 |
INTRODUCTION |
The hepatitis C virus
(HCV)1 is a major human
pathogen for which both vaccines and broadly effective therapeutics are
not available (1-3). HCV has a positive-sense RNA genome of ~9600
bases and expresses a polyprotein of ~3000 amino acids (4) that is
processed by host and viral proteases to yield 10 mature proteins (5, 6). Subgenomic HCV RNAs from which the structural proteins have been
excised but which express non-structural proteins and cis-encoded RNA
elements essential for replication (replicons) (7-10) are capable of
autonomous replication in cell culture (11-13).
An important feature of HCV is its high level of genetic variability,
which is believed to be a consequence of the low fidelity of the viral
polymerase. This variability is underscored by the identification of
six major HCV genotypes (designated 1-6), more than 50 subtypes, and
numerous quasi-species within each subtype (14). Several clones that
are infectious in chimpanzees have been described previously (15-22).
In contrast, replicons derived from only the HCV-con1 and HCV-N
isolates have been shown to replicate robustly in cell culture thus far
(11-13). Efficient replication in cell culture has been invariably
associated with adaptive mutations that dramatically increase the
frequency with which replication is established (12, 23-25). Adaptive
mutations in the HCV-con1 replicon have been localized to various
non-structural genes, although substitutions in NS5A, for example
S232I, appear to be the most effective (23). Similarly a 4-amino acid
insertion in NS5A that is not commonly observed in vivo is
required for the replication in cell culture of an HCV-N replicon (12).
Surprisingly, known infectious clones are not replication-competent
even after the introduction of adaptive mutations (23). Interestingly, a recent study showed that cell culture adaptive mutations that confer
cell culture replication competence to the HCV-con1 replicon abrogate
or dramatically attenuate its infectivity in vivo (26).
Efforts to identify additional HCV sequences that replicate in cell
culture remain a high priority, because availability of these sequences
will not only contribute to a better understanding of HCV biology but
will also facilitate the development of therapeutic agents that are
broadly effective against all clinically relevant HCV genotypes. We
described recently the development of a powerful HCV replication
system, Bla-Rep, that employs HCV replicons expressing the
-lactamase reporter (bla) and subpopulations of Huh7 cells that are
enhanced for HCV replication (27). Here we used this system and a
systematic mutational approach to identify the restriction to
replication of two known infectious HCV isolates. We report that the
amino acid residue at position 470 in domain II of the NS3 helicase
acts in concert with the NS5A-S232I adaptive mutation in determining
the cell culture replication competence of HCV-BK and HCV-H77 isolates
(genotypes 1b and 1a, respectively). The results presented here predict
that substitutions in the NS3 helicase in combination with previously
identified HCV-con1 adaptive mutations can confer replication
competence to diverse HCV isolates.
 |
MATERIALS AND METHODS |
Cell Culture--
Huh7 human hepatoma cells were grown in
Dulbecco's modified minimal essential medium (Cellgro) supplemented
with 2 mM GlutaMAX (Invitrogen), non-essential amino
acids, 100 units/ml penicillin, 100 µg/ml of streptomycin, and 10%
heat-inactivated fetal bovine serum (Invitrogen). Media for
culture of cell lines harboring replicon that expressed neomycin
phosphotransferase were supplemented with G418 as indicated. Cells were
grown at 37 °C and 5% CO2 and were passaged twice per week.
Chimpanzee Infectious HCV-BK--
HCV-BK comprises the genotype
1b BK sequence from the 5'-UTR through the KpnI restriction
site in NS5B derived from a Japanese patient and a 3'-UTR (genotype 1a)
derived from an Italian patient (28). Direct intrahepatic injection of
in vitro transcribed HCV-BK RNA causes HCV infection in
chimpanzees. Inoculation of a second chimpanzee with serum from an HCV
BK-infected chimpanzee also results in infection, demonstrating that
the BK sequences used in this study are infectious (29).
Replicon Constructs--
HCV-con1 replicon constructs that
express the bla reporter were as described previously (27). The HCV-BK
subgenomic replicon was constructed by replacing the NS3 through the
3'-UTR sequence of the HCV-con1 replicon with the corresponding region
from HCV-BK. The HDV ribozyme was added to the 3'-ends of both the
HCV-con1 and HCV-BK replicons to facilitate the generation of native
3'-ends (30). In addition, a unique ClaI restriction site
has been introduced at the NS5B-3'-UTR junction. Mutations were
introduced as indicated using the QuikChange PCR mutagenesis kit
(Stratagene). NS3 amino acid numbering is based on the mature sequence
and does not include the initiator methionine present in the replicon
constructs used.
