Virology 285, 6–11 (2001)

doi:10.1006/viro.2001.0948, available online at http://www.idealibrary.com on

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A Novel Mechanism to Ensure Terminal Initiation by Hepatitis C Virus NS5B Polymerase

Zhi Hong,* ,1 Craig E. Cameron,† Michelle P. Walker,* Christian Castro,† Nanhua Yao,*
Johnson Y. N. Lau,* and Weidong Zhong*

*ICN Pharmaceuticals, 3300 Hyland Avenue, Costa Mesa, California 92626; and †Department of Biochemistry and Molecular Biology,
Pennsylvania State University, University Park, Pennsylvania 16802

Received January 31, 2001; returned to author for revision February 20, 2001; accepted April 10, 2001

Hepatitis C virus (HCV) nonstructural protein 5B (NS5B) RNA-dependent RNA polymerase (RdRp) has acquired a unique
␤-hairpin in the thumb subdomain which protrudes toward the active site. We report here that this ␤-hairpin plays an
important role in positioning the 3⬘ terminus of the viral RNA genome for correct initiation of replication. The presence of this
␤-hairpin interferes with polymerase binding to preannealed double-stranded RNA (dsRNA) molecules and allows only the
single-stranded 3⬘ terminus of an RNA template to bind productively to the active site. We propose that this ␤-hairpin may
serve as a “gate” which prevents the 3⬘ terminus of the template RNA from slipping through the active site and ensures
initiation of replication from the terminus of the genome. This hypothesis is supported by the ability of a ␤-hairpin deletion
mutant that utilizes dsRNA substrates and initiates RNA synthesis internally. The proposed terminal initiation mechanism may
represent a novel replication strategy adopted by HCV and related viruses. © 2001 Academic Press

Infection by hepatitis C virus (HCV) is a significant
human medical problem with an estimated prevalence of
170 million cases globally. Up to four million individuals
may be infected in the United States alone (1). In most
instances, the virus establishes a chronic and insidious
infection that persists for decades. This usually results in
recurrent and progressively worsening liver inflamma-
tion, which often leads to more severe disease states
such as cirrhosis and hepatocellular carcinoma.
HCV, a member of the Flaviviridae family, is a positive-
stranded RNA virus. Its life cycle consists of several
important and interrelated processes that occur primarily
in the cytoplasm of the host cells (19). Among these
intricate processes, RNA replication is the centerpiece of
the viral proliferation cycle. While many viral and/or host
factors are suspected to be involved, one certainty is that
the virally encoded polymerase has a key role in the
entire replication process. The interplay between viral
polymerase and its substrates (template, primer, and
incoming nucleotide) sets the initial and minimal frame-
work for viral replication. For HCV, the nonstructural pro-
tein 5B (NS5B) is the RNA-dependent RNA polymerase
(RdRp) required for genome replication (3, 7, 14). Al-
though the first RdRp was discovered more than 20 years
ago in poliovirus infected cells, relatively little was
1
To whom correspondence and reprint requests should be ad-
dressed at ICN Pharmaceuticals, Inc., 3300 Hyland Avenue, Costa
Mesa, CA 92626. Fax: (714) 668-3141. E-mail: zhihong@icnpharm.com.
0042-6822/01 $35.00
Copyright © 2001 by Academic Press
All rights of reproduction in any form reserved.
known about this class of polymerases until recently.
Structural studies of unliganded poliovirus 3D pol (9) and
HCV NS5B (4, 13) have revealed unique features and
provided useful information for the comparative analysis
of RdRps in relation to other classes of polymerases.
This will in turn help to advance our understanding of
viral replication at the molecular level for positive-
stranded RNA viruses.
A Unique ␤-Hairpin in the Thumb Subdomain of HCV
NS5B Polymerase. One novel structural component
unique to HCV polymerase is a ␤-hairpin found in the
thumb subdomain, which protrudes toward the active
site located at the base of the palm subdomain (yellow
ribbon, Fig. 1A). The ␤-hairpin consists of 12 amino acids
(LDCQIYGACYSI) with a tyrosine (Y) and glycine (G) pair
at the top of the hairpin (position and direction of the
␤-hairpin is shown at the bottom of Fig. 1A). In stark
contrast, this ␤-hairpin is absent in polioviral polymerase
(3D pol) (Fig. 1B). This structural difference suggests that
the ␤-hairpin may serve a special function important for
HCV replication. Poliovirus, a well-studied virus, initiates
its genome replication using a protein primer (VPg, prod-
uct of 3B) (18). Contrary to the inability of HCV NS5B to
bind dsRNA substrates productively as reported previ-
ously (21), polioviral 3D pol can efficiently assemble an
elongation-competent complex with a symmetrical du-
plex RNA substrate (sym/sub), suggesting that the 3D pol
apoenzyme can accommodate preannealed double-
stranded template/primer (2). This observation coincides
with the lack of an analogous ␤-hairpin in 3D pol (Fig. 1B)
6
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FIG. 1. Structural comparison between HCV NS5B RdRp and poliovirus 3D pol RdRp. Molecular surface structures were constructed for both
polymerases (turquoise color for HCV and blue color for poliovirus) and oriented with the palm at the base, the fingers to the left, and the thumb
subdomain to the right. The essential GDD motifs (polymerase motif C; part of the active site) are colored red. In HCV NS5B, the ␤-hairpin comprising
residues 443 through 454 is colored yellow and protrudes toward the active site (4). The sequence of the ␤-hairpin is shown at the bottom of (A). In
both structures, the unique molecular shape of the RdRps with the fully encircled NTP binding site is clear. This encircled active site likely exists in
3D pol because the extreme aminoterminal residues in the crystal structure contact the thumb subdomain, just as in HCV NS5B, before proceeding
to form the fingers subdomain. This figure was produced using the program INSIGHT II (Molecular Simulation Inc., San Diego, CA). In the crystal
structure of poliovirus 3Dpol (9), approximately 30% of the protein residues were unobserved. The availability of HCV NS5B RdRp structure (4) allows
a model of the complete poliovirus 3D pol structure to be constructed. Based on the similar polypeptide fold in regions common to both crystallographic
models, the missing portions of the 3D pol structure were simply transferred from NS5B and the correct side chains were assigned with the most
common rotamer. The junctions between crystallographically observed and inferred residues were adjusted according to local similarity to a
database of polypeptide fragments derived from high-resolution crystal structures.

