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Journal of Virology, August 1999, p. 7044-7049, Vol. 73, No. 8
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Specific Interaction between the Hepatitis C Virus
NS5B RNA Polymerase and the 3' End of the Viral RNA
Ju-Chien
Cheng,1,
Ming-Fu
Chang,2 and
Shin C.
Chang1,*
Institutes of
Microbiology1 and
Biochemistry,2 National Taiwan
University College of Medicine, Taipei, Taiwan, Republic of China
Received 26 January 1999/Accepted 3 May 1999
 |
ABSTRACT |
Hepatitis C virus (HCV) NS5B protein is the viral RNA-dependent RNA
polymerase capable of directing RNA synthesis. In this study, an
electrophoretic mobility shift assay demonstrated the interaction
between a partially purified recombinant NS5B protein and a 3' viral
genomic RNA with or without the conserved 98-nucleotide tail. The
NS5B-RNA complexes were specifically competed away by the unlabeled
homologous RNA but not by the viral 5' noncoding region and very poorly
by the 3' conserved 98-nucleotide tail. A 3' coding region with
conserved stem-loop structures rather than the 3' noncoding region of
the HCV genome is critical for the specific binding of NS5B.
Nevertheless, no direct interaction between the 3' coding region and
the HCV NS5A protein was detected. Furthermore, two independent
RNA-binding domains (RBDs) of NS5B were identified, RBD1, from amino
acid residues 83 to 194, and RBD2, from residues 196 to 298. Interestingly, the conserved motifs of RNA-dependent RNA polymerase for
putative RNA binding (220-DxxxxD-225) and template/primer position
(282-S/TGxxxTxxxNS/T-292) are present in the RBD2. Nevertheless, the
RNA-binding activity of RBD2 was abolished when it was linked to the
carboxy-terminal half of the NS5B. These results provide some clues to
understanding the initiation of HCV replication.
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TEXT |
Hepatitis C virus (HCV) is an
enveloped virus that possesses a single-stranded positive-sense RNA
genome encoding a polyprotein of approximately 3,000 amino acid
residues (7, 20, 29). The conserved 5' noncoding region
(5'NCR) of the HCV genome (4, 13) is highly structured and
contributes to the internal ribosome entry site important for the
translation initiation of HCV RNA (14, 15, 25, 34, 35, 39).
The 3' noncoding region (3'NCR) consists of a short genotype-specific
region and a poly(U)-C(U)n repeat stretch of
variable length followed by a highly conserved tail of 98 nucleotides
(nt) (12, 21, 30, 37).
Studies to understand the molecular mechanism of HCV replication have
been restricted by the lack of a well-established cell culture system,
but studies from other positive-sense RNA viruses may provide some
clues. Upon flavivirus infection, translation of incoming viral genomic
RNA occurs, and replication of the viral RNA begins with the synthesis
of minus-strand RNA which then serves as the template for the synthesis
of progeny genomic RNA. The replication appears to take place at the
perinuclear endoplasmic reticulum and requires virus-encoded proteins
NS3 (proteinase/helicase) and NS5 (polymerase) as components of the
presumed replicative complex (36). In addition, the 3'
terminus of the flavivirus genomic RNA forms a conserved pseudoknot
structure (26). It is generally believed that conserved
sequences and structures at the 3' terminus of viral genomic RNA
function as cis-acting signals that interact with viral and
cellular proteins to initiate the synthesis of minus-strand RNA during
viral replication.
