2003, 77(5):3181. DOI:
and Charles M. Rice
Keril J. Blight, Jane A. McKeating, Joseph Marcotrigiano
Genotype 1a RNAs in Cell Culture
Efficient Replication of Hepatitis C Virus
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JOURNAL OF VIROLOGY, Mar. 2003, p. 3181–3190
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Vol. 77, No. 5
Efficient Replication of Hepatitis C Virus Genotype 1a
RNAs in Cell Culture
Keril J. Blight,* Jane A. McKeating, Joseph Marcotrigiano, and Charles M. Rice*
Laboratory of Virology and Infectious Disease, Center for the Study of Hepatitis C,
The Rockefeller University, New York, New York 10021
Received 5 September 2002/Accepted 5 December 2002
Hepatitis C virus (HCV) genotype 1 (subtypes 1a and 1b) is responsible for the majority of treatment-
resistant liver disease worldwide. Thus far, efficient HCV RNA replication has been observed only for sub-
genomic and full-length RNAs derived from genotype 1b isolates. Here, we report the establishment of efficient
RNA replication systems for genotype 1a strain H77. Replication of subgenomic and full-length H77 1a RNAs
required the highly permissive Huh-7.5 hepatoma subline and adaptive amino acid substitutions in both NS3
and NS5A. Replication could be detected by RNA quantification, fluorescence-activated cell sorting, and
metabolic labeling of HCV-specific proteins. Replication efficiencies were similar for subgenomic and full-
length RNAs and were most efficient for HCV RNAs lacking heterologous RNA elements. Interestingly, both
subtype 1a and 1b NS3 adaptive mutations are surface exposed and present on only one face of the NS3
structure. The cell culture-adapted subtype 1a replicons should be useful for basic replication studies and for
antiviral development. These results are also encouraging for the development of adapted replicons for the
remaining HCV genotypes.
Persistent infection with hepatitis C virus (HCV) is one of
the primary causes of chronic liver disease. Progression to
chronic active hepatitis with cirrhosis occurs in ?20 to 30% of
infected individuals, and HCV-associated liver disease is now
the leading cause of liver transplantation in the United States
(7). Genotypes 1a and 1b, the most prevalent worldwide, have
the poorest rates of response to the present treatment regimen,
a combination of pegylated alfa interferon 2b with ribavirin (4,
HCV, a member of the family Flaviviridae, is a small envel-
oped virus whose genome is a 9.6-kb single-stranded RNA with
positive polarity consisting of a 5? nontranslated region (NTR),
a large open reading frame encoding the virus-specific pro-
teins, and a 3? NTR (reviewed in references 1, 15, and 21). The
5? NTR contains an internal ribosome entry site (IRES) me-
diating translation of a single polyprotein of ?3,000 amino
acids with the structural proteins (C, E1, and E2) located in the
N terminus and the nonstructural proteins (NS2, NS3, NS4A,
NS4B, NS5A, and NS5B) encoded in the remainder. The
NS3-5B coding region is sufficient for RNA replication in cell
culture (17), and these proteins are presumed to function as
components of the HCV replicase. The NS3 protein possesses
serine protease and nucleoside triphosphatase-helicase activi-
ties, NS4A is a cofactor for the NS3 serine protease, and NS5B
is the RNA-dependent RNA polymerase. The functions of
NS4B and NS5A remain elusive, although NS5A, a phosphor-
ylated protein, has been a target for adaptive mutation, allow-
ing efficient initiation of HCV replication in vitro (2, 9, 13).
Amino acid substitutions in the NS3, NS4B, and NS5B proteins
can also enhance replication to various degrees (9, 13, 16).
Initially, only the genotype 1b Con1 RNA sequence was
replication competent in the human hepatoma cell line Huh-7,
and adaptive mutations in the HCV-encoded proteins were
required for efficient HCV replication (2, 9, 13, 16). More
recently, a second genotype 1b isolate, HCV-N, was reported
to replicate in Huh-7 cells; however, unlike the Con1 strain,
the HCV-N infectious clone replicated in the absence of ad-
ditional cell culture-adaptive mutations (10). Attempts to ex-
tend this system to other genotypes have been largely unsuc-
cessful (2, 9, 10, 14). Despite the ability of RNA transcripts
from the genotype 1a H77 infectious clone of HCV to initiate
a robust replication cycle in chimpanzees after direct intrahe-
patic inoculation (11), selectable subgenomic replicons based
on this consensus clone failed to confer antibiotic resistance in
a variety of hepatoma cells, including the Huh-7 cell line (2).
Recently, a Huh-7 subline, Huh-7.5, that was highly permis-
sive for Con1 subgenomic and full-length RNA replication was
isolated (3). This subline facilitated the selection of G418-
resistant colonies supporting replication of H77-derived sub-
genomic replicons carrying a mutation in NS5A shown to en-
hance replication of Con1 replicons (3). Analysis of replicating
HCV RNAs in these cell clones revealed additional adaptation
in the NS3 coding region that increased the replicative capac-
ities of subgenomic and full-length H77 RNAs to comparable
MATERIALS AND METHODS
Cell cultures. Huh-7.5 cell monolayers (3) were propagated in Dulbecco’s
modified Eagle medium (DMEM) supplemented with 10% heat-inactivated fetal
bovine serum (FBS) and 0.1 mM nonessential amino acids (DMEM– 10% FBS).
For cells supporting subgenomic replicons, 750 ?g of G418 (Geneticin; Gibco-
BRL)/ml was added to the culture medium.
