From the Institute of Biochemistry and
** Institute of Microbiology and Immunology, National
Yang-Ming University, Taipei, Taiwan 112, Republic of China
Received for publication, May 1, 2002, and in revised form, October 3, 2002
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ABSTRACT |
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We have demonstrated previously that the core
protein of hepatitis C virus (HCV) exhibits suppression activity on
gene expression and replication of hepatitis B virus (HBV). Here we
further elucidated the suppression mechanism of HCV core protein. We
demonstrated that HCV core protein retained the inhibitory effect on
HBV gene expression and replication when expressed as part of the full length of HCV polyprotein. Based on the substitution mutational analysis, our results suggested that mutation introduced into the
bipartite nuclear localization signal of the HCV core protein resulted
in the cytoplasmic localization of core protein but did not affect its
suppression ability on HBV gene expression. Mutational studies also
indicated that almost all dibasic residue mutations within the
N-terminal 101-amino acid segment of the HCV core protein (except
Arg39-Arg40) impaired the
suppression activity on HBV replication but not HBV gene expression.
The integrity of Arg residues at positions 101, 113, 114, and 115 was
found to be essential for both suppressive effects, whereas the Arg
residue at position 104 was important only in the suppression of HBV
gene expression. Moreover, our results indicated that the suppression
on HBV gene expression was mediated through the direct interaction of
HCV core protein with the trans-activator HBx protein,
whereas the suppression of HBV replication involved the complex
formation between HBV polymerase (pol) and the HCV core protein,
resulting in the structural incompetence for the HBV pol to bind the
package signal and consequently abolished the formation of the HBV
virion. Altogether, this study suggests that these two suppression
effects on HBV elicited by the HCV core protein likely depend on
different structural context but not on nuclear localization of the
core protein, and the two effects can be decoupled as revealed by its
differential targets (HBx or HBV pol) on these two processes of the HBV
life cycle.
Hepatitis C virus (HCV)1
is a major causative agent of non-A, non-B hepatitis and is involved in
the development of both chronic liver disease and hepatocellular
carcinoma (1-2). The viral genome consists of a positive-stranded RNA
of about 9.6 kb that encodes a large polyprotein of 3008-3037 amino
acids (reviewed in Ref. 3). This polyprotein undergoes proteolytic
processing by cellular signalases and viral proteases to yield at least
10 mature viral proteins classified as structural or nonstructural (NS)
proteins (3). The core protein, which is located at the N terminus of the polyprotein, is a component of viral capsid. It is phosphorylated (4), has both nuclear and cytoplasmic localization (reviewed in Ref.
5), and possesses several distinct functions. For example, it acts as a
regulatory protein that positively or negatively modulates the cellular
or viral promoters (5), although the molecular mechanism of this
transaction is still not fully understood. Additionally, it interacts
with a wide spectrum of cellular factors such as apolipoprotein AII
(6), lymphotoxin- Hepatitis B virus (HBV), a member of the hepadnavirus family, is a DNA
virus with partially double-stranded DNA genome held in a circular
conformation by overlapping 5'-ends of the DNA strands (20). It is also
associated with the development of liver cirrhosis and hepatocellular
carcinoma (20). HBV encodes 4 overlapping reading frames that code for
surface proteins (HBsAg), core protein (HBc/HBeAg), polymerase (pol),
and the X protein (HBx). Among them, HBx has received much attention
because it is regarded as a multifunctional protein important for the
viral life cycle and viral-host interactions (reviewed in Refs.
21-24). HBx has been implicated in HBV-mediated hepatocellular
carcinoma by its ability to induce liver cancer in some transgenic mice
(25); it modulates a wide range of cellular functions including
transcription, signal transduction, cell cycle control, genotoxic
stress responses, apoptosis, protein degradation, and carcinogenesis
(reviewed in Refs. 22-24, 26, and 27). In addition, it up-regulates
the expression of HBV genes by trans-activating the HBV
enhancer and promoters (28-30) and interacts with the transcriptional
machinery, such as RPB5 (31), TBP (32), TFIIB (33, 34), or CREB/ATF (35), to facilitate transcriptional activation. Furthermore, HBx also
stimulates transcription by interacting with the components of signal
transduction pathways such as Ras/Raf/mitogen-activated protein kinase
(36-39), protein kinase C (40), Jak1-STAT signaling (41),
stress-activated protein kinase/c-Jun N-terminal kinase (26, 42), and
NF- Despite containing a DNA genome, HBV replicates via reverse
transcription of a linear, terminally redundant RNA pre-genome that is
packaged into a viral capsid (48). This pre-genomic RNA functions
additionally as mRNA for the synthesis of two viral proteins, core
protein and pol, which in turn interact with the package signal (termed
The prevalence of HCV infection in patients with HBV infection has been
examined in several studies (62-65). Interestingly, both clinical and
animal studies have shown that HCV might exert a viral interference
effect that suppresses or terminates the HBV carrier state (66-70).
Still other findings suggest a reciprocal inhibition between these two
viruses in patients coinfected with HBV and HCV (71-73). Along this
line, our previous data indicated that the HCV core protein had the
trans-suppression activity on HBV gene expression and
replication (74). This trans-suppression ability of HCV core
protein was positively regulated by protein kinase A and C through
modulation of the phosphorylation level of its Ser99 and
Ser116 residues (4). Furthermore, the suppression of HBV
encapsidation by the HCV core protein was more severe when compared
with that in the HBV gene expression (4, 74). However, the exact
molecular mechanism for these suppression effects remains to be
determined. Because HCV core protein is a multifunctional protein with
several functional motifs, including basic charged residues and nuclear localization signals (74-76), and because our initial attempt to locate the suppressive domain of HCV core protein suggests the importance of the C-terminal 22-amino acid segment encompassing residues 101-122 (74), in this study we introduced mutations into the
basic residues within NLSs or 22-residue suppressive domains. The
properties of HCV core mutant variants were examined in order to
understand whether the same domain is involved in both suppressive
activities of the HCV core protein. The involvement of HBx in this
suppression effect by the HCV core protein was also explored.
Additionally, the in vitro coimmunoprecipitation method and
streptavidin-agarose affinity chromatography were adapted for the study
of the mutual interaction among the HCV core protein and HBV
encapsidation components, HBV pol and package signal, in an attempt to
elucidate the molecular mechanism of the interference of the HBV
encapsidation by the HCV core protein. Our results shown here strongly
suggest that HCV core protein inhibits the HBV gene expression and
viral replication through interacting with the two important regulators
in HBV life cycle, HBx and HBV pol proteins. Additionally, our results
clearly indicate that the suppressive domain of core protein on HBV
gene expression and viral replication is distinct with the former which
is located mainly on the C-terminal 22 residues, whereas the latter
spans almost the entire region of the HCV core protein, and several Arg
residues (Arg101, Arg113, Arg114,
and Arg115) are found to be essential for both suppressive
activities. Moreover, in this study we also demonstrate that this
suppression of HBV gene expression and replication occurs when HCV core
protein is expressed as part of the HCV polyprotein.
Plasmids--
HCV core protein expression construct pECE/HCVC-KF
was described previously (74). In this construct the structural protein of HCV contains the whole coding region (191 amino acids) of core protein. Plasmid pHCVc-SE, a derivative of pGEM-3Zf(+) harboring the
HCV core and partial E1 region, was constructed by inserting the 1.4-kb
StuI-EcoRI fragment of pECEC/HCVS-EK (74) into
the HindIII (Klenow-filled) and EcoRI-digested
pGEM-3Zf(+) vector. When this construct is linearized with appropriate
restriction endonuclease and transcribed in vitro with SP6
RNA polymerase, the transcripts containing various sizes of HCV core
gene can be produced. Plasmid pET23a/HCVc is a derivative of pET-23a
harboring the 0.6-kb full-length HCV core fragment
(AccI-FspI) (7). When linearized with
SacII or ClaI and transcribed in vitro
with T7 RNA polymerase, the resulting transcript encodes the T7-tagged 101 or 122 amino acid residues of HCV core protein (T7-C101 or T7-122)
with additional 24 amino acids (including 11 amino acids of T7 tag) at
the N-terminal segment of the HCV core protein. Plasmids pGST/HCVc24,
pGST/HCVc101, pGST/HCVc122, and pGST/HCVc195 are derivatives of
pGEX-3KS that direct the synthesis of different lengths of HCV core
protein fused with the C terminus of GST (4, 7). Plasmids
pSR Site-directed Mutagenesis of the HCV Core Protein and HBx
Protein--
The "Altered Sites" system (Promega) was used for
in vitro mutagenesis of the HCV core or HBX gene
as described by the supplier. The construct pSELECT/HCVC, a derivative
of pSELECT-1 containing the full length of HCV core gene, was used for
site-directed mutagenesis (4). The HCV core mutants bearing lysine
and/or arginine residue substitution mutations generated in this study
are listed in Fig. 1. The oligonucleotides used for mutation were
synthesized and indicated by the position of the first mutated amino
acid residue in the HCV core protein (see below). The mutant primers
used are as follows: M9,
5'-GTTACGTTTGGTTGCTGCTTGGGGTTTAGGAT-3'; M12,
5'-GACGGTTGGTGTTAGCTGCGGTTTTTCTTTGGGGTT3'; M17,
5'-TAACGTCCTGTGGGGCAGCGTTGGTGTTACGTTTGG-3'; M39,
5'-CCAACCTGGGGCCCGGCCGGCAACAAGTAAACTC-3';
M50, 5'-CCGCTCGGAAGTCGCCGCAGTCGCACGCACACC-3';
M61,
5'-GGGATAGGTTGTGCCGCTCCACGAGGTTGC-3'; M69,
5'-CCTGCCCTCGGGGGCGGCAGCCTTGGGGATAGG-3';
M101L, 5'-CGAGAGCCGAGGGTGAC-3'; M101K,
5'-GGCCGAGAGCCCTTGGGTGACAGGAGC-3'; M104L,
5'-CAACTAGGCAGAGAGCCG-3'; M104K,
5'-CCAACTAGGCTTAGAGCCGCGGGGTG-3'; M113L,
5'-GACCTACGCAGGGGGTCATT-3'; M113K,
5'-GACCTACGCTTGGGGTCATTAGG-3'; M114L,
5'-CGCGACCTAAGCCGGGGTC-3'; M114K,
5'-ATTACGCGACCTCTTCCGGGGGTCATTAG-3'; M115A,
5'-CAAATTACGCGACGCACGCCGGGGGTC-3'; M115K,
5'-TTACGCGACTTACGCCGGGGG-3'; M117L,
5'-ACCCAAATTAAGCGACCTACG-3'; M117K,
5'-CTTACCCAAATTCTTCGACCTACGCCGG-3'; and M117D,
5'-CTTACCCAAATTATCCGACCTACGCCGG-3'. The
underlined boldface bases are mutated bases. All mutant sequences were
confirmed by sequencing. The 0.7-kb HindIII-EcoRI
fragments of mutant DNA in pSELECT/HCVC derivatives were then subcloned
into HindIII/EcoRI-digested pECE expression
vector, and the resultant pECE derivatives harboring mutated HCV core
genes were used for transfection. To generate HBx null HBV genome, the
three ATG codons at positions 1, 79, and 103 in the open reading frame
of HBX gene were converted into Val codon. This was done by
using the construct pSELECT/HBV for site-directed mutagenesis. This
construct carries a 3.2-kb EcoRI fragment of the full-length
of HBV gene from pSHH2.1. The mutant primers used are as follows:
Met-1/Val, 5'-CATCGTTTCCGTGGCTGCTAG-3'; Met-2/Val 5'-GCACGTCGCGTG-GAGACCAC-3'; and
Met-3/Val, 5'-CTCTCAGCAGTGTCAACGAC-3'. The
underlined boldface bases are mutated bases. All mutant sequences were
confirmed by sequencing. The HBV mutant plasmid pHBV (X Cells and Transfection--
Human hepatoma cell line HuH-7 and
human cervical carcinoma cell line HeLa were cultured as described
previously (7, 74). Ava.5 cells are HuH7-derived cell lines harboring
the autonomously replicating HCV replicon of subgenomic NS3 to NS5B
region (kindly provided by C. M. Rice and Apath) (80). This cell
line was cultured in the presence of 1 mg/ml G418. Cells were subjected
to cotransfection with various plasmid combinations (10-20 µg DNA
each for 9-cm dish or 20-40 µg DNA each for 15-cm dish) by the
calcium phosphate coprecipitation method or by the SuperFect
transfection reagent (Qiagen, Hilden, Germany).
