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R
Virology 309 (2003) 339 –349
www.elsevier.com/locate/yviro
A long HBV transcript encoding pX is inefficiently exported
from the nucleus
Gilad Doitsh and Yosef Shaul*
Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, Israel
Received 12 September 2002; returned to author for revision 10 October 2002; accepted 7 January 2003
Abstract
The longest hepatitis B virus transcript is a 3.9-kb mRNA whose function remained unclear. In this study, we wished to identify the
translation products and physiological role of this viral transcript. This transcript initiates from the X promoter region ignoring the inefficient
and noncanonical viral polyadenylation signal at the first round of transcription. However, an HBV mutant with canonical polyadenylation
signal continues, though with lower efficiency, to program the synthesis of this long transcript, indicating that the deviated HBV
polyadenylation signal is important but not essential to enable transcription of the 3.9-kb species. The 3.9-kb RNA contains two times the
X open reading frame (ORF). The X ORF at the 5⬘-end is positioned upstream of the CORE gene. By generating an HBV DNA mutant
in which the X and Core ORFs are fused, we demonstrated the production of a 40-kDa X-Core fusion protein that must be encoded by the
3.9-kb transcript. Mutagenesis studies revealed that the production of this protein depends on the 5⬘ X ORF ATG, suggesting that the 3.9-kb
RNA is active in translation of the X ORF. Based on these features, the 3.9-kb transcript was designated lxRNA for long X RNA. Unlike
other HBV transcripts, lxRNA harbors two copies of PRE, the posttranscriptional regulatory element that controls the nuclear export of HBV
mRNAs. Unexpectedly, despite the presence of PRE sequences, RNA fractionation analysis revealed that lxRNA barely accumulates in the
cytoplasm, suggesting that nuclear export of lxRNA is poor. Collectively, our data suggest that two distinct HBV mRNA species encode
pX and that the HBV transcripts are differentially regulated at the level of nuclear export.
© 2003 Elsevier Science (USA). All rights reserved.
Introduction
Hepatitis B virus (HBV) is an enveloped virus with a
circular, partially double-stranded DNA genome. HBV is a
major causative agent of liver diseases (reviewed by Arbuthnot and Kew, 2001; Nassal, 1999; Seeger and Mason,
2000). The HBV genome, the smallest among mammalian
viruses, is 3.2 kpb in length and contains four major ORFs;
all are compactly organized and efficiently expressed. Every
nucleotide in the genome, including those of the known
regulatory DNA cis elements, is engaged with at least one of
these ORFs. Upon infection, the genome is imported into
the cell nucleus, where it is repaired to form a covalently
closed circular DNA (cccDNA). The episomal cccDNA
serves as the template for transcription by the host RNA
polymerase II. The HBV genome contains several promot* Corresponding author. Fax: ⫹972-8-934-4108.
E-mail address: [email protected] (Y. Shaul).
ers, and gene expression is largely regulated at the level of
transcription initiation. These promoters are under regulation of two enhancers, one, enhancer-I, located upstream to
the X promoter and the other, enhancer-II, upstream to the
core promoter (reviewed by Huan and Siddiqui, 1993).
Studies in tissue culture and in HBV transgenic mice suggest that enhancer-I plays a major role in controlling HBV
transcription (Shaul et al., 1985; Honigwachs et al., 1989;
Ben-Levy et al., 1989; Guidotti et al., 1995; Fukai et al.,
1997; Shamay et al., 2001).
At present, only five major HBV unspliced transcripts
have been unequivocally demonstrated (Fig. 1). In addition,
a number of spliced variants were identified (Gunther et al.,
1997). Among the five known transcripts, the longest are the
3.5-kb precore mRNA (pcRNA), encoding the HBeAg, and
the 3.4-kb pregenomic mRNA (pgRNA), which encodes the
Core protein. The pgRNA serves as the template for DNA
synthesis, as well. The other three subgenomic RNAs are
the 2.1- to 2.4-kb mRNAs, encoding the S, PreS2, and
0042-6822/03/$ – see front matter © 2003 Elsevier Science (USA). All rights reserved.
