RNA-dependent RNA Polymerase Activity of the Soluble
Recombinant Hepatitis C Virus NS5B Protein Truncated at the
C-terminal Region*
Tatsuya
Yamashita
§,
Shuichi
Kaneko§,
Yukihiro
Shirota
§,
Weiping
Qin
,
Takahiro
Nomura
,
Kenichi
Kobayashi§, and
Seishi
Murakami
¶
From the
Department of Molecular Oncology, Cancer
Research Institute and the § 1st Department of Internal
Medicine, Kanazawa University, 13-1 Takara-Machi,
Kanazawa, Ishikawa, Japan
 |
ABSTRACT |
The hepatitis C virus (HCV) NS5B protein
encodes an RNA-dependent RNA polymerase (RdRP), which is
the central catalytic enzyme of HCV replicase. We established a new
method to purify soluble HCV NS5B in the glutathione
S-transferase-fused form NS5Bt from Escherichia
coli which lacks the C-terminal 21 amino acid residues encompassing a putative anchoring domain (anino acids 2990-3010). The
recombinant soluble protein exhibited RdRP activity in
vitro which was dependent upon the template and primer, but it
did not exhibit the terminal transferase activity that has been
reported to be associated with the recombinant NS5B protein from insect cells. The RdRP activity of purified glutathione
S-transferase-NS5Bt and thrombin-cleavaged non-fused NS5Bt
shares most of the properties. Substitution mutations of NS5Bt at the
GDD motif, which is highly conserved among viral RdRPs, and at the
clustered basic residues (amino acids 2919-2924 and 2693-2699)
abolished the RdRP activity. The C-terminal region of NS5B, which is
dispensable for the RdRP activity, dramatically affected the
subcellular localization of NS5B retaining it in perinuclear sites in
transiently overexpressed mammalian cells. These results may provide
some clues to dissecting the molecular mechanism of the HCV replication
and also act as a basis for developing new anti-viral drugs.
 |
INTRODUCTION |
Hepatitis C virus (HCV)1
is a positive single-strand RNA virus that has been shown to be the
major causative agent of transfusion-associated hepatitis (1, 2). From
the structural similarities, it had been proposed that HCV was related
to the flaviviruses and pestiviruses, but later it was classified as a
separate genus in the Flaviviridae family because of its distinctive
characteristics (3-5). The viral genome encodes a polyprotein
precursor of about 3000 amino acids (aa), which is temporarily
processed by a combination of host and viral proteases, resulting in at
least 10 distinct products as follows:
NH3-C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B-COOH (6-8). The
HCV polyprotein is first cleaved by a host signal peptidase generating
the structural proteins, C/E1, E1/E2, E2/p7, and p7/NS2 (9-11). The
NS2/NS3 site is then cut by an HCV-encoded metalloprotease, NS2, and
the remaining sites are processed by a virus-encoded serine protease,
NS3, with or without NS4A as a cofactor (12-19). NS3 has protease,
NTPase, and RNA helicase activities (20-22), whereas NS5A may be
phosphorylated and act as a putative cofactor of NS5B (23, 24). Since
HCV NS5B contains the "GDD" sequence motif, which is highly
conserved among all RdRPs characterized to date (25), it has been
predicted that the NS5B protein encodes an RdRP, which is the central
catalytic enzyme of the HCV replicase. RdRP activity of the recombinant
NS5B purified from transfected insect cells was recently reported (26,
27).
Since persistent infection of HCV is related to chronic hepatitis and
eventually to hepatocarcinogenesis (28), HCV replication is one of the
targets to eradicate the HCV reproduction and to prevent hepatocellular
carcinoma. HCV is believed to follow a similar replication strategy to
other positive-stranded RNA viruses (29-33). However, the molecular
mechanism of HCV replication remains elusive mainly because the cell
culture systems for HCV reproduction are far too insensitive to dissect
the molecular events of viral replication (34-36). Under these
limitations, we undertook an alternative approach to characterize the
properties of NS5B, an RdRP which is the putative central catalytic
enzyme of HCV RNA synthesis (1).
Here we present the purification method and properties of a soluble
bacterial recombinant HCV NS5B protein which retains activity for
RNA-dependent RNA synthesis in vitro. The
purified bacterial NS5B shares several properties with the protein
expressed in insect cells, but it has no terminal transferase activity.
In addition, we found that the C-terminal 21 amino acid residues of
NS5B harbors a putative anchoring domain that affects its subcellular
localization in mammalian cells, although the region is dispensable for
the RdRP activity.
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EXPERIMENTAL PROCEDURES |
Construction of Plasmids--
A plasmid-derived from pGENK1 (37,
38), pGENKS, was used to express the recombinant HCV NS5B in
Escherichia coli. It encodes the consensus kination site for
protein kinase A, the cleavage site for thrombin, and additional
multiple cloning sites (EcoRI, SacI,
KpnI, XmaI, SalI, and
BamHI) (Fig. 1A). HCV JK1 cDNA (39) harboring
NS5B was subcloned by PCR using the following the set of primers,
NS5BFor and NS5BRev, which have an artificial initiation codon, and
SacI and SalI sites
(Table I, all primer sequences hereafter
are shown in the table). The truncated mutant of NS5B, NS5Bt, which
lacks the 21 amino acid residues at the C-terminal (aa 2990-3010), was
subcloned by PCR with NS5BFor and NS5BtRev. Substitution mutants of
NS5Bt-m1 to -m3 were constructed by PCR mutagenesis with overlap
extension using the designed mutagenizing primers, NS5BFor and NS5BtRev
(38). The resultant NS5Bt-m1 to -m3 proteins have the following
substitution mutations, GDD to VDD (aa 2736-2738), YRHRAR to AAAAAA
(aa 2919-2924), and CGYRRCR to AAAAAAA (aa 2693-2699), respectively.
