Early interactions of marine birnavirus infection in several fish cell lines

Masayuki Imajoh1, Ken-ichi Yagyu2 and Syun-ichirou Oshima1

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


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Marine birnavirus (MABV), a member of the genus Aquabirnavirus, family Birnaviridae, is an unenveloped icosahedral virus with two genomes of double-stranded RNA. The mechanisms of MABV adsorption and penetration are still undetermined. This work examined MABV infection in susceptible and resistant fish cell lines. MABV adsorbed not only onto the cell surfaces of susceptible (CHSE-214 and RSBK-2) cells but also onto resistant (FHM and EPC) cells. Furthermore, the virus entered the cytoplasm through the endocytotic pathway in CHSE-214, RSBK-2 and FHM cells but did not penetrate EPC cells. Thus, restriction of the MABV replication cycle is different between resistant FHM and EPC cells. The virus was found to bind to an around 250 kDa protein on CHSE-214, RSBK-2, FHM and EPC cells. Thus, this 250 kDa protein may be a major MABV receptor that exists in the plasma membranes of all four cell lines examined. This result suggests further that another receptor for virus penetration may exist in CHSE-214, RSBK-2 and FHM cells but not in EPC cells.


   INTRODUCTION
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Marine birnavirus (MABV) is a member of the genus Aquabirnavirus of the family Birnaviridae. MABV is an unenveloped icosahedral virus about 60 nm in diameter with two genomes of double-stranded RNA (Dobos et al., 1979). MABV is the causative agent of ascites and deformity in fish and causes serious losses to the fish-farming industry in Japan in yellowtail fry and fingerling Seriola quinqueradiata (Sorimachi & Hara, 1985; Nakajima et al., 1993; Nakajima & Sorimachi, 1994). Infectious pancreatic necrosis virus (IPNV) is the type species of the genus Aquabirnavirus and was first reported as a pathogenic virus of fish (Wolf et al., 1960). IPNV is an important pathogen associated with an acute and contagious disease in salmonids (Wolf, 1988). MABV can be distinguished from IPNV by serotyping (Kusuda et al., 1993; Hosono et al., 1994) and genogrouping of the amino acid sequences of the junction region for the VP2 and NS polypeptides encoded by the large segment A genome (Hosono et al., 1996).

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).


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Cells and virus.
Four cell lines, CHSE-214, RSBK-2, FHM and EPC, were cultured at 20 °C in Dulbecco's modified Eagle's medium nutrient mixture F-12 HAM (Sigma) supplemented with 10 % FBS (Hyclone) and 100 µg kanamycin ml-1. MABV strain Y-6 from yellowtail S. quinqueradiata with ascites (Kusuda et al., 1993) was used in this study.

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.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Reactivity of anti-MABV Y-6 virion rabbit serum
To verify the reactivity of anti-MABV Y-6 virion rabbit serum, Western blot analysis was done using purified virus proteins and lysates of virus-infected cells. The major bands detected in the purified virus preparation were the two bands of the VP2 and VP3 proteins, the major structural proteins of the virus (Fig. 1, lane 1). Anti-MABV Y-6 virion rabbit serum reacted with these two proteins (Fig. 1, lane 2). Anti-MABV Y-6 virion rabbit serum reacted mainly with the precursor of VP2 (pVP2) and the mature VP2 and VP3 capsid proteins in lysates of virus-infected CHSE-214 and RSBK-2 cells (Fig. 1, lanes 4 and 6) but not in lysates of virus-infected FHM and EPC cells (Fig. 1, lanes 8 and 10). Nonspecific reactions were not detected in lysates of all four mock-infected cell lines (Fig. 1, lanes 3, 5, 7 and 9). These results indicated, therefore, that the anti-MABV Y-6 antiserum was specific in the antigen–antibody reaction.