doi:10.1016/S0042-6822(03)00156-9
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Fig. 1. Transcription map of HBV (adw). Inner circles represent the viral partial dsDNA, wide arrows represent the overlapping viral ORFs, and the outer
thin arrows represent the different viral transcripts, including the 3.9-kb lxRNA (this study), initiating from the X promoter. The viral enhancer-I is also shown
within the minus strand DNA. Nucleotides are numbered according to the unique EcoRI site ⫽ 1. Transcription polyadenylation site P(A)S is located within
the Core gene coding sequence, at position 1919. P, RT-polymerase; DR, direct repeats.
PreS1 viral surface antigens, and the 0.7-kb mRNA that
encodes the regulatory X protein (pX or HBx). An additional and longer transcript of 3.9 kb was previously described (Guo et al., 1991; Kim et al., 1992) that so far has
been poorly characterized. In this report, we describe characterization of this transcript by genetic manipulations and
show that is active in production of the X protein. We
designated this transcript long X-RNA (lxRNA).
We have previously shown that nuclear export of mRNA
is a regulated process (Shaul et al., 1982). The underlying
mechanisms are under extensive investigation and some of
the details have been resolved (reviewed by Kuersten et al.,
G. Doitsh, Y. Shaul / Virology 309 (2003) 339 –349
341
Fig. 2. Different HBV DNA templates are able to program the synthesis of the long RNA. (A) A schematic representation of the HBV-derived (adw subtype)
constructs used in this study. The HBV ⫻ 2 construct contains a head to tail HBV DNA dimer, the HBV ⫻ 1.3 construct contains a single polynucleotide
representing 1.3 copies of HBV DNA, and the circular DNA was generated by self-ligation of a copy of HBV DNA. The X and Core ORFs are designated
by open rectangles. Other HBV genes are not depicted. EnI, enhancer-I; RI, RV, and Taq1 are the EcoRI, EcoRV, and Taq1 unique HBV DNA restriction
sites. (B) Huh7 and HepG2 cells were transfected with the plasmids described in A and total RNA was analyzed by Northern blotting. Cell lines and HBV
constructs are depicted above the corresponding lanes. RNA was extracted 30 h posttransfection. The level of X RNA is underpresented due to the fact that
the whole HBV DNA was used as a probe. The 1.8-kb RNA species was identified as a spliced HBV RNA (data not shown).
2001). The mechanism by which incomplete, spliced HBV
transcripts are exported to the cytoplasm is still controversial. A posttranscriptional regulatory element (PRE) within
the HBV mRNAs has been identified and was shown to be
important in exporting HBV mRNAs to the cytoplasm
(Donello et al., 1996; Huang and Liang, 1993; Huang and
Yen, 1994). The HBV PRE is located in a region that
overlaps with the X gene open reading frame. This region is
included in all known HBV transcripts. The lxRNA includes this region twice, and we therefore studied the subcellular distribution of this transcript. Interestingly, analysis
of two different cell lines revealed that the lxRNA is confined to the nucleus and is barely found in the cytoplasmic
fraction, suggesting that it is subject to specific regulation at
the level of nuclear export.
Results
The 3.9-kb RNA is a genuine HBV transcript
To analyze the HBV transcription pattern, we transfected
cells with three different constructs of HBV DNA (Fig. 2A).
These include a plasmid that harbors a head to tail HBV
dimer and a 1.3⫻ HBV DNA construct. The latter was
previously used to establish HBV transgenic mice with high
efficiency of HBV replication (Guidotti et al., 1995). In the
1.3⫻ HBV DNA construct, the HBV sequence begins from
the enhancer-I-X promoter region. For construction of the
third DNA template, we generated a closed circular HBV
DNA lacking any plasmid flanking sequences to mimic as
accurately as possible the authentic transcription template of
the virus, i.e., cccDNA. Total RNA was extracted from the
transfected Huh7 and HepG2 cells and analyzed by Northern blotting. The transcription patterns obtained following
transfection with each of the constructs were very similar,
suggesting that all three represent authentic HBV DNA
templates (Fig. 2B). A 3.9-kb-long transcript was previously detected by a number of groups (Guo et al., 1991;
Kim et al., 1992; Su et al., 1989; Suzuki et al., 1990).
