Dissociation of a MAVS/IPS-1/VISA/Cardif-IKKepsilon molecular complex from the mitochondrial outer membrane by hepatitis C virus NS3-4A proteolytic cleavage.
ABSTRACT Intracellular RNA virus infection is detected by the cytoplasmic RNA helicase RIG-I that plays an essential role in signaling to the host antiviral response. Recently, the adapter molecule that links RIG-I sensing of incoming viral RNA to downstream signaling and gene activation events was characterized by four different groups; MAVS/IPS-1-1/VISA/Cardif contains an amino-terminal CARD domain and a carboxyl-terminal mitochondrial transmembrane sequence that localizes to the mitochondrial membrane. Furthermore, the hepatitis C virus NS3-4A protease complex specifically targets MAVS/IPS-1/VISA/Cardif for cleavage as part of its immune evasion strategy. With a novel search program written in python, we also identified an uncharacterized protein, KIAA1271 (K1271), containing a single CARD-like domain at the N terminus and a Leu-Val-rich C terminus that is identical to that of MAVS/IPS-1/VISA/Cardif. Using a combination of biochemical analysis, subcellular fractionation, and confocal microscopy, we now demonstrate that NS3-4A cleavage of MAVS/IPS-1/VISA/Cardif/K1271 results in its dissociation from the mitochondrial membrane and disrupts signaling to the antiviral immune response. Furthermore, virus-induced IKKepsilon kinase, but not TBK1, colocalized strongly with MAVS at the mitochondrial membrane, and the localization of both molecules was disrupted by NS3-4A expression. Mutation of the critical cysteine 508 to alanine was sufficient to maintain mitochondrial localization of MAVS/IPS-1/VISA/Cardif and IKKepsilon in the presence of NS3-4A. These observations provide an outline of the mechanism by which hepatitis C virus evades the interferon antiviral response.
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ABSTRACT: Hantaviruses successfully replicate in primary human endothelial cells by restricting the early induction of interferon-β (IFNβ) and interferon stimulated genes (ISGs). Gn proteins from NY-1V, ANDV and TULV, but not PHV, harbor elements in their 142 residue cytoplasmic tails (GnTs) that inhibit RIG-I/MAVS/TBK1-TRAF3 directed IFNβ induction. Here we define GnT interactions and residues required to inhibit TRAF3-TBK1 directed IFNβ induction and IRF3 phosphorylation. We observed that GnTs bind TRAF3 via residues within the TRAF-N domain (392-415) and that binding is independent of the MAVS interactive TRAF-C domain (415-568). We determined that GnT binding to TRAF3 is mediated by C-terminal degrons within NY-1V or ANDV GnTs and that mutations which add degrons to TULV or PHV GnTs confer TRAF3 binding. Further analysis of GnT domains revealed that TRAF3 binding is a discrete GnT function which is independent of IFN regulation, and that residues 15-42 from the NY-1V GnT C-terminus are required for inhibiting TBK1 directed IFNβ transcription. Mutagenesis of the NY-1V GnT revealed that altering tyrosine 627 (Y627A/S/F) abolished GnT regulation of RIG-I/TBK1 directed IRF3 phosphorylation and transcriptional responses of ISRE, κB and IFNβ promoters. Moreover, GnTs from NY-1V, ANDV and TULV, but not PHV, inhibited RIG-I directed IRF3 phosphorylation. Collectively, these findings suggest a novel role for GnTs in regulating RIG-I/TBK1 pathway directed IRF3 phosphorylation and IFN induction, and define virulence determinants within GnTs that may permit the attenuation of pathogenic hantaviruses.Journal of Virology 01/2014; · 4.65 Impact Factor
- ACS Chemical Biology 10/2014; 9(10):2393-2403. · 5.36 Impact Factor
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ABSTRACT: The flaviviral encephalitis has now become a major health concern in global scale. The efficient detection of viral infection and induction of the innate antiviral response by host's innate immune system are crucial to determine the outcome of infection. The intracellular pattern recognition receptors TLRs, RLRs, NLRs and CLRs play a central role in detection and initiation of robust antiviral response against flaviviral infection. Both cytoplasmic RLRs, RIG-I and MDA5 have been shown to be implicated in sensing flaviviral genomic RNA. Similarly among TLRs mainly TLR3 and TLR7 are known to respond in flaviviral infections as they are known to sense dsRNA and ssRNA moiety as their natural cognate ligand. Several studies have also shown the roles of NLRs and CLRs in mounting an innate antiviral response against flavivirus but, it is yet to be completely understood. Until now only few reports have implicated NLRs and CLRs in induction of antiviral and proinflammatory state following flaviviral infection. The current review therefore aims to comprehensively analyze past as well as current understanding on the role of PRRs in flaviviral infections.Virus Research 06/2014; · 2.83 Impact Factor
JOURNAL OF VIROLOGY, June 2006, p. 6072–6083
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Vol. 80, No. 12
Dissociation of a MAVS/IPS-1/VISA/Cardif-IKKε Molecular Complex
from the Mitochondrial Outer Membrane by Hepatitis C Virus
NS3-4A Proteolytic Cleavage†
Rongtuan Lin,1,3* Judith Lacoste,1Peyman Nakhaei,1,2Qiang Sun,1Long Yang,1,3Suzanne Paz,1,2
Peter Wilkinson,1Ilkka Julkunen,4Damien Vitour,5Eliane Meurs,5and John Hiscott1,2,3*
Terry Fox Molecular Oncology Group, Lady Davis Institute for Medical Research,1and Departments of Microbiology and
Immunology2and Medicine,3McGill University, Montreal, Canada; National Public Health Institute and
University of Helsinki, Helsinki, Finland4; and Department of Virology, Pasteur Institute, Paris, France5
Received 29 November 2005/Accepted 4 April 2006
Intracellular RNA virus infection is detected by the cytoplasmic RNA helicase RIG-I that plays an essential
role in signaling to the host antiviral response. Recently, the adapter molecule that links RIG-I sensing of
incoming viral RNA to downstream signaling and gene activation events was characterized by four different
groups; MAVS/IPS-1-1/VISA/Cardif contains an amino-terminal CARD domain and a carboxyl-terminal mi-
tochondrial transmembrane sequence that localizes to the mitochondrial membrane. Furthermore, the hepa-
titis C virus NS3-4A protease complex specifically targets MAVS/IPS-1/VISA/Cardif for cleavage as part of its
immune evasion strategy. With a novel search program written in python, we also identified an uncharacterized
protein, KIAA1271 (K1271), containing a single CARD-like domain at the N terminus and a Leu-Val-rich C
terminus that is identical to that of MAVS/IPS-1/VISA/Cardif. Using a combination of biochemical analysis,
subcellular fractionation, and confocal microscopy, we now demonstrate that NS3-4A cleavage of MAVS/IPS-
1/VISA/Cardif/K1271 results in its dissociation from the mitochondrial membrane and disrupts signaling to
the antiviral immune response. Furthermore, virus-induced IKK? kinase, but not TBK1, colocalized strongly
with MAVS at the mitochondrial membrane, and the localization of both molecules was disrupted by NS3-4A
expression. Mutation of the critical cysteine 508 to alanine was sufficient to maintain mitochondrial localiza-
tion of MAVS/IPS-1/VISA/Cardif and IKK? in the presence of NS3-4A. These observations provide an outline
of the mechanism by which hepatitis C virus evades the interferon antiviral response.
