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INTRODUCTION |
The hepatitis C virus
(HCV)1 is a major cause of
chronic hepatitis around the world (1-3). Chronic infection with HCV
results in liver cirrhosis and often hepatocellular carcinoma (4, 5). HCV belongs to the Flaviviridae family and has a
positive-sense single-stranded RNA genome. The HCV RNA genome is ~9.5
kb in length and consists of a long open reading frame encoding a
polyprotein of ~3,000 amino acid residues and two highly conserved
untranslated regions flanking the 5' and 3' ends of the genome. The
polyprotein is cleaved by host and viral proteases into three
structural proteins and seven nonstructural proteins:
NH2-C-E1-E2-P7-NS2-NS3-NS4A-NS4B-NS5A-NS5B-COOH (6, 7). The
5'-untranslated region of 341 nucleotides contains an internal ribosome
entry site, which consists of four stem-loop structures followed by an
initiation codon for the polypeptide. The 3'-untranslated region of
200-300 nucleotides includes a short variable sequence, a poly(U)
region, a polypyrimidine (U/C) tract, and a highly conserved
X region, and is believed to be important for HCV RNA
replication as reported previously (8).
Studies on HCV replication have been a focus of attention because
inhibition of HCV replication not only may have therapeutic significance for chronic hepatitis but also could reduce the incidence of or even prevent hepatocellular carcinoma. Little is known about the
mechanism involved, however, because of a lack of efficient cell
culture systems (3, 6), although some aspects of the replication
process have been recently elucidated with HCV RNA replicon systems (9,
10). HCV NS5B, located at the most C terminus of the polyprotein, is an
RNA-dependent RNA polymerase (RdRP), the central catalytic
enzyme of HCV replication, as was demonstrated with the recombinant
forms of NS5B expressed and purified from insect cells and
Escherichia coli (6, 11-14). Crystal models of NS5B clearly
confirm that it belongs to the large family of nucleic
acid-dependent nucleic acid polymerases sharing the fingers
and thumb subdomain structure with conserved motifs (15-17). NS5B also
has several unique properties including two loops connecting fingers
and a thick thumb conserved only among HCV isolates.
We previously reported the expression and purification of a soluble
form of NS5B truncated at the C terminus, NS5Bt, which retains RdRP
activity in vitro. A 21-amino acid sequence was deduced to
be the membrane-anchoring domain, which is dispensable for RdRP
activity, but important for perinuclear localization of the full-size
NS5B (12, 18). The subcellular localization of NS5Bt was exclusively
nuclear and distinct from the perinuclear localization of NS5B in the
GFP-fused form transiently overexpressed in mammalian cell lines (14).
The membrane anchoring of NS5B might be important for HCV replication
because most of the HCV NS proteins seem to be involved in the
replication process by forming a dynamic replication complex attached
to membranes. All NS proteins except NS3 have the intrinsic ability to
interact with membranes, and even NS3 is recruited to membrane through
the formation of a complex with NS4A, a cofactor of NS3 protease. In
addition, HCV replication requires direct protein-protein interactions
among NS proteins in a temporal order. HCV RNA and NS proteins have
been reported to interact with many host factors, and some may modulate
HCV replication. Recently, we reported that the RdRP activity of NS5B is regulated by homomeric oligomerization of NS5B and heteromeric interaction with NS5A (19, 20). There may be host protein(s), which
modulate HCV replication by interacting with RdRP, the catalytic enzyme
of HCV replication (21).
The exclusive nuclear localization of NS5Bt, an artificial truncated
form missing the membrane-anchoring domain, may reflect a new property
of NS5Bt to interact with some nuclear component(s), which may affect
NS5B function. Furthermore, the nuclear clusters of NS5Bt seemed to be
similar to the deformed nucleoli described previously (22). Therefore,
we examined whether nuclear NS5Bt is colocalized with nucleoli and the
factor(s) involved in the interaction. Here we report that a nucleolar
protein, nucleolin, can directly bind to NS5B in vivo and
in vitro, even when the full-length NS5B is localized to
perinuclear membranes.
