functional and therapeutic analysis of hepatitis c virus ns3/4a protease control of antiviral immune

25 Pages·2007·4.32 MB·English

JBC Papers in Press. Published on February 8, 2007 as Manuscript M610361200
The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.M610361200
FUNCTIONAL AND THERAPEUTIC ANALYSIS OF HEPATITIS C
VIRUS NS3/4A PROTEASE CONTROL OF ANTIVIRAL IMMUNE
DEFENSE
Cynthia L. Johnson, David M. Owen, and Michael Gale, Jr.
Department of Microbiology, University of Texas Southwestern Medical Center, Dallas,
Texas, 75390
Running title: Structure-function analysis of HCV NS3/4A
Address correspondence to: Michael Gale, Jr., Department of Microbiology, UT Southwestern Medical
Center, 5323 Harry Hines Blvd. NA6.308, Dallas, Texas 75390-9048.
Tel. [phone]. Fax [phone]. E-Mail: [email protected]

Chronic hepatitis C virus (HCV) infection is a restore innate immune defenses to HCV
major global public health problem. HCV infection.
infection is supported by viral strategies to
evade the innate antiviral response, wherein the Hepatitis C virus (HCV) establishes
viral NS3/4A protease complex targets and persistent infections in the majority of exposed
cleaves the interferon promoter stimulator-1 individuals. There are nearly 200 million people D
o
(IPS-1) adaptor protein to ablate signaling of worldwide with chronic HCV infection, and it is a w
n
interferon α/β immune defenses. Here we major cause of hepatitis and liver disease. loa
d
e
examined the structural requirements of Moreover, HCV is associated with the d
NS3/4A and the therapeutic potential of development of liver cancer and is currently the from
NS3/4A inhibitors to control the innate immune leading indication for liver transplants (1,2). HCV http
response against virus infection. The structural is a member of the Flaviviridae family of ://w
composition of NS3 includes an amino-terminal enveloped, single-stranded, positive-sense RNA w
w
serine protease domain and a carboxyl- viruses. The 9.6 kb genome of HCV encodes a .jb
c
terminal RNA helicase domain. NS3 mutants single polyprotein that is post-translationally .org
lacking the helicase domain retained the ability processed into at least 10 structural and b/
y
to control virus signaling initiated by retinoic nonstructural (NS) proteins by a combination of gu
e
s
acid inducible gene-I (RIG-I) or melanoma host and viral proteases (3). In addition to their t o
n
differentiation antigen 5, and suppressed the primary role in HCV genome replication and A
p
downstream activation of interferon regulatory virion maturation, various HCV proteins have ril 7
factor (IRF)-3 and nuclear factor kappa B (NF- been shown to antagonize host immune defenses. , 2
0
κB) through the targeted proteolysis of IPS-1. HCV control of the host response and interferon 19
This regulation was abrogated by truncation of (IFN) α/β antiviral defenses may provide a
the NS3 protease domain or by point mutations cellular foundation for viral persistence (4).
that ablated protease activity. NS3/4A protease The innate antiviral response to RNA
control of antiviral immune signaling was due virus infection is triggered when intermediates of
to targeted proteolysis of IPS-1 by the NS3 viral replication, including viral RNA or protein
protease domain and minimal NS4A cofactor. products, are recognized by specific cellular
Treatment of HCV-infected cells with a NS3 pathogen recognition receptors (5). Retinoic acid
protease inhibitor prevented IPS-1 proteolysis inducible gene-I (RIG-I) and melanoma
by the HCV protease and restored RIG-I differentiation associated gene 5 (MDA5) are
immune defense signaling during infection. cytosolic pathogen recognition receptors that bind
Thus, the NS3/4A protease domain can target to viral RNA and double-stranded RNA (dsRNA),
IPS-1 for cleavage and is essential for blocking albeit with variable efficiency (6,7). Both are
RIG-I signaling to IRF-3 and NF-κB, while the DExD/H-box RNA helicases and encode tandem
helicase domain is dispensable for this action. amino-terminal caspase activation and recruitment
Our results indicate that NS3/4A protease domains (CARDs)(6). RIG-I is an essential
inhibitors have immunomodulatory potential to pathogen recognition receptor for various
Copyright 2007 by The American Society for Biochemistry and Molecular Biology, Inc.
negative-strand viruses and Flaviviridae (8), activity (33,36-38). Moreover, structural studies
including HCV (9,10). RNA binding by RIG-I of full-length NS3 indicate that the C-terminus of
and MDA5 promotes a conformation change that the helicase domain may interact with the protease
potentiates signaling by the CARDs (9,11) to a active site (39), suggesting a structural and
CARD-containing adaptor protein termed IFN-β functional interconnectivity of the two domains.
promoter stimulator 1 [IPS-1; reviewed in (12)], NS3 also encodes an arginine-rich region shown to
located on the mitochondrial membrane. IPS-1 bind other host proteins (40,41). NS3/4A
confers downstream signaling to the latent interaction with its cellular targets could therefore
cytoplasmic transcription factors IFN regulatory occur through a variety of processes mediated by
factor-3 (IRF-3) and nuclear factor-κB (NF-κB) its helicase or protease domains.
