From INSERM U522, Hôpital de Pontchaillou,
35033 Rennes Cedex, France and
CNRS UMR 5535, Institut de
Génétique Moléculaire,
34293 Montpellier Cedex 5, France
Received for publication, September 23, 2002, and in revised form, January 27, 2003
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Chronic hepatitis C virus (HCV) infection
frequently leads to liver cancer. To determine the viral factor(s)
potentially involved in viral persistence, we focused our work
on NS2, a viral protein of unknown function. To assign a role for NS2,
we searched for cellular proteins that interact with NS2. Performing a
two-hybrid screen on a human liver cDNA library, we found that NS2
interacted with the liver-specific pro-apoptotic CIDE-B protein.
Binding specificity of NS2 for CIDE-B was confirmed by cell-free assays associated with colocalization studies and coprecipitation experiments on human endogenous CIDE-B. CIDE-B, a member of the novel CIDE family
of apoptosis-inducing factors, has been reported to show strong cell
death-inducing activity in its C-terminal domain. We show that this
CIDE-B killing domain is involved in the NS2 interaction. NS2
binding was sufficient to inhibit CIDE-B-induced apoptosis because an
NS2 deletion mutant unable to interact with CIDE-B in vitro
lost its capacity to interfere with CIDE-B cell death activity.
Although it has been reported that CIDE-B-induced apoptosis is
characterized by mitochondrial localization, the precise apoptotic
mechanism remained unknown. Here, we show that CIDE-B induced cell
death in a caspase-dependent manner through cytochrome
c release from mitochondria. Furthermore, we found that NS2
counteracted the cytochrome c release induced by CIDE-B. In vivo, the CIDE-B protein level was extremely low in
adenovirus-infected transgenic mice expressing the HCV polyprotein
compared with that in wild-type mice. We suggest that NS2 interferes
with the CIDE-B-induced death pathway and participates in HCV
strategies to subvert host cell defense.
Of the different antiviral defense systems developed by the cell,
programmed cell death, or apoptosis, significantly contributes to the
prevention of viral replication, dissemination, and persistence (1, 2).
To survive, viruses elaborate multiple protective strategies that could
interfere at different levels of the process leading to cell death. The
complex molecular program of apoptosis may be initiated by intrinsic or
extrinsic signals. Recently, the double-stranded
RNA-dependent protein kinase PKR has been shown to play a
critical role as an intracellular stimulus in mediating double-stranded
RNA-induced apoptosis via the activation of the Fas-associated death
domain signaling cascade (3-5). This Fas-associated death
domain-mediated death signaling pathway is also activated by extrinsic
signals such as Fas ligand and tumor necrosis factor- Research on the impact of hepatitis C virus
(HCV)1 infection on host cell
apoptosis is still hampered by the lack of a reliable cell culture
system or of a small animal model that supports HCV replication and
infection (20, 21). Thus, until now, few data have been obtained on
possible strategies used by HCV to thwart apoptosis when used as an
innate cellular antiviral defense. The HCV NS5A
(nonstructural 5A) protein and the
E2 (envelope 2) glycoprotein were recently
proposed to directly interact and inhibit the enzymatic function of the
interferon-induced double-stranded RNA-dependent PKR
(22-24). This viral defense mechanism would prevent both protein synthesis inhibition, normally mediated by phosphorylation of eukaryotic initiation factor 2- Interestingly, our investigations suggest that the HCV NS2
(nonstructural 2) protein
intervenes in the viral defense system against apoptosis. Mature NS2 is
a 23-kDa hydrophobic transmembrane protein anchored to the endoplasmic
reticulum (33). It is generated by proteolytic processing of the HCV
polypeptide in the infected cell (34). Although the biological
relevance of NS2 in polypeptide processing was previously demonstrated
by a study showing that a modified HCV genome (in which mutations were
introduced into the NS2 sequence) encoded an HCV polypeptide that
abolished its infectivity in chimpanzees (35), the biological function
of the cleaved mature NS2 protein is still unknown.
In this study, we identified for the first time a cellular partner of
NS2, the CIDE-B protein, a member of the recently identified pro-apoptotic CIDE family. We show that CIDE-B mediates cytochrome c release from mitochondria and caspase 3 activity and that
NS2 acts as an inhibitor of CIDE-B-induced apoptosis. In
vivo, CIDE-B was suppressed in adenovirus-infected transgenic mice
expressing HCV proteins. We suggest that NS2 interferes with the
CIDE-B-induced death pathway and participates in HCV strategies to
subvert host cell defense.
Plasmid Constructions
Yeast Expression Vectors--
The pAS2 GST Expression Vectors--
The CIDE-B gene was
amplified by PCR from the pACT2-CIDE-B vector using a 5'-primer
containing a BglII site that anneals upstream of the
NcoI site within the pACT2 plasmid and a 3'-primer with an
EcoRI site. The digested fragment was inserted in-frame with GST into the BamHI and EcoRI sites of the pGEX4T1
plasmid (Amersham Biosciences). This cloning led to the introduction of
two additional restriction sites, NcoI and BamHI,
which conferred to pGEX4T1 cloning compatibility with the yeast
expression vectors. Thus, to generate the remaining GST vectors used in
the cell-free assay, the CIDE-B gene was replaced with
full-length NS2 or its deletion mutants or with mutant CIDE-B-(1-117)
through direct subcloning from the yeast expression vectors into our
modified pGEX4T1 vector (pGEX4T1-NB).
Mammalian Expression Vectors--
NS2, NS2-(99-217),
NS2-(140-217), or CIDE-B was subcloned into the KpnI and
EcoRI sites of the pEGFP-C3 plasmid
(Clontech), yielding N-terminally tagged green
fluorescent protein (GFP). The same sites were used to fuse NS2 to
vesicular stomatitis virus G-tagged sequence in pVM6 (Roche
Molecular Biochemicals) and CIDE-B to hemagglutinin-tagged sequence in
pHM6 (Roche Molecular Biochemicals), yielding vectors pVM6-NS2 and
pHM6-CIDE-B, respectively. The DFF45 vector was a kind gift of X. Wang
(13). The DFF45 gene was inserted in-frame with GFP,
yielding the pEGFP-DFF45 vector. The pcDNA3-FLAG vector was a kind
gift of G. Nunez (17). Full-length CIDE-B and its deletion mutants were
inserted in-frame with FLAG downstream of the T7 promoter into the
XbaI and ApaI sites of pcDNA3-FLAG. All DNA
constructs were verified by DNA sequencing (Genome Express S. A.).2
Yeast Two-hybrid System
Two-hybrid screens were performed using the Gal4BD-NS2 or
Gal4BD-NS2-(99-217) expression plasmid and the human liver cDNA library fused to Gal4AD in the pACT2 vector (HL4024AH,
Clontech) according to the manufacturer's
protocol. Interaction specificity was verified by a mating strategy as
described previously (50). Positive clones were isolated, and
interaction specificity was checked for a second time by a
cotransforming approach (52). The cDNAs of clones that were
positive in these two approaches were sequenced (Genome Express
S. A.).
Random Mutagenesis by PCR and Screening of Mutants
The cDNA corresponding to C-terminal amino acids 110-219 of
CIDE-B was amplified by error-prone PCR as described (39). Screening of
the mutant library was performed as described previously (53) using
Gal4BD-NS2-(99-217) as a bait.
