Institutes of Virology1 and Pathology3, School of Veterinary Medicine Hannover, Buenteweg 17, 30559 Hannover, Germany
Federal Research Centre for Virus Diseases of Animals, Institute for Diagnostic Virology, Boddenblick 5a, 17498 Insel Riems, Germany2
Author for correspondence: Irene Greiser-Wilke. Fax +49 511 953 8898. e-mail Irene.Greiser-Wilke{at}tiho-hannover.de
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Abstract |
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Introduction |
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While considerable information is now available about genome structure and viral protein function, there is little and inconsistent information available about the structure and morphogenesis of the virion. Since BVDV is an enveloped virus, budding from the extracellular membrane could be one possible mechanism for the release of mature particles. A prerequisite for virus release by this mechanism is the integration of viral glycoproteins into the membrane of the host cell. In this study, the expression of viral proteins on the surface of infected cells and in soluble and insoluble subcellular fractions was analysed using different approaches. After labelling with monoclonal antibodies (MAbs), no expression of the viral glycoproteins E2 and ERNS was detected on the surface of viable BVDV-infected cells by either confocal laser scanning microscopy (CFM) or FACS analysis. In addition, no viral proteins were immunoprecipitated with the specific MAbs from biotin-labelled cell surfaces of infected cells. Subcellular fractionation of virus-infected cells followed by radioimmunoprecipitation (RIP) with MAbs demonstrated that the two viral glycoproteins E2 and ERNS as well as the non-structural proteins NS2-3 and NS3 were almost quantitatively associated with intracellular membranes. From these results, we conclude that BVDV is released by budding into the cisternae of the endoplasmic reticulum (ER). Due to the fact that all proteins analysed were membrane-bound or membrane-associated, it can be excluded that one or more of them function as soluble factors during BVDV-induced apoptosis. This is in agreement with the findings that in BVDV-infected cell cultures, only cells actively synthesizing viral proteins undergo apoptosis (Grummer et al., 1998 ).
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Methods |
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All cells were grown in Dulbeccos modification of Eagles medium supplemented with 5% bovine serum that had been tested for the absence of BVDV and antibodies against BVDV. Cell cultures were maintained at 37 °C in a 5% CO2 incubator.
Monoclonal antibodies (MAbs).
The E2-specific MAb BVD/CA3, the ERNS-specific MAb BVD/C42 and the NS3-specific MAb BVD/C16 were used (Bolin et al., 1988 ; Cay et al., 1989
; Greiser-Wilke et al., 1991b
). As a positive control, MAb BVD/CA27 was included. This MAb is directed against the putative cellular receptor for BVDV (Schelp et al., 1995
), which is, presumably, the bovine CD46 (boCD46) (Rumenapf et al., 2000
). As an additional control for the immunoprecipitation of biotinylated viral surface proteins, the SFV transmembrane protein p62 was precipitated using MAb SFV/C8 and the nucleocapsid protein was precipitated using MAb SFV/C2 (Greiser-Wilke et al., 1991a
). As a negative control for the immunoprecipitation of radioactively labelled proteins, lysates from BVDV-infected cells were precipitated using the unrelated MAb BM/40, which is specific for Brucella melitensis lipopolysaccharide (Greiser-Wilke & Moennig, 1987
).
Surface and intracellular indirect immunofluorescence (IF) staining
CFM analysis.
For intracellular staining, cells were grown on coverslips and infected with virus at an m.o.i of 1. After 48 h, the cells were fixed for 5 min in ice-cold 80% acetone. After washing with Ca2+- and Mg2+-deficient PBS (PBSM-), the coverslips (infected cells and mock-infected controls) were incubated for 15 min with MAbs (cell culture supernatants diluted 1:10 in PBSM-). After two additional washing steps with PBSM-, the coverslips were incubated for 15 min with the ALEXA488 conjugate (Molecular Probes) diluted 1:500 in PBSM-, washed and analysed by CFM (Zeiss LM510).
To stain the cell surface, coverslips with viable cells were incubated for 30 min on ice with the appropriate MAbs. After two washing steps, the coverslips were fixed with 1% paraformaldehyde (PFA) (Sigma) in PBSM- and incubated with the ALEXA488 conjugate for 30 min on ice. The coverslips were then washed and analysed by CFM. Cell viability was determined using 10-5 M propidium iodide (PI) in PBSM- (Sigma). From preparations with surface-stained cells, only samples with greater than 90% viable cells were evaluated.