The NS5B protein of HCV is a membrane-associated phosphoprotein
(16) that possesses the conserved GDD motif of RNA-dependent RNA polymerase (RdRp) (19). RdRp activity of the HCV NS5B
has been demonstrated in vitro, and several amino acid motifs essential for the enzymatic activity were identified (1, 2, 10, 23,
38). Nevertheless, template specificity of the viral RNA on the
NS5B RdRp activity was not observed. It is reasonable to propose that
following HCV infection, the initiation of minus-strand RNA synthesis
depends on an initial recognition and specific binding of the NS5B RNA
polymerase or replicative complex to the 3' terminus of the viral
genomic RNA. Many specific sequences as well as structures at the 3'
termini of the genomes of positive-sense RNA viruses have been
demonstrated to be important for the synthesis of minus-strand RNA
(11, 18, 22, 27, 28). Previous studies also demonstrated a
specific binding of encephalomyocarditis virus RNA polymerase to the
viral poly(A)-containing 3'NCR (8, 9). However, recent studies indicated that HCV NS5B interacted with the 3' conserved 98 nt
of the viral genome with little specificity (23) and had no
clear preference to utilize the 98-nt RNA as a template in RdRp
activity assay (38). In this study, by performing a
competitive electrophoretic mobility shift assay (EMSA), we have
identified conserved stem-loop structures in the 3' coding region of
HCV genomic RNA important for the binding of NS5B RNA polymerase. In
addition, two RNA-binding domains of the NS5B RNA polymerase were identified.
Expression of the full-length recombinant HCV NS5B protein.
To
evaluate the biological functions of HCV RNA polymerase, a full-length
NS5B cDNA was obtained from a serum sample of an HCV-infected patient
by reverse transcriptase-PCR. The cDNA was cloned into pET15b, which
allows expression of the full-length NS5B protein in Escherichia
coli. Following induction with 1 mM isopropyl-
-D-thiogalactopyranoside (IPTG), a protein
with a molecular mass of 67 kDa was detected in the insoluble fraction
of the bacterial lysate (Fig. 1A, lane
3). Specificity of the protein as an induced recombinant HCV NS5B
protein was confirmed by Western blot analysis using rabbit antibodies
against an N-terminal peptide (NH2-MSYTWTGALITPCAAE-COOH) of the NS5B (Fig. 1B) and the serum of an HCV patient (data not shown).
Similar to the experience of Yuan et al. (40), our efforts to obtain soluble full-length NS5B protein from E. coli
turned out to be unsuccessful regardless of modifications of growth
conditions or expression vectors (data not shown). Therefore, the
insoluble NS5B protein was recovered from sodium dodecyl
sulfate-polyacrylamide gels and was used to examine its biological
functions.
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FIG. 1.
Expression of HCV NS5B protein in E. coli.
(A) Coomassie blue staining. E. coli BL21(DE3) cells were
transformed with plasmids pET15b (lanes 1 and 2) and pET15b-NS5B (lanes
3 and 4) and grown at 37°C. Plasmid pET15b-NS5B bears HCV cDNA from
nt 7258 to 9030 of the coding region that encodes the full-length NS5B
representing the viral polyprotein from amino acid residues 2420 to
3010. Expression and purification of the recombinant NS5B protein were
performed essentially according to the procedures described by the
manufacturer (Novagen). Cell lysates of soluble (lanes 2 and 4) and
insoluble (lanes 1 and 3) fractions were resolved on a sodium dodecyl
sulfate-8% polyacrylamide gel. Coomassie blue staining is shown. (B)
Western blot analysis. The insoluble fractions of protein lysates from
cells transformed with pET15b (lane 1) and pET15b-NS5B (lane 2) were
immunoblotted following the procedures previously described
(6) with the immunoglobulin G fraction of a rabbit antiserum
that was raised against the NS5B peptide
NH2-MSYTWTGALITPCAAE-COOH. Arrowheads indicate the
IPTG-induced recombinant NS5B protein.
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Specific binding of HCV NS5B to the 3' end of viral genomic
RNA.
