Institut für Virologie, Tierärztliche Hochschule Hannover, Bünteweg 17, 30559 Hannover, Germany
Correspondence
Georg Herrler
Georg.Herrler{at}tiho-hannover.de
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ABSTRACT |
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INTRODUCTION |
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Little information is available concerning the late stage of the BVDV replication cycle. Immunostaining of virus-infected cells indicated that glycoproteins E2 and E(rns) are absent from the plasma membrane (Greiser-Wilke et al., 1991; Grummer et al., 2001
; Weiland et al., 1999
). Viral envelope proteins, occasionally detected by surface staining, have been attributed to virus particles accumulating on the cell surface (Weiland et al., 1999
). The absence of BVDV glycoproteins from the plasma membrane is consistent with the failure to detect virus budding from the cell surface (Gray & Nettleton, 1987
) suggesting that BVDV most likely matures at intracellular membranes. So far, no signals that retain the BVDV envelope proteins in intracellular compartments have been determined.
The E2 protein, expressed by recombinant vesicular stomatitis virus (VSV), has been reported to be present on the plasma membrane (Grigera et al., 2000). Similarly, cells infected by recombinant baculovirus were also shown to contain BVDV E2 protein on the cell surface (Kweon et al., 1997
); this is in contrast to the E2 proteins of members of other genera within the family Flaviviridae. The hepatitis C virus E2 protein has been shown to contain a retention signal within the membrane anchor that prevents transport to the cell surface (Cocquerel et al., 1998
).
We have constructed chimeric E2 proteins that contain the membrane anchor (M) and/or the cytoplasmic tail (T) of other viral glycoproteins that are transported to the cell surface. Using different labelling procedures and detection methods we were able to show that the E2 protein M domain contains an intracellular localization signal and that an arginine residue within this hydrophobic domain is an essential element of the sorting signal. Interestingly, the export signal present in the cytoplasmic tail of the VSV G protein was found to overrule the retention signal within the E2 membrane anchor.
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METHODS |
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Construction of plasmids.
The G protein gene of VSV (strain Indiana) was amplified from the pVSV-XN2 vector (kindly provided by John K. Rose, New Haven, USA) by PCR using oligonucleotides a and b (Table 1). The two primers contained an EcoRI and a BamHI restriction site, respectively, which allowed us to clone the PCR product into the respective sites of the pTM1 vector (Moss et al., 1990
) resulting in pTM1-G. The total open reading frame of G was sequenced and found to be identical with the published sequence (accession no. NC_001560).
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Western blot analysis of transfected cells.
BSR-T7/5 cells grown in 35-mm diameter dishes were transfected with 3 µg of plasmid DNA and 6 µl Lipofectamine 2000 Reagent. At 24 h post-transfection, cells were scraped into 1 ml of PBS, pelleted by centrifugation, and lysed in 200 µl of NP40 lysis buffer (50 mM Tris/HCl, pH 7·5, 150 mM NaCl, 0·5 % sodium desoxycholate, 1 % NP40, protease inhibitors). Twofold-concentrated SDS sample buffer was added to the clarified lysate. The samples were run on a 10 % SDS-polyacrylamide gel under non-reducing conditions and transferred to a nitrocellulose membrane. The blots were incubated with either a cocktail of BVD/CA1 and BVD/CA3 monoclonal antibodies (each 1 : 60 in PBS) or with a polyclonal rabbit anti-VSV serum (1 : 1000 in PBS) followed by incubation with biotinylated anti-mouse or anti-rabbit immunoglobulin serum (Amersham; 1 : 1000). Following incubation with a streptavidinperoxidase complex (Amersham; 1 : 1000), the antigens were visualized by chemiluminescence (BM chemiluminescence blotting substrate, Roche Diagnostics) (Zimmer et al., 2001b).
Biotinylation and immunoprecipitation of surface proteins.
At 24 h post-transfection (see above), BSR-T7/5 cells grown in 35-mm diameter dishes were labelled with an N-hydroxysuccinimide ester of biotin (sulfo-NHS-biotin; Pierce), and the virus antigens were immunoprecipitated from the cell lysates according to a published protocol (Zimmer et al., 2001a, b
). For immunoprecipitation, a polyclonal rabbit anti-VSV serum (1 : 200) or a cocktail composed of the BVD/CA1 and BVD/CA3 monoclonal antibodies (1 : 50 each) was used.
Radiolabelling and treatment with endoglycosidases.