Notably, all the three constructs programmed the synthesis
of the long 3.9-kb RNA species, suggesting that this transcript is a genuine HBV mRNA. The fact that a transcript of
this size was produced by the 1.3⫻ HBV DNA (4.2 kbp)
strongly argues that the transcription initiation site of the
3.9-kb transcript may reside very close to the X gene promoter region, in agreement with previous reports (Guo et
al., 1991; Kim et al., 1992).
The deviated poly(A) signal affects the level but not the
transcription pattern of HBV
Synthesis of the 3.9-kb RNA initiates at the X gene
promoter region and the transcript is polyadenylated only
after the second round of transcription. Similar behavior is
shared by other long pg/pcRNA species. This suggests that
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Fig. 3. Introduction of a canonical poly(A) site does not eliminate 3.9-kb RNA biosynthesis. (A) Huh7 cells were transfected with either a wt HBV DNA or Y6N,
an HBV mutant that contains a canonical poly(A) site (AATAAA) as previously described (Paran et al., 2000). Total RNA samples prepared at the indicated time
points and were analyzed by Northern blotting using the X-gene DNA probe for hybridization. Note that the X RNA disappears before the 3.9-kb RNA (compare
days 1.5 to 3). (B) The rate of HBV RNA decay. Huh7 cells were transfected with a wt HBV DNA and after 48 h actinomycin D (Act D) was added to block
transcription, as previously described (Doitsh and Shaul, 1999). Cells were harvested at the indicated time points following actinomycin D addition.
the transcription machinery must ignore the poly(A) signal
at the first transcription round. As HBV contains a deviated
poly(A) signal with relatively low efficiency, it is reasonable to suggest that the inefficiency of this signal plays an
instrumental role in the production of the long transcripts.
To investigate this possibility, we employed an HBV DNA
mutant named Y6N containing a consensus poly(A) signal
(AATAAA; Paran et al., 2000). This mutant expresses all
the known HBV transcripts, including the 3.9-kb RNA (Fig.
3A). However, a noticeable increase in the level of 0.7-kb X
mRNA with a concomitant reduction in the levels of 3.9-kb
RNA and pg/pcRNA is evident. These data suggest that the
3.9-kb transcript may be transcribed even in the presence of
a consensus poly(A) signal, but the abundance of this
mRNA species is increased in the presence of a “leaky”
polyadenylation site.
The level of both 3.9-kb RNA and 0.7 X RNA is sharply
reduced after 3–5 days posttransfection (Fig. 3A). The decay of the 3.9-kb RNA was, however, slower than that of X
RNA (compare lanes 1 and 2). This difference is unlikely to
be the result of changes in transcription rate as both transcripts are expected to be regulated by the same promoter.
Thus, it is possible that the 3.9-kb RNA is more stable than
the 0.7-kb X RNA. To address this the transfected cells
were treated with actinomycin D to block transcription and
RNA was subjected to a time course analysis. The data
obtained are in agreement with the possibility that the
3.9-kb RNA is more stable than the short X RNA (Fig. 3B).
