Resistance Mutations Define Specific Antiviral Effects
for Inhibitors of the Hepatitis C Virus p7 Ion Channel
Toshana L. Foster,1,2* Mark Verow,2,3* Ann L. Wozniak,4Matthew J. Bentham,1Joseph Thompson,3
Elizabeth Atkins,2Steven A. Weinman,4Colin Fishwick,3Richard Foster,3Mark Harris,2and Stephen Griffin1,2
The hepatitis C virus (HCV) p7 ion channel plays a critical role during infectious virus
production and represents an important new therapeutic target. Its activity is blocked by
structurally distinct classes of small molecules, with sensitivity varying between isolate p7
sequences. Although this is indicative of specific protein–drug interactions, a lack of high-
resolution structural information has precluded the identification of inhibitor binding
sites, and their modes of action remain undefined. Furthermore, a lack of clinical efficacy
for existing p7 inhibitors has cast doubt over their specific antiviral effects. We identified
specific resistance mutations that define the mode of action for two classes of p7 inhibitor:
adamantanes and alkylated imino sugars (IS). Adamantane resistance was mediated by an
L20F mutation, which has been documented in clinical trials. Molecular modeling
revealed that L20 resided within a membrane-exposed binding pocket, where drug binding
prevented low pH-mediated channel opening. The peripheral binding pocket was further
validated by a panel of adamantane derivatives as well as a bespoke molecule designed to
bind the region with high affinity. By contrast, an F25A polymorphism found in genotype
3a HCV conferred IS resistance and confirmed that these compounds intercalate between
p7 protomers, preventing channel oligomerization. Neither resistance mutation signifi-
cantly reduced viral fitness in culture, consistent with a low genetic barrier to resistance
occurring in vivo. Furthermore, no cross-resistance was observed for the mutant pheno-
types, and the two inhibitor classes showed additive effects against wild-type HCV. Conclu-
sion: These observations support the notion that p7 inhibitor combinations could be a
useful addition to future HCV-specific therapies. (HEPATOLOGY 2011;54:79-90)
(IFN) and ribavirin (Rib) is inadequate, which, com-
bined with high cost and poor patient compliance, has
driven the demand for new virus-specific drugs.1Future
standard of care will replace IFN/Rib with combina-
epatitis C virus (HCV) infects over 3% of the
population, causing severe liver disease. Cur-
rent therapy comprising pegylated interferon
tions of specific inhibitors, such as seen for human im-
munodeficiency virus (HIV) therapy. However, extensive
HCV variability raises concerns over the ability of rela-
tively few compounds to suppress resistance. Thus, great
effort focuses on expanding the repertoire of HCV drug
targets, expedited by the availability of the Japanese ful-
minant hepatitis clone 1 (JFH-1) infectious isolate.2
Abbreviations: Ama, amantadine; DHPC, 1,2-diheptanoyl-sn-glycero-3-phosphocholine; GT, genotype; HCV, hepatitis C virus; HIV, human immunodeficiency
virus; IFN, interferon; IS, imino sugar; JFH-1, Japanese fulminant hepatitis clone 1; LMPG, lyso-myristoylphosphatidylglycerol; NN-DGJ, N-nonyl
deoxygalactonojirimycin; NN-DNJ, N-nonyl deoxynojirimycin; NS, nonstructural; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; Rib,
ribavirin; Rim, rimantadine.
From the1Section of Oncology and Clinical Research, Leeds Institute of Molecular Medicine, St. James’s University Hospital, Leeds, United Kingdom; the2Institute of
Molecular & Cellular Biology and Astbury Centre for Structural & Molecular Biology, Faculty of Biological Sciences, and3School of Chemistry, University of Leeds,
Leeds, United Kingdom; and the4Department of Internal Medicine, University of Kansas Medical Center, Kansas City, KS.
Received October 21, 2011; accepted April 11, 2011.
Supported by the University of Leeds Biomedical Health Research Centre; a Medical Research Council New Investigator Research Grant (G0700124) and
Yorkshire Cancer Research Grant (PP025) (to S. G.); a Wellcome Trust Ph.D. studentship (to T. L. F.); a Cooperative Awards in Science and Engineering Ph.D.
studentship from the Biotechnology and Biological Sciences Research Council (BBSRC) and Pfizer (to E. A.); and a BBSRC studentship (to M. V.).
*These authors contributed equally to this work.
Address reprint requests to: Stephen Griffin, Section of Oncology and Clinical Research, Leeds Institute of Molecular Medicine, Wellcome Trust Brenner
Building, St. James’s University Hospital, University of Leeds, LS9 7TF, United Kingdom. E-mail: firstname.lastname@example.org; fax: (44)-113-343-8501.
View this article online at wileyonlinelibrary.com.