* Corresponding author. Present address for Keril J. Blight: Depart-
ment of Molecular Microbiology, Center for Infectious Disease Re-
search, Washington University School of Medicine, 660 South Euclid
Ave., Campus Box 8230, St. Louis, MO 63110. Phone: (314) 286-0065.
Fax: (314) 362-1232. E-mail: firstname.lastname@example.org. Mailing ad-
dress for Charles M. Rice: Laboratory of Virology and Infectious
Disease, Center for the Study of Hepatitis C, The Rockefeller Uni-
versity, 1230 York Ave., New York, NY 10021. Phone: (212) 327-7046.
Fax: (212) 327-7048. E-mail: email@example.com.
on June 6, 2013 by guest
Plasmid construction. Standard recombinant DNA technology was used to
construct and purify all plasmids. Primed DNA synthesis was performed with
KlenTaqLA DNA polymerase (W. Barnes, Washington University, St. Louis,
Mo.), and regions amplified by PCR were confirmed by automated nucleotide
sequencing. Plasmid DNAs for in vitro transcription were prepared from large-
scale bacterial cultures and purified by centrifugation in CsCl gradients.
All nucleotides and amino acid numbers refer to the location within the H77
full-length HCV genome p90/HCVFLlongpU (11), commencing with the core
coding region. The selectable replicon, pHCVrep13/Neo (H/SG-Neo), and the
derivative, pHCVrep13(S2204I)/Neo [H/SG-Neo (I)] (Fig. 1), containing the
NS5A mutation, S2204I, have been described (2). For these subgenomic con-
structs, NS5B polymerase-defective derivatives were generated carrying a triple-
amino-acid substitution changing the Gly-Asp-Asp (GDD) motif in the active
site to Ala-Ala-Gly (AAG) (12), and throughout this report they are referred to
as pol?. All plasmids were engineered to carry an HpaI runoff site for RNA
transcription, generating RNA transcripts containing one additional U nucleo-
tide at the 3? terminus. The Con1-derived constructs used in this study have been
described previously (3).
The plasmid pHCVrep90/A1226D?S2204I [H/SG-Neo (D?I)] (Fig. 1), con-
taining the mutation A1226D in NS3, was constructed by ligating the DraIII-
XhoI fragment of a reverse transcription (RT)-PCR product amplified from total
RNA isolated from cell clone H-2 using the primers 2424 (5? GCAGCCTGTT
GACAGGCAATA 3?) and 2419 (5? CATAATAATTTGTGACGAGTG 3?),
together with the KpnI-DraIII and XhoI-NsiI fragments from pHCVrep13
(S2204I)/Neo, into pHCVrep13(S2204I)/Neo cleaved with KpnI and NsiI. To
engineer the mutation P1496L in pHCVrep13(S2204I)/Neo, thus creating pH-
CVrep90/P1496L?S2204I [H/SG-Neo (L?I)] (Fig. 1), nucleotides 4485 to 4861
in NS3 were PCR amplified from pHCVrep13(S2204I)/Neo using the mutant
primer 2616 (5? ACTCAGCCGGAGGGGCGCTCCCCCGGTGCCACAAAT
CTGTAGATGCCTAGCTTCCCCCTGCCAGTCCTGCC 3?) and oligonucleo-
tide 2617 (5? CTATCCCCCTCGAGGTGATCAAG 3?). The PCR product was
digested with XhoI and BglI and, together with the BglI-BamHI and BamHI-
EcoRI fragments from pHCVrep13(S2204I)/Neo, was inserted between the
XhoI-EcoRI sites of pHCVrep13(S2204I)/Neo.
The replicons pNTR-EMCV/HCVrep90(A1226D?S2204I)
EMCV/HCVrep90(P1496L?S2204I) [H/SG-5?HE (D?I) and H/SG-5?HE
(L?I)] (Fig. 1) containing the mutations A1226D and P1496L, respectively, were
constructed by inserting the KpnI-EcoRI fragment from either pHCVrep90/
A1226D?S2204I or pHCVrep90/P1496L?S2204I between the KpnI and EcoRI
sites of pNTR-EMCV/HCVrep13(S2204I) (K. J. Blight, unpublished data).
The plasmid p90/HCVFL(S2204I), containing the full-length H77 genome
with S2204I in NS5A and an HpaI runoff site, was constructed from p90/HCV
FLlongpU (K. J. Blight, unpublished data). The derivatives, p90/FL(A1226D?
S2204I) and p90/FL(P1496L?S2204I) [H/FL (D?I) and H/FL (L?I)] (Fig. 1),
containing the mutations A1226D and P1496L, were generated by replacing the
AvrII-MluI portion of p90/HCVFL(S2204I) with the AvrII-NsiI fragment from
either pHCVrep90/A1226D?S2204I or pHCVrep90/P1496L?S2204I and the
NsiI-MluI fragment from pHCVrep13(S2204I)/Neo.
The selectable biscistronic full-length HCV clone p90/FL-Neo(P1496L?
