Calpain activation by hepatitis C virus proteins inhibits the extrinsic apoptotic signaling pathway.
ABSTRACT An unresolved question regarding the physiopathology of hepatitis C virus (HCV) infection is the remarkable efficiency with which host defenses are neutralized to establish chronic infection. Modulation of an apoptotic response is one strategy used by viruses to escape immune surveillance. We previously showed that HCV proteins down-regulate expression of BH3-only Bcl2 interacting domain (Bid) in hepatocytes of HCV transgenic mice. As a consequence, cells acquire resistance to Fas-mediated apoptosis, which in turn leads to increased persistence of experimental viral infections in vivo. This mechanism might participate in the establishment of chronic infections and the resulting pathologies, including hepatocellular carcinoma. We now report that Bid is also down-regulated in patients in the context of noncirrhotic HCV-linked tumorigenesis and in the HCV RNA replicon system. We show that the nonstructural HCV viral protein NS5A is sufficient to activate a calpain cysteine protease, leading to degradation of Bid. Moreover, pharmacological inhibitors of calpains restore both the physiological levels of Bid and the sensitivity of cells toward a death receptor-mediated apoptotic signal. Finally, human HCV-related tumors and hepatocytes from HCV transgenic mice that display low Bid expression contain activated calpains. CONCLUSION: Calpains activated by HCV proteins degrade Bid and thus dampen apoptotic signaling. These results suggest that inhibiting calpains could lead to an improved efficiency of immune-mediated elimination of HCV-infected cells.
- SourceAvailable from: Jacques Piette[Show abstract] [Hide abstract]
ABSTRACT: Exposure to hepatitis C virus (HCV) typically results in chronic infection that leads to progressive liver disease ranging from mild inflammation to severe fibrosis and cirrhosis as well as primary liver cancer. HCV triggers innate immune signaling within the infected hepatocyte, a first step in mounting of the adaptive response against HCV infection. Persistent inflammation is strongly associated with liver tumorigenesis. The goal of our work was to investigate the initiation of the inflammatory processes triggered by HCV viral proteins in their host cell and their possible link with HCV-related liver cancer. We report a dramatic upregulation of the lymphotoxin signaling pathway and more specifically of lymphotoxin-β in tumors of the FL-N/35 HCV-transgenic mice. Lymphotoxin expression is accompanied by activation of NF-κB, neosynthesis of chemokines and intra-tumoral recruitment of mononuclear cells. Spectacularly, IKKβ inactivation in FL-N/35 mice drastically reduces tumor incidence. Activation of lymphotoxin-β pathway can be reproduced in several cellular models, including the full length replicon and HCV-infected primary human hepatocytes. We have identified NS5B, the HCV RNA dependent RNA polymerase, as the viral protein responsible for this phenotype and shown that pharmacological inhibition of its activity alleviates activation of the pro-inflammatory pathway. These results open new perspectives in understanding the inflammatory mechanisms linked to HCV infection and tumorigenesis.PLoS Pathogens 03/2013; 9(3):e1003234. · 8.14 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Despite major advances in the understanding and treatment of hepatitis C, a preventive vaccine remains elusive. The marked genetic diversity and multiple mechanisms of persistence of hepatitis C virus, combined with the relatively poor immune response of the infected host against the virus, are major barriers. The lack of robust and convenient model systems further hampers the effort to develop an effective vaccine. Advances in our understanding of virus-host interactions and protective immunity in hepatitis C virus infection provide an important roadmap to develop potent and broadly directed vaccine candidates targeting both humoral and cellular immune responses. Multiple approaches to generating and testing viral immunogens have met with variable success. Several candidates have advanced to clinical trials based on promising results in chimpanzees. The ultimate path to a successful preventive vaccine requires comprehensive evaluations of all aspects of protective immunity, innovative application of state-of-the-art vaccine technology and properly designed vaccine trials that can affirm definitive endpoints of efficacy.Nature medicine 07/2013; 19(7):869-78. · 27.14 Impact Factor
