Antiviral Effect and Virus-Host Interactions in Response to Alpha Interferon, Gamma Interferon, Poly(I)-Poly(C), Tumor Necrosis Factor Alpha, and Ribavirin in Hepatitis C Virus Subgenomic Replicons

Abstract

The recently developed hepatitis C virus (HCV) subgenomic replicon system was utilized to evaluate the efficacy of several known antiviral agents. Cell lines that persistently maintained a genotype 1b replicon were selected. The replicon resident in each cell line had acquired adaptive mutations in the NS5A region that increased colony-forming efficiency, and some replicons had acquired NS3 mutations that alone did not enhance colony-forming efficiency but were synergistic with NS5A mutations. A replicon constructed from the infectious clone of the HCV-1 strain (genotype 1a) was not capable of inducing colony formation even after the introduction of adaptive mutations identified in the genotype 1b replicon. Alpha interferon (IFN-alpha), IFN-gamma, and ribavirin exhibited antiviral activity, while double-stranded RNA (dsRNA) and tumor necrosis factor alpha did not. Analysis of transcript levels for a series of genes stimulated by IFN (ISGs) or dsRNA following treatment with IFN-alpha, IFN-gamma, and dsRNA revealed that both IFNs increased ISG transcript levels, but that some aspect of the dsRNA response pathway was defective in Huh7 cells and replicon cell lines in comparison to primary chimpanzee and tamarin hepatocytes. The colony-forming efficiency of the replicon was reduced or eliminated following replication in the presence of ribavirin, implicating the induction of error-prone replication. The potential role of error-prone replication in the synergy observed between IFN-alpha and ribavirin in attaining sustained viral clearance is discussed. These studies reveal characteristics of Huh7 cells that may contribute to their unique capacity to support HCV RNA synthesis and demonstrate the utility of the replicon system for mechanistic studies on antiviral agents.

Full text

JOURNAL OF VIROLOGY, Jan. 2003, p. 1092–1104 Vol. 77, No. 2
0022-538X/03/$08.00⫹0 DOI: 10.1128/JVI.77.2.1092–1104.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Antiviral Effect and Virus-Host Interactions in Response to Alpha
Interferon, Gamma Interferon, Poly(I)-Poly(C), Tumor Necrosis
Factor Alpha, and Ribavirin in Hepatitis C Virus
Subgenomic Replicons
Robert E. Lanford,
1
* Bernadette Guerra,
1
Helen Lee,
1
Devron R. Averett,
2
Brad Pfeiffer,
1
Deborah Chavez,
1
Lena Notvall,
1
and Catherine Bigger
1
Department of Virology and Immunology, Southwest National Primate Research Center, Southwest Foundation
for Biomedical Research, San Antonio, Texas 78227,
1
and Anadys Pharmaceuticals,
San Diego, California 92121
2
Received 12 August 2002/Accepted 22 October 2002
The recently developed hepatitis C virus (HCV) subgenomic replicon system was utilized to evaluate the
efficacy of several known antiviral agents. Cell lines that persistently maintained a genotype 1b replicon were
selected. The replicon resident in each cell line had acquired adaptive mutations in the NS5A region that
increased colony-forming efficiency, and some replicons had acquired NS3 mutations that alone did not
enhance colony-forming efficiency but were synergistic with NS5A mutations. A replicon constructed from the
infectious clone of the HCV-1 strain (genotype 1a) was not capable of inducing colony formation even after the
introduction of adaptive mutations identified in the genotype 1b replicon. Alpha interferon (IFN-␣), IFN-␥,
and ribavirin exhibited antiviral activity, while double-stranded RNA (dsRNA) and tumor necrosis factor alpha
did not. Analysis of transcript levels for a series of genes stimulated by IFN (ISGs) or dsRNA following
treatment with IFN-␣, IFN-␥, and dsRNA revealed that both IFNs increased ISG transcript levels, but that
some aspect of the dsRNA response pathway was defective in Huh7 cells and replicon cell lines in comparison
to primary chimpanzee and tamarin hepatocytes. The colony-forming efficiency of the replicon was reduced or
eliminated following replication in the presence of ribavirin, implicating the induction of error-prone repli-
cation. The potential role of error-prone replication in the synergy observed between IFN-␣ and ribavirin in
attaining sustained viral clearance is discussed. These studies reveal characteristics of Huh7 cells that may
contribute to their unique capacity to support HCV RNA synthesis and demonstrate the utility of the replicon
system for mechanistic studies on antiviral agents.
Chronic hepatitis C virus (HCV) infections are one of the
leading causes of liver disease worldwide (2). The prevalence
of HCV infections is 1 to 2%, although certain geographical
regions, age groups, and ethnic groups have much higher rates
of infection (3). Although symptoms may be mild for decades,
20% of persistently infected individuals may eventually de-
velop serious liver disease including cirrhosis and liver cancer
(2). HCV infection is the leading cause for liver transplanta-
tion in the United States (12). Although the initial use of
interferon (IFN) for treatment of chronic infections yielded
marginal results, the current therapeutic regimen of pegylated
alpha 2b IFN (IFN-␣2b) and ribavirin provides substantially
improved rates of sustained viral clearance of 42 and 82% for
genotype 1 and genotype 2 and 3, respectively (45). Treatment
of acute infections with standard IFN therapy without ribavirin
is highly efficacious and approaches 100% sustained viral clear-
ance (33). Nonetheless, a great need exists for improved anti-
viral agents, since many patients still do not benefit from IFN
therapy, and IFN therapy is associated with undesirable side
effects. The lack of a suitable tissue culture system has previ-
ously hampered the development of antiviral agents, but the
recent development of a replicon system for HCV (43) has
partially fulfilled this need.
HCV is a member of the Flaviviridae family. The genome is
single-stranded, positive-sense RNA (Fig. 1). Since the cloning
of the viral genome (1, 11), rapid advances have been attained
in defining viral functions (reviewed in references 5 and 56).
The 5⬘ noncoding region (NCR) contains an internal ribosome
entry site (IRES). The amino terminus of the viral polyprotein
contains the structural proteins, the capsid and two envelope
proteins, E1 and E2. The function of p7 is not known. NS2/NS3
is a metalloprotease that cleaves NS2 from NS3. NS3 is a serine
protease and the viral helicase. NS4A is a cofactor for the
serine protease. The function of NS4B is unknown. NS5B is
the viral RNA polymerase. NS5A is a phosphoprotein that
contains a sequence known as the IFN sensitivity-determining
region (ISDR). Enomoto et al. (17) first demonstrated a rela-
tionship to sequence variation in this region and resistance to
IFN therapy. Gale and colleagues have shown that this region
interacts with PKR, providing a plausible mechanism for the
modulation of the host response to IFN (22, 23). However, the
precise function of NS5A is still unknown, and whether PKR
binding accounts for viral resistance to IFN is controversial
(54, 61). NS5A induces interleukin 8 synthesis that may con-
tribute to IFN resistance (25, 55), and NS5A has been shown
* Corresponding author. Mailing address: Department of Virology
and Immunology, Southwest Foundation for Biomedical Research,
7620 NW Loop 410, San Antonio, TX 78227. Phone: (210) 258-9445.
Fax: (210) 670-3329. E-mail: rlanford@icarus.sfbr.org.
1092

to interact with grb2 (62) and a SNARE-like protein (66). E2
has been shown to interact with PKR and may be involved in
IFN resistance as well (51, 65). Following the polyprotein open
reading frame is a 3⬘ NCR that has a variable region, a long
poly(U)-polypyrimidine stretch, and a highly conserved 98-
nucleotide terminus.
Although HCV does not replicate in conventional tissue
culture systems, a surrogate system has been created on the
basis of a bicistronic replicon constructed by Lohmann and
coworkers (43). The 5⬘ end of the HCV genome, including the
IRES and 12 codons of the core protein, is fused in frame with
the neomycin phosphotransferase gene, the encephalomyocar-
ditis virus (EMCV) IRES drives translation of the HCV non-
structural proteins, and the construct terminates with the 3⬘
NCR of HCV. When synthetic RNA from this construct is
transfected into the human liver cell line Huh7, G418-resistant
colonies which persistently maintain the replicon RNA can be
isolated. The utility of this system was markedly enhanced
when Blight and coworkers (8) demonstrated that adaptive
mutations arise that permit highly efficient colony formation.
This has been reproduced in a number of studies including this
one (8, 27, 35, 42). A replicon has been developed from a
FIG. 1. Schematic of HCV genome and replicon design. The HCV genome is depicted with the 5⬘ NCR containing an IRES and the 3⬘ NCR
including a variable region (Var), a polyuridine or polypyrimidine stretch (U/PP), and a 98-nucleotide (nt) conserved region (CR). The depiction
of the HCV IRES was adapted from the structural study of Honda et al. (28). The open reading frame of the polyprotein is depicted as a rectangle
with demarcation of the individual viral protein domains, and the positions of some of the viral functions are depicted above. c, capsid protein;
e1 and e2, envelope proteins E1 and E2. The structure of the bicistronic replicon is illustrated below the HCV genome, with the HCV IRES and
EMCV IRES regulating translation of the neomycin phosphotransferase (NEO) gene and the nonstructural proteins of HCV, respectively. A T7
promoter is fused to the HCV 5⬘ terminus for the production of synthetic RNA. A domain of the NS5A protein is expanded to illustrate the ISDR,
PKR binding domain, and the region of the most common adaptive replicon mutations, including the deletion from amino acids 2207 to 2254
observed by Blight et al. (8). The amino acid changes for the adaptive mutations detected in the resident replicons isolated from 10 independent
G418-resistant colonies are indicated at the bottom of the figure, and the sites of NS5A hyperphosphorylation (amino acids 2197, 2201, and 2204)
are indicated by arrows. WT, wild type.
V
OL. 77, 2003 ANTIVIRAL AGENTS AND HCV REPLICONS 1093