To determine the RNA-binding activity of HCV NS5B, an EMSA
was performed as previously described (39). The 3' HCV RNA
designated 3'CNUX, which consists of the coding region of the HCV
polyprotein from nt 8736 to 9030 (3'C) and the entire viral 3'NCR,
including the genotype-specific region (N), a
poly(U)-C(U)n repeat stretch (U), and the
conserved 98-nt tail (X), was in vitro synthesized in the presence of
[
-32P]UTP and used as a substrate. Results clearly
demonstrated an interaction between HCV NS5B protein and the 3'CNUX
RNA. The levels of RNA-protein complexes raised were in agreement with
increasing amounts of the partially purified NS5B (Fig.
2, lanes 1 to 5). The complexes were
diminished in the presence of either the unlabeled 3'CNUX RNA or the
3'CNU RNA that lacks the 3'-terminal 98 nt (Fig. 2, lanes 6 to 11).
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FIG. 2.
Complex formation between HCV NS5B protein and the
3'CNUX RNA. (Top) Structures of the HCV 3'-end RNAs, 3'CNUX, and 3'CNU.
The heavy line that begins with nt 8736 and ends with nt 9030 represents the 3' coding region of the HCV polyprotein. The 3'CNUX RNA
consists of the 295-nt 3' coding region and the entire viral 3'NCR
encompassing a short genotype-specific region, a
(U)33-C(U)n stretch, and a conserved
98-nt tail with stem-loop structure. The 3'CNU RNA contains the 295-nt
3' coding region and a partial 3'NCR as shown. (Bottom) EMSA. The
[-32P]UTP-labeled 3'CNUX RNA was incubated with
increasing amounts (lanes 2 to 5, 0.15, 0.75, 1.5, and 3 ng,
respectively) of the partially purified HCV NS5B protein or with 3 ng
of the NS5B protein in the presence of various amounts of competitor
RNAs as indicated (lanes 7 to 11). Reaction products were resolved in a
4% polyacrylamide gel under nondenaturing conditions. The gel was
dried and subjected to autoradiography. Lanes 1 and 6 represent free
3'CNUX RNA probe to which no NS5B was added in the reaction.
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Previous studies using the RNA filter binding assay indicated that the
interaction between HCV NS5B protein and a 3' viral
RNA bearing the
98-nt tail was nonspecific (
23). Because the
3'NCR of the
viral genome is likely to be the entry site for RNA
polymerase to
initiate genome replication, we attempted to address
the question
further by performing a competitive EMSA using various
fragments of the
HCV genomic RNA as competitors. As shown in Fig.
3, the complex formed between
radiolabeled 3'CNU RNA and HCV NS5B
protein was completely annihilated
by a fivefold molar excess
of the unlabeled 3'CNU RNA (lane 4).
Competition by the 3' (3'X
RNA) conserved 98-nt tail was observed only
at a higher dose of
competitor, and the competition effect was reduced
(lanes 6 to
8). On the other hand, the 5'NCR competitor had no effect
at all
(lanes 9 to 11). Taken together, these results indicated that
the HCV NS5B has a preference to interact with the 3' viral RNA
that
contains a region coding for the carboxyl terminus of the
HCV
polyprotein rather than to interact with the 3' 98-nt tail
or 5'NCR.
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FIG. 3.
Preferential binding of NS5B protein to the HCV 3'CNU
RNA. (Top) Structures of the HCV genomic RNA and RNA fragments used in
the competition analysis. The heavy line represents the coding region
of the HCV polyprotein. The 5'NCR contains a highly ordered structure
341 nt in length, and the 3' NCR consists of the genotype-specific
region, a (U)n-C(U)n
stretch, and the conserved 98-nt tail. (Bottom) Competitive EMSA. EMSA
was performed with HCV NS5B protein and the 32P-labeled
3'CNU RNA in the presence (lanes 3 to 11) or absence (lane 2) of
unlabeled competitor RNAs as indicated. Competitor RNAs used were 1- (lanes 3, 6, and 9), 5- (lanes 4, 7, and 10), and 10-fold (lanes 5, 8, and 11) molar excesses to the labeled 3'CNU RNA. Lane 1 represents free
3'CNU RNA probe to which no NS5B protein was added in the reaction. We
consistently observed two bands of the 3'CNU RNA probe that should
represent the same RNA fragment of different conformations. A single
band was detected when the probe was analyzed on a sequencing gel
containing 8 M urea (data not shown).
