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Journal of Virology, October 2000, p. 9732-9737, Vol. 74, No. 20
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Roles of the AX4GKS and Arginine-Rich Motifs of
Hepatitis C Virus RNA Helicase in ATP- and Viral RNA-Binding
Activity
Shin C.
Chang,1,*
Ju-Chien
Cheng,1,
Yi-Hen
Kou,1
Chuan-Hong
Kao,1
Chiung-Hui
Chiu,1
Hung-Yi
Wu,2 and
Ming-Fu
Chang2
Institutes of Microbiology1
and Biochemistry,2 National Taiwan
University College of Medicine, Taipei, Taiwan, Republic of China
Received 29 November 1999/Accepted 26 July 2000
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ABSTRACT |
The nonstructural protein 3 (NS3) of hepatitis C virus (HCV)
possesses protease, nucleoside triphosphatase, and helicase activities. Although the enzymatic activities have been extensively studied, the
ATP- and RNA-binding domains of the NS3 helicase are not
well-characterized. In this study, NS3 proteins with point
mutations in the conserved helicase motifs were expressed in
Escherichia coli, purified, and analyzed for their effects
on ATP binding, RNA binding, ATP hydrolysis, and RNA unwinding. UV
cross-linking experiments indicate that the lysine residue in the
AX4GKS motif is directly involved in ATP binding, whereas
the NS3(GR1490DT) mutant in which the arginine-rich motif
(1486-QRRGRTGR-1493) was changed to QRRDTTGR bound ATP as well as the wild type. The binding activity of HCV NS3 helicase to the viral RNA was drastically reduced with the mutation
at Arg1488 (R1488A) and was also affected by the K1236E substitution in
the AX4GKS motif and the R1490A and GR1490DT mutations in
the arginine-rich motif. Previously, Arg1490 was suggested, based on
the crystal structure of an NS3-deoxyuridine octamer complex, to
directly interact with the 
-phosphate group of ATP. Nevertheless,
our functional analysis demonstrated the critical roles of Arg1490 in
binding to the viral RNA, ATP hydrolysis, and RNA unwinding, but not in
ATP binding.
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TEXT |
Hepatitis C virus (HCV) is the major
causative agent of posttransfusion and sporadic non-A, non-B hepatitis
(7, 27). Patients with HCV infection often develop chronic
hepatitis that leads to cirrhosis and hepatocellular carcinoma (1,
9, 36). HCV belongs to the family Flaviviridae whose
members are enveloped viruses containing a single-stranded
positive-sense RNA genome. The HCV genome bears a single open reading
frame that encodes a polyprotein encompassing about 3,000 amino
acid residues (8, 23, 42). Proteolytic cleavage of the
polyprotein by host and viral proteases yields at least 10 products as NH2-C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B-COOH (15, 16, 19, 21, 29, 38). HCV nonstructural protein 3(NS3) is a multifunctional protein that possesses three known enzymatic activities in two independent domains. The N-terminal one-third domain contains a serine protease activity. The NS3 protease
and the viral NS2 and NS4A proteins are important for the processing of
the HCV polyprotein to generate nonstructural proteins (2,
10, 14, 15, 20, 43). The C-terminal two-thirds domain of the NS3
protein contains several conserved sequences shared by members of the
DEAD box family of RNA helicases (12, 13, 37). These include
AX4GKS Walker A nucleotide binding motif (motif I), DECH
Walker B nucleoside triphosphate (NTP) binding-hydrolysis motif (motif
II), TAT RNA unwinding motif (motif III), and a QRRGRTGR arginine-rich motif (motif VI) thought to be involved in RNA
binding. NTPase and RNA helicase activities have been demonstrated for both the NS3 helicase domain (24, 40, 41) and the
full-length NS3 protein (11, 18). The kinetics of the ATP
hydrolysis and duplex unwinding have been analyzed (32-34).
In addition, crystal structures of the NS3 helicase have revealed
potential amino acid residues that interact with ATP and RNA substrates
(6, 26, 47). However, RNA substrates were located at
different clefts in the NS3 structures with or without a bound
deoxyuridine octamer. Cocrystal structure of the NS3 helicase and ATP
has not been determined, and whether the arginine-rich motif of the NS3
is involved in ATP or RNA binding remained to be functionally
determined. In this study, we demonstrated that the lysine residue in
the AX4GKS motif is directly involved in ATP binding and
that Arg1488 in the arginine-rich motif (1486-QRRGRTGR-1493)
is important for RNA-binding activity. Arg1490 is critical for
HCV NS3 protein in binding to the viral RNA, ATP hydrolysis, and RNA
unwinding, but not in ATP binding.
Expression and purification of HCV NS3 proteins.
An HCV NS3
protein representing the viral polyprotein from amino acid
residues 1043 to 1635 was produced along with its mutant forms, NS3(K1236E) and NS3(GR1490DT), from plasmids pET15b-NS3, pET15b-NS3(K1236E), and pET15b-NS3(GR1490DT), respectively.