Analysis of HBV-related Antigens and Viral Particles--
The
culture medium collected from day 6 post-transfection was assayed for
HBsAg and HBe/HBcAg by using enzyme immunoassay kits (EverNew or
General Biologicals) (74). The secreted HBV particles were detected by
an assay for endogenous DNA polymerase activity as described previously
(74, 79).
RNA Preparation and Northern Blotting--
Cellular RNA was
extracted by using TRI reagent (Molecular Research Center) according to
the instructions of the supplier (Molecular Research Center). The RNA
samples were electrophoresed in a 1% formaldehyde-agarose gel and then
transferred to nitrocellulose paper. Prehybridization and hybridization
were performed as described previously (81). The HBV DNA probe was
prepared from the 3.2-kb HBV fragment (EcoRI fragment) of
pSHH2.1 by the nick translation method (82).
In Vitro Transcription--
RNAs for in vitro
translation or binding were produced by an in vitro
transcription kit as suggested by the manufacturer (Promega). Recombinant pHCVc-SE, which was intended for generation of full length
(C195, p22) and the truncated (C101, p11) HCV core mRNA, was
linearized at FspI or SacII prior to in
vitro transcription. To produce the transcripts encoding the
full-length (pol) or the truncated forms (pol749 and pol567) of HBV
polymerase, recombinant pHBV97Po was linearized with SmaI,
NcoI, or AccI, prior to in vitro
transcription with T3 RNA polymerase (see Fig. 10). To produce the
biotinylated package signal RNA, EcoRI-linearized pHBV-PS template was subjected to in vitro transcription as
described above, except that 1 mM biotin-16-UTP (Roche
Molecular Biochemicals) (final UTP concentration, 2.5 mM)
was added to the transcription reaction mixture containing SP6 RNA polymerase.
In Vitro Translation--
35S-Labeled HCV core
protein and HBV polymerase or their derivatives were made in rabbit
reticulocyte lysates by translation of their corresponding mRNAs,
according to the manufacturer (Promega). In brief, synthesized RNAs
(8-12 µg each in 4-6 µl) were incubated in a total reaction
volume of 50 µl containing 35 µl of nuclease-treated rabbit
reticulocyte lysate (Promega), 1 µl of RNasin (40 units/µl), 1 µl
of 1 mM amino acid mixture (minus methionine), and 3.5 µl of [35S]methionine (>1000 Ci/mmol, 10 mCi/ml, Amersham
Biosciences) at 30 °C for 1 h. The in vitro
translated HCV core protein (e.g. C101 species) was also
prepared in the reticulocyte lysate containing 14C-labeled
amino acid as described previously (74). For in vitro synthesis of [35S]Met-labeled T7-tagged HCV core variants
(T7-C195, T7-122, and T7-C101), pET23a/HCVc or appropriate restriction
endonuclease (SacII and ClaI)-linearized
pET23a/HCVc was used as a template for the TNT system
(Promega). [35S]Met-labeled HA-tagged HBx was also
produced by the TNT kit using pCMV-HA-HBx as a template.
The in vitro translated products were then processed for
protein or RNA binding analysis.
Expression and Purification of GST Fusion Proteins and in Vitro
Pull-down Binding Assay--
HCV core protein or HBx protein was
expressed individually as GST fusion proteins from the expression
vector pGST/HCVc24, pGST/HCVc101, pGST/HCVc122, pGST/HCVc195, or
pGST/HBx. Expression and purification of the GST fusion proteins were
performed as described (4). For each in vitro binding assay,
15 µl of glutathione-Sepharose 4B beads (Amersham Biosciences) bound
to the appropriate GST fusion protein (4 µg) was incubated with
in vitro translated 35S-labeled HBV polymerase
or HCV core protein with gentle rotation. The binding and washing
conditions were described by Huang et al. (83). Proteins
bound on the beads were eluted by sampling buffer (84),
fractionated by SDS-PAGE, and detected by autoradiography.
In Vitro Coimmunoprecipitation--
In vitro
translated products of HCV core protein and HBV RNA polymerase were
incubated at 4 °C with either anti-HBV pol antibody (supplied by
C. M. Chang) or HCV patient sera (positive for anti-HCV core
protein) (74) which were prebound with protein A-Sepharose (20 µl
packed volume) and suspended in 350 µl of NETN buffer (150 mM NaCl, 1 mM EDTA, 20 mM Tris-HCl
(pH 7.5), 0.5% Nonidet P-40), and the suspension was rocked overnight.
The immunoprecipitates were recovered by centrifugation and washed four
times with NETN buffer, boiled in sampling buffer, and analyzed by
SDS-PAGE (84). The gel was dried and processed for autoradiography, and
if necessary the band intensity was quantified by PhosphorImaging
(Amersham Biosciences).
In Vivo Coimmunoprecipitation--
HeLa or Ava.5 cells (density
of 2 × 106 per 10-cm dish) were cotransfected with
FLAG-tagged HBx (pFLAG-HBx) or HBV polymerase (pFLAG-HBVpol) expression
plasmid together with HCV core protein expression constructs
(pSR Streptavidin Precipitation of Protein-RNA Complexes--
To
analyze the RNA binding activity of HCV core protein and HBV
polymerase, the method described by Pollack and Ganem (53) was followed
with slight modification. In brief, the in vitro translated
HCV core protein or HBV polymerase was preincubated with the
biotinylated HBV package sequence at 30 °C for 30 min. About 20 µl
of packed streptavidin-agarose beads (Invitrogen) in 350 µl of Ipp150
buffer (150 mM NaCl, 10 mM Tris-HCl (pH 7.5), 10 µg/ml yeast tRNA, 40 units/µl RNasin) was added to the mixture, and the solution was rocked at 4 °C for 1 h. The pellet
recovered by centrifugation was washed four times with 1 ml of Ipp150
containing 0.1% Nonidet P-40. The recovered pellet was resuspended in
15 µl of 2× SDS-PAGE sampling buffer (84) and boiled for 5 min. After brief centrifugation, the supernatant was analyzed by SDS-PAGE and processed for autoradiography.
Immunofluorescence--
For analysis of the subcellular
localization of HCV core protein and its variants, HuH-7 cells grown on
coverslips were transfected with HCV core constructs (pECE/HCVC-KF or
its variants) or their control vector pECE (2 µg each). After day 6, cells were fixed with acetone/methanol (1:1) ( Site-directed Mutagenesis of the HCV Core Protein--
Examination
of the deduced amino acid sequence of the HCV core protein revealed
that it has four clusters of basic amino acid residues as follows:
5PKPQRKTKRNTNRRP19;
38PRRGPR43;
58PRGRRQPIPKARRP71;
99SPRGSRPSWGPNDPRRRSR117 (see Fig.
1A). The first three clusters
have been identified as independent nuclear localization signals
(designated NLS1, NLS2, and NLS3) when individually fused to
Most Arg Residues within the 22-Residue Suppressive Domain but Not
Dibasic Residues in NLS Regions of HCV Core Protein Are Important for
Suppression on HBV Gene Expression--
The 24 variants of the HCV
core protein were analyzed for their suppressive activity on the HBV
gene expression. As shown in Table I,
when these substitution variants of HCV core protein expression
construct (as pECE derivatives) were individually cotransfected with
HBV plasmid pSHH2.1, the suppression of HBV antigen HBsAg and HBe/HBcAg
production in the human hepatoma cell line HuH-7 in all the mutant
constructs examined was comparable with that of the wild-type
(2-3-fold suppression), except for substitution mutants M101L, M104L,
M104K, M113L, M113K, M114L, M114K, M115L, and M115K. Northern blot
analysis of HBV-specific transcripts (3.5 and 2.1 kb) gave similar
results (Fig. 2). Therefore, these results indicate that Arg101, Arg104,
Arg113, Arg114, and Arg115, but not
Arg117 and dibasic residues in NLS regions, are crucial for
the inhibitory effect of core protein on the HBV gene expression.
Notably, mutant M101K still retained the wild-type suppressive
activity, whereas this was not the case for M101L mutant, suggesting
that the basic residue is required for this particular Arg residue. It
should be pointed out that Arg101 is within the protein
kinase C recognition motif for Ser99, and phosphorylation
of this serine residue has been demonstrated to be essential for the
suppressive effect of the HCV core protein (4). Thus, our present
results obtained from mutants M101K and M101L are consistent with our
previous study (4), as the loss or retention of the suppressive
activity of the HCV core protein in these two particular mutants
correlates with the functional status of the PKC site at
Ser99.