which, in HCV NS5B polymerase, imposes steric hin-
drance against docking the dsRNA (13). For HCV, assem-
bly of a catalytically competent complex requires the
RNA template to be single-stranded at the 3⬘ terminus
(10, 21). Upon template binding to the polymerase, short
RNA primers (i.e., the initiating nucleotide, one to three
nucleotides in length) basepair with the terminal tem-
plate bases in the active site of the polymerase and
prime RNA synthesis from the 3⬘ terminus of the template
(21). A structural model for the quaternary complex of
HCV polymerase/template/primer/nucleotide has been
proposed, which suggests that the unique ␤-hairpin
plays an important role in positioning the 3⬘ terminus of
viral RNA properly in the polymerase active site for cor-
rect initiation of RNA replication (21). To test this hypoth-
esis, a deletion mutant was constructed to significantly
shorten the ␤-hairpin without disrupting the structural
integrity and the catalytic activity of HCV NS5B.
Shortened ␤-Hairpin Allows Internal Initiation from
Preannealed Duplex RNA. Guided by the crystal struc-
ture of HCV NS5B polymerase (4), four amino acids from
each strand were deleted simultaneously to shorten the
␤-hairpin without potential global structural perturbation
that may affect the RdRp activity. The resulting deletion
mutant, BL⌬8, retained two amino acids at the top of the
␤-hairpin, which were changed from YG to GG to favor
the formation of the observed type I⬘ ␤-turn between the
two ␤-strands (from LDCQIYGACYSI to LGGI). This dele-
tion mutant as well as its parent wild-type NS5B protein
were produced in bacterial cells and purified as de-
scribed previously (9, 13). The purity of the wild-type and
BL⌬8 NS5B is above 95% and comparable to that of the
8 RAPID COMMUNICATION