Nucleotide sequence analysis of both strands of the plasmids indicated
that NS3(K1236E) and NS3(GR1490DT) contain amino acid
substitutions in conserved motifs I (1230-AX4GKS-1237) and
VI (1486-QRRGRTGR-1493), respectively (Fig.
1A; data not shown), and no additional
mutations were generated during the cloning procedures. The
overexpressed NS3 proteins were found predominantly in the insoluble
fractions of the bacterial lysates (Fig. 1B and C). For biochemical
analysis, the NS3 recombinant proteins were purified from sodium
dodecyl sulfate (SDS)-polyacrylamide gels and renatured by dialysis
(Fig. 1B and C). In addition, to examine possible contaminating
activities from host proteins, plasmid pET15b was transformed and
expressed in parallel (data not shown). The total cell lysate was
prepared, and proteins eluted from the corresponding position of the
HCV NS3 protein on SDS-polyacrylamide gels were used as host protein controls in functional analyses.
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FIG. 1.
Expression of recombinant HCV NS3 proteins. (A)
Schematic representation of the HCV NS3 protein. The bar spanning amino
acid residues 1027 to 1657 represents the full-length HCV NS3 protein,
and the shaded region from residues 1043 to 1635 represents the NS3
protein used in this study. Amino acid substitutions in the conserved
motifs 1230-AX4GKS-1237 and 1486-QRRGRTGR-1493
of the NS3 mutant proteins are indicated. To obtain the HCV NS3 cDNA
spanning amino acid residues 1043 to 1635, total RNA was isolated from
serum of an HCV-infected patient, and reverse transcriptase-nested PCR
was performed as described previously (49) with C103
(3120-TGGTTGCATCATCACTAGC-3138; nucleotides are numbered
starting from the translational initiation site of the HCV
polyprotein) and C32
( 4927-TGGTTATGGGGTGCGTGA-4910; the letters indicate sequences of the complementary strand) as the first primer set
and C104 (3126-CATCATCACTAGCCTCACAGG-3146) and C50
(4906-TGACCTCATTTTGGACGGCT-4887) as the second primer
set. The HCV NS3 cDNA was first cloned into pGEM-4Z vector and then
resubcloned into pET15b to generate plasmid pET15b-NS3 encoding a
His-tagged HCV NS3 from amino acid residues 1043 to 1635 of the viral
polyprotein. Plasmid pET15b-NS3(K1236E) encodes an NS3
mutant protein, designated NS3(K1236E), in which a Lys-to-Glu
substitution was generated at the 1230-AX4GKS-1237 motif.
Plasmid pET15b-NS3(GR1490DT) encodes an NS3 protein with the
arginine-rich motif 1486-QRRGRTGR-1493 mutated to
QRRDTTGR, designated NS3(GR1490DT). Both plasmids were
generated by replacing a subdomain of the wild-type NS3 cDNA-containing
plasmid with a cognate PCR fragment bearing the desired mutations. (B)
Coomassie blue staining. Total cell lysates (T) were prepared from
E. coli BL21(DE3) transformed with plasmids pET15b-NS3
(lanes 1 to 4), pETI5b-NS3(K1236E) (lanes 5 to 8), and
pET15b-NS3(GR1490DT) (lanes 9 to 12) and separated into soluble (S)
and insoluble pellet (P) fractions, following three freeze-thaw cycles,
sonication, and centrifugation. For purification of the recombinant NS3
proteins from insoluble fractions, the insoluble proteins were
resuspended and boiled for 5 min prior to SDS-polyacrylamide gel
electrophoresis. SDS-polyacrylamide gel electrophoresis was performed
in Tricine-Tris buffer. Specific bands representing the
isopropyl- -D-thiogalactoside (IPTG)-induced NS3 proteins
were sliced out, recovered through Electro-Eluter (Bio-Rad), and
dialyzed against a buffer containing 25 mM Tris and 192 mM glycine. The
dialysates were used as the partially purified NS3 proteins (E) in
functional analyses. A Bio-Rad protein assay was performed to determine
protein concentrations. (C) Western blot analysis. Western blot
analysis was performed with the immunoglobulin G fraction of a rabbit
serum against the HCV NS3 protein that followed the procedures as
previously described (4). Arrowheads in panels B and C indicate the
IPTG-induced recombinant NS3 proteins. Lane abbreviations (T, S, P, and
E) are defined in the legend to panel B.
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ATPase and RNA helicase activities.
The partially purified NS3
proteins were analyzed for ATPase and RNA helicase activities. As shown
in Fig. 2, ATPase activity of the
wild-type NS3 protein increased in a dose-dependent manner. The
possibility of contaminating host ATPase activity in the partially purified NS3 proteins was eliminated, since no activity was
detected with the host protein control (Fig. 2A) prepared in
parallel as described earlier. ATPase activity was significantly
reduced with mutant proteins NS3(K1236E) and
NS3(GR1490DT) (Fig. 2). These results indicated that both the
AX4GKS and arginine-rich motifs are involved in ATP
hydrolysis.
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FIG. 2.