second genotype 1b strain (27, 32) that did not require adap-
tive mutations for high colony-forming efficiency (32), and
recently replicons containing the full-length genome have been
developed (32, 53).
In this study, we have utilized the replicon system for anal-
ysis of several compounds for potential antiviral effect, includ-
ing IFN-␣, IFN-␥, tumor necrosis factor alpha (TNF-␣),
poly(I)-poly(C), and ribavirin. Although no antiviral effect was
observed with TNF-␣ or poly(I)-poly(C), a synthetic double-
stranded RNA (dsRNA) and known inducer of IFN and IFN-
stimulated genes (ISGs), both IFN-␣ and IFN-␥ exhibited an-
tiviral effects. The antiviral effect of ribavirin in this system
could be ascribed to the induction of error-prone replication
similar to recent findings with GB virus B (GBV-B) (38), a
surrogate model for HCV. The expression levels for a number
of ISGs were monitored before and after antiviral treatments
to begin a characterization of the virus-host interactions in-
volved in this system.
MATERIALS AND METHODS
Replicon constructs. Rep1bNeo was constructed from synthetic oligonucleo-
tides to be an exact copy of the replicon described by Lohmann et al. (43)
(GenBank accession no. AJ242652). The Rep1aNeo construct is identical to the
Rep1bNeo construct except that all HCV components were derived from the
HCV-1 infectious clone (40) (GenBank accession no. AF271632) including the 5⬘
NCR, the region encoding NS3-NS5B, and the 3⬘ NCR. The Rep1b/aNeo con-
struct was derived from Rep1aNeo by substituting 228 nucleotides (positions
1927 to 2155) from Rep1bNeo into Rep1aNeo, which altered 12 of the first 73
amino acids of NS3. Adaptive mutations were detected by direct sequencing of
PCR products from NS3, NS5A, and NS5B which spanned nucleotides 3414 to
5312, 6828 to 7118, and 8855 to 9029, respectively. The specific adaptive muta-
tions NS5A-S2204I and NS3-D1431Y were introduced into replicons using PCR-
directed mutagenesis. Synthetic replicon RNA was prepared from DNA linear-
ized with ScaI (Rep1bNeo) and XbaI (Rep1aNeo and Rep1b/aNeo) using the T7
Megascript kit (Ambion, Austin, Tex.) and was purified by DNase treatment,
RNazol (Leedo, Houston, Tex.) extraction, and ethanol precipitation. RNA was
quantified by optical density, and the concentration and quality were confirmed
by agarose gel electrophoresis.
Cells and transfections. Huh7 cells were cultivated in a 1:1 mixture of Dul-
becco’s modified Eagle’s medium and Ham’s F12 medium supplemented with
10% fetal bovine serum (FBS) and 50 ␮g of gentamicin sulfate per ml. RNA
transfections were performed using DMRIE-C transfection reagent (GIBCO/
BRL, Rockville, Md.) at a ratio of 5 ␮g of lipid to 1 ␮g of RNA. Replicons with
low or no colony-forming efficiency were transfected using 10 ␮g of replicon
RNA per 100-mm-diameter culture dish; however, replicons with adaptive mu-
tations were transfected at 1 to 0.01 ␮g of replicon RNA diluted into Huh7 RNA.
The RNA and lipid were diluted individually into 4.5 ml of medium without FBS,
combined, incubated for 15 min at room temperature, and added to the cultures.
Cultures were washed three times with medium without FBS prior to transfec-
tion and two times with medium containing FBS after transfection. Cultures were
exposed to the RNA-lipid mixture for6hat37°C. Culture medium was supple-
mented with 250 ␮g of G418 per ml beginning 1 day after transfection. This
transfection protocol routinely yielded approximately 45% transfection efficiency
for RNA, as evaluated by transfection of a Sindbis virus replicon expressing
␤-galactosidase (8). Measurement of replicon copy number and antiviral studies
were conducted with cultures 48 h after plating (cultures were approximately
60% confluent). None of the antiviral treatments employed induced noticeable
toxicity by microscopic inspection of the cultures or by measurement of glycer-
aldehyde-3-phosphate dehydrogenase (GAPDH) transcript levels. This was true
even when treatment was extended for 7 days. Primary chimpanzee and tamarin
hepatocytes were cultivated in a hormonally defined, serum-free medium as
previously described (39).
TaqMan quantitative RT-PCR for replicon RNA and host transcripts. Total
cell RNA was isolated from cell cultures using RNazol (Leedo). Replicon RNA
was quantified by a real-time, 5⬘ exonuclease reverse transcription-PCR (RT-
PCR) (TaqMan) assay (40) using the ABI 7700 sequence detector (Perkin-Elmer
Biosystems, Foster City, Calif.). The primers and probe were derived from the 5⬘
NCR and were selected using the Primer Express software designed for this
purpose (Perkin-Elmer Biosystems). The forward primer contains nucleotides
149 to 167 (5⬘-TGCGGAACCGGTGAGTACA-3⬘), the reverse primer contains
nucleotides 210 to 191 (5⬘-CGGGTTTATCCAAGAAAGGA-3⬘), and the probe
contains nucleotides 189 to 169 (5⬘-CCGGTCGTCCTGGCAATTCCG-3⬘). The
fluorogenic probe was labeled with FAM (6-carboxyfluorescein) and TAMRA
(6-carboxytetramethylrhodamine) and was obtained from Synthegen (Houston,
Tex.). The primers and probe were used at 10 pmol per 50-␮l reaction mixture.
The reactions were performed using the Brilliant Plus Single Step RT-PCR kit
(Stratagene, La Jolla, Calif.) and included a 30-min 48°C RT step, followed by 10
min at 95°C, and then 40 cycles of amplification using the universal TaqMan
RT-PCR standardized conditions: 15 s at 95°C for denaturation and 1 min at
60°C for annealing and extension. The standards used to establish genome
equivalents (ge) were synthetic RNAs transcribed from a clone of the 5⬘ NCR of
the HCV-1 strain (40). Synthetic RNA was prepared using the T7 Megascript kit
and was purified by DNase treatment, RNazol extraction, and ethanol precipi-
tation. RNA was quantified by optical density, and 10-fold serial dilutions were
prepared from 1 million to 10 copies using tRNA as a carrier. These standards
were run in all TaqMan RT-PCR assays in order to calculate the number of ge
in the experimental samples. The conditions for quantification of transcripts
from ISGs were identical to those described above for replicon RNA. Most
assays were multiplexed using GAPDH. The primer and probe sets used in these
assays will be presented elsewhere (C. Bigger, B. Guerra, K. M. Brasky, G. B.
Hubbard, M. Beard, and R. E. Lanford, submitted for publication).
Antiviral treatments. Poly(I)-poly(C) was obtained from ICN (Costa Mesa,
Calif.) and Sigma (St. Louis, Mo.). A highly purified preparation of ribavirin
(1-␤-
D-ribofuranosyl-1H-1,2,4-triazole-3-carboximide) was a gift from Schering
Plough Research Institute (Kenilworth, N.J.). Some commercially available
preparations of ribavirin contain trace contaminants that are often toxic in tissue
culture studies. This preparation was specifically purified and selected for low
toxicity in vitro. Human IFN-␣-2b (intron A) was obtained from Schering Plough
Research Institute. IFN-␥ was obtained from R&D Systems (Minneapolis,
Minn.). Antiviral treatments with IFN-␣, IFN-␥, and poly(I)-poly(C) were initi-
ated using subconfluent cultures, but after 24 h, the cultures reached confluency.
Treatment with ribavirin used confluent cultures to reduce the adverse effects of
ribavirin seen on rapidly dividing cells.
In vitro translation. In vitro translations were preformed with 1 ␮g of replicon
RNA prepared as described above, heated to 70°C for 10 min, and cooled on ice.
Translation reactions were performed in a total volume of 25 ␮l using the
Promega (Madison, Wis.) nuclease-treated rabbit reticulocyte lysate systems
supplemented to contain 90 mM KCl to enhance for IRES-driven translation.
Reaction mixtures contained 20 ␮M amino acids lacking methionine, 10 ␮Ci of
[
35
S]methionine (Express
35
S; Perkin-Elmer, Boston, Mass.), and RNasin (Pro
-
mega). Reactions were performed for1hat30°C and the products were exam-
ined directly by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-
PAGE).
PKR assays. For PKR assays, Huh7 cells were pretreated with 100 ␮gof
poly(I)-poly(C) per ml for2horwith 1,000 U of IFN-␣2b per ml for 16 h or left
untreated. Cultures were washed twice in phosphate-buffered saline (PBS), and
cell lysates were prepared using PEB (PBS containing 1% Nonidet P-40 [NP-40],
10% glycerol, 1 mM dithiothreitol, and protease inhibitors) and were clarified in
a microcentrifuge for 20 min at 4°C. PKR was immunoprecipitated for2hat4°C
using protein G beads containing 5 ␮g of monoclonal antibody clone 13 to PKR
(BD Biosciences, San Diego, Calif.). The beads were washed five times with PEB
and once with PKR buffer (20 mM Tris [pH 7.5], 0.1 mM EDTA, 80 mM KCl,
5% glycerol, 2 mM MgCl
2
, 2 mM MnCl
2
, and 0.2 mg of bovine serum albumin
per ml). Kinase reactions were conducted for 15 min at 30°C with PKR bound to
the beads in 50 ␮l of PKR buffer supplemented with 5 ␮Ci of [␥-
32
P]ATP
(Perkin-Elmer). PKR was eluted from the beads with SDS gel sample buffer and
analyzed by SDS-PAGE and phosphorimage analysis.
Northern blot hybridization. For analysis of replicon RNA by Northern blot
hybridization, total cellular RNA was isolated from replicon lines using RNazol.
RNA was analyzed by electrophoresis on a 1% agarose-formaldehyde gel at 30
Vfor16hat16°C and was transferred to Gene Screen Plus hybridization transfer
membranes (Perkin-Elmer) using downward capillary transfer. Membranes were
prehybridized in SDS hybridization buffer {6⫻ SSPE [1⫻ SSPE is 0.18 M NaCl,
10 mM NaH
2
PO
4
, and 1 mM EDTA (pH 7.7)], 10% SDS, 200 ␮g of salmon
sperm DNA per ml, and 50% formamide} for5hat59°C and were hybridized
in the same buffer containing 10
6
counts of
32
P-labeled riboprobe per ml for 16 h
at 59°C. Riboprobes were prepared from a linearized vector containing the
neomycin phosphotransferase gene downstream of a T7 RNA polymerase pro-
moter using the Promega Riboprobe System as described by the manufacturer.
Membranes were washed twice with 1⫻ SSC (1⫻ SSC is 0.15 M NaCl plus 0.015
M sodium citrate) with 0.1% SDS at 23°C for 30 min and twice with 0.1⫻ SSC
1094 LANFORD ET AL. J. VIROL.

