MRC Virology Unit, Institute of Virology, Church Street, Glasgow G11 5JR, UK1
Author for correspondence: Arvind Patel. Fax +44 141 337 2236. e-mail a.patel{at}vir.gla.ac.uk
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Abstract |
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Introduction |
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All three surface proteins undergo post-translational modification. Each carries a partially glycosylated site at Asn-146 of the S domain and glycosylation also occurs in the preS2 region. A potential N-linked glycosylation site (aa 4 or 15) in the preS1 domain of L is not used (Bruss et al., 1996a ; Heermann et al., 1984
). However, L is myristylated at Gly-2 in the preS1 domain, and this fatty acid moiety has a profound effect on the in vitro infectivity of the virion (Bruss et al., 1996b
). Furthermore, the myristylation at Gly-2 is essential for the maintenance of infectivity of HBV in vivo, although Gly-2 to Ala substitution did not have any apparent effect on the presentation of the preS1 domain on the virion surface, or the particle morphology (Bruss et al., 1996b
).
The anchorage and translocation of the surface proteins into the ER are achieved by the signal sequences in the S domain, which have multispanning transmembrane topologies (Berting et al., 1995 ; Eble et al., 1986
, 1987
, 1990
; Gerlich et al., 1993
, Guerrero et al., 1988
, Sheu & Lo, 1994
). Current models predict that S traverses the ER membrane at least twice so that both the termini as well as the region between the transmembrane domains II and III are exposed in the lumen (and thus externally on the mature virion) and the other regions of S are disposed to the cytosolic side (Guerrero et al., 1988
; Löffler-Mary et al., 2000
; Prange & Streeck, 1995
; Stirk et al., 1992
). The topology of the S domain in HBV M and L is predicted to be similar to that of the S protein (Bruss et al., 1996a
; Gerlich et al., 1993
; Prange & Streeck, 1995
; Stirk et al., 1992
). In addition, the preS2 domain of M is thought to be located in the ER lumen (Eble et al., 1990
). Interestingly, L differs from M and S in that in approximately half of the L molecules the preS1 and preS2 (preS) regions remain cytosolic and therefore located internally in the virion providing a scaffolding function by interaction with the nucleocapsid. In the other half of the L molecules the preS region is lumenally disposed and consequently exposed at the surface of the virion (Bruss et al., 1994
, 1996a
; Gerlich et al., 1993
; Ostapchuk et al., 1994
; Prange & Streeck, 1995
). This dual topology of L would be consistent with its presumed multifunctional role, in cell receptor binding by the surface exposed region, and in interaction with viral nucleocapsids and in other intracellular functions by the internalized region of the preS1 domain in infected cells (Bruss & Vieluf, 1995
; Hildt et al., 1996
; Klingmuller & Schaller, 1993
; Neurath et al., 1986
, Rothmann et al., 1998
; Ryu et al., 2000
).
The topology models for HBV surface antigens described above depict no overall differences in the structure of the S domain of L and M compared with S. However, evidence to support this hypothesis has been lacking. In this paper, we provide the first experimental evidence for the existence of structural differences between the S domain of L and the S protein. In addition, we have examined the intracellular distribution of the HBV surface proteins and their interaction with each other.
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Methods |
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Generation of plasmid constructs and recombinant vaccinia viruses expressing HBV surface proteins.
The genes encoding the HBV surface proteins were cloned immediately downstream from a strong synthetic late vaccinia virus promoter into the transfer vector pMJ601 (Davison & Moss, 1990 ) as described below. The portion of the HBV genome encoding L was amplified from the serum of a patient infected with HBV (subtype adw) by PCR. The resulting PCR product carrying the L open reading frame (ORF) was cloned into pMJ601. The nucleotide sequence encoding the M ORF was excised from the L construct described above using appropriate restriction enzymes and inserted into pMJ601. The S gene was subcloned from plasmid pRK5/HBsAg (a kind gift from W. F. Carman, Glasgow, UK) into pMJ601. The HBV sequences in pMJ601 were inserted into the thymidine kinase gene of vaccinia virus strain WR by homologous recombination, and the recombinant viruses vL, vM and vS expressing L, M and S protein, respectively, were isolated as described (Davison & Moss, 1990
).
The nucleotide sequence encoding the HBV preS1 domain (aa 1108) of L was cloned into the bacterial expression vector pQE30 (Qiagen). The resulting plasmid, pQES1, carries sequences encoding aa MRGSHHHHHHGS followed by aa 1108 representing the HBV preS1 domain. This protein was expressed in E. coli, purified, and used to immunize rabbits for generation of antisera.
