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INTRODUCTION |
The hepatitis C virus
(HCV)1 is an RNA virus that
causes acute and chronic liver disease (for reviews see Refs. 1-3). A
high proportion of infected patients fail to clear the virus and
contract chronic infection, which may lead to liver cirrhosis and,
eventually, to hepatocellular carcinoma. Currently it is estimated that
100-200 million people worldwide suffer from a chronic HCV infection. Thus, HCV became a focus of intensive research worldwide.
Based on similarities of genome organization and virus particle
structure with the flaviviruses like yellow fever virus and the animal
pathogenic pestiviruses like the classical swine fever virus (CSFV),
HCV has been classified as the separate genus Hepacivirus in
the family Flaviviridae (4). These viruses have in common an enveloped
virus particle and a single-stranded RNA genome of positive polarity
encoding a polyprotein that is cleaved by host cell signalases and
viral proteinases. In the case of HCV, the genome has a length of
~9600 nucleotides. Its single long open reading frame is flanked at
the 5'- and 3'-ends by nontranslated regions of about 340 and 230 nucleotides in length, respectively. The 5' nontranslated region is
important for efficient translation of the viral polyprotein and
functions as an internal ribosome entry site (5-7). The 3'
nontranslated region most likely required for RNA replication carries a
3'-terminal highly conserved sequence forming stable secondary and
probably also higher order structures (8-11).
HCV polyprotein processing is accomplished by a combination of host and
viral proteinases (for reviews, see Refs. 2, 12, and 13). At least 10 different cleavage products could be identified, which are aligned
within the polyprotein as follows (from the amino to the carboxyl
terminus): NH2-C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B-COOH (14-21). The structural proteins C-E2 are the major constituents of
the virus particle, whereas the nonstructural proteins 2-5B most
likely are required for RNA replication.
While the mechanisms of polyprotein processing have been studied in
great detail, our knowledge about HCV replication is scarce. This is
essentially due to the lack of robust cell culture systems allowing
efficient virus propagation and the lack of an animal model other than
the chimpanzee. By analogy to other plus strand RNA viruses, it is
assumed that after infection of the host cell the viral RNA is
liberated into the cytoplasm and used for synthesis of the viral
proteins. These, most likely in conjunction with cellular proteins,
assemble into a membrane-associated replicase complex responsible for
the multiplication of the viral RNA via a minus strand RNA
intermediate. At least two viral proteins most likely are directly
involved in this reaction: NS3 carrying in the carboxyl-terminal domain
an NTPase/helicase activity (22-25) and NS5B, the
RNA-dependent RNA polymerase (26-30).
In an attempt to set up appropriate in vitro systems
permitting a detailed analysis of HCV replication, we have recently
developed a simple expression and purification system allowing the
production of large quantities of an enzymatically active NS5B protein
carrying a carboxyl-terminal hexahistidine affinity tag (27). In
agreement with reports by other groups, we observed with the purified
protein a primer-dependent RNA polymerase activity able to
processively copy homo- and heteropolymeric templates without
detectable requirement for additional cofactors and without template
specificity (26-30). On heteropolymeric templates, initiation of RNA
synthesis is primed via a "copy-back" mechanism; i.e. 3'
terminal sequences fold back intramolecularly, creating a
template-bound primer, which is elongated to generate an RNA product
twice the length of the input template. Only in the case of
homopolymeric templates or templates with a 3' homopolymeric tract,
which cannot form intramolecular base pairings, the enzyme requires the
addition of complementary exogenous primers (26-30).
Using an intensive mutation analysis, we identified four amino acid
sequence motifs within NS5B crucial for enzymatic activity (27). These
motifs, designated A-D (31), are located in the central NS5B domain
and highly conserved between RdRps of plus strand RNA viruses. Motif A,
most likely involved in NTP binding and catalysis, is most sensitive
toward substitutions (27). Motif B probably is involved in template
and/or primer positioning and characterized by an invariant glycine
residue absolutely essential for enzymatic activity. The well known
motif C, according to the primary sequence also designated the GDD
motif, is important for NTP binding and catalysis, with the first
aspartic acid residue being least tolerant toward substitutions. In
contrast, alterations affecting the glycine and the second aspartic
acid residue are much better tolerated, leading to enzymes with still
well detectable RdRp activities (27). Motif D, probably also involved
in NTP binding and catalysis, for most RdRps and reverse transcriptases is characterized by a highly conserved lysine residue, whereas an
arginine residue is found for most HCV isolates. Interestingly, a
lysine substitution for the arginine increases RdRp activity by about
50% (27).
In this report, we continue our biochemical characterization of
HCV NS5B. We demonstrate that RdRp activity is selectively stimulated
by up to 2 orders of magnitude by high concentrations of GTP. We
present evidence for the specificity of this effect and show that
GTP-mediated RdRp activation is not due to an increase of RNA binding
or the elongation rate, suggesting that high concentrations of GTP
accelerate a rate-limiting step during the early phase of RNA
synthesis. Finally, we found that NS5B of CSFV can be stimulated by GTP
with comparable efficiency but not the 3D polymerase of poliovirus.
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EXPERIMENTAL PROCEDURES |
Materials--
All radiolabeled nucleotides (3000 Ci/mmol) were
purchased from Amersham Pharmacia Biotech, and all unlabeled
ribonucleotides were from Roche Molecular Biochemicals at the highest
quality available. GDP and GMP (disodium salts) and heparin 6000 were obtained from Sigma, homopolymeric RNA templates (average length 400 nucleotides) from Amersham Pharmacia Biotech, and 12-mer RNA oligonucleotides from MWG-Biotech. Mung bean nuclease was purchased from Biolabs, T7 RNA polymerase from Epicenter, and RNasin and RNase-free DNase from Promega.