Western Blot Analysis
GFP-expressing HeLa Cells--
Cells were harvested in 20 µl
of ice-cold lysis buffer (50 mM HEPES (pH 7.5), 150 mM NaCl, 1 mM EDTA, 2.5 mM EGTA,
0.1 Tween 20, 10% glycerol, 0.1 mM
Na3VO4, 1 mM NaF, and 10 mM Yeast Protein Extracts--
Cells were harvested in 400 µl of
2× Laemmli buffer (Sigma) and vortexed in the presence of 400 µl
of acid-washed glass beads (Sigma). The beads were spun down, and 20 µl of each protein sample were analyzed by Western blotting using
anti-Gal4BD antibody LK5C1 (Santa Cruz Biotechnology, Santa Cruz, CA).
All blots were treated with either horseradish peroxidase-labeled goat
anti-rabbit or anti-mouse immunoglobulin (Dako Corp., Carpinteria, CA)
and revealed using the SuperSignal kit (Pierce).
Production of Anti-human CIDE-B Antibody
A rabbit polyclonal antibody raised against a synthetic peptide
mapping the N terminus of human CIDE-B (amino acids 5-19) was
purchased from BioAtlantic (Nantes, France). The antibody was purified
using the MabTrap kit (Amersham Biosciences) according to the
manufacturer's instructions.
Binding Assays
Binding of NS2 to Recombinant CIDE-B--
GST fusion proteins
were expressed in Escherichia coli and purified as described
(54). [35S]Methionine-labeled CIDE-B and
CIDE-B-(118-219) proteins were produced from their appropriate
pcDNA3-FLAG vectors using the TNT T7 coupled
reticulocyte lysate system (Promega, Madison, WI). Two micrograms of
each purified GST protein were incubated overnight at 4 °C with 10 µl of 35S-labeled CIDE-B or CIDE-B-(118-219) in
phosphate-buffered saline containing 1 mM
phenylmethylsulfonyl fluoride and protease inhibitors. Beads were
washed five times with 10 mM Tris-HCl (pH 8.0), 1 mM EDTA, 150 mM NaCl, and 0.1% Tween 20 prior
to their loading on SDS-polyacrylamide gels, and bound labeled
materials were visualized by autoradiography.
Binding of NS2 to Human Endogenous Cellular CIDE-B--
HepG2
cells were lysed and incubated with the different purified recombinant
GST proteins as previously described (55). Human endogenous CIDE-B was
detected with purified anti-CIDE-B antibody.
Cell Lines and Transfection
HeLa and COS-7 cells and the HepG2 human hepatoma cell line were
cultured as described (52). For colocalization studies and analysis of
cytochrome c release, cells were transfected by electroporation as described (52). For apoptosis assays, cells were
transfected using the LipofectAMINE PLUS reagent kit (Invitrogen) according to the manufacturer's instructions.
Indirect Immunofluorescence
Twenty-four hours after electroporation, cells were treated as
previously described (52). For FLAG-CIDE-B staining, permeabilized cells were incubated for 1 h with anti-FLAG antibody M2 (Sigma) diluted in phosphate-buffered saline with 0.2% saponin and 1% bovine
serum albumin, washed, and then treated for 45 min with Texas
Red-conjugated anti-mouse IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA). Cells were washed, mounted on a microscope slide
with fluorescent mounting medium (Dako Corp.), and analyzed by confocal
microscopy using a Leica TCS NT instrument.
Cell Death Assay
HeLa cells (5 × 105) were spread on glass
coverslips and transfected with 4 µg of the indicated mammalian
expression vectors using the LipofectAMINE PLUS reagent kit.
Twenty-four hours later, cells were fixed, and nuclei were stained for
15 min at room temperature with 0.5 µg/ml Hoechst 33342 (Sigma) in
phosphate-buffered saline containing 0.5% saponin. Cells were washed
and covered with microscope slides with the mounting medium, and
apoptotic nuclei were identified on the basis of condensed chromatin
and nuclear fragmentation using a Leica DM RXA instrument. Optical
sections were mounted using Adobe Photoshop software.
Analysis of Mitochondrial Release of Cytochrome c
HeLa cells (2.4 × 107) were transfected with
25 µg of the indicated mammalian expression vectors. After 24 h,
mitochondria were isolated. Briefly, cells were collected, washed, and
resuspended in fractionation buffer containing 250 mM
sucrose, 20 mM Tris-HCl (pH 8.0), 1 mM EDTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, and a protease inhibitor mixture tablet. Cells were homogenized in a Dounce glass homogenizer (B pestle) and centrifuged at
1000 × g for 10 min at 4 °C to remove intact cells
and nuclei. Supernatants were centrifuged at 10,000 × g for 25 min at 4 °C to separate mitochondrial and
cytosolic fractions. The mitochondrial pellet was resuspended in 20 µl of fractionation buffer, and protein concentration were determined
by the Bradford method. A total of 30 µg of mitochondrial protein
were subjected to 15% SDS-PAGE followed by Western blotting using
anti-cytochrome c antibody H104 (Santa Cruz Biotechnology).
To immunoprecipitate cytosolic cytochrome c, 800 µg of
protein issued from each cytosolic fraction were incubated for 3 h at 4 °C with anti-cytochrome c antibody 6H2.B4
(Pharmingen) and 30 µl of protein A-Sepharose beads (Amersham
Biosciences). The beads were washed, and bound cellular proteins were
analyzed by Western blotting using anti-cytochrome c
antibody H104.
Caspase Activity Assay
COS-7 cells were transfected with 2 µg of pEGFP or
pHM6-CIDE-B vector, harvested at 24 h, and lysed in caspase
activity buffer (56). One-hundred micrograms of crude cell lysate were
incubated with 80 µM
substrate-7-amino-4-methylcoumarin for 1 h at 37 °C. Caspase-mediated cleavage of DEVD-7-amino-4-methylcoumarin was measured
by spectrofluorometry (Molecular Devices) at an excitation/emission wavelength pair of 380/440 nm. Caspase activity is indicated in Vmax values.
HCV Transgenic Mice
Transgenic mice with liver-specific expression of RNA encoding
the complete viral polyprotein were produced as described (42).
Adenoviral Infections and Mice were infected with 2.5 × 109
plaque-forming units of adenoviral Liver protein extracts were analyzed by 12% SDS-PAGE followed by
Western blotting using anti-CIDE-B antibody E-19 (Santa Cruz Biotechnology). To ensure comparable loading of the samples, the blot
was incubated with anti-annexin V antibody (kind gift of F. Russo-Marie). Specific bands were quantified by scanning the autoradiographs with a Shimadzu densitometer.
Data Analysis
Statistical analysis was performed using Student's t
test for unpaired data. Data were considered to be significant at
p < 0.05 and p < 0.01.
Identification of CIDE-B as a Cellular Partner of NS2--
Mature
HCV NS2 is a viral protein of unknown function. To elucidate its
biological role, we searched for possible cellular protein partner(s)
that directly interact with NS2 using the two-hybrid system.