S2204I) [H/FL-Neo (L?I)] (Fig. 1) was assembled in a two-step cloning proce-
dure. First, in a four-piece ligation reaction, the XbaI-BsaI fragment from
pHCVrep13(S2204I)/Neo, the BsaI-AatII fragment from pHCVBMFL(S2204I)/
Neo (3), and the AatII-XhoI fragment from p90/HCVFL(S2204I) were cloned
between the XbaI and XhoI sites in pSL1180 (Pharmacia), generating the inter-
mediate plasmid pSL1180/90NTR-C12-Neo-EMCV-CXho. Second, the XbaI-
KpnI portion of p90/FL(P1496L?S2204I) was replaced with the XbaI-XhoI frag-
ment excised from pSL1180/90NTR-C12-Neo-EMCV-CXho, together with a
XhoI-KpnI-digested PCR product generated by extension of the overlapping oli-
gonucleotides 2411 (5? CCACCTCGAGGTAGACGTCAGCCTATCCCCAAGG
CACGTCGGCCCGAGG 3?) and 2412 (5? CCAGTGGTACCCGGGCTGAGCC
To delete the E1-p7 coding region from p90/HCVFL(S2204I), the KpnI-MslI
portion of a PCR product amplified from p90/HCVFL(S2204I) with the forward
primer 2749 (5? GCCCGGGTACCCTTGGCCCCTCT 3?) and the reverse
primer 2748 (5? CCAGAGCACCTCCGTGTCCAGGGCTGAAGCGGGCAC
GGTCAGGCA 3?) and the MslI-BsrGI fragment from p90/HCVFL(S2204I)
were ligated between the KpnI and BsrGI sites of p90/HCVFL(S2204I). The
P1496L mutation and an HpaI runoff site were engineered between the AvrII and
MluI sites in this construct, named p90/FL?E1-p7(S2204I), by inserting the
AvrII-NsiI fragment from pHCVrep90/P1496L?S2204I, together with the NsiI-
MluI fragment from pHCVrep13(S2204I)/Neo, thus creating p90/FL?E1-
p7(P1496L?S2204I) [H/?E1-p7 (L?I)] (Fig. 1).
RNA transcription and electroporation of cultured cells. Plasmid DNAs con-
taining H77 and Con1 sequences were linearized with HpaI and ScaI, respec-
tively. In vitro transcription was performed as described previously (3). DNase-
treated RNA transcripts (0.5 to 1 ?g) were electroporated into 5 ? 106Huh-7.5
cells using a BTX ElectroSquarePorator essentially as described previously (3).
The transfected cells were plated in (i) 150-mm-diameter dishes for selection of
G418-resistant colonies, (ii) 100-mm-diameter dishes for determining the effi-
ciency of G418-resistant colony formation and fluorescence-activated cell sorting
(FACS) analysis, (iii) 35-mm-diameter wells for quantifying HCV RNA and for
metabolic labeling experiments, or (iv) eight-well chamber slides (Becton Dick-
inson) for immunofluorescence studies. For dishes requiring G418 selection, 48 h
after the cells were plated, the medium was replaced with DMEM– 10% FBS
supplemented with 1 mg of G418/ml. Three weeks later, the resultant foci were
either isolated and expanded for further analysis or fixed with 7% formaldehyde,
stained with 1% crystal violet in 50% ethanol, and counted to determine G418
transduction efficiency (3).
FIG. 1. Schematic representation of HCV RNAs used in this study. The 5? and 3? NTR structures are shown, and open reading frames are
depicted as open boxes with the polyprotein cleavage products indicated. The first 21 amino acids of the core coding region (solid box), the neo
gene (Neo; shaded box), and the EMCV IRES (EMCV; solid line) are illustrated. The nomenclature adopted for each construct is displayed on
the right, and throughout this report, the HCV RNAs are prefaced by either H or Con1 to indicate H77- or Con1-derived sequences, respectively.
3182BLIGHT ET AL. J. VIROL.
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RNA extraction, RT-PCR amplification, and quantification of HCV RNA.
Total cellular RNA was isolated from cell monolayers using TRIZOL reagent
(Gibco-BRL) according to the manufacturer’s protocol and precipitated with
isopropanol. The RNA pellet was washed in 80% ethanol and resuspended in
H2O. The NS3-5B coding region was amplified from total cellular RNA ex-
tracted from G418-resistant cell clones in four overlapping fragments by RT-
PCR. Approximately 105molecules of HCV RNA were mixed with 5 pmol of
HCV-specific primer, and the primer was extended at 43.5°C for 1 h in a 5-?l
reaction mixture containing 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM
MgCl2, 10 mM dithiothreitol, 0.5 mM (each) deoxynucleoside triphosphate, 4 U
of RNasin (Promega), and 50 U of Superscript II reverse transcriptase (Gibco-
BRL). The cDNAs were then amplified with KlenTaqLA DNA polymerase using
35 cycles of 95°C for 30 s, 55 to 60°C for 30 s, and 68°C for 4 min. The PCR
products were recovered from preparative low-melting-point agarose gels by
phenol extraction, and ?40 ng of purified PCR product was directly sequenced
with an ABI 9600 automatic DNA sequencer.
HCV-specific RNA levels in total cellular RNA preparations extracted from
transfected Huh-7.5 cell monolayers were quantified using primers specific for
the 5? NTR in a real-time RT-PCR assay as previously described (3).
Protein detection. Transfected cell monolayers were removed from the 100-
mm-diameter dishes, and a single-cell suspension was prepared for FACS anal-
ysis as described previously (3). Briefly, cells were resuspended at 2 ? 106per ml
and fixed for 20 min at room temperature (rt) in a final concentration of 2%
paraformaldehyde in phosphate-buffered saline (PBS). The fixed cells were
washed twice in PBS, resuspended at 2 ? 106per ml, permeabilized for 20 min
at rt in 0.1% saponin– PBS, and stained for 1 h at rt with an HCV-specific
monoclonal antibody to NS3 (10E5/24; 1/500 dilution) kindly provided by Raf-
faele De Francesco, Istituto di Ricerche di Biologia Molecolare, Rome, Italy
(19). Bound monoclonal antibody was detected by incubation for 1 h at rt with
anti-mouse immunoglobulin G (IgG) conjugated to Alexa 488 (Molecular
Probes) diluted 1:1,000 in 3% FBS– 0.1% saponin– PBS. The stained cells were
washed three times with 0.1% saponin– PBS and resuspended in FACSflow
buffer (BD Biosciences) prior to analysis using a FACSCalibur (BD Biosciences).