Replication Assays--
Replicon RNAs were transcribed and
purified as reported previously (27) except that the templates were
digested with XbaI, which cuts downstream of the HDV
ribozyme. The integrity of the transcribed RNAs was checked by
analytical agarose gel electrophoresis, and RNA was quantitated by absorbance.
Replication assays were performed as described previously (27). Bla-Rep
assays were performed using MR2 cells, which are derived from Huh7
cells and are enhanced for HCV subgenomic replication (27). Cells were
plated at 2.5-3 × 105 cells/well in six-well tissue
culture plates and allowed to adhere overnight. Cells were
lipotransfected with DMRIE-C reagent (Invitrogen) at the
indicated concentrations of bla-replicon RNA for 6-8 h and then
incubated overnight. Cells were stained 16-24 h later with CCF4-AM dye
to check for transfection efficiency. Cells were split into 96-well
plates (4000 cells/well) and assayed at day 3 or 4 for replication by
staining with CCF4-AM dye and measuring emission at 460 and 530 nm
using a CytoFluor 4000-plate reader. In this assay format, viable cells
that do not express the reporter fluoresce green (530 nm), whereas
those that do express the reporter fluoresce blue (460 nm). The ratio
of the blue emission to the green emission gives a measure of the
fraction of the cells that harbor replicon. For colony formation
assays, 2 × 105 Huh7 cells were transfected with the
indicated neomycin replicon RNA using DMRIE-C. Medium for selection
experiments was supplemented with 250 µg/ml G418. Colonies were
counted 3 weeks after transfection. A "GAA" HCV-con1 replicon was
used as the negative control for all replication assays. This replicon
contains alanine residues in place of the aspartic acids at positions
318 and 319 in the GDD motif of NS5B that is essential for catalytic activity.
Purification, Reverse Transcription, and Sequencing of Viral and
Replicon RNAs--
HCV RNA was isolated from chimpanzee serum using
the RNeasy RNA purification kit (Qiagen), and total RNA was purified
from 106 replicon-harboring cells by TRIzol (Invitrogen)
extraction followed by spin-column purification using the RNeasy RNA
purification kit. cDNAs comprising the NS3 region were generated by
reverse transcription using Superscript II reverse transcriptase
(Invitrogen). cDNAs were subsequently amplified with Expand High
Fidelity polymerase, subcloned into pSTBlue1 (Novagen), and then
sequenced with an ABI 373 Sequencer.
NS3 Helicase Expression and Purification--
NS3 helicases were
subcloned into the BamHI and HindIII restriction
sites of pET-21B and expressed in BL21(DE3) cells. NS3 helicases were
isolated from clarified bacterial lysates in three steps using
immobilized metal-affinity chromatography, Q-Sepharose (Amersham
Biosciences) chromatography, and poly(U)-Sepharose column chromatography. Purified helicases were stored at
20 °C in 20 mM Tris, pH 7.0, 10% glycerol. Protein concentrations were
determined by absorbance at 280 nm using a calculated molar extinction
coefficient of 47,480 M
1
cm
1.
NS3 Helicase Unwinding Assays--
The
32P-end-labeled, partial duplex DNA helicase substrate was
prepared essentially as described by Levin and Patel (31). Unwinding
assay reactions were performed in 20 µl and contained 25 mM MOPS, pH 6.5, 3 mM MgCl2, 2 mM dithiothreitol, 100 µg/ml bovine serum albumin, 2 nM substrate, and 10 nM helicase. Helicase and
substrate were preincubated for 15 min at 25 °C before initiation of
the reaction by the addition of ATP to 5 mM. Reactions were stopped at various times by the addition of 5 µl of 5× the
termination mixture. Products were resolved using a 10%
acrylamide-Tris borate-EDTA gel and quantified by densitometric
scanning with a Storm 860 PhosphorImager and ImageQuant software
(Molecular Dynamics).
 |
RESULTS |
Subgenomic Replicons Comprising HCV-BK Non-structural Proteins and
3'-UTR Are Not Cell Culture Replication-competent--
We focused our
efforts on HCV-BK and HCV-H77, two isolates that are infectious in
chimpanzees but that have failed to replicate in cell culture. Because
they are infectious, all the activities and cis elements required for
replication that are encoded by both sequences are known to be
functional. We generated replicons that comprise the NS3-NS5B
non-structural genes and the 3'-UTR from HCV-BK to test for cell
culture replication. These constructs were engineered with an HDV
ribozyme, which autocatalytically cleaves itself from the 3'-end of the
replicon to yield replicon RNA transcripts with native 3'-ends.