HBV Viral Replication Is Affected Diversely by HCV Core
Variants--
When these variants of HCV core expression construct
were individually cotransfected with HBV plasmid pSHH2.1, the
suppression of HBV viral particle production in HuH-7, as determined
from the endogenous DNA polymerase assay (see "Experimental
Procedures"), was either lost completely or affected to various
degrees (wild-type about 14-30-fold inhibition, mutants about
2-8-fold inhibition or 1.7-2.9-fold enhancement) in all the
constructs examined, except for mutants M39, M101K, M104L, M104K,
M117L, M117K, and M117D (Fig. 3).
Therefore, our results suggest that integrity of most dibasic residues
in NLS regions and Arg113, Arg114, and
Arg115, but not
Arg39-Arg40, Arg104 and
Arg117, is essential for the suppressive activity of the
HCV core protein on HBV viral particle formation. The loss of the
suppressive activity of M101L mutant but not M101K again implies that
the role of Arg101 in the suppressive activity of HCV core
protein is for modulation of the phosphorylation of Ser99.
Interestingly, our results also suggest that, of these six arginines located within the C-terminal 22-amino acid segment of core protein, the four residues Arg101, Arg113,
Arg114, and Arg115 are essential for both
suppressive effects on HBV gene expression and replication. In
contrast, Arg117 is irrelevant to both suppressive
activities of the HCV core protein, because replacement with neutral
(leucine) or acidic residues (aspartate residue) at this site did not
affect the suppressive activity. The most interesting residue is
Arg104, as this site is only required for the inhibitory
activity on the HBV transcription and gene expression but not for HBV
viral replication (Figs. 2 and 3). Notably, the importance of the two arginine residues, Arg113 and Arg114, in the
suppressive activity of the HCV core protein could not completely
account for their role in modulation of Ser116
phosphorylation. This is inferred from the fact that replacement with
lysine residue in M113K and M114K mutants, which presumably retains the
functional status of PKA site at Ser116, still led to a
loss of the inhibitory activity of the core protein. A considerable
enhancement (about 2.9-fold) of HBV particle production was also noted
in NLS mutant M50 and to a less extent (1.7-1.8-fold) in mutants M9,
M17, and M61 (Fig. 3), although the molecular mechanism for this effect
is still unclear.
Based on these results, it appears that introducing substitution
mutations into the basic residues of NLS regions or the 22-residue suppressive domains impart various effects to the suppressive activity
of the core protein on HBV viral replication.
The Nuclear Localization of HCV Core Protein Is Not Required for
Its Suppression Activity--
Because mutation in the NLS regions
affected the suppression effect of the core proteins on HBV replication
to various degrees but retained a comparable wild-type level of
suppression on HBV gene expression (Figs. 2 and 3 and Table I), it is
pertinent to know whether these mutational effects resulted from the
influence on the subcellular localization of HCV core protein. As shown in Fig. 4, all NLS core mutants displayed
nuclear localization except for the M50/69 mutant, in which cytoplasmic
localization of core protein was observed. This strongly implies that
the bipartite NLS located at residues 50-70, but not other NLSs,
serves as a functional NLS in the full-length HCV core protein. Because
M50/69 mutant retained its suppression activity on HBV gene expression and also conferred considerable degrees of suppression (about 5-fold)
on HBV particle release (Figs. 2 and 3, Table I), it seems that nuclear
localization of core protein is not required for its suppression
activity on HBV gene expression and replication.
HBx Mediates the Suppression of HBV Gene Expression but Not
Replication by the HCV Core Protein--
Next, we elucidated the
suppression mechanism of core protein on HBV gene expression. Because
HBx is the trans-activator for HBV transcription (29), it is
likely that the suppression of HBV gene expression by the HCV core
protein is mediated through HBx. To assess this possibility, we
analyzed the effect of HCV core protein on the HBV gene expression from
HBx null plasmid pHBV(X
The possibility of the complex formation between HCV core protein and
HBx was examined further. As shown in Fig.
6, confocal microscopy analysis using
indirect immunofluorescence staining indicated that the full-length and
truncated forms of HCV core protein (C195, C122, and C101) colocalized
with the FLAG-tagged HBx in both nuclear and cytoplasmic compartments.
Furthermore, by using the GST fusion proteins of HCV core variants
harboring the full-length or N-terminal 122 or 101 amino acid residues
of core protein (GST/HCVc101, GST/HCVc122, and GST/HCVc195) for
pull-down analysis, results indicated that in vitro
translated HA-tagged HBx could be precipitated by these three GST-core
variants (Fig. 7A). A
reciprocal experiment using GST/HBx for pull-down of the in
vitro translated T7-tagged HCV core proteins (T7-C101, T7-C122, or
T7-C195) gave a similar conclusion (Fig. 7, B and
C). Moreover, in vivo coimmunoprecipitation
experiment using anti-FLAG antibody for immunoprecipitation also
confirmed that FLAG-HBx formed a complex with these three forms of HCV
core protein in transfected HeLa cells, albeit the two truncated forms
of core protein exhibited only weak interaction with FLAG-HBx compared
with that of the full-length core protein (Fig. 7D).
All together, our results indicate that HCV core protein associates
with HBx, and this interaction regulates the suppression of HBV gene
expression by the core protein of HCV. Additionally, because the
suppression-impaired mutant C101 still retained its ability to interact
with HBx, this implies that the binding of HBx is necessary but not
sufficient for the suppression effect of HCV core protein on HBV gene expression.
HCV Core Protein Forms a Complex with HBV Polymerase--
The
suppression of HBV encapsidation process by the HCV core protein may be
because of the direct interaction of this protein with the HBV pol. To
test this possibility, a coimmunoprecipitation experiment was
performed. Fig. 8A shows the
in vitro translated, 35S-labeled full-length of
HCV core protein (C195, p22) and HBV pol protein (92 kDa) used in this
study. In addition to the p22 species, minor protein species with a
size of 18 (p18) or 44 kDa (p44) was detected in the translation
products of HCV core protein, which presumably represent the
degradation (p18) or dimer form (p44) of p22 as noted previously (4).
When in vitro translated HCV core protein and HBV pol
protein were incubated together and immunoprecipitated with HCV patient
sera (positive for anti-HCV core protein, see Fig. 8B), HBV
pol was coprecipitated with HCV core protein (Fig. 8B,
lane 2). This coimmunoprecipitation of pol protein depends on the
presence of HCV core protein (Fig. 8B, compare lanes
1 and 2). In vitro binding analysis using
purified GST/HCV core fusion proteins harboring various core domains
for pull-down affinity assay of in vitro translated HBV pol
protein suggested that N-terminal 101- or 122-amino acid fragment of
core protein but not its 24-amino acid fragment could interact with HBV
pol protein (Fig. 8C). An in vivo
coimmunoprecipitation experiment using anti-FLAG antibody for
immunoprecipitation revealed that both the full-length and the
truncated HCV core variants (C195, C122, and C101) could
form a complex with the FLAG-tagged HBV pol protein in transfected HeLa
cells (Fig. 8D). Notably, this complex formation between HCV
core and HBV pol proteins was not mediated by RNA, because RNase
treatment of coimmunoprecipitates did not disrupt their interaction
(Fig. 8D). Furthermore, indirect immunofluorescence
microscopy analysis revealed that all three forms of HCV core protein
colocalized with the GFP-tagged HBV pol in the cytoplasmic compartment
when these expression plasmids were cotransfected into HeLa cells (Fig.
9). Taken together, these results suggest
that both the full-length and truncated (C122 and C101) variant of HCV
core protein can associate with the HBV pol in vitro and
in vivo. Detection of the complex formation between the
full-length HCV core protein and the HBV core-pol fusion protein was
also noted previously (87).
Mapping the Interaction Domains of HCV Core Protein and HBV
Polymerase--
To delineate the interaction domain of HCV core
protein on HBV pol protein, a similar coimmunoprecipitation experiment
was performed on the C-terminal truncated forms of HBV pol generated from in vitro translation of the 3'-end truncated
transcripts of HBV pol gene (see "Experimental Procedures"). The
truncated HBV pol used in this study harbored the N-terminal 749 and
567 amino acid residues, respectively (Fig.
10, A and B).
These two truncated HBV pol were designated as pol749 or pol567 with
sizes around 76 or 60 kDa, respectively (Fig. 10B). We found
that the full-length HCV core protein could associate with the pol749
but not with the smaller truncated form pol567 (Fig. 10C,
compare lane 4 with lane 6). Interestingly, the
truncated pol749 had much stronger binding affinity to HCV core protein
as compared with that of the full-length HBV pol (Fig. 10C,
compare lane 2 with lane 4). These results
demonstrate that HCV core protein binds to the C-terminal central
region of HBV pol through amino acids 567-749, which resides in the
junction region between reverse transcriptase and RNase H domains of
pol protein (88). This interacting region has been shown to be crucial
for RNA encapsidation and reverse transcription activities of HBV pol
protein (53, 88, 89).
HCV Core Protein Inhibits Complex Formation between HBV Polymerase
and
Because HCV core protein has binding affinity for both HBV pol and
To examine whether the presence of increasing amounts of HCV core
protein might impair the binding of HBV pol to its package signal,
various amounts of HCV core protein (0.4-2.0 pmol) were incubated with
a fixed amount of HBV pol (0.2 pmol) and biotin-labeled HCV Core Variant Binds to HBV Polymerase and HCV Core Protein Retains Its Inhibitory Effect on HBV
Gene Expression and Replication When Expressed as Part of the
Full-length Polyprotein--
Our study indicated that expression of
HBV antigens and production of HBV particles were suppressed by HCV
core protein, and this suppression is mediated through the interaction
with HBV two regulatory proteins, HBx and HBV pol proteins. Questions
arise regarding whether these interactions and effects on HBV also
occur in the context of full-length HCV polyprotein. To investigate this, we examined the suppression ability of HCV polyprotein on HBV by
cotransfection of HCV polyprotein expression construct pSR
Next, we examined whether in the presence of other HCV viral proteins
the core protein can associate with HBx or HBV pol protein. In this
experiment we used the HuH7-derived cell lines harboring subgenomic HCV
RNA replicon (NS3 to NS5B) (Ava.5 cells) (see "Experimental Procedures") for cotransfection. The expression of NS3 protein in
Ava.5 cells was detected by immunoblot, which presumably was generated
from proteolytic processing of HCV polyprotein (Fig. 14A). When FLAG-tagged HBx
or HBV pol protein expression construct together with HCV core protein
expression construct (pSR Our laboratory previously demonstrated that the HCV core protein
has the trans-suppression activity on HBV gene expression and replication, and phosphorylation of Ser99 and
Ser116 residues in HCV core protein is essential for this
suppressive effect (4, 74). In this study, we demonstrated that this suppression of HBV gene expression and replication also occurs when HCV
core protein was expressed in the context of the intact HCV polyprotein
(Fig. 13). We also extended our previous work to map the
trans-suppressive domain of HCV core protein responsible for
the suppressive activity on HBV gene expression and replication. According to our earlier work (74), the N-terminal 122-amino acid
fragment, but not the 101-amino acid fragment, of the HCV core protein
retained the same suppressive effect as the full-length core protein.