The 3.9-kb transcript encodes the X protein
The 3.9-kb RNA contains all the viral ORFs and may
potentially encode every one of the viral genes. This RNA
has a unique ORF organization in which the X ORF is
positioned upstream of the Core ORF. We therefore assumed that it is likely that this transcript specifically encodes the X protein. To provide functional evidence for X
protein synthesis, we inserted an extra nucleotide 10 bp
upstream of the X stop codon in the region where the X and
preCore ORFs overlap (Fig. 4). Consequently, this HBV
DNA mutant, designated X-Core virus, contains a (⫺1)
shifted coding frame at the 3⬘ terminus of its X gene and is
expected to encode an X-Core fused product. Notably, this
insertion eliminates HBeAg synthesis by the pcRNA. The
production of an X-Core 40-kDa fusion protein was first
examined in a heterologous expression system. To this end,
the X, the Core, and the fusion mutant were cloned in
expression vectors under the SV40 transcription regulatory
units. Cells were transfected and the proteins were subjected
to Western blot analysis probed with three different antibodies. The 40-kDa protein was detected by all three antibodies used, an anti-X and an anti-Core-specific monoclo-
G. Doitsh, Y. Shaul / Virology 309 (2003) 339 –349
343
Fig. 4. A schematic illustration of the unique genetic organization of the 3.9-kb RNA. The top of the scheme shows the 1.3⫻ copy HBV DNA used for
transfection including all the known ORFs. The wavy lines indicate the two longest HBV transcripts. In the X-Core fusion mutant construct, a single
thymidine was inserted 10 nucleotides upstream of the X-termination codon causing the X ORF at this point to shift into the (⫺1) PreCore ORF.
Consequently, translation of the X gene from the 3.9-kb RNA results in the production of a fused X-Core protein. This insertion disrupts the HBeAg gene
as it resides upstream of the mutated site. Note that the core gene coding sequence is located 76 nucleotides downstream to the frame-shifting point and is
not affected by this mutation.
nal antibody and a polyclonal antibody that was raised
against the unique sequence motif at the fusion point (Fig.
5). Anti-X-C antibody is not highly specific and recognizes
pX as well. These data indicate that in a heterologous
system an X-Core fusion protein is expressed.
Next we examined the production of the X-Core fusion
protein by the intact HBV DNA. To this end, Huh7 cells
were transfected with either a wt or the X-Core mutant
DNA; total RNA and proteins were extracted and analyzed
at various time points. No major differences were revealed
in transcription patterns obtained using these two HBV
plasmids (Fig. 6). In contrast, Western analysis using antiCore-specific antibodies revealed the production of a 40kDa protein only following transfection with the X-Core
HBV DNA. This protein is indistinguishable from the protein encoded by the heterologous expression system. Additionally, although not clearly seen in this figure, the HBV
mutant does not express the 18-kDa HBeAg. These data
provide the first functional evidence for X protein production by the 3.9-kb transcript. To further support this possibility, we constructed an Xko-Core HBV plasmid in which
we introduced a stop codon in the X ORF downstream to the
first ATG. Cells were transfected with X-Core or Xko-Core
HBV DNA and RNA and protein were analyzed over time.
The RNA patterns of these two constructs were very similar; nevertheless, in contrast to the X-Core HBV, the XkoCore mutant failed to express the 40-kDa X-Core fused protein
(Fig. 7). These data provide solid evidence for the 3.9-kb RNA
Fig. 5. Production of the X-Core fusion protein in a heterologous system. 293T cells were transfected with HA-pX- (X), HA-Core- (C), and HA-X-Core (XC)
pSG5 expression plasmids and harvested for protein analysis. The extracted proteins were analyzed using three different blots with three different antibodies,
as indicated under the panels. Anti-X-C antibody was raised against the fused epitope junction sequence, but it recognizes pX as well.
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and Liang, 1993; Huang and Yen, 1994). As PRE is localized within the X ORF, the lxRNA harbors two copies of
this element. To further understand the mode of action of
these sequences, we tested the intracellular distribution of
the lxRNA. To this end, HepG2 and Huh7 cells were transfected with HBV DNA and harvested after 30 h for total or
cytoplasmic RNA analysis. Unexpectedly, lxRNA was four
times less represented in the cytoplasmic fraction (Fig. 8,
lanes 1– 4). However, total RNA analysis of both cell lines
confirmed that lxRNA was indeed produced. Analysis of
total, cytoplasmic, and nuclear RNA fractions of HBVtransfected HepG2 cells revealed that lxRNA was preferentially accumulated in the nucleus (lanes 5–7). These results
suggest that, despite PRE sequences, most of the lxRNA
molecules are not efficiently exported from the nucleus and
might suggest a unique nuclear role for this transcript,
which awaits clarification.