The substituted mutant at the C-terminal of NS5B, NS5B-m4, was
constructed by PCR with NS5BFor and NS5Bm4Rev, which have an artificial
BamHI site. NS5B-m4 has two amino acid substitutions, Leu
and Val, both to Pro at residues 2998 and 3001, respectively (Fig.
1B).
All the mammalian expression vectors were derived from pSG5UTPL (37,
40). The green fluorescent protein (GFP) cDNA was prepared by PCR
using phGFP-S65T (CLONTECH) as a template with the
primer set, GFPNotFor which has the artificial EcoRV and
NotI sites, and GFPEcoRev which has an artificial
EcoRI site. The DNA fragment with an artificial EcoRV
site and BamHI sites was inserted into the pSG5UTPL blunt
and BamHI vector, whose EcoRI site was blunted
using Klenow fragment before BamHI digestion (pGFP). By using this pGFP vector, another mammalian expression vector, pNKFLAG, was constructed. The sequence encoding the FLAG-tag epitope sequence in
the preferable context for translation initiation was generated from
pFLAGHis/p53 (gift from R. Roeder). The fragment generated by PCR with
the primer set, NKFLAGFor which has the artificial NotI
site, the consensus translation initiation site, and p53Rev which has
an artificial BamHI site, was inserted into the pGFP NotI and BamHI vector, generating the
pNKFLAG/p53. Plasmid pNKFLAG was constructed to replace the p53 insert
with the multicloning sites as described above using EcoRI
and BamHI sites. The various kinds of HCV NS5B cDNA were
inserted into the EcoRI and BamHI sites of
pSG5UTPL, pNKFLAG, and pGFP.
Another plasmid series, pGEM3zf(+)/NS5BBg, pGEM3zf(+)/poly(U), and
pGEM3zf(+)/3'X were used for in vitro transcription to prepare the RNA templates. These were constructed by means of PCR with
synthesized oligonucleotide primers as described below. For
pGEM3zf(+)/NS5BBg, a set of primers, 5BBgFor and 5BBgRev, which have an
artificial BamHI and EcoRI site, respectively,
was used in the PCR with pGEM3zf(+)/HCV JK-1 as a template, which contains HCV JK-1 cDNA between the EcoRI and
BamHI sites. For pGEM3zf(+)/poly(U), a set of
oligonucleotides, poly(U)For which has an artificial EcoRI
site and poly(U)Rev which has the artificialBamHI and
BbsI sites, was annealed and subjected to PCR cloning,
generating a fragment containing the poly(U) stretch of HCV. For
pGEM3zf(+)/3'X, a set of oligonucleotides, 3'XFor which has the
artificial EcoRI and BbsI sites and 3'XRev which
has the artificial AflII and BamHI sites, was
annealed and subjected to PCR cloning, generating a fragment containing
3'X (41, 42). The DNA fragments with the artificial EcoRI
and BamHI sites were inserted into the pGEM3zf(+) (Promega) EcoRI and BamHI vector. All the
constructs were sequenced with Taq sequencing kits and a DNA
sequencer (374A, Applied Biosystems).
Expression and Purification of Bacterial Recombinant NS5B
Protein--
GST-fused HCV NS5Bt was expressed as described previously
(43-45). The plasmid pGENKS/NS5Bt was transformed into the E. coli strain BL21 pLysS(DE3), and the transformants were then
cultured in 10 ml of LB medium with 100 µg/ml ampicillin at 30 °C
overnight. Cultures were diluted into 1 liter of LB medium with 100 µg/ml ampicillin and incubated at 30 °C until the
A600 reached 0.6-0.7. These
cultures were then induced overnight with 0.4 mM
isopropyl-
-D-thiogalactopyranoside. The cells (from 1 liter) were harvested by centrifugation and washed once with
phosphate-buffered saline (PBS). The pellet was suspended in 32 ml of
PBS containing 1 mM dithiothreitol (DTT) and 1% Triton
X-100 (buffer A). The suspension was sonicated on ice until it was no
longer viscous and then centrifuged at 15,000 × g for
20 min. The supernatant was placed on ice (supernatant 1, S1), while
the pellet was suspended in 32 ml buffer A containing 1.0 M
NaCl, sonicated again, and the suspension was centrifuged. After the
supernatant was collected (supernatant 2, S2) and mixed with the S1
fraction, the NaCl concentration was adjusted to 0.33 M
using buffer A. This combined supernatant (supernatant 3, S3) was
passed through a DEAE-Sephacel column equilibrated with buffer A. The
flow-through fraction was mixed with 1 ml of glutathione-Sepharose 4B
beads (Amersham Pharmacia Biotech) equilibrated with buffer A, and the
protein was allowed to absorb to the beads for 1 h at 4 °C. The
