Department of Virology, University of Ulm, Albert-Einstein-Allee 11, D-89081 Ulm, Germany1
Author for correspondence: Eva Schmitteckert. Present address: Institute for Pharmacology and Toxicology, University of Wuerzburg, Versbacher Str. 9, D-97078 Wuerzburg, Germany. Fax +49 931 201 3539. e-mail schmitteckert{at}toxi.uni-wuerzburg.de
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Abstract |
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Introduction |
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Due to the transactivating activity of the X-protein, great efforts were undertaken to test whether this viral gene product could be involved in the induction of liver carcinoma, a complication which is frequently seen in patients who are chronically infected with the human hepatitis B virus (HBV). In particular, several transgenic mouse lines were established which expressed the HBV X-protein under authentic or foreign promoter control. One problem which was encountered during these studies was the fact that it proved quite difficult to detect the X-protein in organs of the animals although, as could be proven by the detection of the corresponding mRNA, the transgene was well expressed (Perfumo et al., 1992 ). Until today, the following efforts to detect the X-protein in vivo have been described.
Billet et al. (1995 ) generated transgenic mice under authentic promoter/enhancer control (lines PEX) as well as under the hepatospecific antithrombin III gene regulatory region (lines AX). Balsano et al. (1994)
found a 16·5 kDa band in Western blot analysis in liver tissue of 8-day-old transgenic mice of line AX16, whereas Billet et al. (1995
) only found a positive Northern blot signal. However, a positive Western blot signal was shown in the liver of a 10-day-old mouse of line PEX7 (Terradillos et al., 1997
).
Likewise, Lee et al. (1990 ) detected X-protein in the liver of 4-week-old X-transgenic mice using the human
-1-antitrypsin regulatory region. By immunohistochemistry only weak staining could be noted; in older mice the X-protein was undetectable. In another study a similar Western blot analysis shows a continual X-protein expression level in animals up to 9 months old (Slagle et al., 1996
).
Likewise, Kim et al. (1991 ) show expression of the x-gene under its own regulatory elements in a transgenic mouse only by Northern blot analysis. An 18 kDa band in SDSPAGE could be found in a primary culture of hepatocytes labelled with [35S]methionine (Koike et al., 1994
).
In a different study detection of X-protein by Western blotting succeeded after preparation of a whole tissue homogenate resuspended in Western blotting sample buffer (Moriya et al., 1996 ).
The aim of this study was to establish optimal conditions for the isolation of X-protein from tissue culture cells as well as from organ material. To facilitate this analysis we used a modified X-protein which could be radioactively labelled with high efficiency and specificity. Our data show that most, if not all, of the X-protein expressed from a cytomegalovirus (CMV)-driven plasmid is not soluble in aqueous buffer containing a non-ionic detergent but rather has to be extracted from the insoluble fraction by boiling with a slightly alkaline SDS buffer. With this method we were able to detect the X-protein in transgenic mice expressing this gene under authentic promoter control.
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Methods |
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For generation of a plasmid expressing a modified X-protein with a recognition site for protein kinase A (pCMV-X-PKA, Fig. 1c), the x-gene was amplified from plasmid pMH3/3097 using the primers X-PKA+, 5' CCC GGA TCC ACC ATG GCT GCT AGG CTG TGC 3', and X-PKA-, 5' ACG GAT ATC TTA CAA CGA TAG TCT CCG GGC AGA GGT GAA AAA GTT 3'. The upstream primer introduces a BamHI site (underlined) immediately 5' from the x-gene ATG (shown in bold face), whereas the downstream primer provides the PKA recognition site (shown in bold face) as well as a new stop codon and an EcoRV site (underlined). This fragment was inserted into pcDNAIAmp (Invitrogen) containing the HCMV-P/E and the simian virus 40 poly(A) signal.