Potential conflict of interest: Nothing to report.
C 2011 by the American Association for the Study of Liver Diseases.
HCV is the prototype member of the Hepacivirus
genus within the Flaviviridae.3It is enveloped and pos-
sesses a positive-sense single-stranded RNA genome of
?9.6 kb. An internal ribosome entry site in the 50
untranslated region drives translation of a polyprotein
that is cleaved into 10 mature products. The core and
envelope glycoproteins with the RNA genome comprise
the virion, whereas nonstructural (NS) proteins modu-
late host metabolism and replication of the viral RNA.
JFH-1 has permitted the study of particle production,
and it has become clear that, in addition to canonical
virion components, other viral proteins are required.4-13
HCV p7 forms a cation channel in vitro,14-16and
both deletions and point mutations markedly reduce
the production of infectious virions in culture.4,5It is
comprised of two trans-membrane domains separated
by a cytosolic loop and forms both hexameric and
heptameric complexes.14,17,18We have recently shown
that p7 acts as a proton channel within infected cells,
which is directly required for the production of infec-
tious virions.19p7 is required for HCV to replicate in
chimpanzees20and small molecules block both channel
function in vitro and virion production in culture, ren-
dering it an attractive antiviral target.21,22
Skepticism concerning p7 inhibitors heralds from
trials where p7 inhibitor monotherapy, or combina-
tions with IFN/Rib failed to significantly improve
responses.23However, evidence from meta-analyses24,25
and patient virus loads at early time points26,27sup-
ports a specific antiviral effect, and selection of specific
nonsynonymous mutations occurs within patient iso-
late p7 sequences.28,29Because HCV displays genotype
(GT)-dependent p7 inhibitor sensitivity,21changes in
amino acid sequence could interfere with the binding
of drug molecules, making it likely that the emergence
of resistant quasispecies accounts for trial outcomes.
Here, we identify p7 resistance mutations specific to
adamantane and IS drugs, indicative of a genuine anti-
viral effect that supports their inclusion in future com-
Materials and Methods
p452/JFH-1c6, pJ4(CVL6S), and pGEX-p7(J4/JFH-1/
452) have been described.2,21,30-32pGEX-p7 mutants
were generated by fusion polymerase chain reaction
(PCR). pJFH-1 mutagenesis: a unique BsiWI–KpnI
pLitJFH-B/K and a silent AvrII site introduced 50of
p7: pLitJFH-B/K(A). The BsiWI–KpnI fragment con-
taining the AvrII site was reintroduced into pJFH-1:
Constructs. pJFH-1, pCON-1/JFH-1c3,
JFH(A), which replicated and produced particles as
wild-type (data not shown). Mutations were generated
in pLitJFH-B/K(A) by fusion PCR. pCON-1/JFH-1c3
mutagenesis: a unique BglII–AflII fragment was ligated
into pLitmus28i (NEB): pLitCON-1-B/A. Fusion PCR
was used to generate an L20F amplimer; this was
digested with NotI and ligated into pLitCON-1-B/A.
The BglII–AflII fragment was then reintroduced into
the full-length chimeric sequence. Constructs were con-
firmed by double-stranded DNA sequencing; primers
and details are available on request.
In Silico Structure Modeling and Drug Bind-
ing. p7 channel models were generated as described31
using Maestro (Schro ¨dinger Inc.). Point mutations
were introduced into wild-type structures with subse-
quent reminimization. The Maestro draw function was
used to design molecules that would fit within the
density associated with L20. Molecules were subjected
to free-energy minimization and stable, bound confor-
mations used as templates for rapid overlay of chemi-
cal structures, generating a small panel of molecules
including CD. These and adamantane analogues were
available from commercial libraries (Maybridge). Pdb
files were analyzed and images were captured using
PyMol version 0.9 (Delano Scientific). Drug-binding
studies against full-channel complexes employed Auto-
dock 4 (Scripps Research Inst., San Diego, CA), Glide
Details are available on request.
Bacterial p7 Expression and In Vitro Channel-
Forming Assay. Wild-type and mutant flu antigen–
tagged p7 was expressed as a glutathione S-transferase
fusion in Escherichia coli, then cleaved and purified as
described.17Real-time measurements of channel activ-
ity were performed as described.33
Virus Culture, Drug Inhibition and Live Cell pH
Assays. Huh7 cells were maintained, transfected, and
treated with inhibitors as described.21Intracellular virions
were liberated by freezing/thawing,11and HCV titres
were determined by focus-forming assay.21For live cell
imaging, infected cells seeded onto poly-D-lysine–coated
cover slips were grown overnight, prior to labeling with
Lysosensor Yellow/Blue DND-160 and quantitation of
cytoplasmic vesicle pH as described.19
Protein Analysis. Viral protein western blots of
Huh7 lysates at 72 hours posttransfection were per-
formed as described21using rabbit anti-core (308),
mouse anti-E2 (AP33), rabbit anti-p7 2715 (GT2a) and
1055 (GT1b),21,34rabbit anti-NS2, sheep anti-NS5A,
and mouse anti–glyceraldehyde 3-phosphate dehydro-
genase (6CS, Invitrogen), with appropriate horseradish
peroxidase–conjugated secondary antibodies (Sigma).
