Central Laboratory Animal Facility of the Johannes GutenbergUniversity of Mainz, Obere Zahlbacherstr. 67 Hochhaus am Augustusplatz, D-55101 Mainz, Germany1
Laboratory Animal Research Unit of the University of Ulm, Albert Einstein Allee 11, D-89081 Ulm, Germany2
Heinrich Pette Institute for Experimental Virology and Immunology, Martinistr. 52, D-20251 Hamburg, Germany3
Zentrum für Molekulare Biologie, Im Neuenheimer Feld 282, D-69120 Heidelberg, Germany4
Department of Medicine II, University of Freiburg, Hugstetter Strasse 55, D-79106 Freiburg, Germany5
Author for correspondence: Kurt Reifenberg. Fax +49 6131 39 30220. e-mail reifenbe{at}mail.uni-mainz.de
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Abstract |
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Introduction |
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Using tissue culture experiments it has been demonstrated that pX represents a moderate transcriptional transactivator of the native viral promoters (Colgrove et al., 1989 ; Spandau & Lee, 1988
; Zahm et al., 1988
). However, in contrast to retroviral transactivators, which exert their function specifically on the viral LTR elements, pX was found to act on a broad multitude of cellular genes (for reviews see Murakami, 1999
; Rossner, 1992
). Furthermore, since X-defective hepadnaviral genomes remained replication-competent upon transfection into differentiated hepatoma cell lines (Blum et al., 1992
; Koike et al., 1989
; Yaginuma et al., 1987
), the activity of the HBV promoters could not strictly depend on the coexpression of pX. On the other hand, studies performed in the WHV system have suggested that an intact X gene is required for the successful establishment of hepadnaviral infection in vivo (Chen et al., 1993
; Zoulim et al., 1994
).
Recently, we have generated several lineages of transgenic mice carrying the HBV core gene either alone or in cis arrangement with the X gene (Reifenberg et al., 1997 , 1999
). By comparing the quantity of hepatic core gene expression between the X-intact and the X-deficient mice, we have demonstrated that the X gene product transactivates the expression of HBV core transcript in vivo. However, expression of pX was not mandatory for synthesis of core mRNA in the livers of transgenic mice.
Here we have established a lineage of transgenic mice expressing all HBV gene products except for the X gene product. These transgenic animals enabled us to address the question whether the X protein is required for virus replication and virion export in vivo. We found that in this transgenic mouse model, pX is not essential for HBV replication and virus secretion.
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Methods |
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Northern blot analysis.
For Northern blotting, snap-frozen tissue samples were pulverized in liquid nitrogen with a micro-dismembrator (Braun Biotech International) and total RNA was prepared using the RNEasy Mini Kit (Qiagen). The samples were treated with DNase (Gibco BRL) and the purified RNA was separated on a formaldehydeagarose gel, blotted on to nylon membranes and hybridized with an HBV-specific 32P-labelled probe.
Southern blot analysis.
Liver or kidney tissue samples (20 mg) were transferred into 500 µl of the ATL tissue lysis buffer from the QIAamp DNA Mini Kit (Qiagen) supplemented with 1 mg/ml proteinase K and incubated at 56 °C for 2 h. After centrifugation through a QIAshredder spin column (Qiagen), the DNA was purified by adsorption on to silica columns as recommended by the supplier. Serum DNA was obtained by using the QIAamp Blood Kit (Qiagen) as described in the manufacturer's handbook. Nucleic acids were separated by agarose gel electrophoresis, transferred on to nylon membranes and hybridized with an HBV-specific 32P-labelled probe.
The viral titre was estimated in the sera of transgenic mice by comparing the Southern blot hybridization signal of purified serum DNA with a known quantity of cloned HBV DNA.
Western blot analysis.
Liver or kidney tissue samples (20 mg) were disrupted in 1x Laemmli loading buffer supplemented with 5% -mercaptoethanol. The lysates were passed through QIAshredder spin columns and denaturated by incubating at 95 °C for 5 min. After gel electrophoresis (13% polyacrylamide, 0·1% SDS) the proteins were blotted on to a PVDF transfer membrane (Schleicher & Schuell). The membrane was probed with a polyclonal rabbit antiserum directed against HBcAg (Nassal, 1992
) or pX (antibodies 70606 or anti-X-Ecoli) and bound antibodies were visualized using the ECL Plus Western blotting detection system (Amersham).
Detection of HBeAg and HBsAg in murine serum.
For detection of serum HBeAg, 30 µl of murine serum was diluted with 170 µl foetal calf serum and analysed using a commercial diagnostic assay (Abbott HBe 2 ELISA). HBsAg seropositivity was analysed by using the AUSAB assay (Abbott).
Sequence analysis.
Liver DNA of transgenic mice was amplified using the two HBV-specific oligonucleotides 5' TGC CAT TTG TTC AGT GGT TCG TAG GGC 3' and 5' CCG GCA GAT GAG AAG GCA CAG ACG G 3'. The PCR products were sequenced using the Thermo Sequenase cycle sequencing kit (Amersham) and the 32P-labelled primer 5' CCG GCA GAT GAG AAG GCA CAG ACG G 3'.