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EXPERIMENTAL PROCEDURES |
Construction of Plasmids--
A plasmid derived from pGENK1 (23,
24), pGENKS, was used to express the recombinant HCV NS5B in E. coli, which contains multiple cloning sites (EcoRI and
BamHI) downstream of the sequences encoding GST protein (14,
25). pYFLAG plasmid for expression of FLAG-tagged bacterial recombinant
proteins was derived from pFLAGHis (a gift from R. Roeder) by replacing
the NdeI-HindIII fragment with an insert
containing a multiple cloning site to generate the EcoRI and
BamHI digestion sites (25, 26). The bacterial His-tagged
expression vector, pYUTHis, was derived from pLHis (27) and harbors
EcoRI and BamHI digestion sites.
All of the mammalian expression vectors were derived from pSG5UTPL
(27). The pNKFLAG vector, which was constructed by replacement of the
NotI-BamHI site of pSG5UTPL with a fragment
composed of an artificial initiation codon and sequences encoding FLAG
epitope and multiple cloning sites (EcoRI and
BamHI), was used to express N-terminally FLAG-tagged
proteins (25). The pNKGST vector, a GST-fused protein expression
vector, was constructed by replacement of the
NotI-BamHI site with a fragment encoding GST
protein, thrombin digestion sites, and multiple cloning sites
(EcoRI and BamHI) derived from pGENKS (25). The
pGFP vector was prepared by PCR using phGFPS65T
(Clontech Co. Ltd.) as a template with a set of primers: GFPNotFor, which has the artificial
EcoRV/NotI site, and GFPEcoRev, which has the
artificial EcoRI site. The DNA fragment with artificial
EcoRV and BamHI sites was inserted into the
pSG5UTPL blunt and BamHI vector, in which the
EcoRI site was blunted using the Klenow DNA fragment before
digestion by BamHI. The resulting plasmid, which expresses a
GFP-fused protein in mammalian cells under the control of the SV 40 promoter, pGFP, was used for construction of GFP-fused NS5B protein.
HCV JK-I cDNA (28) harboring NS5B was subcloned by PCR using the
sets of primers NS5BEFor and NS5BBaRev, which have an artificial EcoRI and BamHI sites, respectively (14, 25). The
truncated mutants and substitution mutants of NS5B were constructed as
reported (25). Nucleolin constructs were generated from a human
nucleolin cDNA clone (Ref. 29; generously provided by Sheng-Chung
Lee), which we subcloned into the EcoRI-BamHI
site of pNKFLAG to create pNKFLAG-nucleolin. The truncated mutants of
nucleolin were subcloned by PCR with primers reported previously
(30-32). The sequences of all of the constructions were confirmed by
the dideoxy sequence method.
Expression and Purification of Bacterial Recombinant NS5B and
Nucleolin Proteins--
GST-fused HCV NS5Bt proteins was expressed and
purified as described previously (14). Briefly, the wild-type
pGENKS-NS5Bt was transformed into E. coli strain BL21 pLysS
(DE3) by treatment with 0.4 mM
isopropyl-
-D-thiogalactopyranoside (IPTG) at 30 °C for 8 h. The cells were harvested by centrifugation and suspended in PBST buffer A (phosphate-buffered saline (
), 1% Triton-X, and 1 mM dithiothreitol (DTT)). After centrifugation of the
sonicated lysates, the supernatants were passed through DEAE-Sepharose
and the GST-fused proteins were recovered with glutathione-Sepharose 4B
beads (GST resin) (Amersham Biosciences). The resins were washed, and
the GST-fused proteins were then eluted with glutathione. The eluted
solution was dialyzed against buffer containing 100 mM
Tris-HCl, pH 8.0, 150 mM NaCl, and 1 mM
DTT.
It is impossible to express full-length nucleolin in E. coli
(29). His-tagged nucleolin and FLAG-tagged nucleolin were expressed in
BL21 pLysS (DE3) as reported previously with some modification (32).