80FOSTER ET AL.HEPATOLOGY, July 2011
Channel Oligomerization Native Polyacrylamide
Gel Electrophoresis. Five micrograms of MeOH-solu-
bilized flu antigen–p7 protein was dried by evapora-
tion, then resolubilized overnight at room temperature
in 20 mM sodium phosphate buffer (pH 7.0) contain-
(LMPG) (monomeric) or 100 mM 1,2-diheptanoyl-
incorporating 4 mM rimantadine-HCl (Sigma) or 4
mM N-nonyl deoxynojirimycin (NN-DNJ) (Toronto
Biochemicals); 2? native polyacrylamide gel electropho-
resis (PAGE) loading dye (150 mM Tris-Cl (pH 7.0),
30% glycerol, 0.05% bromophenol blue) was added
and samples were separated on a 4-20% TGX gel (Bio-
rad) prior to staining with Coomassie Brilliant Blue.
Molecular Modeling of p7 Inhibitor Interac-
tions. We have modeled the heptameric GT1b J4 isolate
p7 complex31with lumenal His17.35We extended these
studies to include a low-pH, open form wherein His17
protonation caused p7 protomers to rotate, inducing
channel opening (Fig. 1A). This is consistent with p7
opening being stimulated at low pH,33as well as cellular
proton conductance.19We also generated a GT2a JFH-1
model (Fig. 1B) with similar structural characteristics to
the J4 channel, despite significant sequence diversity.
Autodock 4.0 was used to model binding sites (resi-
due interactions <4 A˚) on J4 and JFH-1 channels for
amantadine (Ama), rimantadine (Rim), and NN-DNJ.
Adamantanes bound to a peripheral, membrane-
exposed region of the channel complex (Fig. 1B, left
panel), preventing channel opening. The location of
this pocket agreed with NMR studies of p7-amanta-
dine interactions36and overlapped with J4 L(50-55)A,
a mutation shown to alter amantadine sensitivity
in vitro.31NN-DNJ did not interact with channel
complexes, instead docking to p7 monomers at the
protomer interface (Fig. 1B, right panel), thus poten-
tially disrupting oligomerization. Accordingly, active
nonyl-IS derivatives were predicted to bind this site
with >10-fold higher affinity than inactive butyl-deriv-
atives15(data not shown). Although relatively well con-
served in other genotypes (Fig. 1C), variation at these
positions may alter compound binding, providing a
basis for genotype-dependent sensitivity.21
Adamantane Resistance Is Conferred by a Muta-
tion Observed in Clinical Trials. J4 and JFH-1 ada-
mantane binding sites contained L20, which mutated
to F20 in GT1b patients unresponsive to IFN/Rib/
Ama.29Comparison of predicted binding affinities
(Autodock) revealed that Rim bound to wild-type
channels with higher affinity compared with Ama,
explaining its increased potency.19,21Ama-resistant JFH-
1 p7 provided a threshold value for effective drug bind-
ing (Kd>7.41 lM). L20F increased predicted Kdvalues
for both Ama and Rim above 7.41 lM (Fig. 2A), with
one exception. We therefore tested JFH-1 L20F p7
Rim sensitivity in vitro, assessing steady state activity
values to measure open channel complexes in drug-
bound equilibrium (Fig. 2B). As predicted, L20F was
Rim resistant, whereas the number of open wild-type
complexes reduced with increasing drug concentration.
JFH-1 and CON-1/JFH-1c3 viruses are Ama resist-
ant, yet susceptible to Rim and NN-DNJ.21At 72
hours posttransfection and through earlier time points
(data not shown), L20F caused no significant defect in
the production of intracellular or extracellular infec-
tious virions, and did not disrupt viral protein expres-
sion or processing (Fig. 2C). JFH-1 L20F p7 showed
slight stabilization compared with wild-type (Fig. 2C),
though this was less apparent in the CON-1/JFH-1
background. Addition of p7 inhibitors at ?IC80con-
centrations had no effect on the levels of intracellular
infectious HCV, consistent with ion channel activity
acting late during infectious virion production. Wild-
type secreted infectivity was reduced by Rim and NN-
DNJ, but not Ama, whereas Rim had no effect on
secreted L20F infectivity (Fig. 2D). L20F NN-DNJ
sensitivity was retained, however, and combining Rim
with NN-DNJ had an additive effect on wild-type
virus but not L20F, supporting separate modes of
action (Fig. 3A). Secreted infectivity could not be
reduced by more than ?2 log10at higher drug con-
centrations (data not shown), indicative of a low level
of ion channel-independent virion production. p7
channel activity therefore enhances, rather than per-
mits, production of infectious HCV.