For expression of the newly modified gene xPKA under the authentic promoter control (pHBV-X-PKA, Fig. 1c), the xPKA fragment was isolated by digestion with NcoI/EcoRV and recloned into plasmid pHBV-X, replacing the x-gene with the newly modified xPKA gene. The HindIIISacI fragment was used for pronuclear injection, generating the lines X-PKA I, IIIVII.
Generation of transgenic mice.
All animal experiments were performed in accordance with the German regulations for the use of animals in biomedical research. Transgenic mice were produced according to standard procedures (Hogan et al., 1994 ). The transgenic lines expressing the wild-type x-gene under authentic promoter control (HBxI and -IV) have been described (Reifenberg et al., 1997
). For generation of transgenic lines expressing the modified X-protein, the HindIIISacI fragment from plasmid pHBV-X-PKA was microinjected into one pronucleus of (C57BL/6JxCBA/Ca) F2 fertilized eggs. Transgene integration was detected by PCR analysis.
Transient expression of X-protein in tissue culture.
HuH-7 cells, a human hepatoma cell line, were grown in six-well tissue culture plates (9 cm2 per well) to 80% confluency in 2 ml Dulbecco's minimal essential medium supplemented with 10% foetal calf serum, 2 mM l-glutamine, 100 IU/ml penicillin G, 0·1 mg/ml streptomycinsulfate. Cells were rinsed once with serum-free medium. Two µg plasmid DNA was diluted into 100 µl serum-free medium; 8 µl lipofectamine (Gibco BRL) was also diluted into 100 µl serum-free medium. The two solutions were combined, mixed gently and incubated at room temperature for 30 min. After adding 800 µl serum-free medium the DNAliposome complexes were overlaid onto the cells. After 6 h of incubation at 37 °C, 1 ml medium containing 20% foetal calf serum was added and the cells were grown for another 42 h, replacing the medium once.
Lysis of tissue culture cells.
One ml 1% Triton lysis buffer [1% Triton X-100, 100 µM PMSF, 0·1% sodium azide in TNE (20 mM TrisHCl pH 8·0, 100 mM NaCl, 1 mM EDTA)] was added to each well. Alternatively, an acidic or alkaline Triton lysis was performed (Fig. 2): 1% Triton X-100 in acidic lysis buffer (C/PNE pH 4·5), containing 2 mM citratephosphate buffer (pH 4·5), 130 mM NaCl, 1 mM EDTA, or 1% Triton X-100 in alkaline lysis buffer containing TNE (pH 8·8).
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Lysis of mouse tissue samples.
Organs were frozen in liquid nitrogen and pulverized in a micro-dismembrator (Braun Biotech). One hundred mg (ovaries 30 mg) of frozen tissue powder was lysed in 2 ml 1% Triton lysis buffer (pH 8·0). After centrifugation the Triton X-100 soluble fraction was dispelled, the insoluble fraction was homogenized by subsequent pulling through 20G and 23G needles in 750 µl 2% SDSME lysis buffer (pH 8·8), boiled, centrifuged and the supernatant was dissolved 1:20 in 1% Triton lysis buffer (pH 8·8). To reduce the background, this solution was cleared again by centrifugation at 20000 g before immunoprecipitation.
Isolation of X-protein by immunoprecipitation.
Fifteen µl of the anti-X specific polyclonal rabbit antiserum 70646 (raised against a recombinant HBxcellular fusion protein expressed in Escherichia coli) was adsorbed to 70 µl pre-swollen protein ASepharose CL-4B (Pharmacia) in 300 µl PBS for 30 min and then added to organ or tissue culture lysates. Immunoprecipitation was carried out overnight at 4 °C.
Radioactive labelling of XPKA protein with PKA and [
-32P]ATP.
The immunoprecipitates were washed three times with PBS buffer and twice with PKA buffer (20 mM TrisHCl, pH 7·0, 100 mM NaCl, 10 mM MgCl2). After adding 30 µl PKA buffer supplemented with 1 mM dithiothreitol (DTT), 20 µCi [-32P]ATP and 4 U PKA (Sigma) reconstituted in deionized water containing 6 mg/ml DTT, samples were incubated three times for 15 min at 30 °C, adding 4 U of the enzyme each time.