Fluorescence-activated cell sorter (FACS) analysis.
Virus-infected cells (25 cm2 flasks, m.o.i of 1) were detached with a trypsin solution and transferred to a U-bottomed 96-well microtitre plate containing 4x104 cells per well.
For intracellular staining, cells were washed twice with PBSM- and fixed with 1% PFA for 15 min on ice. After washing with PBSM-, the cells were permeabilized for 5 min at room temperature with 0·0025% digitonin (Sigma) in PBSM- and washed once with PBSM-. The BVDV-specific MAbs (diluted cell culture supernatants) were added for 15 min and, subsequently, the cells were washed twice with PBSM-. The ALEXA488 conjugate was then added and incubated for 15 min. After two additional washes with PBS, the cells were resuspended in 100 µl PI solution. Only PI-positive cells were evaluated by flow cytometry.
To stain the cell surface (Beer & Wolf, 1999 ), detached cells were washed twice with PBSM- and incubated with BVDV-specific MAbs for 30 min on ice. After two washes, the ALEXA488 conjugate was added for 30 min on ice. After two additional washes with PBS, cells were resuspended in 100 µl PI solution and IF was measured by flow cytometry. Only PI-negative cells were analysed for surface expression of BVDV proteins (5000 cells per sample).
FACS analysis was performed with a FACScan cytofluorometer (Becton Dickinson) using the Cell Quest (Becton Dickinson) and WinMDI (Salk Institute) software.
Isotopic labelling and subcellular fractionation.
Twenty-four h after seeding 3x106 cells per tissue culture dish (10 cm diameter, 3 dishes per experiment), the cells were infected with BVDV or mock-infected. After 48 h, the monolayers were rinsed three times with PBSM- and overlaid with methionine- and cysteine-free medium (Gibco BRL) supplemented with 10% dialysed foetal calf serum (dFCS). Incubation was for 2 h at 37 °C. Labelling was performed by the addition of 5·5x106 Bq trans [35S]methionine (ICN) in 2 ml methionine- and cysteine-free medium with 10% dFCS. The cultures were then incubated for 6 h at 37 °C. The cells were rinsed twice with ice-cold PBSM- and once with bidistilled water (Aqua bidist). The cell monolayers were removed using a rubber policeman, resuspended in 1 ml ice-cold Aqua bidist and centrifuged at 4 °C for 6 min at 800 g. The sedimented cells were pre-swollen at 4 °C for 60 min in 1 ml Aqua bidist containing Complete protease inhibitors (Boehringer). After microscopic examination, the remaining cells were lysed by ultrasonic waves at 35 kHz for 510 s, without damaging the nuclei. Lysed cells were centrifuged at 4 °C for 10 min at 1000 g to remove the nuclei. The resulting supernatants were ultracentrifuged at 4 °C for 60 min at 100000 g in order to separate cellular membranes and soluble components from the cytosol (De Duve, 1971 ). The supernatants (800 µl) were carefully decanted into a new reaction tube, mixed with 200 µl 5x RIPA buffer (1x RIPA comprises 150 mM NaCl, 1·0% Triton X-100, 0·5% deoxycholate, 0·1% SDS, 50 mM TrisHCl, pH 8) and stored for 10 min on ice. The sediments were resuspended by adding 1 ml 1x RIPA buffer and were also incubated for 10 min on ice. For RIP analysis, samples were divided into two equal aliquots and incubated with a mixture of MAbs BVD/C16, C42 and CA3 (immunoaffinity purified) and protein G-Sepharose beads (Pharmacia) overnight. After washing the beads three times with RIPA buffer and eluting the proteins by boiling in sample buffer (2% SDS, 10% glycerol, 100 mM DTT, 60 mM TrisHCl, pH 6·8), SDSPAGE was performed in 7·5% acrylamide stacking gels with 15% acrylamide resolving gels. After electrophoresis, the gels were fixed in 30/12·5% (v/v) methanolacetic acid and dried under vacuum. Dried gels were analysed using a PhosphorImager (Molecular Dynamics).
Duplicate cultures were prepared as described above, but without adding the radioactive label. These samples were used to assess the level of contamination in the fractions containing soluble and membrane-associated cell components, respectively, by measuring the activity of the tracking enzymes.