At 24 h post-transfection (see above), BSR-T7/5 cells grown in 35-mm-diameter dishes were metabolically labelled for 2 h with 100 µCi of [35S]methionine/[35S]cysteine (Tran35S-Label, ICN). The cells were lysed with 600 µl NP40 lysis buffer and the viral proteins were isolated by immunoprecipitation (see above) and eluted in 20 µl of 1 % SDS. A 10 µl aliquot of the immunoprecipitate was diluted with 90 µl of 50 mM sodium acetate buffer, pH 5·5, containing 1 % octylthioglucoside and a protease inhibitor cocktail. Endoglycosidase H (10 mU) (Calbiochem) were added to 30 µl of this dilution, and incubated for 2 h at 37 °C. In parallel, a 10 µl aliquot of the immunoprecipitate was diluted with 90 µl of 50 mM phosphate buffer, pH 7·0, containing 1 % octylthioglucoside and a protease inhibitor cocktail, and 30 µl of this dilution were treated with 1 U of N-glycosidase F (Roche) for 2 h at 37 °C. The samples were run on a 12 % SDS-polyacrylamide gel under reducing conditions and analysed by autoradiography (Zimmer et al., 2002).
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RESULTS |
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Cells transfected with expression plasmids containing either of the different constructs, were subjected to SDS-PAGE under non-reducing conditions and analysed by Western blotting. As shown in Fig. 7, mutant E2(M*Rdel) was not detectable under these conditions (lane d). With the other mutants, both the monomeric and the dimeric forms of E2 were present. Expression of mutants containing a terminal RR motif (lane c) and/or an R/A mutation in the putative membrane anchor (lanes e and f) resulted in stronger signals than expression of the authentic E2 (lane a) or the E2 protein lacking the carboxy-terminal tail (lane b). These differences may reflect differences in the expression rate, in the protein stability, or in the capacity to adopt the native conformation under the Western blot conditions.
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DISCUSSION |
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Interestingly, the chimeric protein E2-G(T) was transported to the cell surface. The cytoplasmic tail of G is known to contain a specific export signal for transport out of the ER. A di-acidic motif (AspXxxGlu, Xxx being any amino acid), has been shown to efficiently recruite G and other proteins to vesicles mediating export from the ER (Nishimura & Balch, 1997). The extended motif TyrThrAspIleGluMet has been reported to further increase the export efficiency and also to be functional on proteins that otherwise only inefficiently exit the ER (Sevier et al., 2000
). As the E2-G(T) protein contains an intracellular localization signal in the membrane anchor and an export signal in the carboxy-terminal domain, our data suggest that the export signal within the cytoplasmic tail of the VSV G protein overrules the ER localization signal within the M domain of the E2 protein.
Retention in the ER has been described for the E2 proteins of viruses belonging to other genera within the family Flaviviridae. The best characterized virus in this respect is the E2 protein of hepatitis C virus (HCV), where the retention signal has also been assigned to the transmembrane domain (Cocquerel et al., 1998). The E2 transmembrane domains of all flaviviruses have a characteristic structure. They consist of two short hydrophobic stretches of amino acids that are separated by one or several hydrophilic residues including at least one charged amino acid (Cocquerel et al., 2000
). This bipartite organization reflects the dual function of this hydrophobic domain. As the E2 protein is synthesized as part of a polypeptide, it requires, at the carboxy terminus, not only a membrane anchor but also a signal peptide to allow signal peptidase to separate E2 in the lumen of the ER from the downstream p7 protein. Whereas the hydrophobic domain of HCV E2 protein is followed by only two terminal amino acids, the carboxy-terminal domain of the corresponding BVDV protein consists of a hydrophilic stretch of seven amino acids (Elbers et al., 1996
; Harada et al., 2000
). The importance of this tail is not known; it is dispensable for intracellular localization of E2 as indicated by the results obtained with the E2(Tdel) mutant. Another difference between the E2 proteins of HCV and BVDV is that the latter virus only contains a single charged residue in the membrane anchor compared to the AspAlaArg tripeptide in the former virus. Replacing the arginine residue in the membrane anchor of BVDV E2 by an alanine results in a protein that is efficiently transported to the cell surface. In the case of HCV, ER retention is abolished more efficiently when in addition to the arginine the aspartic acid is replaced by an alanine (Cocquerel et al., 2000
). The arginine residue may allow the hydrophobic domain to adopt a hairpin-like structure that is required to function both as a membrane anchor and a signal peptide. In such a structure, both the aminoterminal and the carboxy-terminal end of the transmembrane domain are directed to the lumen of the ER. It has been pointed out that this organization may be transient, and that after processing in the ER the tail portion may reorientate in such a way that it faces the cytosolic side of the membrane (Allison et al., 1999
). In the case of the HCV E2 protein, evidence for this reorientation process has been obtained by adding tags to either end and determining the location of the tags (Cocquerel et al., 2002
). With the E2-G(T) mutant we have a function-based assay for the location of the carboxy terminus. The export signal in the tail domain of VSV G protein is functional, if it is exposed at the cytosolic side of the ER membrane. As our results suggest that the export signal in the E2-G(T) protein overrules the ER localization signal in the M domain, the tail of the chimeric protein is expected to face the cytosolic side. Therefore, our data are in agreement with the assumption that the membrane-spanning domain reorientates in such a way that the carboxy-terminal tail faces the cytosolic side of the membrane.