Construction of Plasmids and Generation of Recombinant
Baculoviruses--
Construction of the plasmid used for expression of
an NS5B (residues 2421-3010 of the HCV-BK polyprotein (32)) carrying a
hexahistidine affinity tag at the carboxyl terminus has been described
(27). Plasmid pBacCSFV-5BHis allowing the expression of a
full-length NS5B of the classical swine fever virus (33) under control
of the polyhedrin promoter was obtained by insertion of an
NcoI/XbaI polymerase chain reaction fragment
covering the complete NS5B sequence into a modified baculovirus transfer vector (27) restricted with NcoI and
SpeI. After sequence analysis of the transferred polymerase
chain reaction fragment, the plasmid was used to generate a recombinant
baculovirus as described recently (27). The resulting CSFV NS5B
contained the hexahistidine epitope at the amino terminus.
Cell Culture and Protein Purification--
Expression of NS5B
proteins of HCV and CSFV in ovary cells of Trichoplusia ni
(High 5 cells) and purification of proteins via differential extraction
of cell lysates and subsequent affinity chromatography was performed as
described recently (27). The purity of both proteins was higher than
90% as determined by densitometry scanning of Coomassie Blue-stained
protein gels. Purified 3Dpol of poliovirus was a generous
gift from E. Wimmer and A. Paul (State University of New York, Stony
Brook, NY) and was purified as described elsewhere (34).
Preparation of RNAs for in Vitro Assays--
Full-length HCV RNA
was transcribed from plasmid pAT1-9604 (27) containing a complete HCV
genome downstream of the promoter for the RNA polymerase of
bacteriophage T7. After linearization of the plasmid downstream of the
HCV insert with XbaI, nucleotides corresponding to linker
sequences were removed by treatment with mung bean nuclease as
specified by the manufacturer. The DNA was purified by extraction with
phenol and chloroform and concentrated by precipitation with ethanol,
and 5 µg were used for a 100-µl in vitro transcription
reaction containing 80 mM HEPES, pH 7.5, 12.5 mM MgCl2, 2 mM spermidine, 40 mM dithiothreitol, 3 mM NTPs, 160 units of
RNasin, and 250 units of T7 RNA polymerase. After 2 h at 37 °C,
125 units of T7 RNA polymerase were added; the reaction was incubated
for a further 2 h, treated with 5 units of RNase-free DNase for 15 min, and extracted with phenol/chloroform; and RNA was precipitated
with isopropyl alcohol at room temperature. The pellet was resolved in
50 µl of diethyl pyrocarbonate-treated water, and remaining
unincorporated nucleotides were removed by gel filtration using a G50
NICK column (Amersham Pharmacia Biotech). Integrity of the RNA was
analyzed by denaturing formaldehyde-agarose gel electrophoresis, and
concentration was determined by optical density measurement.
RdRp Assays--
RdRp assays were performed essentially as
described (27). In brief, a 25-µl reaction was set up containing 20 mM Tris-HCl (pH 7.0), 12.5 mM
MgCl2, 10 mM KCl, 1 mM EDTA, 500 µM ATP and UTP, 10 µM CTP, 10-50 µCi of
[
-32P]CTP, 3 mM dithiothreitol, 20 units
of RNasin, 500 ng of heteropolymeric RNA or 0.4 µg of homopolymeric
primer-template mixture, 200 ng of purified NS5B of HCV or 1 µg of
NS5B of CSFV, and various concentrations of GTP. Unless otherwise
stated, samples were incubated for 2 h at 22 °C. To compensate
for the lower specific activity of NS5B mutants and CSFV-5B, analogous
reactions were performed containing only 1 µM CTP. In the
case of titrations of GMP and GDP, GTP concentrations were lowered to 1 µM. When poly(A)-oligo(U)12 was used as
template-primer, reactions contained 10 µCi of
[
-32P]UTP adjusted to a final concentration of 10 µM UTP. For analysis of reaction products by gel
electrophoresis, the enzyme was inactivated by the addition of 50 µl
of PK buffer (300 mM NaCl, 100 mM Tris-HCl (pH
7.5), 1% (w/v) SDS, and 20 µg of tRNA), 25 µl of diethyl
pyrocarbonate-treated water, and 50 µg of proteinase K (Roche
Molecular Biochemicals). After 30 min at 37 °C, samples were
extracted with phenol/chloroform, and RNAs were precipitated with
isopropyl alcohol. Precipitates were resolved in 25 µl of gel buffer
(40 mM MOPS (pH 7.0), 10 mM sodium acetate, 1 mM EDTA, 50% (v/v) formamide, 2.2 M
formaldehyde), heated to 55 °C for 15 min, and chilled on ice, and 1 µl of ethidium bromide (10 mg/ml) was added. After a 10-min
incubation at room temperature, 5 µl of loading buffer (50%
glycerol, 0.25% bromphenol blue, 0.25% xylenecyanol, 1 mM
EDTA) was added, and samples were loaded onto an agarose gel containing
2.2 M formaldehyde, 40 mM MOPS (pH 7.0), 10 mM sodium acetate, and 1 mM EDTA.
Electrophoresis was performed at 5 V/cm. Gels were dried and analyzed
using an BAS 2500 BioImage analyzer system (Fuji) or x-ray films
(Eastman Kodak Co.). To determine incorporation of radioactivity,
reactions were stopped by the addition of 100 µg of calf thymus DNA
and 1 ml of 10% trichloroacetic acid, 0.5% tetrasodium pyrophosphate. After a 30-min incubation on ice, samples were filtered through GF/C
glass microfiber filters (Whatman); filters were washed five times with
1% trichloroacetic acid, 0.1% tetrasodium pyrophosphate; and bound
radioactivity was determined by liquid scintillation counting. RdRp
assays with 3Dpol were done as described elsewhere (35).