beads were then washed thoroughly with buffer A and then with 50 mM Tris-HCl, pH 8.0, and 1 mM DTT. The GST-NS5B
was eluted with 4 ml of buffer B (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 10 mM glutathione, 10 mM DTT, and 0.1% Triton X-100) and then eluted with 4 ml
of buffer B containing 500 mM NaCl before being subjected
to column purification (27). After the NaCl concentration was adjusted
to 150 mM, the eluted solution was applied to a
heparin-Sepharose column (Amersham Pharmacia Biotech) equilibrated with
buffer B. The column was then washed with LG buffer (20 mM
Tris-HCl, pH 7.5, 1 mM EDTA, 5 mM DTT, 20%
glycerol, 0.5% Triton X-100), and the column was eluted with LG buffer
containing from 100 mM to 1 M NaCl. The elution
profile was analyzed by 10% SDS-PAGE, and the GST-NS5Bt was found to
elute broadly from 500 to 900 mM NaCl. These fractions were
collected and diluted with LG buffer to adjust the NaCl concentration
to 150 mM. The solution was then applied to a
poly(U)-Sepharose column (Amersham Pharmacia Biotech) equilibrated with
LG buffer containing 150 mM NaCl. After washing, the column was eluted with LG buffer containing from 200 mM to 1 M NaCl. GST-NS5Bt eluted between 500 and 700 mM
NaCl. The fractions were collected and dialyzed against LG buffer
containing 150 mM NaCl. For the non-fusion NS5Bt, the
protein-bound glutathione resin was washed extensively with thrombin
cleavage buffer (50 mM Tris-HCl, pH 7.5, 150 mM
NaCl, 2.5 mM CaCl2, and 1% Triton X-100). The
GST-fused NS5Bt was cleaved overnight in thrombin cleavage buffer
containing 50 units of thrombin (Amersham Pharmacia Biotech) to release
the NS5Bt protein from the GST-bound glutathione resin. The supernatant containing the NS5Bt protein was collected and dialyzed against LG
buffer containing 150 mM NaCl (45). Samples were collected at various stages in the purification, analyzed by SDS-PAGE, and assayed for polymerase activity. The protein concentration was measured
by the Bradford method or Coomassie staining with bovine serum albumin
as the standard.
Poly(A)-dependent UMP Incorporation Assay and
the Substrate Specificity of the Activity--
The incorporation of
[
-32P]UMP or [
-32P]dTTP was measured
as described previously (26, 27, 46). The standard reaction (10 µl)
was carried out in buffer containing 20 mM Tris-HCl, pH 7.5, 5 mM MgCl2, 1 mM DTT, 1 mM EDTA, 20 units of RNase inhibitor (Wako Chemicals Co.
Ltd.), 2 µCi of [
-32P]UTP (800 Ci/mmol, Amersham
Pharmacia Biotech), 10 µM UTP, 10 µg/ml poly(A), and
oligo(U)14 (1 µg/ml). After a 2 h incubation at
25 °C, the reaction solution was transferred to DE81 filters (Whatman) to stop the reaction. The filters were washed extensively with 0.5 M Na2HPO4, pH 7.0, rinsed
with 70% ethanol, and air-dried before the filter-bound radioactivity
was measured by a scintillation counter. For RdRP activity, both
oligo(U)14 and oligo(dT) were used as primers. For reverse
transcriptase activity, poly(A), oligo(dT), and
[
-32P]dTTP were used as the template, primer, and
substrate, respectively. For RNA polymerase activity, poly(dA),
oligo(U), and [
-32P]UTP were used, whereas for DNA
polymerase activity, poly(dA), oligo(dT), and
[
-32P]dTTP were used. Rifampicin and actinomycin D
(Sigma) were dissolved in ethanol and added to the reactions at the
indicated concentrations. In these experiments the reactions that
contained the same volume of ethanol were used as a standard.
Preparation of RNA Templates for Polymerase Assay--
The
plasmids, pGEM3zf(+)/5BBg, pGEM3zf(+)/poly(U), and pGEM3zf(+)/3'X, were
linearized by digestion with BamHI or EcoRI,
whereas pNKFLAG was linearized by digestion with BglII to
prepare the control RNA. In vitro transcription using these
templates was carried out with T7 RNA polymerase or SP6 RNA polymerase
according to the manufacturer's instructions (Promega). After
incubation, the DNA templates were digested with RNase-free DNase. The
RNA products were extracted with phenol/chloroform (1:1) and passed through a Sephadex G-50 column to remove free nucleotides before ethanol precipitation. The RNA concentrations were measured
spectrophotometrically, adjusted to 1 µg/µl with water, and then
stored at
20 °C. The quality of the RNA samples was confirmed by
electrophoresis in a MOPS-denaturing gel or urea-denaturing
polyacrylamide gel.