Discussion
Fig. 6. The X-Core HBV mutant efficiently produces the fused 40-kDa
protein. Huh7 cells were transfected with plasmids harboring either wt or
X-Core HBV DNA (HBV ⫻ 2) constructs, as indicated. Cells were harvested at the indicated time points, and total RNA and proteins were
extracted, separated on formaldehyde–agarose gel and SDS–PAGE, respectively, and analyzed. For Western analysis, monoclonal mouse antiCore (mAb 22) and anti-␤-tubulin antibodies were used. Note that the
HBeAg appears as a smear under the Core only in the wt construct.
being engaged in pX production in the context of wt HBV
DNA. We therefore designated this RNA lxRNA.
The lxRNA is poorly nuclear exported
PRE sequences are important for the export of HBV
transcripts to the cytoplasm (Donello et al., 1996; Huang
In this study, we describe the characterization of the
longest HBV transcript, designated lxRNA. At least three
independent HBV DNA constructs can encode the biosynthesis of this transcript, including a self-ligated HBV recombinant DNA. This finding suggests that the lxRNA is an
authentic HBV transcript. Notably, the 1.3-kb HBV DNA
plasmid with enhancer-I and X ORF at both termini is
equally productive in both lxRNA transcriptions, reinforcing the fact that this transcript initiates at the X ORF
promoter. In transgenic mice, a similar 1.3xHBV construct,
but not shorter versions 1.2 and 1.1 kb in length and lacking
enhancer-1 at the 5⬘-end position, give rise to virion production (Guidotti et al., 1995). However, lxRNA was not
detected in the HBV transgenic mice. Considering the fact
that the short X mRNA is also not detected in the transgenic
mice, the lack of lxRNA is not surprising. Our preliminary
results show that production of the lxRNA is transient and
disappears after 3–5 days (unpublished data). This transient
expression could explain the poor detection of this transcript
in some studies. In addition, given the fact that lxRNA is
underpresented in the cytoplasm, studies relying on cytoplasmic RNA analysis would be unlikely to detect this RNA
species.
lxRNA may be regarded as an X transcript that escapes
polyadenylation at its first round of transcription (Guo et al.,
1991). Since the noncanonical HBV poly(A) signal is inefficient, it is possible that lxRNA is produced as a transcription “by-product” and as such plays no role in HBV life
cycle. However, this is rather unlikely since the synthesis of
other crucial HBV transcripts, such as pc/pgRNA, involves
escaping polyadenylation, as well. This is perhaps one of
the reasons HBV poly(A) signal activity is regulated by a
number of upstream cis elements (Russnak and Ganem,
1990; Schutz et al., 1996). To investigate the requirement of
a weak poly(A) signal in production of the HBV
G. Doitsh, Y. Shaul / Virology 309 (2003) 339 –349
345
Fig. 7. The lxRNA encodes pX. Huh7 cells were transfected with the HBV ⫻ 1.3 construct harboring either a single X-Core mutation (lanes 1–3) or double
Xko⫹X-Core mutations (lanes 4 – 6). Both mutations were positioned only within the upstream X gene, while the 3⬘ X ORF in both constructs remained intact.
Schemes of the HBV ⫻ 1.3 constructs used are illustrated above the panels. The “Stop” arrow indicates the Xko stop mutation at position 27 of the X gene
and the “(⫺1) ORF” arrows indicate the X-Core (⫺1) frameshift mutation positioned 10 bp upstream of the X gene stop codon. Cells were harvested at the
indicated time points. RNA and proteins were extracted and analyzed.
readthrough transcripts, we generated a mutant HBV DNA
in which this signal was modified to the canonical efficient
poly(A) signal. This mutant was capable of programming
the synthesis of all the viral readthrough transcripts, including lxRNA, although not as efficiently as the wild-type
DNA. Furthermore, the ratio between the lxRNA and pc/
pgRNA levels was similar regardless of the nature of
poly(A) signal, suggesting both to be regulated by the same
molecular mechanism. We therefore suggest that lxRNA
readthrough transcription is a regulated process.