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Fig. 1. Reactivity of anti-MABV Y-6 virion rabbit serum against purified virus and virus-infected cell lysates. Lanes: 1, SDS-PAGE (14 % polyacrylamide gel) profile of purified MABV Y-6 virions stained with Coomassie brilliant blue R-250; 2, Western blot analysis of purified MABV Y-6 virions probed with anti-MABV Y-6 virion rabbit serum; 3–10, Western blot analysis of cell lysates probed with anti-MABV Y-6 virion rabbit serum. Lanes: 3 and 4, lysates of mock- and virus-infected CHSE-214 cells; 5 and 6, lysates of mock- and virus-infected RSBK-2 cells; 7 and 8, lysates of mock- and virus-infected FHM cells; 9 and 10, lysates of mock- and virus-infected EPC cells. Positions of a molecular mass marker (Bio-Rad) are indicated on the left.

 
Virus binding assay
To assess the ability of the virus to bind to the cell surfaces of four cell lines, the localization of the virions was detected in virus-adsorbed cells by immunofluoresence and immunoelectron microscopy. Cells were incubated with virus for 30 min, reacted with anti-MABV Y-6 virion rabbit serum and stained with FITC-conjugated anti-rabbit IgG. MABV virions were clearly visible as bright signals in CHSE-214, RSBK-2, FHM and EPC cells (Fig. 2, right panels). Fluorescence was not observed in mock-infected cells (Fig. 2, left panels). Immunoelectron microscopy was done with 10 nm gold-conjugated goat anti-rabbit IgG. Virions were detected as gold-labelled virions with a diameter of about 60 nm on the surfaces of CHSE-214, RSBK-2, FHM and EPC cells (Fig. 3). These results indicate that MABV can bind to the cell surfaces of all four cell lines used in this study.



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Fig. 2. Localization of MABV Y-6 virions in CHSE-214 (A), RSBK-2 (B), FHM (C) and EPC (D) cells detected with fluorescent antibody. After adsorption for 30 min, cells were fixed in cold acetone, incubated with anti-MABV Y-6 virion rabbit serum and stained with FITC-conjugated anti-rabbit IgG. After washings, cells were counterstained with CYTO 64 RED Fluorescent Nucleic Acid stain for 10 min at room temperature. Mock-infected cells (left panels) were prepared as control for nonspecific binding by the same procedure as that for virus-adsorbed cells (right panels). Magnification, x200.

 


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Fig. 3. Localization of MABV Y-6 virions detected at the plasma membrane in CHSE-214 (A), RSBK-2 (B), FHM (C) and EPC (D) cells by immunoelectron microscopy. Cells were incubated with virus for 30 min at 20 °C. Immunomarking was performed directly on monolayer cells with 10 nm gold-conjugated goat anti-rabbit IgG using a preembedding technique. Bar, 250 nm.

 
Ultrastructural analysis at the early stages of MABV infection
MABV virions first appeared at the plasma membrane in four cell lines after adsorption for 20 min (Fig. 4A–D). Pits beneath the virions were observed at the plasma membrane in CHSE-214 cells (Fig. 4E, F). Small vesicles containing the virions were observed occasionally close to large vesicles containing the virions (Fig. 4G) as well as those fused between these compartments in CHSE-214 cells (Fig. 4H). Virions were more frequently within large vesicles rather than bound to the cell surface or captured in the small pits in CHSE-214 cells after adsorption for 40 min (Fig. 4I). Intact virions seemed to be structurally degraded or transformed within vesicles in CHSE-214 cells (Fig. 4J). In RSBK-2 and FHM cells, similar to CHSE-214 cells, vesicles containing the virions were observed in the cytoplasm (Fig. 4K, L). These results indicate that MABV entered the cytoplasm by an endocytic pathway at the early stages of MABV infection in CHSE-214, RSBK-2 and FHM cells.



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Fig. 4. Electron microscopy analysis of MABV Y-6 entry in CHSE-214, RSBK-2, FHM and EPC cells. (A–D) MABV Y-6 virions appeared at the plasma membrane in all four cell lines after adsorption for 20 min. (E, F) Pits beneath MABV Y-6 virions were observed at the plasma membrane in CHSE-214 cells. (G, H) Small vesicles containing MABV Y-6 virions were occasionally observed close to large vesicles containing MABV Y-6 virions as well as fused between these compartments in CHSE-214 cells. (I) MABV Y-6 virions were within large vesicles more frequently than bound to the cell surface or existed in small pits in CHSE-214 cells after adsorption for 40 min. (J) Intact MABV Y-6 virions seemed to be degraded within vesicles in CHSE-214 cells. (K, L) Vesicles containing MABV Y-6 virions were observed in the cytoplasm in RSBK-2 and FHM cells, similar to CHSE-214 cells. Bars, 250 nm.