RNA-dependent RNA Polymerase
Assay--
RNA-dependent RNA polymerase assay was
performed in a total volume of 40 µl containing 20 mM
Tris-HCl, pH 7.5, 5 mM MgCl2, 1 mM
DTT, 1 mM EDTA, 20 units of RNase inhibitor, 50 µg/ml
actinomycin D, 5 µCi of [
-32P]UTP, and 0.5 mM each of the remaining NTPs (i.e. ATP, CTP,
and GTP) with 10 µg/ml RNA template (26). The concentration of the limiting nucleotide was adjusted to 10 µM. The reaction
mixtures were incubated at 30 °C for 2 h. After incubation, the
reaction was stopped by digestion with 50 µg of proteinase K
(Boehringer Mannheim) in proteinase K buffer (150 mM NaCl,
50 mM Tris-HCl, pH 7.5, and 0.5% SDS) for 30 min. The RNA
products were extracted with phenol/chloroform (1:1) before ethanol
precipitation. After heat denaturation (94 °C, 2 min) these products
were analyzed by 8 M urea, 8% PAGE. The gels were dried
and analyzed using a BAS 1000 BioImage analyzer system (Fuji).
Subcellular Localization of Transiently Expressed
NS5B--
Subcellular localization of NS5B in mammalian cells was
examined by a transient expression system using HepG2, HLE, and COS-1 cells. About 1 × 105 cells were plated on a slide
glass in a Quadriperm microscope slide culture well (Heraeus) 1 day
before transfection with the GFP-NS5B expression plasmid, pGFP/NS5B, or
the FLAG-tagged NS5B expression plasmid, pNKFLAG/NS5B. The cells were
rinsed and fixed with 1.5% paraformaldehyde in PBS for 30 min before
being post-fixed for 5 min in 100% cold methanol. These slides were
then air-dried at
25 °C and stored at
80 °C. GFP-fused
proteins were detected after counterstaining with 0.0005% Evans Blue
in PBS. Samples expressing FLAG-tagged proteins were blocked with 0.5%
bovine serum albumin in PBS and stained overnight with anti-FLAG M2
antibody (Scientific Imaging Systems Co. Ltd.) diluted in PBS
containing 0.5% bovine serum albumin (1:330). Immunostaining was
carried out according to the standard procedure, using absorbed rabbit anti-mouse IgG, biotinylated goat anti-rabbit IgG, and
streptavidin-fluorescein isothiocyanate (Amersham Pharmacia Biotech)
with counter staining by Evans Blue.
The processed slides were examined using a BX-50 fluorescence
microscope (Olympus) with NIBA and WIB filters, and the images were
visualized by digital printing (Pictrography 3000, Fuji). Similar
expression levels of the GFP-fused proteins in HLE cells were
immunologically detected by Western blotting using anti-NS5B IgG and
anti-GFP IgG (CLONTECH) (data not shown).
Preparation of Rabbit Antisera, Human HCV Patient Sera, and
Immunological Detection--
Antisera against NS5B were raised in
rabbits by subcutaneous inoculation with around 200 µg of purified
bacterial hexahistidine-tagged NS5B in complete Freund's adjuvant
(Sigma). The IgG fraction of the antisera was purified using a protein
A-Sepharose column according to the manufacturer's instruction
(Amersham Pharmacia Biotech). Both the antisera and purified IgG
fraction were used at a 1:3000 dilution for Western blotting as
described previously (38, 40).
The human serum used in this study was obtained from a Japanese chronic
HCV (genotype Ib) patient. The IgG fraction was purified as described
above. The antibody was used in Western blotting at a 1:200
dilution.
 |
RESULTS |
Expression and Purification of the Bacterial Recombinant NS5B
Protein--
Several different kinds of vectors including His-tagged
and GST fusion expression plasmids were examined to obtain the soluble recombinant full-size NS5B protein from E. coli
transformants (Fig. 1A). All
the NS5B proteins tested were expressed at a low level, and most were
recovered in the insoluble fractions as described previously (47). With
the prediction programs in Genome net servers2 PSORT and SOSUI, we
found that the C-terminal part of NS5B contains a highly hydrophobic
region (aa 2989-3005) which is predicted to be an anchoring domain
(Fig. 1B, shaded region). Therefore, the effect of the
C-terminal region was examined by constructing a GST fusion expression
plasmid, GST-NS5Bt, which lacked the C-terminal 21 amino acid residues
(aa 2989-3010, see "Experimental Procedures"). A 95-kDa protein
corresponding to the GST-fused NS5Bt was overexpressed in the E. coli transformants by
isopropyl-
-D-thiogalactopyranoside induction and then
recovered in the soluble fractions (Fig.
2A). The soluble protein was
bound to glutathione resin; however, most of the 95-kDa protein
remained on the resin after standard elution procedure (38, 40, 44).
