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Institute of Virology, Department for Infectious Diseases, University of Veterinary Medicine Hannover, Hannover, Germany
Correspondence
Beatrice Grummer
beatrice.grummer{at}tiho-hannover.de
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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The sequences of primers used for the construction of plasmids are available with the online version of this paper.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). The structural proteins comprise the nucleocapsid C protein and three envelope glycoproteins, Erns, E1 and E2, of which at least Erns and E2 are postulated to be responsible for virus attachment and/or cell entry (Donis & Dubovi, 1987; Hulst & Moormann, 1997). E1 is assumed to function as a membrane anchor for E2 (Rümenapf et al., 1993). Erns and E2 form disulphide-linked homodimers, whereas E1 is found as heterodimers in association with E2 (Rümenapf et al., 1993; Weiland et al., 1990). Neither Erns nor E2 is detectable on the cell surface of infected cells, but they are associated with intracellular membranes (Greiser-Wilke et al., 1991; Grummer et al., 2001). Recently, an endoplasmic reticulum (ER) retention signal within the membrane anchor of the E2 protein was identified (Köhl et al., 2004). It is well known that the Erns protein is secreted from infected cells due to the lack of a typical transmembrane anchor (Fetzer et al., 2005; Rümenapf et al., 1993).
The exact function and interaction of the three envelope proteins in virus entry is as yet inconclusive. For CSFV, it has been shown using lentiviral pseudotypes that E1 and E2 are sufficient to mediate virus entry, whilst Erns is dispensable in this process (Wang et al., 2004). On the other hand, it was shown that pestiviral Erns has the ability to bind to glycosaminoglycans, which implies a possible role in initial binding to the surface of permissive cells (Hulst et al., 2001; Iqbal et al., 2000).
To analyse further the essential interactions of the BVDV glycoproteins and their role in virus entry, vesicular stomatitis virus (VSV) pseudotypes containing BVDV glycoproteins were constructed. Because VSV gets its envelope by budding at the plasma membrane, chimeric Erns, E1 and E2 containing the membrane anchor and cytoplasmic tail of glycoprotein G of VSV (VSV-G) were generated to redirect the BVDV envelope proteins efficiently to the plasma membrane. In contrast to infected cells, in transiently transfected cells, BVDV envelope proteins were found in the cytoplasm but also at the plasma membrane. This enabled the generation of pseudotypes containing native as well as chimeric BVDV glycoproteins. Protein interactions in transfected cells were analysed by site-directed mutagenesis. Infection assays with variants of VSV/BVDV pseudotypes were carried out to explore the prerequisites for successful BVDV entry.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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), were grown in selection medium (Eagle's minimal essential medium supplemented with 250 µl hygromycin B ml–1, 1 mg zeocin ml–1 and 5 % FCS). All cell cultures were maintained at 37 °C and 5 % CO2.
Virus.
G-VSV-G is a recombinant VSV with the gene for enhanced green fluorescent protein (EGFP) replacing the glycoprotein G gene (Hanika et al., 2005). For propagation of G-VSV-G, BHK-G43 cells were treated with mifepristone (diluted 1 : 1000) and infected with the recombinant VSV 24 h later. Cell culture supernatants were harvested and cell debris removed by centrifugation at 24 h post-infection (p.i.). The culture supernatants were aliquotted and stored at –80 °C.
Construction of plasmids.
Chimeric Erns, E1 and E2 (Erns–MAT, E1–MAT and E2–MAT) were generated by replacing the putative transmembrane domains of the BVDV envelope proteins with the membrane anchor (MA) and cytoplasmic tail (T) of the VSV-G protein. To generate plasmids containing Erns–MAT, Erns, E1, Flag–E1 and E1–MAT, an overlapping PCR technique was performed as described previously (Köhl et al., 2004). The BVDV Erns and E1 genes were amplified from a full-length clone, pACNR/NADL (Mendez et al., 1998), by PCR. The gene fragments for the relevant parts of the VSV-G protein were amplified using the plasmid pTM1-E2-G(MT) (Köhl et al., 2004). Purified PCR products were mixed in a molar ratio of 1 : 1 : 1 and hybridization was carried out as reported previously (Köhl et al., 2004). After amplifying the hybrid genes, the PCR products were digested with BamHI and XbaI and the fragments ligated into the vector pCG1 (Cathomen et al., 1995). For site-directed mutagenesis for generation of E1-C/A, E1-C/S, Flag–E1-K, Flag–E1-R and Flag–E1-KR, modified sense and antisense primers were used. To generate E2–MAT, E2 and E2-R/A, the genes were amplified from pTM1-E2-G(MT), pTM1-E2 or pTM1-E2(R/A) (Köhl et al., 2004), respectively. BamHI and XbaI restriction sites were added to the 5' ends of the respective sense and antisense primers. After cutting with BamHI and XbaI, the PCR products were ligated into vector pCG1with T4 ligase (Fermentas). The combined protein domains used are shown in Fig. 1. Oligonucleotide sequences are available in Supplementary Table S1 (available in JGV Online).