An additional parameter that authenticates mRNA is its
capacity to be translated. Here we provide strong evidence
that the lxRNA can encode pX. This was demonstrated
using an lxRNA construct encoding a fusion protein of the
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Fig. 8. The lxRNA resides in the nucleus after synthesis. HepG2 and Huh7 cells were transfected with wt HBV DNA. Plates were subjected to total RNA
(TR), cytoplasmic (Cyt), or nuclear (Nuc) RNA isolation 30 h posttransfection and were analyzed using an HBV probe. Although equal amounts of RNA
were loaded per lane the nuclear RNA fraction (lane 7) represents four times more cells. A GAPDH probe (lanes 1– 4) was used to normalize the RNA content
in each lane. Ethidium bromide analysis of rRNA was used to quantify the RNA in each lane (ethidium analysis of lanes 8 –10 corresponds to lanes 5–7,
respectively). The sizes of the separated ribosomal RNA molecules are depicted. Note that the 45S rRNA precursor molecules reside in the total and nuclear
but not in the cytoplasmic RNA fractions. Arrows indicate the viral transcripts.
X and the CORE genes. It appears therefore that during the
HBV life cycle two different mRNA species are produced
for pX production: the 3.9-kb lxRNA and the 0.7-kb X
mRNA. In general, pX functions are difficult to demonstrate
by conventional transfection experiments. Under these conditions, large amounts of viral DNA plasmids are introduced
into the cells and at least some of the roles of pX may be
compromised. A number of reports have shown the importance of pX in HBV replication (Chen et al., 1993; Zoulim
et al., 1994; Sitterlin et al., 2000; Bouchard et al., 2001; Xu
et al., 2002). Nevertheless, the possible requirement for both
putative pX transcripts was not addressed. Recently a cell
line was established that is susceptible to HBV infection
(Paran et al., 2001). This line may enable us to address pX
synthesis and functions in a more physiological way.
lxRNA harbors two copies of the PRE sequence. HBVPRE is similar to the Rev response element (RRE) of human
immunodeficiency virus (HIV) and related lentiviruses
(Cullen, 1998; Hope, 1997) and plays a crucial role in HBV
RNA nuclear export. As the sequence of PRE is found in all
the HBV mRNAs, it was rather unexpected that only the
lxRNA was underpresented in the cytoplasm, suggesting its
inefficient nuclear export. Given the fact that lxRNA is
active in translation there are several possible explanations
for lxRNA expression: (1) a fraction of this transcript may
be exported to the cytoplasm, where it is actively translated;
(2) lxRNA undergoes nuclear processing to yield a fully
spliced RNA which later accumulates in the host cytoplasm
(see below); (3) lxRNA undergoes nuclear translation
(Brogna et al., 2002; Iborra et al., 2001). The latter is an
interesting possibility which might explain the requirement
for two X gene transcripts: lxRNA may supply the nuclear
and the 0.7-kb X RNA the cytoplasmic X protein.
HBV PRE can functionally substitute HIV RRE and
activate the export of intron-containing transcripts (Huang
and Yen, 1995). Furthermore, competitive inhibition of HIV
REV-mediated nuclear export also blocks PRE-mediated
export activity (Roth and Dobbelstein, 1997). However, in
mechanistic terms, the HBV PRE might be closely related to
the constitutive transport element (CTE) of retroviruses.
Leptomycin B (LMB), which specifically inhibits XPO-1/
CRM-1, blocks RRE-mediated nuclear export but not CTEcontaining and PRE-containing RNA (Otero et al., 1998).
Unlike RRE, which binds the HIV rev protein, both CTE
and HBV PRE are functional in the absence of viral proteins. Nevertheless, here we demonstrate a new feature of
the HBV PRE in which two copies per RNA is no more
constitutively active, as expected from an authentic CTE.
The implication of this finding on the mechanism of PRE
activity must be addressed in future.