with 0.1% SDS at 65°C for 30 min. Membranes were analyzed by phosphorimage
analysis and autoradiography.
RESULTS
Isolation of cell lines with genotype 1b replicons with adap-
tive mutations. Our studies were initiated in a manner previ-
ously described using an exact copy of the original genotype 1b
replicon described by Lohmann et al. (43) (designated
Rep1bNeo in these studies). Huh7 cells were transfected with
synthetic RNA produced from the construct using DMRIE-C,
a RNA transfection reagent. The colony-forming efficiency of
this replicon in the presence of G418 selection was frequently
zero and never exceeded five colonies per ␮g of transfected
RNA. Ten colonies from a single experiment were expanded
for further analysis. Replicon RNA was quantified using a
TaqMan, real-time RT-PCR assay with a primer and probe set
directed at the HCV 5⬘ NCR. The level of replicon RNA
varied from 1.7 ⫻ 10
6
to 1.3 ⫻ 10
7
ge/␮g of cellular RNA
(Table 1) or approximately 17 to 130 ge/cell, assuming that
copy number is uniform in cells of the same line. Clone 45 cells
were plated in identical plates at 60% confluency and analyzed
at multiple times over a 10-day period. Although a 50% decline
in replicon RNA (number of ge per microgram of cell RNA)
occurred between days 1 and 2, no further decline was ob-
served over 10 days, suggesting that the differences in replicon
number in different cell lines was not due to minor variations
in culture confluency (data not shown). Three regions (a por-
tion of NS3, NS5A, and NS5B) of the replicon present in each
cell line were PCR amplified and sequenced to determine the
consensus sequence of the resident replicon. Previously, most
adaptive mutations have been detected in a region adjacent to
the ISDR of NS5A (Fig. 1) (8, 27, 35, 42); however, adaptive
mutations have also been detected in NS3, NS4B, and NS5B
(27, 35, 42). In this study, the replicon present in each cell line
contained a single mutation within NS5A, and 2 of the 10
replicons contained mutations in NS3, while no mutations were
noted within NS5B. The NS5A mutations spanned nucleotides
6861 to 6951 and amino acids 2177 to 2204 (nucleotide and
amino acid numbers are based on HCV genomic RNA se-
quence) (Fig. 1 and Table 1). A total of six unique mutations
were observed; two were present in two different replicons
each (S2197F and S2204I), and one was present in three rep-
licons (S2202L). One aspect that was noteworthy was the mu-
tation of two of the serine residues associated with hyperphos-
phorylation of NS5A (amino acids 2197 and 2204) (64); 4 of
the 10 mutations detected involved these two serine residues
(Fig. 1 and Table 1). Clones 35 (NS5A S2204I) and 38 (NS5A
A2199T) also carried mutations in NS3 at I1278L and Q1335E,
respectively. In addition, a mutation at NS3 D1431Y was ob-
served in combination with NS5A S2204I in a cell line not
shown in Table 1. Transfection of total cellular RNA derived
from each cell line resulted in a much higher colony-forming
efficiency than the parental replicon, suggesting that the
changes were adaptive mutations. Extrapolation of the Taq-
Man RT-PCR results for replicon copy number (number of ge
per microgram of cellular RNA) to the number of colonies
formed per microgram of transfected cell RNA revealed that
colony-forming efficiencies ranged from 52,000 to 500,000 col-
onies/␮g of replicon RNA (Table 1). Since limited regions of
each replicon were sequenced, the presence of additional
adaptive mutations cannot be excluded.
Synergy of NS3 mutations with NS5A mutations and failure
of adaptive mutations to enhance the colony-forming efficiency
of genotype 1a replicons. To construct a Rep1bNeo replicon
with high colony-forming efficiency, one of the common NS5A
mutations at amino acid position 2204 (serine to isoleucine;
S2204I) was introduced into Rep1bNeo by PCR-directed mu-
tagenesis. While the parental replicon often yielded no colo-
nies in individual assays, Rep1bNeo-2204 produced up to 800
colonies/␮g of replicon RNA. Although a significant improve-
ment over the parental replicon, the transfection of total cell
RNA (Table 1) from selected colonies suggested that addi-
tional mutations must account for the very high efficiency ob-
served in the adapted replicons. Next, we tested the NS3 mu-
tation at amino acid D1431Y by itself and in concert with the
S2204I mutation. Rep1bNeo-1431 exhibited no significant im-
provement in colony-forming efficiency over the parental rep-
licon, while the NS3 mutation was highly synergistic with the
NS5A mutation (Rep1bNeo-1431/2204 [Table 2]), producing
up to 80,000 colonies/␮g of replicon RNA. This colony-form-
ing efficiency is comparable to that determined for the repli-
cons present in selected colonies and suggests that NS3 muta-
tions complement NS5A mutations in a nonadditive
mechanism.
Previous studies have suggested that genotype 1a replicons
are not capable of forming colonies in Huh7 cells at detectable
levels (8, 27). Since these studies were conducted with a single
strain of genotype 1a (H77), it is not clear whether this is a
general property of genotype1a isolates or unique to this
strain. We constructed genotype 1a replicons using our infec-
tious clone of the HCV-1 prototype sequence (40), the only
other genotype 1a infectious clone. The construct design was
identical to that used for Rep1bNeo, except that all HCV
TABLE 1. NS5A adaptive mutations of Rep1bNeo
a
Clone
Replicon RNA
(10
6
ge/␮gof
cell RNA)
b
Amino acid
c
Colony-forming
efficiency
(10
3
colonies/␮gof
replicon RNA)
d
Position Change
2 1.7 2177 D3H 500
42 10.1 2197 S3F79
45 11.9 2197 S3F87
38 9.5 2199 A3T 167
40 9.2 2200 S3R53
13 7.4 2202 S3L94
39 1.8 2202 S3L 154
43 5.3 2202 S3L52
35 13.1 2204 S3I72
37 2.6 2204 S3I 147
a
Huh7 cells were transfected with Rep1bNeo, and G418-resistant colonies
were established as cell lines.
b
The replicon copy number was determined by TaqMan RT-PCR and ex
-
pressed as ge per microgram of cellular RNA. To determine the number of ge
per microgram of RNA, cells were plated at a low density and harvested 48 h
later, when the cultures were still subconfluent.
c
Regions of NS3, NS5A, and NS5B were amplified and sequenced to deter
-
mine adaptive mutations. The amino acids are given in the single-letter code.
d
To determine the colony-forming efficiency of the adapted replicons, Huh7
cells were transfected with 10 ␮g of total cell RNA isolated from each cell line,
and the colony-forming efficiency in the presence of G418 was determined. The
colony-forming efficiency is presented as the number of colonies per microgram
of transfected replication RNA that was extrapolated from the estimated number
of replicon ge per microgram of cell RNA.
VOL. 77, 2003 ANTIVIRAL AGENTS AND HCV REPLICONS 1095

