National Institute for Medical Research, Division of Membrane Biology, The Ridgeway, Mill Hill, London NW7 1AA, UK1
Department of Medicine2 and Department of Histopathology5, Imperial College School of Medicine, St Marys Campus, South Wharf Road, London W2 INY, UK
National Institutes of Health, Hepatitis Viruses Section, LID, NIAID, Building 7, Room 206, Bethesda, MD 20892, USA3
Division of Medical and Molecular Genetics, Guys, Kings and St Thomass School of Medicine, London SE1 9RT, UK4
Author for correspondence: Frank Henkler. Present address: Institute for Cell Biology and Immunology, University of Stuttgart, Allmandring 31, 70569 Stuttgart, Germany. Fax +49 711 685 7484. e-mail Frank.Henkler{at}po.uni-stuttgart.de
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Abstract |
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Introduction |
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In this study we have analysed expression and intracellular localization of HBx in transfected cells, using a sensitive and highly specific monoclonal antibody (Henkler et al., 1995 ). Our data indicate that compartmentalization of HBx is more complex than thought previously, and we demonstrate that a significant proportion of cytoplasmic HBx is associated with mitochondria.
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Methods |
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Tissue culture and transfection.
A431 cells were cultured under 5% CO2 in DMEM containing 10% FCS, 50 U/ml penicillin and 50 mg/ml streptomycin, split into six-well plates (5x104 cells per well) and grown on 13 mm cover glasses. Transfections of subconfluent cells were carried out using Lipofectamine (1·5 µg pCMVX and 10 µl of Lipofectamine per 1 ml transfection mix), according to the manufacturers (GIBCO-BRL) instructions. Cells were harvested 24 h post-transfection for analysis by immunofluorescence or Western blotting. Treatment of living cells with MitoTracker-Red (40250 nM), MitoTracker Red CM-H2XRos (80300 nM) or LysoTracker (150 nM) was carried out for 30 min, followed by a thorough wash for 15 min in PBS prior to fixing.
Western blotting.
Twenty µg total protein from cell lysates or 1 µg of GST-fusion proteins was separated by SDSPAGE, blotted onto nitrocellulose and blocked in 5% non-fat milk. Blots were incubated with 16F1 hybridoma supernatant (diluted 1:10) or pAB-X (diluted 1:1000) for 1 h and for a further 1 h with HRP-labelled secondary antibodies. Immunocomplexes were visualized using the Enhanced Chemiluminescence (ECL) detection system (Amersham).
Immunofluorescence and confocal microscopy.
Cells were fixed in cold methanolacetone (30:70, v/v), blocked for 20 min with PBS1% BSA, incubated for 1 h with 16F1 hybridoma supernatant and washed in PBS0.1% Tween 20. Cells were then incubated with a donkey anti-mouse FITC-labelled secondary antiserum (Jackson Immunoresearch Laboratories) for 45 min. Biotinylated BMA was diluted into 16F1 supernatant (1:50) and streptavidinTexas Red (50 µg/ml) was applied with secondary antiserum for co-staining of HBx and the endoplasmic reticulum. Fluorescent specimens were analysed using an Olympus microscope with CCD camera, which was operated by Delta Vision computer software (Applied Precision Inc.). Alternatively, stained cells were examined with a Leica DM RXE confocal microscope.
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Results |
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Using high resolution confocal microscopy at high magnification, mitochondrion-like structures were visualized that showed a strong peripheral staining of HBx (Fig. 5D). In contrast, no or very little X-protein was detectable in the centres. This may indicate that HBx is associated either with the organelle surface, or is localized within the membrane system, but not in the mitochondrial matrix. This hypothesis requires further corroboration and the precise suborganellar distribution remains to be established by electron microscopy.
Associations between HBx and mitochondria were further confirmed in Huh-7 cells (Fig. 6CE
). In agreement with our observations in A431 cells, a substantial fraction of cytoplasmic HBx was associated with mitochondria in cultured liver cells. Our data support a very recent report which suggested that associations occur between HBx and mitochondria in these cells (Rahmani et al., 2000
). However, our experiments do not support the concept that the X-protein is primarily a mitochondrial protein. Firstly, HBx maintained a predominantly nuclear distribution in moderately expressing Huh-7 cells where its cytoplasmic distribution was dispersed. Secondly, a major extramitochondrial fraction of HBx was also found in the cytoplasm, suggesting a differential compartmentalization of cytoplasmic HBx.
High-level expression of HBx leads to aggregation and to an abnormal distribution of mitochondria
Detection of HBx in punctate or aggregated structures raised the question of whether these patterns were related to the proposed compartmentalization of HBx in mitochondria. Although granular HBx was rarely observed at moderate expression levels, these structures were occasionally found, but neither cytoplasmic nor nuclear granules were co-stained by MitoTracker dyes. These observations suggest that the granular staining pattern of HBx is initially not related to an association with mitochondria (examples of granular HBx are indicated with blue arrowheads in Fig. 6). In contrast, a partial co-aggregation of HBx and mitochondria was often observed in highly expressing cells, which showed a granular and predominantly cytoplasmic staining pattern (Fig. 7
). However, the distribution of mitochondria was not altered in cytoplasmic areas where no granular or aggregated HBx was detected (Fig. 7B
). Again, this observation indicates a distinction between granular punctate detection of HBx and its localization in mitochondria. In a very recent report, HBx was shown to cause aggregation of mitochondria at the nuclear periphery, which led to induction of apoptosis (Takada et al., 1999
). However, our experiments suggest that co-aggregation of X-protein and mitochondria requires high expression levels and is unlikely to occur when the protein is expressed at the low levels expected in HBV infection.