Full-length NS2 was used as a bait to screen a human liver cDNA
library. Although Western blot analysis showed expression of the bait
(Fig. 1A, lane 3),
no specific interaction could be detected (data not shown). This
observation may be explained by the presence of hydrophobic and
transmembrane domains at the N terminus of NS2 (amino acids 1-98) (33,
36, 37), which might hamper the nuclear translocation of the bait, thus
preventing the detection of any interaction. Therefore, we performed a
second two-hybrid screen using an NS2 mutant with residues 1-98
deleted (NS2-(99-217)) (lane 4). A 1.2-kb cDNA encoding
human CIDE-B, a protein of 219 amino acids, could be isolated. We
further examined the interaction specificity between CIDE-B and NS2
(Fig. 1B). First of all, background binding was excluded
using empty constructs (rows 7, 9, and
10). Because CIDE-B has also been shown to homodimerize (19), this property was used to check the functional integrity of this
cloned CIDE-B (row 8). CIDE-B specifically bound to
NS2-(99-217) (row 2); but, as expected, no interaction
could be revealed when the hydrophobic N terminus was maintained in the
NS2 sequence, probably for the reasons explained above. Any unspecific
binding with CIDE-B was excluded because no interaction was detected
either between CIDE-B and an unrelated protein such as Snf1
(row 3) or between CIDE-B and other viral proteins such as
NS4A and NS3-(1-180) (rows 4 and 5,
respectively). We preferentially used the N-terminal proteinase domain
(NS3-(1-180)) because it was already identified as a valuable prey in
a two-hybrid screen carried out with NS4A as a bait (38). Finally, no
interaction was detected between NS2-(99-217) and Snf4, a non-relevant
yeast protein (row 6). Taken together, our results provide
evidence that the human CIDE-B protein is a specific cellular partner
of the viral NS2 protein.
Characterization of the NS2 Region Involved in Binding to
CIDE-B--
To define the region within NS2 involved in the
interaction with CIDE-B, we tested different truncation mutants of
NS2-(99-217) for their capacity to bind CIDE-B in the two-hybrid
system. The results shown in Fig.
2A indicate that at least a
region encompassing amino acids 135-139 of NS2 supports interaction
with CIDE-B.
The data obtained in the two-hybrid system were confirmed by a
biochemical approach (Fig. 2B). Polypeptides of full-length NS2 and CIDE-B and their deletion mutants were produced as recombinant GST fusion proteins and immobilized on glutathione-agarose beads. The
different GST proteins were tested for their ability to retain in
vitro translated 35S-labeled CIDE-B. Whereas no
background binding could be detected on the GST protein (lane
2), 35S-labeled CIDE-B was retained on GST-CIDE-B
(lane 6). This result validates the system because it
reproduced the known homodimerization of the cellular protein.
Moreover, CIDE-B with its C terminus deleted (GST-CIDE-B-(1-117)) lost
its capacity to dimerize with the wild-type form of the protein
(lane 7), a result that is thus consistent with the
observation that the C-terminal domain is required for CIDE-B
dimerization (19). Interestingly, this cell-free methodology allowed us
to highlight that both the truncated (NS2-(99-217)) (lane
4) and full-length (lane 3) NS2 proteins have the
identical property of binding to CIDE-B. These data support our
hypothesis given above that the lack of interaction observed in the
two-hybrid screen with wild-type NS2 is not due to the inability of the
viral protein to bind to CIDE-B, but instead to the presence of its hydrophobic domains which are unsuited for this methodology.
Furthermore, GST-NS2-(140-217) did not retain labeled CIDE-B
(lane 5), consistent with our results obtained in the
two-hybrid experiment.
The CIDE-B C-terminal Domain Is Required for Both Homodimerization
and NS2 Binding--
To determine the domain in CIDE-B required for
NS2 binding, we generated two deletion mutants: (i) CIDE-B-(1-117),
which contains the CIDE-N domain sharing homology with the N-terminal
regions of DFF40 and DFF45, and (ii) CIDE-B-(118-219), which comprises the CIDE-C domain sufficient for CIDE-B dimerization and death activity
(Fig. 3A) (17). Both deletion
mutants were fused to Gal4AD and analyzed for their binding capacity
with Gal4BD-CIDE-B and Gal4BD-NS2-(99-217) in the AH109 yeast strain.
Gal4BD-NS2-(140-217) was added as a negative control (Fig.
3B, row 4). CIDE-B homodimerization, highlighted
by the interaction between the full-length proteins (row 5),
was maintained only with the deletion form containing the C-terminal
domain of the protein (rows 6 and 7). This domain was revealed to be also involved in the binding to NS2-(99-217) because its presence was required for the interaction with the viral
protein (rows 1-3).
We attempted to corroborate these findings with the cell-free binding
assay, which also permitted testing of full-length NS2. The retention
of in vitro translated 35S-labeled
CIDE-B-(118-219) was tested against different recombinant GST proteins
(Fig. 3C). CIDE-B-(118-219) was adsorbed only on beads
displaying either full-length CIDE-B (lane 6) or NS2
containing residues 99-139 (lanes 3 and 4),
whereas no interaction was revealed for GST, GST-NS2-(140-217), or
GST-CIDE-B-(1-117) (lanes 2, 5, and
7, respectively).
To identify precisely the residues involved in NS2 binding, we randomly
mutagenized CIDE-B C-terminal amino acids 110-219 by error-prone PCR
(39), and we searched for mutants unable to interact with NS2-(99-217)
in the two-hybrid system. We isolated a CIDE-B C-terminal mutant whose
interaction with NS2-(99-217) was impaired by a single point mutation
(Fig. 3D, row 2). This mutant contains
phenylalanine instead of tyrosine at position 160 (Y160F).
Interestingly, this CIDE-B mutant also failed to interact with native
CIDE-B (row 4), illustrating that CIDE-B homodimerization is
impaired in yeast cells. Taken together, these results indicate that
the CIDE-B C-terminal domain seems to be sufficient for both CIDE-B
dimerization and NS2 interaction.
NS2 Associates with CIDE-B in the Cell--
We further
investigated the subcellular localization of NS2 and CIDE-B by
immunofluorescence staining and confocal microscopy analysis in
mammalian cells. NS2 and its deletion mutants were expressed as GFP
chimeras, whereas wild-type and truncated CIDE-B proteins were
FLAG-tagged at their N termini. Cells were cotransfected with the
different plasmids as indicated in Fig.
4. After 24 h, GFPs were revealed by
direct GFP fluorescence, and the cellular distribution of CIDE-B was
examined by staining with an antibody directed against the FLAG
epitope.
As previously described (19), FLAG-CIDE-B showed a punctate
signal, with the presence of discrete dots in a perinuclear region
(Fig. 4, panels b, e, and h). We
detected full-length NS2 fused to GFP in the perinuclear space (see
Fig. 5B, panel b), as reported by Kim et
al. (40). Coexpression of CIDE-B and the NS2 chimera revealed
partial overlapping staining signals in the perinuclear region,
indicating that fractions of both proteins were located in the same
area (Fig. 4A, panel c). Interestingly, the
recombinant GFP-NS2-(99-217) protein, corresponding to the deletion
mutant used as a bait protein in the two-hybrid screen, showed a
localization pattern similar to that of the full-length protein
(panels a and d) and still colocalized with
CIDE-B (panel f). In contrast, the subcellular localization
of the GFP-NS2-(140-217) chimera, comprising the deletion mutant
deficient for CIDE-B in vitro binding, lost the dense
localization of GFP-NS2 and GFP-NS2-(99-217). GFP-NS2-(140-217) was
mostly found in the nucleus, and a weak diffuse signal was also
detected in the cytoplasm (panels g and i).
Notwithstanding, the subcellular distribution of FLAG-CIDE-B was
maintained in part of the perinuclear space (panel h). The deletion mutant FLAG-CIDE-B-(1-117), defective for both dimerization and NS2 binding, showed diffuse cytoplasmic staining, with a
significant fraction accumulating in the nucleus (panel k).
Therefore, no overlapping staining signal with GFP-NS2 was observed
(panel l).