sequences were derived from the HCV-1 clone. The resulting
replicon RNA, Rep1aNeo, was not capable of inducing colony
formation following transfection into Huh7 cells in multiple
attempts (Table 2). Analysis of the in vitro translation pattern
from this RNA in rabbit reticulolysates revealed that expres-
sion of the HCV nonstructural proteins from the EMCV IRES
was very low in comparison to Rep1bNeo, despite the fact that
translation of the neo gene from the HCV IRES was equivalent
for the two constructs (Fig. 2A). A series of chimeric constructs
revealed that a short sequence at the amino terminus of NS3
adjacent to the EMCV IRES was responsible for the differ-
ences in translation efficiencies of the two constructs. To pro-
vide more-efficient translation of the HCV nonstructural pro-
teins from the Rep1aNeo replicon, a portion (228 nucleotides)
of the genotype 1a sequence was replaced with the genotype 1b
sequence to create Rep1b/aNeo, which differed from
Rep1aNeo by 12 amino acids (see Materials and Methods for
details). Presumably, a RNA structure within the genotype 1a
sequence inhibited translation from the EMCV IRES. Al-
though this chimeric construct exhibited in vitro translation
properties equivalent to Rep1bNeo (Fig. 2A), it was not capa-
ble of inducing colony formation in Huh7 cells (Table 2). Since
the Rep1bNeo replicon itself exhibited marginal colony-form-
ing ability prior to acquisition of adaptive mutations, the
NS5A-S2204I mutation was introduced alone or in combina-
tion with D1431Y into Rep1b/aNeo. These mutations did not
increase the colony-forming efficiency of Rep1b/aNeo to de-
tectable levels following transfection of Huh7 cells. These re-
sults suggest that replicons based on the HCV-1 genotype 1a
sequence do not possess the capacity to induce detectable
levels of colony formation in Huh7 cells even with mutations
known to enhance genotype 1b strains. Part of our analysis of
these constructs included in vitro translation, as shown in Fig.
2A. When the replicons containing the NS3 mutations were
compared to the parental replicons and those containing the
NS5A mutation alone, NS3 exhibited a significantly increased
mobility by SDS-PAGE. This was true whether the D1431Y
mutation was present in the Rep1bNeo or Rep1b/aNeo back-
ground and whether the S2204I mutation was present or not
(Fig. 2B). At this time, the reason for the increased mobility of
NS3 is not understood, but it may be indicative of an altered
posttranslational modification, which in turn may play a role in
the synergy of this mutation with the NS5A mutation.
Antiviral effects of IFN-␣, IFN-␥, poly(I)-poly(C), and
TNF-␣. The potential antiviral activities of IFN-␣, IFN-␥,
poly(I)-poly(C), and TNF-␣ were examined using the replicon
system. In these studies, a replicon cell line was treated with
various concentrations of the compound, and the level of rep-
licon RNA in cell lysates was quantified using TaqMan RT-
PCR assays. All assays were multiplexed for GAPDH mRNA
to ensure that the treatment had no overt adverse effect on
cellular mRNA levels. Initially, a time course study was per-
formed with each antiviral agent. Clone 45 cultures (Table 1)
were treated for 24, 48, and 72 h with 100 to 1,000 U of
IFN-␣2b or IFN-␥ per ml, 100 to 1,000 ␮g of poly(I)-poly(C)
per ml, or 200 to 1,000 U of TNF-␣ per ml. Dramatic reduc-
tions in replicon RNA levels were observed at both concentra-
TABLE 2. Colony-forming efficiency of replicon constructs
a
Replicon RNA
Colony-forming efficiency
(colonies/␮g of replicon RNA)
Rep1bNeo ....................................................................... ⱕ5
Rep1bNeo-2204.............................................................. 800
Rep1bNeo-1431.............................................................. 0
Rep1bNeo-1431/2204..................................................... 80,000
Rep1aNeo ....................................................................... 0
Rep1b/aNeo .................................................................... 0
Rep1b/aNeo-2204........................................................... 0
Rep1b/aNeo-1431/2204.................................................. 0
a
Huh7 cells were transfected with various replicons, and the colony-forming
efficiency in the presence of G418 was determined. Replicons were transfected at
10 ␮g/100-mm-diameter dish, except for Rep1bNeo-1431/2204, which was trans-
fected at 10 ng of replicon RNA mixed with 10 ␮g of Huh7 RNA as carrier. Each
replicon was tested in multiple experiments. Rep1bNeo frequently failed to yield
colonies, but in a single experiment yielded 50 colonies/10 ␮g of transfected
RNA, and these colonies were the source of the cell lines in Table 1.
FIG. 2. In vitro translation of bicistronic HCV replicons.
(A) Rep1aNeo (1a), Rep1b/a (1b/a), and Rep1b (1b) synthetic RNAs
were produced by in vitro transcription reactions utilizing vectors lin-
earized at EcoRI, a site downstream of the neomycin phosphotrans-
ferase gene (Fig. 1), or at XbaIorScaI to produce full-length replicon
RNA. RNAs were in vitro translated using rabbit reticulocyte lysates in
the presence of [
35
S]methionine, and the products were analyzed by
SDS-PAGE. Rep1a produced reduced levels of NS3 compared to both
Rep1b and Rep1b/a, while translation of the Neo protein was similar
for all constructs. (B) The impact of the NS3 D1431Y mutation on the
migration of NS3 by SDS-PAGE was examined following in vitro
translation. All replicon RNAs containing the D1431Y mutation ex-
hibited an increased mobility for NS3 regardless of whether the mu-
tation was in the presence of the NS5A S2204I mutation or in the
Rep1b (1b) or Rep1a (1a) background. wt, wild type.
1096 LANFORD ET AL. J. V
IROL.