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Discussion |
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In this study, we analysed the intracellular localization of HBx and showed that the protein is primarily localized in the nucleus at low expression levels. In contrast, at elevated levels HBx accumulated in the cytoplasm, leading to a proportional decrease of its nuclear fraction. This indicates further that the capacity for nuclear import or retention of HBx is limited. Our data are consistent with a recently proposed model which suggested that the nuclear import of HBx is dependent on cellular proteins, such as IB
(Weil et al., 1999
), or on other viral or host cell factors that have not yet been identified. Our finding that HBx is preferentially compartmentalized in the nucleus at low expression levels has wide implications. These experiments indicate a much higher significance of nuclear HBx than suggested in previous localization studies, where the protein was only or predominantly found in the cytoplasm. These localization data support the concept that transactivation by HBx involves binding to transcription factors and nuclear proteins. Further, associations between HBx and DNA repair enzymes have been described (Lee et al., 1995
; Becker et al., 1998
; Groisman et al., 1999
; Jia et al., 1999
). These interactions could also be important, since the HBV replication cycle involves the conversion of its incompletely synthesized viral genome into a covalently closed circular DNA. In addition, nuclear HBx could also interfere with the function of the p53 tumour suppressor protein.
We have further demonstrated that the levels of nuclear HBx remain low, while elevated expression correlated with its accumulation in the cytoplasm. This may further explain the difficulties in detecting nuclear HBx, both in vivo and in cultured cells. In addition, there is evidence that the X-protein functions at a very low expression level. HBx was recently shown to activate the HBV-core promoter in transgenic mice. Interestingly, the magnitude of this effect was much higher than previously reported in transfected cells, but HBx was not detectable in these mice using standard detection methods (Reifenberg et al., 1999 ).
Moderately expressing A431 cells were used as a model to analyse the cytoplasmic compartmentalization of HBx using confocal microscopy. We demonstrated by immunofluorescence staining that a substantial fraction of cytoplasmic HBx is associated with mitochondria. Computer analysis of the HBx amino acid sequence, using PSORT software (University of Tokyo), suggested that the N-terminal sequence of HBx may have features of a putative mitochondrial targeting signal (data not shown). However, Western blotting detected only the wild-type full-length protein when both this and truncated variant proteins were synthesized using expression vectors (Fig. 1). Consistent with this, no cleavable signal peptide was predicted in the primary structure of HBx using the SignalP server (Technical University of Denmark). These observations suggest that the association of HBx with mitochondria, or any other organelle, does not involve processing of the protein. Furthermore, putative mitochondrial targeting sequences in HBx must be weak, since a substantial amount of cytoplasmic X-protein does not accumulate in mitochondria.
Attempts to analyse the subcellular distribution of HBx by differential centrifugation were inconclusive, because the protein co-sedimented with a mitochondrial marker both in the nuclear (1000 g) and mitochondrial (10000 g) fractions. The value of cell fractionation experiments is further limited, because these experiments do not allow a differential analysis of individual cells according to expression levels. This leads to a disproportionate representation of aggregated HBx in strongly expressing cells. Aggregation of HBx has been recently described by Takada et al. (1999) . In this report, HBx and p53 were co-detected in abnormally aggregated mitochondrial structures and it was further suggested that HBx could induce mitochondria-related cell death. Our data do not support all of these conclusions. We have demonstrated here a differential compartmentalization of HBx in both nucleus and cytoplasm, with a fraction associated with mitochondria. However, alterations to the mitochondrial staining or organelle distribution were not apparent in moderately expressing cells.
The functions of HBx in mitochondria are not known. HBx was previously shown to induce or to predispose cells to apoptosis, although there is also evidence for an inhibition of p53-dependent apoptosis (Elmore et al., 1997 ). These proposed apoptotic or cytotoxic effects of HBx could be related to its mitochondrial localization, since this organelle has a major role in regulation of programmed cell death (Martinou, 1999
). However, it is not yet clear whether HBx-mediated cytotoxic effects are significant in the immunopathology of hepatitis B infection and there is no apparent linkage between its pro-apoptotic activities and virus replication. Alternatively, HBx could affect different mitochondrial functions, such as calcium storage, which may be directly related to cytoplasmic signalling events. Very recently, HBx was shown to bind HVDAC3, a mitochondrial ion-channel protein (Rahmani et al., 2000
). Although the functional significance of this interaction is not yet clear, this observation could suggest that HBx may affect the permeability of the mitochondrial membrane for metabolites or ions. The precise functions of mitochondrial HBx remain to be established. However, we speculate here that the demonstrated association of HBx with mitochondria may have a central significance in its biological role and functions.
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Acknowledgments |
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References |
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Received 7 September 2000;
accepted 2 January 2001.