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Antibodies.
The E-specific monoclonal antibody (mAb) BVD/CA3 (Bolin et al., 1988) and the Erns-specific mAb BVD/C12 (Greiser-Wilke et al., 1991) were used. Flag–E1 was detected using an antibody recognizing the Flag epitope (anti-Flag M2; Sigma-Aldrich). For detection of E1–MAT, the anti-VSV-G mAb P5D4 (Sigma-Aldrich) directed against the cytoplasmic tail of the VSV-G protein was used. mAb I1 was directed against VSV (anti-VSV-I1; Lefrancois & Lyles, 1982). A fluorescein isothiocyanate (FITC)-conjugated anti-mouse antibody (Sigma-Aldrich) was used for immunofluorescence analysis and a peroxidase-conjugated anti-mouse antibody (Dako Cytomation) was used for Western blotting.
Immunofluorescence.
Transfected cells were fixed with 3 % paraformaldehyde and permeabilized with 0.2 % Triton X-100 at 20 h post-transfection. Cells were incubated with the mAbs for 1 h at 37 °C, followed by FITC-conjugated anti-mouse IgG (Sigma). Cells were analysed by fluorescence microscopy using a Zeiss Axiophot 2 microscope with 450–490 nm band-pass filters.
Flow cytometry analysis.
Transfected BHK-21 cells were detached with 200 µl Accutase (Sigma-Aldrich) at room temperature for 15 min. The cells were resuspended in 800 µl PBS, transferred to a 1.5 ml reaction tube and centrifuged at 200 g for 3 min at 4 °C. The pellets were resuspended in 100 µl of the appropriate antibodies, transferred to a microtitre plate and incubated for 1 h at 4 °C. Cells were washed three times with 100 µl MIF buffer (1 g BSA and 10 mg sodium azide in 100 ml PBS) and centrifuged at 200 g at 4 °C for 3 min. Subsequently, the cells were incubated with an FITC-conjugated secondary antibody, diluted 1 : 500 in MIF buffer, for 1 h at 4 °C. After additional washing steps, the cells were resuspended in 100 µl MIF buffer and immediately analysed by flow cytometry. As a negative control, BHK-21 cells were stained with the secondary antibody only.
Western blotting.
BHK-21 cells were lysed at 20 h post-transfection in 200 µl NP-40 lysis buffer [50 mM Tris/HCl (pH 7.5), 150 mM NaCl, 0.5 % sodium desoxycholate, 1 % NP-40, Complete protease inhibitor (Roche)]. Proteins were separated by SDS-PAGE using 5 % acrylamide stacking gels and 10–12.5 % acrylamide resolving gels and transferred to PVDF membranes (Millipore). The blots were incubated with the appropriate mAb (diluted 1 : 100 in PBS), followed by incubation with horseradish peroxidase-conjugated anti-mouse antibody (diluted 1 : 1000; DakoCytomation). Proteins were visualized using BM Chemiluminescence Western Blotting Substrate (POD; Roche) or an ECL Advance Western Blotting Detection kit (GE Healthcare).
Pseudotypes.
BHK-21 cells grown in 35 mm diameter dishes were transfected with a total amount of 4 µg plasmid DNA. At 20 h post-transfection, the cells were infected with G-VSV-G (m.o.i. of 10) for 1 h at 37 °C. After appropriate washing with DMEM, the cells were inoculated with mAb anti-VSV-I1 for 1 h at 37 °C to neutralize unabsorbed virus. The cells were washed again and 2 ml culture medium was added. After 20 h, the cell culture supernatants were harvested, clarified by centrifugation and inoculated directly onto cells.
Infectivity of pseudotypes.
FBK, HeLa and BHK-21 cells were infected with the pseudotype viruses (100 µl cell culture supernatant per well of an eight-well chamber slide; Nunc) and incubated for 24 h at 37 °C. The cells were fixed with 3 % paraformaldehyde and the number of infectious units (IU) of virus was determined by counting the number of EGFP-positive cells.