tions of IFN-␣2b at 72 h, i.e., 40- and 172-fold reduction at 100
and 1,000 U/ml, respectively (Fig. 3). Although substantial
antiviral activity was also observed with IFN-␥, the reduction in
replicon RNA was approximately three- to fourfold less (14.4-
and 44.9-fold reduction at 100 and 1,000 U/ml, respectively, at
72 h). No significant reduction was observed with poly(I)-
poly(C), even at 1,000 ␮g/ml (Fig. 3), or with TNF-␣ at 1,000
U/ml (data not shown).
In part to confirm these findings and to determine the max-
imum reduction observed with each antiviral following a more
prolonged treatment, cultures were treated for 7 to 9 days with
each antiviral. Replicon levels were reduced by 2,300- and
23,000-fold at 100 and 1,000 U/ml of IFN-␣2b, respectively
(Table 3). This was not an unanticipated finding, since other
studies have observed a reduction in replicon RNA following
IFN-␣ treatment (8, 20, 27). However, extended treatment
with poly(I)-poly(C), a known inducer of dsRNA response
genes including type 1 IFN genes and ISGs, still had no anti-
viral effect. This was an unanticipated finding, since poly(I)-
poly(C) has a high level of antiviral activity for many viruses,
including GBV-B replication in primary tamarin hepatocytes
(38), which is a closely related surrogate model for HCV.
Poly(I)-poly(C) treatment was performed by direct addition of
the compound to the medium at concentrations as high at
1,000 ␮g/ml, as well as transfection with lipid reagents without
detectable antiviral effect. Although IFN-␥ again exhibited an-
tiviral activity, the prolonged treatment did not result in sig-
nificantly greater reduction of replicon RNA than treatment
for 72 h (100 or 1,000 U/ml of IFN-␥ for 7 days resulted in a
2.7- and 31.9-fold reduction in replicon RNA, respectively).
Host cell response to antiviral treatments. To further ex-
plore the differences in response to IFN-␣ and poly(I)-poly(C),
induction of dsRNA-IFN-stimulated gene was examined using
TaqMan RT-PCR assays for the transcripts of STAT1␣,
STAT1␤, PKR, ISG12, IRF-1, and IP10. Huh7 cells were com-
pared to replicon cells following 24 to 48 h of treatment with
IFN-␣, IFN-␥, or poly(I)-poly(C) (Table 4). In this experiment,
IFN-␣ resulted in 11.8- and 104-fold reduction in replicon
RNA at 24 and 48 h, respectively, while IFN-␥ resulted in 6.1-
and 31.2-fold reduction in replicon RNA at 24 and 48 h.
Poly(I)-poly(C) again failed to induce a significant decline in
replicon RNA. All of the ISGs were elevated in IFN-␣-treated
Huh7 and replicon cells; however, IRF-1 was increased by only
2.3- to 3.3-fold, and it is not clear that these values represent
significant increases over the baseline levels of the untreated
cultures.
Although the basal levels of the ISGs were similar in both
FIG. 3. Kinetics of HCV replicon RNA decline following treat-
ment with IFN-␣, IFN-␥, and poly(I)-poly(C). Clone 45 cells were
treated with IFN-␣ or IFN-␥ at 100 or 1,000 U/ml and with poly(I)-
poly(C) at 100 or 1,000 ␮g/ml and harvested at 24, 48, and 72 h. Results
from 100 (A) and 1,000 (B) U/ml or ␮g/ml. Replicon RNA levels were
quantified by TaqMan RT-PCR and expressed as the fold change from
the levels of untreated cultures harvested at the same time points. All
values are averages from duplicate cultures.
TABLE 3. Antiviral effects of IFN-␣, poly(I)-poly(C), and IFN-␥
a
Treatment Fold reduction
b
GAPDH Ct
c
IFN-␣ (U/ml)
0 0 18.0
100 2,300 17.6
1,000 23,000 17.6
Poly(I)-poly(C) (␮g/ml)
0 0 16.8
50 1.4 16.5
IFN-␥ (U/ml)
0 0 15.9
100 2.7 15.6
1,000 31.9 15.2
a
Replicon lines were treated with IFN-␣, poly(I)-poly(C), or IFN-␥ for7to9
days. In this experiment, clone 8 cells were treated with IFN-␣, while clone 45
cells were treated with IFN-␥ and poly(I)-poly(C). Treatments were initiated on
confluent cultures, and the medium was changed every 2 days. No overt toxicity
was observed.
b
Replicon RNA was quantified by TaqMan RT-PCR, and values are ex
-
pressed as fold reduction compared to the values for untreated cells.
c
The TaqMan RT-PCR assay for replicon RNA was multiplexed for the
GAPDH mRNA to demonstrate a lack of effect of treatments on cellular
mRNA, and all values were normalized for GAPDH. GAPDH values are ex-
pressed as Ct, the amplification cycle at which the values exceeded the back-
ground threshold.
VOL. 77, 2003 ANTIVIRAL AGENTS AND HCV REPLICONS 1097

Huh7 cells and clone 45 cells, some variation was noted in the
degree of induction of ISGs in the two cell types following
treatment with IFN-␣ and IFN-␥. While PKR was induced to
similar levels in the presence and absence of the replicon and
STAT1␣ and STAT1␤ were increased approximately twofold
more in the absence of the replicon, ISG12 and IP10 induction
by IFN-␣ was increased by approximately 8- to 10-fold in the
presence of the replicon (Table 4). The greater induction of
ISG12 and IP10 by IFN-␣ in the presence of the replicon was
not observed following treatment with IFN-␥. As expected,
some of the IFN response genes were induced differentially by
IFN-␣ and IFN-␥. While IRF-1 was minimally induced by
IFN-␣ in comparison to IFN-␥, the opposite was true for PKR.
The increased induction of ISG12 and IP10 by IFN-␣ in
clone 45 cells in comparison to Huh7 cells could be due to
clonal variation of individual cells selected from the Huh7
population; alternatively, it could represent an important dif-
ference induced by either viral dsRNA or an HCV protein. To
determine the degree of variation in the IFN induction of ISGs
in Huh7 cells and different replicon lines, clones 2, 40, and 45
were compared to Huh7 cells following 24 h of treatment with
IFN-␣ and IFN-␥. Stat1␣, ISG12, and IP10 transcripts were
examined by TaqMan RT-PCR assays. Although in all cases
the transcripts were induced in both Huh7 and replicon cell
lines in the presence of IFN-␣ or IFN-␥, the differential induc-
tion of IP10 and ISG12 in cells containing the replicon in
comparison to Huh7 cells was less apparent. The induction of
IP10 transcripts in IFN-␣-treated cells was greater in all rep-
licon lines than in Huh7 cells. In two lines (clones 40 and 45),
it was again increased by ⬎10-fold more than in Huh7 cells, but
in a third line (clone 2), IP10 transcripts were induced only
about 2.5-fold more than in Huh7 cells (Table 5). No pattern
was observed for either Stat1␣ or ISG12, but in general, rep-
licon lines had higher levels of ISG induction in the presence
of IFN-␣. Interestingly, the pattern for IP10 in the two exper-
iments remained constant with regard to Huh7 cells and clone
45 cells treated with IFN-␣ and IFN-␥. IP10 transcripts were
induced to 10-fold-greater levels by IFN-␣ in clone 45 cells in
comparison to Huh7 cells, while IFN-␥ induced IP10 to a
greater extent in Huh7 cells than in clone 45 cells (Table 5).
These data indicate that (i) ISG induction occurs in the pres-
ence of both IFN-␣ and IFN-␥, (ii) some differential regulation
of ISGs is observed between the two IFNs, and (iii) although
some differences were observed in the presence and absence of
the replicon, some of the variation may be due to the clonal
selection of cell lines.
In contrast to IFN-␣ and IFN-␥, poly(I)-poly(C) treatment
did not induce the expression of ISGs in either Huh7 cells or
clone 45 cells (Table 4). These data suggest that the parental
Huh7 cells are altered in regulation of some component of the
dsRNA response pathway, which may explain the lack of an-
tiviral effect by poly(I)-poly(C) on the replicon in these cells.
This contrasts sharply with primary chimpanzee hepatocytes,
which were highly responsive to poly(I)-poly(C). Primary chim-
panzee hepatocytes were treated with either IFN-␣ or poly(I)-
poly(C) and analyzed for the induction of ISGs. In chimpanzee
hepatocytes, 50 ␮g of poly(I)-poly(C) per ml was superior to
100 U of IFN-␣ per ml in the induction of ISGs (Table 4). We
had previously demonstrated the potent antiviral properties of
poly(I)-poly(C) in the GBV-B/tamarin hepatocyte model.
Since this system permitted the direct comparison of two cell
types in which poly(I)-poly(C) had either no antiviral activity
against HCV replicons or potent antiviral activity against an
HCV surrogate, we examined poly(I)-poly(C) treatment in
Huh7 cells and primary tamarin hepatocytes. TaqMan RT-
PCR assays were used to quantify the level of induction of
IFN-␤ transcripts at 2, 7, and 24 h after poly(I)-poly(C) addi-
tion. We chose IFN-␤, since it is known to be responsive to
dsRNA and has potent antiviral activity. No induction of
IFN-␤ transcripts was noted in Huh7 cells, while 12- to 18-fold
induction occurred in tamarin hepatocytes at all three time
TABLE 5. Induction of ISGs by IFN-␣ and IFN-␥ in multiple
replicon lines
Treatment
and cell
a
Fold change
b
Stat-1␣ ISG12 IP10 HCV
IFN-␣
Huh7 4.5 104.5 3.0
Clone 2 6.9 403.6 7.6 9.5
Clone 40 7.0 275.0 52.9 8.1
Clone 45 4.8 130.6 43.0 4.8
IFN-␥
Huh7 8.4 25.0 227.3
Clone 2 8.0 47.1 33.1 7.5
Clone 40 8.2 21.9 109.7 8.7
Clone 45 6.5 15.3 75.2 4.7
a
Huh7 and clone 2, 40, and 45 cells were treated for 24 h with IFN-␣ or IFN-␥
at 1,000 U/ml.
b
Levels of HCV replicon RNA and transcripts for STAT-1␣, IP10, and ISG12
were quantified by TaqMan RT-PCR assays. The data are averages from dupli-
cate cultures and are expressed as the fold change compared to the values for
untreated cultures.
TABLE 4. Induction of ISG transcripts by IFN-␣, IFN-␥, and
poly(I)-poly(C)
a
Cell
b
Treatment
Fold change
c
STAT1␣ STAT1␤ PKR ISG12 IRF-1 IP10
Huh7 IFN-␣ 41.1 38.8 20.3 312 3.1 10.0
Rep IFN-␣ 18.6 24.1 18.7 2,374 3.3 111.5
Huh7 IFN-␥ 80.8 70.6 3.4 80.0 47.9 198.8
Rep IFN-␥ 31.3 33.5 4.5 74.3 45.5 123.1
Huh7 Poly(I)-poly(C) 1.3 ⫺1.3 1.0 1.6 ⫺1.2 ⫺1.6
Rep Poly(I)-poly(C) ⫺1.1 1.1 ⫺1.5 2.2 ⫺1.1 1.3
CH IFN␣ 2.0 3.6 3.8 9.3 1.4 12.6
CH Poly(I)-poly(C) 3.7 8.9 5.3 40.4 2.2 549.0
a
Huh7 and clone 45 cell lines were treated with IFN-␣ (1,000 U/ml), polyIC
(100 ␮g/ml), or IFN-␥ (1,000 U/ml). Primary chimpanzee hepatocytes were
treated with IFN-␣ (100 U/ml) or polyIC (50 ␮g/ml).
b
Rep, replicon clone 45 cell line; CH, chimpanzee hepatocytes.
c
Transcripts for various ISGs were quantified using TaqMan RT-PCR assays.
The data are averages from duplicate cultures and are expressed as the fold
change compared to the values for untreated cultures. The data for IFN-␣ and
IFN-␥ treatment are from the 24-h harvest, since this time point had the highest
values. For polyIC, similar values were obtained for the 24- and 48-h time points.
The 48-h values are shown to demonstrate that a delayed response was not
missed. The chimpanzee hepatocytes were harvested at 72 h for both treatments.
Additional cells were not available to repeat the chimpanzee hepatocyte study at
multiple time points. The level of the replicon RNA was decreased by 104- and
31.2-fold in IFN-␣- and IFN-␥-treated cells at the 48-h time point. No decrease
in replicon RNA was observed following polyIC treatment.
1098 LANFORD ET AL. J. VIROL.