was unable to utilize sym/sub or preannealed dsRNA

FIG. 2. A ␤-hairpin deletion mutant allows productive binding to dsRNA
substrate to initiate RNA synthesis. (A) Expression and purification of HCV
NS5B and poliovirus 3Dpol. NS5B cDNA was isolated from a subgenomic
replicon clone (pFKI389/NS3-3⬘/wt) kindly provided by Dr. Ralf Barten-
schlager (Johannes-Gutenberg University, Mainz, Germany). The region
encoding the C-terminal 21 amino acids was removed to improve the
solubility of NS5B as described previously (7). The BL⌬8 deletion mutant
was created from the C-terminally deleted NS5B by using the Quick-
Change Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA). The
poliovirus 3Dpol was produced as described (8). The purity of each poly-
merase was above 95%. (B) Use of duplex RNA substrate. Single-nucle-
otide incorporation assay was performed using end-labeled sym/sub RNA
as the template/primer pair. AMP incorporation was compared among
wild-type NS5B (lane 2), ␤-hairpin deletion mutant BL⌬8 (lane 3), poliovirus
3D pol (lane 4). No template control is in lane 1. A measure of 2 ␮M of each
enzyme was employed, respectively.
poliovirus 3D pol (Fig. 2A). The BL⌬8 mutant was first
tested for its ability to utilize the dsRNA substrate, sym/
sub. The wild-type NS5B, as shown previously (5, 21),
efficiently to direct RNA synthesis (Fig. 2B, lane 2). In
contrast, the BL⌬8 mutant was able to utilize sym/sub as
efficiently as poliovirus 3D pol (Fig. 2B, compare lanes 3
and 4) under the same assay conditions (described in
Fig. 3 legend). Interestingly, the overall RdRp activity of
BL⌬8 was increased approximately fivefold compared to
the wild-type NS5B (Fig. 4). These results support the
notion that the ␤-hairpin prevents HCV polymerase from
binding to a double-stranded template/primer duplex in a
productive manner, while polioviral polymerase lacking
the ␤-hairpin binds to dsRNA and efficiently extends the
labeled primer. This observation argues that the accom-
modation of the nascent dsRNA (a product of elongation
during replication) by HCV polymerase must be accom-
panied by a conformational change from initiation to
elongation in the polymerase active site, perhaps by
moving the ␤-hairpin away from the active site.
HCV NS5B initiates RNA synthesis from the initiating
nucleotide (de novo) or a dinucleotide at the 3⬘ terminus
of the template (21). The dinucleotide-mediated terminal
initiation assay was selected to characterize the ␤-hair-
pin deletion mutant. The initiating dinucleotide, believed
to be the product of abortive initiation, was shown to
prime RNA synthesis efficiently by HCV NS5B (21), a
phenomenon that was also demonstrated in bacterio-
phage T7 RNA polymerase-mediated RNA synthesis (6).
Terminal initiation is in accordance with the quaternary
structural model wherein the 3⬘ terminus of the RNA
template sterically clashes with the tip of the ␤-hairpin
loop and is unable to slip through the active site (21).