Briefly, E. coli strain BL21 pLysS (DE3) transformed by pYUTHis-nucleolin was grown to an A600 of 0.5 at
37 °C, and protein expression was induced by 1 mM IPTG
at 37 °C for 4 h. The cells were harvested by centrifugation
and resuspended in buffer B (50 mM sodium phosphate, pH
8.0, 300 mM NaCl, 1 mM PMSF, 10 mM
leupeptin and aprotinin, and 1 mM DTT) with DNase I and
lysed by sonication. After centrifugation (30 min at 10,000 × g), the supernatant was subjected to affinity binding to
Ni2+-nitrilotriacetic acid-Sepharose (Qiagen). The resin
was washed several times with buffer B and buffer C (50 mM
sodium phosphate, pH 6.0, 300 mM NaCl, 10% glycerol, 1 mM PMSF, 10 mM leupeptin and aprotinin, and 1 mM DTT). The bound protein was eluted with buffer C
containing 0.5 M imidazole. The eluted solution was
dialyzed against buffer D (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10% glycerol, 1 mM DTT, and 0.1%
Triton X-100). Next, E. coli strain BL21 pLysS (DE3)
transformed by pYFLAG-nucleolin was grown to an
A600 of 0.5 at 37 °C, and protein expression
was induced by 1 mM IPTG at 37 °C for 4 h. The
cells were harvested by centrifugation and resuspended in lysis buffer
(50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% Nonidet P-40, 1 mM PMSF, 10 mM leupeptin and aprotinin, and 1 mM DTT). Incubation of the sonication supernatant with
anti-FLAG M2 resin (Kodak Scientific Imaging System) was followed by
extensive washing with lysis buffer and elution with lysis buffer
containing FLAG peptide (0.1 mg/ml). The eluted solution was dialyzed
against buffer C.
Cell Lines and DNA Transfection--
COS1 cells (a monkey kidney
cell line) and HLE cells (a human hepatoma cell line) were cultured at
37 °C in Dulbecco's modified Eagle's medium containing 10% fetal
calf serum plus 100 units of penicillin and 100 µg/ml streptomycin.
DNA transfection was performed with the calcium phosphate method as
reported (14).
Subcellular Localization of Transiently Expressed
NS5B--
Subcellular localization of NS5B in mammalian cells was
examined with a transient expression system using HLE cells.
Approximately 1 × 105 cells were plated on a slide
glass in a Quadriperm microscope slide culture well (Heraeus Co. Ltd.)
1 day before transfection with the GFP-fused NS5B expression plasmid.
The cells were transfected by the calcium phosphate method. The
transfected cells were rinsed with PBS (
) and fixed with 1.5%
paraformaldehyde in PBS (
) for 30 min. before post-fixation for 5 min
in 100% cold methanol. These slides were air-dried at
25 °C and
stored at
80 °C. GFP-fused proteins were detected after
counterstaining with 0.0005% Evans-Blue in PBS (
). For
immunostaining, the slides were stained with anti-mouse nucleolin
antibody (1:300) (kindly provided by Y. Hirose, Cancer Research
Institute, Kanazawa University) and amplified by horseradish peroxidase-conjugated anti-mouse IgG antibody and detected with streptavidin-conjugated Texas-Red. The processed slides were examined using a confocal laser microscope (LSM 510, Carl Zeiss Co. Ltd.), and
the images were visualized by digital printing (Pictography 4000, Fuji
Co. Ltd.).
Preparation of Cell Extracts, Co-precipitation with GST Resin,
and Western Blot Analysis--
Transient transfections of COS1 cells
were carried out as described previously (26, 33). The transfected
cells were harvested and washed with PBS (
) and sonicated in lysis
buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 10% glycerol, 1 mM PMSF, 10 mM leupeptin and aprotinin, and 1 mM DTT).