The hepatitis C virus (HCV) is an important cause of human
chronic liver diseases (10, 21) and is a major public health
problem. More than 170 million people worldwide are infected
with HCV (38), a virus that is often associated with significant
liver disease, including chronic active hepatitis, cirrhosis, and
hepatocellular carcinoma (3). HCV is an enveloped virus clas-
sified in the Flaviviridae family (33). The positive-stranded viral
RNA genome encodes a single polyprotein precursor that is
processed into structural proteins (core, envelope protein 1,
and envelope protein 2) and nonstructural proteins (p7, NS2,
NS3, NS4A, NS4B, NS5A, and NS5B) by host and viral pro-
teases (reviewed in references 32 and 39).
HCV and many viral infections are detected by the host cell
through the presence of viral nucleic acids, such as single- and
double-stranded RNA (dsRNA), triggering the production of
interferons (IFN) and other cytokines that in turn stimulate
innate and adaptive immune responses (16). Extracellular viral
dsRNA is recognized by the Toll-like receptor 3 (TLR3) (1, 2),
whereas intracellular viral dsRNA is detected by two recently
characterized RNA helicases, RIG-I (41) and/or Mda5 (4, 18).
The importance of the RIG-I pathway was confirmed with the
generation of RIG-I-deficient mice (19), which revealed that
RIG-I and not the TLR system played an essential role in the
IFN antiviral response in most cell types, including fibroblastic,
epithelial, and conventional dendritic cells. In contrast, plas-
macytoid dendritic cells utilize TLR-mediated signaling in
preference to RIG-I.
Upon dsRNA recognition and binding by its RNA helicase
activity, RIG-I dimerizes and undergoes conformational alter-
ations that enable the N-terminal CARD domain to interact
with another downstream adapter protein(s). RIG-I signaling
ultimately engages the IKK kinase complex and the stress-
activated kinases, as well as the IKK-related kinases TBK1 and
IKKε, leading to the phosphorylation and activation of NF-?B,
ATF-2/c-jun, and interferon regulatory factor 3 (IRF-3) tran-
scription factors (27). Coordinated activation of these factors
results in the formation of a transcriptionally competent en-
hanceosome that triggers IFN-? production (30).
How HCV deals with the immediate host intracellular re-
sponse to virus has been an area of intense study. Recent
studies demonstrated that the HCV gene product NS3-4A
protease complex, a multifunctional serine protease, efficiently
blocked the RIG-I signaling pathway and contributed to virus
persistence by enabling HCV to escape the IFN antiviral re-
sponse. Nevertheless, RIG-I was not a direct target of NS3-4A,
and likewise, the kinases TBK1 and IKKε were not subject to
proteolytic cleavage by NS3-4A (8, 14, 37). Interestingly, the
NS3-4A protease appears to target the TRIF/TICAM adapter
of the TLR3 pathway and causes specific proteolytic cleavage
* Corresponding author. Mailing address: Lady Davis Institute for
Medical Research, 3755 Cote Ste. Catherine, Montreal H3T 1E2,
Quebec, Canada. Phone: (514) 340-8222, ext. 5272. Fax: (514) 340-
7576. E-mail for Rongtuan Lin: email@example.com. E-mail for
John Hiscott: firstname.lastname@example.org.
† Supplemental material for this article may be found at http://jvi
of TRIF, although this pathway has a minimal role in triggering
the IFN antiviral response (22a). Additional evidence for the
importance of RIG-I comes from studies demonstrating that
permissiveness for HCV RNA replication in Huh7.5 (6) cells is
due to mutational inactivation of the RIG-I protein (37). Thus,
RIG-I signaling appears to be an essential pathway regulating
cellular permissiveness to HCV replication.
The adapter molecule that links RIG-I sensing of incoming
viral RNA and downstream activation events was recently elu-
cidated by four independent groups (20, 29, 35, 40). MAVS/
IPS-1/VISA/Cardif contains an amino-terminal CARD do-
main and a carboxyl-terminal mitochondrial transmembrane
sequence that localizes this protein to the mitochondrial mem-
brane, thus suggesting a novel role for mitochondrial signaling
in the cellular innate response (35). Under the name of Cardif,
this protein was described by Meylan et al. to interact with
RIG-I and recruits IKK?, IKK?, and IKKε kinases through its
C-terminal region. Importantly, Cardif was cleaved at its C-
terminal end, adjacent to the mitochondrion-targeting domain,
by the NS3-4A protease of hepatitis C virus (29). Li et al.
subsequently demonstrated that NS3-4A cleavage of MAVS/
IPS-1/VISA/Cardif resulted in its dissociation from the mito-
chondrial membrane and disruption of signaling to the antivi-
ral immune response (23).