Because p7 inhibitors specifically block HCV-medi-
ated alkalinization of intracellular vesicles required for
virion production,19we assessed whether L20F pre-
vented Rim inhibition of p7 activity in infected cells
using Lysosensor yellow/blue (Fig. 3B). In accordance
with infectivity data, vesicular pH in JFH-1 L20F–
infected cells was unaffected by increasing Rim concen-
tration, whereas JFH-1–infected cells experienced a
Rim-dependent reacidification. L20F adamantane resist-
ance therefore unequivocally links the antiviral effect of
p7 inhibitors to the prevention of vesicle alkalinization.
Validation of Molecular Models and the Predicted
Adamantane Binding Site Using Novel Inhibitors. The
L20F phenotype provided compelling evidence for the
validity of drug binding predictions, yet the possibility
HEPATOLOGY, Vol. 54, No. 1, 2011FOSTER ET AL. 81
Fig. 1. Modeling p7 complexes and inhibitor interactions. Models for the J4 (GT1b) and JFH-1 (GT2a) heptameric p7 channel complexes were
generated using Maestro as described in the Materials and Methods. Autodock was used to determine energetically favorable drug binding sites
for adamantanes (Ama and Rim) and alkylated imino sugars. Drug binding sites were defined as molecules interacting at a distance of <4 A˚.
(A) Shows the J4 channel structure modeled under neutral (pH 7.0) and acidic (pH 4.0) conditions. Protonation of His17 (blue) induces a con-
formational shift that results in rotation of p7 protomers and resultant opening of the structure. (B) Molecular models indicating positions of p7
inhibitor binding sites (red). Left panel shows the JFH-1 channel complex docked to rimantadine (for simplicity, one out of the seven drug mole-
cules are represented) at a peripheral, membrane-exposed binding site. Right panel shows a monomeric J4 p7 molecule bound to NN-DNJ at
the protomer interface. (C) Alignment of p7 sequences from prototype HCV GT1-3 sequences and representative GT4-7 sequences from the Los
Alamos database. Top panel shows location of residues identified in J4 and JFH-1 (red, bold type) predicted to bind to Ama/Rim (J4: G18, I19,
L20, F44, Y45, W48; JFH-1: N15, G18, L19, L20, F22, W48, P49, L52, L53, plus L47 from adjacent protomer) and the corresponding positions
in other GT are highlighted in bold type. Bottom panel shows the same information for binding of NN-DNJ to monomeric J4 p7 (S21, F22, F25,
F44, Y45, V47, W48, L51). Positions of L20 and F25 are highlighted by a dashed box and sensitivity to inhibitors are indicated: þ, sensitive; -,
resistant; ?, unknown; ?/- unknown but related sequences shown to be resistant in culture.46
82FOSTER ET AL.HEPATOLOGY, July 2011
Fig. 2. Characterization of p7 carrying the L20F mutation. Both J4 and JFH-1 adamantane binding sites contained L20, which could represent
a resistance determinant when mutated to F20. Accordingly, L20F was generated in both JFH-1 and CON-1 backgrounds to investigate its effects
in vitro and in vivo. (A) Predicted Kdvalues generated in Autodock for GT 1b and 2a p7, incorporating a described resistance determinant (L50-
55A)31as well as the L20F mutation. The Ama-resistant JFH-1 protein set a threshold for effective drug binding (>7.41 lM) and predicted re-
sistant channels are shown in bold. (B) Recombinant wild-type and L20F JFH-1 p7 protein was assessed for Rim resistance in vitro using fluo-
rescent dye release from liposomes. Steady state readings were compared (30 minutes following incubation at 37?C) to assess relative
populations of open channels. (C) Wild-type and L20F JFH-1 and CON-1/JFH-1c3 viruses were assayed for virion production and protein expres-
sion 72 hours post-electroporation of Huh7 cells as described in the Materials and Methods. The left panel shows intracellular and extracellular
infectivity for wild-type and mutant viruses at the 72-hour time point. The middle panel shows wild-type/L20F HCV proteins detected by immuno-
blotting with specific antibodies (see Material and Methods). The right panel shows extracellular infectivity at 72 hours posttransfection of JFH-1
(diamonds) or JFH-1 L20F (squares) in cells treated with increasing Rim concentrations. (D) The effects of Rim, Ama, and NN-DNJ at concentra-
tions shown on secreted JFH-1 and CON-1/JFH-1c3 wild-type (gray bars) and L20F (white bars) infectivity were determined 72 hours post-elec-
troporation of Huh7 cells by focus forming assay. Each experimental condition was conducted in triplicate and graphs shown are representative
of at least two independent experiments. Error bars represent one standard deviation.
remained that resistance occurred by an alternate
mechanism. We therefore validated predicted p7–ada-
mantane interactions using drugs as probes for specific-
ity. First, we selected a group of amantadine analogues
in rank order of JFH-1 p7 binding from three docking
programmes (see Materials and Methods) (Fig. 4A).