For metabolic labeling experiments, cell monolayers in 35-mm-diameter wells
were incubated for 10 to 12 h in methionine- and cysteine-deficient minimal
essential medium containing 1/40 the normal concentration of methionine, 5%
dialyzed FBS, and Express35S protein-labeling mix (140 ?Ci/ml; NEN). Cell lysis
and immunoprecipitation under denaturing conditions using HCV-positive pa-
tient serum, JHF (recognizing NS3, NS4B, and NS5A), and Pansorbin cells have
been described previously (3).
Replication of H77-derived subgenomic replicons. Con1 and
HCV-N, both genotype 1b, are the only two isolates reported
to replicate in Huh-7 cells. To test the abilities of Con1 adap-
tive mutations to enhance replication of genotype 1a-derived
replicons, we incorporated the highly adaptive Ser-to-Ile sub-
stitution in NS5A (S2204I) into subgenomic replicons derived
from a chimpanzee-infectious H77 genotype 1a cDNA clone
(11). Although modeled after the Con1 selectable replicons,
these genotype 1a-derived replicons failed to replicate in
Huh-7 cells (2). The lack of replication might have been due to
the Huh-7 cellular environment, requirements for different or
additional adaptive mutations, or a combination of these fac-
tors. Recently, a Huh-7 subline (Huh-7.5) was established by
“curing” a cell clone containing a Con1 subgenomic replicon
by prolonged treatment with alpha interferon (3). A majority
of these cells (?70%) are permissive for HCV RNA replica-
tion after transfection with Con1 replicons harboring certain
adaptive mutations (e.g., S2204I) (3). The increased permis-
siveness of the Huh-7.5 cell line for Con1 replication led us to
reexamine the replicative abilities of H77 subgenomic repli-
cons in these cells.
Two G418-selectable replicons, H/SG-Neo and H/SG-Neo
(I) (Fig. 1), containing wild-type sequence and S2204I in
NS5A, respectively, were evaluated for the ability to replicate
compared to an NS5B polymerase-defective replicon, pol?. In
vitro-synthesized RNA was electroporated into Huh-7.5 cells,
and G418 selection was imposed 48 h later. After 3 weeks, a
few G418-resistant colonies were visible for Huh-7.5 cells
transfected with H/SG-Neo (I), but not with H/SG-Neo and
H/SG-Neo (pol?). From two independent electroporations, 16
individual cell colonies were isolated, and HCV replication was
verified by metabolically labeling cell monolayers and immu-
noprecipitation of HCV antigens. After the separation of la-
beled proteins by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE), NS3, NS4B, and NS5A were
detected in each cell clone, but not in the parental Huh-7.5
cells. Data for three of these clones are shown in Fig. 2. Inter-
estingly, the migration of NS3 differed depending on the clone,
suggesting that a modification(s) within NS3 may have oc-
Identification of adaptive mutations. The low frequency of
cells supporting H77 replication suggested that adaptive mu-
tations were generated, allowing replication at a level sufficient
to confer resistance to G418. To determine if subgenomic
RNAs within individual cell clones had acquired additional
mutations, the NS3-5B coding region was amplified by RT-
PCR from total cellular RNA isolated from three clones (H-1,
H-2, and H-3 [Fig. 2]) and sequenced. The S2204I change in
NS5A was maintained, but in addition, a single-amino-acid
substitution in the helicase domain of NS3 was found in each
clone. For one clone (H-1), a Pro-to-Leu substitution at locus
1496 (P1496L) was identified, while in the other two clones,
H-2 and H-3, Ala at position 1226 was mutated to Asp
(A1226D). For an additional nine cell clones, the NS3-4A
coding region was amplified, and sequence analysis of these
RT-PCR products identified the mutation P1496L in seven
FIG. 2. Detection of HCV proteins in cell clones supporting H77
subgenomic replication. Two days postseeding, monolayers of the cell
clones H-1, H-2, and H-3 and parental Huh-7.5 cells (Mock) were
incubated for 10 h in the presence of [35S]methionine and [35S]cys-
teine. The labeled cells were lysed and immunopreciptated with HCV-
positive human serum (JHF, anti-NS3, NS4B, and NS5A), and the
labeled proteins were separated by SDS– 10% PAGE. The mobilities
of molecular-mass standards are indicated on the left, and the migra-
tions of NS3, NS4B, and NS5A are shown on the right.
VOL. 77, 2003 HCV H77 RNA REPLICATION IN CELL CULTURE3183
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clones and A1226D in the remainder. In addition to P1496L,
one replicon (H-6) encoded a second NS3 mutation (V1355I).
Due to the conservative nature of this substitution, we did not
investigate the effect of the mutation on HCV replication.
Thus, of the 12 independent cell clones sequenced at the pop-
ulation level, only mutations localized within the helicase do-
main of NS3 were identified.