Therefore, we modified the elution procedure to improve the yield of
the soluble GST-NS5Bt and finally succeeded in recovering about 1 mg of
the soluble recombinant protein from a 2-liter cell culture. Protein
was first eluted with glutathione in buffer B containing 150 mM NaCl and next with glutathione in buffer B containing
500 mM NaCl (see "Experimental Procedures"). Several
proteins of around 30 kDa in molecular mass, which were degradation
products containing the GST portion, were eluted with buffer B
containing 150 mM NaCl (data not shown), but most of the
95-kDa protein was eluted with buffer B containing 500 mM
NaCl. The sample recovered in this buffer contained trace amounts of
other proteins including multiple degradation products of GST-NS5Bt at
70-80 kDa and a faint band of 60 kDa in mass (Fig. 2A, lane
4). Therefore two additional affinity chromatography steps,
heparin- and poly(U)-Sepharoses, were introduced to purify further the
GST-NS5Bt (see "Experimental Procedures"). The 60-kDa band was
removed by the poly(U) column chromatography. The final preparation
contained a single 95-kDa band with no other bands detected by
Coomassie Brilliant Blue staining (Fig. 2A, lane
5). The 95-kDa band was identified as the recombinant NS5Bt
protein since it was specifically recognized with both rabbit
anti-NS5Bt IgG and anti-GST IgG on Western blotting (Fig. 2,
B and C). Also the band was immunologically
detected by serum from a human HCV patient but not by that from a
normal blood donor (data not shown). The GST-NS5Bt protein, which bound
to the glutathione resin, was treated with thrombin through the
artificial consensus sequence located at the junction of the GST
portion to obtain the non-fusion NS5Bt (Fig. 1A). New bands
of a doublet with a molecular mass of approximately 63 kDa were
recovered in the unbound fraction of thrombin cleavage (Fig. 2A,
lane 6), and these were specifically recognized by anti-NS5Bt IgG
but not by anti-GST IgG (Fig. 2, B and C, lane
6). The purified GST-fused and non-fused NS5Bt proteins were
dialyzed for 24 h against LG buffer containing 150 mM
NaCl and stored at
80 °C for further examination.

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Fig. 1.
Construction of HCV NS5B expression plasmids.
A, the GST expression vector pGENKS. All the HCV subgenomic
cDNA fragments were inserted into the SacI and the
SalI sites of pGENKS and expressed as GST-fused proteins in
E. coli. Underlined are the thrombin cleavage and kination
sites for the in vitro labeling with
[ -32P]ATP. B, upper part shows
the polyprotein structure of HCV, and the hydrophobic profile (Kyte and
Doolittle) of the NS5B protein is shown below. The
shaded region shows a putative anchoring domain predicted by
the SOSUI program. Expression constructs for the NS5B protein are shown
in the lower part of the figure. Numbers indicate
amino acid positions within the HCV polyprotein precursor. NS5B
contains the full-size protein. NS5Bt has a deletion from aa 2989 to
3010. NS5Bt-m1 has a GDD motif substituted to VDD. NS5Bt-m2 and -m3
have an alanine substitution in the clustered regions of basic residues
as described under "Experimental Procedures." NS5B-m4 has mutations
in the putative anchoring region as described under "Experimental
Procedures."
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Fig. 2.
Purification of the bacterial expressed
GST-NS5Bt protein. A, the GST-NS5Bt protein was expressed in
E. coli and purified as described under "Experimental
Procedures." Samples from the purification steps were separated by
10% SDS-PAGE and stained with Coomassie Brilliant Blue
(CBB). Lanes 1-6 are total cell lysate,
sonication supernatant, the solution after DEAE-Sephacel
chromatography, the eluate from the glutathione-Sepharose 4B column,
the eluate from poly(U)-Sepharose column, and non-fusion NS5Bt after
thrombin cleavage respectively. B, Western blot analysis
using anti-NS5B antibody. C, Western blot analysis using
anti-GST antibody. One-tenth of the protein amounts was transferred to
the nitrocellulose membrane and subjected to Western blotting.
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RNA Synthesis Activity of the Bacterial Recombinant NS5B
Proteins--
RNA synthesis activity of the samples from various
purification steps of the bacterial recombinant NS5Bt protein was
examined by the UMP incorporation assay, namely incorporation of
[
-32P]UMP using poly(A) and oligo(U)14 as
template and primer, respectively (see "Experimental Procedures").
UMP incorporation of purified GST-NS5Bt was detected, and the relative
activity exceeded over 10,000 times that of the starting material
(Table II). The incorporation was
detected in a dose-dependent manner, but no incorporation was observed in the presence of GST alone (Fig.
3C and Table
III). The incorporation was continued for
at least 4 h at 25 °C; however, at 37 °C the rate was low
and it continued for only 2 h when a plateau was reached (Fig.
3B, 25 and 37 °C). The
RNA synthesis activity of both cleaved and uncleaved forms of NS5Bt was
not inhibited by the presence of 20 µg/ml rifampicin or 50 µg/ml
actinomycin D, and it remained unaffected even at concentrations of up
to 200 and 500 µg/ml, respectively (Table III and data not
shown).

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Fig. 3.
RdRP activity of the purified GST-NS5Bt and
thrombin cleaved NS5Bt in the UMP incorporation assay. A,
substrate specificity of the protein was measured by the assays using
poly(A) or poly(dA) as template and oligo(U)14 or oligo(dT)
primer. Incorporation of [ -32P]UMP or
[ -32P]dTTP was measured as described under
"Experimental Procedures." B, time course of the
reaction. The reactions were carried out at two temperatures (25 and
37 °C) using 90 ng of GST-NS5Bt and at 30 °C using 0.5 µg of
thrombin-cleaved NS5Bt. C, dose effect of the GST-NS5Bt.
Different amounts of GST-NS5Bt were added to the reaction.
