the cytoplasm by endocytosis. This idea suggests further that another receptor for virus penetration may exist in the plasma membranes of CHSE-214, RSBK-2 and FHM cells.
The molecular mass of the protein of CHSE-214 cells to which the virus bound was slightly larger than that for three other cell lines. This difference could be explained by the hypothesis that the small difference we observed in the size of the receptor proteins is attributed to a minor structural variation in the process of glycosylation. To prove this hypothesis, further experiments are necessary to characterize this 250 kDa protein, for example, by treatment with N-glycosylation inhibitors (Tulsiani et al., 1982; Steven et al., 1983
) and several proteases (trypsin, proteinase K, pronase or papain). The results of this study could tentatively prove the 250 kDa protein as a candidate MABV receptor in CHSE-214, RSBK-2, FHM and EPC cells.
In summary, we showed that MABV bound to the cell surfaces of not only susceptible CHSE-214 and RSBK-2 cells but also to resistant FHM and EPC cells. MABV entered the cytoplasm of all the cells, except EPC cells, which the virus did not penetrate. Furthermore, we found that MABV bound to an around 250 kDa membrane protein of CHSE-214, RSBK-2, FHM and EPC cells. To identify the 250 kDa protein as a definitive MABV receptor, we need to purify the 250 kDa protein such that antibodies can be raised to test their ability to block MABV adsorption both in vivo and in vitro.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Berger, E. A., Murphy, P. M. & Farber, J. M. (1999). Chemokine receptors as HIV-1 coreceptors: roles in viral entry, tropism, and disease. Annu Rev Immunol 17, 657700.[CrossRef][Medline]
Borrow, P. & Oldstone, M. B. (1992). Characterization of lymphocytic choriomeningitis virus-binding protein(s): a candidate cellular receptor for the virus. J Virol 66, 72707281.[Abstract]
Couve, E., Kiss, J. & Kuznar, J. (1992). Infectious pancreatic necrosis virus internalization and endocytic organelles in CHSE-214 cells. Cell Biol Int Rep 16, 899906.[Medline]
de Verdugo, U. R., Selinka, H. C., Huber, M., Kramer, B., Kellermann, J., Hofschneider, P. H. & Kandolf, R. (1995). Characterization of a 100-kilodalton binding protein for the six serotypes of coxsackie B viruses. J Virol 69, 67516757.[Abstract]
Dobos, P., Hill, B. J., Hallett, R., Kells, D. T., Becht, H. & Teninges, D. (1979). Biophysical and biochemical characterization of five animal viruses with bisegmented double-stranded RNA genomes. J Virol 32, 593605.[Medline]
Espinoza, J. C. & Kuznar, J. (1997). Infectious pancreatic necrosis virus (IPNV) does not require acid compartments for entry into cells. Arch Virol 142, 23032308.[CrossRef][Medline]
Fijan, N., Sulimanovic, D., Bearzotti, M. M., Muzinic, D., Zwillenberg, L. O., Chilmonczyk, S., Vautherot, J. F. & de Kinkelin, P. (1983). Some properties of the epithelioma papulosum cyprini (EPC) cell line from carp Cyprinus carpio. Annales de d'Institut Pasteur. Virology 137, 207220.
Fryer, J. L., Yusha, A. & Pilcher, K. S. (1965). The in vitro cultivation of tissue and cells of Pacific salmon and steelhead trout. Ann N Y Acad Sci 126, 566586.[Medline]
Geraghty, R. J., Krummenacher, C., Cohen, G. H., Eisenberg, R. J. & Spear, P. G. (1998). Entry of alphaherpesviruses mediated by poliovirus receptor-related protein 1 and poliovirus receptor. Science 280, 16181620.
Granzow, H., Weiland, F., Fitchner, D. & Enzmann, P. J. (1997). Studies of the ultrastructure and morphogenesis of fish pathogenic viruses grown in cell culture. J Fish Dis 20, 110.
Gravell, M. & Malsberger, R. G. (1965). A permanent cell line from the fathead minnow (Pimephales promelas). Ann N Y Acad Sci 126, 555565.[Medline]
Hosono, N., Suzuki, S. & Kusuda, R. (1994). Evidence for relatedness of Japanese isolates of birnaviruses from marine fish to IPNV. J Fish Dis 17, 433437.
Hosono, N., Suzuki, S. & Kusuda, R. (1996). Genogrouping of birnaviruses isolated from marine fish: a comparison of VP2/NS junction regions on genome segment A. J Fish Dis 19, 295302.
Imajoh, M. & Suzuki, S. (1999). Apoptosis induced by a marine birnavirus in established cell lines from fish. Fish Pathol 34, 7379.
Jung, S.-J. (1998). Change of infection properties of subcultured marine birnavirus in several fish cell lines. J Fish Dis 11, 8996.