(13-16). IRF-3 activation commences with its Several peptidomimetic NS3/4A protease
virus-induced phosphorylation, dimerization, and active site inhibitors are in preclinical
nuclear translocation whereupon it binds to the development as HCV antiviral drugs (42). Future
promoter region of IRF-3-dependent genes, use of these compounds could represent an
including IFN-β, IFN-stimulated gene (ISG) 15, alternative or supplement to the current treatment
and ISG56 (17-19). Parallel NF-κB activation regimen for HCV infection, IFN-α and ribavirin
induces a variety of proinflammatory genes and combination therapy, which is only effective in
cooperates with IRF-3 to induce IFN-β expression about 50% of patients (43). NS3/4A protease D
o
(20). ISGs have antiviral and immunomodulatory inhibitors may also exhibit immunomodulatory w
n
activity that limit HCV infection (4,21-23), and properties by removing the protease-dependent lo
a
d
their expression marks the effector stage of the blockade to the innate antiviral response during ed
innate antiviral response (24). HCV replication (23,25), although the impact of fro
m
HCV evades the innate antiviral response IPS-1 in this process is not known. The current h
ttp
in part through the actions of the viral NS3/4A study was undertaken to define the domain ://w
protease/helicase complex, which ablates RIG-I structure and function of NS3/4A that regulates w
w
and MDA-5 signaling (6,10,25) through the host cell innate antiviral response. .jb
c
proteolytic cleavage and inactivation of IPS-1 .o
rg
(16,26,27). NS3/4A cleaves IPS-1 at C508, b/
y
thereby releasing it from the mitochondrial g
u
e
membrane and preventing its downstream EXPERIMENTAL PROCEDURES st o
activation of immune effectors (27). However, the n A
p
NIPSS3-/14 Ac lesatrvuacgtue raaln md oltoifsss aonfd RfeIaGtu-Ir esa ntdh atM dDirAec5t MutagenEexspisr e–s spioFnL AGCl oNnSin3g/4 Aa nandd pSFitLe-AdGir eNctSe3d ril 7, 2
0
signaling have not been defined. were generated as described (23). Constructs (Fig. 1
9
NS3/4A is a complex, bifunctional 1) containing NS3 helicase domain truncations at
molecule (Fig. 1) that is essential for NS protein amino acid (aa) 1206 (protease domain alone,
processing and viral RNA replication (28,29). The Prot), 1238, 1486, and 1501, as well as a protease
amino-terminal region of NS3 contains a serine domain deletion (NS3 aa 1207-1658, Hel) were
protease that non-covalently binds its cofactor, made as follows. NS3 mutants were cloned by
NS4A. NS4A is a small peptide necessary for PCR (with primers containing a 5’HindIII site and
efficient processing of the HCV polyprotein by a 3’XbaI site) using BD Advantage-HF 2 PCR kit
NS3 and is thought to be involved in tethering the (BD Biosciences) into pCR2.1 vector (TA cloning
complex to intracellular membranes (30,31). The kit, Invitrogen). These constructs were then
carboxy-terminus of NS3 possesses a DExD/H subcloned into the HindIII and XbaI sites of
box helicase with nucleoside triphosphatase pFLAG-CMV-2 vector (Sigma). A protease
(NTPase) activity believed to participate in RNA active site mutant (NS3/4A S1165A) and deletion
unwinding during viral RNA replication (32). Its of arginine-rich region (aa 1487-1501; NS3/4A
helicase activity was found to be positively Δ1487-1501) were constructed using
modulated by the protease domain and NS4A (33- QuickChange XL site-directed mutagenesis kit
35). Conversely, conserved motif VI of the (Stratagene). Since NS4A forms part of the
helicase domain has been shown to affect protease protease active site and is indispensable for full
2
protease activity (31), a scheme was devised Hyclone), 1X nonessential amino acids, 2 mM L-
allowing expression of NS4A with the NS3 glutamine, antibiotic-antimycotic solution, and 1
mutants. Expressing NS4A from a separate mM sodium pyruvate as described previously (25).
plasmid was not sufficient to result in efficient Huh7.5 cells, a subline of Huh7 cells kindly
protease activity (data not shown). To circumvent provided by C. Rice, contain a defect in RIG-I and
this problem we created a primer containing an do not signal through the RIG-I pathway (10).
amino-terminal KpnI site, 12 amino acids of UNS3/4A and UNS3 cells (a gift from D.
NS4A (aa 21-32; which intercalate into the NS3 Moradpour) are osteosarcoma cells conditionally
protease active site), and a flexible linker (GSGS) expressing HCV 1a NS3/4A or HCV 1a NS3,
region, and subcloned this into the HindIII sites of respectively, and were described previously
the pFLAG NS3 1026-1206, creating a fully- (30,48). HP and K2040 cells are Huh7 cells
functional, single-chain (SC) NS3/4A protease harboring cell culture-adapted subgenomic HCV
domain (SC Protease). This strategy was similar 1b replicons (11,22). Cell culture methods for
to that described by Taremi et al. (44). The UNS3/4A, HP, and K2040 cells have been
flexible linker was also subcloned into pFLAG described elsewhere (11,22,23). For Sendai Virus
NS3 (SC NS3), pFLAG NS3 1026-1238 (SC NS3 infection (Cantell strain, Charles River
1026-1238), pFLAG NS3 1026-1486 (SC NS3 Laboratories), 1x105 or 2x104 cells/well were
1026-1486), and pFLAG NS3 1026-1501 (SC NS3 seeded into 12-well or 48-well dishes respectively,
D
o
1026-1501) to generate the SC constructs and where indicated transfected with expression w
n
indicated in parentheses. NS3/4A, NS3/4A constructs. Twenty-four hours after seeding or lo
a
d
S1165A, and the SC Protease were subcloned into transfection, cells were washed twice in PBS and ed
pEF1/Myc-His B (Invitrogen) to allow high mock-infected or infected with 100 hemagglutinin fro
m
efficiency expression of each. NS3/4A and units (HAU) of Sendai virus per mL of serum-free h
ttp
NS3/4A S1165A were cut with HindIII, blunt- media (SFM) for one hour. Three volumes of ://w
ended with Kleenow fragment, digested with XbaI culture media were then added to wells and cells w
w
and subcloned into the EcoRV and XbaI site of incubated another 17 or 19 hours as described (23). .jb
c
pEF1/Myc-HisB. The SC Protease was subcloned HCV 2a stock was produced as described .o
rg
into the SacI and XbaI sites of pEF1/Myc-His B. previously (27). For HCV infection, b/
y
Primer sequences for all constructs are available approximately 4x104 cells seeded in a 12-well g
u
e
upon request. plate were infected at a MOI of 1 with HCV 2a in st o
Plasmids and Transfection – pFLAG culture media for 3 hours, washed in PBS, and n A
p
NprSev4iAo,u splFyL dAeGsc rNibSe5dA (, 2a3n,d2 5p,4F5L)A. G p NEFSB5Aos/-BR IwGe-rIe, cuunlttiul rceo lmleecdtiioan aadtd tehde. in Cdieclalste dw teirme et hpeonin tisn.c uSbCaHte6d ril 7, 2
0
pEFBos-N-RIG, and pEFBos-C-RIG expression (kindly provided by Schering-Plough)(23) and 1
9
plasmids (7) as well as pEFBos-MDA5, pEFBos- ITMN-C (a gift from Dr. S. Seiwert at Intermune,
N-MDA5, and pEFBos-C-MDA5 (6) are amino- Brisbane, CA) protease inhibitor treatments were
terminally FLAG-tagged. pEFBos, pEFBos-IPS-1, conducted by replacing culture medium with
pMyc-IPS-1, pIRF3-ΔN, pIFN-β-luc, pCMV- medium containing 20μM SCH6 or the indicated
Renilla-luc, and pPRDII-luc were described amount of ITMN-C.