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No incorporation was observed without either primer or template,
indicating that there was no terminal transferase activity in the NS5Bt
protein fractions (Table III). Optimal incorporation was observed over
a broad range of pH values from pH 7.0 to 8.5; however, the
incorporation was inhibited at low or high pH values such as pH 6.0 or
9.0 (Fig. 4A). The
incorporation was most efficient at 30 °C and was reduced at
temperatures lower or higher than 30 °C (Fig. 4B). It was
inhibited when the KCl concentration was more than 100 mM
(Fig. 4C) and was strictly dependent on the Mg2+
ion. The optimal concentration was between 2.5 and 5 mM
(Fig. 4D). The divalent Zn2+ ion could not
replace this requirement (data not shown). Ionic detergents such as
0.01% Sarkosyl or SDS completely inhibited the activity, whereas 0.1%
of non-ionic detergents such as Triton X-100, Nonidet P-40, Tween 20, and CHAPS had little effect (Table IV).

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Fig. 4.
Properties of the RdRP activity of the
purified GST-NS5Bt and thrombin-cleaved NS5Bt. The RdRP activities
of the purified GST-NS5B and thrombin-cleaved NS5Bt were measured by
the UMP incorporation assay under different reaction conditions. The
reactions were carried out using 90 ng of GST-NS5Bt or 0.5 µg of
thrombin-cleaved NS5Bt. The incubation was basically carried out for
2 h at 25 °C. A, the reaction mixtures at different
pH values were prepared by sodium phosphate buffer. Percentage
incorporations of the standard reaction (Tris-HCl buffer, under
"Experimental Procedures") are shown. One hundred percent of
GST-NS5Bt and thrombin-cleaved NS5Bt was 47,284 and 20,841 cpm,
respectively. B, the reaction was carried out at the
indicated temperatures. C, KCl was added to the reaction
mixture at the indicated concentrations. D, the UMP
incorporation assay was carried out in the presence of the different
concentrations of magnesium ion. The reactions were performed with 90 ng of GST-NS5Bt or 0.5 µg of thrombin-cleaved NS5Bt.
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Table IV
Effect of detergent on the activity
100% activity was 25,755 cpm in the standard reaction (no detergent).
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The properties of the GST-NS5Bt polymerase activity were characterized
using different templates and primers. Poly(A)-dependent oligo(U)-primed UMP incorporation in the assay was much higher than
that primed by oligo(dT) (about 2 times), and the purified GST-NS5Bt
retained no reverse transcriptase, RNA polymerase, or DNA polymerase
activities in these reaction conditions (Fig. 3A). Since the
UMP incorporation assay utilizes an artificial template and primer, we
next examined whether GST-NS5Bt could utilize the HCV RNA as template
and primer as reported previously (26, 27) (Fig.
5). For this purpose, the HCV 3'-UTR,
which should serve as replication initiation site, was divided into
three regions as follows: the 5BBg contained the region from the
BglII site in NS5B to 3'-UTR before the poly(U) stretch, the
poly(U) contained the poly(U) stretch, and the 3'X which was recently
discovered and may have an important role in the viral replication (41, 42). These regions of the HCV subgenome RNA were synthesized by
in vitro transcription and examined in the RNA synthesis
assay (Fig. 5A and see "Experimental Procedures").
32P-Labeled RNA bands of the same or a smaller size than
the input RNAs were detected (Fig. 5B). The RNA synthesis
required all four ribonucleotides as substrate, and no incorporation
was detected when UTP or CTP alone was used as substrate (data not
shown). Both HCV RNAs and control RNA could serve as template and
primer (Fig. 5, lane 4). The in vitro RNA
synthesis with 3'X of the HCV 3'-UTR was rather low compared with those
with the other RNA regions (Fig. 5, compare lanes 1 and
4 to 3). The poly(U) stretch of HCV 3'-UTR could
not serve as template and primer by itself (Fig. 5, lane 2).
These results suggest that RNA was utilized as template and primer and
that GST-NS5Bt had no terminal transferase activity as demonstrated
with the UMP incorporation assay.

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|
Fig. 5.
RNA synthesis activity using in
vitro transcribed RNA. A, construction of RNA
templates. 5BBg contained the region from the BglII site in
NS5B to 3'-UTR, poly(U) contained the poly(U) stretch of HCV, and 3'X
contained 3'X of HCV. Control RNA were prepared by
BglII-digested pNKFLAG plasmid. The RNA sizes are described
in the figure. B, RNAs prepared by in vitro
transcription were used as template and primer (see "Experimental
Procedures"), and the reactions were carried out for 2 h at
30 °C. The RNA products were purified by organic extraction followed
by ethanol precipitation and finally separated on 8 M urea,
8% PAGE.
|
|
The GST-NS5Bt proteins purified from transformants of E. coli JM109 and BL21 pLysS had similar properties in the UMP
incorporation assay (data not shown). The glutathione-eluted fraction
purified from BL21 pLysS contained no T7 RNA polymerase since it could not be detected immunologically with an anti-T7 RNA polymerase antibody.3
The possibility that the GST moiety may affect the properties of the
RdRP activity of NS5Bt was addressed by the purified thrombin-cleaved
non-fusion NS5Bt (see "Experimental Procedures"). The activity in
the standard UMP incorporation assay was lower than that of GST-NS5Bt
which was purified using heparin and poly(U) columns (about 25% of
GST-NS5Bt, Table II). No incorporation was observed without either
primer or template, indicating the absence of terminal transferase
activity in the NS5Bt protein fractions (Table III). It was resistant
to rifampicin and actinomycin D which is the same as GST-NS5Bt (data
not shown). The properties of the cleaved protein were almost the same
as those of GST-NS5Bt with regard to optimal temperature, KCl
sensitivity, and optimal magnesium concentration (Fig. 4) except the
RdRP activity of the non-fused NS5Bt was higher in the sodium phosphate
buffer (Fig. 4A compared with Fig. 3B or Fig. 4,
B-D), and its optimal pH was higher that of the
GST-fused form (Fig. 4A). Thrombin-cleaved non-fusion NS5Bt could also utilize RNA as template and primer (data not shown).