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ABSTRACT: Enterovirus 71 (EV71) is a causative agent of an array of childhood diseases with severe neurological manifestations implicated. EV71 infection is known to induce caspase-dependent apoptosis in cell cultures and animal models. However, whether an alternative mode of apoptotic pathway independent of caspase activation can be triggered by EV71 infection has not been explored. In this study, we showed that calcium (Ca(2+))-activated calpains are capable of mediating caspase-independent pathway activation during EV71-induced apoptosis in HeLa cells. Results from subcellular fractionation analysis and confocal imaging indicated that during EV71 infection, apoptosis inducing factor (AIF), a primary mediator of the caspase-independent pathway, became truncated and translocated from the mitochondrion to nucleus. This is accompanied by the release of cytochrome c and sharply decreased mitochondrial membrane potential. AIF knockdown data indicated significant protection against apoptotic cell death, with greater protection provided by the addition of a pan-caspase inhibitor. The Ca(2+)-dependent, calpain isoforms 1 and 2 but not cathepsins were proved crucial for the altered AIF behavior as studied by the pharmacological inhibitor and the knockdown approaches. We then analyzed Ca(2+) dynamics in the infected cells and found elevated levels of mitochondrial Ca(2+). Treatment with ruthenium red, a mitochondrial Ca(2+) influx inhibitor, significantly blocked calpain activations and AIF cleavage. Our conclusion is that calpain activations via Ca(2+) flux plays an essential role in eliciting an AIF-mediated, caspase-independent apoptotic pathway in EV71-infected cells. These findings should be useful for understanding the virus-induced cytopathology and the impact of Ca(2+) homeostasis on EV71 infection.Journal of General Virology 03/2013; · 3.13 Impact Factor
Calpain Activation by Hepatitis C Virus Proteins
Inhibits the Extrinsic Apoptotic Signaling Pathway
Yannick Simonin,1,2Olivier Disson,1,2* Herv´ e Lerat,1,2† Etienne Antoine,1,2Fabien Binam´ e,1,2Arielle R. Rosenberg,3
Solange Desagher,1,2Patrice Lassus,1,2Paulette Bioulac-Sage,4and Urszula Hibner1,2
An unresolved question regarding the physiopathology of hepatitis C virus (HCV) infection
is the remarkable efficiency with which host defenses are neutralized to establish chronic
infection. Modulation of an apoptotic response is one strategy used by viruses to escape
BH3-only Bcl2 interacting domain (Bid) in hepatocytes of HCV transgenic mice. As a
consequence, cells acquire resistance to Fas-mediated apoptosis, which in turn leads to
increased persistence of experimental viral infections in vivo. This mechanism might par-
ticipate in the establishment of chronic infections and the resulting pathologies, including
hepatocellular carcinoma. We now report that Bid is also down-regulated in patients in the
context of noncirrhotic HCV-linked tumorigenesis and in the HCV RNA replicon system.
We show that the nonstructural HCV viral protein NS5A is sufficient to activate a calpain
cysteine protease, leading to degradation of Bid. Moreover, pharmacological inhibitors of
receptor–mediated apoptotic signal. Finally, human HCV-related tumors and hepatocytes
from HCV transgenic mice that display low Bid expression contain activated calpains.
Conclusion: Calpains activated by HCV proteins degrade Bid and thus dampen apoptotic
signaling. These results suggest that inhibiting calpains could lead to an improved efficiency
of immune-mediated elimination of HCV-infected cells. (HEPATOLOGY 2009;50:000-000.)
to clear the virus and become chronic carriers, with a
persistent presence of detectable virus in the serum.1Pa-
tients with chronic hepatitis C are at risk for hepatic fi-
brosis, frequently culminating in hepatic cirrhosis and
hepatocellular carcinoma (HCC).2
FL-N/35 transgenic mice, with hepatocyte-targeted
expression of the entire open reading frame of the geno-
type 1b HCV, are at risk for steatosis and hepatocellular
FL-N/35 hepatocytes are resistant to apoptosis induced
by the Fas/CD95 death receptor stimulation. The lack of
sensitivity to apoptotic stimulation was related to de-
creased expression of BH3-only Bcl2 interacting domain
sis regulators.4Importantly, Bid-deficient hepatocytes are
refractory to T lymphocyte–induced cell death, suggest-
a consequence, to liver pathologies characteristic of
chronic HCV infection.4
Here, we report that Bid is also down-regulated in the
licons and in a subset of HCV-linked human HCC.
ersistent infection with hepatitis C virus (HCV) is
among the most common infectious causes of
chronic liver disease. The majority of patients fail
atitis B virus; HCC, hepatocellular carcinoma; HCV, hepatitis C virus; mRNA,
messenger RNA; NS5A, nonstructural protein 5A; PBS, phosphate-buffered saline;
RT-qPCR, real-time quantitative PCR; SEM, standard error of the mean; shRNA,
short hairpin RNA; TNF-?, tumor necrosis factor alpha; z-VAD-fmk, benzyloxy-
carbonyl-Val-Ala-Asp (OMe) fluoromethylketone.