elsewhere (23,25,27,46). GenElute Endotoxin- Confocal Microscopy – 2x104 cells were
free Plasmid Midiprep kit (Sigma) was used to plated in 4-chamber slides, 200 ng of the indicated
prepare plasmid DNA for transfections. expression plasmids transfected, and cells
Transfections were performed using Fugene 6 collected 12 or 24 hours later. Cells were fixed in
transfection reagent (Roche Molecular 4% paraformaldehyde and blocked in PBS
Biochemicals) or Lipofectamine 2000 (Invitrogen) containing 10% FBS and 1% Triton X-100.
according to manufacturer’s protocol. Antibodies against the FLAG epitope (Sigma),
Cells, Viruses, and Protease Inhibitors – IPS-1 (a gift from Z. Chen), IRF-3 (kindly
Huh7 human hepatoma cells (47), Huh7.5, and provided by M. David), and HCV NS3 NCL
HEK 293 cells were propagated in Dulbecco’s (Novacastra laboratories) were used in
Modified Eagle Medium (DMEM, Cellgro) combination with Alexa 488- or Alexa 594-
supplemented with 10% fetal bovine serum (FBS, conjugated secondary antibodies (Invitrogen).
3
Slides were mounted with Prolong Gold and mutant oligonucleotides were obtained from
(Invitrogen), and examined using a Zeiss Pascal Santa Cruz Biotechnology. Non-radiolabeled,
Laser Scanning confocal microscope and Zeiss cold competitor was used as a negative control.
LSM software. The images shown are ≤ 7 micron Statistical Analysis – Differences among
optical slices. treatments were examined using unpaired t-tests,
Immunoblotting and Immunoprecipitation and were considered statistically significant when
Analysis – Immunoblots were performed as P < 0.02 and were marked with an asterisk.
described (23). Blots were probed with antibodies Statistical differences in which P ≤ 0.001 were
against HCV NS3 A6 (kindly provided by J. Ye), marked with two asterisks.
ISG56 (a gift from G. Sen), ISG15 (kindly
provided by A. Haas), Sendai virus (a gift from I. RESULTS
Julkunen), actin (Santa Cruz Biotechnology), NS3
goat pAb (US Biologicals), Phospho-IκBα (Cell Characterization of NS3/4A constructs. In
Signaling), IκBα (Santa Cruz Biotechnology), order to determine the minimal region of NS3/4A
GAPDH (Santa Cruz Biotechnology), HCV (a gift that could modulate the host response, a series of
from W. Lee), ISG48 (Santa Cruz Biotechnology), FLAG epitope-tagged NS3 truncation and deletion
Myc epitope (Bethyl Laboratories) or those mutants were constructed (Fig. 1). To assess
described above. Anti-human and anti-goat HRP individual domains of NS3 and NS4A in host D
o
conjugated secondary antibodies were obtained response regulation, we produced constructs w
n
from Jackson Laboratories; anti-rabbit and anti- encoding NS4A alone (23), the NS3 protease lo
a
d
mouse HRP conjugated secondary antibodies were domain (Protease), helicase domain (Helicase), or ed
obtained from Perkin Elmer. full-length NS3 lacking the arginine-rich region fro
m
Coimmunoprecipitation was performed using (ΔArg), as well as full-length NS3/4A harboring a h
ttp
FLAG M2 agarose beads (Sigma) as described protease activate site mutation (S1165A) as a ://w
(45) and immunoblot analysis was conducted. negative control. These were placed under the w
w
Promoter Luciferase Reporter Assays – control of the CMV-Immediate-Early promoter. .jb
c
2x104 cells/well were seeded into 48-well dishes Previous studies have identified a twelve amino .o
rg
and cotransfected with 25ng of pIFN-β-luc or acid region of NS4A (aa 21-32) that intercalates b/
y
pPRDII-luc and 5ng pCMV-Renilla along with the into the NS3 protease active site and is g
u
e
indicated amounts of expression constructs. Cells indispensable for full protease activity (31). In st o
n
were then cultured alone or subjected to virus order to express full NS3/4A protease activity A
p
infection and harvested 20 hours later for from a single plasmid, we attached these 12 aa of ril 7
luciferase assay as described (23). Results were NS4A to each NS3 mutant via a flexible linker, , 2
0
calculated relative to Renilla luciferase values. creating a fully-functional, single-chain (SC) 1
9
Electrophoretic Mobility Shift Assay NS3/4A protease encoding full-length NS3 (SC
(EMSA) – EMSA analysis of NF-κB DNA binding NS3), progressive deletions of the NS3 helicase
activity was conducted on UNS3/4A and UNS3 domain (SC 1501, SC 1486, SC 1238), or the
nuclear extracts as described (46). Briefly, cells protease domain alone (SC Protease). Use of
were grown in the presence (-NS3/4A) or absence similar SC constructs as a tool for examining the
(+NS3/4A, +NS3) of 1 mg/ml tetracycline, and NS3/4A protease structure and function has been
protease inhibitor (20 µM SCH6 or 10 µM ITMN- well established in crystallization studies (39) and
C) or DMSO (mock treatment) was added to the studies identifying small molecule protease
media for 24 hours. Cells were then mock- inhibitors (49). Moreover, Taremi et al. found that
infected or infected with 100 HAU/ml of Sendai such SC Protease constructs displayed protease
virus and infection was allowed to proceed 18 activity comparable to wildtype (wt) NS3/4A (44).