points (Fig. 4). Since numerous different approaches to induc-
tion of ISG transcription with poly(I)-poly(C) were attempted
in Huh7 cells without success, we questioned whether poly(I)-
poly(C) was entering the cells and inducing latent dsRNA
binding proteins. To test this, Huh7 cells were treated with
poly(I)-poly(C) for2horwith IFN-␣ for 16 h, and PKR was
immunoprecipitated and used in kinase assays. In the presence
of dsRNA, PKR dimerizes and undergoes autophosphoryla-
tion, and PKR kinase activity can also be induced by IFN-␣.In
comparison to untreated Huh7 cells, both poly(I)-poly(C) and
IFN-␣ dramatically induced PKR kinase activity (Fig. 5).
These data suggest that Huh7 cells are responsive to poly(I)-
poly(C) but that they do not respond in a typical fashion to
dsRNA, since no antiviral activity or induction of ISG tran-
scription was apparent using this compound.
Ribavirin induces error-prone replication of HCV replicon
RNA. Ribavirin has an antiviral effect for a number of viruses
in tissue culture (14, 47, 58). The monophosphate form of
ribavirin is an IMP dehydrogenase (IMPDH) inhibitor, and at
least some of the in vitro antiviral effect has been ascribed to
the reduction of GTP pools by inhibition of IMPDH. Although
this has deleterious effects on cellular metabolism, these effects
are tolerated in short-term studies in nondividing cell cultures.
Ribavirin is used in combination with IFN-␣ for the treatment
of HCV infections and provides a significant improvement in
the rate of sustained viral clearance in comparison to the rate
for IFN-␣ alone. However, ribavirin monotherapy does not
result in a significant reduction in the level of viremia despite
a marked improvement in liver disease (9, 16). One mechanism
suggested to explain the improvement with ribavirin therapy
involves an immunomodulatory activity possessed by ribavirin
that promotes a Th1-biased immune response (19, 30, 50, 60).
However, the mechanism of the synergistic effect that ribavirin
has with IFN-␣ for increasing the percentage of patients with
sustained viral clearance remains uncertain. We recently dem-
onstrated a direct antiviral effect of ribavirin on GBV-B virus
that involves induction of error-prone replication due to riba-
virin triphosphate incorporation (38). The following experi-
ments were conducted to determine whether ribavirin has an
antiviral effect on the HCV replicon and whether this effect
could be ascribed to induction of error-prone replication.
Experiments with ribavirin were conducted with confluent
nondividing cultures to minimize the adverse effects from GTP
pool reduction. In addition, a highly purified preparation of
ribavirin was used; the preparation was devoid of trace con-
taminants that often cause toxicity in tissue culture studies
unrelated to the IMPDH inhibition by ribavirin (see Materials
and Methods). Initially, the level of ribavirin was titrated to
determine the optimal dose for antiviral effect with minimal
cellular toxicity. A replicon cell line was treated with 50 to 400
␮M ribavirin for 9 days, and total cell RNA was examined by
TaqMan RT-PCR assay to determine the effects on replicon
RNA and GAPDH mRNA levels. Little effect was observed at
50 or 100 ␮M, while treatment with 200 and 400 ␮M ribavirin
resulted in 19.3- and 2,900-fold reduction in replicon RNA,
respectively (Table 6). No overt toxicity or decrease in the level
of GAPDH mRNA was observed at any treatment level, but
some decline in the secretion of apolipoprotein B was observed
at the higher ribavirin concentrations (data not shown). A time
course study revealed that treatment of cultures with 400 ␮M
ribavirin reduced replicon RNA from 2.2-fold at 24 h to 24.3-
fold at 72 h when analyzed by TaqMan RT-PCR (Fig. 6C).
Analysis of the same cellular RNAs for replicon RNA levels by
Northern blot hybridization and quantification by phosphorim-
ager analysis of the blot provided essentially identical results
for the percent decrease in replicon RNA in ribavirin-treated
cultures (Fig. 6B). Since only the area of the gel representing
intact replicon RNA is used in phosphorimage analysis, these
results suggest that the replicon RNA was not degraded in
ribavirin-treated cultures. The autoradiogram of the Northern
FIG. 5. PKR activation by poly(I)-poly(C) in Huh7 cells. Huh7
cells were treated with 100 ␮g of poly(I)-poly(C) per ml for2horwith
1,000 U of IFN-␣ per ml for 16 h prior to harvest. PKR was immuno-
precipitated from cell lysates, and kinase reactions were conducted
with [␥-
32
P]ATP with PKR still bound to the antibody beads. Phos
-
phorylated PKR was analyzed by SDS-PAGE and autoradiography
(top) or by phosphorimage analysis (bottom). Phosphorimage values
are expressed as arbitrary units and represent the volume in individual
PKR bands shown in the blot.
FIG. 4. IFN-␤ transcription in response to poly(I)-poly(C). Huh7,
clone 45, and primary tamarin hepatocyte cultures were harvested
after 2, 7, and 24 h of treatment with 100 ␮g of poly(I)-poly(C) per ml.
The levels of IFN-␤ transcripts were quantified in total cell RNA by
TaqMan RT-PCR and were expressed as fold change in comparison to
the levels of untreated cells. All values are averages of duplicate
cultures.
VOL. 77, 2003 ANTIVIRAL AGENTS AND HCV REPLICONS 1099

also demonstrated that the replicon RNA from treated cul-
tures was not detectably degraded (Fig. 6A); however, due to
the 24.3-fold decrease in replicon RNA at 72 h, overexposure
of the autoradiogram was required to confirm that the RNA at
this time point was not degraded (data not shown).
The cellular RNAs from the same time course study were
used to evaluate whether the antiviral effect was due to error-
prone replication. To test this hypothesis, Huh7 cells were
transfected with total cellular RNA derived from treated and
untreated cultures to determine whether a decrease occurred
in the colony-forming capacity of the replicon in treated cul-
tures. This test for error-prone replication avoids the issue of
ribavirin toxicity and is similar to the approach used for
GBV-B (38) where we demonstrated that virus from treated
cultures had a reduced specific infectivity when used to infect
new cultures. In the current studies, cells were transfected with
equivalent numbers of ge of replicon RNA derived from
treated and untreated cultures. For each time point, cells were
transfected with 10 ␮g of total cell RNA from the treated
cultures (the maximum amount of cellular RNA that can be
efficiently transfected), and the RNA from untreated cultures
was adjusted with Huh7 cell RNA such that cells were trans-
fected with equal amounts of cell RNA containing equal rep-
licon copy numbers from treated and untreated cultures (Table
7).
Colony-forming activity for the RNA from treated cultures
was reduced by 3.1- and 6.4-fold at 24 and 48 h of treatment,
respectively, while RNA from cultures treated for 72 h was
devoid of colony-forming activity (Table 7). These data dem-
onstrate a progressive loss in colony-forming activity for the
replicon RNA with increased exposure to ribavirin and support
the conclusion that ribavirin has the capacity to affect HCV
RNA replication through a mechanism of error-prone replica-
tion.
DISCUSSION
The development of a replicon system for HCV has pro-
vided a means to approach a number of biological questions
previously not possible. One of the primary uses of this system
will be the evaluation of antiviral agents and virus-host inter-
actions. Persistence of the replicon is very sensitive to IFN-␣,
confirming that IFN-␣ has a direct antiviral effect on the virus
(8, 20, 27). In this study, we have extended these observations
to include IFN-␥ and ribavirin, and we have demonstrated the
absence of antiviral effect for poly(I)-poly(C) and TNF-␣.
The successful application of the replicon system to HCV
research is dependent in part upon the realization that specific
adaptive mutations dramatically increase the ability of the rep-
licon to persist and induce colony formation (8). Adaptive
mutations have been described by several laboratories (8, 27,
35, 42), and the scope of these mutations was increased in this
study. As in previous studies, the primary adaptive mutations
TABLE 6. Titration of antiviral effect of ribavirin
a
Ribavirin
concn (␮M)
Replicon
(10
6
ge/␮gof
cell RNA)
b
Fold
reduction
c
GAPDH
Ct
d
0 5.8 0 17.4
50 3.4 1.7 17.6
100 2.5 2.3 17.6
200 0.3 19.3 17.6
400 0.002 2,900 17.6
a
Clone 8 cells were plated and allowed to reach confluency for 3 days and then
were treated with ribavirin for 9 days.
b
Replicon RNA was quantified by TaqMan RT-PCR and expressed as copy
number (ge) per microgram of cell RNA.
c
The data are expressed as the fold change compared to the values for
untreated cultures.
d
The TaqMan RT-PCR assay for replicon RNA was multiplexed for the
GAPDH mRNA to demonstrate a lack of overt toxicity or decrease in cellular
mRNA in ribavirin-treated cells. GAPDH values are expressed as Ct, the am-
plification cycle at which the values exceeded the background threshold.
FIG. 6. Antiviral effect of ribavirin on HCV replicon RNA. Clone
24 cells were cultivated with (⫹) or without (⫺) 400 ␮M ribavirin for
24, 48, or 72 h. Replicon RNA was analyzed by agarose gel electro-
phoresis, Northern blot hybridization, and autoradiography (A). RNA
in the same blot was quantified by phosphorimage analysis (B), or the
replicon RNA was quantified by TaqMan RT-PCR (C). Values in
panels B and C are expressed as percentages of untreated, control
cultures at each time point.
1100 LANFORD ET AL. J. V
IROL.