Total cell lysate was diluted 3-fold with lysis buffer, mixed with 10 µl of glutathione-Sepharose 4B beads, and then incubated for 4 h
on a rotator in a cold room. After an extensive wash with lysis buffer,
the bound proteins were eluted, fractionated by SDS-PAGE, transferred
onto nitrocellulose membranes, and subjected to Western blot analysis
with anti-FLAG M2 antibody. The proteins were visualized by enhanced
chemiluminescence according to the instructions from the manufacturer
(Amersham Biosciences). The nitrocellulose membranes used for Western
blot analysis with anti-FLAG M2 antibody were reproved with anti-GST monoclonal antibody (Zymed Laboratories) as recommended by the manufacturer (Amersham Biosciences).
GST Pull-down Assay in Vitro--
Approximately 1 µg of GST or
GST-NS5Bt bacterial recombinant protein immobilized on 10 µl of
glutathione-Sepharose 4B resin was pre-blocked with 1% bovine serum
albumin and then incubated with 0.2 µg of FLAG-tagged nucleolin in
lysis buffer for 4 h on a rotator in a cold room. After an
extensive wash with lysis buffer, the bound proteins were fractionated
by SDS-PAGE and subjected to Western blot analysis with anti-FLAG M2 antibody.
Poly(A)-dependent UMP Incorporation Assay--
The
RdRP activity of GST-NS5Bt was examined by the UMP incorporation assay
reported previously (14, 25). The incorporation of
[
-32P]UMP was measured using poly(A) and
oligo(U)14 as template and primer, respectively. 10 nM GST-NS5Bt was incubated at 25 °C for 2 h in
20-µl reaction mixtures containing 20 mM Tris-HCl, pH
7.5, 5 mM MgCl2, 1 mM DTT, 1 mM EDTA, 40 units of RNase inhibitor, 4 µCi of
[
-32P]UTP (800 Ci/mmol), cold 10 µM UTP,
10 µg/ml poly(A), and 1 µg/ml oligo(U)14. The reaction
was stopped by transferring the reaction solution to DE81 filters
(Whatman), which were then washed extensively with 0.5 M
Na2HPO4, pH 7.0, and briefly rinsed with 70%
ethanol. The filter-bound radioactivity was measured by a scintillation counter. To examine the effect of nucleolin on NS5B RdRP activity, 5, 10, 50, 100, and 250 nM of His-tagged nucleolin was added
before preincubation.
 |
RESULTS |
Nucleolar Localization of NS5B Missing the Membrane-anchoring
Domain, NS5Bt, and Perinuclear Colocalization of the Full-length NS5B
and Nucleolin--
We examined whether the nuclear clusters of NS5B
lacking the C-terminal 21 amino acid residues, NS5Bt, are colocalized
with nucleoli. GFP-fused forms of NS5B and NS5Bt were transiently
overexpressed in mammalian cells (HLE) and then analyzed with a
confocal microscope after immunological staining with anti-nucleolin
antibody. We applied nucleolin as a representative nucleolar marker
(Fig. 1). As reported previously, the
subcellular localization of NS5Bt was exclusively nuclear with nuclear
body-like clusters; however, the full-length NS5B was abundant in
perinuclear regions with granular spots (Fig. 1, A and
B) (14, 34). The GFP signals in the nucleus completely
colocalized with the signals detected by anti-nucleolin antibody,
clearly indicating that the nuclear clusters of NS5Bt are nucleoli
(Fig. 1B). Interestingly, NS5B and nucleolin did not
colocalize in the nucleus, but strong merged signals were observed in
perinuclear regions (Fig. 1A). The results demonstrate that
GFP-NS5B was colocalized with endogenous nucleolin in perinuclear
regions. In the control, nucleolin was exclusively distributed in
nucleoli when GFP alone was overexpressed (Fig. 1C), and the
GFP signals were spread diffusely in both the nucleus and cytoplasm.
The GFP signals and the anti-nucleolin signals were merged in neither
the nucleus nor the cytoplasm. Nucleolar accumulation was observed in
the cells expressing NS5B having substitution mutations in the
membrane-anchoring domain to disrupt the hydrophobic properties of the
domain (data not shown). Therefore, the membrane-anchoring domain is
critical to the perinuclear localization of NS5B, but the
colocalization of nucleolin with NS5B or NS5Bt was not affected by the
domain. These results suggest that NS5B retains the ability to interact
directly or indirectly with nucleolin. The strong perinuclear
distribution of endogenous nucleolin in the NS5B-expressing cells may
imply some role for nucleolin in NS5B function.