Fold stimulation of RNA synthesis was calculated from the ratio of cpm
obtained with NS5B or 3Dpol at a given NTP concentration
and at 10 µM NTP (cpm (X mM
NTP):cpm (10 µM NTP)).
Determination of Elongation Rates--
Elongation rate was
determined as described recently with minor modifications (36). In
brief, RdRp assays were performed as described above with the
difference that NS5B and RNA template were incubated for 5 min at room
temperature in 20 mM Tris-HCl (pH 7.0), 12.5 mM
MgCl2, 10 mM KCl, and 1 mM EDTA
prior to the addition of the reaction mixture. To inhibit further
initiation of RNA synthesis during the reaction, heparin 6000 was
included at a final concentration of 1 mg/ml. Reactions were terminated at various times and processed for denaturing formaldehyde-agarose gel
electrophoresis as described above.
RNA Binding Assay--
75 ng of purified NS5B were incubated
with 160 ng in vitro transcribed full-length HCV RNA
supplemented with the analogous radiolabeled RNA (200,000 cpm; specific
activity ~2.5 × 109 cpm/µg) in 50 mM
HEPES (pH 7.5), 7 mM MgCl2, 5 mM
2-mercaptoethanol, 20 units of RNasin, and various concentrations of
GTP in a total volume of 100 µl. Under these conditions, ~30% of
the input RNA was bound. After a 1-h incubation at 22 °C, samples
were diluted to 1 ml with TE (10 mM Tris-HCl (pH 7.5), 1 mM EDTA) and filtered through 0.45-µm pore size
nitrocellulose filter disks (BA 85; Schleicher & Schüll), which
had been boiled for 30 min in TE prior to use. Bound radioactivity was
determined by liquid scintillation counting. Background as determined
by an analogous titration with bovine serum albumin was subtracted.
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RESULTS |
Expression and Purification of HCV and CSFV NS5B
Polypeptides--
The method we developed for purification of HCV NS5B
carrying a carboxyl-terminal hexahistidine affinity tag is based on the observation that in contrast to most cellular and baculovirus proteins,
NS5B is poorly soluble under physiological conditions. However, it can
be efficiently extracted from cell lysates with buffers containing high
concentrations of salt, glycerol, and detergent (27). Therefore,
lysates of infected insect cells were repeatedly extracted with buffers
containing increasing concentrations of these ingredients, and soluble
proteins contained in the supernatants were discarded (Fig.
1, lanes 2 and
3). After three extractions, NS5B was strongly enriched in
supernatant 3 (S3; lane 4) and used for further purification by affinity chromatography. To compare the
biochemical properties of HCV NS5B with the analogous protein of the
closely related pestivirus CSFV, we constructed two recombinant baculoviruses directing the expression of NS5B of the CSFV Alfort/187 strain (33) carrying a hexahistidine affinity tag either at the amino
or the carboxyl terminus. Both proteins could be purified in an
enzymatically active form using the method developed for HCV-5B.2 As exemplified for
the amino-terminal fusion protein, the differential extraction of the
cell lysate allowed a strong enrichment of the protein in supernatant
3, and it could be further purified by affinity chromatography
(lanes 10 and 12, respectively).
Because the expression level of this protein was much higher than the one of the carboxyl-terminal fusion protein, the amino-terminal fusion
protein was used for the analyses described in this report. Purity of
this protein was >90% as was the case for HCV-5B.

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Fig. 1.
Expression of HCV- and CSFV-5B proteins in
insect cells and purification. Insect cells were infected with
recombinant baculoviruses directing the expression of the given NS5B
proteins. 72 h postinfection, cells were lysed with a hypotonic
buffer. After centrifugation, the supernatant (S1) was
discarded, and the pellet was extracted with a buffer containing
intermediate concentrations of salt, glycerol, and detergent. The
supernatant (S2) obtained after centrifugation was
discarded, and the pellet was extracted with a buffer containing high
concentrations of these components. The resulting supernatant
(S3) was applied to a nickel nitrilotriacetic acid spin
column, and the flow-through (Ft) was discarded. After
several washing steps and a nuclease treatment, proteins were eluted
with 250 mM imidazole (El). of each
fraction, corresponding to 4 × 105 cells, was
analyzed by SDS-11% polyacrylamide gel electrophoresis for HCV-5B or
SDS-10% polyacrylamide gel electrophoresis for CSFV-5B, and gels were
stained with Coomassie Brilliant Blue R-250. In the case of total cell
lysate (T) only was loaded onto the gels.
Numbers between the panels correspond
to the sizes of marker proteins (kDa).
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Stimulation of NS5B RdRp Activity by High Concentrations of
GTP--
We have recently described a detailed determination of the
kinetic constants of HCV-5B (36). During the course of these experiments, we observed a biphasic titration curve for GTP. At low
concentrations of GTP, the enzyme displays a standard Michaelis-Menten kinetic with a Km for GTP of 0.5 µM on
heteropolymeric templates and ~3.0 µM on the
poly(C)-oligo(G)12 homopolymer (36) (Fig.