hours. As a positive control for NF-κB activation, As shown in Fig. 2A, when coexpressed in Huh7
some wells were washed twice in PBS and treated cells, a NS5AB polyprotein substrate was
for 30 minutes with 20 ng/mL TNF-α in SFM. efficiently cleaved by wt NS3/4A, SC Protease,
Antibody supershifts were performed using SC NS3, ΔArg, SC 1238, SC 1486, and SC 1501
antibodies against NF-κB p50 or p65 subunits constructs (middle panel, lanes 4, 7, 8, 14, 16-18,
(Santa Cruz Biotechnology). NF-κB consensus respectively). The NS5AB polyprotein was not
4
cleaved by NS4A, NS3/4A S1165A, Helicase, SC suppresses RIG-I signaling of the host response
NS3 S1165A, or Protease (lanes 3, 5, 6, 9, 15, (25). We verified that our wt NS3/4A construct
respectively), and was only partially cleaved by could suppress RIG-I-enhanced virus signaling as
NS3 alone (lane 2). When expressed from the well as constitutive signaling by ectopic N-RIG
elongation factor-1 promoter expression plasmid (encoding the CARD domains alone) (Fig. 3A).
(pEF), pEF wt NS3/4A, and pEF SC Protease but We then assessed regulation of RIG-I signaling to
not pEF NS3/4A S1165A also mediated NS5AB the IFN-β promoter by a subset of our NS3
cleavage (compare lanes 11-13). These results constructs. As shown in Fig. 3B, the SC Protease
validate the expression and cleavage activity of the and ΔArg constructs suppressed N-RIG signaling
NS3 constructs, and show that efficient NS3/4A of the IFN-β promoter to an extent similar to wt
proteolytic activity requires the protease domain NS3/4A. Expression of NS3 alone, NS4A,
and its NS4A cofactor but does not require the NS3/4A S1165A, or the NS3 helicase domain had
NS3 helicase domain. no significant effect on promoter activation.
The helicase domain is dispensable for Moreover, when expressed in Huh7 cells, wt
inhibition of innate defense signaling. To NS3/4A and the SC Protease suppressed the
determine the NS3 structural requirements for expression of the IRF-3 target genes, ISG56 and
inhibition of host antiviral signaling, we evaluated ISG15, but NS3/4A S1165A and the NS3 Protease
each construct for its ability to regulate IFN-β domain failed to suppress IRF-3 target gene D
o
promoter induction in response to Sendai virus expression (Fig. 3C). w
n
infection. It should be noted that Sendai virus and We also examined NS3 construct loa
d
HCV trigger the host response through similar regulation of MDA5 signaling. In order to ed
mechanisms that are blocked by NS3/4A determine the effect of MDA5 regulation in the from
(10,23,25). Wt NS3/4A significantly suppressed absence of RIG-I, we utilized Huh7.5 cells lacking http
Sendai virus induction of IFN-β promoter activity RIG-I function (10). As shown in Fig. 4A, Huh7.5 ://w
comparable to a dominant-negative IRF-3 control cells failed to activate the IFN-β promoter in ww
construct (Fig. 2B, IRF3-ΔN). However, response to Sendai virus infection, but ectopic .jb
c
constructs lacking NS4A and/or the intact protease MDA5 complemented this defect. Moreover, we .org
domain of NS3 failed to block signaling to the verified that our wt NS3/4A construct could b/
y
g
IFN-β promoter. Of note, the SC NS3 construct, suppress wt MDA5 or N-MDA5 (encoding the u
e
s
similar to that used in crystallization studies (39), CARD domains) signaling to the IFN-β promoter t o
n
mediated a weaker albeit significant suppression in Huh7.5 cells. To determine the minimal A
p
of virus signaling to the IFN-β promoter. Further domain of NS3/4A necessary to block MDA5 ril 7
studies revealed that the SC NS3 protein did not signaling, we evaluated the ability of constructs , 2
0
localize to membrane-bound compartments (data encoding wt NS3/4A (positive control), NS3/4A 19
not shown), suggesting its decreased ability to S1165A (negative control), the SC Protease, and
block viral activation of the IFN-β promoter could Protease to regulate signaling to the IFN-β
be due to aberrant localization. We also assessed promoter by N-MDA5 (Fig. 4B). The SC Protease
Sendai virus-induced ISG expression in cells inhibited promoter activation to the same extent as
transfected with constructs encoding wt NS3/4A, wt NS3/4A and the IRF3-ΔN control, but neither
NS3/4A S1165A, or the SC Protease. As shown the NS3 protease domain alone nor NS3/4A
in Fig. 2C, wt NS3/4A and the SC Protease S1165A blocked promoter signaling. Both wt
suppressed ISG production, but ISGs were NS3/4A and the SC Protease suppressed ISG15
induced in cells expressing the protease-deficient expression in response to ectopic N-MDA5 (Fig.