Thus, it is likely that the region between amino acids 101 and 122 of
the HCV core polypeptide is responsible for the suppressive activity of
the HCV core protein. In this study, we mutated six arginine residues
(Arg101, Arg104, Arg113,
Arg114, Arg115, and Arg117) within
this 22-amino acid segment of the HCV core polypeptide, and we studied
the mutational effects on the core protein's suppressive effect (see
Fig. 1B). Our results (Figs. 2 and 3 and Table I) indicated
that mutation of Arg117 did not interfere with the
trans-suppressive activity of the HCV core protein, and the
role of Arg101 on the trans-suppressive activity
of the core protein was involved in phosphorylation of
Ser99 by protein kinase C, which is essential for the
suppressive activity of the HCV core protein. Arg104 mutant
still blocked HBV encapsidation but did not confer any effect on HBV
gene expression (Figs. 2 and 3 and Table I). However, single
substitution mutation at Arg113, Arg114, or
Arg115 led to the loss of both suppressive effects (Figs. 2
and 3 and Table I). Thus, these results support the notion that the
amino acid segment between 101 and 122 residues is an important domain for the trans-suppression activity of HCV core protein on
HBV transcription and viral encapsidation. Furthermore, our results strongly suggest that both suppressive activities are at least in part
mediated through different amino acid residues of the HCV core protein
(e.g. Arg104), albeit some arginine residues are
crucial for both suppressive effects (e.g.
Arg101, Arg113, Arg114, and
Arg115).
The finding that several arginine residues within the 22-residue
suppressive domain are critical for the inhibitory activity of the HCV
core protein raises the question concerning whether other basic
residues located outside this segment play any role in the suppressive
activity of the HCV core protein. Because the N-terminal 101 residues
of HCV core polypeptide contain three independent NLSs and one
bipartite NLS, in this work we also individually or jointly mutated
dibasic residues located within these NLSs regions including residues
at 9/10, 12/13, 17/18, 39/40, 50/51, 61/62, or 69/70 of the HCV core
protein (see Fig. 1A). We showed that all these NLS mutants
retained the trans-suppressive activity on HBV gene
expression (Fig. 2 and Table I). However, most of their suppressive
activity on HBV virion replication was lost to a different degree (Fig.
3), indicating that the trans-suppressive domains of HCV
core protein involved in inhibiting HBV gene expression and virion
replication are rather different. Whereas the suppressive domain of HCV
core protein on HBV gene expression may be located solely on amino acid
residues 101-122, the important residues for suppression of HBV
encapsidation probably span the entire region (122 amino acid residues)
of the HCV core protein. In addition, consistent with our earlier work,
this study also demonstrated that the core protein exhibited much
stronger suppression activity on the HBV replication (15-30-fold
inhibition) as compared with its inhibitory effect on the HBV gene
expression (2-4-fold). Moreover, it was noted that in some mutants the
multiple mutations of several dibasic residues adversely led to a
partial recovery of the suppression ability of the HCV core protein on
HBV replication but not on HBV gene expression. Specifically, multiple
mutations introduced into dibasic residues at position 50/51 and 69/70
in M50/69 core mutant partially restored the suppression ability of
core protein (5-fold suppression) on HBV replication as compared with
that of mutation at 50/51 (M50; 2.9-fold enhancement) or 69/70 (M69; 2-fold inhibition) (Fig. 3). Similarly, M17/39 core mutant still retained certain strength of suppression ability (8-fold suppression), whereas M17 core mutant barely had the suppression ability (1.7-fold enhancement) (Fig. 3). Taken together, our results imply that the
ability to suppress HBV replication is more sensitive to mutations within the NLS region of core protein and that the structural context
of the core protein rather than the amino acid residue itself is more
important for its suppression on HBV replication.
In this study, we also examined the functional NLS of HCV core protein
by mutational analysis (see Fig. 1A). Based on
immunofluorescence study (see Fig. 4), we found that the functional NLS
governing the nuclear entry of the HCV core protein actually resembles
the bipartite configuration (85) consisting of two clusters of basic residues separated by a 17-amino acid spacer and is located within residues 50-70 (50RKTSERSQPRGRRQPIPKARR70).
The characteristics of core protein NLS differ from the prototypic NLSs
consisting of short stretches of basic amino acids as found in NLS1,
NLS2, or NLS3 (75, 76) (Fig. 1A). This discrepancy could be
due to the different methods used for the identification of NLS. As
noted, mapping the NLS by deletion analysis, as reported by other
groups, presumably unmasks cryptic NLS. This bipartite NLS of HCV core
protein has a property similar to a number of viral proteins, such as
HDAg of HDV (92), Tof protein of HTLV-1 (93), Bel 1 protein of human
foamy virus (94), and tegument pp65 (UL83) of HCMV (95). Moreover, it
should be noted that M50/69 core mutant lost the nuclear transport
activity but to some extent retained its suppression activity on HBV,
suggesting the dispensability of nuclear targeting for the inhibitory
ability of HCV core protein.
One of the major findings in this work is the observed inability of HCV
core protein to inhibit HBV gene expression in HBx null mutant
background (see Fig. 5A; Table II). This result, together with the detection of the complex formation between HCV core protein and HBx (see Figs. 6, 7, and 14B), provides an important
clue for the HCV core protein-mediated inhibitory effect on HBV gene
expression. Given that HBx can trans-activate HBV enhancer
and promoters (28-30), it becomes appealing that the HCV core-mediated
inhibition of HBV transcription acts through directly interacting with
this key trans-activator of HBV. Confocal microscopy
analysis of HCV core protein and FLAG-tagged HBx coexpressed cells
showed the colocalization of HBx and HCV core protein in both nuclear
and cytosolic compartments (see Fig. 6). However, in line with our results indicating that the nuclear transport of core protein is not a
prerequisite for inhibition of HBV gene expression and the findings
that HBx regulates transcription at either subcellular compartment (24,
27, 96), likely the suppression of HBV gene expression by HCV core
protein may take place via forming a complex with the cytoplasmic HBx.
However, the involvement of nuclear HBx-HCV core complex in this
suppression effect is not formally excluded. Furthermore, when
considering that both HBx and HCV core proteins are the promiscuous
regulators affecting a plethora of cellular activities or interacting
with a long list of cellular proteins involved in transcription, cell
growth, and apoptotic cell death (reviewed in Refs. 5, 23, and 24), presumably the presence of these two viral proteins or their complex formation during dual infection of both hepatitis viruses may aggravate
or counteract their individual activity or cellular functions.
Apparently, a more comprehensive survey and comparison of their
activities or targets may shed some light on this issue. Along this
line, it is noted that both viral proteins have the same cellular
targets, such as p53 and 14-3-3 (12, 16, 97-103). However, in contrast
to HCV core protein (12), HBx down-regulates apoptosis and
transcriptional activation mediated by this tumor suppressor (97-101).
In the case of 14-3-3, both viral proteins stimulate Raf-1 kinase or
Ras/Raf-1/mitogen-activated protein kinase pathway through direct
targeting to this scaffold protein or its associated complex including
MEKK1, SEK1, and stress-activated protein kinase (16, 102).
Additionally, a recent study (103) has demonstrated that among more
than 10 viral proteins of HCV and HBV including HBx, the core protein
of HCV is the most potent signal transducer on several intracellular
signals, especially NF- Assembly of the replication-competent HBV nucleocapsid involves the
complex association of at least three different components, including
pre-genomic RNA, core protein, and HBV polymerase (49, 50, 111, 112).
In this study, we have shown that the full-length HCV core protein can
complex with HBV polymerase to prevent the binding of the pol protein
to its package signal (Figs. 8, 10, and 11). Because binding of the HBV
pol to In the present study, the in vitro analysis of the mutual
interaction between the C-terminal truncated form of the HCV core protein (C101) and the HBV encapsidation components strongly suggests that, unlike the full-length core protein, this mutant form of the core
protein cannot compete with HBV pol for binding to the package signal,
although it can form a complex with HBV pol protein or with the Mapping studies reveal that the junction region between reverse
transcriptase and RNase H domains of the HBV pol is important for
binding to HCV core protein (see Fig. 10). These two regions are the
most critical for HBV pre-genomic RNA encapsidation and reverse
transcriptional reaction (53, 88, 89, 119). Several studies (53, 88,
89, 119) have suggested that mutation at these two regions severely
impairs the HBV virion formation including the pre-genomic RNA
packaging. Considering the importance of these two regions in HBV pol
function, it is perhaps not surprising that the inhibition of the
binding of pol protein to Within the last few years, considerable progress has been made in
understanding the roles of the HBV pol and cellular factors in HBV
nucleocapsid assembly. It is interesting to note that translation and
package of HBV pol are two intimately coupled events in hepadnaviruses (50, 120), and a more recent study invokes a role of Hsp90 and its
partner p23 or Hsp60 as participants in the interaction of the HBV pol
with Our observations presented here confirm and extend previous studies on
the suppression of HBV gene expression and encapsidation by the HCV
core protein and establish the in vitro mechanistic model
for the inhibitory mechanism of the HCV core protein on these two
processes of HBV. This study further suggests that binding with HBV
trans-activator HBx or encapsidation components per
se is not a guarantee of the suppression activity.
Additionally, the functional domains for suppressive activity of
the HCV core protein are distinct in such a way that it is more
stringent on the core protein structure for inhibition on HBV
replication as compared with that for inhibition on HBV gene
expression. Moreover, HCV core protein bears the
trans-suppression ability on gene expression of the HBV and
several cellular and viral promoters (reviewed in Ref. 5), but so far
it exhibits much stronger suppression activity on the HBV encapsidation
process. Further understanding of the molecular mechanisms involved in
both suppression activities of the HCV core protein will be helpful for
designing this protein as an antiviral drug specifically against HBV replication.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
receptor (7-9), tumor necrosis factor-
type 1 receptor (10), heterogeneous nuclear ribonucleoprotein K (11), p53
(12), RNA helicase (13, 14), LZIP (15), 14-3-3 (16), and p21/WAF1 (17);
and in most cases the core protein also affects the biological
functions of its targeted proteins. Moreover, the core protein is
capable of transforming primary rat embryo fibroblasts in cooperation with Ras (18) and causes hepatocellular carcinoma in certain strains of
transgenic mice (19).