HCV-con1 replicons that are transcribed with the HDV ribozyme show the
same replication activity as replicons that are transcribed from a
ScaI digested template without the ribozyme (data not
shown). Replicons were generated either with the bla reporter for
transient replication assays or with the neomycin phosphotransferase
gene for selection experiments. In addition, the S232I mutation in NS5A
that confers cell culture adaptation to the HCV-con1 replicon (23) was
engineered into the HCV-BK replicon. As shown in Fig.
1, the wild-type replicon derived from
the BK chimpanzee infectious genome did not replicate in the transient
replication assay (Fig. 1). The activity of the HCV-BK replicon
containing the S232I mutation is not significantly different from that
of the replication-deficient HCV-con1 replicon, indicating that this
adaptive mutation does not confer replication competence to HCV-BK
(Fig. 1). Similar analyses were performed by colony formation assay
using corresponding neor replicons. The wild-type HCV-BK
replicon did not yield any colonies. Introduction of the S232I adaptive
mutation conferred modest replication competence to HCV-BK (~100
colonies/µg) compared with corresponding HCV-con1 replicons (~5000
colonies/µg). These results indicate that the introduction of the
NS5A-S232I adaptive mutation is not sufficient to confer robust
replication competence to the HCV-BK replicon.

View larger version (8K):
[in this window]
[in a new window]
|
Fig. 1.
Restriction to efficient replication of
HCV-BK subgenomic replicons maps to NS3. MR2 cells were
transfected with 5 µg of the indicated replicon RNAs and then assayed
for replication by Bla-Rep at day 4. The regions derived from HCV-BK
are shown in black and those from HCV-con1 in
white. The results are averages of three or more replicates.
Em460/Em530, ratio of fluorescence emission at 460 and
530 nm.
|
|
Restriction to Cell Culture Replication of HCV-BK Replicon Maps to
NS3--
To identify the block to replication in the BK replicons, we
generated chimeras in which the various non-structural proteins of
HCV-BK and HCV-con1 were swapped. These swaps were initially made using
HCV-BK replicons that had the S232I mutation, because this mutation
modestly improved BK replicon replication in the colony formation assay
described above. Substitution of NS4A, NS4B, NS5A, or NS5B either alone
or in combination had modest or no effects on the replication of either
replicon, suggesting that these regions did not account for the
differences in replication competence (data not shown). However, as
shown in Fig. 1, replacement of the NS3 coding region in the BK
replicon with the con1 NS3 resulted in a replicon that replicated with
essentially the same activity as the HCV-con1 replicon with the
NS5A-S232I mutation. Conversely, introduction of the HCV-BK NS3 into
the HCV-con1 replicon essentially abolished replication activity (Fig.
1). In contrast, none of the other con1 regions yielded any improvement
in replication of the HCV-BK replicon (data not shown).
Mutations in NS3 Helicase Confer Cell Culture Replication
Competence to HCV-BK--
The results obtained from the chimera
experiments demonstrated that the block to HCV-BK replication in cell
culture maps to NS3. An alignment of the HCV-BK and HCV-con1 sequences
revealed that there are 12 amino acid differences in NS3, with 1 mutation mapping to the protease domain and the remaining 11 mapping to the helicase domain. To identify which of the amino acid differences accounted for the dramatic differences in replication efficiency, each
of the residues in the HCV-BK NS3 that differed from that in HCV-con1
was individually mutated to the residue found in HCV-con1. The
resulting replicons were then tested for replication activity by
Bla-Rep. As shown in Fig. 2, two
mutations restored replication competence to the BK replicon. The
introduction of an R470M mutation into the HCV-BK NS3 helicase resulted
in a replicon that had significantly higher replication efficiency than
the HCV-con1 S232I replicon. The NS3-S196T mutation also enhanced
HCV-BK replicon activity but with lower efficiency than the R470M
mutation. At all other positions tested, introduction of the
corresponding HCV-con1 residue had only modest effects. When the
NS3-S196T and R470M mutations were combined, a modest but reproducible
additivity in transduction efficiency was observed (data not shown). To
determine the nature of the higher level of reporter gene expression
observed in the HCV-BK replicons containing the R470M mutation, we
measured the fraction of the cell population harboring replicon. We
then calculated reporter activity normalized to the number of
replicon-harboring cells (27). The increase in reporter gene
expression was found to be because of both increases in the frequency
with which replication is established (the fraction of cells that
expressed reporter) and to a modest (<2-fold) increase in the replicon
copy number per cell (data not shown).