 
We counted in triplicate the numbers of pits beneath the virions and vesicles containing the virions per cell in more than 100 cells (Table 1). The numbers of pits averaged 1·91, 1·61 and 1·82 per cell in CHSE-214, RSBK-2 and FHM cells, respectively, and were not statistically different. The numbers of vesicles averaged from 2·53 to 5·09 in CHSE-214 cells, from 2·95 to 4·73 in RSBK-2 cells and from 2·61 to 4·69 in FHM cells after adsorption for 30–60 min and were not statistically different. However, in EPC cells, pits beneath the virions or vesicles containing the virions were not observed until adsorption for 60 min. These results indicate that MABV virions failed to enter the cytoplasm of EPC cells only after adsorption for 60 min.


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Table 1. Numbers of pits beneath MABV Y-6 virions and vesicles containing MABV Y-6 virions

The numbers of pits after adsorption for 20 min and vesicles containing MABV Y-6 virions in each cell after adsorption for 30, 40, 50 or 60 min were counted using electron microscopy. More than 100 cells of each sample were counted in triplicate and compared using ANOVA and Tukey's test (P<0·01).

 
Identification of the virus binding protein by VOPBAs
A number of cell receptors for a variety of viruses have been identified successfully by VOPBA analysis (Borrow & Oldstone, 1992; de Verdugo et al., 1995; Ludwig et al., 1996; Mizukami et al., 1996; Salas-Benito & del Angel, 1997). Cellular proteins were prepared and used in VOPBAs to identify specially MABV-bound proteins. Virions were found to bind to a protein with a molecular mass of around 250 kDa from CHSE-214, RSBK-2, FHM and EPC cells (Fig. 5B, panel 1). The molecular size of the binding protein of CHSE-214 was slightly larger than those of RSBK-2, FHM and EPC cells. When virions were omitted from the reaction process, the 250 kDa band was not seen (Fig. 5B, panel 2). This indicated that binding to a protein of around 250 kDa was a specific interaction.



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Fig. 5. Analysis of MABV Y-6 virion binding proteins by VOPBA. Cellular proteins (25 µg) from each of four cell lines were separated by reducing SDS-PAGE (5 % polyacrylamide gel) and transferred electrophoretically onto a nitrocellulose membrane. The gel was stained with silver and the membrane was processed for VOPBA. (A) SDS-PAGE profile of the cell proteins of each of four cell lines. (B) VOPBA results. The virus was omitted from the first incubation as a control for nonspecific binding (panel 2).

 

   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
CHSE-214 and RSBK-2 cells are susceptible to MABV infection but FHM and EPC cells are resistant to MABV infection (Jung, 1998). The first interaction between host cell and virus is virus adsorption onto the plasma membrane through specific virus receptors. The replication cycle of MABV infection may be restricted at the adsorption step in FHM and EPC cells. In this study, we examined if MABV binds to the cell surfaces of two susceptible and two resistant cell lines by immunofluorescence and immunoelectron microscopy. By immunofluorescence microscopy, virions were observed after adsorption for 30 min as a specific fluorescence in all four cell lines. Gold-labelled virions were also detected at the plasma membranes in these four cell lines by immunoelectron microscopy. These results showed that MABV bound to all four cell lines, indicating that MABV can bind to the cell surfaces of resistant FHM and EPC cells. We showed previously that the original inoculated virus concentration was isolated as a cell-associated form throughout the incubation period despite the fact that no progeny virus was released in FHM and EPC cells after MABV infection (Imajoh & Suzuki, 1999). This obviously supports the evidence that MABV bound to the cell surfaces of FHM and EPC cells in this study. Thus, the replication cycle of MABV infection appears to be restricted after the virus adsorption step in FHM and EPC cells.

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.


   ACKNOWLEDGEMENTS
 
We are grateful to Dr T. Taniguchi, Kochi Medical School, for his sincere advice.


   REFERENCES
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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Received 30 September 2002; accepted 13 March 2003.