ATPase activity of HCV NS3 proteins. Increasing
amounts (0.4 to 160 ng) of the partially purified recombinant HCV NS3
proteins were analyzed for ATPase activity as described previously
(46) except that the reaction was performed at pH 7.6 for 30 min. Twenty nanograms of a purified dog pancreatic ATPase (Sigma)
and 160 ng of a host protein preparation (H) were analyzed in parallel
as positive and negative controls, respectively. An autoradiogram is
shown (A). The conversion rates of ATP hydrolysis were calculated with
a PhosphorImager (STORM 840; Molecular Dynamics) and plotted as a
function of the input proteins (B). Each point represents the average
of three independent experiments. Standard deviation bars are shown.
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RNA helicase activity of the NS3 mutants was examined by using a
partially double-stranded RNA substrate. The wild-type NS3
protein completely unwound the RNA substrate in the presence of
ATP,
but mutations in NS3(K1236E) and NS3(GR1490DT) completely
abrogated the activity (Fig.
3). In
addition, consistent with
the energy requirement, RNA-unwinding
activity of the wild-type
NS3 protein was annihilated in the absence of
ATP (Fig.
3, lane
4). The effects on ATPase and helicase activities
with mutations
in the AX
4GKS and arginine-rich motifs have
been demonstrated
previously (
18,
25), but whether the
mutated amino acids are
directly involved in ATP binding has not been
demonstrated. In
addition, the role of the conserved motifs involved in
RNA binding
remained to be elucidated.
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FIG. 3.
RNA-unwinding activity of HCV NS3 proteins. An unwinding
activity assay was performed with 1 µmole of the NS3 proteins as
indicated and 0.2 pmol of a -32P-labeled partially
double-stranded RNA substrate in the presence (+) or absence ( ) of
ATP for 30 min at 37°C, in a buffer containing 20 mM Tris [pH 7.0],
1.5 mM MgCl2, 2 mM dithiothreitol, 4 µg of bovine serum
albumin (BSA), 40 U of RNasin, and 2.5 mM ATP. For preparation of the
partially double-stranded RNA, plasmid pGEM-4Z was independently
digested with HindII and Asp718. The
HindII-linearized pGEM-4Z was used as a template to perform
in vitro transcription with SP6 RNA polymerase in the absence of
[-32P]UTP, and the Asp718-linearized
template was used to perform transcription with T7 RNA polymerase in
the presence of [-32P]UTP. These resulted in an
unlabeled 42-nucleotide SP6 transcript and a radiolabeled 51-nucleotide
T7 transcript. An annealing reaction to generate partially
double-stranded RNA substrate essentially followed the procedure as
previously described (22) except that the RNA substrate was
further purified by resolution in a 15% nondenaturing polyacrylamide
gel. The RNA-unwinding reaction was stopped by adding a buffer
containing 3% SDS and 150 mM EDTA (pH 8.0). The reaction products were
analyzed on a 12% nondenaturing polyacrylamide gel
(acrylamide:bisacrylamide weight ratio of 19:1) and visualized by
autoradiography. In lane 1, the RNA substrate was heat-denatured to
release the 32P-labeled single-stranded RNA product.
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ATP-binding activity of NS3 mutants.
To learn whether the
decline of ATPase activity of the NS3 mutant proteins resulted from
a reduction in ATP binding, UV cross-linking experiments were
carried out with [-32P]ATP. Equal amounts of
the purified wild-type and mutant NS3 proteins were analyzed
together with a purified dog pancreatic ATPase. The amount of
[-32P]ATP cross-linked to the NS3(GR1490DT)
protein was similar to the level of the wild-type NS3 protein but was
decreased to less than 30% for NS3(K1236E) as measured by a
PhosphorImager (STORM 840; Molecular Dynamics) (Fig.
4). These results indicated that the
conserved lysine residue in the AX4GKS motif is critical
for ATP binding of HCV NS3 protein, whereas the Arg1490 residue in the
arginine-rich motif (1486-QRRGRTGR-1493) is dispensable for ATP binding.
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FIG. 4.
The conserved Lys1236 residue, but not Arg1490, is
critical for the ATP-binding activity of HCV NS3 protein. To determine
the ATP-binding activity, UV cross-linking experiments were performed
at 4°C with [-32P]ATP (3,000 Ci/mmol) and 120 µg
each of the partially purified NS3 proteins as indicated. In
addition, 2 µg of a purified dog pancreatic ATPase (Sigma)
was analyzed in parallel as a positive control (lane 1). The
reaction mixtures in a buffer containing 20 mM
morpholinepropanesulfonic acid (MOPS)-KOH [pH 7.0], 25 mM NaCl, 2.5 mM MgCl2, and 25% glycerol were irradiated at 254 nm from
a distance of 3 cm for 20 min and subjected to SDS-8% polyacrylamide
gel electrophoresis. Coomassie blue staining (A) and
autoradiography (B) are shown. Arrowheads indicate the purified dog
pancreatic ATPase.
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The AX
4GKS motif is the Walker A nucleotide binding motif
(motif I) of RNA helicases (
37,
45). Mutation at the lysine
residue in the conserved motif of eIF-4A abrogated nucleotide
binding,
ATP hydrolysis, and RNA helicase activities (
31,
35).