SDSPAGE and detection of XPKA protein by autoradiography.
After four washing steps with PBS, the precipitated proteins were dissolved by boiling the immunocomplexes for 5 min in 40 µl of reducing protein sample buffer (200 mM TrisHCl pH 8·8, 0·5 M sucrose, 5 mM EDTA, 0·1% bromophenol blue; adding 3% SDS, 2% ME shortly prior to use) and separated on a 15% discontinuous SDSpolyacrylamide gel, with a constant current of 20 mA/cm2, using prestained protein molecular mass standards (Gibco BRL) as marker proteins. Subsequently the gel was dried and exposed to a film. Corresponding to the calculated molecular mass a specific band should be expected at 17 kDa.
Western blotting.
The proteins separated in the SDSpolyacrylamide gel were transferred to a nitrocellulose membrane (Schleicher & Schuell) in transfer buffer (25 mM TrisHCl, 0·2 M glycine, 20% methanol; overnight transfer, 60 V, 4 °C) according to the method of Towbin et al. (1979) . The membrane was briefly washed in PBS and unspecific protein binding sites were saturated with PBS1% BSA0·1% sodium azide over 6 h. The polyclonal anti-X antiserum 70646 was added to a dilution of 1:2000 and incubated overnight at room temperature. After three washing steps (PBS, PBS0·1% Triton X-100, PBS) the specifically bound antibodies were detected by protein Aalkaline phosphatase conjugate (15 U) in PBS1% BSA0·1% sodium azide for 3 h. After another three washing steps the membrane was incubated twice for 15 min in 100 mM TrisHCl (pH 7·5), 150 mM NaCl and briefly rinsed in 100 mM TrisHCl (pH 9·2), 100 mM NaCl, 50 mM MgCl2. Ninety µl NBT (nitro blue tetrazolium) solution (75 mg/ml NBT, 70% dimethylformamide) and 70 µl BCIP (5-brome-4-chloro-3-indolyl phosphate) solution (50 mg/ml BCIP in dimethylformamide) were added to 20 ml 100 mM TrisHCl (pH 9·2), 100 mM NaCl, 50 mM MgCl2 for the colour reaction (stopped with 10 mM TrisHCl, pH 8·0, 1 mM EDTA).
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Results |
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In order to examine these points we decided to produce a construct expressing a slightly modified X-protein which can be detected with high sensitivity. To this end, a short sequence of 15 nucleotides was attached to the 3' end of the x-gene by PCR mutagenesis and the gene thus modified was cloned downstream of the HCMV major immediate-early promoter/enhancer (pCMV-X-PKA; Fig. 1). The five amino acids encoded by the extra sequence represent a specific recognition site for PKA. Since the PKA phosphorylation using [
-32P]ATP is both efficient and specific, it allows the detection of even trace amounts of a protein. This method has previously been used for the detection of the HBV polymerase in a vaccinia virus system (Bartenschlager et al., 1991
).
In the first experiment, HuH-7 cells, transiently transfected with pCMV-X-PKA, were lysed with buffer containing 1% Triton X-100 and subjected to immunoprecipitation with an X-specific antiserum. The immunoprecipitated proteins were incubated with PKA and [-32P]ATP and then analysed by SDSPAGE and subsequent autoradiography. Surprisingly, no specific band could be detected in the samples obtained from the cells transfected with pCMV-X-PKA in comparison to untransfected cells (data not shown).
In the next experiment we therefore decided to analyse not only the soluble but also the non-soluble fraction of the cells and also to test the effects of different pH values. As is shown in Fig. 2, again no specific protein band could be detected in the cell lysates, regardless of whether an acidic or an alkaline lysis buffer was used (Fig. 2
, lanes 13). However, by boiling the cell pellets with a slightly alkaline buffer containing 2% SDS and 2% ME a protein with the expected size of about 17 kDa could be extracted from the transfected cells which was specifically phosphorylated by PKA (Fig. 2
, lanes 9 and 10). Interestingly, this band was not observed if an acidic extraction buffer was used (lane 8).