NS3/4A S1165A mutant. Together these results 4C). Taken together, these results demonstrate
indicate that the fully-functional NS3 protease that the SC Protease is sufficient to block signaling
domain with its minimal NS4A cofactor is by both RIG-I (Fig. 3) and MDA5 (Fig. 4) in a
necessary for suppressing the host cell signaling of process that requires the NS3 protease domain and
IFN-β promoter expression. its NS4A cofactor. The NS3 helicase domain,
The functional NS3/4A protease domain however, is dispensable for host response
inhibits signaling by RIG-I and MDA5. NS3/4A regulation.
5
The SC Protease blocks activation of IRF- Protease inhibited activation of the PRDII
3 and exhibits a subcellular localization pattern promoter element in response to both Sendai virus
similar to wt NS3/4A. In order to verify that the infection and ectopic N-RIG. Similar results were
SC Protease was sufficient for inhibition of IRF-3 observed when the PRDII promoter element was
activation, we examined IRF-3 and SC Protease stimulated by ectopic N-MDA5 (data not shown).
distribution in transfected Huh7 cells. Sendai We also examined the influence of NS3/4A upon
virus infection of cells caused the redistribution of virus-induced phosphorylation of the NF-κB
IRF-3 from the cytoplasm to the nucleus (23), inhibitor, IκBα. As shown in Fig. 6D, Sendai
consistent with its virus-induced activation, but virus infection of HEK 293 cells induced the
this response was blocked in control cells phosphorylation of IκBα within 20 hours post
expressing wt NS3/4A (Fig. 5). Expression of the infection, but expression of either wt NS3/4A or
SC Protease also blocked IRF-3 activation, the SC Protease but not NS3/4A S1165A
maintaining it in a cytoplasmic context during prevented virus-induced IκBα phosphorylation
Sendai virus infection. Notably, despite the lack (compare lanes 2, 4, 6, 8). These results link HCV
of the putative amino-terminal membrane control of NF-κB function with NS3/4A protease
anchoring motif of NS4A (30), the SC Protease domain regulation of RIG-I and MDA5.
localized to a subcellular compartment similar to The protease domain of NS3/4A is
wt NS3/4A. Thus, the NS3 protease domain and a sufficient to target and cleave IPS-1. RIG-I and
D
o
minimal NS4A cofactor are sufficient to confer MDA5 signal IRF-3 and NF-κB activation through w
n
proper localization and inhibition of IRF-3 the IPS-1 adaptor protein (13), which is targeted lo
a
d
activation. and cleaved by NS3/4A to inactivate signaling ed
Regulation of NF-κB by NS3/4A. RIG-I (16,27). To determine if the functional NS3/4A fro
m
and MDA5 signal the parallel activation of IRF-3 protease domain was sufficient to physically target h
ttp
and NF-κB (6), and we sought to examine the and cleave IPS-1, we examined complex ://w
effect of NS3/4A and its requirement for protease formation between IPS-1 and the SC Protease or a w
w
activity on regulation of NF-κB function. We protease-defective derivative (Fig. 7A, wt and SA, .jb
c
performed EMSA to measure NF-κB binding to a respectively) in transfected Huh7 cells. .org
target probe in nuclear extracts of cells treated Immunoprecipitation studies revealed that the b/
y
with tumor necrosis factor-α (TNF-α; control) or functional SC Protease and a SC Protease S1165A gu
e
infected with Sendai virus. TNF-α or Sendai virus mutant each bound to IPS-1 (Fig. 7A lanes 2 and st o
n
infection triggered NF-κB binding (Fig. 6A, lanes 3). When compared to the SC Protease, we A
p
1 and 5, respectively). Expression of NS3/4A observed an apparent reduction in the amount of ril 7
(lane 11) but not NS3 alone (lane 17) blocked SC Protease S1165A mutant bound to IPS-1 in , 2
0
these experiments. While this could be due to 1
Sendai virus-induced NF-κB binding. The 9
protein expression or stability differences between
NS3/4A blockade of NF-κB binding was relieved
the SC Protease constructs, it could also be
when cells were treated with SCH6 or ITMN-C
explained by potential changes in the active site
peptidomimetic active site inhibitors of the
conformation caused by the serine to alanine
NS3/4A protease (lanes 13 and 15, respectively).
switch, possibly resulting in decreased affinity for
These results were verified by luciferase reporter
cleavage substrates. Importantly, the interaction
assay, in which wt NS3/4A suppressed Sendai
between IPS-1 and the SC Protease was not
virus induction of an NF-κB-dependent PRDII-
disrupted by ITMN-C protease inhibitor treatment,
luciferase promoter and its enhancement by
and the SC Protease maintained an interaction
ectopic RIG-I or MDA5 (Fig. 6B). To determine
with the cleaved form of IPS-1 (Fig. 7A, lanes 3
the domain of NS3/4A necessary for this effect,
and 4). Ectopic IPS-1 is a potent trigger of the
HEK 293 cells were transiently transfected with
host response (13), and we found that it triggered
the indicated NS3 construct followed by Sendai
endogenous ISG15 expression in Huh7 cells (Fig.