Transmembrane domains usually consist of 2025 hydrophobic amino acids. At least 16 leucines are required to form a transmembrane helix (Monne et al., 1999
). The two hydrophobic stretches in the membrane anchor of BVDV E2 consist of 11 and 12 amino acids, respectively. Therefore, they are too short to form
helices by themselves. This may explain the failure to detect E2(M*Rdel) after Western blotting. The protein is synthesized, as seen by immunofluorescence analysis, but the conformation of this protein is not stable enough to be detected after blotting onto the membrane. The E2 protein may require the stable anchoring in the membrane to adopt the correct conformation. Whether the deletion mutants are secreted into the cell supernatant has to be determined in future studies. The two hydrophobic domains of the E2 proteins of hepatitis C virus are also too short to form
helices by themselves. Therefore, it has been proposed, for this protein, that the hairpin-like structure of the hydrophobic domain is maintained as long as it is associated with the translocon that transfers the nascent polypeptide from the cytosol to the lumen of the ER. Once the host signal peptidase has cleaved between E2 and p7, the hydrophobic domain may reorientate to an extended structure that allows integration into the lipid bilayer and exposes the carboxy terminus at the cytosolic side of the ER (Op de Beeck & Dubuisson, 2003
). From our results and from the short length of the hydrophobic domain, we propose that the model described for hepatitis C virus E2 also applies to the BVDV E2 protein. On the other hand, the E2 protein of flaviviruses has a longer hydrophobic domain and may have a different topology (Zhang et al., 2003
).
The arginine residue within the membrane anchor of E2 plays a role not only for the formation of a hairpin-like structure during association with the translocon, but is also important for the intracellular retention of E2. The E2(R/A) mutant was efficiently transported to the cell surface. A hydrophilic amino acid within the membrane anchor has been shown to be essential for intracellular localization not only of flavivirus proteins but also for cellular proteins retained in the ER (Bonifacino et al., 1991). Interestingly in Western blot analysis, the E2(R/A) mutant gave a stronger signal than the parental protein. It is not known whether the arginine residue affects ectodomain conformation detected by the antibody or whether it renders the protein more sensitive to proteolytic degradation. A different effect was observed when arginine residues were added to the membrane anchor. This modification only had a marginal effect on intracellular retention of E2. Analysis of the mutant E2(TdelRR) by Western blot resulted in a stronger signal than the parental protein. At this amino acid position, the presence of arginine residues is obviously favourable for optimal detection of E2. Many membrane proteins contain a charged amino acid at the transition of the membrane anchor to the cytoplasmic tail (Dalbey, 1990
). The reason why a charged amino acid at this location is absent in the E2 protein may be related to the function of the transmembrane domain as a signal peptide. Possibly, arginine residues at the end of the membrane anchor have a negative effect on the cleavage by signal peptidase or on reorientation of the hydrophobic domain.
Our data on the intracellular retention of the E2 protein are in agreement with results reported for the homologous protein of other members of the family Flaviviridae. In the case of another pestivirus, classical swine fever virus, a chimeric E2 protein containing the membrane anchor/cytoplasmic tail of an influenza A virus haemagglutinin was incorporated into recombinant influenza virions, suggesting that this chimera was transported to the plasma membrane, the site of influenza virus maturation (Zhou et al., 1998). Our data are also consistent with the information available for BVDV-infected cells. However, they are in contrast with data on BVDV E2 protein expressed by recombinant VSV or baculovirus. Using these expression systems it has been reported that E2 protein is expressed on the cell surface (Kweon et al., 1997
; Grigera et al., 2000
). The reason for this discrepancy is not clear. One reason may be that upon overexpression of the E2 protein the cellular machinery for intracellular retention may be saturated and some E2 protein may be transported to the cell surface. This phenomenon has been reported also for E1 and E2 proteins of hepatitis C virus (Bartosch et al., 2003
; Drummer et al., 2003
; Hsu et al., 2003
).