Neutralization assays.
Neutralization assays were performed with a bovine BVDV antiserum, mAb BVD/CA3 directed against the E2 protein and anti-VSV-I1 antibody directed against VSV. The infectious pseudotypes were incubated with an equal volume of the antibodies for 1 h at 37 °C and 200 µl was then inoculated onto FBK cells seeded in eight-well chamber slides (Nunc).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) and FACS analysis (Fig. 2b). Similar to the observations made with cells infected with wild-type BVDV (Grummer et al., 2001), all proteins could be detected in the cytoplasm of transiently transfected cells. Additionally, native Erns and E2, as well as their chimeric counterparts Erns–MAT and E2–MAT and E2-R/A, were found at the plasma membrane.
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G-VSV-Erns-E1-E2 and G-VSV-E1-E2 were able to infect BVDV-permissive cells (Fig. 3). In contrast, none of the VSV/BVDV pseudotypes containing chimeric Erns, E1 and/or E2 could infect susceptible cells.
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To analyse the influence of the Flag tag on E1 functionality, an infection assay with the pseudovirus G-VSV-Flag-E1-E2 was carried out and revealed the same infectious properties as G-VSV-E1-E2 in that FKN cells could be infected but HeLa cells could not (data not shown).
Neutralization of pseudoviruses
Neutralization assays were carried out to confirm that infectivity of the pseudoviruses was mediated by the BVDV envelope proteins. The pseudotyped viruses were incubated with bovine antiserum to BVDV, with mAb BVD/CA3 or with a neutralizing anti-VSV antibody prior to inoculation of BVDV-susceptible cells. As expected, no fluorescence was detected after pre-incubation with the bovine antiserum and mAb BVD/CA3, indicating that the viruses G-VSV-Erns-E1-E2 and G-VSV-E1-E2 were neutralized, whilst the anti-VSV antibody could not prevent infection of bovine cells. In contrast, the BVDV-specific antibodies were not able to inhibit infection of the positive-control G-VSV-G, but the infectivity of this pseudovirus was eliminated by pre-incubation with the anti-VSV antibody (data not shown).
Influences on heterodimer formation
To clarify the non-infectious nature of pseudoviruses with chimeric BVDV envelope proteins, cells were co-transfected with different combinations of native and chimeric E1 and E2 and analysed by Western blotting. E1–E2 heterodimers could be detected, as well as E2 monomers and dimers (Fig. 4a). There was no heterodimer formation if one of the proteins was chimeric (Fig. 4b). In contrast to the monomeric form of E2 represented by a single band, E2–MAT monomers appeared as a double band of approximately 50 kDa. These most likely represent different glycosylation forms of the protein, due to the efficient transport of E2–MAT to the plasma membrane.
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Analysis of amino acids involved in heterodimer formation
To analyse the conditions for heterodimer formation, the cysteine residue at position 668 located in the hypothesized membrane anchor of E1 was substituted for alanine (E1-C/A) and serine (E1-C/S). The charged amino acids located at positions 671 and 674 within the transmembrane domains of E1 were replaced by alanine, respectively. Replacement by alanine and linkage with a Flag epitope resulted in the constructs Flag–E1-K, Flag–E1-R and Flag–E1-KR (Fig. 1). Furthermore, mutant E2-R/A, where the central arginine within the membrane anchor was replaced by alanine (Köhl et al., 2004), was included in the study. Subsequently, the mutated proteins were analysed in co-transfection studies for their ability to form heterodimers. Regardless of the combination, the monomeric as well as the dimeric forms of E2 and E2-R/A were found (Figs 5a and 6a). Similar to E2–MAT, E2-R/A monomers were represented by a double band of about 50 kDa, most likely reflecting different glycosylation forms of the protein. This also resulted in a higher molecular mass for E2-R/A homodimers compared with homodimers of the native E2.
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). However, in cells co-expressing E2 and Flag–E1, heterodimer formation was the dominating event. In contrast, in cells co-expressing the mutated Flag–E1 and E2 proteins, the monomeric and dimeric form of E2 were mainly detectable. In particular, in cells co-expressing the Flag–E1-KR protein, heterodimer formation was clearly reduced (Fig. 5a). In cells co-expressing E2-R/A and Flag–E1, no heterodimer formation was detectable (Fig. 6a).
