The Nucleolar Phosphoprotein B23 Interacts with
Hepatitis Delta Antigens and Modulates the Hepatitis Delta Virus RNA
Replication*
Wen-Hung
Huang
,
Benjamin Y. M.
Yung§,
Wan-Jr
Syu¶, and
Yan-Hwa Wu
Lee
From the
Institute of Biochemistry and
¶ Institute of Microbiology and Immunology, National Yang-Ming
University, Taipei, 112 and the § Department of
Pharmacology, College of Medicine, Chang Gung University,
Taipei, Taiwan, Republic of China
Received for publication, November 6, 2000, and in revised form, April 10, 2001
 |
ABSTRACT |
Hepatitis delta virus (HDV) encodes two isoforms
of delta antigens (HDAgs). The small form of HDAg is required for HDV
RNA replication, while the large form of HDAg inhibits the viral
replication and is required for virion assembly. In this study, we
found that the expression of B23, a nucleolar phosphoprotein involved
in disparate functions including nuclear transport, cellular
proliferation, and ribosome biogenesis, is up-regulated by these two
HDAgs. Using in vivo and in vitro experimental
approaches, we have demonstrated that both isoforms of HDAg can
interact with B23 and their interaction domains were identified as the
NH2-terminal fragment of each molecule encompassing the
nuclear localization signal but not the coiled-coil region of HDAg.
Sucrose gradient centrifugation analysis indicated that the majority of
small HDAg, but a lesser amount of the large HDAg, co-sedimented with
B23 and nucleolin in the large nuclear complex. Transient transfection
experiments also indicated that introducing exogenous full-length B23,
but not a mutated B23 defective in HDAg binding, enhanced HDV RNA
replication. All together, our results reveal that HDAg has two
distinct effects on nucleolar B23, up-regulation of its gene expression
and the complex formation, which in turn regulates HDV RNA replication.
Therefore, this work demonstrates the important role of nucleolar
protein in regulating the HDV RNA replication through the complex
formation with the key positive regulator being small HDAg.
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INTRODUCTION |
Hepatitis delta virus (HDV) is a negative-strand RNA virus and is
also a subviral satellite of hepatitis B virus
(HBV)1 (1). Patients
co-infected with HDV and HBV are at high risk for developing fulminant
hepatitis, liver cirrhosis, and hepatocellular carcinoma (2). This
virus has a single-stranded circular RNA genome of 1.7 kb which is
folded into an unbranched rod-like structure (for review, see Ref. 3
and the references therein). Apart from its RNA genome, the HDV
particle also contains hepatitis delta antigen (HDAg), the only known
protein encoded by HDV, and an envelope provided by the surface antigen
(HBsAg) of HBV. However, HDV RNA replication is independent of its HBV
helper and is dependent on the presence of HDAg (4).
The HDAg exists as two protein species of 27 (214 amino acid residues)
and 24 (195 amino acid residues) kDa, known as large and small HDAg,
respectively (5). These two isoforms are translated from the same
initiation codon of a single open reading frame, but the large
HDAg contains an additional 19 amino acids at its carboxyl terminus,
which is the result of RNA editing of the termination codon of small
HDAg during the viral replication (6). The functional domains of HDAg
have been well characterized (for review, see Refs. 3 and 7 and
references therein). These include an NH2-terminal
coiled-coil domain (amino acid residues 31-52) for HDAg
oligomerization (7-10), two independent nuclear localization signals
(NLSs) (amino acid residues 35-44 and 68-88) for nuclear transport of
HDAg and HDV RNA (11-13), and a central HDV RNA-binding domain (amino
acid residues 95-146) (14-16). In addition, the COOH-terminal portion
of large HDAg contains a prenylation site, which is important for viral
particle assembly with HBsAg (17, 18). Although containing similar
sequences and functional domains, these two isoforms of HDAgs have
different roles during HDV life cycle. Small HDAg functions to
facilitate HDV RNA replication in a manner that is not yet understood
(4), while large HDAg acts as a potent trans-dominant
inhibitor for HDV replication (19, 20) and interacts with HBsAg during
HDV viral particle assembly (18, 21, 22). Both HDAgs are phosphoprotein
(23-26) and phosphorylation regulates HDV RNA replication but not
viral assembly (25, 27). More recent studies suggested that HDAg has a
RNA chaperone activity (28) and can interact with nucleolin for
targeting to nucleolus (29). The trans-suppression ability
of both HDAgs on RNA polymerase II-dependent transcription
(30) and the observed trans-activation ability of the large
HDAg on certain promoters (31), imply that HDAg has the ability to
modulate host cellular gene expression.
The exact molecular mechanism for HDV RNA replication is still obscure.
A double rolling-circle mechanism for HDV replication has been proposed
whereby the host-encoded polymerase undergoes RNA-dependent
RNA synthesis to produce a circular unit-length complement of the
genomic RNA, termed the antigenome (for review, see Ref. 3 and
references therein). The antigenomic RNA, which is also the coding
strand for HDAg, serves as a template for the synthesis of genomic RNA
via the same rolling-circle mechanism. Since HDAg does not possess any
RNA polymerase activity, the host-encoded RNA polymerase is required
for HDV replication in a RNA-directed RNA synthesis manner. However,
the identity of the responsible polymerase and the nature of the
essential role of the small HDAg in the HDV replication remain unclear.
Although host RNA polymerase II is suspected to be involved (32-34), a
more recent study by Modahl et al. (35) suggested that this
might be not the case. Notably, the finding that the HDAg-associated
cellular factors, delta-interacting protein A and nucleolin, can
modulate HDV RNA replication (29, 36), suggests that cellular factors
other than RNA polymerase are involved in HDV replication.
In this study, we found that the mRNA level of the human nucleolar
protein B23 gene is up-regulated in both HDV-replicating and
HDAg-producing cell lines. B23 is a major nucleolar phosphoprotein that
performs a plethora of activities. This protein-like nucleolin is
present in abundance in both tumor cells and proliferating cells and
constitutes a common signal for cell proliferation (37-39). It
localizes in granular regions of nucleoli (40, 41), is associated with
preribosomal particles (42, 43), and forms hexamers that may be
important for the assembly of ribosomes (44, 45). B23 has the ability
to shuttle between the nucleus and the cytoplasm (46), and binds to
nuclear localization signal containing peptides (47), and thus serves
as a shuttle protein in the nuclear import. For example, it forms a
specific complex with the nucleolar protein, p120 (48), nucleolin (49),
and several viral proteins such as Rex of human T-cell leukemia virus (50), the Rev (51) and Tat (52) proteins of human immunodeficiency virus (HIV). Its function is to facilitate their nuclear import. Interestingly, in this study we also found that the B23 protein can
directly interact with the two isoforms of HDAg and the interaction domains of B23-HDAg reside within the fragment containing the nuclear
localization signal of HDAg, suggesting that B23 may serve as shuttle
protein for transport of HDAg into the nucleus. Additionally, our
results also indicated that the small HDAg is found in a complex of B23
and nucleolin with a size larger than 700 kDa, and the interaction of
small HDAg with B23 regulates HDV RNA replication. Therefore, the
association of the major nucleolar protein B23 and nucleolin with the
small HDAg may represent an important mechanism for HDV RNA replication.