From the1Centre National de la Recherche Scientifique (CNRS), UMR 5535,
InstitutdeGe ´ne ´tiqueMole ´culairedeMontpellier,Montpellier,France;2Universite ´
deMontpellier2andUniversite ´deMontpellier1,Montpellier,France;3Universite ´
Paris Descartes, Institut National de la Sante ´ et de la Recherche Me ´dicale (IN-
SERM), Equipe Avenir “Virologie de l’he ´patite C”, Institut Cochin, Paris, France;
and4Ho ˆpital Pellegrin, Service d’Anatomie Pathologique, Centre Hospitalier Uni-
versitaire Bordeaux, Bordeaux, France.
Received March 10, 2009; accepted July 7, 2009.
*Current address: Institut Pasteur, 25 rue du Dr Roux, 75015 Paris, France.
†Current address: INSERM, U955, Ho ˆpital Henri Mondor, 94010 Cre ´teil,
Recherche sur le SIDA (U.H.), Association pour le Recherche sur le Cancer (ARC)
Address reprint requests to: Urszula Hibner, Institut de Ge ´ne ´tique Mole ´culaire
de Montpellier CNRS UMR 5535, 1919 route de Mende, 34293 Montpellier
cedex 5, France. E-mail: email@example.com; phone: ?33 467 613 656.
Copyright © 2009 by the American Association for the Study of Liver Diseases.
Published online in Wiley InterScience (www.interscience.wiley.com).
Potential conflict of interest: Nothing to report.
Additional Supporting Information may be found in the online version of this
Mechanistically, the nonstructural viral protein NS5A is
sufficient for Bid degradation, which occurs via the acti-
vation of a calpain, a cellular cysteine protease. Pharma-
cological inhibition of calpains restores the physiological
levels of Bid, suggesting that calpains may be interesting
molecular targets for therapies aimed at increasing the
efficiency of immune elimination of HCV-infected cells.
Patients and Methods
Animals. FL-N/35 transgenic animals3were bred and
maintained according to the institutional guidelines.
Nine-month-old males were used in these experiments.
Patients and Tissue Samples. Twenty-three HCC
and corresponding nontumoral tissues were obtained
sity Hospital, Bordeaux, France. Small liver pieces from
tumoral and nontumoral livers were immediately frozen
in liquid nitrogen and stored at ?80°C until use. In par-
tive sections were processed for diagnostic purposes. In
this study, the series included 17 patients infected with
HCV and six patients infected with HBV. Their clinical
data such as sex and age, as well as fibrosis stage of the
nontumoral liver according to Metavir score,5are re-
ported in Table 1. Informed consent was obtained ac-
cording to the institutional regulations.
Cell Culture and Treatments. Cells were cultured in
Dulbecco’s modified Eagle medium supplemented with
10% fetal bovine serum, 100 ng/mL streptomycin, and
100 U/mL penicillin and 400 ?g/mL of G418 for cells
harboring the Nneo/C-5B and Nneo/3-5B replicons.
When appropriate, cells were treated with lactacystin (50
?M), benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoro-
MDL-28170 (100 ?M) for 24 hours and staurosporine
(1 ?M), tumor necrosis factor-alpha (TNF-?) (0.5 ng/
mL), and actinomycin D (0.5 ?g/?L) for 14 hours. All
drugs were from Sigma (St. Louis, MO).
Apoptosis Test. Subconfluent cultures were treated
with TNF-? (0.5 ng/mL) and actinomycin D (0.5 ?g/
mL). Where appropriate, MDL-28170 (100 ?M) was
added 6 hours before apoptotic inducers. Apoptosis was
assayed after 14 hours on pooled adherent and floating
cells using the Annexin V-Cy3 Apoptosis Detection Kit
(Abcam, Cambridge, UK) according to the manufactur-
er’s instruction. The cells were analyzed on a FACSCali-
bur flow cytometer using CellQuestPro software (BD
Zymography. Zymograms were run according to pre-
viously described methods6,7with minor modifications.
Resolving gels contained 0.2% of casein (Hammerstein,
USB Corp., Cleveland, OH). Cells were homogenized in
150 ?L of extraction buffer (20 mM Tris-HCl [pH 7.5],
5 mM ethylene diamine tetraacetic acid (EDTA), 5 mM
ethylene glycol tetraacetic acid, 1 mM dithiothreitol) and
centrifuged for 15 minutes at 10,000g at 4°C. Protein
concentrations were measured using the bicinchoninic
(BCA) assay. Samples (25 ?g) were electrophoresed at
100 V for 4 hours at 4°C, the gels were rinsed and incu-
bated for 18 hours at 20°C in Ca2?containing buffer,
stained for 2 hours with Coomassie brilliant blue, then
placed in boiling water for 8 minutes. Protease activity is
visualized as a clear band on the gel.