ATP
serves as an energy source and is essential for RNA-unwinding
activity.
The effect of the lysine substitution on the ATP-binding
activity is
likely to be the major cause that the RNA helicase
activity of
NS3(K1236E) was abolished. Recent studies of the crystal
structure
of the HCV NS3 helicase indicated that the AX
4GKS motif
forms a phosphate-binding loop for binding to the
-phosphate
group
of ATP (
26). In addition, the AX
4GKS motif is
located
at the N terminus of an
-helix structure and in close
proximity
to the conserved aspartic acid residue of the DEXH motif
(
47).
This allows the lysine residue of the
AX
4GKS motif to make an
additional contact with the
aspartic acid residue of the DEXH
motif (
6,
26,
47). DEXH is
the Walker B NTP binding-hydrolysis
motif (motif II) that interacts
with the magnesium ion of Mg-ATP.
The reduced ATPase activity of
NS3(K1236E) (Fig.
2) may reflect
a
combined effect of the single amino acid substitution in the
AX
4GKS motif on binding of ATP and interaction with the
conserved
DEXH
motif.
So far, the crystal structure of the HCV NS3 helicase-ATP complex has
not been determined. A recent study of the cocrystal
structure of
PcrA DNA helicase and a nonhydrolyzable ATP analog
(adenylyl
imidodiphosphate [ADPNP]) indicated that Arg610 in motif
VI
(599-EERRLAYVGITRA-611) (
39) of PcrA contacts
with the
-phosphate
group of ADPNP (
44). In the
"inchworm" unwinding model (
48)
of HCV NS3 helicase,
Arg1490 in motif VI (1486-QRRGRTGR-1493)
was proposed to
directly contact the
-phosphate group of a bound
molecule of ATP
(
26,
28). Nevertheless, our functional analysis
with the
NS3(GR1490DT) mutant protein demonstrated that Arg1490
is not
critical for ATP binding to the NS3 protein (Fig.
4).
However,
the reduction in the ATPase activity of
NS3(GR1490DT) (Fig.
2)
did indicate that the arginine-rich motif is
involved in ATP
hydrolysis.
RNA-binding activity of NS3 mutants.
RNA-binding activity of
the NS3 mutant proteins was examined by filter binding assay and
Northwestern analysis with an HCV 3'-end RNA (3'CNU RNA) (5)
as the probe. The relative RNA-binding activity of NS3(GR1490DT) to
the wild-type NS3 protein was about 10% by filter binding assay
(Fig. 5A) and 26% by Northwestern analysis (Fig. 5B). Although the values of 10 versus 26% in the two
assays differed by two- to threefold, the results were reproducible and
clearly demonstrated that Arg1490 in the arginine-rich motif is
important for HCV NS3 protein to bind to the viral RNA. In addition,
amino acid substitution at Lys1236 [NS3(K1236E)] decreased RNA
binding to about 30% (by the filter binding assay) to 40% (by
Northwestern analysis) of the wild-type level (Fig. 5). These results
suggested a sequential binding mechanism of ATP and RNA to the HCV NS3
helicase. Substitution at Lys1236 drastically reduced the ATP-binding
activity and rendered NS3 helicase in a conformation not suitable for
RNA binding. This is similar to the mechanisms proposed for eIF4A
(30) and PcrA (44); binding of ATP results in a
conformational change in the helicases that allows RNA binding and
induces ATP hydrolysis.
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FIG. 5.
Both mutations at Lys1236 and Arg1490 affected the
RNA-binding activity of HCV NS3 protein. (A) Filter binding assay. An
RNA-binding reaction was performed as previously described
(17) with 1 µmole of 32P-labeled HCV 3'CNU RNA
(5) and 10 nmole of proteins NS3 (sample 1), NS3(K1236E)
(sample 2), NS3(GR1490DT) (sample 3), BSA (sample 4), and a host
protein preparation (Host) (sample 5) as indicated. Input RNA (10%),
10% of the total reaction mixture spotted directly onto a
nitrocellulose membrane; Bound RNA, 90% of the reaction mixture from
which the unbound RNA has been removed. Radioactivity on the
nitrocellulose membranes was counted with a scintillation counter
(Beckman LS6000TA). The percentage of HCV 3'CNU RNA bound to the
wild-type NS3 protein was normalized to 100%, and the relative
RNA-binding activity of each of the NS3 mutants and control proteins
was calculated. Each column represents the average of three independent
experiments. Standard deviation bars are shown. (B) Northwestern
analysis. Two identical sets of the indicated proteins were resolved on
an SDS-polyacrylamide gel in Tricine-Tris buffer and analyzed,
respectively, by Coomassie blue staining (top) and Northwestern
analysis with the [-32P]UTP-labeled HCV 3'CNU RNA
(bottom). Northwestern analysis was performed as previously described
(3). Radioactivity of the bound RNA was measured by a
PhosphorImager (STORM 840; Molecular Dynamics), and the relative
RNA-binding activity of the mutant and control proteins to the
wild-type NS3 protein was calculated as indicated.