Immunoprecipitations.
Cells were infected at an m.o.i. of 10 p.f.u. per cell with recombinant vaccinia viruses. Where required, cells were incubated with 10 µg/ml tunicamycin for 3 h prior to infection to inhibit glycosylation. For radiolabelling, cells were washed with PBS at 5 h post-infection and incubated in methionine-free Eagles medium containing 50 µCi/ml [35S]methionine for 16 h. The culture supernatant was aspirated and clarified at 13000 r.p.m. in a benchtop centrifuge to remove cellular debris. The clarified medium containing radiolabelled proteins was adjusted to 20 mM TrisHCl, pH 7·4, 150 mM NaCl; 1 mM EDTA, 0·5% Triton X-100 (final concentrations) and used in immunoprecipitation assays described below. Cell monolayers were washed three times with PBS and the cells were dislodged and pelleted by brief centrifugation. The cells were resuspended in lysis buffer (see above) and incubated on ice for 30 min. The cell nuclei were removed by centrifugation and the lysates used for immunoprecipitation of proteins as follows. The clarified radiolabelled medium of cell lysate was incubated with appropriate monoclonal or polyclonal antibodies at 4 °C overnight. The immune complexes were then precipitated using protein ASepharose. Following washing with lysis buffer, the immune complexes were released from protein ASepharose using SDSPAGE denaturation buffer and boiled for 3 min. Samples were fractionated by SDSPAGE (10% polyacrylamide) and the labelled proteins detected using a Bio-Rad Personal FX phosphorimager.
Generation of anti-preS1 antisera.
Two female New Zealand White rabbits were immunized intramuscularly with 100 µg of bacterially expressed and purified preS1 domain in Freunds complete adjuvant. The rabbits were boosted at 14 day intervals with 100 µg of the preS1 domain in Freunds incomplete adjuvant. Ten days after the last boost, the rabbits were bled out and antisera (R142 and R143) prepared. Antiserum R143 was used in experiments described in this paper.
Monoclonal antibodies.
The anti-S MAb 6B1 (a generous gift from C. McCaughey and H. ONeill, Queens University, Belfast, UK) was generated in mice immunized with the anti-HBV vaccine Engerix (GlaxoSmithKline) essentially as described previously (Coyle et al., 1992 ). MAbs H35, H53 and H166 (Chen et al., 1996
) were a kind gift from R. Decker, Abbott Laboratories, USA. MAbs 2-12F2 (anti-M) and G1/93 (anti-ERGIC p53) (Schweizer et al., 1991
) were kindly supplied by W. H. Gerlich (Institute of Medical Virology, Giessen, Germany) and H.-P. Hauri (Dept of Pharmacology, University of Basel, Switzerland), respectively.
Confocal microscopy.
Cells were grown on coverslips to 5060% confluence prior to experimental procedures. They were infected with vaccinia virus strain WR or recombinant viruses expressing HBV surface proteins at a multiplicity of 0·5 p.f.u. per cell and incubated overnight at 37 °C. The cell monolayers were washed with PBS, fixed with methanol at -20 °C and washed with PBS containing 0·05% Tween 20 (PBST). The permeabilized cells were then incubated with appropriate primary antibodies described in the text. The bound antibodies were detected with appropriate secondary antibodies conjugated to FITC (Sigma) or FluorLink Cy5 (Amersham). Concanavalin AFITC (Sigma) was used at appropriate dilution to stain internal cellular membranes. The coverslips carrying the labelled cells were mounted on glass slides with a drop of Citifluor anti-fade reagent, and examined with a Zeiss laser scanning microscope; the images were analysed using the LSM5150 software.
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Results |
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To confirm the interaction between L and S, we performed immunoprecipitation assays. HepG2 cells were infected with vaccinia viruses (m.o.i. of 10 p.f.u. per cell) and the expressed proteins labelled with [35S]methionine. Radiolabelled proteins from infected cell lysates and medium were immunoprecipitated using MAb 6B1 or anti-preS1 polyclonal antiserum R143 followed by SDSPAGE. The anti-preS1 antiserum R143 readily immunoprecipitated the 42 kDa L from vL-infected cells (Fig. 3 A, lane 2). Not surprisingly, L was not found in the medium of vL-infected cells, as when expressed alone it is retained in the ER (Fig. 3A
, lane 6). Interestingly, both L and the two forms of S proteins were co-immunoprecipitated by R143 from the medium as well as lysates of cells co-infected with vL+vS (Fig. 3A
, lanes 7 and 3, respectively), indicating that the interaction between these proteins is required for secretion of L from the cells. These results are consistent with our immunofluorescence data, which showed that both L and S co-localize in the cytoplasm.