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EXPERIMENTAL PROCEDURES |
Plasmids--
Plasmid pSVL-d2g contains a head-to-tail
tandem dimer of the full-length HDV cDNA (as 1.7-kb XbaI
fragment) inserted at the XbaI site of the vector plasmid
pSVL (Amersham Pharmacia Biotech) (25). On transfection into mammalian
cells, this plasmid can direct replication of the viral genome and
expression of HDAg. Plasmids pGEM3L and pGEM3S contain a
ScaI-EcoRI fragment (1.1 kb) of HDV cDNA
driven by the SP6 or the T7 phage promoter of pGEM-3Zf(
) (53), which
can allow the production of strand-specific probes by in
vitro transcription and large or small HDAg by in vitro
translation. Plasmids pGEX-3X-L and pGEX-3X-S, kindly provided by
S. J. Lo (Yang-Ming University, Taiwan), contains the
SstII-EcoRI fragment of the large or small HDAg
open reading frame in-frame inserted into the plasmid pGEX-3X, which
can direct the expression of either a GST-fused large HDAg (GST-DAg) or
small HDAg (GST-dAg). Plasmid pCR3-FLAG-B23 is an expression construct
of B23 in mammalian cells with an additional FLAG-tag at the
NH2 terminus. This construct was generated by polymerase
chain reaction using a NH2-terminal primer
5'-ACCATGGACTACAAAGACGATGACAAGCTTATGGAAGATTCGATGGAC-3' and
COOH-terminal primer 5'-CGCCGCGGATCCTTAAAGAGACTTCCTCCACT-3' with a B23
cDNA clone pET-T7 as the template (54). The amplified polymerase
chain reaction product was inserted into the vector pCR3.1
(Invitrogen). Plasmid pCR3-FLAG-B23-(1-127) is a mutant derivative of pCR3-FLAG-B23 that can direct the expression of the
NH2-terminal amino acid residues 1-127 of B23 fused with
the FLAG-tag. The construction of the plasmid
pCR3-FLAG-B23-(1-127) was carried out in a similar manner to
pCR3-FLAG-B23 except that a different COOH-terminal polymerase chain
reaction primer 5'-CCTCCTCTAGATTAATCTTCTGACTCTGCATC-3' was used. The
COOH-terminal truncated variants of the in vitro translated
B23 were produced by the TNT system (Promega) from the plasmid of
pCR3-FLAG-B23 digested with the restriction enzymes EarI,
BstNI, HhaI, or BglII, allowing
generation of the NH2-terminal 127, 144, 190, and 285 amino
acid residues of B23, respectively. Plasmids expressing the His-tagged
HDAg polypeptides, which included the wild-type full-length small HDAg,
dAg-(1-195), and its deletion mutants NdAg-(1-88),
NMdAg-(1-143), MdAg-(89-143), and CdAg-(89-195) (28), were kindly
supplied by H.-N. Wu (Academia Sinica, Taiwan). Plasmids pMT-DAg and
pMT-dAg contain a 1.1-kb ScaI-EcoRI fragment of
the large or small HDAg cDNA sequence under human metallothionein (MT) promoter control. Plasmid pSVL-d contains a 1.1-kb
ScaI-EcoRI fragment of the small HDAg cDNA
sequence under SV40 late promoter control. Plasmid pdAg-
(17-56), a
derivative of pGEM-3Zf(
), can direct the expression of in
vitro translated small HDAg variant dAg-
(17-56) with an
in-frame deletion of residues 17-56 when prepared by the TNT system
(Promega).
Cell Lines, Transfection, and Subcellular
Fractionation--
Human hepatocellular carcinoma cell lines HuH-7 and
HepG2 were cultured as described in Yeh et al. (25).
SVLD3-N1 cells (designated as N1) were HepG2 stable clones integrated
with trimeric HDV cDNA and constitutively expressing both isoforms
of the HDAgs and both HDV genomic and antigenomic RNA (55). L7 and L10
are the stable cell lines constitutively expressing the large HDAg in
HepG2 cells, while S2 and S7 are the stable HepG2 cell lines expressing
the small HDAg. These cell lines were established by transfected HepG2
cells (2 × 106 cells) with plasmid DNA of pMT-DAg or
pMT-dAg (5 µg) together with pSV2-neo (0.5 µg) by electroporation.
After 4 weeks selection with 0.8 mg/ml G418 (Sigma), cell colonies were
picked up, cultured for another 2 weeks, and analyzed for the
expression of HDAg by Western blotting. Detection of HDAg was performed
with human anti-HDAg antiserum as the primary antibody and horseradish
peroxidase-conjugated goat anti-human antiserum as the secondary
antibody using the enhanced chemiluminescence detection method (ECL,
Pierce). G418-resistant, HDAg-negative cell colonies (C4 and C5) were
also picked up, expanded in G418-supplemented medium, and were used as
control cell lines. For the transient transfection experiment, plasmid
DNAs were transfected into HuH-7 cells by EffecteneTM
transfection reagent (Qiagen). For preparation of total cell lysates
and nuclear extracts, cells were harvested, lysed, and treated as
described by You et al. (56, 57).
RNA Preparation, Northern Blotting, and mRNA Differential
Display Method--
Total RNAs from each cell lines were extracted by
using TRI reagent (Molecular Research Center) according to the
instructions of the supplier. For Northern blot analysis, 20 µg of
total cellular RNA samples was subjected to electrophoresis on a 6%
formaldehyde, 1% agarose RNA gel and then transferred into a
nylon filter. The filter were pre-hybridized and then hybridized
according to the standard method (58). For detection of genomic or
antigenomic HDV RNA, the strand-specific HDV RNA probe was prepared by
in vitro transcription of pGEM3L using the SP6/T7
transcription kit (25). Differential display of mRNA was performed
according to the instructions of the supplier using the RNAimage kit
(GeneHunter Co.).