View larger version (8K):
[in this window]
[in a new window]
|
Fig. 2.
Point mutations S196T and R470M in NS3
helicase confer replication competence to BK replicon. Each of the
11 amino acids that differ in the NS3 helicases of HCV-BK and HCV-con1
were mutated by PCR mutagenesis. MR2 cells were transfected with HCV-BK
replicon RNAs containing the indicated NS3 helicase mutations and
NS5A-S232I and then assayed by Bla-Rep 3 days later. HCV-con1
subgenomic replicons containing the NS5A-S232I mutation with or
without the inactivating GAA mutation in NS5B were tested
in parallel. The results are averages of three or more independent
experiments. Em460/Em530, the ratio of
fluorescence emission at 460 and 530 nm.
|
|
Although the NS3-R470M mutation dramatically enhances the replication
activity of HCV-BK replicons, different residues are observed at this
position in other isolates. To determine whether arginine at this
position is incompatible with replication or rather that the methionine
at this position is unique in conferring cell culture replication
competence, HCV-BK replicons containing R470P and R470G mutations were
tested. Proline, glycine, and leucine are frequently seen in genotype 1 HCV isolates at this position (Table I).
As shown in Fig. 3, replicons containing
either proline or glycine in place of Arg-470 replicated albeit with
modestly lower efficiency those with methionine at this position.

View larger version (11K):
[in this window]
[in a new window]
|
Fig. 3.
Proline and glycine at position 470 in NS3
helicase are compatible with efficient replication. HCV-BK
subgenomic replicons were engineered to contain substitutions at NS3
helicase position 470 with residues frequently seen in other isolates.
Replication was determined by Bla-Rep at day 3 after transfection.
Results are averages of three or more independent experiments.
Em460/Em530, ratio of fluorescence emission at 460 and
530 nm.
|
|
To assess the effect of BK residues at positions 196 and 470 on
HCV-con1 replication fitness, corresponding HCV-con1 replicons were
engineered to have the T196S or M470R mutation or both and were tested
in Bla-Rep. As shown in Fig. 4,
T196S and M470R caused a modest and dramatic reduction in
replication activity, respectively, whereas the combination of both
mutations essentially abrogated replication activity. These results
demonstrate that residues 196 and 470 both contribute to the
replication of HCV-con1 replicons.

View larger version (12K):
[in this window]
[in a new window]
|
Fig. 4.
Residues present at positions 196 and 470 in
HCV-BK NS3 attenuate replication of the HCV-con1 replicon. T196S,
M470R, or both mutations were introduced into the NS3 of the HCV-con1
replicon. MR2 cells were transfected with replicon RNAs and assayed by
Bla-Rep 3 days later. Results are averages of three or more
experiments. Em460/Em530, ratio of fluorescence
emission at 460 and 530 nm.
|
|
Mutations in NS3 Are Not Sufficient to Confer Cell Culture
Replication Competence--
As mentioned above, the S232I adaptive
mutation in NS5A had minimal effects on the replication competence of
the BK replicon. However, robust replication of BK replicons was
observed when this mutation was combined with R470M and to a lesser
extent with S196T-NS3 helicase mutations. The corresponding replicons
without the S232I mutation in NS5A were tested in transient replication assays to assess whether S196T or R470M helicase mutations could by
themselves confer replication competence to HCV-BK replicons. Replicons
containing either of the NS3 helicase mutations but not the S232I-NS5A
adaptive mutation did not replicate (Fig.
5A), demonstrating that cell
culture replication of both con1 and BK replicons is dependent on both
the NS3 and NS5A adaptive mutations. Additional analyses using a colony
formation assay confirmed the results shown in Fig. 5A but
also indicated that residue 470 makes a significantly greater
contribution to replication activity than residue 196 (Fig.