Kusuda, R. & Kawarasaki, A. (1993). Establishment and characterization of a cell line derived from the kidney of red sea bream, Pagrus major. Suisanzoshoku 41, 455460.
Kusuda, R., Kado, K., Takeuchi, Y. & Kawai, K. (1989). Characteristics of two virus strains isolated from young Japanese flounder Paralichthys olivaceus. Suisanzoshoku 37, 115120.
Kusuda, R., Nishi, Y., Hosono, N. & Suzuki, S. (1993). Serological comparison of birnaviruses isolated from several species of marine fish in south west Japan. Fish Pathol 28, 9192.
Kusuda, R., Nagato, K. & Kawai, K. (1994). Characteristics of a virus isolated from red sea bream, Pagrus major showing exophthalmos. Suisanzoshoku 42, 145149.
Laquerre, S., Argnani, R., Anderson, D. B., Zucchini, S., Manservigi, R. & Glorioso, J. C. (1998). Heparan sulfate proteoglycan binding by herpes simplex virus type 1 glycoproteins B and C, which differ in their contributions to virus attachment, penetration, and cell-to-cell spread. J Virol 72, 61196130.
Ludwig, G. V., Kondig, J. P. & Smith, J. F. (1996). A putative receptor for Venezuelan equine encephalitis virus from mosquito cells. J Virol 70, 55925599.[Abstract]
McFerran, J. B., McNulty, M. S., McKillop, E. R., Connor, T. J., McCracken, R. M., Collins, D. S. & Allan, G. M. (1980). Isolation and serological studies with infectious bursal disease virus from fowl, turkeys and ducks: demonstration of a second serotype. Avian Pathol 9, 395404.
Mizukami, H., Young, N. S. & Brown, K. E. (1996). Adeno-associated virus type 2 binds to a 150-kilodalton cell membrane glycoprotein. Virology 217, 124130.[CrossRef][Medline]
Montgomery, R. I., Warner, M. S., Lum, B. J. & Spear, P. G. (1996). Herpes simplex virus-1 entry into cells mediated by a novel member of the TNF/NGF receptor family. Cell 87, 427436.[Medline]
Nakajima, K. & Sorimachi, M. (1994). Serological and biochemical characterization of two birnaviruses; VDV and YAV isolated from cultured yellowtail. Fish Pathol 29, 183186.
Nakajima, K., Maeno, Y., Arimoto, M., Inouye, K. & Sorimachi, M. (1993). Viral deformity of yellowtail fingerings. Fish Pathol 28, 125129.
Nieper, H. & Müller, H. (1996). Susceptibility of chicken lymphoid cells to infectious bursal disease virus does not correlate with the presence of specific binding sites. J Gen Virol 77, 12291237.[Abstract]
Ogawa, M., Yamaguchi, T., Setiyono, A., Ho, T., Matsuda, H., Furusawa, S., Fukushi, H. & Hirai, K. (1998). Some characteristics of a cellular receptor for virulent infectious bursal disease virus by using flow cytometry. Arch Virol 143, 23272341.[CrossRef][Medline]
Salas-Benito, J. S. & del Angel, R. M. (1997). Identification of two surface proteins from C6/C36 cells that bind dengue type 4 virus. J Virol 71, 72467252.[Abstract]
Sieczkarski, S. B. & Whittaker, G. R. (2002). Dissecting virus entry via endocytosis. J Gen Virol 83, 15351545.
Sorimachi, M. & Hara, T. (1985). Characteristics and pathogenicity of virus isolated from yellowtail fingerlings showing ascites. Fish Pathol 19, 231238.
Steven, P. G., Shaper, J. H. & Schnaar, R. L. (1983). Tunicamysin inhibits ganglioside biosynthesis in neuronal cells. Proc Natl Acad Sci U S A 80, 15511555.[Abstract]
Suzuki, S., Nakata, T., Kamakura, M., Yoshimoto, M., Furukawa, Y., Yamashita, Y. & Kusuda, R. (1997). Isolation of birnavirus from Agemaki (jack knife clam) Sinonovacura constricta and survey of the virus using PCR technique. Fish Sci 63, 563566.
Suzuki, S., Kamakura, M. & Kusuda, R. (1998). Isolation of birnavirus from Japanese pearl oyster Pinctada fucata. Fish Sci 64, 342343.
Tulsiani, D. R., Harris, T. M. & Touster, O. (1982). Swainsonine inhibits the biosynthesis of complex glycoproteins by inhibition of Golgi mannosidase II. J Biol Chem 257, 79367939.
Wolf, K. (1988). Infectious pancreatic necrosis. In Fish Viruses and Fish Viral Diseases, pp. 115157. New York: Cornell University Press.
Wolf, K., Snieszko, S. F., Dunbar, C. E. & Pyle, E. (1960). Virus nature of infectious pancreatic necrosis in trout. Proc Soc Exp Biol Med 104, 105108.
Received 30 September 2002;
accepted 13 March 2003.