virus infection (Fig. 6C, left panel) or were
7B). This reaction was blocked by both the SC
cotransfected with a construct encoding
Protease and wt NS3/4A concomitant with IPS-1
constitutively active N-RIG to trigger PRDII
cleavage. Thus, the SC Protease is sufficient to
promoter signaling (Fig. 6C, right panel). The SC
target and cleave IPS-1. We also found that the
6
SC Protease suppressed IFN-β promoter induction to HCV, we examined ITMN-C treatment of Huh7
by ectopic IPS-1 as well as wt NS3/4A (Fig. 7C). cells harboring a HCV 2a replicon or infected with
Further, the SC Protease blocked NF-κB- the JFH1 strain of HCV 2a. Treatment of HCV 2a
dependent induction of the PRDII promoter replicon cells resulted in alteration of endogenous
element by ectopic IPS-1, but neither NS3 nor the IPS-1 from a predominantly cleaved form to its
protease or helicase domains affected promoter native, full-length form concomitant with
induction (Fig. 7D). Thus, the targeted cleavage induction of ISG56 expression over a 48 hour time
of IPS-1 by the NS3/4A protease domain inhibits course (Fig. 9A and B). To verify these results in
downstream signaling to IRF-3 and NF-κB. the context of an actual HCV infection, Huh7 cells
NS3/4A protease inhibitor prevents were infected with JFH1 (50) at an MOI of 1 for
cleavage and restores localization of IPS-1 To 48 hours followed by 24, 36, or 48 hours ITMN-C
determine the influence of the NS3/4A protease treatment (Fig. 9C). After 48 hours of HCV
and protease inhibitor treatment on IPS-1 infection, endogenous IPS-1 levels were reduced
localization and function, we examined the with a large portion of the remaining protein
subcellular distribution of IPS-1 in the presence of present as the cleaved form, consistent with
NS3/4A alone and in the context of HCV RNA previously published data (27). Within 24 hours
replication when cells were treated with ITMN-C. of ITMN-C treatment and throughout the time
As shown in Fig. 8A, IPS-1 showed a course, we observed a recovery of full-length IPS- D
o
characteristic thread-like appearance in the cell 1 levels with corresponding induction of ISG56 w
n
cytoplasm, consistent with its mitochondrial expression and reduction in viral protein loa
d
membrane localization (27). However, in the abundance (Fig. 9C). ed
presence of NS3/4A, this localization was fro
m
disrupted, and IPS-1 exhibited the diffuse DISCUSSION h
ttp
cytoplasmic staining pattern previously described ://w
(26,27). When the cells were exposed to the Our results show that the NS3 protease w
w
ITMN-C protease inhibitor for 24 hours, IPS-1 domain and minimal NS4A cofactor are sufficient .jb
c
was restored to its proper localization in the to inhibit antiviral signaling through RIG-I and .org
presence of NS3/4A (Fig. 8A). We further MDA5 and block activation of the downstream b/
y
examined this regulation in the context of HCV transcription factors IRF-3 and NF-κB. This is gu
e
RNA replication in two cell lines harboring accomplished by protease domain targeting and st o
genetically distinct HCV replicons that are cleavage of IPS-1. Our data indicate that the NS3 n A
p
d(Kif2fe0r4e0n tiraelplyli croensi)s ttaon tt h(eH Pa nrteivpilricaol na)c tainodn ss eonfs iItFivNe hreeslpicoanssee droemgualiant iodno esa ndn oti napclatiyv aati orno leo f inI PhSo-1st. ril 7, 2
0
therapy (11,22). In the presence of NS3, IPS-1 Moreover, infection studies demonstrated for the 1
9
membrane localization was disrupted, whereas first time that NS3/4A protease inhibitor therapy
K2040 cells with no visible NS3/4A staining of HCV infection can effectively remove the
typically retained a normal pattern of IPS-1 NS3/4A blockade to the host response, restoring
localization (Fig. 8B, mock treated). When the RIG-I signaling to IPS-1 and activation of ISG
cells were treated with ITMN-C, IPS-1 returned to expression in infected cells.
a native distribution within a 24 hour time course The protease domain of NS3 has been
even in the presence of NS3/4A (Fig. 8B, +ITMN- shown to mediate internal interactions with the
C). Moreover, during ITMN-C treatment the carboxyl terminal helicase domain, in which the
colocalization of NS3/4A with IPS-1 became latter has been suggested to potentiate protease
apparent, suggesting they are initially present in activity (37-39). We found that the helicase
the same subcellular milieu and that NS3/4A domain was dispensable for NS3/4A control of the
cleavage of IPS-1 disperses it from this host response and signaling by RIG-I and MDA5.
environment (Fig. 8A, B). That the helicase domain is not involved in host
ITMN-C restores the endogenous host response control is further supported by the
response triggered by HCV RNA replication and observations that the SC Protease, lacking the
HCV infection. To examine the effect of protease complete helicase domain of NS3, mediated
inhibitor treatment on IPS-1 and the host response binding and cleavage of IPS-1 similar to wt
7
NS3/4A. Taken together, these results imply that (39,54,55). Indeed, the TRIF adaptor protein has
the NS3 helicase domain does not play a role in been identified as an in vitro substrate of NS3/4A
directing NS3/4A interactions with IPS-1. proteolysis (56). In this case residues within the
Importantly, our results show that neither full- 3/10 helix of NS3 located adjacent to the protease
length NS3 nor the NS3 protease domain alone active site bind to a region of TRIF near the
could block signaling by RIG-I or MDA5 when cleavage site (57). This shared relationship
expressed in the absence of NS4A; however, between protein interaction and cleavage could
NS4A alone does not regulate the host response. indicate that NS3/4A may target and interact with
In contrast, each NS3 construct efficiently TRIF and IPS-1 through similar processes. The
suppressed signaling when expressed as a SC mechanics of the NS3/4A-IPS-1 interaction are
construct with aa 21-32 of NS4A, encoding the currently under investigation.