Adaptive values of mutations identified in NS3. To analyze
whether A1226D and P1496L in NS3 conferred an adaptive
phenotype, these mutations were independently introduced
into H/SG-Neo (I) (Fig. 1) to produce H/SG-Neo (D?I) and
H/SG-Neo (L?I), respectively. Their replication efficiencies
were compared in Huh-7.5 cells by measuring the number of
cells transduced to G418 resistance. The frequency of Huh-7.5
cells able to support H/SG-Neo (L?I) replication was
?30,000-fold higher than for H/SG-Neo (I), while H/SG-Neo
(D?I) demonstrated an ?800-fold increase in the number of
G418-resistant colonies (Fig. 3A). However, the replicative
capacities of H/SG-Neo (L?I) and H/SG-Neo (D?I) were
consistently lower than that of Con1/SG-Neo (I) (?7- and
?34-fold, respectively) (Fig. 3A).
Next, we addressed whether these NS3 mutations could con-
fer a replicative advantage in the absence of the NS5A S2204I
mutation. G418-resistant colonies were selected after transfec-
tion of Huh-7.5 cells with the selectable replicon H/SG-Neo
(L) carrying only the P1496L substitution but not the replicon
containing A1226D [H/SG-Neo (D)] (Fig. 3B). The calculated
G418 transduction efficiency for H/SG-Neo (L) was ?0.008%,
a 250-fold reduction compared to that of H/SG-Neo (L?I)
(Fig. 3B), demonstrating that P1496L, together with S2204I, is
required for efficient H77 subgenomic RNA replication. The
low frequency of colonies generated after transfection of
H/SG-Neo (L) suggests that additional adaptive mutations
may have arisen, which we are investigating further.
Previously, it was demonstrated that replication of Con1
subgenomic and genomic RNAs containing adaptive mutations
could be monitored soon after RNA transfection of Huh-7.5
cells without the need for G418 selection (3). H77-derived
subgenomic and full-length RNAs containing either P1496L or
A1226D, together with S2204I, were transfected into Huh-7.5
cells; total RNA was extracted 96 h later, and HCV RNA levels
were quantified by RT-PCR. The replication-defective repli-
FIG. 3. Colony-forming abilities of H77 subgenomic RNAs containing mutations in NS3. (A) Huh-7.5 cells were electroporated with 0.5 ?g
(each) of the subgenomic replicons H/SG-Neo (L?I), H/SG-Neo (D?I), Con1/SG-Neo (I), and H/SG-Neo (pol?). Forty-eight hours later, the cells
were subjected to G418 selection, and the resulting colonies were fixed and stained with crystal violet. Representative dishes after 2.5 ? 104cells
were plated are illustrated. The percentage below each dish refers to the calculated G418 transduction efficiency of the replicon that was
determined by serially titrating transfected cells from 2 ? 105to 1 ? 103cells per 100-mm-diameter dish, together with feeder cells electroporated
with the pol?replicon. The resulting G418-resistant foci were counted for at least three cell densities, and the relative G418 transduction efficiency
was expressed as a percentage after dividing the number of colonies by the number of electroporated Huh-7.5 cells initially plated. (B) One
microgram (each) of the subgenomic RNAs H/SG-Neo (L?I), H/SG-Neo (L), H/SG-Neo (D), and H/SG-Neo (pol?) was transfected into Huh-7.5
cells, and after 3 weeks of G418 selection, the transduction efficiency was determined as described for panel A. Dishes seeded with 2 ? 105
electroporated cells are depicted, with the relative transduction efficiencies shown below.
3184BLIGHT ET AL.J. VIROL.
on June 6, 2013 by guest
con, H/SG-Neo (pol?), was transfected in parallel to allow
discrimination between input RNA and that generated by pro-
ductive replication. HCV RNA levels relative to the pol?con-
trol were higher for the subgenomic replicons (H/SG-Neo and
H/SG-5?HE) containing P1496L and S2204I than for those
harboring the A1226D and S2204I combination (Fig. 4; com-
pare sample 4 to 5 and 7 to 8). The levels of H/FL (L?I) and
H/FL (D?I) RNAs were ?120- and ?9-fold higher than that
of pol?(Fig. 4, samples 10 and 11), consistent with the repli-
cative ability of subgenomic RNAs carrying the same muta-
tions. However, H77 subgenomic RNA levels were ?5 to ?13-
fold lower than those observed for Con1-derived replicons
[Con1/SG-Neo (I) and Con1/SG-5?HE (I)] (Fig. 4, compare
sample 4 to 3 and 7 to 1), whereas H77 and Con1 full-length
replication rates were comparable (Fig. 4, samples 9 and 10).
We also examined HCV protein expression by immunopre-
cipitation and FACS analysis. The levels of35S-labeled NS3 in
Huh-7.5 cells transfected with H/SG-Neo (L?I) and H/SG-
5?HE (L?I) were lower than those of the corresponding Con1
subgenomic replicons (Fig. 4, compare lane 4 to 3 and 7 to 1),
whereas NS3 was undetectable in cells transfected with repli-
cons carrying the A1226D change or pol?(Fig. 4). NS4B and
NS5A were visible above background only in Con1/SG-5?HE
(I) and Con1/SG-Neo (I) RNA-transfected cells (Fig. 4, lanes
1 and 3). The frequency of HCV antigen-positive cells quan-
tified by FACS analysis yielded trends similar to those noted in
the replicon RNA levels, where 17% of cells stained positive
for NS3 after transfection of H/SG-5?HE (L?I) RNA com-
pared to 3% for H/SG-5?HE (D?I) RNA (Fig. 4). As ex-
pected, a higher percentage of cells transfected with Con1/SG-
5?HE (I) expressed NS3 (74 and 65%) (Fig. 4 and 5B). For
full-length RNAs, a lower frequency of NS3-positive cells was
evident in H/FL (D?I) than in H/FL (L?I) (2 versus 17%),
and35S-labeled NS3 was detectable only in Huh-7.5 cells trans-
fected with H/FL (L?I) (Fig. 4, lanes 10 and 11). Similar
frequencies of NS3 antigen-positive cells were observed after
transfection of Huh-7.5 cells with full-length and subgenomic
H77 replicons carrying the P1496L and S2204I mutations, and
they were comparable to those seen with Con1-derived full-
length RNA [Con1/FL (I); ?14%] (Fig. 4 and 5B). These
results are consistent with our earlier observation that sub-
genomic and full-length H77 RNAs containing P1496L and
S2204I have the highest replicative capacities in Huh-7.5 cells.