Determination of enzyme activity.
The activity of the following enzymes was determined for each of the fractions: (a) the mitochondrial enzymes succinate dehydrogenase (SDH) and glutamate dehydrogenase (GluDH), which are specific for the membrane fraction (Singer, 1974 ; Ludtke et al., 1975
) and (b) glycerol-3-phosphate dehydrogenase (GAPDH) and lactate dehydrogenase (LDH), which are specific for the cytosolic fraction (Gonzalez-Cerezo & Dalziel, 1975
; Lee et al., 1982
). The activity of these enzymes was determined using the Ultrospec 2000 photometer (Pharmacia). The enzyme kinetics were evaluated using the Swift Reaction Kinetics 1.01 software (Pharmacia).
Biotinylation of proteins in cell lysates.
Intracellular biotinylation of viral proteins was performed using the Cellular Labelling and Immunoprecipitation kit (Roche), as instructed by the manufacturer.
Biotinylation of cell surfaces.
MDBK cells were infected 24 h after seeding into 6-well plates with BVDV or mock-infected with medium. As a control, BHK-21 cells were infected with SFV or mock-infected. After 24 h, the cell monolayers were rinsed three times with ice-cold PBS and overlaid with 0·5 ml biotin solution (0·5 mg sulpho-N-hydroxysuccinimidebiotin (Pierce) per ml of PBS, pH 7·2). The cells were then labelled for 1 h at 4 °C. The biotin solution was removed and the cells were rinsed once with ice-cold 0·1 M glycine solution to stop the reaction. After that, the cells were washed for 15 min with 0·1 M glycine at 4 °C. The last washing step was performed with ice-cold PBS. Proteins were solubilized by the addition of 1 ml RIPA buffer and virus proteins were immunoprecipitated as described above. For this, 500 µl aliquots were incubated overnight with a pool of MAbs (BVD/C16, CA3 and C42 or SFV/C2 and C8, respectively).
After immunoprecipitation and SDSPAGE (7·5% acrylamide stacking gels with 15% acrylamide resolving gels), the proteins were transferred to PVDF membranes (Millipore). The membranes were incubated for 1 h at room temperature with a streptavidin peroxidase-conjugated antibody (Amersham) diluted 1:1000. Proteins were detected using the BM chemiluminescence blotting substrate POD (Roche).
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Results |
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Localization of viral proteins in subcellular fractions
Subcellular fractionation was performed to assess if, and to what extent, the envelope glycoproteins ERNS and E2 and the non-structural proteins NS2-3 (ncp BVDV) and NS3 (cp BVDV) are membrane-associated. For this, it was found to be sufficient to analyse the membrane and cytosolic fractions, since this would be enough to establish whether the viral proteins of interest are soluble or not. As demonstrated by IF analysis, BVDV replicates in the cytoplasm of infected cells only; therefore, the nuclear fraction was discarded. The cytoplasmic components were fractionated by differential centrifugation to yield one fraction containing organelles with membrane-bound or membrane-associated proteins and one fraction with the soluble components of the cytoplasm. An additional step to separate the mitochondria was not performed, since these organelles are not involved in viral protein synthesis. To estimate the degree of contamination of each of the fractions, the specific activity of the tracking enzymes GAPDH and LDH (cytosolic fraction) and SDH and GluDH (membrane fraction) was determined. As expected, only a low level of contamination between the two fractions was detected, comprising about 315% of the total enzyme activity only (Table 1). To detect the BVDV proteins in each of the two fractions, equal amounts of radioactive counts (d.p.m.) were incubated with pools of the three MAbs BVD/C42, CA3 and C16 (Fig. 4
). In the fractions of mock-infected cells, no specific bands corresponding to BVDV proteins could be detected. In contrast, in the membrane fraction of cells infected with ncp BVDV, prominent bands with molecular masses of about 48 (ERNS), 56 (E2) and 125 kDa (NS2-3) were seen; a minor band with a molecular mass of about 20 kDa was also detected. In the corresponding cytosolic fraction, no bands corresponding to the NS2-3 protein or the E2 glycoprotein were detectable, while a weak band corresponding to the ERNS protein was clearly visible. Similar results were found for the subcellular fractions of cells infected with cp BVDV. Here, both the NS2-3 and the NS3 proteins characteristic for cp BVDV were detected in the membrane fraction only. In this fraction, the 20 kDa protein and an additional protein with a molecular mass of about 60 kDa were also visible. In the cytosolic fraction, only weak bands corresponding to the E2 and ERNS proteins could be precipitated.