Immunofluorescence--
Immunofluorescence was performed by a
method modified from the published procedures (57, 59). The
localization of HDAg and FLAG-tagged B23 protein in transfected cells
was examined by fluorescence microscopy or confocal laser scanning
microscopy (Leica TCS-NT). For immunofluorescence staining, the cells
were fixed with acetone/methanol (1:1) (
20 °C) and probed
with human anti-HDAg antiserum or mouse monoclonal anti-FLAG M2
antibody (Kodak), followed by fluorescein isothiocyanate-conjugated
rabbit anti-human IgG or rhodamine-conjugated rabbit anti-mouse IgG.
In Vitro Binding Analysis of HDAg and B23--
The GST-HDAg
fusion proteins expressed from pGEX-3X-L and pGEX-3X-S vectors were
purified as described elsewhere (60, 61). The His-tagged HDAg fusion
proteins expressed from various expression vectors (dAg-(1-195),
NdAg-(1-88), NMdAg-(1-143), MdAg-(89-153), and CdAg-(89-195)) (28)
were affinity purified using a His-bound resin column according to the
instructions from the supplier. For binding with the endogenous B23,
HeLa cell nuclear extracts (200 µg) were incubated with 20 µl of
glutathione-Sepharose 4B beads prebound with GST or GST-HDAg (4 µg)
or His resins prebound with His-tagged HDAg variants (4 µg) at
4 °C overnight under gentle agitation. The beads were washed four
times with phosphate-buffered saline containing 0.3% Nonidet P-40.
Proteins bound on beads were eluted using the sample buffer and
resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE, 12.5% polyacrylamide gel), and processed for Western blot
analysis. Detection of B23 was performed with goat anti-B23 antiserum
(Santa Crutz) as the primary antibody and horseradish
peroxidase-conjugated anti-goat antiserum as the secondary antibody
using the ECL method. For binding with the in vitro
translated B23, the B23 variants (5 µl) were prepared from the
various restriction enzyme-digested pCR3-FLAG-B23 by the TNT system
(Promega) and incubated with GST or GST-HDAg bound
glutathione-Sepharose 4B beads (20 µl) at 4 °C overnight and the
resins were washed four times with phosphate-buffered saline containing
0.5% Nonidet P-40. Proteins bound on beads were eluted by the sample
buffer and resolved by SDS-PAGE (12.5% gel), and detected by autoradiography.
In Vitro and in Vivo Co-immunoprecipitation--
For the
in vivo co-immunoprecipitation (co-IP) of HDAg and B23, the
nuclear extracts of HepG2 cells (500 µg) and N1 cells (200 µg) were
immunoprecipitated with protein A-Sepharose (20 µl packed volume)
conjugated with human anti-HDAg antiserum (20 µl) or anti-FLAG M2
affinity gel (Kodak) as a negative control. For the in vitro
co-IP of HDAg and B23, the in vitro translated B23 or HDAgs
(5 µl each) were immunoprecipitated with protein A-Sepharose (20 µl
packed volume) conjugated with human anti-HDAg antiserum (20 µl).
Alternatively, the in vitro translated FLAG-tagged B23 or
HDAg variants (10 µl each) were immunoprecipitated with protein
A-Sepharose (20 µl packed volume) conjugated with mouse monoclonal
anti-FLAG antibody (M2) (7.5 µl). The immunoprecipitates obtained
from both experiments were washed with phosphate-buffered saline
containing 0.1% Nonidet P-40 (in vitro co-IP) or 0.3%
Nonidet P-40 (in vivo co-IP) and processed for immunoblot
analysis or autoradiography.
Sucrose Gradient Centrifugation--
The nuclear extracts of the
HDAg producing cell lines (L10 or S7) and their parental HepG2 cells
were prepared for sucrose gradient centrifugation. Nuclear extracts
(0.6 ml) (500 µg) were loaded on 10.8 ml of 10-60% (w/v) sucrose
gradient prepared in NET buffer (50 mM Tris-HCl, pH 7.9, 150 mM NaCl, and 0.5 mM EDTA). Samples were
subjected to centrifugation at 38,000 rpm in a SW41 rotor
(Beckman) for 16 h at 4 °C. The gradients were aliquoted into
0.6-ml fractions from the top and analyzed for B23 and HDAg by
immunoblot. The protein standards thyroglobulin (669 kDa) and catalase
(232 kDa) were also run on sucrose gradients in parallel with samples.
 |
RESULTS |
The Expression of Nucleolar Phosphoprotein B23 Is Up-regulated in
Both HDV- and HDAg-producing Cells--
To search for the cellular
target genes affected by HDV, we performed an mRNA differential
display assay (see "Experimental Procedures"). The total RNAs from
both HDV-producing N1 cells and its parental HepG2 cells were isolated
and the differentially expressed RNAs were analyzed by the RNAimage kit
(GeneHunter Co.). The identity of differentially expressed cDNA
clones was determined by DNA sequencing and through sequence alignment,
we found that one of the candidate genes was the human nucleolar
phosphoprotein B23 (also known as nucleophosmin, NO38, or numatrin)
(62-64). Northern blot analysis confirmed the up-regulation of the B23
transcript in the HDV-producing N1 cell line (Fig.
1A). To find out whether this
enhancement of B23 expression resulted from HDAg, we also examined B23
expression in large and small HDAg-producing stable cell lines (L7 and
L10 cell lines for large HDAg, S2, and S7 cell lines for small HDAg),
parental HepG2 cells, and control cell lines which failed to show HDAg
expression (C4 and C5) (see "Experimental Procedures").
Interestingly, although the expression level of the large HDAg is not
comparable with that of the small HDAg in their respective producing
cells as shown by Western blot analysis (Fig. 1B), the
enhancement of B23 mRNA expression occurred to a similar extent as
that found in the N1 cell line (about 3-4-fold increase) (Fig.
1A). As noted, there is about a 2-fold increase in the
protein level of B23 in these HDV- or HDAg-producing cells with nuclear
nucleolin as a loading control (Fig. 1B), further supporting
the notion that B23 expression is up-regulated in both the HDV- and the
HDAg-producing cell lines. However, the lack of apparent correlation of
the expression level of two isoforms of HDAg and the extent of B23
expression is intriguing. Clearly, the observed up-regulation of B23
expression in these HDAg-positive cells is HDAg-specific but not due to
clonal selection against G418, since both G418-resistant, HDAg-negative
cell lines C4 and C5 did not have any effect (Fig. 1B,
compare lanes 8 and 9 with lanes
2-6). Presumably, this may reflect the differential
trans-activation ability of two isoforms of HDAg, a property
noted previously (31).