NS4A central core region integral to NS3 protease Our study demonstrates that NS3/4A
activity (31). Previous work has associated NS4A proteolysis of IPS-1 blocks signaling by both RIG-
aa 2-19 with localizing NS3 to intracellular I and MDA5, and ablates virus activation of
membranes (30). Despite lacking this domain of downstream IRF-3 and NF-κB, thus confirming
NS4A, our SC constructs effectively cleaved that IPS-1 serves as the essential adaptor for RIG-I
NS5AB and blocked host response signaling. and MDA5 signaling. In the case of NF-κB, we
Moreover, when expressed in Huh7 cells, the SC found that the NS3/4A or SC Protease blockade of D
o
Protease construct demonstrated a subcellular NF-κB activation was concomitant with w
n
distribution pattern equivalent to wt NS3/4A that lo
suppression of IκB-α phosphorylation, and that a
d
tshimati lwarhlyil es uNppSo4rAte di sI PrSe-q1u itraerdg eftoinrg f. u lWl ep rcootenocllyutdiec cNeFll-sκ Bw aecreti vattrieoante dc ouwldit hb e af ulNlyS 3r/e4sAto repdr owtehaesne ed from
action of NS3, localization and IPS-1 targeting by h
inhibitor. Thus, the RIG-I/MDA5 axis may signal ttp
NS3/4A are controlled by residues located within NF-κB activation through a canonical process of ://w
the NS3 protease domain and/or the NS4A central w
kinase phosphorylation of IκB and unmasking of w
core. The amino-terminal 21 aa of NS3 consist of NF-κB DNA-binding activity (58). Similar to the .jbc
several highly hydrophobic residues, and crystal .o
control of IRF-3 signaling (23,25,27), these rg
structure analysis showed that this region extends b/
processes of NF-κB signaling are disrupted by y
away from the rest of the protease (51), suggesting g
u
IPS-1 proteolysis. As a signaling adaptor protein, e
iAt ddmitaioyn ablley , inav orlveceedn t ins tumdeym bdreamneo nsttertahteerdi nga. IPS-1 may serve to recruit factors of IRF-3 and/or st on A
NF-κB activation. This idea is supported by p
mthiet occhoonntdexrita l omf emHbCraVn e RloNcaAli zarteipolni coafti oNnS 4(A5 2i)n, swtuitdhi evsa rthioaut sh saivgen adleinmgo ncostmrapteodn eInPtSs,- 1in icnlutedriancgti othnes ril 7, 20
implicating NS4A in directing NS3 to the 1
9
IKKε protein kinase (59), TRAF3 (60), and FADD
mitochondria and site of interaction with IPS-1.
(13) into a complex with IPS-1. In the context of
These possibilities are presently being examined.
HCV infection, an IPS-1 signaling complex would
We found that the SC Protease could bind
possibly be disrupted through the targeted
IPS-1 and that this interaction was maintained in
proteolysis of IPS-1 by NS3/4A to ablate a wide
the presence of the peptidomimetic protease active
range of immune signaling action.
site inhibitor ITMN-C, which abrogated IPS-1
NS3/4A protease inhibitors are being
proteolysis (Fig. 7A). Under these conditions
developed for use as future HCV therapeutics (42)
ITMN-C is expected to occupy the protease active
and our studies define a novel role for these
site and catalytic residues (53). Thus, the SC
compounds as immunomodulatory agents.
Protease may bind IPS-1 independently of
NS3/4A cleavage of IPS-1 during HCV RNA
substrate occupancy in the protease active site.
replication and infection results in IPS-1 release
This suggests a model in which NS3/4A may first
from the mitochondrial membrane and diffuse
interact with IPS-1 through protease domain
redistribution throughout the cytoplasm with
residues outside the catalytic pocket, thereby
associated loss of host immune signaling (27). We
positioning it for subsequent proteolysis. NS3/4A
found that ITMN-C treatment of cells harboring
exhibits a shallow substrate binding cleft that
distinct HCV 1b replicons mediated a potent
might accommodate other protein substrates
8
inhibition of NS3/4A, restoring the native of NS3/4A protease function permits a rapid
distribution of endogenous IPS-1 as early as 12 restoration of IPS-1 function, host response
hours after treatment, regardless of the differential signaling, and immune action directly within
sensitivities of each HCV replicon to IFN-α HCV-infected cells; 2) endogenous HCV RNA is
therapy (11,22). Moreover, protease inhibitor a potent trigger of RIG-I signaling in infected cells
treatment of Huh7 cells harboring an HCV 2a (9); and 3) RIG-I-responsive genes, including
replicon or infected with the HCV 2a JFH1 clone ISG56 (22), may directly suppress HCV
caused the accumulation of IPS-1 from a NS3/4A- replication. We conclude that NS3/4A protease
cleaved form to a full-length form and related inhibitors possess immunomodulatory activity
induction of ISG56 expression. These results through prevention of IPS-1 proteolysis and
indicate that 1) ITMN-C treatment and inhibition restoration of RIG-I signaling.