Because we could not exclude in these experiments that the observed
colocalization patterns were due to the ability of two overexpressed
proteins to form aggregates in the cell, we further investigated the
interaction of NS2 with endogenous CIDE-B. For this purpose, we
developed a novel antibody raised against a synthetic peptide mapping
the N terminus of human CIDE-B (amino acids 5-19). As expected, this
antibody recognized purified GST-CIDE-B-(1-117), as demonstrated in
Fig. 4B (lane 1). To determine whether NS2 associates with endogenous CIDE-B, we incubated HepG2 lysates with
purified GST, GST-NS2, or GST-NS2-(140-217) immobilized on glutathione-agarose beads. Bound proteins were analyzed by Western blotting with anti-CIDE-B antibody. Endogenous CIDE-B bound to GST-NS2
(lane 4), but not to GST (lane 3) or
GST-NS2-(140-217) (lane 5), the deletion mutant deficient
for CIDE-B interaction in the two-hybrid system (Fig.
2B).
NS2 Inhibits CIDE-B-induced Apoptosis--
Because CIDE-B is
involved in apoptosis, it was important to study whether NS2
binding to CIDE-B could interfere with the death signal mediated by
CIDE-B. We analyzed the apoptotic state of cells transfected with
GFP-CIDE-B along with either a non-relevant protein ( NS2 Interferes with the Mitochondrial Cytochrome c Release Induced
by CIDE-B--
Although it was reported that mitochondrial
localization and dimerization are required for CIDE-B to induce cell
death (19), the precise apoptotic mechanism remained unknown. We
wondered whether CIDE-B might be involved in the mitochondrial
death pathway and thus might induce apoptosis in a
caspase-dependent manner (for review, see Ref. 41). We
first studied whether CIDE-B induces caspase-3 activity. Fig.
6A shows that the
Vmax values of CIDE-B-expressing cells were
3-fold higher than those of GFP-expressing control cells, indicating
enzymatic activity of caspase-3 in the presence of CIDE-B.
Because caspase-3 activity can be the result of cytochrome c
release from mitochondria during apoptosis (for review, Ref. 41), we
further wondered whether CIDE-B could be associated with the cytochrome
c release. For this investigation, we prepared mitochondrial
and cytosolic fractions from HeLa cells transfected with either an
empty plasmid or a recombinant plasmid expressing CIDE-B. Cytochrome
c was directly detected by Western blotting in the
mitochondrial fraction or was first immunoprecipitated from the
cytosolic fraction. Obviously, as shown in Fig. 6B, CIDE-B induced cytochrome c release as shown by the decreased
amount of cytochrome c detected in the mitochondrial
fraction and its increased level observed in the cytosolic fraction of
the CIDE-B-expressing cells compared with the control cells. These
results demonstrate that CIDE-B-induced apoptosis is accompanied by
cytochrome c release.
We further wondered whether NS2 counteracts the cytochrome c
release induced by CIDE-B to protect cells from apoptosis. To answer this question, we assessed the mitochondrial cytochrome c levels in HeLa cells transiently transfected with
FLAG-tagged CIDE-B or vesicular stomatitis virus G-tagged NS2 or
cotransfected with both proteins (Fig. 6C). The signal
revealed in cells expressing vesicular stomatitis virus G-tagged NS2
(lane 4) was comparable to that obtained in the control
cells (lane 1), whereas only a weak band was detected in
mitochondria isolated from FLAG-CIDE-B-expressing cells (lane
2). When CIDE-B and NS2 were coexpressed (lane 3), cytochrome c release was partly avoided because the signal
intensity was intermediate to those detected in the control (lane
1) and CIDE-B-expressing (lane 2) cells. This result
suggests that NS2 may protect cells from the cytochrome c
release induced by the apoptotic CIDE-B protein.
CIDE-B Expression Is Sensitive to Viral Infection, and Its Protein
Content Is Altered in HCV Transgenic Mice--
To examine whether
CIDE-B expression is sensitive to HCV proteins in vivo, we
used a previously described HCV transgenic mice model (42) that permits
the study of NS2 activity within the context of the entire viral
polyprotein rather than in isolation. Indeed, interactions between
viral proteins can significantly modulate their effects in
vivo (42) and thus explain the conflicting results obtained in
transgenic mice expressing single HCV proteins (43).
We first analyzed the CIDE-B protein content in liver tissues from
wild-type and HCV transgenic mice by Western blotting. Fig.
7 (A and B) shows
that the CIDE-B protein content was similar between HCV transgenic and
wild-type mice. Because HCV proteins in the transgenic mice are not
recognized as foreign proteins inducing an immune response, we decided
to analyze the CIDE-B protein content in the context of viral
infection. We compared the CIDE-B protein content in liver extracts
from non-transgenic and HCV transgenic mice, both infected with an
adenovirus. As shown in Fig. 7 (C and D), the
amount of CIDE-B in the livers of adenovirus-infected wild-type mice
was reduced to 35% of that in the livers of normal mice.
Interestingly, when the HCV transgenic mice were infected with an
adenovirus, the CIDE-B protein level was dramatically lowered to 1% of
that in wild-type mice.
Chronic HCV infection frequently leads to liver cancer. Because
the liver is the major HCV replication site, HCV seems to have
developed strategies to promote its survival in this organ. This study
points out the interaction between the viral NS2 protein and
liver-specific CIDE-B, a cellular protein involved in apoptosis, as a mechanism contributing to the prevention of apoptosis. The interaction of NS2 with CIDE-B was evidenced by yeast two-hybrid and
cell-free assays associated with colocalization studies and coprecipitation experiments of human endogenous CIDE-B. The NS2-binding site was determined to be the CIDE-B cell death domain. This
binding is sufficient to inhibit CIDE-B-induced apoptosis because an
NS2 deletion mutant unable to interact with CIDE-B in vitro
lost its capacity to interfere with CIDE-B cell death activity.
Additional interest is provided by the binding specificity for CIDE-B.
Indeed, CIDE-A, the second member of the CIDE protein family, which
presents a widespread tissue distribution except in the liver (17),
does not bind to NS2 (data not shown).
The pro-apoptotic CIDE-B protein can be divided into two distinct
domains: the N-terminal (CIDE-N) and C-terminal (CIDE-C) domains. The
CIDE-N domain shares homology with the N-terminal regions of DFF40 and
DFF45, a nuclease and its inhibitor, respectively. The CIDE-B
C-terminal region is necessary and sufficient for the intrinsic
apoptotic activity of CIDE proteins (17). Interestingly, we determined
that the interaction of NS2 takes place within the CIDE-B C-terminal
domain, whereas this domain is also involved in the
homodimerization of CIDE-B (this study and Ref. 19). In addition,
tyrosine 160 in the CIDE-B C-terminal domain is necessary for both
CIDE-B dimerization and NS2 interaction, suggesting a putative binding
competition between NS2 and CIDE-B for CIDE-B itself. Because
homodimerization is required for CIDE-B cell death activity, we suggest
that NS2 binding might affect CIDE-B-induced apoptosis by interfering
with its dimerization.
Although apoptosis induced by the CIDE proteins has been characterized
by DNA fragmentation and counting of adherent cells undergoing
apoptosis (17), the molecular mechanism supporting this biological
effect has not been completely elucidated. The requirement of CIDE-B
homodimerization and its mitochondrial localization for cell death
induction has been recently reported (19). Here, we analyzed the effect
of CIDE-B overexpression on the mitochondrial death pathway. We have
shown that CIDE-B induces cytochrome c release from the
mitochondria. Given that cytochrome c release from
mitochondria during apoptosis is associated with the delivery of
caspase-activating molecules (for review, see Ref. 41), we further
studied the influence of CIDE-B on caspase activity. We have shown that
CIDE-B induces caspase-3 activity. Taken together, our results favor
the idea that CIDE-B induces apoptosis through the cytochrome
c/caspase-9/caspase-3 pathway.