B (43, 44). Notably, this protein is essential for the viral
infection in woodchucks (45, 46) but dispensable for virus replication
in transfected culture cells (47).
) that appeared at the 5'-end of the pre-genomic RNA to initiate the
RNA encapsidation process (49, 50). Encapsidation of the RNA template
is under stringent control, because only the pre-genomic RNA is
selectively encapsidated. In HBV, the
package signal of the
pre-genomic RNA is characterized by the presence of a stem-loop
structure that is believed to serve as the docking site for the binding
of the pol and is essential for both packaging and DNA priming
(49-54). Synthesis of the two viral DNA strands occurs within the
nucleocapsid, and it is sequential in the way that minus strand DNA
synthesis occurs first by using the pol protein itself as a primer, and
followed by the plus strand DNA synthesis via the concerted actions of
the reverse transcriptase and RNase H activities of pol protein
(54-58). Recently, it was found that the interaction of molecular
chaperones (Hsp90 and Hsp60) with HBV pol is critical for the
maintenance of the enzyme in a conformation competent for its functions
(59-61).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/HCVc101, pSR
/HCVc122, and pSR
/HCVc195, the mammalian
expression constructs for different lengths of HCV core protein,
were described previously (14). Plasmid pSR
/HCV-FL is the mammalian
expression construct for the full length of HCV polyprotein. This
plasmid was constructed by insertion of the 1.1-kb HindIII
(filled in)-KpnI fragment consisting of the pSR
promoter
region (HindIII-PstI fragment, 0.8 kb) and 0.3-kb
of HCV core coding region into the XbaI (filled
in)/KpnI-digested p90/HCVFLlongpU (kindly provided by
Apath). Plasmid pSHH2.1 contains a tandem dimer of the HBV genome
inserted at the EcoRI site of the vector plasmid pSV08 (77).
Plasmid pHBV-PS, a derivative of pGEM-3Zf(+), was constructed by
excising the 264-bp BglII (filling-in the overhangs with
Klenow enzyme)-HindIII fragment of HBV package signal
sequence from plasmid pHBV3.5 (78) and then inserting it into the
SmaI/ HindIII-digested pGEM-3Zf(+). When
linearized with EcoRI and transcribed in vitro
with SP6 RNA polymerase, pHBV-PS produces a 264-nucleotide transcript
containing the HBV package signal, spanning HBV nucleotides 1722-1986
(HBV adw subtype, with nucleotide positions numbered from
the unique EcoRI site of HBV). Plasmid pHBV97Po, a
derivative of pBluescript II KS+/
harboring the 2.89-kb of
full-length HBV polymerase gene fragment, was provided by C. M. Chang (National Yang-Ming University, Taiwan). In this construct, the
polymerase gene fragment (AluI-SacI fragment) was derived from pMH3/3097 (79). When this construct is linearized with
appropriate restriction endonucleases and transcribed in vitro with T3 RNA polymerase, transcripts coding for various
length of HBV polymerase can be produced. Plasmid pHEX-X1, the
mammalian expression construct for HBx under the HBX
promoter control, was provided by S. J. Lo (National Yang-Ming
University, Taiwan). Plasmid pGST-HBx, which can direct the expression
of the full-length HBx protein fused with the C terminus of GST, was
constructed by insertion of the 462-bp PCR-generated
EcoRI/XhoI fragment of the HBX gene
derived from plasmid pMH3/3097, into the
EcoRI/XhoI-digested pGEX-5X-1 (Amersham
Biosciences). To construct plasmid pCMV-HA-HBx for generation of
HA-tagged HBx, the same 462-bp EcoRI-XhoI
fragment of HBX gene was subcloned into the
EcoRI/XhoI-digested pCDNA-3-HA vector
(Invitrogen). Plasmid pFLAG-HBx, the mammalian expression construct for
FLAG-tagged HBx, was constructed by insertion of the 462-bp
PCR-generated HindIII-EcoRI fragment of the
HBX gene from plasmid pMH3/3097 into the
HindIII/EcoRI-digested pFLAG-CMV-2 (Eastman
Kodak). Plasmid pGFP-HBVpol, which can express green fluorescent
protein (GFP)-tagged HBV polymerase protein, was cloned by insertion of
the 3-kb EcoRV-SmaI pol gene fragment from the pMH-
C-BE (provided by S. J. Lo, National Yang-Ming University, Taiwan) into the EcoRI (filled in)-digested pEGFP-C3 vector
(Clontech). The plasmid pFLAG-HBVpol was generated
by inserting the 3-kb HindIII-SmaI DNA fragment
of plasmid pGFP-HBVpol into HindIII/SmaI site of pFLAG-CMV-2.
)
was then created by subcloning a 3.2-kb EcoRI fragment of
the HBx null HBV genome in the pSELECT/HBV as a tandem dimer into vector plasmid pSV08, and the resulting mutant construct was used for transfection.
/HCVc195, pSR
/HCVc122, or pSR
/HCVc101). After 24-48 h,
cells were washed twice with ice-cold phosphate-buffered saline and
collected by centrifugation. Whole cell extracts were prepared from
transfected cells by lysis in PBS containing 0.5% Nonidet P-40 and 1×
protease inhibitor mixture (CompleteTM, Roche Molecular
Biochemicals) for 30 min on ice. The extracts were cleared by
centrifugation at 10,000 × g for 20 min. Supernatants were incubated for overnight at 4 °C with anti-FLAG M2
antibody-conjugated agarose beads (Sigma) in binding buffer (50 mM Tris-HCl (pH 8.0), 100 mM NaCl, 5 mM MgCl2, 20% glycerol) or binding buffer
containing RNase A (10 µg/ml). Beads were washed four times with
binding buffer, and bound proteins were separated by SDS-PAGE and
analyzed by Western blotting.
20 °C) and probed
with rabbit anti-HCV core antiserum, followed by fluorescein
isothiocyanate (FITC)-conjugated goat anti-rabbit IgG. For confocal
immunofluorescence microscopy, HeLa cells grown on coverslips were
cotransfected with pFLAG-HBx or pGFP-HBVpol together with HCV
core-expressing plasmids (pSR
/HCVc195, pSR
/HCVc122, or
pSR
/HCVc101) or its control vector pSR
(1 µg each). Twenty four
hours after transfection, cells were washed three times with PBS, fixed
with 4% paraformaldehyde in PBS for 30 min at room temperature,
permeabilized for 15 min with 0.5% Triton X-100 in PBS, and incubated
with human anti-HCV core patient sera (4) or anti-FLAG M2 antibody
(Sigma) and then stained with rhodamine-conjugated goat anti-human IgG
antibody (The Jackson Laboratories) and FITC-conjugated goat anti-mouse
IgG antibody (The Jackson Laboratories).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-galactosidase (75), whereas by deletion analysis the second basic
amino acid cluster (NLS2) was found to be as important as an NLS in the
truncated core protein containing N-terminal 123 amino acid residues
(76). We also noticed a putative bipartite NLS (85) located at residues 50-70 (RKTSERSQPRGRRQPIPKARR; designated Bipartite NLS, Fig.
1A) of the HCV core protein. Thus, it is not clear which NLS
represents the critical NLS in the natural context of HCV core protein.
It is also not clear whether the nuclear localization of this protein is essential for its suppression activity on HBV replication and gene
expression. When considering that basic residues are important for NLS
function (86) and the 22-amino acid region spanning residues 101-122
of the core polypeptide, which is crucial for the suppressive activity
of the core protein (74), also contains six arginine residues (residues
101, 104, 113, 114, 115, and 117), site-directed mutagenesis of
individual or consecutive arginine/lysine residue or various
combinations thereof were performed in order to assess their role in
the suppression activity of the core protein. In the first set of 11 NLS mutants, consecutive basic residues appearing at NLS regions
(residues 9/10, 12/13, 17/18, 39/40, 50/51, 61/62, and 69/70) were
replaced by alanine residue (see Fig. 1A; designated as M9,
M12, M17, M39, M50, M61, M69, M12/39, M17/39, M50/69, and M12/39/61,
respectively). In the second set of core mutants, the six arginine
residues located at residues 101-122 as described above were mutated
to lysine or neutral ones (leucine in most cases or alanine).
Additionally, aspartate residue was also used to replace arginine
residue at 117. All together, 13 mutants in the second set of core
variants were obtained (see Fig. 1B, designated as M101L,
M101K, M104L, M104K, M113L, M113K, M114L, M114K, M115A, M115K, M117L,
M117K, M117D, respectively).
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Fig. 1.
Mutants of HCV core protein used in this
study. All HCV core mutants carried either a single, double, or
multiple mutation designed for site-specific replacement of basic
residues at NLS regions (A) or amino acid residues 101-122
(B) with alanine (A), leucine (L),
lysine (K), or aspartate residue (D). The amino
acid sequence of the HCV core protein used for the analysis was adapted
from Takeuchi et al. (121), and only the relevant amino acid
sequences are displayed. The NLSs in the HCV core protein are
shown.
Expression of HBsAg and HBeAg/BcAHg in HuH-7 cells after
cotransfection with cloned HBV DNA and various HCV core mutant
constructs
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Fig. 2.
Northern blot analysis of HBV transcription
in HuH-7 cells cotransfected with HBV plasmid and various HCV core
mutants. HuH-7 cells were cotransfected (20 µg each) with HBV
plasmid pSHH2.1 and HCV core expression construct pECE/HCVC-KF
(WT) and its various mutant derivatives that carried the
mutations as indicated. Total cellular RNAs were prepared from cells at
day 6 post-transfection and probed with 32P-labeled HBV DNA
as described under "Experimental Procedures." The same blot was
rehybridized with 32P-labeled 18 S or
glyceraldehyde-3-phosphate dehydrogenase gene fragment
(G3PDH). The positions of 3.5- and 2.1-kb HBV-specific
transcripts are indicated by arrows. Mock,
without transfection; control, cells transfected with
pSHH2.1 and vector pECE. The designations for HCV core mutants are
shown in Fig. 1.
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Fig. 3.