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In agreement with our results of the roles of the arginine-rich motif
of HCV NS3, previous studies have demonstrated that
the conserved motif
of eIF4A is required for ATP hydrolysis and
RNA binding
(
30). In addition, crystal structure analysis and
computer
graphics modeling indicated that the guanidinium groups
of Arg1487,
-1490, and -1493 in the arginine-rich motif of the
HCV NS3 helicase are
ideally situated to bind the phosphate groups
of an RNA substrate
(
6,
47). However, an electrophoretic
mobility shift assay
performed in a previous study with the C-terminal
three-fourths of the
HCV NS3 protein and a nonviral RNA probe
in vitro transcribed from a
PvuII-linearized pGEM3 suggested that
Arg1488 is the
only arginine residue in motif VI (1486-QRRGRTGR-1493)
involved in RNA binding (
25).
To understand the possible role of Arg1488 in binding to HCV RNA,
plasmids pET15b-NS3(R1488A) and pET15b-NS3(R1490A)
encoding
NS3 proteins with single amino acid substitutions at Arg1488
[designated
NS3(R1488A)] and -1490 [designated
NS3(R1490A)], respectively,
were further generated and expressed
in
Escherichia coli BL21(DE3)
(data not shown). Mutant
proteins NS3(R1488A) and NS3(R1490A)
were purified from
the insoluble fractions of the bacterial lysates
(Fig.
6A and
B) and analyzed for binding activity to
the HCV 3'CNU
RNA (
5). When compared to the wild-type NS3
protein, NS3(R1490A)
demonstrated a moderate binding activity
whereas the Arg1488 mutation
reduced the viral RNA-binding activity to
15% of that of the wild
type (Fig.
6C).
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FIG. 6.
The Arg1488 residue is important for the RNA-binding
activity of HCV NS3 protein. HCV NS3 mutant proteins NS3(R1488A)
and NS3(R1490A) were produced from plasmids
pET15b-NS3(R1488A) and pET15b-NS3(R1490A), respectively.
Expression and purification of the His-tagged mutant proteins followed
the procedures described in the legend to Fig. 1B. Three identical sets
of the BSA control and purified wild-type and mutant NS3 proteins as
indicated were resolved on an SDS-polyacrylamide gel and analyzed by
Coomassie blue staining (A), Western blot analysis with a monoclonal
antibody against the 6XHis-tag (CLONTECH) (B), and Northwestern
analysis with the [-32P]UTP-labeled HCV 3'CNU RNA
probe described in the legend to Fig. 5B (C).
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Previous analyses of crystal structure and computer graphics modeling
have suggested that Arg1490 is involved in RNA binding
(
6,
47). However, the studies did not reveal the important
role of
Arg1488 in interacting with RNA substrate, which conflicted
with the
functional study indicating that Arg1488 was the only
arginine residue
in the motif binding to a nonviral RNA (
25).
Although
nonspecific RNAs have been used to study RNA-binding
characteristics of
many viral proteins, the use of a specific
viral RNA probe should
provide a condition better mimicking the
natural environment. Our
results indicate that both Arg1488 and
1490 are involved in binding to
the HCV RNA. Taken together, the
present study clearly demonstrated
that the conserved lysine residues
in the AX
4GKS motif
is critical for ATP binding, whereas Arg1488
and -1490 in the
arginine-rich motif (1486-QRRGRTGR-1493) are
important for
the HCV NS3 protein to interact with the viral
RNA.
Nucleotide sequence accession number.
The cDNA
sequence encoding the HCV NS3 protein has been deposited in EMBL
under accession no. AJ238652.
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ACKNOWLEDGMENTS |
We thank Shu-Chen Chu and Jui-Hung Yen for technical assistance.
This work was supported in part by research grants
NSC84-2331-B-002-015-MH and NSC87-2314-B-002-184 to S.C.C. from the
National Science Council of the Republic of China and NHRI-GT-EX89S723L to M.-F.C. from the National Health Research Institutes of the Republic
of China.
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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:
886-2-23123456, 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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REFERENCES |
| 1.
|
Alter, H. J.,
R. H. Purcell,
J. W. Shih,
J. C. Melpolder,
M. Houghton,
Q.-L. Choo, and G. Kuo.
1989.
Detection of antibody to hepatitis C virus in prospectively followed transfusion recipients with acute and chronic non-A, non-B hepatitis.
N. Engl. J. Med.
321:1494-1500[Abstract].
|
| 2.
|
Bartenschlager, R.,
L. Ahlborn-Laake,
J. Mous, and H. Jacobsen.
1993.
Nonstructural protein 3 of the hepatitis C virus encodes a serine-type proteinase required for cleavage at the NS3/4 and NS4/5 junctions.
J. Virol.
67:3835-3844[Abstract/Free Full Text].
|
| 3.
|
Chang, M.-F.,
S. C. Baker,
L. H. Soe,
T. Kamahora,
J. G. Keck,
S. Makino,
S. Govindarajan, and M. M. C. Lai.
1988.
Human hepatitis delta antigen is a nuclear phosphoprotein with RNA-binding activity.