We further demonstrated that NS2 protects cells from the cytochrome
c release induced by CIDE-B. Indeed, the cytochrome
c level remaining in the mitochondrial fraction of cells
coexpressing NS2 and CIDE-B was higher than that in the mitochondrial
fraction of cells expressing CIDE-B alone. Interestingly, experiments
performed with HCV transgenic mice expressing the core, E1, E2, and NS2 proteins in the liver showed that Fas-mediated apoptosis is blocked at
the mitochondrial level as illustrated by the inhibition of cytochrome
c release (31). We therefore favor the idea that NS2 may
contribute to this previously observed anti-apoptotic effect.
Until now, CIDE-B-induced apoptosis has been exclusively demonstrated
ex vivo by overexpression of the protein in different established cell lines (17, 19). In this study, we have shown, in
vivo, that the amount of CIDE-B in normal mice was lowered (3-fold) after viral infection. This result is consistent with the idea
that the suppression of pro-apoptotic CIDE-B may cause a defective
apoptotic system and thus may be a general mechanism of viral evasion
from the host cell defense. It is noteworthy that the effect of viral
infection on the reduction of CIDE-B levels was potentiated
(35-fold) in transgenic mice expressing HCV proteins. This result is in
accordance with the idea that NS2 may be involved in the dramatic
CIDE-B protein inhibition, although we cannot exclude that other viral
proteins might also be involved in this phenomenon.
Altogether, these observations raise the question of the molecular
mechanism by which NS2, a transmembrane protein anchored to the
endoplasmic reticulum (33), may bind to and inhibit the death activity
of a cytosolic CIDE-B protein, which translocates to mitochondria to
fulfill its killing activity. Given that the exact transmembrane
topology of NS2 is still not elucidated (33, 37, 44, 45), we suggest
that the NS2 C-terminal region harboring the identified CIDE-B-binding
site has to be directed to the cytosol. Whether NS2 may trap CIDE-B in
the endoplasmic reticulum via binding to its C-terminal domain and thus
hindering its mitochondrial localization required for its apoptotic
activity or whether NS2 can interfere with CIDE-B dimerization at the
mitochondrial site remains to be determined. The latter hypothesis
implies that at least some NS2 proteins may also have a cytosolic
localization. Further investigations are needed to confirm these hypotheses.
Several interactions between different HCV proteins and cellular
partners implicated in apoptosis has already been documented, e.g. the core protein and the complement receptor gC1qR
(46), the core protein and members of the tumor necrosis factor
receptor family (47, 48), E2 and PKR (24, 49), and NS5A and PKR (22,
23). Whereas all these interactions concern nonspecific hepatic
proteins involved in apoptotic mechanisms, our study describes the
relation between the NS2 protein of a hepatotropic virus and a
liver-specific cellular protein, CIDE-B. We therefore propose that this
interaction between NS2 and CIDE-B may be one mechanism that
contributes to the viral persistence observed in HCV pathogenesis.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(6-8). The
binding of Fas ligand to its Fas/APO-1/CD95 cell-surface death receptor
triggers the activation of caspase-8, which thereupon can directly
cleave procaspase-3 in its activated form (9). Alternatively, caspase-8
is also able to activate Bid, a pro-apoptotic member of the Bcl-2
family that triggers the release of cytochrome c from
mitochondria (10, 11). Cytosolic cytochrome c forms a
complex with Apaf-1 and activates caspase-9, the substrate of which is
procaspase-3 (12). Then, the activated caspase-3 targets the
DNA fragmentation factor, a
heterodimeric protein complex consisting of the inhibitory chaperone
protein DFF45 and the DFF40 nuclease (13). Cleavage of DFF45 by the protease results in the release of DFF40 from its inhibited state and
allows nucleosomal DNA degradation, which is the ultimate stage of
apoptosis (13-16). Both proteins of the DNA fragmentation factor
complex share a homologous region at their N terminus called the CIDE-N
(cell death-inducing DFF45-like
effector) domain. This peculiar region, involved in
the interaction between DFF40 and DFF45, is also found in the novel
CIDE family of cell death activators (17). This common sequence
explains the inhibitory control exerted by DFF45 on CIDE proteins,
probably via homophilic interaction between their respective CIDE-N
domains (17, 18). Proteins of this CIDE family differ from DFF40 by
their specific C-terminal domain, called CIDE-C, which is responsible
for their cell death property, which requires homodimerization and
mitochondrial localization to fulfill their killing activity (17,
19).
(25), and apoptosis induced through the Fas-associated death domain-mediated death signaling pathway (3-5). The HCV core protein has been reported also to interfere with the Fas-mediated death signaling pathway. However, its
role as an apoptosis enhancer or inhibitor is still in debate (26-32).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-NS3 and
pAS2
-NS4 vectors were a kind gift of P. Legrain (38, 50). The
cloned HCV-H genome was provided by G. Inchauspé (51). The
NS2 gene was amplified from the HCV-H genome by PCR and
subcloned into the NcoI and BamHI sites of
pAS2
(50). Full-length CIDE-B and deletion mutants of NS2, NS3,
and CIDE-B were amplified by PCR and inserted into the NcoI
and BamHI sites of vectors pAS2
and pACT2
(Clontech, CA).
-glycerophosphate with freshly added 0.1 mM phenylmethylsulfonyl fluoride, 10 mM dithiothreitol, and a protease inhibitor mixture tablet (Roche Molecular Biochemicals)). After 15 min, lysates were sonicated. Protein
concentration was determined by the Bradford method (Bio-Rad), and 30 µg were subjected to 12.5% SDS-PAGE followed by Western blotting
using anti-GFP antibody (Clontech).
-Galactosidase Assays
-galactosidase (Rous
sarcoma virus-
-galactosidase) (57) by tail intravenous injections.
Liver biopsies were collected 21 days post-infection. Crude protein
extract from 0.5 µg of liver powder was tested for
-galactosidase
activity using the luminescent
-galactosidase assay
(Clontech) according to the manufacturer's instructions.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (36K):
[in a new window]
Fig. 1.
Specific interaction of the HCV NS2 C
terminus with the human CIDE-B protein in the two-hybrid system.
A, shown are the results from Western blot analysis of the
different bait proteins. Yeast extracts were prepared from the AH109
yeast strain transformed with each of the indicated Gal4BD
hybrid proteins (lanes 2-4) and analyzed using anti-Gal4BD
antibody. Lane 1 contains non-transfected
(N.T.) cells. B, the AH109 reporter yeast strain
expressing the pairs of indicated hybrid proteins fused to Gal4BD and
Gal4AD was analyzed for histidine auxotrophy. AH109 double
transformants were patched on medium containing histidine
(+His) and then replica-plated on histidine-free medium
( His). Yeast growth in the absence of histidine indicates
interaction between hybrid proteins. For the mutant NS2-(99-217), the
hydrophobic region at the N terminus of NS2 from amino acids 1-98 was
omitted (corresponding to amino acids 810-908 in the HCV polypeptide).
Binding specificity was assessed using different yeast (SNF1 and SNF4)
and viral (NS4A and NS3-(1-180)) proteins (rows
3-6). The known homodimerization of human CIDE-B was used as a
positive binding control (lane 8).
View larger version (27K):
[in a new window]
Fig. 2.
Characterization of NS2 binding to human
CIDE-B. A, mapping of the NS2 domain interacting with
CIDE-B in the two-hybrid assay. The AH109 yeast strain was
cotransformed with Gal4AD-CIDE-B and each Gal4BD-NS2 deletion mutant.
