Institut für Virologie, Universitätsklinikum Essen, Hufelandstraße 55, 45122 Essen, Germany
Correspondence
M. Lu
mengji.lu{at}uni-essen.de
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ABSTRACT |
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Present address: Institute for Medical Microbiology and Immunology, University of Bonn, Sigmund-Freud-Strasse 25, 53105 Bonn, Germany.
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INTRODUCTION |
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The short N-terminal preS2 region on the M-sAg of mammalian hepadnaviruses has various glycosylation sites. Indeed, M-HBsAg and M-sAg of woodchuck hepatitis virus (WHV) possess N- and O-glycans (Tolle et al., 1998; Schmitt et al., 1999
). Although the precise function of the glycosylation of the M-sAg of WHV (M-WHsAg) has not been studied, it appears that impaired glycosylation of M-WHsAg may inhibit virus assembly. Block et al. (1998)
showed that an inhibitor of
-glucosidase was able to block M-WHsAg processing and intracellular trafficking, thus leading to inhibition of virus assembly. Treatment with an
-glucosidase inhibitor suppressed the production of enveloped virions in woodchucks chronically infected with WHV (Block et al., 1998
). Therefore, the glycosylation of viral proteins may represent a target for antiviral therapies.
The sites of glycosylation on the preS2 region of M-WHsAg (WHVpreS2 region) have not been precisely identified. The asparagine (Asn) residue at amino acid (aa) 3 is followed by glutamine-threonine (Gln-Thr) and therefore represents a potential site for N-glycosylation (Tolle et al., 1998). In this work, a substitution of Thr to Asn at aa 5 of the WHVpreS2 region on the M-WHsAg by site-directed mutagenesis eliminated this potential N-glycolysation site. It could be shown that this amino acid substitution impaired glycosylation of M-WHsAg and led to an aberrant accumulation of the mutated protein in the cellular compartment. These results confirmed the assumption that the sequence Asn-Gln-Thr at aa 35 of the WHVpreS2 region represents an N-glycosylation site. Furthermore, correct N-glycosylation of M-WHsAg appears to be essential for its intracellular trafficking.
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METHODS |
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In vitro translation.
In vitro translation of the chimeric proteins from pMCS3, pMCS3Ngl and pMCS3preS2 was carried out with the TNT-coupled reticulocyte lysate system (Promega) following the manufacturer's instructions. The translations were performed with and without the microsomal fraction (Promega) to study post-translational modifications of the in vitro-translated products. Further controls were performed in the presence of 2 µg tunicamycin ml-1 for inhibition of N-glycosylation (Sigma) or 2 mM p-nitrophenyl N-acetyl
-D-galactosaminide (Sigma) for inhibition of O-glycosylation.
Transfection of cell lines, indirect immunofluorescence (IF) staining and laser-scanning microscopy.
HepG2 or HeLa cells were cultivated in chamber slides (Nunc) and transfected with the recombinant DNA constructs pMCS3, pMCS3Ngl and pMCS3preS2 with the Effectene Transfection Reagent (Qiagen) following the manufacturer's instructions. For each well of a two-well chamber slide, 2 µg plasmid DNA was used for transfection. Transfected cells were incubated for 2448 h in an incubator at 37 °C and 5 % CO2. Cells were then washed once with PBS, fixed with 50 % methanol (v/v) for 30 min at 4 °C, washed twice with PBS and air-dried. Antibodies against HBsAg and markers of the Golgi apparatus (Golgi-Zone; BioTrend) and the endoplasmic reticulum (ERAB, ER antibody binding protein; BioTrend) were used for IF staining at a dilution of 1 : 100, as recommended by the manufacturer. Cells were incubated with primary antibodies at 37 °C for 1 h, washed twice with PBS and further incubated with appropriate secondary antibodies. Secondary antibodies against rabbit, goat or mouse IgGs labelled with Cy2 or Cy3 (Dianova) were used at a 1 : 200 dilution in PBS containing 0·1 % Evan's blue. After an additional incubation for 1 h at 37 °C, cells were washed twice and covered with mounting medium. HBsAg stained red and the Golgi stained green (see Fig. 4
). Additionally, the ER was stained blue with 100 nM of the ER blue/white tracker (Molecular Probes) 30 min before fixation, according to the manufacturer's protocol. Stained cells were analysed by confocal laser-scanning microscopy (Carl Zeiss).
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Fractionation of cellular compartments.
HepG2 cells were transfected as described above and harvested 4872 h post-transfection. The cells were pelleted at 3000 r.p.m. for 10 min in an Eppendorf 5416 centrifuge at room temperature and the pellet was washed twice with ice-cold PBS. The cells were lysed under native conditions by dounce homogenization and sonification and mounted on to a sucrose gradient (2060 %, w/v) following the recommendations of Rickwood (1992). The cellular compartments were separated by ultracentrifugation at 100 000 g for 4 h. The fractions were collected after centrifugation. For each fraction, both the refractive index and the total amount of protein were determined. According to their density, the fractions were assigned to the cellular compartments ER, Golgi or plasma membrane and spotted on to a nitrocellulose membrane to detect the chimeric proteins by Western blot analysis. The membranes were saturated with fat-free milk powder (10 % in PBS), washed twice with PBS and incubated with primary antibodies against HBsAg, the ER and the Golgi for 1 h at 37 °C. The membranes were washed twice with PBS-T (0·05 % Tween-20 in PBS) and incubated for 1 h with a secondary antibody coupled to alkaline phosphatase. Detection of bound antibodies was carried out using NBT/BCIP.