With one exception, these molecules behaved as
expected; those predicted to bind equally or better
than Rim inhibited JFH-1 p7 in vitro (Fig. 4B) and
achieved equivalent or improved results in culture
when added at Rim IC50(Fig. 4C). Those predicted
to bind less well than Rim had no effect. The excep-
tion (compound D) indicated that although our mod-
els provide a reliable guide to compound binding, they
(and molecular docking programmes) are not 100%
accurate. Interestingly, effective analogues were not
affected by the L20F mutation, despite adamantyl
moieties interacting identically with the Ama/Rim
binding pocket. However, extended analogue side
chains formed additional interactions with A41 and
G46, which presumably overcame disruption caused
We next designed nonadamantane molecules using
the ‘‘Draw’’ function in Maestro with a high pre-
dicted affinity for the J4 and JFH-1 binding sites.
These were screened in a subgenomic replicon for
effects on HCV RNA replication and cell viability
(data not shown).21Compound CD (Fig. 5A) both
inhibited GT1b p7 activity in vitro and showed an
equivalent antiviral effect to Rim, to which L20F vi-
rus was resistant (Fig. 5B,C). To our knowledge, CD
is the first molecule designed entirely against a de
novo molecular model to display an antiviral effect
IS Resistance of GT3a HCV Supports an Antivi-
ral Effect Targeting Channel Oligomerization. GT3a
452 isolate p7 displays resistance to NN-DNJ in vitro
and in culture.21This provided an excellent basis to
Fig. 3. Specificity of the L20F
adamantane resistance mutation.
(A) Huh7 cells transfected with
wild-type (gray bars) or L20F (white
bars) JFH-1 RNA were treated with
?IC80concentrations of Rim and/
or NN-DNJ as indicated. Effects on
mined at 72 hours by focus form-
ing assay, and cell lysates were
tested for NS5A expression by
western blot analysis. *Statistically
significant improvement of dual
treatment compared with individual
molecules (P < 0.05) as deter-
mined by Student t test. Results
are representative of two experi-
mental repeats with conditions in
triplicate. Error bars represent one
(white circles) or JFH-1 L20F (black
circles) infected cells were labeled
with Lysosensor blue/green in the
rimantadine concentration (horizon-
tal axis, lM). Effects on intracellu-
lar vesicle pH (vertical axis) were
determined by quantitation of the
340/440 nm and 380/510 nm
using a 410nm dichroic as
The cytoplasms of
100 cells were quantified for each
condition with baseline determined
by a region on the same images
adjacent to the cell in question.
Error bars represent one standard
84FOSTER ET AL. HEPATOLOGY, July 2011
investigate whether IS targeted oligomerization and to
identify resistance polymorphisms. DHPC induces oli-
gomerization of IS-sensitive J4 p7 in vitro, inducing
heptameric complexes equivalent to liposomes.31We
therefore assessed whether IS or Rim blocked oligomeri-
zation of J4 and 452 p7. NN-DNJ abrogated J4 p7 oli-
gomerization and channel activity, yet 452 p7 activity
was insensitive to this drug and oligomerization was not
Fig. 4. Activity of rank-ordered
amantadine analogues compared
with Rim. (A) Several amantadine
analogues targeting the predicted
Ama/Rim binding site of JFH-1 p7
were selected in rank order by com-
parison of their binding scores
using three in silico docking pro-
grams: Autodock, E-Hits, and Glide.
D, G, and H were predicted to bind
10-100? more avidly than Rim, E
with approximately the same affinity
as Rim and both F and J binding
100? less. (B) Compound activity
was tested in vitro (40 lM) versus
JFH-1 p7 with effects on resultant
real-time channel activity as indi-
cated by colored lines. (C) Ana-
logues were tested at the Rim
equivalent IC50 concentration (40
lM) for effects on secreted infectiv-
ity 72 hours post-electroporation of
JFH-1 (black bars) or JFH-1 L20F
(gray bars) by focus forming assay.
Each condition was performed in
duplicate and results are represen-
tative of three independent experi-
ments. Error bars represent one
HEPATOLOGY, Vol. 54, No. 1, 2011 FOSTER ET AL.85
affected (Fig. 6A). Rim did not affect oligomerization,
but it inhibited channel activity in both cases, confirm-
ing separate modes of action for these inhibitor classes.