J. Virol.
62:2403-2410[Abstract/Free Full Text].
|
| 4.
|
Chang, M.-F.,
C.-Y. Sun,
C.-J. Chen, and S. C. Chang.
1993.
Functional motifs of delta antigen essential for RNA binding and replication of hepatitis delta virus.
J. Virol.
67:2529-2536[Abstract/Free Full Text].
|
| 5.
|
Cheng, J.-C.,
M.-F. Chang, and S. C. Chang.
1999.
Specific interaction between the hepatitis C virus NS5B RNA polymerase and the 3' end of the viral RNA.
J. Virol.
73:7044-7049[Abstract/Free Full Text].
|
| 6.
|
Cho, H.-S.,
N.-C. Ha,
L.-W. Kang,
K. M. Chung,
S. H. Back,
S. K. Jang, and B.-H. Oh.
1998.
Crystal structure of RNA helicase from genotype 1b hepatitis C virus: a feasible mechanism of unwinding duplex RNA. J. Bio1.
Chem.
273:15045-15052.
|
| 7.
|
Choo, Q.-L.,
G. Kuo,
A. J. Weiner,
L. R. Overby,
D. W. Bradley, and M. Houghton.
1989.
Isolation of a cDNA clone derived from blood-borne non-A, non-B viral hepatitis genome.
Science
244:359-362[Abstract/Free Full Text].
|
| 8.
|
Choo, Q.-L.,
K. H. Richman,
J. H. Han,
K. Berger,
C. Lee,
C. Dong,
C. Gallegos,
D. Coit,
A. Medina-Selby,
P. J. Barr,
A. J. Weiner,
D. W. Bradley,
G. Kuo, and M. Houghton.
1991.
Genetic organization and diversity of the hepatitis C virus.
Proc. Natl. Acad. Sci. USA
88:2451-2455[Abstract/Free Full Text].
|
| 9.
|
Di Bisceglie, A. M.
1995.
Hepatitis C and hepatocellular carcinoma.
Semin. Liver Dis.
15:64-69[Medline].
|
| 10.
|
Failla, C.,
L. Tomei, and R. De Francesco.
1994.
Both NS3 and NS4A are required for proteolytic processing of hepatitis C virus nonstructural proteins.
J. Virol.
68:3753-3760[Abstract/Free Full Text].
|
| 11.
|
Gallinari, P.,
D. Brennan,
C. Nardi,
M. Brunetti,
L. Tomei,
C. Steinkuhler, and R. De Francesco.
1998.
Multiple enzymatic activities associated with recombinant NS3 protein of hepatitis C virus.
J. Virol.
72:6758-6769[Abstract/Free Full Text].
|
| 12.
|
Gorbalenya, A. E., and E. V. Koonin.
1993.
Helicases: amino acid sequence comparisons and structure-function relationships.
Curt. Opin. Struct. Biol.
3:419-429.
|
| 13.
|
Gorbalenya, A. E.,
E. V. Koonin,
A. P. Donchenko, and V. M. Blinov.
1989.
Two related superfamilies of putative helicases involved in replication, recombination, repair and expression of DNA and RNA genomes.
Nucleic Acids Res.
17:4713-4730[Abstract/Free Full Text].
|
| 14.
|
Grakoui, A.,
D. W. McCourt,
C. Wychowski,
S. M. Feinstone, and C. M. Rice.
1993.
A second hepatitis C virus-encoded proteinase.
Proc. Natl. Acad. Sci. USA
90:10583-10587[Abstract/Free Full Text].
|
| 15.
|
Grakoui, A.,
D. W. McCourt,
C. Wychowski,
S. M. Feinstone, and C. M. Rice.
1993.
Characterization of hepatitis C virus-encoded serine proteinase: determination of proteinase-dependent polyprotein cleavage sites.
J. Virol.
67:2832-2843[Abstract/Free Full Text].
|
| 16.
|
Grakoui, A.,
C. Wychowski,
C. Lin,
S. M. Feinstone, and C. M. Rice.
1993.
Expression and identification of hepatitis C virus polyprotein cleavage products.
J. Virol.
67:1385-1395[Abstract/Free Full Text].
|
| 17.
|
Gwack, Y.,
D. W. Kim,
J. H. Han, and J. Choe.
1996.
Characterization of RNA binding activity and RNA helicase activity of the hepatitis C virus NS3 protein.
Biochem. Biophys. Res. Commun.
225:654-659[CrossRef][Medline].
|
| 18.
|
Heilek, G. M., and M. G. Peterson.
1997.
A point mutation abolishes the helicase but not the nucleoside triphosphatase activity of hepatitis C virus NS3 protein.
J. Virol.
71:6264-6266[Abstract].
|
| 19.
|
Hijikata, M.,
N. Kato,
Y. Ootsuyama,
M. Nakagawa, and K. Shimotohno.
1991.
Gene mapping of the putative structural region of the hepatitis C virus genome by in vitro processing analysis.
Proc. Natl. Acad. Sci. USA
88:5547-5551[Abstract/Free Full Text].
|
| 20.
|
Hijikata, M.,
H. Mizushima,
T. Akagi,
S. Mori,
N. Kakiuchi,
N. Kato,
T. Tanaka,
K. Kimura, and K. Shimotohno.
1993.