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RESULTS |
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The construct pMCS3 encoded the wild-type WHVpreS2 sequence containing the potential glycosylation site Asn-Gln-Thr at aa 35 (Fig. 1). In the construct pMCS3Ngl, the codon for Thr at aa 5 was changed to Asn by site-directed mutagenesis, thus eliminating the potential Asn-Gln-Thr glycosylation site. As an additional control, the preS2 start codon was mutated by site-directed mutagenesis in pMCS3
preS2, which thus encoded a chimeric protein comprised of aa 1116 of WHsAg and aa 121226 of HBsAg.
Mutation of the WHVpreS2 region affects the glycosylation of M-sAg
The chimeric proteins encoded by plasmids pMSC3, pMSC3Ngl and pMSC3preS2 were in vitro translated using the TNT-coupled reticulocyte lysate system (Promega). Microsomal fractions were added to the reactions to study post-translational modification of translation products. In addition, translation reactions were carried out in the presence of the glycosylation inhibitors tunicamycin and p-nitrophenyl-N-acetyl-
-D-galactosaminide, which block N- and O-glycosylation, respectively. The in vitro translation products were subjected to SDS-PAGE and visualized by autoradiography (Fig. 2
).
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The mutated M-sAg has an abnormal intracellular distribution
Glycosylation plays an important role in intracellular trafficking and localization of proteins. Therefore, we examined whether mutation of the glycosylation site in the WHVpreS2 domain had any influence on the intracellular localization of the hepadnaviral M-sAg. HepG2 or BHK cells were transfected with the plasmids pMCS3, pMCS3Ngl and pMCS3preS2. The cells were fixed and stained with an anti-HBs mAb or a polyclonal antiserum to the WHVpreS2 region (Fig. 3
). IF staining of MCSAg3 with antibodies to the a determinant or to the WHVpreS region (Fig. 3
, first row) showed a diffuse distribution of the chimeric proteins in the cytoplasm, which is regularly observed for wild-type middle surface antigens of hepadnaviruses (Zheng et al., 2002
). Incubation of transfected cells with the glycosylation inhibitors tunicamycin and p-nitrophenyl N-acetyl
-D-galactosaminide led to an abnormal distribution of the wild-type protein MCSAg3 within cells, with the formation of granular structures (Fig. 3
, second row). A similar cellular distribution of mutated MCSAg3Ngl expressed by pMCS3Ngl was found in transfected cells (Fig. 3
, third row). These results strongly suggest that the absence of glycosylation on the WHVpreS2 region has a significant impact on the intracellular trafficking and localization of hepadnaviral surface proteins. The plasmid pMCS3
preS2 expressed only small surface antigens, which were positively stained by anti-HBs antibodies but not by antibodies to the WHVpreS2 region (Fig. 3
, fourth row). Small CSAg3 showed a diffuse distribution within cells, similar to the wild-type MCSAg3.
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Fig. 4 shows the results of the confocal microscopic analysis. In the first row, staining of wild-type MCSAg3, ER and Golgi is shown. MCSAg3 was evenly distributed throughout the ER and Golgi. Small CSAg3 showed a similar distribution (Fig. 4
, pMCS3
preS2). Again, an inhibition of glycosylation, either by inhibitors or by mutation, led to clustering of MCSAg3 in cells (Fig. 4
, pMCS3+inhibitor and pMCS3Ngl). The prominent white colour in the merged staining indicated that the majority of the MCSAg3 was localized in the ER-cis-Golgi region.
The intracellular distribution of MCSAg3 and MCSAg3Ngl was further examined by fractionation of cellular compartments by isopycnic centrifugation on a sucrose gradient. Transient transfected cells expressing wild-type and mutated MCSAg3 were harvested after 48 or 72 h, lysed and loaded on to a 2060 % sucrose gradient. After centrifugation, fractions containing particular cellular compartments the ER, Golgi and plasma membranes were selected and analysed for the presence of wild-type and mutated MCSAg3 (Table 1). While wild-type MCSAg3 was detected in association with all three fractions, mutated MCSAg3Ngl was enriched in the Golgi fraction.
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DISCUSSION |
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Based on these results, it appears that the mutant MCSAg3 did not simply misfold leading to aggregation within the cytoplasm, since the synthesized proteins were transported though the ER. In addition, the mutant MCSAg3Ngl was detected by anti-HBs mAbs. The binding of anti-HBs mAbs to the HBsAg a determinant is sensitive for misfolding of surface antigens. Thus, this finding supports the conclusion that the folding of MCSAg3 was unimpaired by the introduced mutation.
We attempted to analyse the secretion of wild-type and mutant MCSAg3 from transient transfected cells. However, the production and secretion of MCSAg3 was at a relatively low level (Zheng et al., 2002). Though the secretion of the mutant MCSAg3 appeared to be reduced, we could not make an accurate quantitative determination of the amount of secreted protein.
Taken together our results suggest that hepadnavirus middle surface antigens contain glycosylation modifications that are essential for their biosynthesis and intracellular transport to their final destiny. Consistent with previously published data from other authors, inhibition of N-glycosylation leads to aggregation of proteins and therefore might inhibit virus assembly (Lu et al., 1997; Mehta et al., 1997
). Thus, inhibition of glycosylation could serve as a novel therapeutic target, as demonstrated by Block et al. (1998)
.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 23 September 2003;
accepted 12 December 2003.
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