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Fig. 1.
Colocalization of HCV NS5B protein and
endogenous nucleolin. A, HLE cells were
transfected with a plasmid expressing GFP-NS5B. Two days after
transfection, the cells were fixed. Immunofluorescence staining was
performed using the anti-mouse nucleolin as primary antibody. GFP-NS5B
and endogenous nucleolin were visualized by confocal laser scanning
microscopy. Green fluorescence indicates GFP or GFP-fused
protein, whereas red (Texas Red) indicates endogenous
nucleolin. Yellow (merged) indicates colocalization of
GFP-NS5B protein and endogenous nucleolin in perinuclear regions.
B, HLE cells were transfected with a plasmid expressing
GFP-NS5Bt. The transfected cells were subjected to the same processes
as in A. GFP-NS5Bt and endogenous nucleolin were colocalized
in nucleoli. C, HLE cells were transfected with a plasmid
expressing GFP alone. The transfected cells were subjected to the same
processes as in A.
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HCV NS5B Protein Interacts with Endogenous and Ecotropically
Expressed Nucleolin--
To examine whether NS5B and NS5Bt can
directly interact with nucleolin in vivo, NS5B and NS5Bt in
their GST-fused forms were transiently expressed in COS1 cells and the
cell lysates were subjected to GST pull-down assay using
glutathione-Sepharose 4B resin. The bound proteins were fractionated by
SDS-PAGE and immunologically detected by Western blotting with
anti-nucleolin antibody. As shown in Fig.
2A, the endogenous nucleolin
bound to GST-NS5B and GST-NS5Bt but not to GST alone. Similarly, the
specific binding of ecotropically expressed nucleolin and NS5B or NS5Bt
was observed when FLAG-tagged nucleolin was transiently coexpressed
with GST-NS5B or GST-NS5Bt in COS1 cells (Fig. 2B). The
result was confirmed using a different combination, GST-nucleolin and
FLAG-NS5B or FLAG-NS5Bt (Fig. 2C). To confirm that the
interaction between nucleolin and NS5B in vivo does not
require RNA, the lysate of COS1 cells transiently co-transfected with
FLAG-nucleolin and GST-NS5B was treated with RNase A. The RNase
treatment had no effect on interaction between nucleolin and NS5B (data
not shown). These results clearly demonstrate that NS5B and NS5Bt
specifically bind to both endogenous and ecotropically expressed
nucleolin in vivo. Apparently the binding between NS5B and
nucleolin is weaker than that between NS5Bt and nucleolin. The result
was much exaggerated by the less efficient recovery of nucleolin with
NS5B, which is mostly insoluble and difficult to subject to pull-down analyses.

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Fig. 2.
Interaction of HCV NS5B with nucleolin.
A, COS1 cells were transiently transfected with a plasmid
expressing GST-NS5B (lane 1), GST-NS5Bt
(lane 2), or GST alone (lane
3). Total lysate was separated by 8% SDS-PAGE and subjected
to Western blotting with anti-mouse nucleolin antibody (left
side, input). Proteins bound to glutathione-Sepharose 4B
were washed with washing buffer, fractionated by 8% SDS-PAGE, and
detected by Western blotting with anti-mouse nucleolin antibody
(right side, bound fraction). B, GST
pull-down assay using plasmids expressing GST, GST-NS5B, GST-NS5Bt, and
FLAG-human nucleolin. Total lysate and bound protein were separated by
8% SDS-PAGE and detected with anti-FLAG antibody. C, GST
pull-down assay using plasmids expressing GST, GST-human nucleolin, and
FLAG-NS5B, FLAG-NS5Bt. Total lysate and bound protein were separated by
10% SDS-PAGE and detected with anti-FLAG antibody.