Comparing NN-DNJ binding sites revealed varia-
tion between J4 and 452 (Fig. 1C), however alignment
with other p7 sequences revealed an F25A polymor-
phism to be covariant with IS resistance. F25 is located
on a predicted bulge in the p7 N-terminal helix, which
may link with adjacent protomers, but is also pre-
dicted to interact with IS head groups (Fig. 1B). We
previously showed that J4 F(22, 25, 26)/A p7 formed
hyperactive channels in vitro that retained Ama sensi-
tivity.31We therefore tested whether this mutant or
F25A in isolation could rescue p7 oligomerization
from NN-DNJ. Both J4 mutant proteins and JFH-1
F25A p7 were insensitive to NN-DNJ in vitro and dis-
played hyperactive channel phenotypes, consistent with
a more open-form channel structure (Fig. 6B). Native
PAGE again correlated IS resistance with the formation
of drug-resistant oligomeric complexes (Fig. 6C). Inter-
estingly, the major species formed by JFH-1 F25A p7
oligomer migrated more rapidly than other proteins, yet
was stable in the presence of NN-DNJ; some heptameric
JFH-1 F25A protein was also apparent. All mutant pro-
teins remained sensitive to Rim in vitro (data not
shown). We next tested F25A in cell culture and, despite
a modest decrease in particle production, the mutant was
resistant to both NN-DNJ and N-nonyl deoxygalactono-
jirimycin (NN-DGJ), but not Rim (Fig. 6D).
This study revealed the mode of action for adaman-
tane and IS p7 ion channel inhibitors and confirmed
that single amino acid changes confer resistance to these
drugs. The low fitness cost for these mutations observed
in culture implies that a minimal genetic barrier to their
selection would exist in vivo, explaining the perceived
lack of efficacy for p7 inhibitors in clinical trials.
HCV IFN/Rib resistance is a multifactorial phe-
nomenon, involving virus and host-associated factors.
This is distinct to resistance against direct-acting
STAT-C antivirals, which are host-independent and
According to quasispecies theory, all possible single
variants exist within an HCV-infected individual, with
selection dependent on fitness. Generation of dual, tri-
ple, and further variants becomes exponentially less
Fig. 5. A novel p7 inhibitor targeting the predicted adamantane binding site. CD was selected from a pool of novel molecules by excluding
nonspecific effects on cell viability or HCV genome replication in a JFH-1 subgenomic replicon assay.21(A) Structure of CD [1,3diben-
zyl5(2H1,2,3,4tetraazol5yl)hexahydropyrimidine] and its binding to the predicted adamantane binding site of the JFH-1 p7 channel model. (B)
Liposome dye-release assay for CD on GT1b J4 p7. L, liposomes only; S, solvent control; CD concentration as indicated. (C) Huh7 cells trans-
fected with wild-type (grey bars) or L20F JFH-1 (white bars) RNA were treated with CD, Rim, and NN-DNJ at concentrations shown (lM) and
secreted infectivity assessed at 72 hours by focus forming assay. Wild-type HCV was susceptible to all three drugs, whereas L20F HCV was
observed to be resistant to both CD and Rim while remaining sensitive to the action of NN-DNJ. Results are representative of two experimental
repeats with conditions in triplicate. Error bars represent one standard deviation.
86 FOSTER ET AL.HEPATOLOGY, July 2011
Fig. 6. GT3a p7 IS resistance is mediated by an F25A polymorphism. (A) To confirm previous observations that GT3a p7 was resistant to the
action of imino sugars (NN-DNJ), recombinant p7 proteins were tested for ion channel activity and their ability to oligomerize in the presence of
both IS and adamantane p7 inhibitors. Left panel: sensitivity of GT3a p7 (452 isolate) and GT1b J4 p7 to IS and Rim was assessed in vitro
(inhibitors at 10 lM). Both proteins were susceptible to Rim, whereas only J4 p7 displayed sensitivity to NN-DNJ. Right panel DHPC-native PAGE
of recombinant protein was used to assess p7 oligomerization. Both proteins solely formed stable heptameric oligomers (7mers) in the presence
of DHPC, but not LMPG where primarily 1/2/3mers and a minor proportion of 7mers were present. Complexes were stable in the presence of
Rim, whereas NN-DNJ disrupted J4, but not 452 channel complexes (Rim at 4 mM, NN-DNJ at 0.5 and 4 mM). þ, 100 mM DHPC; ?, 100 mM
LMPG; ND, no drug; 7mer, heptamer; 1/2/3mer, monomer/dimer/trimer. (B) Activity of IS (40 lM) versus wild-type/F25A p7 proteins and J4
F(22, 25, 26)/A was assessed in vitro. Liposomes Meth, liposomes with 5% MeOH solvent control, other reactions as indicated in legends. (C)
DHPC-native PAGE was used to assess effects of IS on wild-type/mutant p7 oligomerization. Annotations as above; FFF/AAA, J4 F(22, 25, 26)A
mutation. (D) Wild-type and F25A JFH-1 were tested for IS susceptibility by measuring secreted infectivity 72 hours post-electroporation in the
presence of increasing IS concentrations (horizontal axes, lM). Left panel: wild-type JFH-1 sensitivity to NN-DNJ and NN-DGJ, Middle panel: JFH-
1 F25A sensitivity to NN-DNJ and NN-DGJ. Right panel: sensitivity of both JFH-1 wild-type and F25A to 80 lM Rim (white bars). Results are rep-
resentative of three experimental repeats with conditions in triplicate. Error bars represent one standard deviation.