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Discussion |
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In agreement with previously published observations (Cheng et al., 1986 ; Chisari et al., 1987
; Persing et al., 1986
; Xu et al., 1997
), our confocal microscopy and immunoprecipitation data show that L localizes predominantly to the ERGIC compartment and is not secreted. In keeping with their secretory characteristic (Huovila et al., 1992
; Molnar-Kimber et al., 1988
), the intracellular distribution of S and M proteins was distinct from that of L in that both S and M co-localized with intracellular membranes, as defined by co-staining with Con A.
The unique characteristic of the anti-S MAb 6B1 allowed us to investigate the intracellular distribution of L and S proteins. In co-expressing cells, L re-localized from the ERGIC to the membrane-associated S protein indicating that these proteins may interact with each other. Presumably this interaction occurs via the disulfide linkages in the transmembrane regions of the S domains and is required for secretion of L. This was confirmed in immunoprecipitation assays where the anti-preS1 antiserum specifically co-immunoprecipitated S from the medium and lysates of cells co-expressing L and S. These results are consistent with those published previously where the surface antigens have been shown to form heteromultimeric complexes upon co-expression (Cheng et al., 1986 ; Chisari et al., 1987
; Molnar-Kimber et al., 1988
; Wunderlich & Bruss, 1996
). Interestingly, the anti-S MAb 6B1 failed to co-immunoprecipitate SL complex from co-expressing cells, indicating that the recognition of S by MAb 6B1 is abrogated when S and L are interacting. This suggests that the binding site of the MAb 6B1 may be occluded or altered (possibly due to conformational changes) when S is interacting with L protein.
To our knowledge, this is the first report that describes an immunological reagent capable of selectively discriminating S from the S domain of L (but not M). The recognition by MAb 6B1 of the conformational epitope on S is not dependent on glycosylation. When expressed in the presence of tunicamycin, S, but not L, was efficiently detected by the antibody both in immunofluorescence and immunoprecipitation assays (data not shown). Thus, the ability of MAb 6B1 to recognize S but not L is not due to possible differential glycosylation of the two proteins. The topology models for HBV surface antigens proposed by various groups do not predict overall differences in the S domain of L and M to the S protein (Bruss et al., 1996a ; Gerlich et al., 1993
; Prange & Streeck, 1995
; Stirk et al., 1992
). However, based on the results presented here, we postulate that the S domain in L has a different topology to the S protein. The topological differences are likely due to a rearrangement of the transmembrane regions in the S domain of L, resulting in a difference in the conformational epitopes displayed by L, and therefore resulting in non-recognition by MAb 6B1. It is possible that the insertion of the preS1 domain into the lipid bilayer at Gly-2 via the myristic acid moiety (Bruss et al., 1996b
) may affect the spatial arrangement of the transmembrane regions, therefore contributing to a difference in the conformation of S and L upon interaction of these two proteins. It is also feasible that the arrangement of the transmembrane
-helices in S and L differ, but the association of the disulphide linkages forming the intermolecular bonds remains conserved to allow oligomerization of the envelope proteins. It is unlikely that the failure of MAb 6B1 to recognize L is due to occlusion of its epitope by the preS region, since in this case the dual topology of L would ensure MAb 6B1 recognition of at least 50% of the L molecules. Interestingly, Paulij et al. (1999)
recently identified a unique anti-S monoclonal antibody epitope within the so-called transmembrane domain III, leading the authors to propose that this domain may not span the membrane at all, but rather is exposed in the lumen. More recently, using protease protection and immunological assays, Grgacic et al. (2000)
showed that the presumed cytoplasmic loop between transmembrane 1 and 2 of duck HBV is actually membrane embedded and protrudes to the particle surface.
In conclusion, numerous studies on the biosynthesis and maturation of hepadnaviral surface antigens have been performed over the years, leading researchers to propose topology models of these proteins. However, in the absence of any X-ray crystallographic data to support a model of the structure, the nature of the presentation of these proteins on the virion surface remains unclear. The availability of MAb 6B1, with its novel characteristics, should further facilitate the study of the conformational nature of these proteins, without the use of potentially disruptive techniques such as the insertion of epitope tags or fluorescent protein domains into the surface antigens.