2A). Surprisingly, a strong
stimulation of RdRp activity was found when GTP concentrations were
raised to beyond 100 µM. Using an in vitro
transcribed HCV full-length RNA ~9600 nucleotides in length as
template, a linear increase of RNA synthesis was found up to 5 mM GTP (Fig. 2A). Stimulation of RNA synthesis
was maximal at 10 mM GTP, whereas higher concentrations led
to an inhibition of the enzyme. Taking the amount of radioactive
incorporation at 10 µM GTP as reference (6 × 103 cpm), an ~40- and 55-fold stimulation of RdRp
activity was found at 5 and 10 mM GTP, respectively. It
should be noted that radioactive incorporation at 10 µM
GTP was rather low and somewhat variable. Therefore, stimulation rates
obtained in different experiments varied between 50 and 100 (see
below).

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Fig. 2.
Activation of NS5B RdRp activity by high
concentrations of GTP. A, a standard RdRp assay was
performed using an in vitro transcribed genomic HCV RNA and
increasing concentrations of GTP. After a 2-h incubation, of
the reaction was withdrawn, and, after trichloroacetic acid
precipitation on glas fiber filters, radioactive incorporation was
determined by liquid scintillation counting. For comparison, the
determination of the Km value for GTP (36) is given
in the insert in the upper right.
B, of the reaction was processed for denaturing
formaldehyde-agarose gel electrophoresis, and radiolabeled RNAs were
visualized by autoradiography. Numbers to the
left refer to the sizes of RNA marker (nucleotides).
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In agreement with the copy-back priming mechanism, analysis of the
reaction products on denaturing formaldehyde-agarose gels revealed that
up to 1 mM GTP the size of the majority of radiolabeled RNAs was larger than the input template (Fig. 2B,
lane 4). With higher GTP concentrations in
addition to an increase of these products, RNAs smaller than the input
became prominent. At GTP concentrations beyond 12.5 mM, the
sizes of all radiolabeled RNAs were significantly reduced, and only RNA
products about the size of the input template and RNAs ~2500-500
nucleotides in length were found (lanes 9-11).
This pattern indicated an inhibition of elongation or processivity at
very high concentrations of GTP with template-sized RNA products being
generated by "abortive" copy-back priming from the input template
and the small RNAs either from RNA primers present in the NS5B or the
RNA template preparation or, less likely, by de novo
initiation. Only under conditions of lower GTP concentrations would the
enzyme have been processive enough to copy the template completely
during the 2-h incubation period.
Several possibilities could be envisaged for the RdRp inhibition at
very high GTP concentrations: (i) a competitive inhibition because of
the high excess of GTP over the other NTPs; (ii) impurities present in
low amounts in the GTP preparation, or (iii) an insufficient concentration of Mg2+ due to the excess of GTP over
Mg2+. Because the latter possibility was the most likely,
the GTP titration was repeated, but this time for every GTP
concentration a 12.5 mM excess of MgCl2 was
used. Under these conditions, an almost identical stimulation of RdRp
activity with a maximum at 12.5 mM GTP was found (not
shown). At higher GTP concentrations, RNA synthesis still was reduced
but to a lower extent, suggesting that inhibition of RdRp activity at
high GTP concentrations in part was due to the excess of GTP over
Mg2+. Attempts to overcome this inhibition by further
increasing the Mg2+ concentration were unsuccessful,
because the enzyme is strongly inhibited by high concentrations of
MgCl2 (36).
To exclude the possibility that the observed effects were due to
contaminants in the GTP preparation used, several batches as well as
GTP preparations from other suppliers were tested including lithium and
sodium salts. In all cases, comparable stimulation rates were found
(not shown). In summary, these results show that RdRp activity of
HCV-5B is stimulated by high concentrations of GTP and that the
inhibition found at GTP concentrations >10 mM in part was
due to an imbalance of the GTP:Mg2+ concentrations. Since a
linear increase of RNA synthesis was found with GTP only up to 5 mM, most subsequent titrations were performed up to this
value using a constant concentration of MgCl2 (12.5 mM) shown to be optimal for our purified enzyme (36).
Selectivity of RdRp Stimulation--
To analyze whether
stimulation of RNA polymerase activity could also be mediated by other
NTPs, RdRp reactions were carried out with increasing concentrations of
ATP, CTP, and UTP. As shown in Fig.
3A, none of these nucleotides
could enhance RNA synthesis to the extent of GTP. Maximum stimulation
was found with ATP (~4-fold), whereas only a 3-fold increase of
enzymatic activity was observed with CTP and UTP (Fig. 3B).
Analysis of the reaction products by agarose gel electrophoresis
revealed that comparable with the results obtained with GTP, under
conditions of high NTP concentrations products about the size of the
input template and short RNAs (500-1500 nucleotides) accumulated
during the 2-h incubation period (Fig. 3C, lanes
4, 8, and 12). Thus, efficient
stimulation of RNA synthesis can only be achieved with GTP, whereas the
inhibition of the enzyme is found with high concentrations of either
NTP, further supporting the conclusion that it is due primarily to an
imbalance of the GTP:MgCl2 concentrations.

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Fig. 3.
Lack of RdRp stimulation by ATP, CTP, and
UTP. A, standard RdRp assays were performed with given
concentrations of ATP ( ), CTP ( ), UTP ( ), or GTP ( ). After
a 2-h incubation, incorporation of radioactivity was determined by
trichloroacetic acid precipitation and liquid scintillation counting.
B, RdRp assays were performed as in A with given
concentrations of ATP ( ), CTP ( ), or UTP ( ). of the
reaction mixture was subjected to trichloroacetic acid precipitation
and liquid scintillation counting, and was processed for
denaturing formaldehyde-agarose gel electrophoresis (C).
Radiolabeled products were visualized by autoradiography. In the case
of ATP and UTP titrations, reactions were performed with
[ -32P]CTP, while for the CTP titration
[ -32P]UTP was used as the radiolabeled nucleotide.
Numbers to the left refer to the sizes of RNA
markers (nucleotides). Note the different scales of the ordinates in
A and B.