5B).

View larger version (11K):
[in this window]
[in a new window]
|
Fig. 5.
NS3 helicase mutations are not sufficient to
confer cell culture replication competence. HCV-BK replicons
containing the indicated substitutions in NS3 with and without the
NS5A-S232I adaptive mutation were assayed by Bla-Rep using replicons
containing the bla reporter (A) and by colony selection
using replicons expressing neor (B).
A, MR2 cells were transfected with the indicated replicon
and assayed 3 days later. The results are averages of three or more
independent experiments. B, for colony selection
experiments, 2 × 105 Huh7 cells were transfected with
the indicated RNA. After 24 h, cells were placed under selection
with 250 µg/ml G418 for 3 weeks, at which time the colonies were
counted. Em460/Em530, ratio of fluorescence emission at
460 and 530 nm.
|
|
NS3 Helicase Domain Mutations at Positions 196 and 470 Confer
Replication Competence to HCV-H77 (Genotype 1a)
Replicons--
Replicons derived from the chimpanzee infectious
genotype 1a isolate HCV-H77 also failed to replicate efficiently in
cell culture with the introduction of the single adaptive mutation S232I in NS5A (3, 12). The results obtained with HCV-BK replicons prompted us to address whether mutations at positions 196 and 470 would
confer cell culture replication competence to HCV-H77 replicons as
well. The wild-type HCV-H77 NS3 helicase has a serine at position 196 and a proline at position 470, a combination that is compatible with
efficient replication in HCV-BK replicons as described above. We
generated a series of HCV-H77 replicons containing either the wild-type
serine or a threonine at position 196 and either the wild-type proline,
methionine, or leucine at position 470 in NS3 with and without the
S232I adaptive mutation in NS5A. These replicons were assayed by
Bla-Rep, and the data are summarized in Fig.
6. The H77 replicon containing Ser-196
and Pro-470 failed to replicate as did replicons containing the point
mutations S196T and P470M in combination with S232I. However, HCV-H77
replicons containing both P470L and NS5A-S232I became
replication-competent, thus demonstrating the importance of this region
of NS3 helicase in the cell culture adaptation of HCV-H77. Although the
S196T had essentially no effect in isolation, the introduction of this substitution into the HCV-H77 replicon containing P470L further enhanced replication activity. Similar data were obtained in colony formation assays using H77 replicons that express
neor. No colonies were observed with either
wild-type replicons or replicons that contained either single NS3 or
NS5A mutations in isolation. Replicons containing the combination of the NS3-P470L and NS5A-S232I mutations yielded 140 colonies/µg of replicon RNA. Addition of the S196T mutations to this replicon further improved colony formation efficiency to 460 colonies/µg of
replicon RNA. For comparison in the same sets of experiments, HCV-con1
replicons containing the NS5A-S232I mutation yielded ~3000
colonies/µg of RNA. These data indicate that the NS3 helicase residues at positions 196 and 470 influence the replication potential of genotype 1a replicons.

View larger version (13K):
[in this window]
[in a new window]
|
Fig. 6.
NS3 helicase domain mutations at positions
196 and 470 confer replication competence to HCV-H77 subgenomic
replicons. HCV-H77 subgenomic replicons containing the indicated
mutations in NS3 and the NS5A-S232I mutation were assayed by Bla-Rep 3 days after the transfection of MR2 cells. The results are averages of
three or more independent experiments. Em460/Em530,
ratio of fluorescence emission at 460 and 530 nm.
|
|
NS3 Helicase Mutations Do Not Affect Helicase Unwinding
Activity--
To explore the effects of the cell culture adaptive
mutations on helicase unwinding activity, HCV-BK NS3 helicase domains with and without the S196T and R470M mutations were expressed and
purified to homogeneity from Escherichia coli. Helicases
were then compared in unwinding assays using double-stranded DNA
substrates. As shown in Table II, the
rates of unwinding were comparable for each protein. This fact, with
the fact that both NS3 mutations map to the protein surface, suggests
that these mutations mediate interactions with other viral- or
host-encoded proteins involved in viral replication.