Effect of HCV core protein mutants on HBV
endogenous DNA polymerase activity in HuH-7 cells. HuH-7 cells
were cotransfected with HBV plasmid pSHH2.1 and various HCV core
constructs (wild-type (WT) or various core mutants), and the
released HBV particles in the medium fraction on day 6 post-transfection were assayed as described under "Experimental
Procedures." The abbreviations for the HCV construct used in the
cotransfection experiment (indicated at the top of each
lane) are identical to those described in the legend to Fig. 2. The
positions of relaxed circular (RC) and linear (L)
forms of HBV DNA are indicated by arrows. The relative
intensity of relaxed circular and linear in each lane is normalized to
the signal of the control and is indicated at the bottom of
each lane.
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Fig. 4.
Subcellular localization of HCV core NLS
mutants. HuH-7 cells transiently transfected with vector pECE
(control), HCV core expression construct pECE/HCVC-KF
(WT), or its mutant derivatives were grown on glass
coverslips and fixed at day 6 post-transfection. HCV core proteins were
visualized with rabbit anti-HCV core sera and FITC-conjugated goat
anti-rabbit IgG antibody. The abbreviations for the HCV mutant
construct used in the transfection experiments are identical to those
described in Fig. 1.
) (see "Experimental
Procedures"). As shown in Fig.
5A (lanes 1-5) and
Table II, in contrast to the case of
pSHH2.1 harboring the wild-type HBV genome, HCV core protein did not
exhibit any inhibition on the production of HBsAg, HBeAg, or HBV
transcript derived from this HBx null plasmid pHBV(X
).
Moreover, when HBx expression construct (pHEX-X1) was cotransfected with this pHBV(X
) plasmid, the suppression of HCV core
protein on HBV gene expression was recovered (Fig. 5A,
lanes 6 and 7; Table II). However, the suppression of HBV particle production was retained in this
pHBV(X
) and HCV core plasmid cotransfected cells (Fig.
5B), suggesting that the mechanism for suppression of HBV
gene expression and replication by the HCV core protein is
decoupled.
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Fig. 5.
Effects of HBx on HCV core protein
suppression ability. HuH-7 cells were cotransfected with HBV
plasmid (pSHH2.1 or pHBV(X )) and HCV core
expression construct pECE/HCVC-KF (core) (or vector pECE) or HBx
expression construct pHEX-X1 (HBx) if applicable. At day 6 post-transfection, total cellular RNA and the released HBV particles
were isolated for Northern blot analysis (A), and HBV
endogenous polymerase activity assay (B), respectively. All
experimental conditions are similar to those described in the legends
to Fig. 2 and 3 except the amount of plasmid used for transfection are
20 µg each for the indicated plasmid in lanes 2-5 of
A and lanes 2-5 of B but were 14 µg
each for lanes 6 and 7 of A.
Expression of HBsAg and HBeAg/HBcAg in HuH-7 cells after
cotransfection with cloned wild-type or HBx null HBV DNA and HCV
core expression construct
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Fig. 6.
Both FLAG-HBx and HCV core proteins
colocalize inside a cell. HeLa cells grown on glass coverslips
were cotransfected with pFLAG-HBx (FLAG-HBx) and vector (SR )
(a-c) or various forms of HCV core construct including
pSR
/HCVc101(C101) (d-f), pSR
/HCVc122 (C122)
(g-i), or pSR
/HCVc195 (C195) (j-l) as
indicated. After 24 h post-transfection, cells were fixed and
stained with mouse anti-FLAG M2 monoclonal antibody and human anti-HCV
core sera, followed by FITC-conjugated goat anti-mouse IgG or
rhodamine-conjugated goat anti-human IgG. The right panels
c, f, i, and l show the
merged image of colocalization of HBx and HCV core protein.
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Fig. 7.
HBx interacts with HCV core protein both
in vitro and in vivo.
A, in vitro binding assay of GST-HCV core fusion
protein and in vitro translated HBx. Glutathione-Sepharose
beads (15 µl) bound to GST (lane 2), GST/HCV-c101
(lane 3), GST/HCV-c122 (lane 4), and GST/HCV-c195
(lane 5) (3-5 µg) were incubated with the in
vitro translated [35S]Met-labeled HA-tagged HBx (15 µl). After extensive washing, proteins that bound on resins
were eluted with sampling buffer and analyzed by SDS-PAGE and
autoradiography (bottom panel). Lane 1, input
in vitro translated [35S]Met-labeled HA-tagged
HBx (2 µl). Coomassie Blue staining of the HCV core variants used in
the binding assay is also shown in the top panel.
B, purified GST (lane 1) and GST/HBx (lane
2) (3-5 µg) used for the binding assay. C, in
vitro binding assay of GST/HBx fusion protein and in
vitro translated HCV core variants. Glutathione-Sepharose beads
(15 µl) bound to GST (lanes 4-6) and GST/HBx (lanes
7-9) (4 µg) were incubated with the in vitro
translated [35S]Met-labeled T7-tagged HCV core variants
C101 (lanes 4 and 7), C122 (lanes 5 and 8), and C195 (lanes 6 and 9) (10 µl). After extensive washing, proteins bound on the beads were eluted
with sampling buffer and analyzed by SDS-PAGE and autoradiography.
Lanes 1-3, input in vitro translated
[35S]Met-labeled T7-tagged HCV core variant.
D, in vivo coimmunoprecipitation of HBx and HCV
core protein. HeLa cells were cotransfected with FLAG-tagged HBx
together with the HCV core expression constructs (see "Experimental
Procedures"). The cells extracts prepared from the transfected cells
were immunoprecipitates (IP) by anti-FLAG (M2)
antibody-conjugated agarose resins (lanes 1-5), and
immunoprecipitates were analyzed by SDS-PAGE followed by immunoblotting
(WB) with human anti-HCV core sera (top panel) or
monoclonal anti-FLAG antibody (middle panel) for the
detection of HCV core variants or FLAG-HBx. The relative expression of
HCV core variants in cell lysates determined by immunoblotting was also
shown at the bottom of the panel.
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Fig. 8.
HCV core protein forms a complex with HBV
polymerase in vitro and in vivo.
A, analysis of in vitro translated, full-length
HCV core protein and HBV polymerase. The
[35S]Met-labeled, full-length HCV core protein
(lane 1) and HBV pol (lane 2) were prepared by
in vitro transcription and translation (see "Experimental
Procedures") and analyzed by 12% SDS-PAGE and autoradiography.
B, in vitro binding analysis of HCV core protein
and HBV polymerase. The in vitro translated HCV core (C195)
(0.5 pmol) and HBV polymerase (pol) (0.2 pmol) were immunoprecipitated
with sera-conjugated protein A-Sepharose from the HCV patient, and the
immunoprecipitates were released by boiling in sampling buffer and then
resolved on SDS-PAGE (see "Experimental Procedures"). The positions
of HBV polymerase (pol), and HCV core protein
(core) are indicated. C, in vitro
binding assay of GST-HCV core fusion protein and in vitro
translated HBV pol (pol). Glutathione-Sepharose beads (15 µl) bound to GST (lane 2), GST/HCV-c24 (lane
3), GST/HCV-c101 (lane 4), and GST/HCV-c122 (lane
5) (4 µg) were incubated with the in vitro translated
[35S]Met-labeled HBV pol (10 µl). After extensive
washing, proteins that bound on resins were eluted with sampling buffer
and analyzed by SDS-PAGE and autoradiography. Lane 1, input
in vitro translated [35S]Met-labeled pol (2 µl). D, coimmunoprecipitation of HBV pol and HCV core
constructs in vivo. HeLa cells were cotransfected with
FLAG-tagged HBV pol (FLAG-pol) together with the HCV core expression
constructs (see "Experimental Procedures"). The cell extracts
prepared from the transfected cells were immunoprecipitates
(IP) by anti-FLAG (M2) antibody-conjugated agarose resins
(lanes 1-5), and immunoprecipitates (IP) were
analyzed by SDS-PAGE followed by immunoblotting (WB) with
human anti-HCV core sera (top panel) or monoclonal anti-FLAG
antibody (middle panel) for the detection of HCV core
protein or FLAG-pol. The coimmunoprecipitation experiment was performed
in the presence of RNase A (10 µg/ml). Also shown at the
bottom of the panel is the immunoblotting of transfected
cell extracts with anti-HCV core sera without
immunoprecipitation.
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Fig. 9.
HBV polymerase and HCV core protein
colocalize in cytoplasm. HeLa cells grown on glass coverslips were
cotransfected with GFP-tagged HBV pol expression construct
pGFP-HBVpol (GFP-HBVpol) and vector (SR ) (a-c) or
various forms of HCV core construct including
pSR
/HCVc101(C101) (d-f), pSR
/HCVc122 (C122)
(g-i), or pSR
/HCVc195 (C195) (j-l) as
indicated. After 24 h post-transfection, cells were fixed and
stained with human anti-HCV core sera, followed by rhodamine-conjugated
goat anti-human IgG. The right panels c,
f, i, and l show the merged image of
colocalization of HBV pol and HCV core protein.
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Fig. 10.
Domain mapping between HBV polymerase and
HCV core protein. A, schematic representations of the
functional domains of HBV polymerase and DNA templates used for
generation of the full-length and C-terminal truncated HBV polymerase.
The approximate locations of terminal protein (TP), the
spacer region, the reverse transcriptase domain, and the RNase H domain
are shown (89, 119). Also shown at the bottom of this panel
is the position of the interaction domain of HBV polymerase and HCV
core protein obtained from this study. The restriction sites
(SmaI, NcoI, and AccI) used to
linearized plasmid pHBV97Po for the in vitro transcript
reaction are indicated. These linearized plasmids were transcribed by
T3 RNA polymerase, and the protein products subsequent to translation
of their run-off transcripts are shown. The numbers refer to the
expected total amino acid residues for the various versions of HBV
polymerase. B, analysis of in vitro translated,
full-length (pol) and truncated forms (pol749 and pol567) of HBV
polymerase. In vitro translation reaction was performed in
the presence of [35S]methionine as described under
"Experimental Procedures." The protein products obtained after
in vitro translation were subjected to SDS-PAGE and
autoradiography. C, in vitro binding analysis of
HCV core protein and the various forms of HBV polymerase. The
full-length and truncated forms of 35S-labeled HBV pol (0.2 pmol) were incubated either with 35S-labeled core protein
(C195) of HCV (lanes 2, 4, and 6) (0.5 pmol) or with buffer (lanes 3, 5, and
7). Following incubation, complexes were immunoprecipitated
(IP) with sera from HCV patients and analyzed SDS-PAGE and
autoradiography. Lane 1, HCV core protein alone
immunoprecipitated with HCV patients sera.