Two distinct proteinase activities required for the processing of a putative nonstructural precursor protein of hepatitis C virus.
J. Virol.
67:4665-4675[Abstract/Free Full Text].
|
| 21.
|
Hijikata, M.,
H. Mizushima,
Y. Tanji,
Y. Komoda,
Y. Hirowatari,
T. Akagi,
N. Kato,
K. Kimuran, and K. Shimotohno.
1993.
Proteolytic processing and membrane association of putative nonstructural proteins of hepatitis C virus.
Proc. Natl. Acad. Sci. USA
90:10773-10777[Abstract/Free Full Text].
|
| 22.
|
Hirling, H.,
M. Scheffner,
T. Restle, and H. Stahl.
1989.
RNA helicase activity associated with the human p68 protein.
Nature
339:562-564[CrossRef][Medline].
|
| 23.
|
Kato, N.,
M. Hijikata,
Y. Ootsuyama,
M. Nakagawa,
S. Ohkohi,
T. Sugimura, and K. Shimotohno.
1990.
Molecular cloning of the human hepatitis C virus genome from Japanese patients with non-A, non-B hepatitis.
Proc. Natl. Acad. Sci. USA
87:9524-9528[Abstract/Free Full Text].
|
| 24.
|
Kim, D. W.,
Y. Gwack,
J. H. Han, and J. Choe.
1995.
C-terminal domain of the hepatitis C virus NS3 protein contains an RNA helicase activity.
Biochem. Biophys. Res. Commun.
215:160-166[CrossRef][Medline].
|
| 25.
|
Kim, D. W.,
J. Kim,
Y. Gwack,
J. H. Han, and J. Choe.
1997.
Mutational analysis of the hepatitis C virus RNA helicase.
J. Virol.
71:9400-9409[Abstract].
|
| 26.
|
Kim, J. L.,
K. A. Morgenstern,
J. P. Griffith,
M. D. Dwyer,
J. A. Thomson,
M. A. Murcko,
C. Lin, and P. R. Caron.
1998.
Hepatitis C virus NS3 RNA helicase domain with a bound oligonucleotide: the crystal structure provides insights into the mode of unwinding.
Structure
6:89-100[Medline].
|
| 27.
|
Kuo, G.,
Q.-L. Choo,
H. J. Alter,
G. L. Gitnick,
A. G. Redeker,
R. H. Purcell,
T. Miyamura,
J. L. Dienstag,
M. J. Alter,
C. E. Stevens,
G. E. Tegtmeier,
F. Bonino,
M. Colombo,
W.-S. Lee,
C. Kuo,
K. Berger,
J. R. Shuster,
L. R. Overby,
D. W. Bradley, and M. Houghton.
1989.
An assay for circulating antibodies to a major etiologic virus of human non-A, non-B hepatitis.
Science
244:362-364[Abstract/Free Full Text].
|
| 28.
|
Lin, C., and J. L. Kim.
1999.
Structure-based mutagenesis study of hepatitis C virus NS3 helicase.
J. Virol.
73:8798-8807[Abstract/Free Full Text].
|
| 29.
|
Lin, C.,
B. D. Lindenbach,
B. M. Pragai,
D. W. McCourt, and C. M. Rice.
1994.
Processing in the hepatitis C virus E2-NS2 region: identification of p7 and two distinct E2-specific products with different C termini.
J. Virol.
68:5063-5073[Abstract/Free Full Text].
|
| 30.
|
Pause, A.,
N. Methot, and N. Sonenberg.
1993.
The HRIGRXXR region of the DEAD box RNA helicase eukaryotic translation initiation factor 4A is required for RNA binding and ATP hydrolysis.
Mol. Cell. Biol.
13:6789-6798[Abstract/Free Full Text].
|
| 31.
|
Pause, A., and N. Sonenberg.
1992.
Mutational analysis of a DEAD box RNA helicase: the mammalian translation initiation factor elF-4A.
EMBO J.
11:2643-2654[Medline].
|
| 32.
|
Porter, D. J. T.
1998.
A kinetic analysis of the oligonucleotide-modulated ATPase activity of the helicase domain of the NS3 protein from hepatitis C virus: the first cycle of interaction of ATP with the enzyme is unique.
J. Biol. Chem.
273:14247-14253[Abstract/Free Full Text].
|
| 33.
|
Porter, D. J. T.,
S. A. Short,
M. H. Hanlon,
F. Preugschat,
J. E. Wilson,
D. H. Willard, Jr., and T. G. Consler.
1998.
Product release is the major contributor to kcat for the hepatitis virus helicase-catalyzed strand separation of short duplex DNA.
J. Biol. Chem.
273:18906-18914[Abstract/Free Full Text].
|
| 34.
|
Preugschat, F.,
D. R. Averett,
B. E. Clarke, and D. J. T. Porter.
1996.
A steady-state and pre-steady-state kinetic analysis of the NTPase activity associated with the hepatitis C virus NS3 helicase domain.