In Vitro Bid Degradation Assay. Recombinant hu-
man Bid (50 ng) was incubated with 50 ?g of cellular
extract for 3 hours at room temperature in 50 ?L calpain
assay buffer (50 mM Tris-HCl, 100 mM NaCl [pH 7.5],
20 mM dithiothreitol, 5 mM CaCl2). Protease inhibitors
(lactacystin (50 ?M), z-VAD-fmk (10 ?M) or MDL-
28170 (100 ?M) were included, as indicated. For tBid
control assays, 50 ng of human rBid was incubated for 45
minutes at 37°C in the presence or absence of 10 ng
recombinant human active caspase 8 in the buffer con-
taining 25 mM HEPES [4-(2-hydroxyethyl)-1-pipera-
Table 1. Clinical and Pathological Features of Patients with
HCV and/or HBV
ND, not determined.
*According to Metavir score (ref. 9) : A? activity: mild (A1) ; moderate (A2) ;
** wd (well) ; md (moderately) ; pd (poorly) differentiated
2SIMONIN ET AL.HEPATOLOGY, Month 2009
zine ethanesulfonic acid; pH 7.4], 5 mM dithiothreitol,
50 mM NaCl, 5% sucrose, 0.5 mM EDTA.
Statistical Analysis. Experiments were performed at
least three times. Data are presented either from a repre-
sentative experiment or as mean ? standard error of the
mean (SEM). Comparisons between groups were ana-
lyzed by Student t test. Significance values: P ? 0.01 (*),
P ? 0.001 (**). The statistical significance of data in Fig.
1 was calculated by the test of Bernoulli.8
Bid Decrease in HCV-Related HCCs. We have pre-
viously reported that Bid protein expression is down-reg-
ulated in hepatocytes of the FL-N/35 mice,4which
express roughly homogenous amounts of all HCV pro-
teins in each cell.9In contrast, a minor and variable pro-
portion of liver parenchymal cells stain positive for viral
proteins in chronically infected human patients.10,11Be-
to infected cells, biopsies from patients with hepatitis C
mouse data in human pathology. In the search for a hu-
man in vivo clonal population derived from an HCV-
infected cell, we concentrated on HCV-related tumors.
Whereas a vast majority of HCV-linked HCCs develop
appear on quasi-healthy tissues.12We reasoned that these
in which the initial steps of oncogenesis would have been
driven by the virus, rather than by the tumor-prone cir-
rhotic environment.13,14We analyzed Bid protein expres-
sion levels in 23 surgical specimens of HCC and the
from chronic infection with HCV or HBV (Table 1).
rhosis, and the remaining eight had varying fibrosis
scores. Interestingly, the only group of patients in whose
tumors Bid was consistently down-regulated (eight of
eight patients) was the HCV-positive group bearing tu-
mors on noncirrhotic livers (Fig. 1A, see also Fig. 5B).
Similar to the transgenic mouse model, Bid protein level
did not correlate with the messenger RNA (mRNA) ex-
pression, suggesting a posttranslational effect (Fig. 1B).
As predicted by our hypothesis, only a minor fraction
(one of nine patients) of HCV-linked tumors that ap-
1C). Finally, in the limited number of HBV-positive tu-
mors analyzed, there was a tendency for an increased Bid
Fig. 1. Bid is down-regulated in a
subset of human hepatocellular car-
cinoma. Proteins and RNA were ex-
tracted from frozen specimens of
human tumors and the correspond-
ing nontumoral liver tissues. Protein
expression was analyzed by immu-
noblotting with an anti-Bid antibody,
and the RNA was assessed by RT-
qPCR. Expression of a house keeping
dehydrogenase (GAPDH), served as a
loading control. (A) Western blot and
corresponding quantification of Bid
level in six HCCs that have arisen on
HCV-positive noncirrhotic livers. (B)
RT-qPCR analysis of Bid mRNA in the
same patients as in (A). (C) Bid pro-
tein expression and corresponding
quantification in tumor and peritu-
moral regions in samples from HCV-
positive patients with cirrhosis. (D) Bid
protein expression and corresponding
quantification in tumor and peritu-
moral region of HBV-positive patients.
Representative immunoblots of at
least three independent experiments
are shown. N, nontumoral; T, tumor.
HEPATOLOGY, Vol. 50, No. 10, 2009SIMONIN ET AL.3
(Fig. 1D). These data suggest that the HCV-linked tu-
ularly distinct from the majority of HCCs. Indeed, the
probability that the difference of phenotypes observed in
HCV-linked HCC in noncirrhotic versus cirrhotic livers
was due to chance is low (P ? 2 ? 10?8). Although not
ilarly to the mouse model, Bid is regulated by the HCV
proteins in infected human hepatocytes in vivo.