Using a combination of biochemical analysis, subcellular
fractionation, and confocal microscopy, we demonstrate that
the virus-inducible IKKε, but not TBK1, was strongly recruited
to the mitochondria via MAVS/IPS-1/VISA/Cardif. Further-
more, stable expression of the NS3-4A complex in Huh8 HCV
replicon cells or transient expression in Cos-7 cells disrupted
the molecular complex between the MAVS/IPS-1/VISA/Cardif
adapter and IKKε, resulting in relocation from the mitochon-
drial membrane to the cytosolic fraction and ablation of sig-
naling to the antiviral immune response. These observations
provide the outline of the mechanism for HCV evasion of the
IFN signaling pathway.
MATERIALS AND METHODS
Plasmid constructions and mutagenesis. Plasmids encoding IKKε and TBK1,
P2(2)-TK pGL3, IFNB/pGL3, IFNA14/pGL3, and pRLTK, have been described
previously (25, 36). The cDNA encoding HCV NS3-4A was amplified from
NS3-4A pcDNA3 plasmid (provided by M. Gale) and cloned into Flag
pcDNA3.1/Zeo (Flag–NS3-4A). The cDNA encoding full-length human K1271
was amplified from a T-cell cDNA library and cloned into MYC pcDNA3.1/Zeo
(Myc-KIAA1271) or Flag pcDNA3.1/Zeo (Flag-K1271). The point mutant A508
was generated by overlap PCR-mediated mutagenesis. The K1271 deletion mu-
tants, including amino acids (aa) 1 to 508 [K1271(aa 1–150)], aa 1 to 467, aa 151
to 540, and aa 1 to 150 and a deletion of aa 150 to 467 (?150–467) and aa 150
to 502 (?150–502), were generated by PCR. DNA sequencing was performed for
confirmation of mutations.
Cell culture, transfections, and luciferase assays. Human hepatoma Huh7
cells have been previously described (8). They were cultured in Dulbecco’s
modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum
(FBS), 50 ?g/ml gentamicin, and nonessential amino acids (1?). The same
medium, also containing 500 ?g/ml neomycin as a selection marker, was used to
culture Huh8 cells, which are derived from Huh7 cells and stably express the
HCV replicon. Human lung carcinoma A549 cells were cultured in F12K media
supplemented with 10% FBS and 50 ?g/ml gentamicin. Where indicated, A549
cells were infected with 10 multiplicities of infection of vesicular stomatitis virus
(VSV) for 1 hour. Transfections for the luciferase assay were carried out in
human embryonic kidney 293 (HEK293) cells grown in DMEM (GIBCO-BRL)
supplemented with 10% fetal bovine serum, glutamine, and antibiotics. Subcon-
fluent HEK293 cells were transfected by the calcium phosphate coprecipitation
method with 100 ng of pRLTK reporter (Renilla luciferase for internal control),
100 ng of pGL-3 reporter (firefly luciferase, experimental reporter), 200 or 1,000
ng of ?RIG-I, K1271, IKKε, or TBK1 expression plasmid, and 125, 500, or 2,000
ng of pcDNA3, Flag NS3-4A pcDNA3 zeo, or Flag A20 pcDNA3 zeo plasmid as
indicated. The reporter plasmids were IFNB pGL3, ISRE-luc, P2(2)-TK pGL3,
and IFNA14 pGL-3 reporter genes; the transfection procedures were previously
described (24). At 24 h after transfection, reporter gene activity was measured by
the Dual-Luciferase reporter assay, according to the manufacturer’s instructions
(Promega). Where indicated, cells were treated with Sendai virus (40 hemagglu-
tinating units/ml) for the indicated time or 15 h for luciferase assays. Transfec-
tions for microscopy analyses were performed in African green monkey kidney
cells (COS-7) cultured in DMEM containing 10% FBS and 50 ?g/ml gentamicin.
Briefly, cells were seeded on glass coverslips (12-mm diameter) and grown
overnight to 50% confluence. At that point, cells were transfected with FuGENE
6 (Roche Diagnostics, Indianapolis, IN), using equal amounts (total of 0.4 ?g
DNA) of K1271-myc, IKKε-myc, and Flag–NS3-4A expression plasmids; as in-
dicated, in the individual experiments, coverslips were harvested and processed
for immunofluorescence staining 18 h later.
Generation of NS3-4A-expressing cell lines. Plasmids pcDNA3 zeo and Flag–
NS3-4A pcDNA3 zeo were introduced into HEK293T cells by the calcium
phosphate method. Cells were selected beginning at 48 h for approximately 3
weeks in DMEM containing 10% heat-inactivated calf serum, glutamine, anti-
biotics, and 100 ?g/ml zeocin (Invitrogen).
Antibodies. K1271(aa 1–150) was expressed in Escherichia coli as a glutathione
S-transferase fusion protein and purified by glutathione-Sepharose column chro-
matography. The recombinant proteins were injected into rabbits or guinea pigs
to produce antisera against KIAA1271(aa 1–150). Similarly GST-K1271(aa 157–
540) fusion proteins were injected into rabbits to produce antisera against
KIAA1271(aa 157–540). Mouse anti-IKKε antibody was obtained from BD Bio-
Sciences (Mountain View, CA). Rabbit anti-TBK1 antibody was obtained from
Upstate Biotech Inc. (Lake Placid, NY). The green (donkey anti-rabbit or anti-
mouse immunoglobin [Ig] conjugated with AlexaFluor 488) and the red (donkey
anti-guinea pig Ig conjugated with AlexaFluor 546) fluorochromes were obtained
from Invitrogen/Molecular Probes.
Coimmunoprecipitation and Western blot analysis. Transient transfection,
coimmunoprecipitation, and Western blot analysis were performed as previous
described (26). In some experiments, mitochondria were isolated using the
reagents and protocols of the mitochondria isolation kit (Pierce, Brockville,
Immunofluorescence staining and confocal microscopy. Cells were seeded on
glass coverslips and grown overnight to 50% confluence. Staining of mitochon-
dria was achieved by placing the cells in 25 nM MitoTracker Orange CMTMRos
(MTO; Invitrogen/Molecular Probes, Eugene, OR), a red fluorochrome, for 30
min in a CO2incubator. Excess MTO was removed by further culturing the cells
in fresh media for another 30 min. Coverslips were washed twice in warm culture
media, fixed (37°C for 15 min) in a warm buffered fixation solution (3.7%
paraformaldehyde and 10% FBS in phosphate-buffered saline [PBS]), and
washed three times in PBS. Further steps were carried out at room temperature.