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Mapping of the specific NS5B binding sequences on the 3'CNU
RNA.
To define more precisely the sequences of 3'CNU RNA essential
for the binding of HCV NS5B protein, a series of competition experiments were performed with RNA competitors derived from the 3'CNU
RNA as shown in Fig. 4. Both 3'CNU and
3'CN RNAs cover the 3' region of the HCV genomic RNA that has been
predicted to possess four conserved stem-loop structures (data not
shown and reference 12), SL1 to SL4. The 3'C and 3'N
RNA represent RNA domains of the 3' coding region and genotype-specific
region, respectively, of the 3'CN RNA. The 3'C'N RNA represents a 5'
deletion of the 3'CN RNA up to the beginning of SL3. Interestingly, we
found that both 3'CN and 3'C RNA competed efficiently the binding of
NS5B to the 3'CNU RNA (Fig. 4, lanes 2 to 7), whereas 3'N and 3'C'N RNA
had little effect (lanes 8 to 13), indicating that the coding region 5'
to the SL3 is important for the binding. In addition, although 3'CN RNA
competed more effectively than the 3'C RNA (compare lane 2 to lane 5),
the presence of both 3'C and 3'N competitors that added together cover
the sequences of 3'CN RNA, did not demonstrate a competition effect
comparable to the 3'CN RNA (lanes 14 to 16). These indicated that the
3'N RNA must be covalently linked to the 3'C RNA for the binding of
NS5B to occur and may imply a role of the SL2 to enhance the
interaction between HCV RNA and NS5B. Consistent with the results of
the competitive EMSA, we found that among radiolabeled 3'C, 3'N, and
3'C'N RNAs, only 3'C RNA formed complex with NS5B (data not shown). In
addition, the formation of 3'C RNA-NS5B complex required a higher dose
of NS5B than that of 3'CNU RNA (data not shown).
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FIG. 4.
Mapping of the binding domains of NS5B on the HCV 3'CNU
RNA. (Top) Structures of the HCV RNAs used in the competitive EMSA and
their characteristics in forming a complex with HCV NS5B protein.
Nucleotide residues flanking the RNA termini are numbered according to
the coding region of HCV polyprotein. RNA fragments that form conserved
stem-loop structures (SL) are indicated by filled boxes. SL4 and SL3
contain nt 8875 to 8918 and nt 8980 to 9010, respectively, of the
coding region of HCV polyprotein. SL2 contains the 3'-terminal 12 nt of
the coding region plus a downstream 11 nt, and SL1 is located within
the genotype-specific region (12). Binding activities are
indicated by plus and minus signs. (Bottom) Competitive EMSA. The
competition analysis was conducted with a gel-purified 3'CNU RNA probe
and HCV NS5B protein in the presence of unlabeled 3'CN (lanes 2 to 4),
3'C (lanes 5 to 7), 3'N (lanes 8 to 10), 3'C'N (lanes 11 to 13), and a
mixture of 3'C and 3'N RNA (lanes 14 to 16) at 1-, 5-, and 10-fold
molar excesses to the [-32P]UTP-labeled 3'CNU RNA
probe. Lane 1 represents the reaction in which no competitor RNA was
added.
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Mapping of the binding domains of NS5B to the 3'CNU RNA.
To
define domains of the NS5B that conferred RNA binding, recombinant NS5B
deletion mutants with His tag were produced as the full-length NS5B.
All the mutant proteins were present in the insoluble fractions (data
not shown) and were purified by polyacrylamide gel electrophoresis and
electroelution as for the full-length NS5B. The purified proteins were
analyzed by Coomassie blue staining (Fig.