Retention of viral glycoproteins and virus morphogenesis at intracellular compartments may provide some advantage for pestiviruses. The absence of viral glycoproteins from the cell surface makes it more difficult for the immune system to detect foreign antigen and possibly prolongs the survival time of infected cells. Maybe this also contributes to the characteristic property of BVDV to cause intrauterine infection (Bolin et al., 1985). In future studies we plan to analyse mutant glycoproteins that are transported to the cell surface in the context of a BVDV infection. It will be interesting to see whether such mutations affect (i) the site of virus maturation, (ii) the incorporation into virus particles, (iii) the survival time of infected cells, and (iv) the capacity to cause intrauterine infections.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Baker, J. C. (1995). The clinical manifestations of bovine viral diarrhea infection. Vet Clin North Am Food Anim Pract 11, 425445.[Medline]
Bartosch, B., Dubuisson, J. & Cosset, F. L. (2003). Infectious hepatitis C virus pseudo-particles containing functional E1-E2 envelope protein complexes. J Exp Med 197, 633642.
Bolin, S. R., McClurkin, A. W. & Coria, M. F. (1985). Frequency of persistent bovine viral diarrhea virus infection in selected cattle herds. Am J Vet Res 46, 23852387.[Medline]
Bolin, S. R., Moennig, V., Kelso-Gourley, V. & Ridpath, J. (1988). Monoclonal antibodies with neutralising activity segregate isolates of bovine viral diarrhea virus into groups. Arch Virol 99, 117123.[Medline]
Bonifacino, J. S., Cosson, P., Shah, N. & Klausner, R. D. (1991). Role of potentially charged transmembrane residues in targeting proteins for retention and degradation within the endoplasmic reticulum. EMBO J 10, 27832793.[Abstract]
Buchholz, U. J., Finke, S. & Conzelmann, K.-K. (1999). Generation of bovine respiratory syncytial virus (BRSV) from cDNA: BRSV NS2 is not essential for virus replication in tissue culture, and the human RSV leader region acts as a functional BRSV genome promoter. J Virol 73, 251259.
Cocquerel, L., Meunier, J. C., Pillez, A., Wychowski, C. & Dubuisson, J. (1998). A retention signal necessary and sufficient for endoplasmic reticulum localisation maps to the transmembrane domain of hepatitis C virus glycoprotein E2. J Virol 72, 21832191.
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 localisation, and assembly of these envelope proteins. J Virol 74, 36233633.
Cocquerel, L., Op de Beeck, A., 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, 28932902.
Compans, R. W. & Herrler, G. (1999). Epithelial cells and viral infection. In Mucosal Immunology, pp. 671683. Edited by P. L. Ogra, J. Mestecky, M. E. Lamm, W. Strober, J. Bienenstock & J. R. McGhee. New York: Academic Press.
Dalbey, R. E. (1990). Positively charged residues are important determinants of membrane topology. Trends Biochem Sci 15, 253257.[CrossRef][Medline]
Donis, R. O., Corapi, W. V. & Dubovi, E. J. (1988). Neutralizing monoclonal antibodies to bovine viral diarrhoea virus bind to the 56K to 58K glycoprotein. J Gen Virol 69, 7786.[Abstract]
Drummer, H. E., Maerz, A. & Poumbourios, P. (2003). Cell surface expression of functional hepatitis C virus E1 and E2 glycoproteins. FEBS Lett 546, 385390.[CrossRef][Medline]
Elbers, K., Tautz, N., Becher, P., Stoll, D., Rumenapf, T. & Thiel, H. J. (1996). Processing in the pestivirus E2-NS2 region: identification of proteins p7 and E2p7. J Virol 70, 41314135.[Abstract]
Gray, E. W. & Nettleton, P. F. (1987). The ultrastructure of cell cultures infected with border disease and bovine virus diarrhoea viruses. J Gen Virol 68, 23392346.[Abstract]
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, 20152019.[Abstract]
Grigera, P. R., Marzocca, M. P., Capozzo, A. V. E., Buonocore, L., Donis, R. O. & Rose, J. K. (2000). Presence of bovine viral diarrhea virus (BVDV) E2 glycoprotein in VSV recombinant particles and induction of neutralizing BVDV antibodies in mice. Virus Res 69, 315.[CrossRef][Medline]
Grummer, B., Beer, M., Liebler-Tenorio, E. & Greiser-Wilke, I. (2001). Localisation of viral proteins in cells infected with bovine viral diarrhoea virus. J Gen Virol 82, 25972605.