Bid Decrease in the Replicon System. Human hep-
atoma Huh7 cells harboring the full-length genome
(Nneo/C-5B) or the subgenomic (Nneo/3-5B) HCV
RNA replicons express, respectively, the full complement
of viral proteins or only the nonstructural proteins (NS3
through NS5B) of a genotype 1b viral isolate.15Both
replicon-containing cell lines expressed less Bid than the
parental Huh7 cells (Fig. 2A-C), the decrease being more
pronounced for the full-length replicon. This effect ap-
of the Bcl-2 family were expressed similarly in the three
cell lines (Supporting Fig. 1). Importantly, as in human
samples (Fig. 1B) and in the transgenic mouse model,4
Bid mRNA levels were not affected by the presence of
viral gene products (Fig. 2D).
In the course of apoptosis, full-length Bid undergoes a
proteolytic cleavage; the truncated protein, tBid, is the
active form that initiates the mitochondrial step of apo-
replicon-containing cells (Fig. 2E), suggesting that in this
for the disappearance of the full-length protein.
Inhibitors of Calpains Restore Bid Expression. In-
creased protein turnover could explain the decreased level
of Bid observed in the presence of the HCV proteins. We
cells with a series of cell-permeable pharmacological pro-
tease inhibitors and looked for the rescue of the Bid
down-regulation phenotype. Proteasome and caspase in-
hibitors, lactacystin and z-VAD, respectively, had no ef-
fect on Bid levels (Supporting Fig. 2). In contrast, Bid
expression was restored in the replicon cell lines grown in
(Fig. 3A,B). Of note, inhibition of calpains had no dis-
cernible effect on Bid expression in the parental Huh7
to account for Bid degradation, we incubated purified
recombinant Bid (r-Bid) protein with soluble extracts of
trol, we used recombinant active caspase 8, which, as ex-
pected, acted on the full-length r-Bid to give rise to tBid
under these conditions. Whereas exogenous Bid was sta-
Fig. 2. Bid is down-regulated in HCV replicons. Proteins and RNA extracted from exponentially growing Huh7 cells, from the full-length (Nneo/C-5B)
or the subgenomic (Nneo/3-5B) replicon-containing lines were analyzed, respectively, by immunoblotting and by RT-qPCR. Bid expression was
normalized to GAPDH. Representative (A) western blot and (B) quantification of Bid protein and (C) immunofluorescence analysis of Bid expression
in the parental cells (Huh7) and subgenomic (3-5B) and full-length (C-5B) replicon-expressing cells. (D) RNA expression in four independent
experiments. Data are presented as mean ? SEM. *P ? 0.01, **P ? 0.001. (E) Western blot analysis of full-length and truncated Bid in apoptotic,
staurosporine-treated 293T cells, exponentially growing Huh7 cells, and exponentially growing replicon-containing cells. No tBid accumulation was
observed in the replicon-expressing cells.
4 SIMONIN ET AL.HEPATOLOGY, Month 2009
of the experiment, the Nneo/C-5B extract contained a
proteolytic activity which led to r-Bid degradation. It was
prevented by a calpain inhibitor, but not by proteasome
or caspase inhibitors (Supporting Fig. 3), suggesting that
Bid is a direct target of a soluble active calpain present in
cells expressing HCV proteins.
Activation of Calpains in the Replicon System and
in HCV-Related HCC. Modulation of calpain activity
could be due either to changes in their expression, their
activation, or both. We found no variation in expression
of two ubiquitously expressed calpains, calpain 1 or cal-
pain 2, in the replicon-containing cell lines compared to
the parental Huh7 cells (Supporting Fig. 4A). These data
were consistent with our quantitative reverse transcrip-
14 known and putative catalytic and two regulatory cal-
pain subunits.17Nine catalytic and both regulatory sub-
units had detectable levels of expression in the Huh7-
derived cells; none of them were significantly altered in
the presence of HCV proteins (Supporting Fig. 4B).
Next, we tested the hypothesis that the viral proteins in-
fluence calpain enzymatic activity. Zymography assays
performed under conditions compatible with calpain ac-
tivity detected a single protease band (Fig. 4A). A weak
signal was detected in the parental Huh7 cells, and both
activity. To assay calpain activation in the Nneo/C-5B
and the Nneo/3-5B cells using an alternative approach,
we next looked for the presence of calpastatin, an endog-
enous calpain inhibitor that is cleaved upon calpain acti-
vation.18As expected, cells harboring either replicon
displayed diminished calpastatin levels, consistent with
calpain activation by the viral proteins (Fig. 4B).