Furthermore, we found that subgenomic and full-length RNAs
replicate to similar levels, in contrast to Con1, where sub-
genomic replicons establish replication in a greater proportion
FIG. 4. Detection of HCV proteins and RNA in Huh-7.5 cells transiently transfected with subgenomic and full-length HCV RNAs. (Top)
Ninety-six hours after RNA transfection of Huh-7.5 cells, the monolayers were labeled with35S protein-labeling mixture and lysed, and NS3, NS4B,
and NS5A were analyzed by immunoprecipitation, SDS– 10% PAGE, and autoradiography. The positions of the molecular-mass standards are
given on the left, and HCV-specific proteins are indicated on the right. (Middle) Total cellular RNA was extracted 96 h posttransfection, and HCV
RNA levels were quantified as described in Materials and Methods. The ratio of HCV RNA to the pol?negative control is shown (HCV
RNA/pol?). (Bottom) Ninety-six hours after transfection, cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% saponin, stained
for HCV NS3, and analyzed by FACS. The percentages of cells expressing NS3 relative to an isotype-matched irrelevant IgG are displayed. Values
of ?1% were considered negative (?). ND, not determined.
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FIG. 5. Replication of HCV RNAs with and without heterologous elements. (A) Huh-7.5 cells were transfected with 1 ?g (each) of full-length
and subgenomic RNAs, and 2 ? 105cells were plated in 35-mm-diameter wells. Ninety-six hours posttransfection, the Huh-7.5 cells were labeled
with [35S]methionine and [35S]cysteine for 10 h. The cells were lysed, and HCV proteins were isolated by immunoprecipitation using a patient
serum specific for NS3, NS4B, and NS5A. HCV proteins and the positions of protein molecular-mass standards (in kilodaltons) are shown. In lane
1, half the amount of immunoprecipitated sample was loaded (0.5x). The ratio of HCV RNA relative to the pol?negative control (HCV
RNA/pol?) is shown below each lane. (B) Transfected Huh-7.5 cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% saponin,
and the frequency of cells expressing NS3 antigen was quantified by FACS. The percentage of cells expressing NS3 relative to pol?-transfected cells
3186 BLIGHT ET AL.J. VIROL.
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and an isotype-matched IgG was determined and is shown in the upper left corner of each plot. The median fluorescence intensity of the gated
positive cells is shown in the upper right corner of each plot. FSC-H, forward scatter; FL1-H, fluorescence. (C) One microgram (each) of H/FL-Neo
(L?I) and H/SG-Neo (pol?) RNA was electroporated into Huh-7.5 cells, and 2 ? 106cells were plated on 100-mm-diameter dishes. G418 selection
was applied 48 h posttransfection, and after 3 weeks in culture, G418-resistant foci were fixed and stained with crystal violet. The G418 transduction
efficiency, displayed below each dish, was determined as described in the legend to Fig. 3.
VOL. 77, 2003HCV H77 RNA REPLICATION IN CELL CULTURE3187
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of cells than constructs containing the complete coding se-
quence (Fig. 4) (3).
Replicative abilities of subgenomic and full-length RNAs.
Both H77- and Con1-derived subgenomic replicons lacking the
neo gene appear to initiate replication more efficiently than
selectable subgenomic RNAs (Fig. 4) (3). To investigate these
observations further, we compared the replication efficiencies
of a number of subgenomic and genomic H77 RNAs in the
presence or absence of heterologous elements. Besides the
selectable bicistronic replicons (SG-Neo [Fig. 1]) and the rep-
licons in which the HCV 5? NTR was fused to the encephalo-
myocarditis virus (EMCV) IRES (SG-5?HE [Fig. 1]), a repli-
con was constructed in which the 5? NTR was followed by the
entire core sequence fused directly to the NS2-NS5B coding
region and the 3? NTR such that cleavage at the core-NS2
junction would be mediated by signal peptidase and translation
was under the control of the homologous IRES [H/?E1-p7
(L?I)] (Fig. 1). In parallel, we tested the H77 full-length
monocistronic RNA [H/FL (L?I)] (Fig. 1) and a bicistronic
derivative [H/FL-Neo (L?I)] (Fig. 1), where the HCV 5? NTR
mediates neo gene translation and the EMCV IRES drives
core-NS5B expression. Both subgenomic and genomic con-
structs were engineered to carry P1496L and S2204I. Ninety-six
hours after the transfection of Huh-7.5 cells, the relative levels
of HCV RNA and protein were measured as described above.