To provide additional evidence that the 17 kDa band was in fact due to the phosphorylated modified X-protein, HuH-7 cells were transfected with expression plasmids containing either the modified or the wild-type x-gene under the control of the HCMV-P/E (Fig. 3). For transfection, 2 µg plasmid DNA (pCMV-X-PKA, lanes 1 and 2; pCMV-X, lanes 5 and 6) and 10 µl (lanes 1 and 5) or 2 µl (lanes 2 and 6) lipofectamine was added to 9 cm2 cell culture wells. Cells treated with 10 µl lipofectamine showed morphological signs of toxicity, therefore 8 µl lipofectamine was used per each 9 cm2 well in the following experiments. After 2 days of expression the cells were lysed, the soluble fraction was discarded and the cell pellet extracted with the alkaline SDSbuffer (pH 8·0) as described above. As is shown in Fig. 3
, a protein of the expected molecular mass could only be detected in the cells which had been transfected with the plasmid expressing the modified x-gene and thus does in fact represent the XPKA protein (Fig. 3
, lanes 1 and 2). From these data we conclude that an alkaline buffer with a strong ionic detergent is required to extract the X-protein from the cells. This fact was probably at least one reason why our former efforts to detect the X-protein had failed and might also explain the negative results obtained by other groups.
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As is obvious from Fig. 4, a protein only slightly heavier (5 additional amino acids) than the bacterially expressed X-protein (lane 2) was detected in the transfected cells (lane 4). The fact that no such band could be observed in our previous experiments shows that the special extraction procedure described here was essential for isolation of the X-protein. However, the liver samples (lane 7 and 8) were negative, suggesting that the Western blot was not sensitive enough. Therefore, as far as our transgenic mice are considered, probably not only was the method which we had used previously not suitable but also there was not enough X-protein present.
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As shown in Fig. 5, phosphorylated X-protein was clearly detectable in the cells transfected with pHBV-X-PKA (lanes 4 and 5). Compared to the pCMV-X-PKA transfections (lanes 1 and 2), it can be estimated that the authentic x-promoter is about 40-fold (as calculated by Image Quant) less efficient. However, this appeared to be sufficient for our purposes.
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Detection of the modified X-protein in vivo
One transgenic F1 offspring of each of the six remaining founders was sacrificed. Liver tissue (100 mg) was prepared and homogenized in 1% Triton lysis buffer. The soluble fraction was discarded and the pellet analysed for X-protein as described above. As shown in Fig. 6, the phosphorylated X-protein could be found in the livers of five of the six offspring animals tested as well as in the liver of the sterile founder. As a positive control one-quarter of the phosphorylated immunoprecipitate of a transfected tissue culture (1 well) was used (lane 1). Altogether, six transgenic lines could be established with the construct HBV-X-PKA, out of which five lines (X-PKA I, IV, V, VI, VII) expressed the XPKA protein in the liver.
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Discussion |
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To date, there have been extensive studies in which the transacting potential of the X-protein on autologous viral as well as heterologous viral and cellular promoters has been examined by in vitro transfection assays (Rossner, 1992 ). However, information about the regulation and function of the X-protein in vivo is still limited. A major problem in investigation of the X-protein in vivo is the necessity of an efficient and sensitive technique for X-protein detection in tissue probes.
Studies on X-transgenic mice lack convincing data concerning the detection of the X-protein and the published data are often not consistent. Balsano et al. (1994 ) show a positive Western blot analysis using only 10 mg liver tissue of transgenic mice (line AX16), yet immunohistology in the same animals gave only a weak signal. Billet et al. (1995
) show no protein detection, but only X-mRNA in livers of these animals.