Cells were permeabilized for 30 min in PBS containing 0.2% Triton X-100 and
3% IgG-free bovine serum albumin (BSA; Jackson ImmunoResearch, West
Grove, PA). From this point on, all steps were done in PBS containing 0.5%
IgG-free BSA. Coverslips were washed twice before being exposed to the pri-
mary antibodies for 60 min. Anti-K1271 antibody was diluted at 1:200, anti-IKKε
antibody was used at 1 ?g/ml, and anti-TBK1 antibody was diluted at 1:500. The
primary antibody was washed off twice, before the coverslips were in the presence
of fluorochromes (2 ?g/ml, red and/or green, as indicated) for 60 min. Finally,
coverslips were washed twice in BSA-PBS, washed once in water, and mounted
on slides using ImmuMount (Thermo Electron Corporation, Pittsburgh, PA).
Samples were analyzed on an inverted Axiovert 200 M Zeiss microscope
equipped with an LSM 5-Pa confocal imaging system (Carl Zeiss Canada, Mon-
treal, Quebec, Canada). Confocal images (0.3- to 0.5-?m slices) were acquired
with a Plan-Apochromat ?63 oil objective, using the argon and HeNe laser lines
(488 nm and 543 nm, respectively).
K1271 activates NF-?B and IRF-3/IRF-7 to induce inter-
feron antiviral response. The observations that RIG-I, TBK1,
and IKKε are not proteolytic substrates of NS3-4A indicated
that an unidentified adapter(s) between RIG-I and the kinases
may be a target for NS3-4A cleavage (8, 14). To this end,
mda-5 and RIG-I protein sequences were separated into do-
VOL. 80, 2006MAVS CLEAVAGE BY HCV NS3-4A 6073
FIG. 1. Characterization of the RIG-I adapter. (A) Alignment of the CARD domains of Mda-5, RIG-I, and K1271. A BLAST alignment
reveals CARD domain homology among the N-terminal sequences of RIG-I, Mda-5, and K1271. C-terminal alignment of the Leu-Val-rich region
of K1271 and Bcl-xL is also shown. (B) Schematic representation of K1271. The 540-aa MAVS/IPS-1/VISA/Cardif molecule is shown schemat-
ically. The location of the CARD domain-containing, proline-rich region that interacts with TRAF6 and the C-terminal mitochondrial
brane region (TM) is shown. Also illustrated is the region adjacent to the TM containing cysteine 508, the target residues for the HCV NS3-4A
protease. The different N- and C-terminal deletions used in this study are shown below the schematic. (C) N- and C-terminal
6074 LIN ET AL.J. VIROL.
mains, and the domains were analyzed separately, creating a
regular expression that represents the patterns of the domain.
Next, a search program was written in python language (http:
//www.biopython.org); a database search was performed that
identified an uncharacterized protein, KIAA1271(K1271),
containing a single CARD-like domain at the N terminus and
a Leu-Val-rich C terminus (Fig. 1A). Interestingly, analysis of
the C terminus of K1271 revealed high homology with the C
terminus of the antiapoptotic protein Bcl-xl. Following PCR
amplification and subcloning, a series of K1271 deletion con-
structs were generated and tested for the capacity to stimulate
the IFN-? promoter (Fig. 1B). Coexpression of K1271 strongly
activated the IFN-?-dependent promoter and appeared to be
the adapter linking RIG-I sensing to downstream signaling
events. Both the N and C termini were essential for transacti-
vation function, since deletion of either end of the protein
eliminated transactivation (Fig. 1C). Interestingly, an expres-
sion construct that contained the N- and C-terminal domains
but lacked the region between aa 150 and aa 467 also stimu-
lated the IFN-? promoter (Fig. 1C), whereas a similar internal
deletion construct removing the region between aa 150 and aa
502 was inactive, indicating that the region of K1271 between
aa 467 and aa 502 was important for transactivation function.
A direct correlation between downstream activation of the
IFN-? promoter and subcellular localization was observed
when the different K1271 deletion constructs were evaluated;
wtK1271 and the active internal deletion ?150–467 were local-
ized to the insoluble fraction of cytoplasmic extracts (Fig. 1D,
lanes 1, 2, 7 and 8), whereas the C-terminal truncation of aa 1 to
508 was localized exclusively in the soluble cytoplasmic extract
(Fig. 1D, lanes 3 and 4). Other constructs, including the inactive
internal deletion ?150–502 and the deletions of aa 1 to 467, aa 1
to 180, and aa 151 to 540, redistributed to both the soluble and
insoluble cytoplasmic fractions (Fig. 1D, lanes 5, 6, and 9 to 14).
Four independent groups reported that the same protein,
alternately named MAVS/VISA/IPS-1-1/Cardif, acted down-
stream of RIG-I and stimulated the expression of IFN-?
through the activation of NF-?B and IRF-3 (20, 29, 35, 40).
The C-terminal Leu-Val domain was recognized as a mito-
chondrial transmembrane domain, and localization to the mi-
tochondrial outer membrane was required for its function (35).
Thus, MAVS/VISA/IPS-1-1/Cardif/K1271 is a critical adapter
of the RIG-I pathway that links the “sensing” of incoming viral
particles by RIG-I with downstream TBK1/IKKε kinases.
K1271 is a direct target of HCV protease NS3-4A. As shown
by Meylan et al. (29), K1271 is a direct target of the HCV
protease NS3-4A, with cleavage occurring adjacent to the
transmembrane domain at Cys508. Increasing levels of coex-
pression of NS3-4A dramatically blocked K1271-driven gene
expression from the IFN-? promoter (Fig. 2A), whereas the
protease-inactive form of NS3-4A did not affect K1271-driven
reporter gene expression. Concomitant with the inhibition of
IFN-?-dependent gene expression, the size of K1271 was re-
duced and the amount of K1271 in the insoluble fraction of
domains of K1271 are required for IFN-? promoter activation. HEK293 cells were transfected with 100 ng of pRLTK control plasmid and 100 ng
of IFN-?–pGL3 reporter plasmid, with increasing amounts (200 ng and 1,000 ng) of different truncated forms of Myc-K1271 expression constructs
as indicated. Luciferase activity was analyzed at 24 h posttransfection by the Dual-Luciferase reporter assay as described by the manufacturer
(Promega). Relative luciferase activity was measured as activation (n-fold; relative to the basal level of reporter gene in the presence of pcDNA3
vector after normalization with cotransfected renilla luciferase activity); values are means ? standard deviations from three experiments. (D)
Localization of active forms of K1271 to the insoluble fraction of the cytoplasm. The soluble lysates and insoluble fractions were prepared from
the HEK293 cells used for panel C, equilibrated to the same volumes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis loading buffer,
and analyzed by immunoblotting with anti-Myc antibody 9E10.