likely and forms the basis for the successful application
of combination therapies. Combination of IFN/Rib
with single STAT-C molecules targeting replication
therefore suppresses HCV replication through distinct
mechanisms. As such, IFN/Rib-resistant HCV will
rapidly become resistant to a third STAT-C drug,
depending on fitness cost and drug potency, because it
is essentially a monotherapy.
For virus assembly inhibitors, resistance would be
expected to arise all the more rapidly in IFN/Rib-
resistant viruses as no suppression of genome replica-
tion occurs. Combinations of assembly inhibitors,
however, can suppress RNA virus resistance.37Our
demonstration of distinct, specific antiviral effects for
two classes of p7 inhibitor therefore supports that
combination with STAT-C therapies, rather than IFN/
Rib, may enhance patient responses, because the
genetic barrier to dual resistance would be significantly
raised. Given that prototype p7 inhibitors have been
trialed in patients (amantadine, rimantadine, UT-231b
[IS] and BIT225 [amiloride]), these could be rapidly
deployed alongside other STAT-C compounds.
Our approach was necessarily based on molecular
modeling of p7 ion channel complexes. Models com-
prised a lumenal N-terminal helix with a conserved
His17 proton sensor, analogous to M2 His37. Cu2þ-
mediated inhibition confirms His17 as lumenal,35and
lowered pH activates GT1b p7.33Accordingly, model-
ing p7 under acidic conditions where His17 is proto-
nated induced an opening of the structure (Fig. 1A).
We recently showed that p7 induces vesicle alkaliniza-
tion, protecting intracellular virions from reduced
pH.19Because low pH induces the fusogenic action of
HCV glycoproteins,38p7 may act analogously to M2
from certain influenza A virus strains, where it prevents
such change in hemagglutinin.39Interestingly, secreted
HCV virions are acid resistant,19,40meaning that an
as-yet unidentified maturation event occurs at a late
stage of virion production where particles are acid-
stabilized. Accordingly, p7 inhibitors do not reduce
intracellular infectivity (Fig. 2D), supporting a post-
assembly role for p7 proton channel function.
Three separate drug docking programs predicted the
same adamantane binding site located on the p7 chan-
nelperiphery. This reconciles
NMR36investigations of inhibitor binding, although
Ama was also modeled within the H77 p7 lumen,41
following classical models for M2.42M2 NMR stud-
ies, however, revealed a peripheral binding site,43
although this is much debated.44,45For GT1b/2a p7
sequences, the residue composition of the Ama/Rim
binding site varied, which caused differences in affinity
dependent on both protein sequence and the com-
pound in question; Rim bound significantly more
avidly than Ama, explaining Ama’s poor antiviral effect
in several studies.19,21,22,46Sequence variation therefore
provides a structural basis for altered drug susceptibil-
ity.21In GT1b and 2a sequences, the adamantane bind-
ing site contains L20, which was mutated to F20 in
unresponsive GT1b IFN/Rib/Ama trial patients.29L20
does not occur in other HCV genotypes, and GT1a
patients in the same study showed no discernable resist-
ance changes, perhaps associated with reduced H77
Ama sensitivity.21,22Nevertheless, L20F conferred ada-
mantane resistance to GT1b and 2a in vitro and in cul-
ture, and protected proton channel function from Rim
in live cells. As resistance denotes specific antiviral
effects, this confirms L20F as a genuine resistance
mutation arising during natural infection in response to
Ama-driven selection. Interestingly, a recent study
describing an NMR-based model for monomeric GT1b
p7 showed no effect of Ama on channel activity,47yet
of the four amino acid positions where this protein var-
ied from the J4 sequence, two (J4: I19, F44, changed
to L19, L44) occurred within the predicted adamantane
binding site. These and other variations may affect in-
hibitor binding to this pocket either directly or indi-
rectly through changes to adjacent residues altering
pocket density; further Ama patient studies have
observed alternative mutations occurring in this region
of the protein.28No sequence analysis exists on patients
receiving IFN/Rib/Rim, yet it would be of interest to
determine whether a more potent inhibitor in this set-
ting could drive resistance in other HCV genotypes.