Immunohistology.
Tissue specimens were fixed in 4% formaldehyde solution in PBS (pH 7·2). HBV antigen-specific immunostaining was performed with paraffin sections using the avidinbiotin complex method (Hsu et al., 1981 ). Paraffin sections were treated with a commercial target unmasking fluid' (Dianova) in a microwave oven before antibody incubation. The sections were incubated overnight at 4 °C with a 1:4000 diluted polyclonal rabbit antiserum specific for HBc/eAg (Schlicht & Schaller, 1989
) or for pX (antibodies 70606 or anti-X-Ecoli), both diluted 1:1000 to 1:4000. Specifically bound antibodies were detected with a biotinylated secondary antibody and subsequent incubation with phosphatase-conjugated streptavidin (Biogenex) and staining with naphthol AS-BI phosphate in combination with hexazotized new fuchsine (Merck). Endogenous avidin-binding activity was reduced by pretreatment of the sections with avidin and biotin solutions (Zymed Laboratories). Negative controls comprised naive rabbit serum and tissues from non-transgenic mice.
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Results |
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Hepadnaviral gene expression
For analysis of hepadnaviral gene expression, the organs of X-deficient 1.3HBV-Xstop-3 transgenic mice were investigated using Northern and Western blotting and immunohistochemical labelling techniques. Since it is a well-established fact that hepadnaviral gene expression of transgenic mice is not restricted to hepatocytes (Guidotti et al., 1995 ; Reifenberg et al., 1997
), these studies were performed with the livers and kidneys of transgenic mice.
In Northern blots prepared with total X-intact 1.3HBV (data not shown) and X-deficient 1.3HBV-Xstop transgenic liver RNAs we could demonstrate expression of the 3·5 kb core- and the 2·4 kb and 2·1 kb surface-specific hepadnaviral transcripts (Fig. 2, lane 2). As depicted in Fig. 3
, the amount of core protein detectable in the livers of the X-negative transgenic mice (Fig. 3
, lane 2) by Western blotting was comparable to that of the X-positive control (Fig. 3
, lane 4). Comparable amounts of HBcAg could also be detected in the kidneys of the X-positive and X-negative HBV transgenic mice (Fig. 3
). To investigate the cellular distribution pattern of core protein expression, paraffin sections of formaldehyde-fixed liver and kidney specimens of X-intact and X-deficient mice were immunostained with an HBcAg-specific polyclonal antibody. As depicted in Fig. 4
, the HBcAg staining pattern was similar in the livers of the X-intact (Fig. 4A
) and X-deficient (Fig. 4B
) mice. In both types of transgenic animals, HBcAg-positive hepatocytes were predominantly located in the centrolobular liver regions and only a few labelled cells were detectable in the periphery of the liver lobules. A considerable but inter-individually varying proportion (maximally 50%) of labelled hepatocytes also exhibited a positive cytoplasmic immunostaining as well as the nuclear immunoreactivity.
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HBV replication and virion export
To answer the question of whether pX is central to hepadnavirus replication in transgenic mice, Southern blots were prepared from hepatic and renal DNA isolated from X-deficient mice and hybridized with a 32P-labelled HBV-specific probe. Interestingly, we could detect HBV replicative intermediates in the livers (Fig. 5, lane 2) and kidneys (Fig. 5
, lane 1) of the X-deficient mice demonstrating that these murine tissues support HBV replication even in the absence of pX. To exclude the possibility that efficient hepadnavirus replication observed in the X-deficient transgenic organs might be due to a remutation restoring the X-gene, we determined the sequence of a PCR-amplified DNA fragment generated from an X-deficient liver DNA template. These investigations confirmed the presence of the stop mutation at codon 8 in the X ORF (data not shown).
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Discussion |
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Importantly, our data have demonstrated that X-negative HBV-transgenic mice are capable of HBV replication and of virion export (Fig. 5). The viral DNA detectable in the serum of the transgenic animals was of the relaxed circular species, clearly indicating that it could not have resulted from cell death and the release of intracellular core particles but originated from the export of mature enveloped viral particles. Hence, pX is not central for these two steps of the viral life-cycle in vivo, which is in accordance with previous results obtained with transfected cells (Blum et al., 1992
; Koike et al., 1989
; Yaginuma et al., 1987
). Furthermore, it must be stated that hepadnavirus replication and virion export in the absence of X coexpression is no special feature of the transgenic lineage described in the present paper, but could also be achieved in another murine transgene model (Christa Kuhn, unpublished observations). On the other hand, two independent research groups (Chen et al., 1993
; Zoulim et al., 1994
) have reported that X-deficient WHV genomes injected into woodchuck livers failed to establish hepadnaviral infection, whereas injection of X-intact control DNA resulted in viraemia. These studies clearly indicate an essential role for the X protein in the natural life-cycle of WHV. In the light of our results, we assume that the failure to establish WHV infection by injecting X-deficient viral genomes is most probably not due to a replication defect of the X-deficient WHV virus particles.
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Acknowledgments |
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References |
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Received 30 October 2001;
accepted 18 December 2001.