The expression of Flag–E1, Flag–E1-K, Flag–E1-R and Flag–E1-KR was confirmed by detection with an anti-Flag antibody (Fig. 5b and Fig. 6b).
Role of E1–E2 heterodimers during pseudovirus entry
To confirm the requirement of heterodimers for BVDV entry, VSV pseudotypes with E2-R/A and E1 were generated. Focus was put on the E2-R/A protein because it was completely unable to form heterodimers (Fig. 6a). In contrast, the mutated E1 proteins displayed reduced heterodimer formation (Fig. 5a). As a positive control, cells were transfected with a plasmid encoding VSV-G, whilst mock-transfected cells were used as a negative control. Cell culture supernatants were harvested at 20 h p.i. and incubated with BVDV-permissive cells. Specific fluorescence signals could only be observed with VSV/BVDV pseudotypes bearing the native proteins, whereas pseudoviruses with E2-R/A failed to infect the cells (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) and severe acute respiratory syndrome-associated coronavirus (Ren et al., 2006). For efficient redirection of the BVDV envelope proteins to the plasma membrane, chimeric Erns, E1 and E2 proteins were generated containing the membrane anchor and cytoplasmic tail of the VSV-G protein. In addition, the native BVDV envelope proteins were also included in this study. Immunofluorescence and FACS analyses of transiently transfected cells revealed that native Erns and E2, as well as Erns–MAT, E1–MAT and E2–MAT, were localized intracellularly. Additionally, all proteins could be detected on the cell surface except for E1–MAT. This was totally unexpected for the native proteins, as in cells infected with the wild-type virus all proteins are exclusively localized intracellularly (Greiser-Wilke et al., 1991; Grummer et al., 2001). However, a similar phenomenon has been reported for HCV E1 and E2 proteins in transfected cells, as well as for proteins of plant cells, and is most likely due to saturation/leakiness of ER retention following overexpression (Bartosch et al., 2003; Crofts et al., 1999; Hsu et al., 2003).
Interestingly, all VSV pseudotypes containing the chimeric envelope proteins failed to infect permissive cells, whereas pseudotypes with the native BVDV envelope proteins were infectious. By neutralizing the infectivity of these pseudoviruses with anti-E2 antibodies or sera from BVDV-infected animals, it was clearly demonstrated that infectivity was indeed mediated by the BVDV envelope proteins. As observed for the closely related porcine pestivirus CSFV, Erns seems to be dispensable for the virus entry process (Wang et al., 2004). This could not have been anticipated for BVDV, as porcine and ruminant pestiviruses might have completely different mechanisms in entering cells and show different tropisms: whilst BVDV infects cell cultures and animals of different species, CSFV is strictly restricted to porcine cell lines and animals of the family Suidae. Despite these differences, our results obtained with the bovine pestivirus BVDV confirmed the observations of Wang et al. (2004).
The non-infectious nature of VSV/BVDV pseudotypes with chimeric envelope proteins led to the conclusion that the correct function of E1–MAT and E2–MAT was altered – most probably due to the modified transmembrane domains. Indeed, for most membrane proteins, the transmembrane domain is more than just an anchor to the membrane (Cocquerel et al., 2000). The transmembrane domains of HCV envelope proteins E1 and E2 possess a signal sequence function in their C-terminal half, play a major role in ER localization of E1 and E2, and are potentially involved in the assembly of these envelope proteins (Cocquerel et al., 2000). This may explain why human immunodeficiency virus pseudotypes expressing HCV E1–E2 envelope proteins that lack these multifunctional transmembrane domains are also non-infectious (Dubuisson, 2000). HCV and BVDV as related members of the family Flaviviridae both have anchor domains that are composed of two stretches of hydrophobic residues separated by a short segment containing at least one fully conserved charged residue (Cocquerel et al., 2000). These parallels in amino acid sequence may also reflect similar functions. The charged residues within the transmembrane domains of HCV E1 and E2 have been identified to play a major role in biogenesis of the non-covalently linked E1–E2 heterodimers (Ciczora et al., 2005, 2006; Cocquerel et al., 2000). In contrast to the non-covalent interaction of HCV E1 and E2, it has been postulated that the glycoproteins of pestiviruses such as CSFV and BVDV form disulfide-linked heterodimers and homodimers, respectively (Thiel et al., 1991; van Rijn et