The N- and C-terminal extremities of each NS2 deletion mutant are
indicated by numbers corresponding to the amino acid
positions in the NS2 sequence. AH109 double transformants were analyzed
for histidine auxotrophy. A plus sign indicates
transactivation of the reporter gene (HIS3). B,
interaction of full-length NS2 and its mutants expressed as GST fusion
proteins with the CIDE-B protein in a cell-free assay. In
vitro translated 35S-labeled CIDE-B was incubated with
equal amounts of purified GST (lane 2), GST-NS2
(full-length) (lane 3) or GST-fused truncated recombinant
NS2 proteins (lanes 4 and 5), previously
immobilized on glutathione-agarose beads. Bound labeled material was
analyzed by SDS-PAGE and autoradiography. Immobilized GST-fused
full-length (lane 6) and truncated (lane 7)
CIDE-B proteins were used as binding controls. One-fifth of the
35S-labeled CIDE-B input for the binding assay was run in
lane 1.
View larger version (32K):
[in a new window]
Fig. 3.
Identification of the NS2-binding domain in
the human CIDE-B protein. A, schematic representation
of the CIDE-B protein and its deletion mutants. The CIDE-N and CIDE-C
domains are represented as gray and black boxes,
respectively. B, mapping of the CIDE-B domain interacting
with the NS2 C terminus in the two-hybrid assay. The AH109 reporter
yeast strain expressing the pairs of indicated hybrid proteins fused to
Gal4BD and Gal4AD was analyzed as described in the legend to Fig.
1A. The AH109 yeast strain cotransfected with Gal4BD-CIDE-B
and each of the indicated Gal4AD hybrid proteins (rows 5-7)
was used as binding controls. C, binding of
CIDE-B-(118-219) to GST-fused full-length and truncated NS2 proteins
in a cell-free assay. In vitro translated
35S-labeled CIDE-B-(118-219) (lanes 2-7) was
incubated with equal amounts of the different purified GST fusion
proteins as described in the legend to Fig. 2B. Bound
labeled material was analyzed by SDS-PAGE and autoradiography.
Immobilized GST-fused full-length (lane 6) and truncated
(lane 7) CIDE-B proteins were used as binding controls.
One-fifth of the 35S-labeled CIDE-B-(118-219) input for
the binding assay was run in lane 1. D,
identification of a CIDE-B C-terminal mutant deficient for NS2
interaction. Shown is a schematic representation of the
CIDE-B-(110-219)Y160F mutant. The CIDE-B C-terminal cDNA was
randomly mutagenized by error-prone PCR, and the mutants unable to
interact with NS2-(99-217) were analyzed by DNA sequencing. The
interaction of the CIDE-B-(110-219)Y160F mutant with NS2 and CIDE-B
was analyzed as described for B.
View larger version (27K):
[in a new window]
Fig. 4.
NS2 associates with CIDE-B in the cell.
A, HeLa cells were cotransfected with 12 µg of
pcDNA3-FLAG-CIDE-B or pcDNA3- FLAG-CIDE-B-(1-117) along with
12 µg of the different GFP vectors (panels a,
d, g, and j). After 24 h, cells
were fixed, permeabilized, treated with anti-FLAG monoclonal antibody,
and stained with a Texas Red-conjugated Fab fragment recognizing mouse
IgG. Subcellular localization of GFP proteins (panels a,
d, g, and j) and FLAG-tagged proteins
(panels b, e, h, and k) was
analyzed by confocal microscopy, and colocalization was visualized by
overlay (panels c, f, i, and
l). B, shown is the binding of NS2 to
endogenous CIDE-B. HepG2 lysates were incubated with equal amounts of
purified GST (lane 3), GST-NS2 (lane 4), and
GST-NS2-(140-217) (lane 5). Bound proteins were resolved by
SDS-PAGE, and CIDE-B association was analyzed by Western blotting.
Purified GST-CIDE-B-(1-117) (lane 1) and crude cell lysate
(lane 2) were used as controls for specific anti-CIDE-B
antibody recognition. Scale bar, 10 µm.
-galactosidase)
or GFP-fused full-length or truncated NS2 (Fig.
5A). The morphological
features of apoptosis in GFP-positive adherent cells were evaluated
after nuclear Hoechst staining. The percentage of apoptotic cells
(identified by condensed nuclei and fragmented chromatin) was
calculated against the total number of GFP-positive cells. Typical
apoptotic and live cells are shown in Fig. 5B
(panels a and b, respectively). Whereas GFP alone
had only a weak apoptotic background activity (15%) (Fig.
5A), GFP-CIDE-B induced cell death in ~60% of transfected
cells, a level previously observed by others (19). As a control, we
used GFP-fused DFF45, which is known to be a specific cellular
inhibitor of CIDE-B (17). DFF45, NS2, NS2-(99-217), and
NS2-(140-217), each fused to GFP and transfected individually,
generated an apoptotic background comparable to that obtained with GFP
alone. Interestingly, cells coexpressing GFP-CIDE-B and GFP-NS2 or
GFP-NS2-(99-217) were partially protected from CIDE-B-induced
apoptosis (~30%) at a level similar to that observed for cells
coexpressing GFP-CIDE-B and GFP-DFF45. By contrast, cells coexpressing
GFP-NS2-(140-217), which was demonstrated to be deficient for CIDE-B
in vitro binding, did not affect CIDE-B-induced apoptosis.
The correct expression of all GFP chimeras used was verified by Western
blot analysis, excluding that the absence of inhibition observed for
GFP-NS2-(140-217) was due to a lack of protein expression (Fig.
5C). These results suggest a striking correlation between
the ability of NS2 and its deletion mutants to bind to CIDE-B and to
inhibit CIDE-B-induced apoptosis, suggesting that the interaction of
NS2 with CIDE-B interferes with the death signal mediated by CIDE-B.
View larger version (28K):
[in a new window]
Fig. 5.
The NS2 protein inhibits CIDE-B-induced
apoptosis. A, quantification of apoptotic GFP-positive
adherent cells. HeLa cells were cotransfected with the indicated
amounts (0 or 2 µg) of different mammalian expression vectors. After
24 h, cells were stained with Hoechst 33342 and observed under a
fluorescence microscope. Apoptotic GFP-expressing cells with condensed
nuclei and fragmented chromatin were counted, and the percentage of
cell death was calculated against the total number of GFP-positive
cells (n = 150). Error bars represent
means ± S.D. of three independent experiments. B,
nuclear morphological analysis of GFP-positive adherent cells. HeLa
cells were treated as described for A. The nuclear
morphology of non-transfected non-apoptotic cells is indicated
by white arrows. Scale bar = 10 µm
(magnification × 800). C, Western blot analysis.
Lysates from cells expressing the indicated GFP fusion proteins were
analyzed using anti-GFP antibody. -GAL,
-galactosidase; N.T., non-transfected cells.
View larger version (30K):
[in a new window]
Fig. 6.
NS2 prevents CIDE-B-induced apoptosis in
mitochondria. A, CIDE-B induces caspase activity. Cells
were transfected with 2 µg of pEGFP or pHM6-CIDE-B vectors.
After 24 h, cell lysates were assayed for caspase-3 activity.
Enzymatic activity is indicated in Vmax values.
B, CIDE-B induces cytochrome c release from
mitochondria. HeLa cells were transfected with 20 µg of pcDNA3
(control) or pcDNA3-FLAG-CIDE-B vector. After 24 h, cells were
homogenized, and mitochondrial and cytosolic fractions were prepared.