FIG. 2. Cleavage of K1271 and inhibition of K1271-mediated trans-
activation by NS3-4A. (A) HEK293 cells were transfected with 100 ng
of pRLTK control plasmid, 100 ng of IFN-?–pGL3 reporter plasmid,
and 200 ng of K1271 (A) or K1271(A508) (B) Myc-tagged expression
plasmid, together with increasing amounts (250 ng, 500 ng, and 1,000
ng) of protease-active NS3-4A or inactive NS3-4A(A139) Flag-tagged
expression plasmid as indicated. Luciferase activity was analyzed at
24 h posttransfection. (C and D) The soluble lysates and insoluble
fractions were prepared from HEK293 cells used for panels A and B,
equilibrated to the same volumes by sodium dodecyl sulfate-polyacryl-
amide gel electrophoresis loading buffer, and analyzed by immuno-
blotting with anti-Flag antibody M2 and anti-Myc antibody 9E10.
VOL. 80, 2006MAVS CLEAVAGE BY HCV NS3-4A 6075
cytoplasmic extracts was decreased (Fig. 2C), whereas expres-
sion of the protease-inactive point mutant A139 did not alter
the mobility or the amount of K1271. In contrast, the K1271
(C508A) point mutation remained localized in the insoluble cy-
toplasmic fraction, thus illustrating that K1271(C508A) was not
cleaved by NS3-4A (Fig. 2D). However, K1271(C508A) IFN-?-
driven gene activity was still decreased when NS3-4A was ex-
pressed, although with about 10-fold decreased efficiency, sug-
gesting that NS3-4A may also target an undefined protein
downstream of K1271.
The RIG-I RNA sensing molecule is highly inducible in
response to virus, IFN, or retinoic acid treatment. To examine
the inducibility and localization of K1271, a new rabbit poly-
clonal K1271 specific antibody was generated; detection of
K1271 revealed that the adapter localized exclusively to the
mitochondrial fraction. Furthermore, expression of K1271 was
constitutive and expression of other components of the RIG-I
pathway, including IKKε and RIG-I itself, or Sendai virus
infection did not significantly alter the expression or mitochon-
drial localization of K1271 (Fig. 3).
Disruption of the mitochondrial localization of K1271 by
NS3-4A. To examine the cleavage and mitochondrial localiza-
tion of K1271 in human hepatoma cells, expression of K1271
was evaluated in both human hepatoma Huh7 and the HCV
replicon-expressing Huh8 cells (5). As shown in Fig. 4A, full-
length K1271 was detected in extracts of Huh7 cells, whereas
the predominant form of K1271 in Huh8 cells was truncated.
To further delineate the localization of K1271, mitochondrial
extracts were prepared from HEK293 cells coexpressing
NS3-4A (Fig. 4B). K1271 was easily detected in the mitochon-
drial but not cytosolic fraction of HEK293 cells (Fig. 4B, lanes
1 and 3), whereas in mitochondrial extracts from cells express-
ing NS3-4A, K1271 was not detected but rather was observed
in the cytosol (Fig. 4B, lanes 2 and 4). Similar results were
obtained when mitochondrial extracts were prepared from
Huh7 and Huh8 cells; K1271 was detected in the mitochondria
of Huh7 cells (Fig. 4B, lane 5), whereas in the NS3-4A-express-
ing HCV replicon cells, K1271 was detected almost exclusively
in the cytosolic fraction (Fig. 4B, lane 8). Curiously, cyto-
chrome c was not a good marker for mitochondrial isolation,
since in the presence of NS3-4A, cytochrome c, but not mito-
chondrial hsp70, levels were reduced by NS3-4A expression
(Fig. 4B, lanes 1, 2, 5, and 6).
Confocal microscopy was also used to visualize the localiza-
tion of K1271 in Huh7 cells (Fig. 5A to C) and in Huh8 cells
(Fig. 5D to F). K1271 staining was localized predominantly to
perinuclear bodies (Fig. 5A) that were costained with the mi-
tochondrial marker MTO (Fig. 5B); merging of the signals
demonstrated a strong colocalization of K1271 and mitochon-
FIG. 3. Localization of K1271 to the mitochondria. HEK293 cells
were transfected with combinations of 200 ng of IKKε and RIG-I; in
some cases, cells were also infected with Sendai virus (40 hemaggluti-
nating units/ml) at 24 h posttransfection for a total of 12 h. Mitochon-
drial extracts were prepared, and 50 ?g of extract was run on 7.5%
sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immu-
noblotted with anti-K1271 antibody.
FIG. 4. Endogenous K1271 is cleaved in Huh8 HCV replicon cells.
(A) Whole-cell extracts (50 ?g) prepared from uninfected or Sendai
virus-infected Huh7 or Huh8 cells were subjected to sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and probed
with anti-K1271 and antiactin antibodies. (B) Mitochondrial and cy-
tosolic fractions (50 ?g) from HEK293T cells transfected with 200 ng
of NS3-4A expression plasmid (lanes 1 to 4) were subjected to SDS-
PAGE and analyzed by immunoblotting with anti-K1271, antitubulin,
anti-cytochrome c, and anti-hsp70. Mitochondrial and cytosolic frac-
tions (50 ?g) from Huh7 (H7) and Huh8 (H8) cells (lanes 5 to 8) were
also subjected to SDS-PAGE and analyzed by immunoblotting with
anti-K1271, antitubulin, anti-hsp70, and anti-cytochrome c.
6076 LIN ET AL.J. VIROL.
dria (Fig. 5C). In contrast, staining of K1271 in NS3-4A-express-
ing Huh8 cells revealed a diffuse staining pattern (Fig. 5D) that
did not reflect the pattern of mitochondrial staining (Fig. 5E).