FIG. 3. The ␤-hairpin deletion mutant initiates RNA synthesis internally. (A) Internal initiation from a dinucleotide primer. “TER” and “INT” template RNAs
were employed to distinguish terminal vs internal initiation from the primer 33pGpG. RdRp activities of wild-type NS5B (lanes 2 and 3), deletion mutant BL⌬8
(lanes 4 and 5) were compared. “⫺” in lane 1 represents a control without the template RNA. “P” represents the dinucleotide pGpG and “P ⫹ 1” is the
trinucleotide product as a result of single nucleotide addition. (B) Internal initiation from a preannealed primer. A preannealed template and primer (P, an
end-labeled primer 33pCGGGCC) were tested for single-nucleotide (AMP) incorporation. Lane 1, no template control “⫺”; lane 2, wild-type NS5B; lane 3, BL⌬8
mutant; lane 4, poliovirus 3Dpol. The standard nucleotide incorporation reaction was carried out in a volume of 20 ␮l containing 50 mM HEPES (pH 7.3), 10
mM ␤-mercaptoethanol, 50 mM NaCl, 5 mM MgCl2, 2 ␮M of end-labeled sym/sub RNA, or 10 ␮M of “TER” or “INT” template RNA and 10 ␮M of end-labeled
dinucleotide, 2 ␮M of polymerase (HCV NS5B or poliovirus 3Dpol), and 100 ␮M of CTP or ATP (2, 21). The product was separated on a 23–25% PAGE/6 M
urea/1⫻ TBE gel and exposed to X-ray film. Individual bands were quantified using a Phosphorimager (Typhoon 8600, Molecular Dynamics, Sunnyvale, CA).
All synthetic RNA templates and primers were chemically synthesized (Oligos, Etc., Wilsonville, OR).
 RAPID COMMUNICATION
FIG. 4. RdRp activity of the wild-type and the BL⌬8 mutant NS5Bs on
in vitro transcribed HCV RNA. (A) Structure of the minigenome RNA
used for RdRp assay. Numbers refer to nucleotide positions within the
HCV subgenomic replicon (15). An internal deletion between two KpnI
sites at positions 1684 and 7477 reduced the size of the minigenome
RNA to about 2.1 kb. (B) Comparison of the RNA products from the HCV
minigenome RNA template. RdRp reactions were initiated by adding 1
␮g of each protein: lanes 2–7, wild-type NS5B; lanes 8–13, BL⌬8 NS5B.
Reactions were terminated by the addition of SDS-containing buffer
and proteinase K at 10 (lanes 3 and 8), 20 (lanes 4 and 9), 30 (lanes 5
and 10), 50 (lanes 2, 6, 11, and 13), or 70 (lanes 7 and 12) min after the
reactions started. Control reactions contained no nucleotides (lanes 2
and 13) for 50 min. Samples were analyzed by denaturing guanidinium-
isothiocyanate-1% agarose gel electrophoresis. Minigenome monomer
size marker was obtained by in vitro transcription of the HCV mini-
genome in the presence of ␣-[ 33P]UTP (lane 1). (C) Comparison of the
RNA products from the homopolymeric RNA template/primer, poly(A)/
oligo(dT), using increasing enzyme concentrations. Reactions were
initiated by the addition of 0.5 (lanes 1 and 4), 1.0 (lanes 2 and 5), or 2.0
(lanes 3 and 6) ␮g of enzyme and terminated after 45 min at 30°C by
the addition of phenol/chloroform.
Indeed, our results (Fig. 3A) demonstrate that HCV NS5B
is unable to utilize the “INT” template (lacking the termi-
nal dicytidylate bases) to initiate RNA synthesis from the
diguanylate primer ( 33pGpG; lane 4), whereas the enzyme
utilized the “TER” template with the terminal dicytidylate
bases very efficiently (lane 5). Clearly, the wild-type HCV
NS5B could not initiate nucleotide incorporation from the
internal dicytidylate (CC) template bases. However, the
␤-hairpin deletion mutant BL⌬8 could utilize the INT
template to initiate RNA synthesis (Fig. 3A, lanes 2 and
9
3), indicating that the shortened ␤-hairpin allowed inter-
nal initiation of RNA synthesis by permitting the 3⬘ ter-
minus of the template to slip through the active site.
Poliovirus 3D pol, which lacks the ␤-hairpin, allowed inter-
nal initiation (Fig. 3B, lane 4) from a primer (P) prean-
nealed to the middle of a template, an activity that could
be duplicated by the BL⌬8 mutant but not by the wild-
type NS5B polymerase (Fig. 3B, compare lanes 2 and 3).
The above results suggest that the apo form of the HCV
polymerase is unable to initiate efficient RNA synthesis
internally due to the presence of the ␤-hairpin, whereas
poliovirus, although capable of internal initiation, has
acquired a discrete mechanism to ensure terminal initi-
ation from the poly(A) stretch at the end of the viral
genome (18).
The BL⌬8 Mutant NS5B Produces Predominantly Trun-
cated RNA Products. To compare the RdRp activity of the
wild-type and the BL⌬8 mutant NS5Bs on a natural HCV
template, an HCV minigenome was constructed that con-
tains authentic 5⬘ and 3⬘ termini and portions of the
coding sequence (pMinigenome) (Fig. 4A) (15). In vitro
transcribed minigenome RNAs were used as templates
in a standard RdRp assay. Products larger than the input
RNA, particularly of dimer size, were apparent for both
wild-type and BL⌬8 mutant NS5Bs (Fig. 4B, lanes 3–7 for
the wild-type NS5B; lanes 8–12 for the BL⌬8 mutant
NS5B). This is consistent with the observation that NS5B
is capable of initiating RNA synthesis via the copy-back
mechanism at the 3⬘ terminus (3, 14). Also in agreement
with previous reports (14, 17), the near dimer-size hairpin
product appeared 15–20 min after the start of the reac-
tion, corresponding to an incorporation rate of approxi-