al., 1994; Weiland et al., 1990). As disulfide bonds are based on cysteine residues, this could be the reason for the inability of E1–MAT to form heterodimers. By generating the chimeric protein E1–MAT, a cysteine residue at position 668 was deleted together with the hypothesized membrane anchor. To explore whether this cysteine is essential for heterodimer formation, it was substituted with alanine and serine, respectively. As the mutations had no effect on heterodimerization, we concluded that other amino acids within the transmembrane domain of E1 must be responsible for this process. This hypothesis was additionally supported by the fact that, although the generation of E2–MAT was not linked to the loss of a cysteine residue, the protein was not able to form heterodimers. We therefore focused on the charged amino acids in the transmembrane domains and replaced them with alanine. Because both residues in the transmembrane domain of E1 (lysine and arginine) and the amino acid in the transmembrane domain of E2 (arginine) are positively charged, a direct interaction through an ion pair can be excluded. For BVDV E2, it has been supposed that the transmembrane domain initially forms a hairpin-like structure in which both the N-terminal and C-terminal ends are oriented towards the lumen of the ER and the charged residue faces the cytosol (Köhl et al., 2004). Signal sequence cleavage at the C terminus of the proteins leads to reorientation of the C-terminal end, which is subsequently directed towards the cytosolic face of the membrane with the charged residue in the middle of the membrane spanning sequence (Cocquerel et al., 2002; Köhl et al., 2004). Thus, it is likely that the lack of interaction is due to conformational changes of the transmembrane domains of the mutated BVDV proteins. These results led us to the conclusion that these domains are needed for heterodimer formation. Beyond this, we demonstrated that pseudoviruses bearing mutated BVDV envelope proteins, which were unable to form heterodimers, failed to infect BVDV-permissive cells, indicating that E1–E2 heterodimers play a key role in BVDV entry.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Bolin, S., Moennig, V., Kelso Gourley, N. E. & Ridpath, J. (1988). Monoclonal antibodies with neutralizing activity segregate isolates of bovine viral diarrhea virus into groups. Arch Virol 99, 117–123.[CrossRef][Medline]
Cathomen, T., Buchholz, C. J., Spielhofer, P. & Cattaneo, R. (1995). Preferential initiation at the second AUG of the measles virus F mRNA: a role for the long untranslated region. Virology 214, 628–632.[CrossRef][Medline]
Ciczora, Y., Callens, N., Montpellier, C., Bartosch, B., Cosset, F. L., Op, D. B. & Dubuisson, J. (2005). Contribution of the charged residues of hepatitis C virus glycoprotein E2 transmembrane domain to the functions of the E1E2 heterodimer. J Gen Virol 86, 2793–2798.
Ciczora, Y., Callens, N., Penin, F., Pecheur, E. I. & Dubuisson, J. (2006). The transmembrane domains of HCV envelope glycoproteins: residues involved in E1E2 heterodimerization and involvement of these domains in virus entry. J Virol 81, 2372–2381.[CrossRef][Medline]
Cocquerel, L., Wychowski, C., Minner, F., Penin, F. & Dubuisson, J. (2000). Charged residues in the transmembrane domains of hepatitis C virus glycoproteins play a major role in the processing, subcellular localization, and assembly of these envelope proteins. J Virol 74, 3623–3633.
Cocquerel, L., Op, D. B., Lambot, M., Roussel, J., Delgrange, D., Pillez, A., Wychowski, C., Penin, F. & Dubuisson, J. (2002). Topological changes in the transmembrane domains of hepatitis C virus envelope glycoproteins. EMBO J 21, 2893–2902.[CrossRef][Medline]
Crofts, A. J., Leborgne-Castel, N., Hillmer, S., Robinson, D. G., Phillipson, B., Carlsson, L. E., Ashford, D. A. & Denecke, J. (1999). Saturation of the endoplasmic reticulum retention machinery reveals anterograde bulk flow. Plant Cell 11, 2233–2248.
Donis, R. O. & Dubovi, E. J. (1987). Characterization of bovine viral diarrhoea-mucosal disease virus-specific proteins in bovine cells. J Gen Virol 68, 1597–1605.
Dubuisson, J. (2000). Folding, assembly and subcellular localization of hepatitis C virus glycoproteins. Curr Top Microbiol Immunol 242, 135–148.[Medline]
Fetzer, C., Tews, B. A. & Meyers, G. (2005). The carboxy-terminal sequence of the pestivirus glycoprotein Erns represents an unusual type of membrane anchor. J Virol 79, 11901–11913.
Greiser-Wilke, I., Dittmar, K. E., Liess, B. & Moennig, V. (1991). Immunofluorescence studies of biotype-specific expression of bovine viral diarrhoea virus epitopes in infected cells. J Gen Virol 72, 2015–2019.