A 280-fold increase in H/?E1-p7 (L?I) RNA over pol?was
observed (Fig. 5A), whereas modest increases in HCV RNA
were evident for H/SG-Neo (L?I) and H/SG-5?HE (L?I)
(?60- and ?140-fold) (Fig. 5A). A higher frequency of Huh-
7.5 cells expressed NS3 antigen after electroporation with
H/?E1-p7 (L?I) (29%) than after electroporation with H/SG-
5?HE (L?I) (18%) and H/SG-Neo (L?I) (8%) (Fig. 5B). NS3
antigen levels in the H77 RNA-transfected cells, as determined
by the median fluorescence intensity of the gated antigen-
positive cells, were similar, suggesting comparable levels of
RNA translation and/or protein stability per cell (Fig. 5B). The
relative amounts of immunoprecipitated35S-labeled NS3 par-
alleled both the frequency of NS3-positive cells and the relative
HCV RNA levels (Fig. 5A). After transfection of H/FL (L?I)
RNA into Huh-7.5 cells, HCV RNA levels increased 110-fold
relative to pol?(Fig. 5A), 14% of cells expressed NS3 (Fig.
contrast, HCV RNA levels for H/FL-Neo (L?I) were no
greater than those of the pol?control (Fig. 5A), and NS3
expression was not detectable by FACS (Fig. 5B) or metabolic
labeling (Fig. 5A, lane 7), suggesting that this construct was
replication defective. In spite of these results, G418-selected
colonies were detectable with a relative transduction efficiency
of 0.03% (Fig. 5C). Taken together, these findings suggest that
H77 RNA replication is more efficient for subgenomic and
genomic constructs that lack the neo gene and the EMCV
35S-labeled NS3 was visible (Fig. 5A, lane 5). In
HCV replicons derived from the genotype 1b isolates Con1
and HCV-N are replication competent in Huh-7 cells (2, 3, 9,
10, 13, 16, 17). Earlier efforts to select stable colonies after
transfection of Huh-7 cells with H77-derived subgenomic
RNAs were unsuccessful, despite the inclusion of the highly
adaptive S2204I change in NS5A that was identified in the
Con1 genetic background (2). We reasoned that additional or
alternative adaptive mutations might be required for replica-
tion in these cells or that Huh-7 cells were not permissive for
H77 replication. In this study, we demonstrated that replica-
tion of subgenomic and full-length RNAs derived from the
H77 infectious clone was dependent upon using the Huh-7
subline Huh-7.5. For reasons that are not yet clear, these cells
possess a cellular environment highly permissive for the initi-
ation of Con1 replication (3). The ability of H77-derived rep-
licons to replicate in Huh-7.5, but not in the Huh-7 parental
cell line, further emphasizes the importance of cellular factors
for HCV replication.
Although a comprehensive study remains to be done, our
results suggest that efficient H77 replication in Huh-7.5 cells
may require at least two adaptive mutations. Initially, G418-
resistant colonies were seen only with transfected subgenomic
H77 RNAs containing the S2204I adaptive change in NS5A. In
all cell clones analyzed, replicating RNAs had acquired a sec-
ond amino acid substitution in the helicase domain of NS3
(A1226D or P1496L). Both these mutations, when combined
with NS5A S2204I, enhanced the colony-forming ability of
subgenomic H77 RNA and allowed the detection of HCV
RNA and proteins 96 h after RNA transfection of either sub-
genomic or genomic replicons. Interestingly, replication was
greatest for H77 RNAs carrying P1496L in NS3 and S2204I in
NS5A. Although S2204I in NS5A increased the replicative
capacity of H77-derived replicons, we were able to select
G418-resistant colonies following transfection of subgenomic
RNAs carrying only P1496L in NS3. Although the P1496L
substitution may be sufficient for replication, the low frequency
of G418-resistant colony formation with these transcripts sug-
gests that an additional adaptation(s) may be required. Exper-
iments are in progress to explore this possibility, with the hope
of finding more optimal combinations of adaptive mutations
for subtype 1a replication. Interestingly, NS3 P1496 is poorly
conserved among HCV isolates, with Met and Gly found at this
position for genotype 1b isolates Con1 and HCV-N, respec-
tively. Given the apparent flexibility of this residue, it will be
interesting to test the effects of other substitutions at this
Adaptive mutations in NS3 have been reported in two other
studies (13, 16). Lohmann et al. and Kreiger et al. identified
and analyzed several spontaneous NS3 mutations individually
and together for effects on replication. The mutations R1283G,
E1383A, and K1609E had 7-, 2-, and 3.5-fold increases in RNA
replication, respectively, compared to the wild-type Con1 sub-
genomic replicon, while K1577R had a modest decrease of
?2-fold. However a replicon that harbored all four mutations
displayed a 10-fold increase in RNA levels over the wild-type
sequence. Kreiger and coworkers isolated two other mutations,
E1202G and T1280I, which displayed 14- and 7-fold increases,
respectively, compared to the parental wild-type sequence. A
replicon containing both mutations (E1202G and T1280I) dis-
played an additive effect, with a 25-fold increase in RNA levels.
Interestingly, the three studies (including this one) failed to
find a mutation at the same position within the protein; how-
ever, seven of the eight mutations are located within the heli-
case domain of NS3. These NS3 mutations generally act syn-
ergistically with mutations in NS4B or NS5A to enhance
3188 BLIGHT ET AL. J. VIROL.
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replication. In an attempt to determine if these adaptive mu-
tations cluster within the three-dimensional structure, the lo-
cation of each position was mapped onto the structure of
NS3-4A (23) (Fig. 6). As noted earlier (13), mutant residues
were located on the solvent-accessible surface, with the excep-
tion of 1280, which is buried. Interestingly, residue 1280 makes
several van der Waals contacts with the solvent-exposed resi-
due 1283, also identified as a position for an adaptive mutation.