Finally, RNA splicing plays a minor role in increasing
the repertoire of HBV transcripts (Chang and Sharp, 1989;
G. Doitsh, Y. Shaul / Virology 309 (2003) 339 –349
Su et al., 1989; Suzuki et al., 1989; Wu et al., 1991; Huang
et al., 2000). Previous studies marked the pgRNA as the
unique splicing precursor of the virus. However unspliced
precursors such as lxRNA are often inferior in nuclear
export. Thus, it is possible that lxRNA is a precursor to a set
of HBV spliced transcripts that encode protein X. This
interesting possibility is currently under study.
Materials and methods
Cell culture and DNA transfections
The cells employed were cultured in Dulbecco modified
Eagle’s minimal essential medium (D5796, Sigma) containing 100 U of penicillin and 100 ␮g of streptomycin per
milliliter, supplemented with 8% fetal bovine serum
(GIBCO Laboratories). Transfection was carried out by the
CaPi method as previously described (Haviv et al., 1995).
Cells were seeded in 10-cm plates 6 – 8 h prior to transfection and reached 60% confluence at the time of transfection.
Each transfection reaction contained a constant amount of
25 ␮g of DNA per 10-cm dish, consisting of 15 ␮g of HBV
plasmid.
Plasmid constructions
HBV constructs
The HBVx2 plasmid contains two head to tail copies of
HBV full-length DNA (subtype adw), which were ligated
into pGEM-3Z (Promega, No. p2151) via the unique EcoRI
site as previously described (Doitsh and Shaul, 1999). For
HBVx1.3 construction, an over-length HBV genome of
4195 bp was produced, harboring a 5⬘ terminus containing
the unique EcoRV site (nt 1043) and a 3⬘ terminus of the
unique TaqI site (nt 2017). This EcoRV–TaqI HBV fragment was inserted between the SmaI–AccI unique sites of
pGEM-3Z, respectively. To obtain a circular genomic
DNA, 40 ␮g of linear HBV DNA was excised by EcoRI and
self-ligated in a large volume (50 ml) ligation mix to encourage intramolecular ligation. All HBV constructs were
confirmed by restriction enzyme analysis.
HBV mutants
The XKO HBV was constructed by inserting a
stop codon at position 27 of the X gene as was previously described (Ori et al., 1998). To generate the
X-Core fusion HBV mutant, a single thymidine was
inserted at position 1878 of the HBV sequence by twostep PCR-based mutagenesis using PWO DNA
polymerase (Boehringer Mannheim) and the following primers: 5⬘-GCGCTGAATCCCGCGGACGACC-3⬘;
5⬘-GGCAGAGGTGAAAAAAGTTGCATGG3⬘; 5⬘CCATGCAACTTTTTTCACCTCTGCC-3⬘; and 5⬘-CAGTGAGAGGGCCCACAAATTG-3⬘. The constructed mutants were confirmed by DNA sequencing.
347
HA-X-Core expression plasmid
A DNA fragment containing both the X and Core genes
was amplified from the X-Core fused HBV mutant using the
following primers: 5⬘-GCGAATTCGGCATGGCTTACCCATACGATGTTCCAGATTACGGTGGTGCTGCTAGGCTGTACTGCCAACTGGATCCTTCG-3⬘ and 5⬘-ATGAATTCACCCTAACATTGAGATTCCCGAG-3⬘. The sense
primer was designed to contain an additional HA sequence
upstream to the X ORF. Both primers were flanked by an
EcoRI restriction sequence. The amplified fragment was inserted into a pSG5 vector (Stratagene, No. 216201) and analyzed by sequencing.