Harada, T., Tautz, N. & Thiel, H. J. (2000). E2-p7 region of the bovine viral diarrhea virus polyprotein: processing and functional studies. J Virol 74, 94989506.
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 U S A 100, 72717276.
Kweon, C.-H., Yoon, Y.-D., An, S.-H. & Lee, Y.-S. (1997). Expression of envelope protein (E2) of bovine viral diarrhea virus in insect cells. J Vet Med Sci 59, 415419.[CrossRef][Medline]
Magar, R., Minocha, H. C. & Lecomte, J. (1988). Bovine viral diarrhea virus proteins: heterogeneity of cytopathogenic and noncytopathogenic strains and evidence of a 53K glycoprotein neutralization epitope. Vet Microbiol 16, 303314.[CrossRef][Medline]
Meyers, G. & Thiel, H. J. (1996). Molecular characterisation of pestiviruses. Adv Virus Res 47, 53118.[Medline]
Monne, M., Nilsson, I., Elofsson, A. & von Heijne, G. (1999). Turns in transmembrane helices: determination of the minimal length of a "helical hairpin" and derivation of a fine-grained turn propensity scale. J Mol Biol 293, 807814.[CrossRef][Medline]
Moss, B., Elroy-Stein, O., Mizukami, T., Alexander, W. A. & Fuerst, T. R. (1990). Product review. New mammalian expression vectors. Nature 348, 9192.[CrossRef][Medline]
Nishimura, N. & Balch, W. E. (1997). A di-acidic signal required for selective export from the endoplasmic reticulum. Science 277, 556558.
Op de Beeck, A. & Dubuisson, J. (2003). Topology of hepatitis C virus envelope glycoproteins. Rev Med Virol 13, 233241.[CrossRef][Medline]
Orban, S., Liess, B., Hafez, S. M., Frey, H.-R., Blindow, H. & Sasse-Patzer, B. (1983). Studies on transplacental transmissibility of bovine virus diarrhoea (BVD) vaccine virus. I. Inoculation of pregnant cows 15 to 90 days before parturition (190th to 265th day of gestation). Zentbl Vetmed Reihe B 30, 619634.
Rümenapf, T., Unger, G., Strauss, J. H. & Thiel, H.-J. (1993). Processing of the envelope glycoproteins of pestiviruses. J Virol 67, 32883294.[Abstract]
Sevier, C. S., Weisz, O. A., Davis, M. & Machamer, C. E. (2000). Efficient export of the vesicular stomatitis virus G protein from the endoplasmic reticulum requires a signal in the cytoplasmic tail that includes both tyrosine-based and di-acidic motifs. Mol Biol Cell 11, 1322.
Weiland, F., Weiland, E., Unger, G., Saalmuller, A. & Thiel, H. J. (1999). Localisation of pestiviral envelope proteins ERNS and E2 at the cell surface and on isolated particles. J Gen Virol 80, 11571165.[Abstract]
Wengler, G., Bradley, D. W., Collett, M. S., Heinz, F. X., Schlesinger, R. W. & Strauss, J. H. (1995). Flaviviridae. In Virus Taxonomy. Sixth Report of the International Commitee on Taxonomy of Viruses, pp. 415427. Edited by F. A. Murphy, C. M. Fauquet, D. H. L. Bishop, S. A. Ghabrial, A. W. Jarvis, G. P. Martelli, M. A. Mayo & M. D. Summers. New York: Springer.
Zhang, W., Chipman, P. R., Corver, J. & 7 other authors (2003). Visualization of membrane protein domains by cryo-electron microscopy of dengue virus. Nat Struct Biol 10, 907912.[CrossRef][Medline]
Zhou, Y., König, M., Hobom, G. & Neumeier, E. (1998). Membrane-anchored incorporation of a foreign protein in recombinant influenza virions. Virology 246, 8394.[CrossRef][Medline]
Zimmer, G., Budz, L. & Herrler, G. (2001a). Proteolytic activation of respiratory syncytial virus fusion protein. Cleavage at two furin consensus sequences. J Biol Chem 276, 3164231650.
Zimmer, G., Trotz, I. & Herrler, G. (2001b). N-glycans of F protein differentially affect fusion activity of human respiratory syncytial virus. J Virol 75, 47444751.
Zimmer, G., Conzelmann, K.-K. & Herrler, G. (2002). Cleavage at the furin consensus sequence RAR/KR(109) and presence of the intervening peptide of the respiratory syncytial virus fusion protein are dispensable for virus replication in cell culture. J Virol 76, 92189224.
Received 20 October 2003;
accepted 13 January 2004.