The seven helicase mutations appear to be confined to one
surface of the helicase domain (compare Fig. 6A and B with
C). Although these mutations are not located within the seven
conserved sequence motifs of RNA helicases (Fig. 6), they do
appear to reside on adjacent surfaces of the active site.
The mechanism by which the changes in NS3 and NS5A act
individually and often synergistically to enhance HCV replica-
tion is unclear. Since the function of NS5A in RNA replication
is unknown, possibilities range from direct alterations in rep-
licase activity (via interaction with other HCV nonstructural
proteins or cellular factors) to modulation of the cellular en-
vironment, including host antiviral defense pathways (22). For
NS3, although the role of the HCV RNA helicase activity in
replication is not known, adaptive mutations could affect RNA
binding, enzymatic activity of the helicase, or higher-order
interactions in the replicase that enhance the efficiency of
productive replication. In future studies, it will be interesting to
examine the effects of different adaptive mutations on the
relative syntheses of positive- and negative-strand RNAs.
However, to understand the mechanism(s) of cell culture ad-
aptation, it is clear that more work is needed to define the
components and the higher-order structure of an active HCV
For H77-derived subgenomic replicons, the inclusion of the
neo gene or the EMCV IRES generally decreased the ability of
RNAs to initiate replication. For instance, transient analyses
H/?E1-p7 (L?I) ? H/SG-5?HE (L?I) ? H/SG-Neo (L?I),
demonstrating that the EMCV IRES is not a requirement for
efficient expression of the HCV replicase region and replica-
tion in Huh-7 cells. A similar trend has also been seen for
Con1-derived replicons, where Con1/SG-5?HE (I) initiates
replication more efficiently than Con1/SG-Neo (I) (this report
and reference 3). In the case of replicons containing the full-
length polyprotein, addition of the neo-EMCV IRES module
also decreases replication efficiency, although G418-resistant
colonies can be selected for both H77 and Con1 bicistronic
derivatives (this work and references 3 and 20). Hence, both
subgenomic and full-length HCV RNA replication can now be
studied in the absence of heterologous sequences. Further-
more, inclusion of these or other adaptive mutations, as well as
minimizing or eliminating non-HCV sequences, may help to
enhance replication of other, as yet nonreplicating HCV ge-
notypes or strains.
Full-length Con1 RNA replicated in ?15% of Huh-7.5 cells
FIG. 6. Locations of NS3 adaptive mutations. Solvent-accessible
surface of the NS3/4A crystal structure (23) highlighting the locations
of several adaptive mutations. Adaptive mutations described in this
paper are colored blue, while published mutations from references 13
and 16 are colored red and green, respectively. The seven conserved
motifs of the RNA helicase are colored cyan. The numbering corre-
sponds to the genotype 1 sequences. (A) The NS4A peptide and
protease domain are on the right, with the helicase domain on the left.
(B and C) Rotations (90 and 180°, respectively) about a vertical axis
(represented by the arrow in panel A).
VOL. 77, 2003 HCV H77 RNA REPLICATION IN CELL CULTURE3189
on June 6, 2013 by guest
compared to ?65% for the subgenome [Con1/SG-5?HE (I)],
whereas the replication efficiencies of H77 subgenomic [H77/
SG-5?HE (L?I)] and genomic HCV RNAs were comparable
(15 to 18%) (Fig. 5B). Moreover, the levels of NS3 per cell
(mean fluorescence intensity [Fig. 5B]) were similar for all
RNAs except Con1/SG-5?HE (I), where it was twice that of the
other RNAs. This surprising difference in replicative capacity
between H77 and Con1 subgenomic and full-length RNAs
underscores the importance of studying more than one isolate.
Thus far, there is no evidence for HCV particle assembly
and release from Huh-7 cells supporting replication of Con1
(3, 20) or HCV-N (10) full-length RNAs. Although Huh-7 cells
may be nonpermissive for one or more of these steps, it is not
known whether this will be generally true for all HCV geno-
types. The well characterized H77 strain is highly infectious in
chimpanzees and replicates to high titers, suggesting that it
may be a good candidate for establishing a complete replica-
tion cycle in cell culture. Since the E1E2 glycoproteins are
retained in the endoplasmic reticulum (ER) via retention sig-
nals in their C-terminal hydrophobic tails (6, 8), assembly (pre-
sumably in the ER or an ER-Golgi intermediate compartment)
and release of HCV particles should be associated with move-
ment of viral particles and their surface glycoproteins through
the secretory pathway. This should result in transit through the
Golgi and the acquisition of complex N-linked glycans. Al-
though endoglycosidase-H sensitivity of the HCV H77 glyco-
proteins has not been examined, the E2 staining pattern is
consistent with ER retention in cells transfected with full-
length cell culture-adaptive RNAs (data not shown). Whether
a low level of particle release from these cells is occurring is
In conclusion, we have established a cell culture system for
replication of HCV RNAs derived from the infectious H77
isolate. These genotype 1a subgenomic and full-length repli-
cons should be useful for basic studies of HCV RNA replica-
tion and HCV-host cell interactions and for antiviral drug
screening and evaluation.
We thank Arash Grakoui for helpful discussion and Brett Linden-
bach for critical reading of the manuscript. We are also grateful to
Raffaele De Francesco and Jean Dubuisson for providing HCV-spe-
cific monoclonal antibodies.
This work was supported in part by grants from the Public Health
Service (CA57973 and AI40034) and the Greenberg Medical Research
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