Grummer, B., Beer, M., Liebler-Tenorio, E. & Greiser-Wilke, I. (2001). Localization of viral proteins in cells infected with bovine viral diarrhoea virus. J Gen Virol 82, 2597–2605.
Hanika, A., Larisch, B., Steinmann, E., Schwegmann-Weßels, C., Herrler, G. & Zimmer, G. (2005). Use of influenza C virus glycoprotein HEF for generation of vesicular stomatitis virus pseudotypes. J Gen Virol 86, 1455–1465.
Hsu, M., Zhang, J., Flint, M., Logvinoff, C., Cheng-Mayer, C., Rice, C. M. & McKeating, J. A. (2003). Hepatitis C virus glycoproteins mediate pH-dependent cell entry of pseudotyped retroviral particles. Proc Natl Acad Sci U S A 100, 7271–7276.
Hulst, M. M. & Moormann, R. J. (1997). Inhibition of pestivirus infection in cell culture by envelope proteins Erns and E2 of classical swine fever virus: Erns and E2 interact with different receptors. J Gen Virol 78, 2779–2787.[Abstract]
Hulst, M. M., Van Gennip, H. G., Vlot, A. C., Schooten, E., De Smit, A. J. & Moormann, R. J. (2001). Interaction of classical swine fever virus with membrane-associated heparan sulfate: role for virus replication in vivo and virulence. J Virol 75, 9585–9595.
Iqbal, M., Flick-Smith, H. & McCauley, J. W. (2000). Interactions of bovine viral diarrhoea virus glycoprotein Erns with cell surface glycosaminoglycans. J Gen Virol 81, 451–459.
Köhl, W., Zimmer, G., Greiser-Wilke, I., Haas, L., Moennig, V. & Herrler, G. (2004). The surface glycoprotein E2 of bovine viral diarrhoea virus contains an intracellular localization signal. J Gen Virol 85, 1101–1111.
Lagging, L. M., Meyer, K., Owens, R. J. & Ray, R. (1998). Functional role of hepatitis C virus chimeric glycoproteins in the infectivity of pseudotyped virus. J Virol 72, 3539–3546.
Lefrancois, L. & Lyles, D. S. (1982). The interaction of antibody with the major surface glycoprotein of vesicular stomatitis virus. II. Monoclonal antibodies of nonneutralizing and cross-reactive epitopes of Indiana and New Jersey serotypes. Virology 121, 168–174.[CrossRef][Medline]
Mendez, E., Ruggli, N., Collett, M. S. & Rice, C. M. (1998). Infectious bovine viral diarrhea virus (strain NADL) RNA from stable cDNA clones: a cellular insertion determines NS3 production and viral cytopathogenicity. J Virol 72, 4737–4745.
Ren, X., Glende, J., Al Falah, M., Schwegmann-Wessels, C., Qu, X., Tan, L., Tschernig, T., Deng, H., Naim, H. Y. & Herrler, G. (2006). Analysis of ACE2 in polarized epithelial cells: surface expression and function as receptor for severe acute respiratory syndrome-associated coronavirus. J Gen Virol 87, 1691–1695.
Rümenapf, T., Unger, G., Strauss, J. H. & Thiel, H. J. (1993). Processing of the envelope glycoproteins of pestiviruses. J Virol 67, 3288–3294.
Thiel, H. J., Stark, R., Weiland, E., Rumenapf, T. & Meyers, G. (1991). Hog cholera virus: molecular composition of virions from a pestivirus. J Virol 65, 4705–4712.
van Rijn, P. A., Miedema, G. K., Wensvoort, G., van Gennip, H. G. & Moormann, R. J. (1994). Antigenic structure of envelope glycoprotein E1 of hog cholera virus. J Virol 68, 3934–3942.
Wang, Z., Nie, Y., Wang, P., Ding, M. & Deng, H. (2004). Characterization of classical swine fever virus entry by using pseudotyped viruses: E1 and E2 are sufficient to mediate viral entry. Virology 330, 332–341.[CrossRef][Medline]
Weiland, E., Stark, R., Haas, B., Rumenapf, T., Meyers, G. & Thiel, H. J. (1990). Pestivirus glycoprotein which induces neutralizing antibodies forms part of a disulfide-linked heterodimer. J Virol 64, 3563–3569.
Received 4 March 2008;
accepted 15 May 2008.
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