Merging of the fluorescence signals demonstrated that the local-
ization of K1271 and mitochondria was no longer coincidental
(Fig. 5F). Altogether, these results demonstrate that NS3-4A ex-
pression results in the cleavage of K1271 and the disruption of its
localization to the mitochondrial membrane.
Distinct subcellular localizations of TBK1 and IKK?: re-
cruitment of IKK? to K1271 localized at the mitochondrial
membrane. To determine whether localization and/or recruit-
ment of the virus-activated kinases TBK1 and IKKε could be
observed in association with mitochondrion-localized K1271,
endogenous K1271 and IKKε were visualized by confocal mi-
croscopy in lung epithelial A549 cells (Fig. 6). In control cells,
IKKε was difficult to visualize due to its low level of expression,
although K1271 was readily detected (Fig. 6, upper panels);
however, in cells that were infected with VSV for 1 h, perinu-
clear IKKε was detected that colocalized with K1271 (Fig. 6,
lower panels). The merging of the respective signals illustrated
a strong colocalization of the two proteins. Interestingly, VSV-
induced IKKε localized almost exclusively with mitochondria
(detected by MTO) in the reticulotubular mitochondrial net-
work (Fig. 7, upper panels), whereas no similar colocalization
was observed between TBK1 and mitochondria (Fig. 7, lower
panels). In fact, preliminary data suggest that TBK1 is partially
associated with the early endosome marker EEA1, indicating
an endosomal association of TBK1. Furthermore, the intensity
and/or localization of TBK1 does not change with time after
VSV infection (data not shown). Together, these data indicate
distinct cytoplasmic localizations of IKKε and TBK1: the virus
inducible IKKε is recruited to the mitochondrial network in
association with K1271, while the constitutively expressed
TBK1 localizes to a distinct cytoplasmic compartment.
Cleavage of the K1271-IKK? complex from the mitochon-
dria by NS3-4A. To address the fate of the K1271-IKKε com-
plex in the presence of the NS3-4A protease, transient expres-
sion studies of primate Cos-7 cells were undertaken (Fig. 8).
As observed for the endogenous molecules shown in Fig. 6,
costaining of transfected K1271 and IKKε revealed a strong
colocalization in a typical mitochondrial pattern (Fig. 8, upper
panels). Importantly, NS3-4A protease coexpression was suf-
ficient to disrupt the mitochondrial localization of K1271-IKKε
(Fig. 8, lower panels), resulting in a diffuse cytoplasmic staining
pattern. When K1271 (C508A) was used in similar experi-
ments, K1271-IKKε remained associated with the mitochon-
drial membrane (see Fig. S1 in the supplemental material).
Similarly, a protease-inactive form of NS3-4A was unable to
cleave the K1271-IKKε molecular complex from the mitochon-
drial surface (data not shown).
Inhibition of downstream IFN activation. The effects of
NS3-4A on downstream gene activation events were next eval-
uated. As demonstrated in Fig. 2, coexpression of increasing
amounts of NS3-4A strongly inhibited K1271-mediated activa-
tion of the IFN-? promoter (Fig. 9A), whereas a truncated
form of K1271 [K1271(aa 1-508)] was unable to stimulate the
IFN-? promoter and was also not inhibited by NS3-4A coex-
pression. A mutated form of K1271, K1271(C508A), with an
alanine substitution at aa 508 which altered the site of NS3-4A
cleavage was completely resistant to cleavage (Fig. 2). How-
ever, coexpression of the NS3-4A complex still inhibited
K1271(C508A)-mediated transactivation more than 10-fold
To verify whether in vivo phosphorylation of IRF-3 and
IRF-7 occurred in response to K1271 expression, an antibody
directed against a phosphopeptide spanning Ser 477/479 of
IRF-7 was raised as a complement to the previously generated
IRF-3 Ser396 antibody (34). Using these phosphospecific an-
FIG. 5. Endogenous K1271 is cleaved in Huh8 HCV replicon cells. Huh7 (A to C) and Huh8 (D to F) cells were treated with MTO (B and
E), fixed in paraformaldehyde, and stained for K1271 (A and D). Confocal fluorescent images were merged (C and F). K1271 was detected using
rabbit anti-K1271 C-term and anti-rabbit AF488 antibodies.
VOL. 80, 2006MAVS CLEAVAGE BY HCV NS3-4A6077
tibodies, it was possible to demonstrate that expression of
?RIG-I, K1271, or TBK1 induced IRF-7 Ser 477/479 and
IRF-3 Ser396 phosphorylation in vivo (Fig. 9B, lanes 4, 7, and
10). Coexpression of the ubiquitin-editing protein A20 (26a) or
the viral NS3-4A protease completely inhibited ?RIG-I- and
K1271-induced IRF-3 and IRF-7 phosphorylation (Fig. 9B,
lanes 5, 6, 8, and 9). On the other hand, TBK1-mediated
phosphorylation of IRF-3 and IRF-7 was not inhibited by A20
FIG. 6. Colocalization of endogenous IKKε and K1271. Human lung epithelial A549 cells, either control (upper panels) or VSV infected (lower
panels), were costained for IKKε (in green) and K1271 (in red), and confocal fluorescent images were merged. IKKε was detected using mouse
anti-IKKε and anti-mouse AF488 antibodies, while K1271 was detected using guinea pig anti-K1271 N-term and anti-guinea pig AF546 antibodies.
FIG. 7. Distinct subcellular localization of IKKε and TBK1. VSV-infected A549 cells were treated with MTO (in red), fixed, and stained for
either IKKε (upper panels) or TBK1 (lower panels). The confocal fluorescent images were merged. IKKε was detected as described above, while
TBK1 was detected using rabbit anti-TBK1 and anti-rabbit AF488 antibodies.
6078 LIN ET AL. J. VIROL.
and NS3-4A, indicating that both of these inhibitory activities
acted upstream of TBK1 (Fig. 9B, lanes 10 to 12) and confirm-
ing previous findings that NS3-4A has no effect on the ability of
TBK-1 to phosphorylate IRF-3 (8).