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Most Amino Acid Substitutions in NS5B Reducing RdRp Activity Also
Affect GTP-mediated Stimulation of RNA Synthesis--
The experiments
described thus far suggest a direct stimulation of RdRp activity by
high concentrations of GTP. However, since the NS5B preparations used
in this study still contained very low amounts of contaminating
cellular proteins, we could not exclude the possibility that the
activation effect was related to a copurified contaminant. For example,
a GTPase in the enzyme preparation could lower the actual GTP
concentration, although in this case we would have been unable to
determine the Km for GTP (0.5 µM on
heteropolymeric templates; Fig. 2A; Ref. 36). Alternatively, a terminal
transferase activity, present in low amounts in the enzyme preparation
(27), might be stimulated by GTP, generating primer-templates that
might be used efficiently by NS5B. To exclude these possibilities, we
tested a panel of mutations in polymerase motifs B and C with reduced
RdRp activity and an arginine substitution in motif D that increased
enzymatic activity ~1.5-fold (Table I
and Fig. 4A; Ref. 27). A
significant reduction of GTP-mediated stimulation was found with all
substitutions affecting the GDD motif, whereas the mutation in motif D
had no effect (Table I). Two opposite phenotypes were found with the
mutations in motif B. While the valine substitution for the
nonconserved threonine at position 286 did not affect stimulation by
GTP, the cysteine substitution for the highly conserved threonine at
position 287 completely abolished this property.

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Fig. 4.
A single amino acid substitution in
polymerase motif B of NS5B abolishes GTP-mediated stimulation of RNA
synthesis. A, a schematic presentation of NS5B
indicating the positions of polymerase motifs A-D is given at the
top. The amino acid sequences of individual motifs highly
conserved among HCV isolates are shown below.
Capital letters indicate amino acid residues that
are highly conserved among RdRps of plus strand RNA viruses, and
underlined and boldface capital
letters refer to residues that are highly conserved among
all viral RdRps. Numbers above the
letters indicate the amino acid positions of NS5B,
arrows below the letters refer to
substituting residues (see Table I). B, standard RdRp assays
were performed with synthetic HCV genomic RNA and GTP concentrations
given above the lanes (mM). After
2 h at 22 °C, samples were processed for denaturing agarose gel
electrophoresis. Numbers between the
gels refer to the sizes of RNA markers (nucleotides).
5BC-His refers to the parental NS5B with the
carboxyl-terminal hexahistidine affinity tag.
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To further characterize the latter mutant, RdRp assays were performed
in parallel with the parental enzyme and an NS5B, which was
enzymatically inactive due to an asparagine substitution for the
invariant 318 aspartic acid. Analysis of the reaction products by
agarose gel electrophoresis revealed that RdRp activity of 5B287C was severely impaired and not stimulatable by GTP
(Fig. 4B). Only RNA products corresponding to the input
template and ~700 nucleotides in length were found, indicating a
strong processivity or elongation defect. This pattern was similar to
the one obtained with the parental NS5B at GTP concentrations of
20
mM GTP (Fig. 2B), and it suggested that the
~10-kilobase RNA corresponds to the input RNA template labeled at the
3'-end by abortive copy-back priming. The small RNAs probably represent
short copy-back primed molecules or molecules initiated from exogenous
primers or by de novo synthesis. In summary, the results
obtained with these altered NS5B proteins strongly argue against the
contribution of a cellular contaminant in the enzyme preparation, and
they suggest that high concentrations of GTP directly stimulate RNA synthesis by NS5B. Furthermore, the fact that all mutations affecting highly conserved residues assumed to contribute to the active site of
the enzyme can no longer be or can only poorly be stimulated by GTP
suggests that the same NTP-binding site is used by GTP for stimulation
and incorporation.
Evidence for Incorporation of the Stimulating GTP--
In the
experiments described so far, we always used an RNA template
corresponding to the HCV genome. To analyze whether GTP-mediated stimulation was dependent on this particular template, a panel of
hetero- and homopolymeric RNAs was characterized in the same way.
Comparable stimulation rates were found with an ~400-nucleotide-long HCV RNA corresponding to the 3'-end of the genome and with an in
vitro transcribed lacZ RNA (data not shown) showing
that GTP enhances RNA synthesis on very different heteropolymeric
templates. In the case of homopolymeric RNAs, a stimulation was only
found with poly(C)-oligo(G)12, whereas no activation was
observed with poly(A)-oligo(U)12 even at 5 mM
GTP (Fig. 5A) or UTP (data not shown), suggesting that the stimulating GTP has to be incorporated into
the RNA chain.

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Fig. 5.
Evidence that the stimulating nucleotide has
to be incorporated into the RNA. A, RdRp assays were
performed as described under "Experimental Procedures" using
poly(C)-oligo(G)12 ( ) or poly(A)-oligo(U)12
( ) and given concentrations of GTP. Incorporation of radioactivity
was determined by trichloroacetic acid precipitation and liquid
scintillation counting. Because of the isotope dilution, GTP
concentrations higher than 1 mM were not tested with the
poly(C)-oligo(G)12 template. B, effect of
increasing concentrations of GMP ( ) and GDP ( ) on RdRp activity
using synthetic full-length HCV RNA and 1 µM GTP (see
"Experimental Procedures").
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To further substantiate this assumption, we analyzed whether
stimulation of RNA synthesis also could be achieved with GMP or GDP.
Therefore, RdRp assays were performed with increasing concentrations of
GMP or GDP. As shown in Fig. 5B, instead of a stimulation of
RNA synthesis a strong reduction was found with GMP and, in particular,
with GDP, corroborating that the stimulating nucleotide also has to be
incorporated and that GMP and GDP compete with GTP for the same
nucleotide binding site.