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HCV NS5B-binding Region Was Mapped near the C Terminus of Nucleolin
in Vivo and in Vitro--
To map the NS5B-binding region of nucleolin,
a series of deletion mutants of nucleolin were constructed (Fig.
3A). NS5Bt was used to dissect
the interaction of nucleolin because of the poor recovery of NS5B in
soluble form. GST-NS5Bt and a deletion mutant of FLAG-nucleolin were
transiently coexpressed in COS1 cells, after which the lysates were
subjected to GST pull-down assay, and the bound proteins were
immunologically detected by anti-FLAG M2 antibody. As shown in Fig.
3B, the full-size nucleolin and the truncated mutants
deleted of the N terminus could bind to NS5Bt, although the smallest
construct of the C-terminal region, nucleolin-R, could not. In
contrast, the N terminus of nucleolin failed to bind to NS5B (Fig.
3B) and nucleolin-1234, -123, -234, and -23 could not bind
either. Therefore, the minimum region necessary for binding NS5B is
within nucleolin-4R, which harbors RNA-binding domain 4 and the RGG
domain. To confirm the direct binding of NS5B and nucleolin, we tried
to express the full-length nucleolin in E. coli, but the
full-length and the N-terminal region of nucleolin could not be
expressed (29). Thus, several FLAG-tagged truncated forms of nucleolin
harboring the C-terminal half were bacterially expressed and purified
with M2-bound agarose (Fig. 3C). FLAG-nucleolin-1234R and
nucleolin-4R were pulled down with GST-NS5Bt, whereas
FLAG-nucleolin-1234 was not recovered with GST-NS5Bt. The mapping
result in vitro is consistent with that in mammalian cell
lysates. Taken together, the data indicate that NS5B can directly bind
nucleolin through the 4R region.

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Fig. 3.
A, structures of wild-type and deletion
mutant forms of human nucleolin and characteristics of NS5B binding.
Boxes represent the structure of different constructs of
human nucleolin protein with the boundaries of amino acid residues
indicated above. The activity of each nucleolin mutant to interact with
NS5B is indicated by a plus or minus
sign. NLS, nuclear localization signal;
RBD, RNA binding domain. B, GST-NS5Bt and the
full-length or deleted FLAG-tagged nucleolin were transiently
co-transfected in COS1 cells. GST pull-down assay and Western blot
analysis were carried out with anti-FLAG antibody. C,
interaction of deletion mutants of FLAG-nucleolin with GST-NS5Bt
in vitro by GST pull-down assay. Partially purified deleted
FLAG-tagged nucleolin was incubated with GST-NS5Bt or GST protein and
pulled down with glutathione-Sepharose 4B resin. The bound proteins
were washed with washing buffer, fractionated by 12% SDS-PAGE, and
subjected to Western blot analysis with anti-FLAG antibody.
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At Least Two Sequences Are Important for Nucleolin
Binding--
Next, we addressed the amino acid sequences of NS5B,
which are responsible for the binding of nucleolin. Recently, we have constructed a series of clustered alanine substitution mutants of NS5B
in which 7 amino acid residues in a row were changed to AAASAAA as
reported previously (25). COS1 cells were transiently cotransfected
with plasmids expressing wild-type or mutated FLAG-NS5Bt and
GST-nucleolin or GST alone, and then the cell lysates were subjected to
GST-pull down assay. The results showed that all FLAG-NS5Bt mutant
proteins except NS5Bt-m211 and -m2 could be recovered with
GST-nucleolin, but not with GST alone (Fig.
4C). They indicate that two
amino acid sequences are critical for the nucleolin-binding. The two
sequences, WKSKKNP and WRHRARS in NS5Bt-m211 and -m2, respectively, are
enriched in basic amino acid residues.

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Fig. 4.
Interaction of NS5B and nucleolin proteins
expressed in mammalian cells. COS1 cells were transiently
co-transfected with plasmids expressing FLAG-tagged NS5B and
GST-nucleolin. A, total lysate was separated by 10%
SDS-PAGE and subjected to Western blot analysis with anti-FLAG
antibody. B, proteins bound to glutathione-Sepharose 4B
resin were washed with washing buffer, fractionated by 10% SDS-PAGE,
and detected by Western blot analysis with anti-FLAG antibody.