Thirty micrograms of each mitochondrial fraction were subjected to
Western blotting using anti-cytochrome c antibody H104
(upper panel). Cytochrome c released in cytosolic
fractions was immunoprecipitated with equal amounts of anti-cytochrome
c antibody 6H2.B4 prior to Western blot analysis
(middle panel). Immunoprecipitation (IP) was
standardized by the detection of the heavy and light chains of the
anti-cytochrome c antibody used (lower panel).
C, NS2 inhibits CIDE-B-induced cytochrome c
release from mitochondria. Cells were cotransfected with 5 µg of
pcDNA3 or pcDNA3-FLAG-CIDE-B along with 20 µg of pVM6-NS2 or
pcDNA3 vector. Equal amounts of mitochondrial fractions were
analyzed by Western blotting. Control, mitochondrial
fraction of non-transfected cells.
View larger version (27K):
[in a new window]
Fig. 7.
CIDE-B protein content in the livers of HCV
transgenic mice compared with wild-type mice without and with
adenoviral infection. A, Western blot analysis was
performed with 45 µg of liver extract from animals using anti-CIDE-B
and anti-annexin V antibodies. B, quantitated results are
expressed as means ± S.D. of five HCV transgenic and five
wild-type (wt) mice. The values obtained with the HCV
transgenic mice are not statistically different from those obtained
with the wild-type mice (p = 0.8864). C,
Western blot analysis was performed as described for A. D, quantitated results are expressed as means ± S.D.
of five wild-type mice, and five adenovirus-infected wild-type mice,
and five adenovirus-infected HCV transgenic mice. The values obtained
with the virus-infected wild-type mice are statistically different from
those obtained with the wild-type mice (p = 0.0041) and
the virus-infected HCV transgenic mice (p = 0.0132). *,
p < 0.05; **, p < 0.01.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Roselyne Primault for confocal microscopy analysis and Virginie Vallet-Erdtmann for critical reading of the manuscript. We thank P. Legrain, G. Inchauspé, F. Russo-Marie, G. Nunez, and X. Wang for the kind gifts of reagents.
![]() |
FOOTNOTES |
---|
* This work was supported in part by INSERM and grants from the Ligue Nationale contre le Cancer (Comités d'Ille et Vilaine and Côte d'Armor).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Postdoctoral Fellow of the Association pour la Recherche sur le Cancer. To whom correspondence should be addressed: INSERM U522, Hôpital de Pontchaillou, 2, rue Henri le Guilloux, 35033 Rennes Cedex, France. Tel.: 33-2-9954-3737; Fax: 33-2-9954-0137; E-mail: lars.erdtmann@univ-rennes1.fr.
¶ Supported by a scholarship from the Ministry of Culture, National Education, and Research of Luxembourg (reference number 01/20).
Published, JBC Papers in Press, February 20, 2003, DOI 10.1074/jbc.M209732200
2 Detailed information about the primers used to amplify the different coding sequences are available upon request.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: HCV, hepatitis C virus; GST, glutathione S-transferase; GFP, green fluorescent protein; Gal4BD, Gal4 DNA-binding domain; Gal4AD, Gal4 activation domain.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. |
O'Brien, V.
(1998)
J. Gen. Virol.
79,
1833-1845 |
2. | Everett, H., and McFadden, G. (1999) Trends Microbiol. 7, 160-165[CrossRef][Medline] [Order article via Infotrieve] |
3. |
Balachandran, S.,
Kim, C. N.,
Yeh, W. C.,
Mak, T. W.,
Bhalla, K.,
and Barber, G. N.
(1998)
EMBO J.
17,
6888-6902 |
4. |
Balachandran, S.,
Roberts, P. C.,
Kipperman, T.,
Bhalla, K. N.,
Compans, R. W.,
Archer, D. R.,
and Barber, G. N.
(2000)
J. Virol.
74,
1513-1523 |
5. | Gil, J., and Esteban, M. (2000) Apoptosis 5, 107-114[CrossRef][Medline] [Order article via Infotrieve] |
6. | Hiramatsu, N., Hayashi, N., Katayama, K., Mochizuki, K., Kawanishi, Y., Kasahara, A., Fusamoto, H., and Kamada, T. (1994) Hepatology 19, 1354-1359[Medline] [Order article via Infotrieve] |
7. | Seino, K., Kayagaki, N., Takeda, K., Fukao, K., Okumura, K., and Yagita, H. (1997) Gastroenterology 113, 1315-1322[Medline] [Order article via Infotrieve] |
8. | Kondo, T., Suda, T., Fukuyama, H., Adachi, M., and Nagata, S. (1997) Nat. Med. 3, 409-413[Medline] [Order article via Infotrieve] |
9. | Nagata, S. (1997) Cell 88, 355-365[Medline] [Order article via Infotrieve] |
10. | Li, H., Zhu, H., Xu, C. J., and Yuan, J. (1998) Cell 94, 491-501[Medline] [Order article via Infotrieve] |
11. |
Salvesen, G. S.,
and Dixit, V. M.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
10964-10967 |
12. | Li, P., Nijhawan, D., Budihardjo, I., Srinivasula, S. M., Ahmad, M., Alnemri, E. S., and Wang, X. (1997) Cell 91, 479-489[Medline] [Order article via Infotrieve] |
13. | Liu, X., Zou, H., Slaughter, C., and Wang, X. (1997) Cell 89, 175-184[Medline] [Order article via Infotrieve] |
14. | Enari, M., Sakahira, H., Yokoyama, H., Okawa, K., Iwamatsu, A., and Nagata, S. (1998) Nature 391, 43-50[CrossRef][Medline] [Order article via Infotrieve] |
15. | Sakahira, H., Enari, M., and Nagata, S. (1998) Nature 391, 96-99[CrossRef][Medline] [Order article via Infotrieve] |
16. | Sakahira, H., Takemura, Y., and Nagata, S. (2001) Arch. Biochem. Biophys. 388, 91-99[CrossRef][Medline] [Order article via Infotrieve] |
17. |
Inohara, N.,
Koseki, T.,
Chen, S.,
Wu, X.,
and Nunez, G.
(1998)
EMBO J.
17,
2526-2533 |
18. | Lugovskoy, A. A., Zhou, P., Chou, J. J., McCarty, J. S., Li, P., and Wagner, G. (1999) Cell 99, 747-755[Medline] [Order article via Infotrieve] |
19. |
Chen, Z.,
Guo, K.,
Toh, S. Y.,
Zhou, Z.,
and Li, P.
(2000)
J. Biol. Chem.
275,
22619-22622 |
20. |
Lohmann, V.,
Korner, F.,
Koch, J.,
Herian, U.,
Theilmann, L.,
and Bartenschlager, R.
(1999)
Science
285,
110-113 |
21. |
Bukh, J.,
Pietschmann, T.,
Lohmann, V.,
Krieger, N.,
Faulk, K.,
Engle, R. E.,
Govindarajan, S.,
Shapiro, M.,
St. Claire, M.,
and Bartenschlager, R.
(2002)
Proc. Natl. Acad. Sci. U. S. A.
99,
14416-14421 |
22. | Gale, M. J., Jr., Korth, M. J., Tang, N. M., Tan, S. L., Hopkins, D. A., Dever, T. E., Polyak, S. J., Gretch, D. R., and Katze, M. G. (1997) Virology 230, 217-227[CrossRef][Medline] [Order article via Infotrieve] |
23. |
Gale, M., Jr.,
Blakely, C. M.,
Kwieciszewski, B.,
Tan, S. L.,
Dossett, M.,
Tang, N. M.,
Korth, M. J.,
Polyak, S. J.,
Gretch, D. R.,
and Katze, M. G.