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Acknowledgments |
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References |
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Bruss, V. & Ganem, D. (1991). The role of envelope proteins in hepatitis B virus assembly. Proceedings of the National Academy of Sciences, USA 88, 1059-1063.[Abstract]
Bruss, V. & Vieluf, K. (1995). Functions of the internal pre-S domain of the large surface protein in hepatitis B virus particle morphogenesis. Journal of Virology 69, 6652-6657.[Abstract]
Bruss, V., Gerhardt, E., Vieluf, K. & Wunderlich, G. (1996a). Functions of the large hepatitis B virus surface protein in viral particle morphogenesis. Intervirology 39, 23-31.[Medline]
Bruss, V., Hagelstein, J., Gerhardt, E. & Galle, P. R. (1996b). Myristylation of the large surface protein is required for hepatitis B virus in vitro infectivity. Virology 218, 396-409.[Medline]
Bruss, V., Lu, X., Thomssen, R. & Gerlich, W. H. (1994). Post-translational alterations in transmembrane topology of the hepatitis B virus large envelope protein. EMBO Journal 13, 2273-2279.[Abstract]
Chen, Y. C., Delbrook, K., Dealwis, C., Mimms, L., Mushahwar, I. K. & Mandecki, W. (1996). Discontinuous epitopes of hepatitis B surface antigen derived from a filamentous phage peptide library. Proceedings of the National Academy of Sciences, USA 93, 1997-2001.
Cheng, K. C., Smith, G. L. & Moss, B. (1986). Hepatitis B virus large surface protein is not secreted but is immunogenic when selectively expressed by recombinant vaccinia virus. Journal of Virology 60, 337-344.[Medline]
Chisari, F. V., Filippi, P., Buras, J., McLachlan, A., Popper, H., Pinkert, C. A., Palmiter, R. D. & Brinster, R. L. (1987). Structural and pathological effects of synthesis of hepatitis B virus large envelope polypeptide in transgenic mice. Proceedings of the National Academy of Sciences, USA 84, 6909-6913.[Abstract]
Coyle, P. V., Wyatt, D., McCaughey, C. & ONeill, H. J. (1992). A simple standardized protocol for the production of monoclonal antibodies against viral and bacterial antigens. Journal of Immunological Methods 153, 81-84.[Medline]
Davison, A. J. & Moss, B. (1990). New vaccinia virus recombination plasmids incorporating a synthetic late promoter for high-level expression of foreign proteins. Nucleic Acids Research 18, 4285-4286.[Medline]
Eble, B. E., Lingappa, V. R. & Ganem, D. (1986). Hepatitis B surface antigen: an unusual secreted protein initially synthesized as a transmembrane polypeptide. Molecular and Cellular Biology 6, 1454-1463.[Medline]
Eble, B. E., Macrae, D. R., Lingappa, V. R. & Ganem, D. (1987). Multiple topogenic sequences determine the transmembrane orientation of hepatitis-B surface-antigen. Molecular and Cellular Biology 7, 3591-3601.[Medline]
Eble, B. E., Lingappa, V. R. & Ganem, D. (1990). The N-terminal (pre-S2) domain of a hepatitis B virus surface glycoprotein is translocated across membranes by downstream signal sequences. Journal of Virology 64, 1414-1419.[Medline]
Gerlich, W. H., Lu, X. & Heermann, K. H. (1993). Studies on the attachment and penetration of hepatitis B virus. Journal of Hepatology 17, S10-14.[Medline]
Grgacic, E. V., Kuhn, C. & Schaller, H. (2000). Hepadnavirus envelope topology: insertion of a loop region in the membrane and role of S in L protein translocation. Journal of Virology 74, 2455-2458.
Guerrero, E., Gavilanes, F. & Peterson, D. L. (1988). Model for protein arrangement in HBsAg particles based on physical and chemical studies. In Viral Hepatitis and liver Disease , pp. 606-613. Edited by A. J. Zuckerman. New York:Liss.
Heermann, K. H. & Gerlich, W. H. (1991). Surface proteins of hepatitis B viruses. In Molecular Biology of Hepatitis B Virus , pp. 109-143. Edited by A. McLachlan. Boca Raton, FL:CRC Press.