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Fig. 1.
Enhancement of cellular B23 expression in HDV
and HDAg producing cell lines. Panel A, total cellular
RNA of HepG2 (G2), and HDV-producing N1 cells (N1), or HDAg-producing
L7, L10, S2, and S7 cells (L7, L10, S2, and S7) was extracted, and 20 µg of these RNAs were analyzed by Northern blot using
32P-labeled B23 cDNA as a probe (see "Experimental
Procedures"). The ethidium bromide-stained rRNAs shown were used as a
loading control. The results were normalized for the level of B23
expressed in HepG2 cells. Panel B, nuclear extracts (20 µg
in lanes 1-6; 10 µg in lanes 7-9) of HepG2
(G2) and its derived cell lines (see "Experimental Procedures")
were analyzed by immunoblot using goat anti-B23 (Santa Crutz), mouse
monoclonal anti-nucleolin antibody (97) (kindly provided by N.-H. Yeh),
and human anti-HDAg antisera for detection. The results were normalized
to the expressed level of B23 in HepG2 cells. DAg, large
HDAg; dAg, small HDAg.
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HDAg Co-localizes with the B23 Protein in the
Nucleoli--
Previous studies have demonstrated the nuclear or
nucleolar localization of HDAg (12, 23, 65, 66). Since B23 is also abundant in nucleolus, we investigated the possibility of
co-localization of these two proteins. Consistent with previous
findings, both HDAg and B23 were nucleolar proteins when expressed in
HeLa cells by transiently transfected with the HDV replication plasmid
pSVL-d2g or the plasmid FLAG-tagged B23 and detected by the indirect
immunofluorescence staining using the anti-HDAg or the anti-FLAG
antibody (Fig. 2A, panels a-f). Furthermore, when the expression
plasmids of HDAg (pMT-DAg or pMT-dAg) and FLAG-tagged B23 were
co-transfected into HeLa cells, confocal microscopy analysis using the
indirect immunofluorescence staining indicated that both forms of HDAgs
co-localized with the FLAG-B23 in nucleoli (Fig. 2B, panels
j-o). Similar results were obtained in cells transiently
co-transfected with the HDV-replication plasmid pSVL-d2g and
FLAG-tagged B23 (Fig. 2B, panels g-i).
Therefore, it appears that both HDAgs and B23 co-localize in
nucleoli.

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Fig. 2.
Both FLAG-B23 and HDAg co-localize inside a
cell. HeLa cells were transfected with pSV-d2g
(panels a-c) or FLAG-tagged B23 construct pCR3-FLAG-B23
(panels d-f) or co-transfected pCR3-FLAG-B23 together with
various forms of HDV construct including pSVL-d2g (panels
g-i), pMT-DAg (panels j-l), pMT-dAg (panels
m-o) as indicated. The distributions of B23 and HDAg were assessed
by indirect immunofluorescence staining (see "Experimental
Procedures"). For double immunofluorescence staining, cells were
stained with human anti-HDAg antiserum and mouse anti-FLAG M2
monoclonal antibody, followed by fluorescein isothiocyanate-conjugated
goat anti-human IgG or rhodamine-conjugated goat anti-mouse IgG. Panels
c and f are the phase-contrast images of
panels a, b, d, and e. Cell nuclei were also
visualized by Hoechst 33258 staining. Preparations in panels
a-f were examined by fluorescence microscopy, while in
panels g-o the immunofluorescence patterns were
recorded by confocal laser scanning microscopy. The left panels
g, j, and m show the merged image of the
co-localization of HDAg with FLAG·B23 in the nucleoli.
Bars, 10 µm.
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Interaction of HDAgs and B23--
Co-localization of B23 with HDAg
suggests that HDAg may interact with B23. To examine this possibility,
a protein binding assay of GST-HDAg fusion protein to in
vitro translated 35S-labeled B23 was performed. As
shown in Fig. 3, A and
B, the in vitro translated B23 bound to both
forms of GST-HDAg fusion protein (GST-DAg and GST-dAg) but not GST. We
further tested the interaction of B23 and HDAg by
co-immunoprecipitation of in vitro translated
35S-labeled B23 and HDAgs (Fig.
4A). As shown in Fig.
4B, B23 together with both large and small HDAg could be
co-immunoprecipitated by the anti-HDAg antisera (lanes
5-7). This co-precipitation was specific, since no B23 was
precipitated in the absence of HDAg (Fig. 4B, lane 2). The
interaction of HDAg with cellular B23 was also investigated using a
GST-HDAg pull-down analysis. As shown in Fig. 3C, when HeLa
cell nuclear extracts were incubated with partially purified GST-HDAg
fusion proteins, and the bound proteins were detected by anti-B23
antibody, results indicated that cellular B23 protein was pulled-down
by both isoforms of GST-HDAg fusion protein. An in vivo
co-immunoprecipitation experiment using anti-HDAg antibody suggested
that B23 in HDV-producing N1 cells as detected by immunoblot was
co-precipitated by the small HDAg (Fig. 4, C and D,
lanes 5 and 6). This co-immunoprecipitation experiment was specific, since no such co-precipitation was observed when using
the anti-FLAG antibody for immunoprecipitation (Fig. 4, C
and D, lanes 3 and 4). Taken together, based on
the four different approaches, our results indicated that both isoforms
of HDAg could interact with B23 both in vitro and in
vivo.

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Fig. 3.
Analysis of the interaction between HDAg and
B23. Panel A, purified GST, GST-large HDAg
(GST-DAg), GST-small HDAg (GST-dAg) (4 µg each)
used for GST pull-down assay. Panel B, in vitro
binding assay of GST-HDAg fusion protein and in vitro
translated B23. Glutathione-Sepharose beads (20 µl) bound to GST
(lane 2), GST-DAg (lane 3), or GST-dAg
(lane 4) (4 µg) were incubated with the in
vitro translated [35S]Met-labeled B23 (5 µl). The
beads were then washed, and proteins on the beads were eluted with
sampling buffer and analyzed by SDS-PAGE and autoradiography.
Lane 1, input in vitro translated
[35S]Met-labeled B23 (5 µl) without incubation with
glutathione-Sepharose beads. Panel C, in vitro
binding assay of GST-HDAg fusion proteins and cellular B23.