GTP Most Likely Accelerates an Early Step of RNA
Synthesis--
Template-dependent synthesis of a nucleic acid
by an RNA polymerase can be divided into at least four consecutive
steps: binding of the enzyme to the template, initiation of RNA
synthesis, elongation, and termination. To find out which of these
steps might be affected by GTP, the following experiments were carried out.
To analyze whether GTP has an effect on RNA binding, a radiolabeled RNA
corresponding to the HCV genome was incubated with purified NS5B in the
presence of increasing GTP concentrations, and bound RNA was
quantitated by liquid scintillation counting (Fig.
6A). Using the assay
conditions given under "Experimental Procedures," no influence of
GTP on RNA binding was found. This result corroborates the finding that
GTP does not stimulate RNA synthesis from the
poly(A)-oligo(U)12 template and that the stimulating nucleotide has to be incorporated into the RNA product.

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Fig. 6.
Stimulation of RNA synthesis by GTP is not
due to an enhancement of RNA binding or an increase of the elongation
rate. A, RNA binding assays were performed with a
radiolabeled HCV genomic RNA in the presence of increasing
concentrations of GTP. RNA-NS5B complexes were immobilized on
nitrocellulose filters, and bound radioactivity was determined by
liquid scintillation counting. B, influence of increasing
GTP concentrations on the elongation rate. RdRp assays were performed
as described under "Experimental Procedures" using the in
vitro transcribed HCV genomic RNA. After the given incubation
times, RNAs contained in the reaction mixtures were purified by
SDS/proteinase K digestion and subjected to an RNase treatment under
conditions allowing only cleavage of single-stranded RNA. Protected
RNAs were analyzed by denaturing formaldehyde-agarose gel
electrophoresis and radiolabeled products were visualized by
autoradiography. Numbers to the right refer to
the sizes of RNA markers (nucleotides).
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Alternatively, GTP might increase the elongation rate. To validate this
assumption, RdRp assays were carried out with the HCV genomic RNA and
increasing concentrations of GTP. After an initial 5-min incubation of
NS5B and template, the reaction was started by the addition of NTPs
together with an excess of heparin. The latter was included to prevent
further initiation of RNA synthesis during the incubation allowing the
measurement of the elongation rate of synchronously primed molecules.
The determination of the sizes of the reaction products was complicated
by the fact that most RNAs were primed by a copy-back mechanism and
therefore covalently linked to the template. Given the lengths of these
molecules, the sizes of newly synthesized RNAs could not be accurately
determined. Therefore, RNA products taken at different time points
during the reaction were treated with RNases under high salt
conditions, allowing the cleavage of only single-stranded but not
double-stranded RNA. In this way, single-stranded loops at the 3'-end
of the template connecting the input RNA and product are cleaved
allowing a separation of template and newly synthesized RNA by
denaturing formaldehyde-agarose gel electrophoresis. As shown in Fig.
6B, no significant difference of the lengths of RNA products
was found when assays were performed with 50, 250, or 1000 µM GTP (lanes 1-3 and
7-9), whereas a strong reduction of their sizes was found
with higher GTP concentrations (lanes 4-6 and
10-12). Thus, high concentrations of GTP have two opposite
effects on RdRp activity: a strong stimulation of RNA synthesis and a
strong reduction of the elongation rate, the latter being due to the
excess of GTP over Mg2+. In summary, these data demonstrate
that stimulation of RdRp activity by GTP is not due to an enhancement
of RNA binding or an increase of the elongation rate. Based on these
exclusion criteria, high concentrations of GTP (up to ~10
mM) appear to stimulate initiation of RNA synthesis.
GTP-mediated Stimulation of RNA Synthesis by NS5B of a Pestivirus
but Not by 3Dpol of Poliovirus--
As described in the
Introduction, HCV belongs to the genus Hepacivirus and is
most closely related to the pestiviruses, a group of animal pathogenic
viruses to which CSFV belongs. This close evolutionary relationship led
us to speculate that GTP-mediated stimulation of RNA synthesis might be
a biochemical trait conserved between these virus groups and raised the
question of whether it would also be found with the more distantly
related poliovirus. To address these questions, we analyzed CSFV-5B and
3Dpol of poliovirus for their ability to be stimulated by
high concentrations of GTP. As described above, the CSFV enzyme was
purified from insect cells (Fig. 1), whereas 3Dpol of
poliovirus was expressed in Escherichia coli and purified as
described elsewhere (34). GTP-mediated stimulation of RNA synthesis was
measured for these two enzymes in parallel with NS5B using the HCV
genome length in vitro transcript as described under
"Experimental Procedures." A strong stimulation was found both with
NS5B of HCV and the analogous enzyme of CSFV (Fig.
7A). Enhancement of RNA
synthesis for the pestiviral polymerase was linear between 0.1 and 5 mM GTP, and the stimulation rate was well comparable to the
one of HCV NS5B. Only at low GTP concentrations did these enzymes
behave somewhat differently in that RdRp activity of CSFV NS5B was
lower but showed a stronger stimulation between 10 and 100 µM GTP. In contrast, no stimulation was found with 3Dpol of poliovirus, both with the full-length synthetic
HCV RNA template (Fig. 7A) and with
poly(C)-oligo(G)12 (data not shown). Attempts to detect a
significant stimulation by varying reaction conditions (in particular
MgCl2 or MnCl2 concentrations) were not
successful (data not shown).

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Fig. 7.