Sequence--
The HCV core protein has RNA binding ability
(90). To determine whether the strong suppression of HBV encapsidation
process by the HCV core protein may be the result of the binding of HCV core protein to HBV package signal (
), we studied the binding affinity of the HCV core protein to the
sequence using the
streptavidin-biotin-mediated binding assay (53; also see
"Experimental Procedures"). When in vitro translated HCV
core protein was incubated with the biotin-labeled HBV
sequence,
the presumed RNA-core protein complex could be bound to the
streptavidin-agarose (Fig.
11A). The binding of the HCV
core protein to the
sequence was found to be enhanced in a
concentration-dependent manner (Fig. 11A,
lanes 3-6). Moreover, this binding of HCV core protein on the
streptavidin-agarose was HBV
sequence-specific because in the
absence of biotinylated
RNA core protein was not retained by the
streptavidin-agarose (Fig. 11A, lane 1), and the
binding signal of HCV core protein could be ablated by an excess of
unlabeled
sequence but not by the unrelated Escherichia
coli cei RNA (354 nucleotides) of ColE7 operon (91)
(Fig. 11B, lanes 2-4). Notably, both the
full-length (p22) and truncated forms (p18) of HCV core protein could
bind the
sequence (Fig. 11A, lanes 5 and
6). Because p18 was present in a lesser amount in the
in vitro translated products (Fig. 8A), the
display of similar intensity of both forms of the HCV core protein in
the precipitate implies that the affinity of p18 species to the
sequence is much stronger as compared with that of the full-length p22
species.
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Fig. 11.
Competition binding analysis of the HCV core
protein to HBV encapsidation components, HBV polymerase and
RNA. A, analysis of the
RNA-binding affinity of the full-length HCV core protein. The binding
affinity of the HCV core protein to the biotin-labeled
RNA was
examined by the streptavidin-biotin-mediated binding assay (see
"Experimental Procedures"). In this RNA-protein binding reaction,
the biotin-labeled
RNA (Bio-
) (60 pmol) was incubated with
increasing amounts of in vitro translated HCV core protein,
ranging from 0.4 to 3.0 pmol (lanes 3-6), and the HCV core
protein-
RNA complexes were precipitated with streptavidin-agarose
beads. The bound labeled proteins were released by boiling in sampling
buffer and detected by SDS-PAGE and autoradiography. Lane 1,
binding assay in the absence of Bio-
; lane 2, binding
assay in the absence of core protein. The positions of the full-length
(p22) and the truncated form (p18) of the HCV core protein are
indicated. B,
RNA-binding specificity of the full-length
HCV core protein. In this RNA-protein binding reaction, the Bio-
(30 pmol) was incubated with in vitro translated HCV core
protein (1.0 pmol) in the presence or absence of 6-fold excess
competitor RNA as indicated and the HCV core protein-
RNA complexes
were precipitated with streptavidin-agarose beads and detected by
autoradiography. C,
RNA-binding specificity of the HBV
polymerase (pol). In this RNA-protein-binding reaction, the
experimental conditions were similar to that described in B
except that the Bio-
(30 pmol) was incubated with in
vitro translated HBV pol (0.2 pmol) in the presence or absence of
6-fold excess competitor RNA as indicated. D, binding of HBV
polymerase to biotinylated
RNA. The in vitro translated
HBV pol (0.2 pmol) was incubated with increasing amounts of
biotinylated
RNA (Bio-
), ranging from 7.5 to 120 pmol
(lanes 3-7) or without Bio-
(lane 2) in a
total of 350 µl of reaction buffer (see "Experimental
Procedures"), and the RNA-protein complexes were precipitated with
streptavidin-agarose beads and displayed by SDS-12% PAGE.
E, competition binding assays. Adding the increasing amounts
of HCV core protein (0.4-2 pmol) to the HBV pol (0.2 pmol) and
biotinylated
RNA (60 pmol) binding reactions (final volume 350 µl), the truncated form (p18) of HCV core protein was detected in the
pol-RNA complexes, which in turn decreased the complex formation of HBV
pol-
RNA (compare lanes 3 and 4 with
lanes 1 and 2) as detected by
streptavidin-agarose affinity chromatography (see "Experimental
Procedures"). F, the supernatant recovered from the
binding reaction mixtures of lanes 2 and 4 in
E was further precipitated with antiserum against HBV pol
(see "Experimental Procedures"), and the precipitates were analyzed
by SDS-PAGE and autoradiography. Lanes 1 and 2,
immunoprecipitates obtained from supernatants of binding reactions in
lanes 2 or 4 of E, respectively;
lanes 3 and 4, immunoprecipitates of HCV core
protein or HBV pol by antiserum against HCV core protein or HBV pol,
respectively. The positions of the HCV core protein (p22 and p18) and
HBV pol (pol) are indicated.
sequence, it may compete for the complex formation between HBV pol and
sequence, resulting in the suppression of the virus encapsidation
process. To assess this possibility, we studied the competition of HCV
core protein with HBV pol for binding to
sequence. As shown in Fig.
11C, in vitro translated HBV pol
bound to
sequence with sequence specificity because the binding
signal of HBV pol in the precipitates of streptavidin-agarose could be blocked by an excess of the unlabeled
sequence but not by the unrelated E. coli cei RNA (Fig.
11C, lanes 2-4). A pilot analysis for the
determination of the saturation amount of
sequence that could bind
to a fixed amount of HBV pol was conducted prior to the competition
experiment. As shown in Fig. 11D, a saturation level was
achieved when 60 pmol of biotin-labeled
sequence was present in the
binding reaction mixture containing 0.2 pmol of in vitro
translated HBV pol (Fig. 11D, lane 6). It is
noted that this similar amount of biotinylated
sequence could bind
about 1.5 pmol of in vitro translated, full-length of HCV
core protein (see Fig. 11A, lane 5).
RNA (60 pmol). The remaining HBV pol bound to the biotin-labeled
RNA was
then detected by the streptavidin-agarose binding assay (Fig.
11E). As predicted, addition of the full-length HCV core protein to the reaction mixture containing HBV pol and
RNA reduced the pol bound to
in a concentration-dependent manner
(Fig. 11E, lanes 2-4). It was found that at the
highest concentration examined (molar ratio of HCV core/HBV pol about
10-folds), HCV core protein reduced the pol signal more than 50% (Fig.
11E, lane 4). Only the truncated form of HCV core
protein, p18, was coprecipitated by the streptavidin-agarose beads
(Fig. 11E, lanes 3 and 4). The
full-length HCV core protein (p22) was present as a complex with HBV
pol in the supernatant recovered from the binding assay because it
could be coimmunoprecipitated by the antiserum against HBV pol (Fig. 11F, lanes 1 and 2). All together, our
results indicate that the full-length HCV core protein interferes with
the ability of HBV pol to bind to
RNA.
Sequence but
Cannot Disrupt the pol-
Complex Formation--
Earlier works (74)
indicate that the N-terminal 101-amino acid segment (C101) of the HCV
core protein do not have the suppression ability in the HBV
encapsidation process. To examine whether this loss of the suppressive
effect in the shorter construct of HCV core protein correlates with the
loss of binding ability to HBV pol protein or
sequence, a similar
coimmunoprecipitation or RNA binding assay was performed on this
truncated C101 species. To circumvent the low level of
[35S]methionine labeling in C101 species (only one Met at
the first initiation codon), 14C-labeled C101 or the
35S-labeled T7-C101 core fusion protein (see
"Experimental Procedures") was used in these experiments. As shown
in Fig. 12, A and
B, the data suggest that this truncated form of the HCV core
protein (C101 or T7-C101) retained its ability to bind with HBV pol and
sequence. We next examined the ability of this
suppression-defective core protein to compete with HBV pol for binding
of
sequence. Fig. 12C indicated that the presence of
increasing amounts of the C101 species (molar ratio 7-35-fold) did not
disrupt the complex formation of HBV pol-
because the pol signal in
the precipitates of streptavidin-agarose remained unchanged. This loss
of the competition activity of the HCV core mutant protein did not
result from its N-terminal fusion with T7 tag, because similar fusion
did not affect the competition ability of the full-length of the HCV
core protein (data not shown). Surprisingly, C101 species (T7-C101) was
also found in the precipitates (Fig. 12C, lanes
3-5). Therefore, although the suppression-impaired mutant of the
HCV core protein could form a complex with HBV pol or
sequence,
they did not affect the HBV pol for binding to
sequence. This
result is consistent with our previous study of the suppressive effect
of this core mutant in vivo (74).
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Fig. 12.
HCV core protein variant could bind to HBV
polymerase and RNA but could not eliminate
the pol-
complex formation. A,
interaction of HCV core protein truncated variant with HBV pol. All
binding assays were identical to the experimental conditions described
in the legend to Fig. 8B except that the truncated version
of 14C-labeled HCV core protein (C101) obtained by in
vitro translation (see "Experimental Procedures") was used in
this study. B, analysis of the
RNA-binding affinity of
the truncated version of HCV core protein. All experimental conditions
were identical to those of Fig. 11A legend except that the
binding affinity of the HCV core protein to the biotin-labeled
RNA
was examined on the truncated version of 35S-labeled
T7-C101 (1.4-7.0 pmol) (lanes 3-5). Lane 1,
binding assay in the absence of Bio-
; lane 2 binding
assay in the absence of T7-C101. C, competition binding
assay. The experimental conditions were identical to those of Fig.
11E legend, except that the in vitro translated
T7-C101 (1.4-7.0 pmol) was used for competition analysis. Lane
1, binding assay in the absence of both Bio-
and T7-C101
(lane 1) or T7-C101 (lane 2). The positions of
the truncated form of the HCV core protein (C101 or T7-C101) and HBV
pol (pol) are indicated.
/HCV-FL
(see "Experimental Procedures" for plasmid construction) with HBV
plasmid pSHH2.1. As shown in Fig.
13A, both HBV antigens, HBe/HBcAg and HBsAg, were reduced 3-fold by the HCV polyprotein. A
strong inhibitory effect (about 20-fold) on HBV particle release was
also observed in pSR
/HCV-FL and pSHH2.1 cotransfected cells (Fig.