Glutathione-Sepharose beads (20 µl) bound to GST (lane 2),
GST-DAg (lane 3), or GST-dAg (lane 4) (4 µg
each) were incubated with nuclear extracts (NE) (200 µg)
of HeLa cells and the bound fractions were analyzed by SDS-PAGE
followed by immunoblotting with antibody against B23. Lane
1, immunoblot of HeLa nuclear extract (50 µg) with anti-B23
antibody.
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Fig. 4.
Co-immunoprecipitation of B23 and
HDAg in vitro and in vivo. Panel
A, in vitro translated [35S]Met-labeled
B23 (lane 1), large HDAg (DAg, lane 2), small
HDAg (dAg, lane 3), B23 and DAg (lane 4), B23 and
dAg (lane 5), or B23 together with DAg and dAg (lane
6) (5 µl each) was prepared and used for immunoprecipitation
experiments. Panel B, in vitro
co-immunoprecipitation of B23 and HDAg. For immunoprecipitation
(lanes 2-7), the in vitro translated products
(10 µl each) as indicated in panel A were
immunoprecipitated (IP) by human anti-HDAg sera and immunoprecipitates
were analyzed by SDS-PAGE followed by autoradiography (see
"Experimental Procedures"). Lane 1, input in
vitro translated B23 without immunoprecipitation. Panels
C and D, in vivo co-immunoprecipitation of
B23 and HDAg. Nuclear extracts of the HepG2 (G2) and N1 cell lines were
immunoprecipitated by anti-FLAG antibody (lanes 3 and
4) or human anti-HDAg sera (lanes 5 and
6) and immunoprecipitates were analyzed by SDS-PAGE followed
by immunoblotting with antibody against B23 (panel C) or
HDAg (panel D) (see "Experimental Procedures").
Lanes 1 and 2, immunoblots of nuclear extracts
with antibody against B23 or HDAg.
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Mapping the Interaction Domains of B23 and HDAg--
The
interaction domain of B23 within HDAg was mapped using the His-tagged
HDAg fragments harboring the full-length (dAg, 1-195 residues), the
NH2 terminus (NdAg, 1-88 residues), the middle (MdAg,
89-143 residues), the NH2 terminus plus the middle (NMdAg, 1-143 residues), and the COOH terminus (CdAg, 89-195 residues) of
small HDAg (Fig. 5A). As shown
in Fig. 5C, apart from the full-length protein, only the
HDAg variants containing the NH2-terminal fragment, NdAg
and NMdAg, but not those harboring the COOH-terminal (CdAg) or middle
fragment (MdAg), could bind to the cellular B23, suggesting that the
interaction domain of B23 is within amino acid residues 1-88 of HDAg.
Since this NH2-terminal fragment harbors a coiled-coil domain (residues 31-52) responsible for HDAg oligomerization (8-10) and two independent NLSs (residues 35-44 for NLS1 and residues 68-88
for NLS2) for targeting HDAg to the nucleus (11, 12), it was of
interest to further define the interaction region within HDAg. As shown
in Fig. 5E, like the wild-type full-length protein, the
in vitro translated small HDAg variant dAg-(17-56) which
lacks the regions of coiled-coil and NLS1 but retains the intact NLS2, could be co-immunoprecipitated with the in vitro translated
FLAG-B23 (lanes 4 and 5). This suggests that the
interaction of B23 is not mediated through the coiled-coil domain and
NLS1 of HDAg, but rather through the NLS2 region.

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Fig. 5.
Mapping of the B23-interacting region in
HDAg. Panel A, schematic diagram of the His-tagged
small HDAg (dAg) and its various derivatives used for
in vitro binding assay. These His-tagged small HDAg variants
are designated by the amino acid residues of small HDAg fragment. The
binding ability of small HDAg and its derivatives with B23 are
indicated by a plus or a minus sign. Panel
B, partial purified His-tagged small HDAg (dAg) or its
various derivatives NdAg, NMdAg, MdAg, and CdAg (5 µg each) used for
the in vitro binding assay. All His-tagged dAg derivatives
were purified as described (see "Experimental Procedures") and
separated by SDS-PAGE and detected by Coomassie Blue (CB)
staining. Panel C, HeLa nuclear extracts (NE)
were incubated with His resins (lane 2) or His resins
prebound with small HDAg derivatives (lanes 3-7) as
indicated, and proteins bound on the beads were eluted with sampling
buffer and analyzed by SDS-PAGE followed by immunoblotting with
anti-B23 antibody (see "Experimental Procedures"). Lane
1, immunoblot of HeLa nuclear extracts (50 µg) with antibody
against B23. Panel D, in vitro translated
[35S]Met-labeled small HDAg (dAg, lane 1),
small HDAg mutant dAg- (17-56) (lane 2), FLAG-tagged B23
(lane 3), FLAG-tagged B23 and small HDAg (lane
4), or FLAG-tagged B23 together with dAg- (17-56) (lane
5) (5 µl each) was prepared and used for immunoprecipitation
experiments. Panel E, for immunoprecipitation, the in
vitro translated products (10 µl each) as indicated in
panel D were immunoprecipitated with mouse anti-FLAG M2
monoclonal antibody prebound with protein A-Sepharose and
immunoprecipitates were analyzed by SDS-PAGE followed by
autoradiography (see "Experimental Procedures").
|
|
The interaction domains of both HDAgs within the B23 protein was also
determined by the in vitro binding analysis using the GST-HDAg fusion proteins (GST-DAg or GST-dAg) and a series mutants of
in vitro translated B23. As shown in Fig.
6, after deletion of the COOH-terminal
fragment of B23 in B23-(1-285) and B23-(1-190), the proteins still
bound to both HDAgs. However, when the size of deletion was increased
in B23-(1-144) variant the binding activity of the mutated B23 was
dramatically reduced. Yet further deletion to give the B23-(1-127)
construct resulted in a complete loss of the binding activity,
suggesting that the region between residues 127 and 190 in B23 is
important for its binding with HDAg. Thus, it can be suggested that the
interaction of B23 and HDAg is mediated through their
NH2-terminal fragments and the regions encompassing amino
acid residues 1-88, but not the coiled-coil region, of HDAg and amino
acid residues 1-190 of B23 are sufficient for the protein-protein interaction.

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Fig. 6.