These results show that HCV NS5Bt in the GST-fused and non-fused forms
from E. coli retain their RdRP activity, which requires a
template and primer in the UMP incorporation assay, but they can
utilize the HCV RNAs and control RNA as template and primer and have
neither terminal transferase activity nor any other kind of
polymerizing activity.
RdRP Activity of Mutant NS5Bt--
The results of the RNA
synthesis activity detected in the two assays indicate that the NS5Bt
protein retains RdRP activity. To prove this, we examined the
importance of the GDD motif of NS5B, which is highly conserved among
viral RdRPs (25). The mutated GST-NS5Bt protein, NS5Bt-m1, in which the
GDD sequence was substituted to VDD, was purified from the E. coli transformed cells (48). The GST-NS5B-m1 protein did not
exhibit any RdRP activity in the UMP incorporation assay (Table
V). This result shows that the RNA
synthesis activity of the GST-NS5B protein absolutely requires the
presence of the GDD motif, indicating that the purified bacterial
recombinant NS5B protein exhibits the RdRP activity of HCV NS5B.
We also evaluated the other mutant proteins, NS5Bt-m2 and -m3, with
substitutions in clustered basic residues. The GST-NS5Bt-m2 and -m3,
which were purified by the same method as for GST-NS5Bt, did not
exhibit any RNA synthesis activity or any inhibitory effect on the RNA
synthesis activity of the wild-type GST-NS5Bt protein in the same
reaction mixture (Table V and data not shown).
The C-terminal Part of NS5B Encodes a Putative Anchoring Domain
That Affects the Subcellular Localization of NS5B--
Since the
C-terminal 21 amino acid residues is dispensable for the RdRP activity
(Fig. 2 and Table I), we addressed the role of the C-terminal region on
the subcellular localization of NS5B in mammalian cells (Fig.
6). Mammalian expression plasmids of NS5B
in the green fluorescent protein (GFP)-fused forms were introduced by
transient expression to a hepatoma cell line, HLE, and the subcellular
localization of the GFP was examined by fluorescence microscopy (see
"Experimental Procedures"). The GFP-NS5B protein was predominantly
distributed in perinuclear regions and diffusely in the cytoplasm,
although the GFP alone was localized diffusely in both the cytoplasmic
and nuclear regions (Fig. 6, A and D). Localization of the GFP signal was dramatically affected by truncation of the C-terminal region, and the GFP-NS5Bt protein was predominantly concentrated in the nuclei (Fig. 6B). The fluorescent
nuclear signal of the GFP-NS5Bt was detected as several clusters that were huge spherical and irregular forms. The GFP-NS5B-m4, harboring two
amino acid substitutions in the anchoring domain, which was constructed
to cancel the likelihood of an anchoring domain in the prediction
programs, was localized in the nuclei as multiple clusters and
diffusely in cytoplasm (Fig. 6C). A similar role for the
anchoring domain on the subcellular localization was observed with
COS-1- and HepG2-transfected cells which expressed the GFP-fused or
FLAG-tagged NS5B proteins (data not shown). These results strongly suggest that the C-terminal region of NS5B, especially the putative anchoring domain, has a role in retaining the protein in the
cytoplasm.

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|
Fig. 6.
Effect of the putative anchoring domain on
the subcellular localization of NS5B. The GFP expression plasmids
of the full-size and mutated NS5B were introduced into a hepatoma cell
line, HLE, by transient transfection. The cells were processed and
examined with a fluorescent microscope as described under
"Experimental Procedures." Expressed proteins were GFP-NS5B
(A), GFP-NS5Bt (B), GFP-NS5B-m4 (C),
and GFP alone (D), respectively.
|
|
 |
DISCUSSION |
The persistent property of the HCV infection has been explained by
its ability to escape from the host immune surveillance through
hypermutability of the exposed regions in the envelope protein E2 (49,
50). Persistent infection of HCV causes chronic inflammation in liver,
liver cirrhosis, and eventually increases the risk of hepatocellular
carcinoma (28). HCV replication or the RNA-dependent RNA
polymerase, a central catalytic enzyme of replication, is one of the
possible targets to prevent hepatocarcinogenesis by blocking HCV
infection.