In a transgenic mouse line which expresses the X-protein under the control of the human -1-antitrypsin regulatory region, Lee et al. (1990
) detected X-protein in the liver only in 24-week-old transgenic mice in Western blot analysis, whereas Slagle et al. (1996
) found a continuous expression level in mice of the same transgenic line from 4 weeks old up to 9 months old.
Because of the technical problems in detecting X-protein in tissue samples, Kim et al. (1991 ) as well as Koike et al. (1994
) tried to detect X-protein in livers of transgenic mice by using a [35S]methionine labelling technique either in vivo or in vitro after isolation of transgenic mouse hepatocytes with collagenase.
A recent study compared 11 mono- and polyclonal anti-X antibodies from five different laboratories (Su et al., 1998 ). The antiserum used to detect the XPKA protein in our study proved to react strongly and also highly specific with X-protein not only in Western blots of recombinant X-protein but also in immunohistochemistry of human livers with HBV-associated disease. Thus, none of the antibodies tested showed any reaction with lysates of these explanted human livers in Western blot analyses.
The great variation of antibodies against X-protein in addition to different extraction procedures and detection techniques applied in previous studies of X-transgenic mice might explain in part the varying success in detection of X-protein. Likewise, the level of X-protein expression due to different promoter elements may influence the formation of aggregates and thus the solubility of X-protein.
In the study presented here we show in particular that the Western blotting technique is not sensitive enough to detect the X-protein in tissue samples in which it is expressed rather weakly under authentic promoter control.
In order to establish a method to solubilize the X-protein in tissue samples to make it accessible to immunoprecipitation a reliable detection technique was necessary. Therefore a modification was introduced into the 3' end of the x-gene which allowed us to radioactively label the X-protein at a newly introduced phosphorylation site. Incubation with PKA and [-32P]ATP enabled detection of the immunoprecipitated proteins at a very high sensitivity and specificity.
A method for the detection of proteins which are expressed at a very low level by introducing an artificial PKA site was developed by Li et al. (1989 ) for the detection of human interferon-
. It has also been used for the detection of HBV polymerase (Bartenschlager et al., 1991
). Here we show for the first time that the radioactive labelling at a newly introduced PKA site is a valuable technique for detection of proteins in transgenic mice.
To obtain a high expression in cell culture, the modified gene xPKA was cloned downstream of the major immediate-early 1 promoter of HCMV. After transient expression of this plasmid in tissue culture cells it was proven that the XPKA protein could be specifically immunoprecipitated (Fig. 3) and detection of the radiolabelled XPKA protein was possible with an extremely high sensitivity. In these assays it became clear that the X-protein is not soluble in aqueous buffers (Fig. 2
) but can be solubilized by boiling in 2% SDS2% ME under slightly alkaline conditions.
In the next experiment we tried to use the technique developed in tissue culture for detection of the X-protein in liver tissue of transgenic mice (lines HBxI and -IV). As positive controls XPKA protein expressed in tissue culture as well as bacterially expressed X-protein were used. All efforts led to negative results. Even with the improved cell lysing technique it was impossible to detect the authentic X-protein after immunoprecipitation in livers of transgenic mice by Western blotting (Fig. 4). Therefore we generated new transgenic mice which expressed the modified xPKA gene with the artificial PKA site instead of the authentic x-gene. Thereby a substantial improvement of the detection sensitivity seemed to be possible. After transfection of cell culture cells with pCMV-X-PKA, 104 cells were sufficient to clearly detect the XPKA protein after radioactive labelling (data not shown). This result allowed us to estimate the detection sensitivity in mouse liver: 100 mg liver tissue contains about 108 hepatocytes. So it could be expected that an expression level by a factor of 104 lower compared to pCMV-X-PKA-transfected cells should be sufficient to allow detection of the X-protein.