Heermann, K. H., Goldmann, U., Schwartz, W., Seyffarth, T., Baumgarten, H. & Gerlich, W. H. (1984). Large surface proteins of hepatitis B virus containing the pre-s sequence. Journal of Virology 52, 396-402.[Medline]
Hildt, E., Saher, G., Bruss, V. & Hofschneider, P. H. (1996). The hepatitis B virus large surface protein (LHBs) is a transcriptional activator. Virology 225, 235-239.[Medline]
Huovila, A. P., Eder, A. M. & Fuller, S. D. (1992). Hepatitis B surface antigen assembles in a post-ER, pre-Golgi compartment. Journal of Cell Biology 118, 1305-1320.[Abstract]
Klingmuller, U. & Schaller, H. (1993). Hepadnavirus infection requires interaction between the viral pre-S domain and a specific hepatocellular receptor. Journal of Virology 67, 7414-7422.[Abstract]
Löffler-Mary, H., Dumortier, J., Klentsch-Zimmer, C. & Prange, R. (2000). Hepatitis B virus assembly is sensitive to changes in the cytosolic S loop of the envelope proteins. Virology 270, 358-367.[Medline]
Molnar-Kimber, K. L., Jarocki-Witek, V., Dheer, S. K., Vernon, S. K., Conley, A. J., Davis, A. R. & Hung, P. P. (1988). Distinctive properties of the hepatitis B virus envelope proteins. Journal of Virology 62, 407-416.[Medline]
Nemeckova, S., Kunke, D., Press, M., Nemecek, V. & Kutinova, L. (1994). A carboxy-terminal portion of the preS1 domain of hepatitis B virus (HBV) occasioned retention in endoplasmic reticulum of HBV envelope proteins expressed by recombinant vaccinia viruses. Virology 202, 1024-1027.[Medline]
Neurath, A. R., Kent, S. B., Strick, N. & Parker, K. (1986). Identification and chemical synthesis of a host cell receptor binding site on hepatitis B virus. Cell 46, 429-436.[Medline]
Ostapchuk, P., Hearing, P. & Ganem, D. (1994). A dramatic shift in the transmembrane topology of a viral envelope glycoprotein accompanies hepatitis B viral morphogenesis. EMBO Journal 13, 1048-1057.[Abstract]
Ou, J. H. & Rutter, W. J. (1987). Regulation of secretion of the hepatitis B virus major surface antigen by the preS-1 protein. Journal of Virology 61, 782-786.[Medline]
Paulij, W. P., de Wit, P. L., Sunnen, C. M., van Roosmalen, M. H., Petersen-van Ettekoven, A., Cooreman, M. P. & Heijtink, R. A. (1999). Localization of a unique hepatitis B virus epitope sheds new light on the structure of hepatitis B virus surface antigen. Journal of General Virology 80, 2121-2126.
Persing, D. H., Varmus, H. E. & Ganem, D. (1986). Inhibition of secretion of hepatitis B surface antigen by a related presurface polypeptide. Science 234, 1388-1391.[Medline]
Prange, R. & Streeck, R. E. (1995). Novel transmembrane topology of the hepatitis B virus envelope proteins. EMBO Journal 14, 247-256.[Abstract]
Rothmann, K., Schnolzer, M., Radziwill, G., Hildt, E., Moelling, K. & Schaller, H. (1998). Host cell-virus cross talk: phosphorylation of a hepatitis B virus envelope protein mediates intracellular signaling. Journal of Virology 72, 10138-10147.
Ryu, C. J., Cho, D. Y., Gripon, P., Kim, H. S., Guguen-Guillouzo, C. & Hong, H. J. (2000). An 80-kilodalton protein that binds to the pre-S1 domain of hepatitis B virus. Journal of Virology 74, 110-116.
Schweizer, A., Matter, K., Ketcham, C. M. & Hauri, H. P. (1991). The isolated ERGolgi intermediate compartment exhibits properties that are different from ER and cis-Golgi. Journal of Cell Biology 113, 45-54.[Abstract]
Sheu, S. Y. & Lo, S. J. (1994). Biogenesis of the hepatitis B viral middle (M) surface protein in a human hepatoma cell line: demonstration of an alternative secretion pathway. Journal of General Virology 75, 3031-3039.[Abstract]
Stirk, H. J., Thornton, J. M. & Howard, C. R. (1992). A topological model for hepatitis-B surface-antigen. Intervirology 33, 148-158.[Medline]
Ueda, K., Tsurimoto, T. & Matsubara, K. (1991). Three envelope proteins of hepatitis B virus: large S, middle S, and major S proteins needed for the formation of Dane particles. Journal of Virology 65, 3521-3529.[Medline]
Wunderlich, G. & Bruss, V. (1996). Characterization of early hepatitis B virus surface protein oligomers. Archives of Virology 141, 1191-1205.[Medline]
Xu, Z. C., Bruss, V. & Yen, T. S. B. (1997). Formation of intracellular particles by hepatitis B virus large surface protein. Journal of Virology 71, 5487-5494.[Abstract]
Received 12 February 2001;
accepted 18 April 2001.