C, structures of full-size, truncated forms and clustered
substitution mutants of NS5B and characteristics of nucleolin binding.
NS5Bt-M211 and -m2 have an alanine substitution in the clustered
regions of basic residues as described under
"Results."
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Clustered Substitution Mutants, NS5Bt-m211 and -m2, Cannot Enter
Nucleoli--
If the direct binding between nucleolin and NS5B is
mainly responsible for the exclusive nucleolar localization of NS5Bt, then two clustered mutants may affect the subcellular localization of
NS5Bt. GFP-NS5Bt-m211 and -m2 were transiently expressed in HLE cells,
and the cells were stained by anti-nucleolin antibody and subjected to
confocal microscope analysis (Fig. 5).
The GFP signals of GFP-NS5Bt-m211 and -m2 were not distributed in
nuclei, but abundant in perinuclear regions; however, endogenous
nucleolin was localized in nucleoli. These results clearly indicate
that two sequences critical for direct nucleolin binding are
indispensable for the nucleolar localization of NS5Bt. The nucleolar
targeting of NS5Bt is a result of the direct interaction of NS5B and
nucleolin.

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Fig. 5.
Subcellular localization of NS5Bt-m211 and
NS5Bt-m2. HLE cells were transfected with plasmid expressing
GFP-NS5Bt-m211 (panel A) and GFP-NS5Bt-m2
(panel B). Transfected cells were fixed and
stained as described in the legend to Fig. 1.
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Nucleolin Has an Inhibitory Effect on NS5B RdRP Activity--
The
direct binding of nucleolin and NS5B may affect the function of NS5B.
First, we addressed whether nucleolin affects the RdRP activity of NS5B
in vitro. The purified soluble bacterial recombinant NS5Bt
was used for an UMP incorporation assay with poly(A) and
oligo(U)14 as template and primer, respectively. We tried
but failed to express bacterial or insect recombinant full-length nucleolin. The C-terminal parts of nucleolin with or without the NS5B-binding region (Fig. 3A) were then purified in
His-tagged forms, because FLAG-tagged truncated nucleolin proteins were
expressed at much lower level for biochemical analyses.
His-nucleolin-1234R was difficult to purify in large amounts. As shown
in Fig. 6, both His-nucleolin-1234R and
-4R, but not His-nucleolin-123 or -23, exhibited an inhibitory effect
on the activity of NS5B RdRP in a dose-dependent manner.
The nculeolin-1234R retains stronger inhibitory ability on the RdRP
activity than nucleolin-4R did, and the NS5B-binding negative
constructs (Fig. 6). The result strongly suggests that the truncated
nucleolin protein requires the NS5B-binding ability for inhibiting RdRP
activity in vitro.

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Fig. 6.
His-nucleolin modulates NS5B RdRP
activity. Purified GST-NS5Bt (10 nM) in the presence
of 0, 5, 10, 50, 100, and 250 nM different deletion mutants
of His-nucleolin were examined by poly(A)-dependent UMP
incorporation assay as described under "Experimental Procedures."
Bar, S.E. of three independent experiments.
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 |
DISCUSSION |
HCV replication, RNA-dependent RNA synthesis, is
distinct from the host macromolecular processes and has been regarded
as a target for intervention to block chronic infection and eventual hepatocarcinogenesis (4, 5). HCV replication consists of minus strand
replication and plus strand replication. Both processes seem to happen
at membranous structures where NS proteins dynamically assemble as a
replication complex with HCV RNA. Recent results with HCV RNA replicon
systems strongly suggest that HCV RNA replication is under regulation
in which NS5A and NS5B are involved (10, 35, 36), although the exact
molecular mechanism remains unclear. Recently we proposed that NS5B
RdRP activity could be regulated at the level of protein-protein
interactions, homomeric oligomerization, and heteromeric interaction
between NS5B and NS5A (14, 19, 20).