We next investigated if calpain activation was also as-
mice, where Bid down-regulation by the HCV proteins
was originally described,4calpastatin degradation accom-
panied the presence of the transgene and the lower Bid
expression level (Fig. 5A). Next, we analyzed calpain ac-
tivity in extracts of tumors and surrounding tissues ob-
tained from human liver biopsies. All of the HCV-
positive tumors from noncirrhotic livers displayed
Fig. 3. Pharmacological calpain inhibi-
tors restore physiological level of Bid ex-
pression. Parental (Huh7) and replicon
(3-5B and C-5B)-containing cells were
grown for 24 hours in the absence or in the
presence of protease inhibitors, as indi-
cated. Protein extracts were analyzed for
Bid expression by immunoblotting. (A) Two
calpain inhibitors (E64D, 50 ?M, and MDL-
28170, 100?M) partially or completely
rescued Bid expression in the replicon-ex-
pressing cell lines. (B) Quantification of the
calpain inhibitor’s effect from three inde-
pendent experiments. Data were normalized
to the expression of GAPDH. For each ex-
periment, the relative expression of Bid in
the Huh7 parental cells was arbitrarily set
as 1. (C) Recombinant human Bid (50 ng)
was incubated with soluble extracts of pa-
rental (Huh7) or full-length replicon (Nneo/
C-5B) cell lines or recombinant active
caspase 8. Calpain inhibitor (MDL-28170,
100 ?M) was added, as indicated. rBid
degradation was analyzed by immunoblot-
ting. (D) Corresponding quantification of
rBid level from three independent experi-
Fig. 4. Calpain activation in HCV replicon-containing cells. (A) Calpain
activity was measured by zymography in independently prepared sam-
ples of parental cells (n ? 2) and both replicon-expressing lines (n ?
3) and by calpastatin cleavage (B). Commercial human calpain 1 was
used as a positive control for zymography.
HEPATOLOGY, Vol. 50, No. 10, 2009SIMONIN ET AL.5
diminished calpastatin levels, in parallel to the decrease of
Bid expression (Fig. 5B). A single tumor of our collection
a low level of calpastatin (Fig. 5C), which in this case did
not correlate with low Bid levels (Fig. 1C).
Our data from the transgenic mouse model, the sub-
genomic and genomic replicons and from the analysis of
human patient samples, all point to the calpains as HCV-
activated mediators of proteolytic degradation of Bid.
NS5A Activates Calpains and Lowers Bid Expres-
tural proteins. In vivo, they are organized in an
endoplasmic reticulum–associated multiprotein com-
activities that may be relevant to the physiopathology of
the viral infection. To determine if the phenotype of cal-
pain activation and Bid down-regulation could be as-
cribed to a specific viral protein, we established stable
populations of Huh7 cells in which the expression of a
single protein, or a combination of two proteins, was
driven by a heterologous promoter. Concentrating on vi-
ral proteins previously described as major regulators of
host cell physiology,13we tested the effect of core, NS3,
NS4A, and NS5A. Retrovirus-driven expression of viral
proteins was controlled by immunoblotting (Supporting
NS5A mimicked the entire complement of viral proteins,
both in its effect on Bid and on calpain activity (Fig.
ily (Supporting Fig. 6). Similarly to the data obtained in
the replicon model (Fig. 2E), decreased Bid expression
was not associated with Bid activation (Fig. 6C). In addi-
tion, a pharmacological inhibitor of calpains, the MDL
28170 compound, partially restored Bid expression (Fig.
6B). Calpains 1 and 2 were probably not responsible for
the observed phenotype, because their partial (capn 2) or
tin levels in the NS5A-expressing cells (Fig. 6D). To de-
termine if Bid down-regulation participated in the
resistance to death receptor–mediated cell death, we as-
sayed the effect of calpain inhibition on apoptosis. TNF-
?–induced apoptosis was significantly reduced in the
presence of NS5A (Fig. 6E; Supporting Fig. 7). Impor-
tantly, down-regulation of Bid expression by shRNA
(Supporting Fig. 8) gave rise to a similar protection (Fig.
6E). As expected from the different modes of action lead-
ing to Bid down-regulation, pharmacological inhibition
of calpains restored the apoptotic response of the NS5A-
expressors, but not of the shRNA-treated cells.
on calpains and, as a consequence, on Bid expression, also
tests performed on Huh7.5.1 cells productively infected
with either the JFH1 isolate virus20,21or the Con1/C3 chi-
the NS5A sequence is highly variable among HCV geno-
types, we hypothesized that this result could be due to a
functional difference between NS5A genotypes. To address
this point, we compared the effects of expression in Huh7
no effect on either the calpain activity or on Bid expression
These results prompted us to reanalyze the data shown
in Fig. 1A. Interestingly, the three patients characterized
by the most profound decline of Bid expression (patients
Fig. 5. Calpain activation in HCV-
linked HCC. (A) Calpastatin and Bid
protein expression in nontumoral liv-
ers of three HCV transgenic mice
(FL-N/35) and three control litter-
mates. (B) Calpastatin protein ex-
pression in tumors and peritumoral
regions of three HCV-positive pa-
tients without cirrhosis (left panel).