We demonstrated here the expression and purification of soluble
bacterial recombinant HCV NS5B in a GST-fused form. The GST-NS5Bt protein exhibited RNA synthesis activity in the UMP incorporation assay, which is completely dependent upon the template and primer and
absolutely requires the GDD motif, a conserved sequence among viral
RdRPs (25). The thrombin-digested non-fused form of the wild NS5Bt
exhibited the similar properties of RdRP activity except for its
behavior in sodium phosphate buffer. The purified mutated GST-NS5Bt
proteins (m1 to m3) had no RdRP activity. These results indicate that
the bacterial recombinant NS5B protein retains RdRP activity. The
bacterial NS5B reported here and the baculovirus-expressed NS5B seem to
share similar properties in their RdRP activity, including the
requirements for template and primer, Mg2+ dependence, and
optimal conditions for their reactions (26, 27). However, the bacterial
recombinant NS5B did not exhibit terminal transferase activity which
has been demonstrated in the insect-expressed NS5B. The GST moiety
located at the N terminus is not the reason for the absence of terminal
transferase activity because the thrombin-cleavaged NS5B also had no
terminal transferase activity. At present, the reason for this
discrepancy remains unclear. However, recently, another group has
suggested that terminal transferase activity detected may be due to
contamination of host protein (51).
The truncation of the C-terminal part of NS5B was first designed to
prepare the soluble GST-fused form of NS5B in E. coli. However, later we found that this region also affected the subcellular localization of NS5B in mammalian cells. The C-terminal part includes two regions, an anchoring domain spanning aa 2989-3003 and a
C-terminal tail (aa 3004-3010), the former is highly conserved among
the HCV isolates in the data bases. This study is the first to propose the presence of an anchoring domain at the C-terminal region of NS5B.
NS5Bt in the GFP-fused form was exclusively distributed in the nuclei
which is in contrast to the perinuclear localization of the full-length
NS5B protein (52, 53). NS5B-m4, which contained mutations in the
anchoring domain, was localized in the nuclei and cytoplasm. Thus it is
plausible that the anchoring domain at the C-terminal has a role in
retaining the protein in the cytoplasm. RNA replication of many plus
strand RNA viruses requires viral membrane proteins which may
facilitate assembly of the replication complex anchored to cell
membranes (5, 29, 30), and the C-terminal region of NS5B may have a
similar role in HCV replication.
NS5B has no typical signal for nuclear targeting, thus the nuclear
localization of NS5Bt may be through its ability to interact with
nuclear components. As GST-NS5Bt exhibits RNA-binding ability as
detected by UV cross-linking or electrophoretic mobility shift assays,4 the RNA binding may
be contributing to the nuclear localization of the NS5Bt protein.
Interestingly, the several huge clustered forms of the GFP-NS5Bt seem
to be unique and distributed in nucleolar regions (54, 55). The role of
the C-terminal tail flanking the anchoring domain still remains to be
addressed. It should be stressed that the C-terminal region is
dispensable for the RdRP activity which was detected by two assays and
also for the interaction of NS5A and NS5B both in vitro and
in vivo.4
The method applied here is sensitive enough to detect the RdRP
catalytic activity of HCV NS5B, but it could not detect specific initiation of HCV replication since the RdRP activities detected in
these systems neither require the 3'X sequence located at the end of
the genome nor a specific primer as reported previously (26, 27, 51).
Plus strand RNA virus replication requires a precise initiation site
and a specific primer, either protein or pre-existing RNA (32, 56-58).
Therefore, the RdRP activity associated with the NS5B proteins simply
reflects the catalytic activity of the central protein of replication.
Specific and efficient viral replication could be achieved by the help
of the other non-structural proteins such as NS3, NS4A, NS4B, and NS5A
which may form a dynamic and higher ordered protein complex(es),
together with viral RNA and probably host factors or subcellular
compartments (33, 59-62). In this context, it is noteworthy that we
could observe only a weak stimulating effect of the bacterial
recombinant HCV NS5A on the RdRP activity of NS5B in the in
vitro assay, although modulation of HCV replication through NS5A
has been suggested and the physical interaction of NS5A and NS5B is
detectable in vitro and in vivo.4
The bacterial expression and purification methods we described here are
simple and can efficiently purify recombinant NS5B. They will not only
provide a useful tool to evaluate the effects of putative metabolic
competitors on HCV RdRP activity, but will also facilitate mutation
analysis of NS5B in future.
 |
ACKNOWLEDGEMENTS |
We are grateful to M. K. Yi, M. Honda,
and Y. Nakamoto for providing HCV JK1 plasmid cDNA; and to Y. Lin,
D. Dorjsuren, and N. Hayashi for encouraging discussion. We are
grateful to B. A. Heinz for critical comments and communicating
unpublished results. We thank M. Takamatsu, C. Matsushima, F. Momoshima, M. Yasukawa, and K. Kuwabara for their technical
assistance.
 |
FOOTNOTES |
*
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.
¶
To whom correspondence should be addressed. Tel.:
81-76-265-2731; Fax: 81-76-234-4501; E-mail:
semuraka{at}kenroku.ipc.kanazawa-u.ac.jp.
1
The abbreviations used are: HCV, hepatitis C
virus; RdRP, RNA-dependent RNA polymerase; DTT,
dithiothreitol; PBS, phosphate-buffered saline; aa, amino acid(s); GFP,
green fluorescent protein; PCR, polymerase chain reaction; PAGE,
polyacrylamide gel electrophoresis; GST, glutathione
S-transferase; UTR, untranslated region; MOPS, 4-morpholinepropanesulfonic acid; CHAPS,
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.
2
The on-line address for PSORT and SOSUI is as
follows: http://www.genome.ad.jp.
3
B. A. Heinz, personal communication.
4
Y. Shirota, manuscript in preparation.
 |
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