5A) and Western blot analysis using a
monoclonal antibody against the His tag (Fig. 5B). The specificity of
the purified proteins was also examined with serum of an HCV patient (data not shown). RNA-binding activities for the NS5B deletion mutants
were examined by Northwestern analysis using radiolabeled 3'CNU RNA as
the substrate in the presence of 50 mM (Fig. 5C) or 150 mM (Fig. 5D)
NaCl. Under the conditions examined, we found that the HCV 3'CNU RNA
bound to the deletion mutants NS5B(1-298), NS5B(1-194), and
NS5B(196-298) even more strongly than the full-length NS5B protein,
but deletion mutants NS5B(196-591), NS5B(300-509), and NS5B(1-82)
failed to interact with the HCV RNA. In addition, the HCV NS5A protein
that was purified from the insoluble lysate of pET15b-NS5A-transformed
cells could not interact directly with the viral 3'CNU RNA (Fig. 5C).
Plasmid pET15b-NS5A bears the HCV cDNA from nt 5917 to 7527 of the
coding region of the viral polyprotein and encodes the full-length NS5A
protein. These results were further confirmed by EMSA. As shown in Fig.
6, the full-length NS5B and deletion
mutants NS5B(1-298), NS5B(1-194), and NS5B(196-298) formed complexes
with the radiolabeled 3'CNU RNA, whereas no complex was detected with
proteins NS5A, NS5B(196-591), NS5B(300-509), and NS5B(1-82). In
light of these results, it appeared that HCV NS5B RNA polymerase
possesses two independent RNA-binding domains (RBDs), from amino acid
residues 83 to 194 (RBD1) and from residues 196 to 298 (RBD2).
Nevertheless, the RNA-binding activity of RBD2 is completely
annihilated when it is linked to the downstream sequences of the NS5B
protein. The reasons why NS5B(196-591) protein failed to interact with
HCV RNA are not clear. It is possible that the downstream sequences
disrupted a proper folding essential for the binding activity of RBD2.
Alternatively, the NS5B(196-591) protein may form a less stable
complex with HCV RNA that was not detected under the standard
conditions used in this study.
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FIG. 5.
Binding of HCV 3'CNU RNA to the recombinant NS5B
deletion mutants. Partially purified full-length NS5A and NS5B and NS5B
deletion mutants as indicated were analyzed by sodium dodecyl
sulfate-15% polyacrylamide gel electrophoresis in Tricine-Tris buffer
and stained with Coomassie blue (A). Equal amounts of the recombinant
proteins as determined by Bio-Rad protein assay were subjected to
Western blot analysis with 6× His monoclonal antibody (ClonTech) (B)
and Northwestern analysis as previously described (5) with
[-32P]UTP-labeled 3'CNU RNA in the presence of 50 mM
(C) and 150 mM (D) NaCl. The control lanes represent protein lysates
prepared from E. coli transformed with the pET15b plasmid.
In panel C, a signal at a position beyond the molecular sizes of the
recombinant NS5B proteins was detected with the control lysate. Its
identity is not known. Panel E shows the structures of the NS5B
recombinant proteins and summarizes their characteristics in binding
3'CNU RNA.
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FIG. 6.
Complex formation between NS5B deletion mutants and the
3'CNU RNA. An EMSA was performed with
[-32P]UTP-labeled 3'CNU RNA, and equal amounts of the
NS5A and NS5B recombinant proteins as indicated. The gel was dried and
subjected to autoradiography.
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Conclusions.
This study investigated the RNA-binding
specificity of the HCV NS5B RdRp. HCV NS5B preferentially interacted
with a 3' viral RNA containing sequences coding for the carboxyl
terminus of the HCV polyprotein. The binding domains of NS5B to the HCV
RNA were mapped to amino acid residues 83 to 194 and 196 to 298.