A second measure of molecular model accuracy and
the validity of the predicted Ama/Rim binding site
involved correlating analogue predicted binding with
antiviral activity. With one exception, analogue activity
in vitro and in culture correlated with their predicted
binding scores relative to Rim, providing further sup-
port for the predicted binding pocket. Interestingly,
the L20F mutation did not confer resistance to these
molecules, indicating that additional binding to p7
through side groups may overcome L20F-mediated
disruption of the Ama/Rim binding site. This may
represent a viable strategy for raising the genetic bar-
rier to resistance against novel p7 inhibitors. Binding
of a bespoke nonadamantane molecule, CD, designed
targeting the J4 and JFH-1 binding site was disrupted
by L20F, despite being an entirely different chemotype,
because its major stabilizing contacts were present
within the original pocket. These experiments demon-
strate the subtlety and complexity inherent to p7/
inhibitor interactions and explain why variations in
88FOSTER ET AL.HEPATOLOGY, July 2011
protein sequence or inhibitor structure can result in
different experimental outcomes. Such studies will,
however, inform the future development of more
potent compounds through iterative refinement and
improvement of rational drug design.
From a therapeutics perspective, alkylated IS p7
inhibitors acted through a mechanism distinct from
that of adamantanes, providing scope for the develop-
ment of parallel yet complimentary p7 inhibitor series.
In agreement with previous studies,15,22docking pro-
grams predicted that nonylated IS bound p7 proto-
mers >10-fold more avidly than those with butyl side
chains, occluded more of the p7 protomer interface
and so disrupted channel oligomerization. IS com-
pounds disrupted J4 p7 oligomerization and channel
activity, but not that of resistant 452 protein.21F resi-
dues have been purported to stabilize p7 oligomeriza-
tion through hydrophobic interactions48and F25 is
predicted to interact with IS head groups in GT1b p7.
In 452 p7, F25 is changed to A, and this polymor-
phism was shown to mediate IS resistance both in vitro
and in culture while remaining Rim sensitive. F25A
mutants also formed hyperactive channel complexes in
vitro which, in the case of JFH-1, appeared to be less
stable and migrated differently by native PAGE.
Nevertheless, F25A HCV genomes were viable in cul-
ture, again showing a low fitness cost for the develop-
ment of p7 inhibitor resistance.
Resistance to p7 inhibitors mediated by single
amino acid changes with little consequence for virus
fitness readily explains their ineffectiveness in clinical
trials combined with IFN/Rib. Virus rebound has
been noted during amantadine mono-26and triple
therapy.27In addition, relatively high IC50values com-
pared with other STAT-C molecules and a maximal
reduction in virus production of ?2log10 even for
combinations of p7 inhibitors understandably gener-
ates skepticism over their usefulness. However, Rim
and IS IC50values in HCV culture are similar to those
in influenza A virus and HIV in vitro/culture systems,
where they progressed to clinical and trial-stage use in
humans, respectively. Given the relatively high degree
(?30% of patients) of breakthrough in trials combining
NS3 inhibitors with IFN/Rib,49the recent success of
expanding the STAT-C repertoire should be an immedi-
ate and ongoing priority. The availability of prototype
p7 inhibitors could rapidly expedite this process, and
future p7 inhibitors could complement STAT-C thera-
pies as these are implemented over the next decade as
an understanding of the molecular basis of resistance
assists in the design of novel, more potent compounds.
(National Institute for Infectious Diseases, Tokyo,
Japan), Charles Rice (Rockefeller University, New
York, NY) & Apath LLC (Brooklyn, NY), Jens Bukh
(Hvidovre University Hospital, Hvidovre, Denmark),
Ralf Bartenschlager (University of Heidelberg, Heidel-
berg, Germany) and Thomas Pietschmann (University
of Hannover, Hannover, Germany) for the provision
of full-length HCV constructs. The rabbit anti-core
(308) antibody was a gift from John McLauchlan
(Centre for Virus Research, Glasgow, UK) and the
mouse anti-E2 (AP33) antibody was a gift from
Arvind Patel (Centre for Virus Research, Glasgow,
UK) and Genentech Inc. (San Francisco, CA). The
rabbit anti-NS2 antibody was a gift from Gholamreza
Haqshenas (Monash University, Victoria, Australia).
We are also grateful to Andrew Macdonald and David
Rowlands (University of Leeds, Leeds, UK) for critical
reading of the manuscript and informative discussion.
We are grateful to Takaji Wakita
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