The ubiquitous, strong HCMV-P/E is not suitable for directing expression of the x-gene in transgenic mice. It can lead to embryonic death or sterility of the founder animals (unpublished data). Therefore the HCMV-P/E was exchanged for the HBV x-promoter and the HBV-enhancers I and II. The DNA construct used for pronuclear injection was gained by BglII digestion and religation of a construct used for generation of mice expressing the preC/core gene as well as the x-gene. These mice, described by Reifenberg et al. (1997 ), showed expression of the transgene at a level which is comparable to a normal HBV infection in humans. This construct contains both enhancer elements of HBV, the enhancer II, which is located within the x-gene, as well as the enhancer I, which lies about 400 bp upstream of the enhancer II (Fig. 1 a
; Shaul et al., 1985
; Tognoni et al., 1985
; Wang et al., 1990
; Yuh & Ting, 1990
). By BglII digestion and religation the major part of the core gene was deleted, without influencing the X expression unit (Fig. 1a
). The PKA site was introduced afterwards by PCR mutagenesis (Fig. 1c
).
In cell culture experiments it could be proven that the new construct was suitable for expression of the XPKA protein and, as calculated by scanning densitometry, the relative expression level of the pHBV-X-PKA construct was a factor of 40 lower compared to the pCMV-X-PKA construct (Fig. 5).
After pronuclear injection of the HBV-X-PKA construct we obtained seven transgenic founder mice, from which six transgenic lines could be established (X-PKA I, III, IV, V, VI, VII). One male founder, which had no offspring, was sacrificed to find out if the transgene was expressed in vivo. The mRNA of the transgene XPKA could be detected in liver and kidneys by RTPCR (data not shown). Therefore the transgenic animals seemed to be suitable for the detection of the XPKA protein in vivo, applying the detection technique developed in tissue culture.
In the sterile founder as well as in five out of six heterozygous offspring from the six transgenic XPKA lines the detection of the XPKA protein in 100 mg liver tissue by radiolabelling of the immunoprecipitated proteins was indeed successful (Fig. 6). One line, X-PKA III, expressed no XPKA protein in the liver, which could be due to a `silent' integration of the transgene.
To date, there are only limited studies concerning the tissue-specificity of the x-promoter. Billet et al. (1995 ) proved that in line PEX7, in which enhancers I and II are present, the x-promoter shows no tissue specificity but rather is active in all 11 tissues tested. However, the core promoter was liver-specific in line PEX7, in which the enhancer I is present, whereas it is active in various tissues of the SV28 and AX lines, which lack the enhancer I. These results suggested that enhancer I directs tissue specificity of the core promoter associated with enhancer II, but not of the x-promoter. These findings were consistent with Guo et al. (1991
), who showed that the ubiquitous herpes simplex virus thymidine kinase promoter associated with enhancer I is active only in hepatic cell lines whereas the x-promoter associated with enhancer I is active in hepatic as well as in non-hepatic cell lines.
Our mice allowed us, for the first time, to investigate the tissue-specificity of the x-promoter by direct detection of the X-protein in vivo. In two out of the five transgenic lines expressing the XPKA protein in the liver the tissue distribution of the transgene expression was investigated. This promised to yield decisive information about the tissue specificity of the x-promoter in a construct containing the two HBV enhancers at the authentic positions.
One male, 12-week-old mouse of line X-PKA I (Fig. 7, upper panel) and one female, 21-week-old mouse of line X-PKA IV (Fig. 7
, lower panel) were sacrificed and the X-protein expression was investigated quantitatively in 14 different tissues. The highest expression level was found in lung, kidney and brain, and in line X-PKA I also in pancreas. Surprisingly, the liver showed only a medium expression level. Low amounts or no expression were found in heart, colon, gonads and skeleton muscle. Overall, the female, 21-week-old mouse of line IV showed higher expression levels than the male, 12-week-old mouse of line I. Consequently, the XPKA protein could be found in many different organs, irrespective of the transgenic line, the age or the sex of the animal. This finding strongly suggests that the x-promoter is not tissue-specific but rather is active in many different cell types.
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Acknowledgments |
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References |
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Received 10 March 1999;
accepted 3 June 1999.
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