K1271 activates endogenous ISG expression and the antivi-
ral state. As a measure of the induction of the antiviral state,
the constitutively active form of RIG-I, ?RIG-I, K1271, or the
TRIF adapter was used to assess the induction of endogenous
IFN-stimulated gene 56 (ISG56) expression. Coexpression of
?RIG-I, K1271, or TRIF resulted in a strong induction of
endogenous ISG56 expression (Fig. 10A, lanes 5, 9, and 17).
Expression of the point-mutated K1271(C508A) also induced
ISG56 expression (Fig. 10A, lane 13). The cellular A20 protein
completely inhibited RIG-I- and K1271-induced ISG56 expres-
sion (Fig. 10A, lanes 6, 10, and 14) but did not affect TRIF-
induced ISG56 expression (Fig. 10A, lane 18). NS3-4A also
totally blocked ?RIG-I- and K1271-mediated induction of
ISG56 (Fig. 10A, lanes 7 and 11); however, NS3-4A only
weakly interfered with K1271(C508A)-mediated induction of
ISG56 (Fig. 10A, lane 15) because NS3-4A failed to cleave
K1271(C508A). In contrast, molecular weight reduction of
K1271 as a result of NS3-4A cleavage was easily detected (Fig.
10A, lane 11). NS3-4A also failed to inhibit TRIF-mediated
ISG56 expression. In all cases, expression of the dominant-
negative form of IRF3, IRF3?N, inhibited ISG56 expression
(Fig. 10A, lanes 8, 12, 16, and 20), demonstrating that ISG56
induction occurred through an IRF3-dependent pathway. As a
further confirmation of the generation of an antiviral state,
HEK293 cells expressing K1271 or IKKε were infected with
VSV and the kinetics of viral protein expression was measured.
Viral protein production was significantly inhibited from 6 to
9 h in control cells to 12 to 24 h in cells expressing K1271 or
IKKε protein; furthermore, ISG56 expression was constitu-
tively detected in K1271- or IKKε-expressing cells (Fig. 10B),
thus demonstrating the generation of an antiviral state by
K1271 that blocked VSV replication.
It has quickly become clear that the RNA helicase RIG-I
pathway plays an essential role in the sensing of incoming virus
infection and directly relays regulatory signals to the host an-
tiviral response. The adapter molecule providing a link be-
tween RIG-I sensing of incoming viral RNA and downstream
activation events was recently elucidated; four independent
groups used high-throughput screening and/or database search
analyses to identify the new signaling component, which has
alternatively been termed MAVS/IPS-1-1/VISA/Cardif (20, 29,
35, 40) and is termed K1271 in this study. The fact that MAVS/
IPS-1-1/VISA/Cardif localizes to the mitochondrial membrane
suggests linkage among recognition of viral infection, the de-
velopment of innate immunity, and mitochondrial function
(35). The IKK-related kinases TBK1 and IKKε are critical
downstream components of the activation of the interferon
antiviral response through their ability to phosphorylate the
C-terminal domains of IRF-3 and IRF-7 (13, 28, 36). In the
present study, we demonstrate that VSV infection of lung
epithelial A549 cells results in the induction and recruitment of
IKKε to the mitochondrial membrane, in association with
K1271 (MAVS/IPS-1-1/VISA/Cardif). Furthermore, the hepa-
titis C virus NS3-4A protease activity specifically cleaves the
MAVS/IPS-1-1/VISA/Cardif-IKKε molecular complex from
the mitochondria as part of its immune evasion strategy. Point
mutation of Cys508 to Ala, the target residue of NS3-4A,
created a MAVS/IPS-1-1/VISA/Cardif protein that not only
was resistant to cleavage and subcellular relocation but also
maintained the association with IKKε in the presence of pro-
tease activity. Disruption of the mitochondrial localization of
FIG. 8. Disruption of the IKKε-K1271 complex localization by expression of NS3-4A. COS-7 cells transfected with IKKε and K1271 expression
plasmids, without (upper panels) or with (lower panels) a construct encoding NS3-4A, were costained for IKKε (in green) and K1271 (in red), and
confocal fluorescent images were merged. IKKε and K1271 were detected as described above.
VOL. 80, 2006MAVS CLEAVAGE BY HCV NS3-4A6079
MAVS/IPS-1-1/VISA/Cardif-IKKε also ablated downstream
signaling to the IFN antiviral response. These observations
provide the outline of the mechanism by which HCV evades
the IFN antiviral response.
The strong and apparently selective recruitment of IKKε to
the mitochondria in association with MAVS/IPS-1-1/VISA/
Cardif is enigmatic, given that TBK1 is involved principally in
downstream signaling to IRF-3 and IRF-7 phosphorylation
and development of the antiviral response (13, 28, 36). Studies
of TBK1 and IKKε knockout mice demonstrate a clear role for
TBK1 in the generation of the antiviral response, with an
accessory role associated to date with IKKε (17, 31). Given that
FIG. 9. Inhibition of IRF and IFN-? activation by NS3-4A. (A) Inhibition of the IFN-? promoter. HEK293 cells were transfected with 100 ng
of pRLTK control plasmid, 100 ng of IFN-?–pGL3 reporter plasmid, and 200 ng of ?RIG-I and wild-type or mutated forms of K1271 expression
plasmids, together with increasing amounts of NS3-4A expression plasmid (125 ng, 500 ng, and 2,000 ng) as indicated. Luciferase activity was
analyzed at 24 h posttransfection by the Dual-Luciferase reporter assay as described by the manufacturer (Promega). Relative luciferase activity
was measured as activation (n-fold; relative to the basal level of reporter gene in the presence of pcDNA3 vector after normalization with
cotransfected renilla luciferase activity); values are means ? standard deviations from three experiments. (B) A20 and NS3-4A inhibit K1271- and
?RIG-I-mediated activation of IRF-3 and IRF-7. HEK293 cells were cotransfected with 1 ?g of IRF-7, IRF-3, Myc-?RIG-I, Myc-K1271, or
GFP-TBK1 and 2 ?g of Flag-A20 or Flag–NS3-4A expression construct as indicated. Whole-cell extracts (50 ?g) were resolved by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis and analyzed by immunoblotting for IRF-7 pSer477/479, IRF-3 pSer396, Flag-A20, Flag–NS3-4A,
Myc-?RIG-I, Myc-K1271, GFP-TBK1, and actin.