13B). Thus, the same level of suppressive effect was
observed by the HCV polyprotein expression construct pSR
/HCV-FL and
by the HCV core expression construct pECE/HCVC-KF (see Table I and Figs. 2 and 3). Western blot analysis indicated that the expression level of HCV core protein generated from proteolytic processing of the
polyprotein in pSR
/HCV-FL transfected cells was comparable with that
of core protein expressed by HCV core expression construct pECE/HCVC-KF
(Fig. 13C). These findings imply that the inhibitory effect
is due predominantly to the core protein, and the contribution of the
other HCV viral proteins, if any, is probably minor. Thus, the core
protein expressed as part of the full-length polyprotein also has the
suppressive effects on HBV gene expression and replication.
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Fig. 13.
Effect of intact HCV polyprotein on
expression of HBV antigens and HBV endogenous DNA polymerase activity
in HuH-7 cells. HuH-7 cells were cotransfected with HBV plasmid
pSHH2.1 (2.5 µg) and HCV polyprotein expression construct
pSR /HCV-FL (17 µg) or pSR
vector (5 µg). The total amount of
plasmid DNA used for transfection was 19.5 µg each by adding
pGEM-3Zf(
) plasmid DNA, if applicable. The medium fraction on day 6 post-transfection was detected for HBsAg and HBeAg/HBcAg (A)
and HBV endogenous DNA polymerase activity (B) (see
"Experimental Procedures"). S/N ratio, sample
versus negative control. The positions of relaxed circular
(RC) and linear (L) forms of HBV DNA are
indicated by arrows. C, analysis of HCV core
protein expression in pSR
/HCV-FL or pECE/HCVC-KF transfected cells.
HuH-7 cells (6-well) were cotransfected with pSHH2.1 (0.5 µg) and HCV
core protein expression construct pECE/HCVC-KF (3.6 kb, 1 µg)
(lane 2) or HCV polyprotein expression construct
pSR
/HCV-FL (13.5 kb, 3.75 µg) (lane 3). The total
amount of plasmid DNA used for transfection was 4.25 µg each by
adding pGEM-3Zf(
) plasmid DNA, if applicable. The detection of HCV
core protein expression in transfected cells was examined by immunoblot
using anti-HCV core sera.
/C195) were cotransfected into Ava.5 cells,
in vivo coimmunoprecipitation experiments using anti-FLAG
antibody for immunoprecipitation revealed that HCV core protein was
coprecipitated with the FLAG-tagged HBx or HBV pol protein in Ava.5
cells (Fig. 14, B and C, lanes 4 and
5). RNase A treatment of the coimmunoprecipitates did not eliminate their interaction (compare lanes 4 and
5), suggesting that the complex formation between HCV core
protein and HBx or HBV pol is not mediated by RNA. Therefore, our
observations of the interaction as well as the effects on HBV elicited
by HCV core protein as presented above likely reflect the context of full-length HCV polyprotein.
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Fig. 14.
HCV core protein can associate with HBx or
HBV pol in the presence of HCV nonstructural proteins (NS3 to
NS5B). A, analysis of NS3 expression in Ava.5 cells.
The cell extracts prepared from HuH-7 or Ava.5 cells were analyzed by
SDS-PAGE followed by immunoblotting with rabbit anti-HCV NS3 sera for
the detection of NS3. The amounts of protein loaded are as follows: 25 µg for lanes 1 and 4; 50 µg for lanes
2 and 5; and 75 µg for lanes 3 and
6. B and C, in vivo
coimmunoprecipitation of HBx or HBV pol and HCV core protein. Ava.5
cells were cotransfected with FLAG-tagged HBx (B) or
FLAG-tagged HBV pol expression construct (C) together with
the HCV core expression construct pSR /HCVc195 (see "Experimental
Procedures") (10 µg each). The cells extracts prepared from the
transfected cells were immunoprecipitates (IP) by anti-FLAG
(M2) antibody-conjugated agarose resins (lanes 1-5), and
immunoprecipitates were analyzed by SDS-PAGE followed by immunoblotting
(WB) with human anti-HCV core sera for the detection of HCV
core protein. All experimental conditions are similar to the legend of
Fig. 7D and Fig. 8D except that the
coimmunoprecipitation experiments of lanes 1-4 of
B and C were performed in the presence of RNase A
(10 µg/ml). Also shown at the bottom (lanes
1-5) or right of the panels (lanes 6-9) is
the immunoblotting of transfected cell extracts with anti-FLAG or
anti-HCV core sera without immunoprecipitation.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B, AP-1, and serum response element
(SRE)-associated pathways. Because activation of these signaling
pathways by HBx or HCV core proteins plays an important role in liver
injury, cirrhosis, and hepatocellular carcinoma (104-107), this
may partially account for the clinical observation of increasing
severity of liver disease in patients with dual infection of these two
viruses (69, 108-110).
is a prerequisite for HBV pre-genomic RNA packaging (49,
111), we anticipated that the full length of HCV core protein would
inhibit encapsidation of the pre-genomic RNA into a nucleocapsid, thus
confirming the observation made with in vivo transfection
experiments (4, 74). The possibility that the suppression of HBV
encapsidation by the HCV core protein is in part due to complex
formation with another encapsidation component-HBV core protein was
also explored. However, our preliminary results provide no support for
this hypothesis, because similar coimmunoprecipitation experiments on
the HCV and HBV core proteins failed to detect their association (data
not shown). Another alternative hypothesis suggested from the present study (see Fig. 11) could imply that the binding of processed forms of
HCV core protein (e.g. p18) to the HBV package signal may
preclude the binding of the HBV pol to the
sequence, which in turn
also affects the encapsidation process. In viewing that the amount (60 pmol) of HBV
sequence used in this study was in large excess to
that of HBV pol (0.2 pmol) or HCV core protein (about 0.4 to 2 pmol),
this possibility seems less likely. Thus, based on our results shown
here, it appears that the major cause for suppression of HBV
encapsidation by HCV core protein is due to inhibition of HBV pol
binding to the
sequence by the formation of inactive HBV pol-HCV
core protein complex. The essence of this model is that of all three
essential encapsidation components, only the HBV pol presents as trace
amounts, thus the selection of HBV pol protein instead of HBV core
protein or
sequence as a target for suppression of HBV
encapsidation by the HCV core protein appears as the most promising mechanism.
sequence. Loss of the competition ability of this core variant appears
to be consistent with the loss of the in vivo suppressive
effect of this variant on HBV encapsidation (74). Additionally, results
obtained from the suppression-defective mutant (C101) also imply that
the retention of C101 species in the encapsidation components (see Fig.
12), if occurring in vivo as in vitro, presumably
does not impair HBV encapsidation. One potential explanation for the
different competitive behavior in this HCV core variant is that the
amino acid segment beyond amino acid residue 101 may be important for
the interference effect on the HBV pol function. Along this line, our
results as shown here indicate that mutation at the Arg113,
Arg114, or Arg115 residue of HCV core protein
severely impairs its suppression ability on HBV encapsidation, and the
fact that phosphorylation of Ser99 and Ser116
also modulates the suppression ability of HCV core protein on HBV
encapsidation (4) all support this explanation. Intriguingly, in this
study we also found a stimulating effect of HBV replication (1.7-2.9-fold) in several core mutants like M50, M9, M17, and M61 (see
Fig. 3). The exact molecular mechanism for this effect is not clear. In
view of the fact that the conformation of HBV pol or HBV pol-
complex dictates the efficiency of the HBV pre-genome RNA encapsidation
process (113-116), presumably the presence of these HCV core variants
directly or indirectly induces the formation of a more competent,
productive conformation of HBV pol or HBV pol
complex for the
encapsidation process. If this notion is correct, one may predict that
the effect of HCV core protein on HBV replication may depend on the
structural context of the HCV core protein. This may well explain the
variation or even the lack of the interference effect between these two
viruses in some clinical cases (73, 117) or in the transgenic mice
model (118). As noted, HCV core protein used for our experiment is
genotype 1b, and in general the different genotype of HCV may impart
amino acid substitution on HCV core protein, even though this protein is highly conserved.
is mediated through the interaction of
the HCV core protein with the reverse transcriptase/RNase H domains of
the pol protein. A relevant question arises whether this complex
formation may also inhibit the reverse transcriptase and RNase H
activities of the pol protein.
(59-61). In view of these results, it is particularly
interesting that the suppression of HBV encapsidation by the HCV core
protein may take place during the translation of HBV pol before its
interaction with other cellular factors for nucleocapsid assembly. The
question of whether the suppression of HBV encapsidation by HCV core
protein has to be mediated via Hsp90, Hsp60, or other cellular factors
remains to be clarified.
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ACKNOWLEDGEMENTS |
---|
We thank C. M. Rice and Apath for generously providing the HCV plasmid p90/HCVFLlongpU and Ava.5 cells. We also thank S. J. Lo, C. M. Chang, and C. K. Chak for providing plasmids or antibodies. We are grateful to M. T. Hsu for critical reading and comments on this manuscript.
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FOOTNOTES |
---|
* This work was supported by National Health Research Institute Grants DOH85-HR-502, DOH86-HR-502, DOH87-HR-502, DOH88-HR-502, NHRI-GT-EX89B502L, NHRI-GT-EX90-9002BL, and NHRI-GT-EX91-9002BL (to Y.-H. W. L.) and in part by National Science Council Grants NSC89-2320-B-010-141 and NSC90-2320-B010-083, and Ministry of Education Grant (Program for Promoting Academic Excellence of Universities) 89-B-FA22-2-4.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.
§ Both authors contributed equally to this work.
¶ Present address: Dept. of Molecular Genetics, University of Texas, M. D. Anderson Cancer Center, Houston, TX 77030.
Present address: Dept. of Biochemistry, Taipei Medical
College, Taipei, Taiwan, Republic of China.
Present address: Dept. of Molecular and Cellular
Biology, Baylor College of Medicine, Houston, TX 77030.
§§ To whom correspondence should be addressed: Institute of Biochemistry, National Yang-Ming University, Taipei, Taiwan 112, Republic of China. Tel.: 8862-2826-7124; Fax: 8862-2826-4843; E-mail: yhwulee@ym.edu.tw.
Published, JBC Papers in Press, October 24, 2002, DOI 10.1074/jbc.M204241200
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ABBREVIATIONS |
---|
The abbreviations used are:
HCV, hepatitis C
virus;
GFP, green fluorescence protein;
GST, glutathione
S-transferase;
HA, hemagglutinin;
HBV, hepatitis B virus;
NLS, nuclear localization signal;
, package signal;
pol, polymerase;
FITC, fluorescein isothiocyanate;
PBS, phosphate-buffered saline;
NS, nonstructural.
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REFERENCES |
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