were localized to a small domain adjacent to the ISDR and
PKR binding sites of NS5A (Fig. 1), although adaptive muta-
tions have been observed in NS3, NS4B, and NS5B (27, 35, 42).
We found that mutations in NS3 frequently arise in conjunc-
tion with the NS5A mutations. Although the NS3 mutation at
D1431Y did not increase colony-forming efficiency of the pa-
rental replicon, it was highly synergistic with the S2204I muta-
tion in NS5A. The synergy between NS3 and NS5A mutations
has been observed previously (35), but this specific combina-
tion of mutations has not been previously observed. Previously
described mutations in NS3 span a region of 407 amino acids
(positions 1202 to 1609) (27, 42) within the helicase domain of
NS3. In our studies, a single NS5A S2204I mutation was asso-
ciated with NS3 mutations separated by 153 amino acids (po-
sitions 1278 to 1431). The mechanisms of adaptive mutations
and the synergy between NS3 and NS5A mutations are not
currently understood but may involve changes in the interac-
tions among viral proteins and in the interactions of viral
proteins with specific host factors. The NS3 D1431Y mutation
increased the mobility of NS3 by SDS-PAGE, whether it was in
the genotype 1b or 1a background, which is suggestive of an
altered posttranslational modification. The adaptive mutations
for genotype 1b did not confer an increased colony-forming
ability to genotype 1a replicons in Huh7 cells, and they did not
extend the host range of the genotype 1b replicon to cell lines
other than Huh7 (data not shown). Previous studies that ex-
amined genotype 1a replicons were restricted to the use of the
H77 strain (8, 27). In this study, we extended the apparent
inactivity of genotype 1a replicons to include the infectious
clone derived from the HCV-1 sequence (40), suggesting that
this may be a general attribute of genotype 1a strains in Huh7
cells, rather than an isolated observation with a single clone.
Currently, the replicon model is limited to the original geno-
type 1b strain employed by Lohmann et al. (43) and one other
genotype 1b isolate (27, 32), the HCV-N infectious clone (6).
Replicons based on the HCV-N sequence do not require adap-
tive mutations for high colony-forming efficiency. A four-ami-
no-acid insertion in the ISDR appears to be responsible for the
inherent colony-forming ability of this clone (32). In addition
to being restricted to two genotype 1b isolates, colony forma-
tion with HCV replicons is limited to a single human liver cell
line, Huh7. Undoubtedly, these limitations will not persist for
long. Recently, full-length bicistronic and monocistronic con-
structs that replicate in Huh7 cells have been developed (32,
53), but the production of infectious particles has not been
demonstrated.
In this study, we have extended our observations on the
antiviral activity of ribavirin to include the subgenomic repli-
cons of HCV. We previously demonstrated a reduction in
specific infectivity for GBV-B produced in the presence of
ribavirin that was attributed to the incorporation of ribavirin
triphosphate by the GBV-B polymerase and an accompanying
increase in error-prone replication (38). This effect has also
been observed with poliovirus (13). HCV replicon RNA ob-
tained from cultures exposed to ribavirin exhibited a decrease
in colony-forming efficiency when transfected into untreated
cells, and the decrease was proportional to the duration of
ribavirin exposure. Recent in vitro observations with HCV
NS5B protein suggest that the HCV RNA polymerase can
incorporate ribavirin triphosphate utilizing a synthetic tem-
plate (44).
Because ribavirin monotherapy does not result in a signifi-
cant reduction in viral load, the improvement in liver disease
realized during ribavirin monotherapy cannot be attributed to
the antiviral properties of ribavirin demonstrated in these stud-
ies. The immunomodulatory activity of ribavirin may be in-
volved in both the improvement of liver disease during mono-
therapy and the increased rate of sustained viral clearance
during combination therapy (for a recent review on ribavirin,
see reference 41). However, the possibility that the synergism
with IFN-␣ is due in part to ribavirin-induced error-prone
replication cannot be dismissed. The small increase in error
rate that may occur at the levels of ribavirin used in the clinic
may result in too few lethal mutations to impact a large pool of
replicating viral RNA. In contrast, this error rate may have a
significant impact on HCV survival once IFN has reduced the
viral load to the extent where the viral RNA is undetectable by
RT-PCR and thus facilitate sustained viral clearance (37). This
would suggest that three phases are involved in sustained viral
clearance in IFN-␣–ribavirin therapy. The initial rapid decline
of virus (phase I) is most likely due to the direct antiviral effect
of IFN-␣. A second more gradual and variable decline in viral
load (phase II) may be due to the death of infected hepato-
cytes. Ribavirin does not appear to play a significant role in
either of the first two phases. A third phase in which ribavirin
induces biologically significant mutagenesis may occur after
replicating viral RNA has been reduced to very low levels. If
this hypothesis is correct, then treatment with higher levels of
ribavirin may lead to an increase in sustained viral clearance;
however, at higher levels, the toxic side effects of ribavirin
necessitate short-term therapy. Indeed, induction of error-
prone replication following short-term, high-dose, intravenous
ribavirin therapy may be the mechanism responsible for the
successful treatment of hemorrhagic fevers induced by arena-
viruses (Lassa, Junin, and Machupo viruses) and bunyaviruses
(Hantaan virus) (18, 29, 34, 48). For HCV, improved ribavirin
therapy may be possible by specific targeting of ribavirin to the
liver to decrease extrahepatic toxicity and potentially increase
efficacy if error-prone replication is indeed a mechanism for
synergy with IFN-␣. The results of future clinical studies with
TABLE 7. Effect of ribavirin on replicon colony-forming efficiency
a
Harvest
time (h)
Ribavirin
b
Transfected
replicon (ge)
Colony-forming efficiency
c
(colonies/␮g of replicon)
24 ⫺ 2.6 ⫻ 10
8
140,000
⫹ 2.6 ⫻ 10
8
45,333
48 ⫺ 8.3 ⫻ 10
7
93,750
⫹ 8.3 ⫻ 10
7
14,583
72 ⫺ 3.1 ⫻ 10
7
138,888
⫹ 3.1 ⫻ 10
7
0
a
Clone 24 cells were treated with or without 400 ␮M ribavirin for 24 to 72 h.
Cell RNA was harvested at each time point, and the replicon copy number was
determined (Fig. 6). RNA from untreated cultures was adjusted with Huh7 RNA
such that transfections of treated and untreated RNAs were performed with
identical replicon copy number (ge) and such that each transfection was per-
formed with 10 ␮g of total cell RNA per 100-mm-diameter dish.
b
Symbols: ⫺, no ribavirin; ⫹, 400 ␮M ribavirin.
c
Colony-forming efficiency was expressed as the number of colonies per mi
-
crogram of transfected replicon RNA which was extrapolated from the ge of
replicon transfected at each time point as shown in the table.
VOL. 77, 2003 ANTIVIRAL AGENTS AND HCV REPLICONS 1101