6080 LIN ET AL. J. VIROL.
IKKε knockout mice do not appear to have major defects in
IFN induction, the targeting of an IKKε-dependent pathway
does not explain the inhibition of IFN induction by the hepa-
titis C protease. Selective recruitment of IKKε may reflect a
distinct functional role for this kinase activity in the host re-
sponse to virus infection, perhaps at the level of coordinating
mitochondrion-dependent cell death in virus-infected cells.
Ongoing biochemical studies have demonstrated that a small
proportion of total TBK1 is present in highly purified mito-
chondrial fractions (Q. Sun, unpublished data); whether this
fraction is enzymatically active and/or physically associated
with MAVS/IPS-1-1/VISA/Cardif remains to be determined.
Although colocalization of TBK1 with the mitochondria and
MAVS/IPS-1-1/VISA/Cardif may be transient and may not be
easily detected by confocal analysis, the observation that IKKε
is so intimately associated with MAVS/IPS-1-1/VISA/Cardif is
not easily resolved at this point. However, in support of a
functional role of IKKε in HCV pathogenesis, previous ex-
FIG. 10. (A) NS3-4A and A20 inhibit K1271-mediated activation of endogenous ISG56 gene expression. HEK293 cells were cotransfected with
1 ?g of pcDNA3, Myc-?RIG-I, Myc-KIAA1271, Myc-KIAA1271(C508A), or Myc-TRIF and 2 ?g of IRF-3?N, Flag-A20, or Flag–NS3-4A
expression construct as indicated. Whole-cell extracts (50 ?g) were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and
analyzed by immunoblotting using antibodies to ISG56, Myc, Flag, IRF-3, and actin. (B) K1271 inhibits VSV replication. HEK293 cell lines
expressing K1271 or IKKε were infected with VSV (1 multiplicity of infection), and viral protein expression was measured at different times after
infection by immunoblotting. Expression of the Flag-tagged transgene and the endogenous ISG56 was also monitored.
VOL. 80, 2006MAVS CLEAVAGE BY HCV NS3-4A 6081
periments have demonstrated that IKKε overexpression, but
not the expression of TBK1 or other signaling adapters,
partially reversed HCV protease-mediated inhibition of IFN
The mechanism of recruitment of IKKε to the mitochondria
remains unclear at present. However, given that NS3-4A is still
able to block IFN-? promoter activity when the Cys508-to-Ala
point-mutated MAVS/IPS-1-1/VISA/Cardif protein is ex-
pressed, albeit with a 10-fold-reduced efficiency, it is possible
that another adapter downstream of MAVS/IPS-1-1/VISA/
Cardif may be targeted by NS3-4A. Alternatively, an additional
mechanism involved in the recruitment of IKKε to the MAVS/
IPS-1-1/VISA/Cardif mitochondrial reticulotubular network
may be affected by the HCV protease. We have also identified
significant changes in mitochondrial reticulotubular architec-
ture following VSV infection and the recruitment of IKKε to
the mitochondria, suggesting that IKKε may be contributing to
Seth et al. identified the localization of MAVS to the mito-
chondrial membrane and also showed that MAVS moved into
a detergent-resistant mitochondrial fraction upon viral infec-
tion (35). The fact that MAVS functionality requires mito-
chondrial association suggests linkage among recognition of
viral infection, the development of innate immunity, and virus-
induced mitochondrion-dependent cell death. In fact, knock-
down of MAVS gene expression by small interfering RNAs
increased apoptosis, possibly hinting at a protective role for
MAVS during the early stages of viral infection. Potentially,
the activation of other components of the mitochondrial mem-
brane could also contribute to signaling to the antiviral re-
sponse. In support of this idea, MAVS colocalizes in the same
detergent-insoluble fraction as the antiapoptotic protein Bcl-
xL. Among the many CARD-containing proteins with roles in
apoptosis and immunity (Apaf1, NOD1, NOD2 RIP2, and
RIG-I), MAVS is unique (7, 11, 12). The localization of this
CARD domain-containing adapter to the mitochondrial mem-
brane is highly strategic and may help the host cell to sense
incoming viral challenge and coordinate an immune or apop-
totic response, depending on the pathogen. Many viruses rep-
licate in intracellular organelles, such as the endoplasmic re-
ticulum; a good example is HCV, which replicates in the
membranous web that connects the endoplasmic reticulum to
the mitochondria. dsRNA structures, possibly within replicat-
ing ribonucleoprotein complexes, may be recognized by RIG-I
and/or Mda-5, resulting in downstream signaling through
MAVS. Mitochondria may be at the center of a delicate bal-
ancing act between the host immune response and virus-in-
duced apoptosis. In the case of HCV infection, cleavage of
MAVS by the NS3-4A protease appears to tip the balance,
resulting in disruption of innate immune responses and the
establishment of chronic HCV persistence (15).
The identification of MAVS/IPS-1/VISA/Cardif and its role
in innate signaling and its characterization as the physiologi-
cally relevant target of the NS3-4A protease are important
steps in the complete understanding of the mechanisms by
which HCV evades the early host response. The essential lo-
calization of this CARD domain-containing adapter to the
mitochondria furthermore suggests an important function in
the coordination of the innate immune and apoptotic re-
sponses. The implications for the study of HCV pathogenesis
are particularly profound, given that experimental compounds,
such as BILN2061 and VX-950, that block NS3-4A protease
activity may accomplish two goals: inhibition of virus multipli-
cation and processing and restoration of the early innate im-
mune response that is critical to the development of a robust
adaptive response in patients (9, 22).
We thank Hong-Bing Shu, Zhijian Chen, Ganes Sen, Wen-Zhe Ho,
and Michael Gale for reagents used in this study and members of the
Molecular Oncology Group, Lady Davis Institute, for helpful discus-
This research was supported by grants from the Canadian Institutes
of Health Research (J.H. and R.L.), CANVAC, and the Canadian
Network for Vaccines and Immunotherapeutics (J.H.) and by the
National Cancer Institute of Canada, with the support of the Canadian
Cancer Society (J.H.). D.V. was supported by l’Agence Nationale de la
Recherche sur le Sida (ANRS). R.L. was supported by an FRSQ
Chercheur-boursier and J.H. by a CIHR Senior Investigator award.
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