In an attempt to clarify the exclusive nuclear localization of NS5Bt, a
form of NS5B deleted of the C-terminal membrane-anchoring domain, here
we report that NS5Bt is recruited to nucleoli through the association
of nucleolin with NS5B. Despite a report that various forms of NS5B
exist in the serum of HCV-infected patients (37), there is no evidence
of cleavage at the junction of the anchoring domain in hepatocytes.
Therefore, the nucleolar localization of NS5Bt is an artificial
phenomenon. Importantly, the full-length NS5B enriched in perinuclear
regions colocalized with endogenous nucleolin, indicating that the
ability of NS5B to bind nucleolin resulted in the change in
distribution of endogenous nucleolin. The binding of NS5B requires at
least two stretches of amino acids 208-214 and amino acids 500-506.
These two sequences are at the bottom of the palm and outer part of the
thick thumb, respectively, and are not close to the pocket for
catalytic activity, but rather exposed. There are three basic amino
acid residues clustered in both sequences, which are consistent with
the proposed nucleolin-binding motifs rich in basic amino acids (38).
The anchoring domain at the C terminus plays a role in recruiting the
NS5B protein to the cytoplasmic membrane and also functions to
redistribute host nucleolar proteins, at least nucleolin. The outcome
of this redistribution of host protein(s) remains to be understood, but it may seem to modulate HCV replication or/and host functions. Preliminary experiments demonstrated that the redistribution of nucleolin seen in GFP-NS5B expressing cells did not affect the cell
cycle by flow cytometric analyses as were not seen with GFP- or
GFP-NS5Bt-expressing cells (data not shown).
We applied nucleolin as a representative nucleolar marker, although
nucleolin has been also well documented to act as a shuttling protein
of RNA, or RNA chaperon, RNA trafficking (39-41). Nucleolin is
involved in shuttling between not only the cytoplasm and nucleus, but
also the cell surface and cytoplasm. Nucleolin on cell membrane has
some role in cell to cell interaction and in viral entry (42, 43). Here
we demonstrated the direct binding of nucleolin and NS5B in
vitro with the bacterial recombinant purified proteins, indicating
that the direct binding does not require the presence of RNA. The
NS5B-binding abilities of the nucleolin truncated constructs are well
correlated to their inhibitory effects on the RdRP activities, strongly
suggesting that the direct binding to NS5B is the reason of the
inhibition. Nucleolin is also involved in the reproduction of several
viruses (38, 44). In poliovirus, nucleolin relocalizes to the cytoplasm
after infection occurs and increases the efficiency of viral gene
expression by enhancing viral RNA translation or RNA replication (44).
One report recently demonstrated that nucleolin stimulated viral
internal ribosome entry site-mediated translation both in
vitro and in vivo (45). This ability to bind RNA seems
to be important for the switching from replication to translation as
the two processes cannot occur at the same time during the life cycle
of RNA viruses. In this context, our results of an inhibitory function
on RdRP activity may have a relevance to the switching mechanism. It
remains highly possible that the outcome of the direct interaction
between nucleolin and NS5B in vivo would be opposite to the
inhibitory effect we observed because we could address its role only
with the truncated forms in vitro. However, nucleolin-1234R
covers almost all functional roles of nucleolin reported so far.
We and other groups have reported that NS5B can be oligomerized, which
is a prerequisite for RdRP activity. We identified residue Glu-18 in
the long loop and His-502 in the thick thumb, as critical for the
oligomerization (19, 46). His-502 is within the sequence mutated in cm2
that is defective in nucleolin-binding. Although exact roles of the
three arginine residues and histidine residue in the sequence covering
cm2 remain to be examined for the nucleolin binding, one interesting
possibility is that nucleolin inhibits the oligomerization of NS5B. The
actual role of nucleolin cannot be addressed biochemically, because a
full-length nucleolin is not available at present using any expression
systems. To understand the role of nucleolin, further experiments are
definitely necessary using HCV replicon systems in which effect of the
full-length nucleolin could be addressed on HCV replication where NS5B
is associated with the other non-structural proteins as replication complex.