View this table:
[in this window]
[in a new window]
|
Table II
NS3 helicase mutations that confer cell culture replication competence
do not affect helicase unwinding activity
|
|
NS3 Residue 470 Substitutions Required for Replication in
Cell Culture Are Not Observed in the Serum of an Infected
Chimpanzee--
It was of interest to investigate whether revertants
containing substitutions in NS3 470 and or NS3 196 could be found in the sera of HCV-BK infected chimpanzees. Sequence analyses of 10 NS3
sequences rescued from the serum of an HCV BK-infected chimpanzee (5780 genome equivalents/ml) (29) did not reveal substitutions in these
positions relative to the parental clone, consistent with the
suggestion that substitutions at these positions are specifically
required for cell culture adaptation.
 |
DISCUSSION |
An unbiased mutagenesis approach was used to identify the block to
cell culture replication of subgenomic replicons derived from two HCV
isolates that have been shown to replicate in chimpanzees. We report
that combination of the previously identified con1 NS5A adaptive
mutation S232I with substitutions in NS3 helicase residue 470 is
necessary to confer cell culture replication competence to the 1b-BK
and 1a-H77 HCV replicons. Residue 470 maps to the surface of the NS3
helicase and is not in the nucleotide, metal binding, or nucleic acid
binding sites, suggesting that this position might be part of an
interaction interface for binding to other viral proteins or
host-encoded factors (32-34). Furthermore the demonstration that
mutations at this position do not affect the in vitro
unwinding activities of purified NS3 helicase is consistent with a role
in mediating interactions with other factors.
Residue Ser-196 is located in domain I of the NS3 helicase and appears
to play a secondary role compared with residue 470. Mutations in domain
I that map near to this position in the helicase have previously been
shown to improve transduction efficiency of the HCV-con1 subgenomic
replicon. For example, mutations E177G and T255I increase the
transduction efficiency of HCV-con1 replicons severalfold either alone
or in combination with the NS5A adaptive mutation S225P (25). Likewise
the mutations R258G and K583E were shown to increase the efficiency of
colony formation in G418 selection experiments with HCV-con1 replicons,
either with or without the NS5B adaptive mutation R465G (24). In these
previous examples, the improvement in replication efficiency was
relatively modest, resulting in up to 10-fold increases in colony
formation efficiency. Similarly, in G418 selection experiments using
HCV-BK neomycin replicons, colony formation efficiency improved nearly 8-fold for replicons containing both the NS3-S196T and NS5A-S232I adaptive mutations compared with those with only NS5A-S232I (Fig. 5B). These results suggest that the NS3-S196T mutation has
modest effects, comparable with those engendered by the previously
reported domain I mutations and that it may act by a similar mechanism.
In contrast, residue 470 is localized to domain II and its critical
involvement in cell culture replication has not been recognized previously. Methionine, proline, and glycine substitutions at this
position are all able to confer replication competence to HCV-BK
replicons. Interestingly, the 1b HCV-N isolate, which exhibits a high
transduction efficiency, also has a glycine at this position and a
4-amino acid insertion in NS5A that appears to be functionally equivalent to the S232I adaptive mutation. Surprisingly, we find that
neither methionine nor proline substitutions at this position are
compatible for replication in the context of HCV-H77 replicons but
instead a leucine substitution conferred replication competence to this
isolate. These results suggest that the residue required at this
position for optimal replication is context-dependent and
may vary among different genotypes. More detailed analyses are likely
to identify additional NS3 helicase domain II residues that are part of
an interface and which may also influence cell culture replication activity.
The biological activity attributed to NS3 helicase residues 196 and 470 is absolutely dependent on the presence of the NS5A adaptive mutation
S232I. In turn the ability of the NS5A S232I mutation to confer
replication competence is also dependent on its cooperation
compatibility with NS3 helicase. The observation that the optimal
residue at position 470 appears to be isolate-specific suggests that
this residue might influence viral replication via a direct interaction
with another viral protein. An alternative interpretation to these
observations is that there are potentially two independent restrictions
to cell culture replication, one of which can be addressed by the
introduction of a mutation in residue 470 of NS3 helicase. The other
restriction requires an adaptive mutation in either NS4B, NS5A, or
NS5B. Additional work will be required to better understand the
mechanism by which substitutions in these residues can confer
replication activity in cell culture. Regardless of the mechanism by
which these mutations mediate cell culture adaptation, the findings
described here provide a rational and simple approach to generate and
characterize isolates of other genotypes that are replication-competent
in cell culture. The availability of clinically relevant isolates that
replicate in cell culture will facilitate the development of broadly
active HCV therapeutic agents.