drugs that separate the antiviral and immunomodulatory ef-
fects of ribavirin likely will resolve this mechanistic uncertainty.
In this study, we also observed antiviral activity with IFN-␣
and IFN-␥ but not with poly(I)-poly(C) and TNF-␣. A number
of laboratories have observed the direct antiviral effect of
IFN-␣ in the replicon system (8, 20, 27), and while this paper
was under review, a publication by Frese et al. (21) demon-
strated the antiviral effect with IFN-␥, as well. This antiviral
effect was independent of the production of nitric oxide or the
depletion of tryptophan, pathways known to be involved in the
antiviral activity of IFN-␥ in some systems. We examined
IFN-␥ because of its known antiviral effect in many systems
and our observations that IFN-␥ transcripts are increased in
the livers of chimpanzees undergoing HCV viral clearance
(R. E. Lanford, unpublished data). Following binding to dis-
tinct receptors, both types of IFNs utilize the JAK-STAT signal
transduction pathway to activate ISG transcription (for re-
views, see references 10, 57, and 59). The primary IFN-␣/␤
response is mediated through the binding of ISRE promoter
elements by the transcription factor ISGF3, a complex of
STAT-1, STAT-2, and IRF-9, while the primary type II IFN
response is accomplished by the binding of GAS promoter
elements by GAF, a homodimer of STAT-1. Many similarities
exist in the signal transduction pathways induced by type I and
II IFNs, including the induction of transcription of large, par-
tially overlapping sets of ISGs (15), most of which have poorly
understood or no known function. Thus, although the best-
characterized antiviral mechanisms of type I and II IFNs differ,
in some systems they may actually involve similar ISG re-
sponses. IFN-␥ can activate ISGF3, and this response is de-
pendent upon activation of the type I IFN pathway (63). We
initiated studies into the common induction of genes in Huh7
cells by IFN-␣ and IFN-␥. The basal level of ISG transcripts
did not differ significantly between Huh7 cells and replicon
lines, suggesting a lack of significant response to the presence
of viral dsRNA. Most ISGs were induced by both IFN-␣ and
IFN-␥, although some differences were observed. For example,
IRF-1 transcription is known to be induced to a greater extent
by IFN-␥ than by IFN-␣ (15, 49), and this difference was
observed in this study in the presence or absence of the repli-
con. IFN-␣ induced IP10 to a much greater extent in replicon
lines than in Huh7 cells; however, some of the differences in
the level of induction of specific genes may be due to the clonal
nature of the replicon lines. At this time, the exact mechanisms
of antiviral activity of IFN-␣ and IFN-␥ in the replicon system
remain unresolved.
We had previously demonstrated a potent antiviral activity
for poly(I)-poly(C) in GBV-B-infected primary tamarin hepa-
tocytes (38). The finding that the replicon system was sensitive
to IFN-␣, but not to poly(I)-poly(C), prompted an exploration
of the dsRNA response in these cells. ISG transcript levels did
not increase in Huh7 cells or replicon lines following poly(I)-
poly(C) treatment. In contrast, the induction of ISGs in pri-
mary chimpanzee hepatocytes was greater for poly(I)-poly(C)
than for IFN-␣. Since poly(I)-poly(C) has a high level of anti-
viral activity against GBV-B in primary tamarin hepatocytes,
we compared this model system directly with Huh7 cells for the
induction of IFN-␤ transcripts following poly(I)-poly(C) treat-
ment. Although tamarin hepatocytes exhibited 12- to 18-fold
increases in IFN-␤ transcripts 2 to 24 h after poly(I)-poly(C)
treatment, no significant increase in IFN-␤ transcripts was
noted in Huh7 cells. Some response to dsRNA was detected in
Huh7 cells and replicon lines, since PKR became phosphory-
lated following poly(I)-poly(C) treatment, so the defect in
dsRNA-induced transcription of ISGs in Huh7 cells is inde-
pendent of, or downstream of, PKR activation. After the initial
review of this manuscript, Pflugheber et al. (52) published
similar observations with regard to poly(I)-poly(C)-induced
phosphorylation of PKR in Huh7 cells, as well as activation of
IRF-1. Although some induction of ISG transcription was ob-
served in their study, the antiviral impact of poly(I)-poly(C) on
replicon RNA was not examined. It is of interest that the
poly(I)-poly(C)-induced degradation of HBV transcripts in
transgenic mice replicating HBV was not altered in PKR and
IRF-1 knockout mice, indicating that each of these antiviral
pathways is dispensable in this system (26).
Our findings indicate that Huh7 cells are defective in some
portion of the signaling pathway for dsRNA but that this defect
does not prevent response to IFN-␣ or IFN-␥. Whether this
defect is related to the permissiveness of Huh7 cells for HCV
replicons is not known, but the defect in dsRNA response
suggests that information gained from the replicon system with
regard to the antiviral mechanism of IFN-␣ may not be entirely
applicable to HCV-infected individuals. Microarray analysis of
serial liver samples from an acute resolving HCV infection in
the chimpanzee model demonstrated high levels of ISG tran-
script induction within 2 days of infection (7). We interpreted
this response to be indicative of a robust dsRNA response in
infected cells that resulted in the secretion of type I IFNs,
which in turn would create zones of cells with ISG induction
adjacent to each infected cell. Since cells within each zone
would be resistant to HCV infection, a reduction in available
replication space would occur as the infection spread in the
liver (for greater discussion of these observations, see refer-
ences 7 and 37). In this manner, the innate antiviral response
may control HCV infections until a T-cell response can elim-
inate infected cells.
The cellular response to dsRNA has been extensively char-
acterized but is still only partially understood. Microarray anal-
ysis of the response to dsRNA revealed that 175 genes exhib-
ited increased expression and 95 genes exhibited decreased
expression (24). This study was conducted in a cell type defec-
tive for the production of type 1 IFNs; therefore, an autocrine
loop from secreted IFN did not complicate interpretation of
the results. Several latent proteins are activated by dsRNA.
The antiviral pathway of 2⬘,5⬘ oligoadenylate synthetase is ac-
tivated by dsRNA, and it in turn activates RNase L, which
possesses potent antiviral activity. MxA possesses antiviral ac-
tivity and is activated by dsRNA, but MxA does not appear to
be involved in the antiviral activity of IFN-␣ in the replicon
system (20). PKR is activated by dsRNA, which leads to the
phosphorylation and inactivation of I␬B, the inhibitor of NF-
␬B. Activation of NF-␬B is required for the dsRNA induction
of IFN-␤ and some ISGs (36, 68). Under some conditions, the
response to dsRNA requires both IRF-1 and STAT1␣ (4) but
is not dependent on PKR (68) or ISGF3 (4). At least some of
the activity of dsRNA is conveyed by the latent dsRNA-acti-
vated factor transcription factor, or DRAF1, which is com-
prised, in part, of IRF-3 and CBP/p300 (67). Recent studies
have also demonstrated a requirement for p53 in the dsRNA
1102 LANFORD ET AL. J. VIROL.

response (31). At this time, the defect in the dsRNA response
pathway in Huh7 cells is not known, and it is not known
whether this influences the permissiveness for HCV replicons.
In some replicon lines, IP10 and ISG12 were induced to a
greater extent by IFN-␣ in comparison to Huh7 cells. These
data imply that IFN-␣ and the presence of the replicon may act
together to increase the level of expression of these genes. The
role of the replicon in this induction could be the presence of
dsRNA, since IFN-␣ and dsRNA are known to behave syner-
gistically (46). As with the PKR activation by poly(I)-poly(C),
this would imply that at least portions of the dsRNA response
pathway are intact in Huh7 cells, at least under the conditions
of IFN-␣ stimulation.
Our studies did not detect suppression of ISG induction by
IFN-␣ in replicon cells in comparison to Huh7 cells. Although
in some studies sequence variation in NS5A has been impli-
cated in the degree of IFN-␣ responsiveness in patients, the
exact mechanism by which NS5A impacts IFN-␣ responsive-
ness has not been determined. In vitro studies have suggested
that NS5A suppresses the IFN response by virtue of its inter-
action with PKR (23) or by induction of interleukin 8 expres-
sion (25, 55). The lack of detectable suppression of the IFN-␣
response in the replicon cells may have been due to a number
of factors, including the high level of IFN exposure, deficien-
cies in the dsRNA response in Huh7 cells, or the effects of
adaptive mutations present in the replicons. However, the re-
sults of these studies do suggest that further investigation of
the interaction of subgenomic replicons with the cellular fac-
tors involved in viral resistance may improve our understand-
ing of the mechanism(s) of IFN resistance among different
genotypes of HCV. Examination of the newly developed full-
length replicons (32, 53) will be important as well, since mul-
tiple HCV proteins may modulate the host response to dsRNA
and IFNs.
ACKNOWLEDGMENTS
This work was supported in part by NIH grants U19 AI40035, RO1
AI49574, and P51 RR13986.
We thank Stuart Ray and David Thomas for insightful discussions
on the potential antiviral role of ribavirin once the replicating viral
population has been reduced by IFN treatment.
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