Mapping the HDAg-interacting region in
B23. Panel A, the in vitro translated,
[35S]Met-labeled B23 variants used for in
vitro pull-down assay. The 35S-labeled COOH-terminal
truncated variants of B23 (lanes 1-4) (5 µl each)
prepared by TNT reactions (Promega) were analyzed by SDS-PAGE and
autoradiography (see "Experimental Procedures"). The B23 variants
were designated by their amino acid residues. Panels B-D,
binding analysis of GST-HDAg fusion proteins and in vitro
translated B23 variants. Glutathione-Sepharose beads (20 µl) prebound
with GST (panel B), GST-DAg (panel C), or GST-dAg
(panel D) (5 µg each) were incubated with the in
vitro translated B23 variants as indicated (5 µl), and proteins
bound on the beads were eluted with sampling buffer and analyzed by
SDS-PAGE and autoradiography (see "Experimental Procedures").
|
|
HDAg Forms a Large Complex with B23 in Vivo--
In the nucleolus,
the protein B23 is expected to be largely in the hexameric form with a
molecular mass of 230-255 kDa (44). Since B23 can associate with a
wide spectrum of cellular factors, it was necessary to investigate the
status of in vivo complex formation between these two
nuclear proteins. To this end, the sedimentation behavior of B23 and
HDAg in the nuclear fractions of both types of HDAg-producing cells was
examined using the sucrose gradient centrifugation. As shown in Fig.
7A, the nuclear B23 in all the
cell lines examined, including L10, S7 cells, and the parental HepG2
(G2) cells, sedimented in two predominant peaks at fractions 5-8 (peak
I) and fractions 15-17 (peak II). Based on the sedimentation profiles
of two standard proteins, catalase (fraction 7, 232 kDa) and
thyroglobulin (fraction 10, 699 kDa), it seems that the nuclear B23 is
either present as a hexameric form (peak I, molecular mass 230 kDa) or
associated with other cellular components (peak II). Intriguingly, the
small HDAg in HDAg-producing S7 cells co-sedimented with the peak II
complex of B23, while in the large HDAg-producing L10 cells, HDAg was distributed more broadly from fraction 12 to 17 and peaked at fractions
12-14. Thus, large HDAg was only partially superimposed with the B23
profiles at peak II. This reflects a partial separation of the large
HDAg from a B23-associated complex. Notably, the small HDAg in S7 cells
also appeared as a minor species at fraction 13, unassociated with B23.
These results strongly suggest that unlike the case of large HDAg, the
cellular small HDAg predominantly co-sedimented with a high molecular
weight form of B23 in vivo. Additionally, when considering
that both HDAg and B23 can associate with another nucleolar protein
nucleolin (29, 49), it was important to examine the status of nucleolin
in the sucrose gradient centrifugation. Our results indicated that
apart from its monomeric form (~100 kDa) which appeared at fractions
4-6, about half the population of nucleolin co-sedimented with B23 and
HDAg at fractions 15-17 (Fig. 7A). Co-immunoprecipitation
indicated that the co-sedimentation of these three nucleolar proteins
(HDAg, B23, and nucleolin) at fractions 15-17 of sucrose gradients of
L10 or S7 cells is not coincidental because both B23 and nucleolin in
these two HDAg-expressing cells, but not in the control cells, were
co-immunoprecipitated by anti-HDAg antibody (Fig. 7B, lanes
2-4 and 6-8). This supports the notion that both
forms of HDAg form a large complex with B23 and nucleolin in
vivo.

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Fig. 7.
Analysis of in vivo complex
formation between HDAg and B23 by sucrose gradient centrifugation.
Panel A, the nuclear extracts of HepG2 (G2), and its
HDAg-producing cells (L10 or S7) (500 µg) were prepared and analyzed
by sucrose gradient centrifugation (see "Experimental Procedures").
Aliquots of each fraction were analyzed by immunoblot using goat
anti-B23, human anti-HDAg antisera, or mouse monoclonal anti-nucleolin
antibody and the ECL method used for detection. Protein standards
(catalase, 232 kDa; thyroglobulin, 669 kDa) were run in a parallel
experiment and their positions are indicated. Panel B, the
nuclear extracts (lanes 2 and 6, 20 µg each) or
sucrose gradient fractions 15-17 (lanes 3-4 and
7-8) (300 µl) obtained from L10 (lanes 2 and
3), S7 (lanes 6 and 7) or G2
(lanes 4 and 8) cells as described in panel
A, were immunoprecipitated with rabbit anti-HDAg antibody. The
immunopreciptates were analyzed by SDS-PAGE followed by immunoblotting
with antibody against HDAg, B23, or nucleolin. Lanes 1 and
5, immunoblots of the nuclear extracts of L10 (lane
1, 20 µg) or S7 (lane 5, 10 µg) cells with antibody
against HDAg, B23, or nucleolin.
|
|
B23 Enhances the HDV Replication--
The above results show that
HDAg elicits two effects on B23, the up-regulation of its expression
and the formation of a complex. Thus, it is rather important to know
whether these two features of HDAg on B23 have any role in regulation
of HDV replication. To explore this possibility, the effect of B23 on
HDV replication was examined by introducing increasing amounts of B23
expression construct together with the HDV-replication plasmid pSVL-d2g
into HuH-7 cells and the level of HDV replication was then analyzed by
examining HDAg expression and HDV genomic/antigenomic RNA production. As shown in Fig. 8A,
introducing 0.2 or 1.0 µg of the FLAG-B23 construct enhanced the
production of the small HDAg as well as increasing the HDV
genomic/antigenomic RNA (about 2-4-fold) in a
dose-dependent manner. Notably, this is not the case for
cells co-transfected with the B23 mutant construct FLAG-B23-(1-127) which is defective in binding with HDAg (Fig. 8B). This
enhancement of HDV replication by B23 is also not due to the effect of
B23 on the promoter activity of the SV-40 late promoter used to drive the HDV replication plasmid pSVL-d2g, since no such enhancement of HDAg
production was observed for the HDAg gene under the SV40 late promoter
control in the construct pSVL-d (Fig. 8C). Therefore, our
results clearly indicate that B23 modulates HDV replication through the
formation of a complex with HDAg.

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Fig. 8.