Isolation and analysis of viral RNAs
Total RNA was extracted from transfected cells at various
time points after transfection by Tri Reagent (MRC, Inc.) as
was previously described (Doitsh and Shaul, 1999). Nuclear
and cytoplasmic RNA were prepared by Nonidet P-40 (NP-40)
lysis as described (Sambrook et al., 1989). In brief, transfected
cells were washed twice with ice-cold phosphate-buffered saline (PBS ⫺Ca2⫹ ⫺MgCl). The cell pellet was resuspended in
0.6 ml of NP-40 lysis buffer (0.14 M NaCl, 1.5 mM MgCl2, 10
mM Tris– hydrochloride, pH 8.6, 0.5% NP-40, 40 u of RNase
inhibitor) and immediately centrifuged at 1000 g for 5 min at
4°C. The supernatant (0.4 ml) was transferred to an Eppendorf
tube for isolation of cytoplasmic RNA. The pellet from the
NP-40 lysis and centrifugation step was washed twice in 0.3 ml
of ice-cold PBS (⫺Ca2⫹ ⫺MgCl) and then sedimented for
isolation of nuclear RNA. All RNA samples were treated with
RNase-free DNasel (Boehringer Mannheim) for 15 min at
37°C and stored in 75% ethanol until used. Northern analysis
was carried out using agarose–formaldehyde gels according to
published protocols (Sambrook et al., 1989). RNA quality and
quantity were monitored by UV absorption and by ethidium
bromide staining. About 25 ␮g of RNA per sample was separated on a 1% agarose–formaldehyde gel and blotted to a
Hybond N nylon membrane (Amersham). Membranes were
prehybridized and hybridized according to the manufacturer’s
instructions. HBV radioactive probes were prepared using either a full-length HBV DNA or the X gene region. For the
GAPDH probe, a 1.3-kb GAPDH cDNA fragment was used.
Probes were labeled by a random priming protocol using the
desired DNA template and [␣-32P]dCTP (Amersham, 3000
Ci/mmol). About 106 cpm (10 ng of DNA) labeled DNA
fragments were used per 1 ml of hybridization buffer. After
hybridization, the membrane was washed for 60 min at 65°C in
a 0.1% SSC, 0.1% SDS buffer and exposed to an X-ray film
for autoradiography. Densitometry was performed by a Fujix
Bas 2500 phosphorimager (Fuji).
Antibodies
For preparation of polyclonal mouse anti-X-Core antibodies, a synthetic peptide encompassing amino acids 147–
151 of the X gene and the three residues from preCore
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ORF
(NH2-Pro-Cys-Asn-Phe-Phe-His-Leu-Cys-COOH)
was synthesized, purified by HPLC column, and lyophilized. The peptide was resuspended in PBS, coupled to a
keyhole limpet hemocyanin (KLH, No. 77106, PIERCE)
carrier, and injected into mice. The sera of five injected
mice and one nonimmune mouse were diluted to 1:6000 and
screened for X-Core-specific recognition.
Protein analysis
Proteins were extracted from cells by TRI-REAGENT
(MRC, Inc.) according to the manufacturer’s instructions
and solubilized for 30 min at 50°C in Laemmli sample
buffer containing 4 M urea. Soluble proteins were boiled for
10 min and subsequently fractionated on a 10% SDS–
polyacrylamide gel. For Western blot analysis, gels were
electroblotted onto a nitrocellulose membrane for 1 h at 200
mA. After blotting, membrane filters were stained with
Ponceau S and soaked for 2 h at RT in blocking solution
[PBS containing 10% nonfat milk and 0.01% v/v Tween 20
(Sigma)]. All further incubation steps were performed in the
same solution. Filters were incubated for 1–2 h at RT in the
presence of monoclonal mouse anti-HBcAg (mAb 22, generated as previously described (Paran et al., 2001)), polyclonal mouse anti-X-Core (described above), monoclonal
mouse anti-X (Haviv et al., 1998), or anti ␤-tubulin (clone
No. TUB2.1, Sigma) antibody and washed three times with
PBS ⫹ 0.01% Tween 20. Protein A or goat antimouse
conjugated with horseradish peroxidase (HRP) (ICN Laboratories) was added (diluted 1:10,000 in blocking solution)
and incubation allowed to proceed for an additional 1 h
followed by three rounds of washes. Antibody–antigen
complexes were visualized by the ECL chemiluminescent
detection system (Pierce) according to the manufacturer’s
instructions on an X-ray film.
Acknowledgments
We thank S. Budilovsky for excellent technical assistance. This research was supported by the MINERVA Foundation, Germany, by a Research Grant from the Burstein
Family Foundation, and by a grant from the Israel Ministry
of Health. Y. Shaul holds the Oscar and Emma Getz Professorial chair.
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