GTP stimulates RNA synthesis by HCV and CSFV
NS5B but not by 3Dpol of poliovirus. A,
RdRp assays were performed with the in vitro transcribed HCV
genomic RNA and HCV-5B ( ), CSFV-5B ( ), or 3Dpol
( ). After 2 h at 22 °C, incorporation of radioactivity was
determined by trichloroacetic acid precipitation and liquid
scintillation counting. B, analysis of the reaction products
by denaturing formaldehyde agarose gel electrophoresis.
Numbers between the gels refer to the
size of RNA markers (nucleotides). GTP concentrations are given above
the lanes (mM).
|
|
An analysis of the reaction products by agarose gel electrophoresis
revealed that the RNAs generated by all three enzymes were larger than
the input suggesting that copy-back priming is a common mechanism (Fig.
7B). However, the overall sizes of the products were
somewhat different. Compared with HCV-5B, RNAs generated by CSFV-5B
were shorter and products obtained with 3Dpol were longer,
indicating different elongation rates or processivities. Furthermore,
in contrast to the GTP-mediated stimulation observed with the NS5Bs,
the poliovirus enzyme was instead inhibited by high concentrations of
GTP. At
2.5 mM, much lower amounts of RNA products were
found, and these RNAs were shorter in size than the ones obtained at
lower GTP concentrations (Fig. 7B; compare, for example,
lane 14 with lane 18). This
pattern was reminiscent of the one found with HCV-5B at GTP
concentrations of
15 mM (Fig. 2B) and could
similarly be explained at least in part by the excess of GTP over
Mg2+. In summary, these results clearly show that RdRp
activities of HCV and CSFV NS5B but not of poliovirus 3Dpol
are stimulated by high concentrations of GTP.
 |
DISCUSSION |
Studies aimed at elucidating the mechanism of HCV replication are
limited by the lack of robust and reliable cell culture systems and
animal models other than the chimpanzee. Therefore, we have begun to
characterize the biochemical properties of NS5B RdRp, most likely the
key player of the viral replicase complex. During the course of the
determination of the Km value for GTP, we observed a
strong stimulation of RNA synthesis by high concentrations of GTP. It
is toward a detailed analysis of this effect that the present study was undertaken.
Using synthetic full-length HCV RNA and purified NS5B, we found a
50-100-fold stimulation of RNA synthesis by GTP. This stimulation was
selective and was not obtained with ATP, CTP, UTP, GMP, or GDP. As
shown by the reduced stimulation of several NS5B mutants, GTP most
likely directly acts on NS5B and occupies the same binding site that is
used for RNA synthesis. The stimulation was found with every
heteropolymeric template tested and with poly(C)-oligo(G)12 but not with the poly(A)-oligo(U)12 homopolymer. All of
these data taken together demonstrate a selective GTP-mediated
stimulation of RNA synthesis by NS5B.
The way this stimulation is accomplished currently is not known. GTP
has no effect on the overall RNA binding properties or the elongation
rate. Based on these observations the acceleration of a rate-limiting
step during initiation of RNA synthesis is the most likely explanation.
In a simple model, GTP would bind to NS5B and induce an
"initiation-competent state," e.g. by the induction or
stabilization of a conformational change required for efficient
initiation. The "activated" GTP-NS5B complex would then bind to the
template and initiate RNA synthesis. Alternatively, NS5B might first
bind to the template and then to GTP. In either case, according to this
model template binding in the absence of GTP would be nonproductive,
i.e. NS5B binds to the RNA but in most cases falls off
without initiation of RNA synthesis. Once the initiation is completed,
the enzyme processively copies the template and is no longer stimulated
by high concentrations of GTP.
In this context, it should be noted that an alteration or stabilization
of the enzyme conformation by high concentrations of GTP has been
suggested for 3Dpol of poliovirus (37). Using cross-linking
of oxidized GTP to purified 3Dpol, it was found that NTP
binding protects RdRp activity from heat denaturation (37).
Interestingly, GTP protected the enzyme to a greater extent than the
other three NTPs indicating a tighter binding. Although
Km for GTP of 3Dpol is very low, high
concentrations (~2 mM) were required for stoichiometric cross-linking and maximum protection from heat denaturation (37). Two
NTP binding sites were mapped, one near the amino terminus (residues
57-74) outside of the polymerase core region and the other in the
central region overlapping with motif B (residues 266-286) (38). Only
the amino-terminal NTP binding site was shown to be crucial for RNA
replication in that a leucine substitution for the highly conserved
lysine residue at position 61 completely abolished RdRp activity (39,
40). For NS5B, the results described here with the various mutations
suggest that an NTP binding site in the core of the enzyme is required.
As deduced from the x-ray crystal structure of 3Dpol (41),
the invariant carboxylates of motifs A and C of NS5B would be clustered
in the "palm" (for a review see Ref. 42). Since substitutions of
these residues both reduce RdRp activity and stimulation by GTP, the
NTP binding site in the core appears to be involved in both reactions.
However, currently we cannot completely rule out the possibility that
these amino acid substitutions caused structural alterations affecting
an NTP binding site somewhere else in NS5B. Furthermore, we also found
a mutant in motif B (5B286V) with a very low RdRp activity
that could be stimulated with an efficiency comparable with the wild
type. However, this mutation is the only one affecting a nonconserved
residue, suggesting that this particular substitution affected a
property of the enzyme not directly involved in NTP binding and/or
catalysis. It remains to be established whether NS5B has more than one
NTP binding site and, if so, which of these is required for enzymatic
activity and/or stimulation by GTP.