REFERENCES

1. Alter, H. J. and Seeff, L. B. (2000) Semin. Liver Dis. 20, 17-35
2. Brown, R. S. (2005) Nature 436, 973-978
D
o
w
n
3. Reed, K. E. and Rice, C. M. (1998) Molecular characterization of hepatitis C virus. In Reesink, H. lo
a
d
W., editor. Hepatitis C virus, Karger, Basel e
d
fro
m
4. Gale, M., Jr. and Foy, E. M. (2005) Nature 436, 939-945 h
ttp
://w
5. Saito, T. and Gale, M., Jr. (2007) Curr. Opin. Immunol. 19, 17-23 w
w
.jb
6. Yoneyama, M., Kikuchi, M., Matsumoto, K., Imaizumi, T., Miyagishi, M., Taira, K., Foy, E., c.o
Loo, Y. M., Gale, M., Jr., Akira, S., Yonehara, S., Kato, A., and Fujita, T. (2005) J. Immunol. 175, brg/
y
2851-2858 g
u
e
s
t o
7. Yoneyama, M., Kikuchi, M., Natsukawa, T., Shinobu, N., Imaizumi, T., Miyagishi, M., Taira, K., n
A
Akira, S., and Fujita, T. (2004) Nat. Immunol. 5, 730-737 p
ril 7
, 2
8. Kato, H., Takeuchi, O., Sato, S., Yoneyama, M., Yamamoto, M., Matsui, K., Uematsu, S., Jung, 0
1
9
A., Kawai, T., Ishii, K. J., Yamaguchi, O., Otsu, K., Tsujimura, T., Koh, C. S., Reis e Sousa,
Matsuura, Y., Fujita, T., and Akira, S. (2006) Nature 441, 101-105
9. Saito, T., Hirai, R., Loo, Y. M., Owen, D., Johnson, C. L., Sinha, S. C., Akira, S., Fujita, T., and
Gale, M., Jr. (2007) Proc. Natl. Acad. Sci. U. S. A 104, 582-587
10. Sumpter, R., Jr., Loo, Y. M., Foy, E., Li, K., Yoneyama, M., Fujita, T., Lemon, S. M., and Gale,
M., Jr. (2005) J. Virol. 79, 2689-2699
11. Sumpter, R., Jr., Wang, C., Foy, E., Loo, Y. M., and Gale, M., Jr. (2004) J. Virol. 78, 11591-
11604
12. Johnson, C. L. and Gale, M., Jr. (2006) Trends Immunol. 27, 1-4
13. Kawai, T., Takahashi, K., Sato, S., Coban, C., Kumar, H., Kato, H., Ishii, K. J., Takeuchi, O., and
Akira, S. (2005) Nat. Immunol. 6, 981-988
9
14. Seth, R. B., Sun, L., Ea, C. K., and Chen, Z. J. (2005) Cell 122, 669-682
15. Xu, L. G., Wang, Y. Y., Han, K. J., Li, L. Y., Zhai, Z., and Shu, H. B. (2005) Mol. Cell 19, 727-
740
16. Meylan, E., Curran, J., Hofmann, K., Moradpour, D., Binder, M., Bartenschlager, R., and
Tschopp, J. (2005) Nature 437, 1167-1172
17. Fitzgerald, K. A., McWhirter, S. M., Faia, K. L., Rowe, D. C., Latz, E., Golenbock, D. T., Coyle,
A. J., Liao, S. M., and Maniatis, T. (2003) Nat. Immunol. 4, 491-496
18. Yoneyama, M., Suhara, W., and Fujita, T. (2002) J. Interferon Cytokine Res. 22, 73-76
19. Grandvaux, N., Servant, M. J., Tenoever, B., Sen, G. C., Balachandran, S., Barber, G. N., Lin, R.,
and Hiscott, J. (2002) J. Virol. 76, 5532-5539
20. Hayden, M. S. and Ghosh, S. (2004) Genes Dev. 18, 2195-2224
D
21. Pflugheber, J., Fredericksen, B., Sumpter, R., Jr., Wang, C., Ware, F., Sodora, D. L., and Gale, o
w
M., Jr. (2002) Proc. Natl. Acad. Sci. U. S. A 99, 4650-4655 nlo
a
d
e
d
22. Wang, C., Pflugheber, J., Sumpter, R., Jr., Sodora, D. L., Hui, D., Sen, G. C., and Gale, M., Jr. fro
(2003) J. Virol. 77, 3898-3912 m
h
ttp
23. Foy, E., Li, K., Wang, C., Sumpter, R., Jr., Ikeda, M., Lemon, S. M., and Gale, M., Jr. (2003) ://w
w
Science 300, 1145-1148 w
.jb
c
.o
24. Sen, G. C. (2001) Annu. Rev. Microbiol. 55, 255-281 rg
b/
y
g
25. Foy, E., Li, K., Sumpter, R., Jr., Loo, Y. M., Johnson, C. L., Wang, C., Fish, P. M., Yoneyama, ue
s
M., Fujita, T., Lemon, S. M., and Gale, M., Jr. (2005) Proc. Natl. Acad. Sci. U. S. A 102, 2986- t o
n
2991 A
p
ril 7
26. Li, X. D., Sun, L., Seth, R. B., Pineda, G., and Chen, Z. J. (2005) Proc. Natl. Acad. Sci. U. S. A , 2
0
1
102, 17717-17722 9
27. Loo, Y. M., Owen, D. M., Li, K., Erickson, A. K., Johnson, C. L., Fish, P. M., Carney, D. S.,
Wang, T., Ishida, H., Yoneyama, M., Fujita, T., Saito, T., Lee, W. M., Hagedorn, C. H., Lau, D.
T., Weinman, S. A., Lemon, S. M., and Gale, M., Jr. (2006) Proc. Natl. Acad. Sci. U. S. A 103,
6001-6006
28. Bartenschlager, R. (1999) J. Viral Hepat. 6, 165-181
29. Rosenberg, S. (2001) J. Mol. Biol. 313, 451-464
30. Wolk, B., Sansonno, D., Krausslich, H. G., Dammacco, F., Rice, C. M., Blum, H. E., and
Moradpour, D. (2000) J. Virol. 74, 2293-2304
31. De Francesco, R., Pessi, A., and Steinkuhler, C. (1998) Antivir. Ther. 3, 99-109
32. Kwong, A. D., Kim, J. L., and Lin, C. (2000) Curr. Top. Microbiol. Immunol. 242, 171-196
10