Expression of Bid in the same sam-
ples (right panel). (C) Calpastatin
protein expression in tumors and
peritumoral regions of HCV-positive
patients with cirrhosis. GAPDH ex-
pression served as a loading control
in all western blots.
6 SIMONIN ET AL.HEPATOLOGY, Month 2009
6741, 3218, and 1866) were all infected with genotype 1
Viruses are under a strong evolutionary pressure to
develop strategies of resistance to their host’s innate and
acquired immune responses. HCV is particularly success-
of acute infections progress to chronicity.1On top of sev-
production and response, antigen presentation, and cell-
Fig. 6. NS5A induces calpain activation, Bid cleavage, and apoptosis resistance. Huh7 cells were transduced with single, or a combination of, retroviral
vector(s) coding for the HCV1b proteins: core, NS3, NS4A, and NS5A, as indicated. (A) Calpastatin and Bid protein expression were assayed by
immunoblotting. (B) Parental and NS5A-expressing cells were grown for 24 hours in the absence or presence of a pharmacological calpain inhibitor
(MDL-28170), and the protein extracts were assayed by immunoblotting. Shown are a representative western blot (left panel) and quantification of data from
three independent experiments (right panel). (C) Western blot analysis of full-length and truncated Bid in staurosporine-treated 293T cells, exponentially
growing parental and NS5A-expressing Huh7 cells, in the presence or absence of a pharmacological calpain inhibitor, as indicated. (D) NS5A-expressing and
parental Huh7 cells were transduced with retroviral vectors encoding shRNA for calpain 1 and 2. Protein extracts were analyzed by immunoblotting. (E)
Parental and NS5A-expressing Huh7 cells were transduced with retroviral vectors coding for Bid or firefly luciferase shRNA. Where indicated, cells were
pretreated with MDL-28170 for 6 hours. TNF? (0.5 ng/mL) and actinomycin D (0.5 ?g/mL) were added where indicated for an additional 14 hours.
Apoptosis was assayed by Annexin-V labeling detected by flow cytometry. (F) Calpastatin and Bid protein expression was analyzed in parental cells and Huh7
cells expressing NS5A of 1b, 1a, and 2a HCV genotypes. Where appropriate, results are presented as mean ? SEM of three separate experiments (*P ?
0.01; **P ? 0.001).
HEPATOLOGY, Vol. 50, No. 10, 2009SIMONIN ET AL.7
optosis of the infected cell.25We previously reported that
the HCV viral proteins decrease the expression of the
BH3-only member of the Bcl-2 family.16In some cell
types, including hepatocytes, it is an essential component
of the apoptotic signaling pathway originating at the so-
called death receptors at the cell surface. In the FL/N-35
immunocompetent mice, decreased Bid expression and
the associated apoptotic defect lead to an increased per-
sistence in the liver of an unrelated virus, suggesting that
the resistance to cell death opposes the T cell–mediated
report that Bid deficiency, and presumably its conse-
quences, is involved in at least some cases of human pa-
thology related to HCV infection.
Because very little of the viral proteins is present in a
chronically infected human hepatocyte, it is difficult to
accurately estimate the fraction of infected cells in patient
liver biopsies.10,11Consequently, extracts from biopsies
for tracking the effect of viral proteins on Bid. Because
biochemical assays are the most reliable way of quantify-
ing both RNA and proteins, we attempted to identify a
cellular population in vivo that is homogenous with re-
derived from an infected cell might correspond to this
tory environment, with its associated oxidative stress, and
cirrhosis, which are the context of a vast majority of
HCV-related HCCs, provide favorable conditions for an
lack of strong oncogenes among the HCV proteins, their
direct impact on hepatocyte cell cycle and apoptosis reg-
ability of tumor initiation in infected cells.13It follows
that a non-negligible proportion of HCV-related tumors
tumors of such origin were likely to be particularly abun-
dant in the minor fraction of HCV-linked HCCs that
appear in livers that are not cirrhotic and, even more
infrequently, have a low fibrotic index. The data pre-
sented here are consistent with such a hypothesis. Indeed,
in contrast to HCV-positive HCCs arising on cirrhotic
livers, but similarly to cells from the HCV-positive trans-
genic mouse livers and from the replicon model, tumors
from the patients without cirrhosis had a decreased ex-
pression of Bid. This phenotype is thus clearly not a gen-
eral feature of HCC. However, it does appear to be
Fig. 1D) or in tumors unrelated to viral infection (not
shown). Thus, whereas we do not think that these data
they support the contention that the HCV affects Bid
expression in hepatocytes of chronically infected human
in our experimental systems, we turned our attention to
protein stability. Many proteases can participate in the
turnover of cellular proteins. Moreover, Bid is a target of
several proteolytic enzymes.16The full-length Bid has a
long half-life,28which is probably why we detected little
in parental Huh7 cells. In contrast, in cells harboring
either the subgenomic or the full-genome-length HCV
inhibition of calpains. Although it is notoriously difficult
to eliminate the possible cross-specificities in the use of
pharmacological enzyme inhibitors, the fact that two
chemically unrelated calpain inhibitors29,30had a similar
activity in our assay argues for these proteases’ involve-
ment in Bid degradation.