mately 100–140 nucleotides per minute. Interestingly, the
BL⌬8 mutant produced RNA products varying in size,
which appeared as a smear by denaturing agarose gel
electrophoresis (Fig. 4B, lanes 8–12). Many of these
heterogeneous RNA products could occur by internal
initiation as a result of the ␤-hairpin deletion, consistent
with the notion that the ␤-hairpin is necessary to ensure
terminal initiation. The overall RNA synthesis activity of
the BL⌬8 mutant was at least five times greater than that
of the wild-type NS5B (Fig. 4B, compare lanes 8–12 with
lanes 3–7). In concert, enhanced RdRp activity by the
BL⌬8 mutant was observed when using the homopoly-
meric RNA template/primer, poly(A)/oligo(dT), in a stan-
dard RdRp assay (Fig. 4C). Much of the enhanced RdRp
activity resulted from initiation from internally annealed
primers, leading to smaller than template-sized products
(Fig. 4C, compare lanes 1–3 with lanes 4–6). Again, these
results support the proposed function of the ␤-hairpin.
Identification of a Conserved Arginine Critical for De
Novo Initiation from the 3⬘ Terminus. A structural model
was constructed to further define the molecular require-
ments at the active site for efficient assembly of the
template RNA and the short initiating dinucleotide (21).
Based on this model, a conserved arginine (R 386) was
10
FIG. 5. A conserved arginine critical for de novo initiation. A critical arginine at position 386 was identified to play a role in stabilizing the initiating
nucleotide(s). (A) Effect on RNA synthesis from the dinucleotide primer, pGpG. Lane 1, no template control “⫺”; lane 2, wild-type NS5B; lane 3, NS5B
with R 386Q mutation; lane 4, BL⌬8 mutant; lane 5, BL⌬8 with R 386Q mutation. “P” represents the end-labeled primer and “P ⫹ 1” represents the extended
primer. (B) Effect on RNA synthesis from the preannealed primer, sym/sub. The lane assignment is the same as (A).
identified and proposed to play a role in stabilizing the
initiating nucleotide (for de novo synthesis) or the dinu-
cleotide (for pGpG-mediated synthesis), which would
otherwise be unable to basepair efficiently with the tem-
plate base(s). Interestingly, our previous mutational anal-
ysis had demonstrated that the equivalent Arg residue
(R 518) in bovine viral diarrhea virus (BVDV) NS5B was
important for de novo RNA synthesis (12). Indeed, site-
directed mutagenesis revealed that R 386 was also impor-
tant for the dinucleotide-initiated RNA synthesis, since
mutation to glutamine (R 386Q) significantly reduced the
ability to utilize the dinucleotide for both the wild-type
and BL⌬8 mutant NS5B (Fig. 5A, compare lanes 2 vs 3
and 4 vs 5). However, this mutation had little effect on
RNA synthesis from a preannealed duplex RNA (sym/
sub) catalyzed by the deletion mutant BL⌬8 (Fig. 5B,
lanes 4 and 5). These observations support the hypoth-
esis that R 386 plays a critical role in stabilizing the inter-
action between the 3⬘ terminus of the RNA template and
the initiating nucleotide(s). We believe that the ␤-hairpin
and R 386 coordinate RNA synthesis from an initiating
nucleotide (de novo) or the initiating dinucleotide. The
dinucleotide is probably an abortive transcript of de novo
initiation that can prime efficient RNA synthesis when
annealed to a bound template in the active site.
Concluding Remarks. Terminal initiation is a critical
first step for many positive-stranded RNA virus replica-
tions. In the absence of repetitive sequence motifs, strict
terminal initiation ensures the integrity of a viral genome.
For poliovirus, a prototype virus representing the Picor-
naviridae family, precise terminal initiation is achieved by
a protein primer which assembles the replication com-
plex at the 3⬘ poly(A) region and initiates RNA synthesis
via a uridylylated protein primer (VPg) (18). This uridyly-
lation is highly specific as it requires specifically 3D pol
activity, UTP, and poly(A), which coincides with the ob-
RAPID COMMUNICATION
servation that the 3⬘ terminal poly(A) region of the polio-
virus genome is the origin of RNA replication (20). HCV,
an RNA virus in the Flaviviridae family, appears to have
adopted a different strategy for de novo RNA replication
(16, 22). This study shows that an intrinsic structural
element is employed by HCV for precise terminal initia-
tion of the viral genome replication.
A recent study demonstrated that BVDV NS5B poly-
merase specifically recognizes the terminal template
base(s) for de novo RNA synthesis (11), providing evi-
dence that terminal initiation of RNA replication is also
required by this related virus. Thus, the identification of a
novel and intrinsic structural element (such as the ␤-hair-
pin in HCV NS5B) for terminal initiation may have a
broader impact on our understanding of viral replication,
as it may represent a replication strategy employed by
related RNA viruses.
ACKNOWLEDGMENTS
C.E.C. is a recipient of a Howard Temin Award (CA75118) and is
supported in part by NIAID Grant AI47350. We thank Ralf Barten-
schlager for providing the valuable subgenomic HCV replicon DNA. We
also acknowledge the technical assistance of Shannon Dempsey.
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