(1998)
Mol. Cell. Biol.
18,
5208-5218 |
24. |
Taylor, D. R.,
Shi, S. T.,
Romano, P. R.,
Barber, G. N.,
and Lai, M. M.
(1999)
Science
285,
107-110 |
25. | Levin, D., and London, I. M. (1978) Proc. Natl. Acad. Sci. U. S. A. 75, 1121-1125[Abstract] |
26. | Ruggieri, A., Harada, T., Matsuura, Y., and Miyamura, T. (1997) Virology 229, 68-76[CrossRef][Medline] [Order article via Infotrieve] |
27. | Dumoulin, F. L., van dem Bussche, A., Sohne, J., Sauerbruch, T., and Spengler, U. (1999) Eur. J. Clin. Invest. 29, 940-946[CrossRef][Medline] [Order article via Infotrieve] |
28. | Honda, A., Arai, Y., Hirota, N., Sato, T., Ikegaki, J., Koizumi, T., Hatano, M., Kohara, M., Moriyama, T., Imawari, M., Shimotohno, K., and Tokuhisa, T. (1999) J. Med. Virol. 59, 281-289[CrossRef][Medline] [Order article via Infotrieve] |
29. | Hahn, C. S., Cho, Y. G., Kang, B. S., Lester, I. M., and Hahn, Y. S. (2000) Virology 276, 127-137[CrossRef][Medline] [Order article via Infotrieve] |
30. | Honda, A., Hatano, M., Kohara, M., Arai, Y., Hartatik, T., Moriyama, T., Imawari, M., Koike, K., Yokosuka, O., Shimotohno, K., and Tokuhisa, T. (2000) J. Hepatol. 33, 440-447[CrossRef][Medline] [Order article via Infotrieve] |
31. |
Machida, K.,
Tsukiyama-Kohara, K.,
Seike, E.,
Tone, S.,
Shibasaki, F.,
Shimizu, M.,
Takahashi, H.,
Hayashi, Y.,
Funata, N.,
Taya, C.,
Yonekawa, H.,
and Kohara, M.
(2001)
J. Biol. Chem.
276,
12140-12146 |
32. | Kalkeri, G., Khalap, N., Garry, R. F., Fermin, C. D., and Dash, S. (2001) Virology 282, 26-37[CrossRef][Medline] [Order article via Infotrieve] |
33. | Santolini, E., Pacini, L., Fipaldini, C., Migliaccio, G., and Monica, N. (1995) J. Virol. 69, 7461-7471[Abstract] |
34. | Lohmann, V., Koch, J. O., and Bartenschlager, R. (1996) J. Hepatol. 24, 11-19[Medline] [Order article via Infotrieve] |
35. |
Kolykhalov, A. A.,
Mihalik, K.,
Feinstone, S. M.,
and Rice, C. M.
(2000)
J. Virol.
74,
2046-2051 |
36. | Pieroni, L., Santolini, E., Fipaldini, C., Pacini, L., Migliaccio, G., and La Monica, N. (1997) J. Virol. 71, 6373-6380[Abstract] |
37. |
Pallaoro, M.,
Lahm, A.,
Biasiol, G.,
Brunetti, M.,
Nardella, C.,
Orsatti, L.,
Bonelli, F.,
Orru, S.,
Narjes, F.,
and Steinkuhler, C.
(2001)
J. Virol.
75,
9939-9946 |
38. | Flajolet, M., Rotondo, G., Daviet, L., Bergametti, F., Inchauspe, G., Tiollais, P., Transy, C., and Legrain, P. (2000) Gene (Amst.) 242, 369-379[CrossRef][Medline] [Order article via Infotrieve] |
39. | Cadwell, R. C., and Joyce, G. F. (1992) PCR Methods Applications 2, 28-33[Medline] [Order article via Infotrieve] |
40. | Kim, J. E., Song, W. K., Chung, K. M., Back, S. H., and Jang, S. K. (1999) Arch. Virol. 144, 329-343[CrossRef][Medline] [Order article via Infotrieve] |
41. |
Wang, X.
(2001)
Genes Dev.
15,
2922-2933 |
42. | Lerat, H., Honda, M., Beard, M. R., Loesch, K., Sun, J., Yang, Y., Okuda, M., Gosert, R., Xiao, S. Y., Weinman, S. A., and Lemon, S. M. (2002) Gastroenterology 122, 352-365[CrossRef][Medline] [Order article via Infotrieve] |
43. | Grakoui, A., Hanson, H. L., and Rice, C. M. (2001) Hepatology 33, 489-495[Medline] [Order article via Infotrieve] |
44. | Mak, P., Palant, O., Labonte, P., and Plotch, S. (2001) FEBS Lett. 503, 13-18[CrossRef][Medline] [Order article via Infotrieve] |
45. |
Yamaga, A. K.,
and Ou, J. H.
(2002)
J. Biol. Chem.
277,
33228-33234 |
46. |
Kittlesen, D. J.,
Chianese-Bullock, K. A.,
Yao, Z. Q.,
Braciale, T. J.,
and Hahn, Y. S.
(2000)
J. Clin. Invest.
106,
1239-1249 |
47. | Matsumoto, M., Hsieh, T. Y., Zhu, N., VanArsdale, T., Hwang, S. B., Jeng, K. S., Gorbalenya, A. E., Lo, S. Y., Ou, J. H., Ware, C. F., and Lai, M. M. (1997) J. Virol. 71, 1301-1309[Abstract] |
48. |
Zhu, N.,
Khoshnan, A.,
Schneider, R.,
Matsumoto, M.,
Dennert, G.,
Ware, C.,
and Lai, M. M.
(1998)
J. Virol.
72,
3691-3697 |
49. |
Pavio, N.,
Taylor, D. R.,
and Lai, M. M.
(2002)
J. Virol.
76,
1265-1272 |
50. | Fromont-Racine, M., Rain, J. C., and Legrain, P. (1997) Nat. Genet. 16, 277-282[Medline] [Order article via Infotrieve] |
51. | Inchauspé, G., Zebedee, S., Lee, D. H., Sugitani, M., Nasoff, M., and Prince, A. M. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 10292-10296[Abstract] |
52. | Erdtmann, L., Janvier, K., Raposo, G., Craig, H. M., Benaroch, P., Berlioz-Torrent, C., Guatelli, J. C., Benarous, R., and Benichou, S. (2000) Traffic 1, 871-883[CrossRef][Medline] [Order article via Infotrieve] |
53. |
Liu, L. X.,
Margottin, F.,
Le Gall, S.,
Schwartz, O.,
Selig, L.,
Benarous, R.,
and Benichou, S.
(1997)
J. Biol. Chem.
272,
13779-13785 |
54. | Frangioni, J. V., and Neel, B. G. (1993) Anal. Biochem. 210, 179-187[CrossRef][Medline] [Order article via Infotrieve] |
55. | Le Gall, S., Erdtmann, L., Benichou, S., Berlioz-Torrent, C., Liu, L., Benarous, R., Heard, J. M., and Schwartz, O. (1998) Immunity 8, 483-495[Medline] [Order article via Infotrieve] |
56. |
Stennicke, H. R.,
and Salvesen, G. S.
(1997)
J. Biol. Chem.
272,
25719-25723 |
57. | Klonjkowski, B., Gilardi-Hebenstreit, P., Hadchouel, J., Randrianarison, V., Boutin, S., Yeh, P., Perricaudet, M., and Kremer, E. J. (1997) Hum. Gene Ther. 8, 2103-2115[Medline] [Order article via Infotrieve] |