Another unresolved question is why the stimulation is selectively
exerted by GTP. Perhaps the initiating nucleotide has to be a guanylate
as is the case for many RNA polymerases like the one from bacteriophage
T7 or the plus strand RNA virus BMV (43, 44). For example, the
DNA-dependent RNA polymerases of E. coli and
T7 possess at least two distinct NTP-binding sites: one that recognizes
only the initiating GTP in a Mg2+-independent manner and
the other used for elongation, binding to all four NTPs in a reaction
requiring Mg2+ (43). In case of BMV, initiation of RNA
synthesis can be stimulated up to ~10-fold by GMP or the dinucleotide
GpG, corresponding to the penultimate and antepenultimate nucleotide of
the viral template (45). The fact that this stimulation is only found
with low concentrations of GTP suggests the existence of two
NTP-binding sites: one for the initiation nucleotide, binding
preferentially GTP or GMP, and one for the elongation nucleotide,
binding each of the four NTPs with a comparable Km.
For NS5B of HCV we did not detect a stimulation by GMP, and the
Km for GTP was ~100-fold lower than the one
reported for BMV (50 µM; Ref. 45), suggesting that a
different mechanism is responsible for the stimulation of RNA synthesis
by high concentrations of GTP.
Another possibility of how GTP stimulates RNA synthesis by NS5B would
be an enhancement of the transition from initiation to elongation. For
the well studied DNA-dependent RNA polymerases, it was
shown that transcription can be functionally separated into several
distinct steps: template binding, promoter localization, melting of the
DNA to form a transcriptionally open complex, nucleotide substrate
binding, formation of the first phosphodiester bond, abortive RNA
synthesis, promoter clearance, and processive elongation and
termination (for a review, see Ref. 46). A characteristic feature of
these enzymes is the inefficient transition from initiation to
elongation resulting in the synthesis of abortive products. These
oligonucleotide products dissociate from the initiated complex when
they are not elongated. A similar phenomenon has been described for the
RdRp of BMV, resulting in the synthesis of an 8-mer oligonucleotide in
a 10-fold molar excess over the full-length RNA product (47, 48).
Interestingly, GTP appears to stabilize the RdRp-RNA interaction and in
this way increases efficiency of RNA synthesis (49). Although a similar
mechanism might be responsible for stimulation of RNA synthesis with
NS5B, it should be kept in mind that BMV RdRp initiates RNA synthesis
de novo, whereas NS5B, at least under the in
vitro conditions used here, is primer-dependent. If
abortive RNA products would be made, then they probably will not be
released but remain covalently bound to the 3'-end of the primer. On
the other hand, currently we cannot exclude the possibility that in addition to a stimulation of primer-dependent RNA
synthesis, a certain fraction of the RNA molecules synthesized at high
GTP concentrations are initiated de novo. Further studies
will be required to clarify these issues.
The relevance of RNA synthesis stimulation by GTP for the in
vivo situation currently is not known. The fact that
unphysiologically high concentrations of GTP are required for maximal
stimulation clearly suggests that a different mechanism may operate in
the infected cell. One simple explanation would be that the effect we
observe here is due to the enzyme preparation containing largely misfolded or thermosensitive proteins and that GTP binds to and activates or stabilizes these molecules and in this way increases the
number of enzymatically active NS5B proteins. However, the results
obtained with the panel of NS5B mutants and with the NS5B of CSFV
suggest that this is not the case. Furthermore, the findings that GTP
does not enhance poly(U) synthesis from the poly(A) template and that
GMP inhibits RNA synthesis indicate that GTP does not act
allosterically on RdRp but rather has to be incorporated into the RNA.
An alternative explanation would be that high concentrations of GTP
substitute for a function normally executed by a cellular or viral
protein. For example, in the case of poliovirus it was shown that VPg
and in particular the VPg precursor 3AB enhance RNA synthesis by
3Dpol 50-100-fold (50, 51). Similar to what we describe
here for NS5B, RNA synthesis stimulated by 3AB is
primer-dependent. 3AB has a dual function; it can interact
with 3Dpol (52), and it is an RNA-binding protein able to
interact with the template-primer (53). Mutational ablation of RNA
binding also reduces 3Dpol stimulation, suggesting that 3AB
enhances interaction between the template and 3Dpol (51,
53). An additional function of VPg is to act as an acceptor of UMP
added by 3Dpol to the OH group of a tyrosine residue within
VPg in a template-dependent reaction (54). Uridylylated VPg
then is used as the primer for transcription of viral RNA. Whether RNA
synthesis by NS5B can be stimulated by a cellular or viral protein
(e.g. NS3 containing the helicase activity (22-25)) remains
to be established.
The finding that RNA synthesis by NS5B of HCV and CSFV but not by
3Dpol is stimulated by high concentrations of GTP
demonstrates the conservation of this biochemical trait between
hepaciviruses and pestiviruses and probably reflects the evolutionary
distance between this virus group and the picornaviruses. It also might
correlate with different mechanisms responsible for initiation of RNA
synthesis. All of these RdRps are strictly
primer-dependent. As described above, in the case of
poliovirus it was shown that the primer for RNA synthesis by
3Dpol is the VPg (or its precursor 3AB), which is
uridylylated by 3Dpol in a template-dependent
reaction (54). For pestiviruses and hepaciviruses, it is not known
whether these enzymes also use a protein as a primer. The difference
from 3Dpol with respect to the GTP-mediated stimulation,
together with the observation that high concentrations of GTP probably
enhance initiation of RNA synthesis, indicates that a different
mechanism is responsible. Possibilities are de novo
initiation of RNA synthesis or priming by a nucleic acid (which is more
difficult to imagine with linear templates). In any case, the results
presented here describe an interesting biochemical property of NS5B,
and they should facilitate further functional analyses of this enzyme.
Since NS5B also is an attractive target for antiviral therapy, our
findings may have important consequences for the development of potent inhibitors.