Binding of RdRp to viral genome is the key step to initiate replication
of positive-sense RNA viruses. Previous studies have
demonstrated
specific sequences and structures at the 3' termini
of positive-sense
RNA viruses involved in the binding of RNA polymerase
and/or initiation
of replication. Examples include a poly(A)-containing
3'-terminal RNA
of encephalomyocarditis virus (
8,
9) and
a murine
coronavirus (
22), a non-base-paired 3' ACC sequence
of a
tRNA-like structure of turnip yellow mosaic virus (
11,
27),
a stem-loop structure of turnip crinkle virus (
28), and
a
pseudoknot structure of polioviruses (
18). The 3'NCR of HCV
genome consists of a short genotype-specific region and a stretch
of
poly(U)-C(U)
n repeats followed by a highly
conserved
98-nt tail (
12,
21,
30,
37). The conserved 98-nt
tail
forms a conserved secondary structure (
3,
17,
21,
31)
and is likely to be involved in the initiation of viral replication.
Cellular proteins, including polypyrimidine tract binding protein,
were
recently demonstrated to specifically interact with the stem-loop
structure of the conserved 98 nt (
17,
33), but no direct
interaction
between the 3' 98-nt tail and 5'NCR of the HCV genomic RNA
was
indicated (
3). In addition, earlier studies with RNA
filter
binding assay failed to demonstrate the specific interaction
between
HCV NS5B and a viral RNA containing the 98-nt sequences
(
23).
In this study, by performing a competitive EMSA, we observed specific
complex formation between HCV NS5B protein and the 3'CNU
RNA that lacks
the 98-nt tail but contains 295 nt upstream and
54 nt downstream from
the stop codon of the viral genomic RNA
(Fig.
3). The 3'X RNA competed
the complex formation poorly, and
the 5'NCR competitor had little
effect. In addition, the poly(U)
stretch in 3'NCR was not essential for
the binding but the short
genotype-specific region may facilitate the
binding of HCV NS5B
to the viral RNA (Fig.
4). Nevertheless, the
genotype-specific
region of 3'NCR did not seem to interact with HCV
NS5B by itself
(Fig.
4). Interestingly, the binding activity of HCV
NS5B to the
various 3'-terminal regions of the viral genomic RNA
correlated
very well with the RdRp activity of the NS5B RNA polymerase.
Using
various HCV 3' RNAs as templates and primers, Yamashita et al.
(
38) demonstrated that the level of in vitro RNA synthesis
with
the 98-nt RNA was relatively low compared to that of a 480-nt
RNA
containing 436 nt of the coding region of HCV polyprotein
plus a
downstream genotype-specific region, whereas a 106-nt poly(U)
stretch
failed to serve as template/primer. Taken together, these
results
indicate that the conserved 3'-terminal tail and
poly(U)-C(U)
n stretch of HCV genome may not be
good substrates or templates
by themselves for NS5B binding and
subsequent RNA replication;
a coding region of about 300 nt immediately
before the stop codon
of the viral genome may play an important role in
the viral replication.
Highly conserved stem-loop structures have been
predicted within
the 200-nt region upstream from the U stretch of the
HCV genomic
RNA (Fig.
4) (
12). Whether these conserved
structures are involved
in the genome replication of HCV remains to be
elucidated. However,
our results do support the idea that the SL2 made
up from sequences
of the 3' coding region and 3'NCR across the stop
codon of HCV
polyprotein is important for the viral RNA to interact
with NS5B
(Fig.
4). The binding activity of NS5B to the 3'CN RNA was
annihilated
when the RNA transcript was divided into two fragments (3'C
and
3'N) that disrupted the formation of SL2. In addition, SL4 is
required for the RNA-binding activity; HCV RNA transcript 3'C'N
in
which the SL4 had been deleted failed to interact with NS5B
RNA
polymerase (Fig.
4).