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Discussion |
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In our experiments using CFM and FACS analysis, we obtained only strong intracellular but no extracellular IF signals. In addition, immunoprecipitation of biotinylated cell surface proteins from virus-infected cells with BVDV-specific MAbs showed that neither the glycoproteins ERNS and E2 proteins nor the NS3 and NS2-3 proteins were detectable on the cell surface. Although this is in agreement with earlier experiments using the same MAbs to detect the antigens on the surface of viable BVDV-infected cells by IF assay (Greiser-Wilke et al., 1991b ), it contradicts more recent reports (Weiland et al., 1999
). Here, IF microscopy on live adherent cells as well as fluorescent cytometry analysis of viable cells led to the detection of the BVDV ERNS and E2 glycoproteins on the cell surface. However, in further investigations by immune electron microscopy, Weiland et al. (1999)
showed that the plasma membrane remained completely unlabelled and concluded that the positive IF signal was probably mediated by the binding of MAbs to virions adhering to the extracellular membrane and not to proteins integrated in the plasma membrane. Moreover, Weiland et al. (1999)
did not use the very specific PI exclusion as a marker for cell viability during their FACS analysis, making it likely that the cells were damaged and thus permeabilized for the MAbs and, therefore, generating false-positive signals.
Localization of viral proteins in the soluble and membranous fractions of BVDV-infected cells showed that the proteins analysed (NS3, NS2-3, ERNS and E2) appeared to be mainly membrane-bound, or at least membrane-associated, in cells infected with both cp and ncp BVDV biotypes. Interestingly, two additional proteins of about 20 and 60 kDa were precipitated from BVDV-infected cell lysates using the E2-specific MAb BVD/CA3. These proteins had been observed before in lysates of infected cells labelled under hypertonic initiation block (HIB) conditions (Donis & Dubovi, 1987 ). In RIP analysis using hyperimmune serum, the 20 kDa protein was visible after a prolonged exposure, whereas the 60 kDa protein was only detectable in lysates of cells labelled under HIB. The latter protein is probably the uncleaved E2p7 protein (Elbers et al., 1996
), whereas the origin of the 20 kDa protein remains unclear.
In the cytosolic fractions, no NS2-3, but small amounts of E2 and ERNS, could be detected. This may be due to the contamination of the soluble fraction with membranous components. On the other hand, it is conceivable that at least ERNS is also present in a soluble form, as large amounts of the protein are shed into the medium by BVDV-infected cells (Rumenapf et al., 1993 ). It remains to be determined whether the BVDV proteins analysed are membrane-bound or merely membrane-associated.
These results show similarities to the distribution of hepatitis C virus (HCV) proteins. The HCV structural proteins E1 and E2 and the core protein accumulate in the ER (Kim et al., 1999 ). The NS2 protein derived from processing at the NS2-3 site is a transmembrane polypeptide, with the C terminus translocated in the lumen of the ER and the N terminus located in the cytosol (Santolini et al., 1995
). The remaining non-structural proteins are accumulated in the ER and the Golgi region (Kim et al., 1999
). In contrast to BVDV NS3, HCV NS3 was detected in the nucleus as well as in the cytoplasm (Muramatsu et al., 1997
). Ultrastructural studies of cells infected with HCV showed that, in some instances, the outer membranes of the particles were connected to the ER membrane, indicating possible virus budding into the cisternae (Serafino et al., 1997
).
In summary, we conclude that mature BVDV particles are not released by budding from the plasma membrane but by budding at the membranes of the ER, similar to HCV. Since none of the viral proteins analysed was detected on the cell surface, this is not in agreement with the concept that the cell association of pestiviruses is due to firm adhesion of virions to the cell surface (Weiland et al., 1999 ). In addition, as the only currently known marker protein for cytopathogenicity (Donis & Dubovi, 1987
), namely the NS3 protein, was found to be membrane-bound, or at least membrane-associated, to cells infected with both virus biotypes, there seems to be no correlation between the location of this protein and its function in the cell.
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Received 8 May 2001;
accepted 13 July 2001.