B23 modulates the HDV replication
activity. Panel A, HuH-7 cells (2 × 106) were co-transfected with plasmids pSVL-d2g (1 µg)
and increasing amounts of FLAG-B23 (0.2 or 1.0 µg) (see
"Experimental Procedures"). The expression level of HDAg or HDV
genomic/antigenomic RNA in the transfected cells at 2 days (for HDAg
detection) or 6 days (for HDV RNA detection) post-transfection were
determined (see "Experimental Procedures") and their relative
intensities were quantitated by a PhosphorImager. Panel B,
all experimental conditions were similar to panel A, except
that FLAG-B23-(1-127) expression plasmid pCR3-FLAG-B23-(1-127) was
used for co-transfection. Panel C, all experimental
conditions were similar to panel A, except that plasmid
pSVL-d was used for co-transfection.
|
|
 |
DISCUSSION |
This study has shown that the steady state level of B23 mRNA
and protein is increased in both HDV-replicating and HDAg-expressing cells (Fig. 1). Overexpression of B23 mRNA is usually found in proliferative or tumor cells (37-39) and thus our results strongly support the notion that HDV infection may reflect a similar status of
proliferation. However, the mechanism by which HDAg up-regulates the
B23 gene expression is still unclear. According to previous studies,
the large HDAg but not its small isoform has a
trans-activation ability on certain promoters (31) or
conversely, both HDAgs have a trans-suppression ability on
RNA polymerase II-dependent transcription (30). Therefore,
it would appear that the mechanism involved in the up-regulation of B23
expression by these two isoforms of HDAg is distinct from those
mechanisms (Fig. 1). When considering that the transcriptional
regulatory region of human B23 gene contains a transcription factor
YY1-binding site (67) and protein B23 can associate with this
transcriptional factor and modulate its transcriptional activity (68),
it is likely that the up-regulation of B23 gene expression by HDAg is
mediated through an effect on the transcriptional activity of YY1.
Apart from the up-regulation of B23 expression, the current study based
on four different lines of evidence reflecting both in vivo
and in vitro situations, has demonstrated that both isoforms of HDAg can associate with the major nucleolar protein B23. The confocal microscopy analysis showed the co-localization of B23 and HDAg
in nucleoli (Fig. 2). The immunoprecipitation experiment in
HDV-replicating N1 cells transiently transfected with the expression plasmid of FLAG-B23 provided further support for the in vivo
binding of B23 and HDAg (Fig. 4, C and D).
Additionally, by using both co-immunoprecipitation (Fig. 4,
A and B) and the GST-HDAg fusion protein
pull-down assay (Fig. 3), both in vitro translated and cellular B23 have been shown to interact with HDAgs. The in
vitro binding studies utilizing deletion mutants of B23 or HDAg
defined the binding sites of these two proteins as the
NH2-terminal regions of both molecules (Figs. 5 and 6).
Interestingly, this region of HDAg (NH2-terminal 88 amino
acid residues) consists of a coiled-coil region (residues 31-52)
important for HDAg oligomerization (7-10), and three discontinuous
stretches of basic amino acids that have been identified as the two
independent NLSs (residues 35-44 and 69-88 for NLS1 and NLS2,
respectively) or nucleolin-binding sites (NBSs) (residues 39-42 and
60-63 for core sequences of NBS1 and NBS2, respectively) and are
required for nuclear or nucleolar localization of HDAg (11, 12, 29).
Deletion of residues 17-56 of HDAg, which contains the coiled-coil
domain overlapped with NLS1 or NBS1, did not lose the binding activity
to B23 (Fig. 5E), suggesting that the interaction region of
B23 likely resides in the segment of HDAg containing an intact NLS2 or
NBS2. Notably, this observation is in accordance with the findings that
NLS2 or NBS2, but not NLS1 or NBS1, is more important in controlling the nuclear or nucleolar transport of HDAg (11, 12, 29). Conceivably,
binding with B23 may be related to nuclear or nucleolar localization of
HDAg. B23 has also been reported to bind the arginine-rich basic region
of the human T-cell leukemia virus protein Rex (50), and the HIV
proteins Rev (51, 69) and Tat (52). These arginine-rich regions serve
as the RNA-binding and nucleolar localization domains for these viral
proteins. However, unlike these targeted viral proteins, B23 did not
interact with the arginine-rich regions of the middle one-third
fragment of HDAg (residues 97-107 or 136-146) which is involved in
RNA binding (16), since the subfragment of HDAg (MdAg) spanning
residues 89-147 failed to bind B23 (Fig. 5C). Therefore,
charge alone does not appear to be sufficient to determine the binding
of HDAg by B23. Similar results have been reported when the binding of
B23 to the nuclear localization signals in nucleolar protein p120 and
nucleolin are analyzed (48, 49). Presumably, the conformation of the
domain is the major factor contributing to its binding ability.
Protein B23 is multifunctional and exhibits nucleic acid binding,
ribonuclease activity, and molecular chaperone activity (70-74). These
three activities reside in nearly independent but partially overlapping
segments of the polypeptide chain (75). The NH2-terminal
nonpolar region and the acidic region of the middle segment of B23 is
important for the chaperone activity of protein B23 (75). The
COOH-terminal 76-residue is essential for nucleic acid binding, while
the central portion of the molecule is required for ribonuclease
activity (75). Interestingly, the analysis of the binding sites of
targeting proteins on B23 have revealed that most of them reside in the
COOH-terminal portion of the molecule. For example, B23 binds to the
nucleolar localization signal of nucleolar proteins p120 (48),
nucleolin (49), and Tat (52) through a fragment of B23 involving amino
acids 187-215 or 194-239. This contrasts with the interaction of HDAg
with B23, which is probably through the two highly acidic regions
(residues 120-132 and 161-188) in the middle segment of the molecule
(46, 76). The NH2-terminal one-third segment of B23 (127 residues) does not bind HDAg. The B23 variant containing residues
1-190 with both two acidic regions has much stronger activity compared with the B23 with residues 1-147 which contains only one acidic region
(Fig. 6). Thus, both acidic regions would seem to be involved in the
binding. The binding region of B23 for viral protein Rex has also been
localized to these acidic regions (50). Therefore, these results
suggested that the interaction of B23 with HDAg is similar to the
reported interaction of B23 to the viral Rex protein, but is different
from the interaction of B23 with the nucleolar protein p120, nucleolin,
and Tat. It has been reported that the oligomerization domain of B23 is
localized in the NH2-terminal 82 amino acids which is also
essential for the molecular chaperone activity (75). Judging from the
sedimentation profiles of B23 in HDAg-expressing cells, it seems that
the interaction of HDAg with B23 does not disrupt the oligomerization
status of B23 (Fig. 7A). Instead, HDAg and B23 formed a
large complex which is bigger than 700 kDa. Notably, a recent study has
indicated that HDAg can bind to nucleolin (29). An interesting question
arises whether this HDAg-nucleolin interaction is mediated through B23,
since nucleolin can also bind B23 (49). As mentioned earlier, since both HDAg and nucleolin bind to a different portion of the B23 molecule, presumably these three molecules can form a ternary complex
with B23 as a bridging or adaptor molecule. Consistent with this,
sucrose sedimentation analysis in combination with the
co-immunoprecipitation experiment indicated that HDAg co-sediments with
both B23 and nucleolin as a large complex (Fig. 7, A and B). Notably, when considering that the cellular expressed
HDAg or B23 molecule has oligomerization property and readily
associates with host proteins to form a high molecular weight complex
(44, 45, 75, 77, 78), likely the HDAg or B23 in this B23-nucleolin-HDAg containing complex is in the oligomer form.