Sequence analysis has revealed several motifs that are conserved among
RdRp (
24). Judging from the prediction of secondary
structure, it was proposed that four of the motifs, each of which
provides one conserved amino acid residue, may constitute a
prerequisite
polymerase module involved in the template binding and
subsequent
polymerization (
24). Of the four motifs of HCV
NS5B, site A
(220-DxxxxD-225) and sites C (317-GDD-319) and D
(R-345) were
predicted to be involved in RNA and nucleoside
triphosphate binding,
respectively, and possibly catalysis,
whereas site B (282-S/TGxxxTxxxNS/T-292)
was thought to be
important for template/primer position. Nevertheless,
previous mutational analysis at these conserved amino acid residues
failed to demonstrate their involvement in RNA binding (
23).
In this study, by performing Northwestern analysis (Fig.
5) and
an EMSA
(Fig.
6) with deletion mutants, we identified two RNA-binding
domains
of the HCV NS5B RNA polymerase from amino acid residues
83 to 194 (RBD1) and 196 to 298 (RBD2). Interestingly, consistent
with the
results of our RNA-binding study, the putative RNA-binding
motif
(site A) and template/primer position motif (site B) of
the polymerase
module are located in the RBD2. In addition, several
clusters of basic
amino acid residues, 151-KGGRKPAR-158, 209-KSKK-212,
and
277-RRCR-280, were found in the RNA-binding domains. Whether
these
clusters play roles in the RNA-binding activity of NS5B
has not yet
been examined. But the observation that mutations
at the cluster
274-CGYRRCR-280 abolished the RdRp activity of
a carboxy-terminal
truncated NS5B (
38) may imply that the RNA-binding
activity
of the RBD2 is essential for the RdRp activity of
NS5B.
Replication of viral genome is a complicated event that requires not
only polymerase but also additional viral and cellular
factors to form
a functional replicative complex. Our RNA-binding
studies indicated
that there was no direct interaction between
HCV NS5A protein and the
viral RNA (Fig.
5 and
6). However, preliminary
data do suggest there is
cross talk between NS5A and NS5B (data
not shown). HCV NS5A is a
phosphoprotein (
32) and was predicted
to play roles in HCV
replication. NS5A was also found to interact
with cellular factors
(data not shown). But components that constitute
a functional
replicative complex to direct replication of HCV
genome with
specificity are not fully understood. In this report,
we have
demonstrated a specific interaction between HCV NS5B RNA
polymerase and
a 3' region of the viral genomic RNA and identified
the interacting
domains of the NS5B. The information provided
here should aid in
understanding the mechanisms of HCV
replication.
Nucleotide sequence accession number.
The cDNA sequence
encoding the full-length HCV NS5B protein has been deposited in GenBank
under accession no. AF145454.
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ACKNOWLEDGMENTS |
We thank Jui-Hung Yen and Ying-Tai Peng for technical assistance.
We are grateful to Bon-Chu Chung, Jen-Yang Chen, Shin-Lian Doong, and
Won-Bo Wang for helpful discussions and comments.
This work was supported by research grants NSC85-2331-B-002-080-MH,
NSC86-2315-B-002-012-MH to S.C.C. from the National Science Council of
the Republic of China and DOH87-HR-723 to M.-F.C. from the Department
of Health, Executive Yuan of the Republic of China. J.-C. C. was the
recipient of a fellowship from the National Science Council (no. 32317D).
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FOOTNOTES |
*
Corresponding author. Mailing address: No. 1, Sec. 1, Jen-Ai Rd., Institute of Microbiology, National Taiwan University
College of Medicine, Taipei, Taiwan, Republic of China. Phone:
886-2-23970800, ext. 8290. Fax: 886-2-23915293. E-mail:
scchang{at}ha.mc.ntu.edu.tw.
Present address: School of Medical Technology, China Medical
College, Taichung, Taiwan.
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Journal of Virology, August 1999, p. 7044-7049, Vol. 73, No. 8
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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