J. Biol. Chem.
271:24449-24457[Abstract/Free Full Text].
|
| 35.
|
Rozen, F.,
J. Pelletier,
H. Trachsel, and N. Sonenberg.
1989.
A lysine substitution in the ATP-binding site of eukaryotic initiation factor 4A abrogates nucleotide-binding activity.
Mol. Cell. Biol.
9:4061-4063[Abstract/Free Full Text].
|
| 36.
|
Saito, I.,
T. Miyamura,
A. Ohbayashi,
H. Harada,
T. Katayama,
S. Kikuchi,
Y. Watanabe,
S. Koi,
M. Onji,
Y. Ohta,
Q.-L. Choo,
M. Houghton, and G. Kuo.
1990.
Hepatitis C virus infection is associated with the development of hepatocellular carcinoma.
Proc. Natl. Acad. Sci. USA
87:6547-6549[Abstract/Free Full Text].
|
| 37.
|
Schmid, S. R., and P. Linder.
1992.
D-E-A-D protein family of putative RNA helicases.
Mol. Microbiol.
6:283-292[Medline].
|
| 38.
|
Selby, M. J.,
Q.-L. Choo,
K. Berger,
G. Kou,
E. Glazer,
M. Eckart,
C. Lee,
D. Chien,
C. Kuo, and M. Houghton.
1993.
Expression, identification and subcellular localization of the proteins encoded by the hepatitis C viral genome.
J. Gen. Virol.
74:1103-1113[Abstract/Free Full Text].
|
| 39.
|
Subramanya, H. S.,
L. E. Bird,
J. A. Brannigan, and D. B. Wigley.
1996.
Crystal structure of a DExx box DNA helicase.
Nature
384:379-383[CrossRef][Medline].
|
| 40.
|
Suzich, J. A.,
J. K. Tamura,
F. Palmer-Hill,
P. Warrener,
A. Grakoui,
C. M. Rice,
S. M. Feinstone, and M. S. Collett.
1993.
Hepatitis C virus NS3 protein polynucleotide-stimulated nucleoside triphosphatase and comparison with the related pestivirus and flavivirus enzymes.
J. Virol.
67:6152-6158[Abstract/Free Full Text].
|
| 41.
|
Tai, C.-L.,
W.-K. Chi,
D.-S. Chen, and L.-H. Hwang.
1996.
The helicase activity associated with hepatitis C virus nonstructural protein 3 (NS3).
J. Virol.
70:8477-8484[Abstract].
|
| 42.
|
Takamizawa, A.,
C. Mori,
I. Fuke,
S. Manabe,
S. Murakami,
J. Fujita,
E. Onishi,
T. Andoh,
I. Yoshida, and H. Okayama.
1991.
Structure and organization of the hepatitis C virus genome isolated from human carriers.
J. Virol.
65:1105-1113[Abstract/Free Full Text].
|
| 43.
|
Tomei, L.,
C. Failla,
E. Santolini,
R. De Francesco, and N. La Monica.
1993.
NS3 is a serine protease required for processing of hepatitis C virus polyprotein.
J. Virol.
67:4017-4026[Abstract/Free Full Text].
|
| 44.
|
Velankar, S. S.,
P. Soultanas,
M. S. Dillingham,
H. S. Subramanya, and D. B. Wigley.
1999.
Crystal structures of complexes of PcrA DNA helicase with a DNA substrate indicate an inchworm mechanism.
Cell
97:75-84[CrossRef][Medline].
|
| 45.
|
Walker, J. E.,
M. Saraste,
M. J. Runswick, and N. J. Gay.
1982.
Distantly related sequences in the - and -subunits of ATP synthase, myosin, kinases and other ATP-requiring enzymes and a common nucleotide binding fold.
EMBO J.
1:945-951[Medline].
|
| 46.
|
Warrener, P.,
J. K. Tamura, and M. S. Collett.
1993.
RNA-stimulated NTPase activity associated with yellow fever virus NS3 protein expressed in bacteria.
J. Virol.
67:989-996[Abstract/Free Full Text].
|
| 47.
|
Yao, N.,
T. Hesson,
M. Cable,
Z. Hong,
A. D. Kwong,
H. V. Le, and P. C. Weber.
1997.
Structure of the hepatitis C virus RNA helicase domain.
Nat. Struct. Biol.
4:463-467[CrossRef][Medline].
|
| 48.
|
Yarranton, G. T., and M. L. Gefter.
1979.
Enzyme-catalyzed DNA unwinding: studies on Escherichia coli rep protein.
Proc. Natl. Acad. Sci. USA
76:1658-1662[Abstract/Free Full Text].
|
| 49.
|
Yen, J.-H.,
S. C. Chang,
C.-R. Hu,
S.-C. Chu,
S.-S. Lin,
Y.-S. Hsieh, and M.-F. Chang.
1995.
Cellular proteins specifically bind to the 5'-noncoding region of hepatitis C virus RNA.
Virology
208:723-732[CrossRef][Medline].
|
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Copyright © 2000, American Society for Microbiology. All rights reserved.
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