An intriguing finding emerged from this study was the preferential
association of the small HDAg with B23 in vivo (Fig.
7A). In vitro, recombinant large and small HDAgs
could bind to B23 (Figs. 3-6), while in vivo about half of
the large HDAg-containing complexes were not associated with B23 (Fig.
7A). This difference in the sedimentation profiles of large
and small forms of HDAg-containing complexes present in the nuclear
extracts may reflect that they associate with different cellular
partners depending on their relevant biological functions. In view of
the essential role of small HDAg in HDV RNA replication, the strong
co-localization of the small HDAg with the two nucleolar proteins, B23
and nucleolin, suggests a potential functional role for this complex in
replication. Although the precise components making up this
B23-nucleolin-small HDAg-containing complex and its biochemical
activity are yet to be determined, it is tempting to speculate that
this complex is the active one involved in HDV RNA replication.
Consistent with this, we found that exogenous B23 appears to have a
stimulatory effect on HDV RNA replication (Fig. 8A) and the
deletion of the HDAg-binding site impaired this effect (Fig.
8B). Similarly, the observation that introducing the
exogenous nucleolin enhances HDV RNA replication (29) agrees with this
hypothesis. If this is the case, an important question arises as
regarding the role of these two nucleolar proteins in HDV replication.
In view of the known functions of B23 and nucleolin (for review, see
Ref. 79), several hypotheses can be put forward. First, that B23 or
nucleolin binding to HDAg may be the mechanism by which HDAg enters the
nuclei/nucleolus. In this regard, there is evidence that the HDV RNA
replication occurs in the nucleus (13, 29, 65, 80-84). In this
scenario, B23 and its binding molecule nucleolin may well play a role
in HDV RNA replication by transporting HDAg from cytoplasm to
nuclei/nucleoli. Second, it can be suggested that since both B23 and
nucleolin are involved in several aspects of nuclear structure and
transcriptional regulation (46, 68, 79, 85-88), one possible role for
B23 or nucleolin binding is to target HDAg to sites of transcription.
This hypothesis is consistent with previous work indicating that there
are interactions between B23 and transcriptional proteins (68, 89) and
also agrees with the fact that nucleolin can serve as a transcriptional
factor (86, 88). Therefore, B23 or nucleolin may tether HDAg to the transcription complex, which in turn confers a regulatory role for HDAg
on HDV RNA replication. More significantly, nucleolin or B23 may act as
an assembly factor to recruit, by protein-protein interaction, other
factors including HDAg into the HDV replication process. Indeed, the
possibility that the HDV antigenomic RNA synthesis may be mediated by
the nucleolar RNA polymerase I, as suggested by a recent study of
Modahl et al. (35), fits the roles one may expect from B23
or nucleolin, since these two nucleolar proteins are known to be
involved in ribosome biogenesis as directed by nucleolar RNA polymerase
I (40, 43, 79, 90-92). Alternatively, when considering that both
nucleolar proteins have RNA binding, reannealing, and the RNA duplex
destabilizing activity (72, 73, 79, 93-95), they may provide a RNA
chaperone activity for HDV RNA replication. Furthermore, the molecular
chaperone activity of B23 (75) may facilitate the proper assembly of
the HDV replication machinery or aid the transport of HDV
ribonucleoprotein particles. Finally, our previous findings that HDV
RNA replication but not viral particle assembly is regulated by casein
kinase II (25, 27), and the observations that both B23 and nucleolin
are nuclear matrix-associated (62, 86) and casein kinase II-regulated (47, 69, 95), may not be coincidental. It implies that these two
nucleolar proteins together with HDAg, casein kinase II, and other
cellular factors (e.g. RNA polymerase or transcription
factor) may interact within the nuclear matrix and participate in the HDV RNA replication cooperatively in a similar manner to the SWAP complex, which contains B23, nucleolin, poly(ADP-ribose) polymerase, and SWAP-70 and is involved in B-cell DNA recombination (96).
In summary, we have found that HDAg can up-regulate the nucleolar
protein B23 expression, and both in turn form a complex which
facilitates HDV RNA replication. This study together with previous work
on nucleolin-HDAg interaction reinforces the important role of
nucleolar proteins in HDV replication. However, further insights into
the replication machinery of HDV are required to define the precise
role of nucleolar proteins in HDV RNA replication.
 |
ACKNOWLEDGEMENTS |
We thank H.-N. Wu and S. J. Lo for
generously providing plasmids and N.-H. Yeh for the supply of
anti-nucleolin monoclonal antibody. T.-S. Yeh kindly assisted with the
sucrose gradient centrifugation experiment. We are also grateful to R. Kirby for critical reading and comments on the manuscript.
 |
FOOTNOTES |
*
This work was supported by grants DOH87-HR-502,
DOH88-HR-502, NHRI-GT-EX89B502L, NHRI-GT-EX89B502Ls, and
NHRI-EX90-9002BL from the National Health Research Institute and in
part by Grants NSC88-2315-B-010-007MH and NSC89-2315B-010-006MH from
the National Science Council, and a grant from Medical Research and
Advancement of Foundation in memory of Dr. Chi-Sheu Tsou (to Y.-H.
W. L.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Recipient of a National Chair Award from the Ministry of
Education (1998-2001) and Distinguished Investigator of National Science Council (1996-2002), Republic of China. To whom correspondence should be addressed: Institute of Biochemistry, National Yang-Ming University, Taipei, Taiwan. Tel.: 886-2-2826-7124; Fax:
886-2-2826-4843; E-mail: yhwulee@ym.edu.tw.
Published, JBC Papers in Press, April 17, 2001, DOI 10.1074/jbc.M010087200
 |
ABBREVIATIONS |
The abbreviations used are:
HDV, hepatitis delta
virus;
GST, glutathione S-transferase;
HDAg, hepatitis delta
antigen;
HBV, hepatitis B virus;
IP, immunoprecipitation;
NLS, nuclear
localization signal;
NBS, nucleolin-binding site;
kb, kilobase(s);
PAGE, polyacrylamide gel electrophoresis;
MT, metallothionein;
HIV, human immunodeficiency virus.
 |
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