NS5A, an HCV protein involved in regulation of
many cellular signal transduction pathways,31has been
reported to activate calpains.32We have confirmed and
extended these results: while a strong increase of calpain-
like activity occurred upon the expression both of the full
specifically the NS5A of the 1b and the 1a HCV geno-
types, the 2a genotype protein had no effect either on
calpain activity or on the stability of the Bid protein.
Interestingly, these viral genotypes with documented di-
pathologies of different prognosis.33Because a dimin-
ished sensitivity to apoptotic stimuli is a hallmark of a
transformed cell, the association of the genotype 1 with
HCV-related tumors34might be of particular interest in
the context of these results.
Several proteases, including calpains, have been re-
ported to process Bid into its active form, the tBid.35In
contrast to caspase 8, extracts from replicon-harboring
cells did not give rise to processed tBid in our experimen-
tal set-up. Moreover, in agreement with our previous re-
shRNA-mediated Bid knockdown, the presence of HCV
proteins (and in particular of NS5A) and the associated
calpain activation correlate with a decreased sensitivity to
death receptor stimulation. Importantly, whereas inhibi-
tion of calpains restores Bid activity and apoptosis sensi-
tivity in NS5A-expressing cells (Figs. 3, 6A; Supporting
Fig. 7), it has no effect on cell death when Bid is down-
regulated by shRNA. Therefore, we believe that Bid deg-
radation is a physiologically relevant consequence of
8 SIMONIN ET AL.HEPATOLOGY, Month 2009
HCV-related calpain activation. In this context, it is
worth noting that calpains 1 and 2, which have been
described as Bid activators, do not appear to be involved
in the Bid degradation initiated by HCV (Fig. 6F). Phar-
macological inhibition of calpains partially restores Bid
levels and the cell’s sensitivity to apoptotic signaling ini-
tiated by TNF-? treatment. These results strongly argue
that, in addition to the previously described effect of
NS5A on the initial steps of TNF receptor signal trans-
duction,36Bid is a physiologically relevant target of this
Although high protease activities are found in many
solid tumors,37the use of a stringent assay of calpastatin
degradation and the limiting of our analysis to HCCs
arising on noncirrhotic livers allowed us to correlate cal-
pain activation with a Bid decrease in human HCC. Al-
though more clinical samples need to be analyzed to
substantiate our interpretation, these data are consistent
with the idea that the activation of calpains by HCV and
served in several animal and cellular models, are also rel-
evant to human pathology.
If true, these results might open new perspectives for
consequences. A strong prediction of our model is that
inhibition of the virus-activated calpains should lead to
in turn would facilitate their elimination in the course of
the physiological immune response. Calpains have al-
ready been investigated as possible therapeutic targets in
human disease, mainly in the context of neurological and
muscular disorders.38,39Our data suggest that it might be
worthwhile to pursue the identification of calpain iso-
forms activated by the HCV and to search for their spe-
cific inhibitors to boost the efficiency of the immune
elimination of the infected cells.
Lemon for the gift of the FL-N/35 transgenic mice and
the N/neoC-5b and N/neo3-5b cell lines, to Dr. Takaji
Wakita for the gift of the clones of the JFH1 and
Con1/C3 viruses, and Drs. Mark Harris and Robert Lan-
ford for clones of different genotypes of NS5A. We thank
Thierry Gostan for help with the statistical analysis,
Krishna Damodar for technical assistance, Charles Bala-
baud and Jean Michel Pawlotsky for their interest and
support, and all members of the U. Hibner lab for com-
ments and discussions.
We are indebted to Stanley M.
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