MasterCARD: a priceless link to innate immunity



MasterCARD: a priceless link to innate immunity
TRENDS in Molecular Medicine
Vol.12 No.2 February 2006
Research Focus
MasterCARD: a priceless link to innate immunity
John Hiscott, Rongtuan Lin, Peyman Nakhaei and Suzanne Paz
Lady Davis Institute for Medical Research - Jewish General Hospital, Departments of Microbiology & Immunology and Medicine,
McGill University, Montreal, QC, H3T 1E2, Canada
Intracellular viral infection is detected by the cytoplasmic RNA helicase RIG-I, which has an essential role
in initiating the host antiviral response. The adaptor
molecule that connects RIG-I sensing of incoming viral
RNA to downstream signaling and gene activation has
recently been elucidated by four independent research
groups, and has been ascribed four different names:
MAVS, IPS-1, VISA and Cardif. The fact that MAVS/IPS1/VISA/Cardif localizes to the mitochondrial membrane
suggests a link between viral infection, mitochondrial
function and development of innate immunity. Furthermore, the hepatitis C virus NS3/4A protease specifically
cleaves MAVS/IPS-1/VISA/Cardif as part of its immuneevasion strategy. These studies highlight a novel role for
the mitochondria and for caspase activation and
recruitment domain (CARD)-containing proteins in
coordinating immune and apoptotic responses.
Initiating antiviral immunity
Upon recognition of early-replication intermediates of
viruses, the host cell activates multiple signaling cascades
that orchestrate the production of interferons (IFNs) and
other cytokines, which in turn initiate innate and adaptive
immune responses [1]. Although microbial pathogens are
usually detected by the Toll-like receptor (TLR) family as a
consequence of defined pathogen-associated molecular
patterns (PAMPs), viral infection is often detected through
the presence of viral nucleic acids, such as single-stranded
RNA (detected by TLR-7) and double-stranded RNA
(dsRNA) (detected by TLR-3), in addition to CpG-containing DNA (detected by TLR-9). Extracellular viral dsRNA
is recognized by TLR-3 [2,3], whereas intracellular viral
dsRNA is detected by two recently characterized RNA
helicases, retinoic acid-inducible gene I (RIG-I) [4] and/or
melanoma differentiation-associated gene 5 (mda-5) [5].
The importance of the RIG-I pathway in antiviral
immunity was confirmed by the generation of RIG-Ideficient mice [6], which revealed that RIG-I and not the
TLR system has an essential role in the IFN-mediated
antiviral response in most cell types, including
fibroblastic, epithelial and conventional dendritic
cells. By contrast, plasmacytoid dendritic cells (pDCs)
use TLR-mediated signaling in preference to
RIG-I-mediated signaling.
Structurally, RIG-I contains two caspase activation and
recruitment domains (CARDs) at its N-terminus and RNA
Corresponding author: Hiscott, J. ([email protected]).
Available online 6 January 2006
helicase activity within its C-terminal portion [4]. The
RNA helicase domain requires ATPase activity and is
responsible for dsRNA recognition and binding, which
leads to dimerization and structural alterations of RIG-I
that enable the CARD domain to interact with other
downstream adaptor protein(s). RIG-I signaling ultimately engages the IkB kinase (IKK) complex (IKKa/b/g),
the stress-activated kinases and the IKK-related kinases
TBK-1 and IKK3, which leads to phosphorylation and
activation of nuclear factor kB (NF-kB), activating
transcription factor 2 (ATF2)–c-JUN and interferon
regulatory factor 3 (IRF3), respectively. Coordinated
activation of these transcription factors results in the
formation of a transcriptionally competent enhanceosome
(a complex of transcription factors that assembles
cooperatively at an enhancer) that triggers IFN-b
production [7].
Characterization of the RIG-I adaptor
The adaptor molecule that provides a link between RIG-I
sensing of incoming viral RNA and downstream activation
events was elucidated recently by four independent
groups [8–11], who used high-throughput screening and/
or database-search analyses to identify this exciting new
signaling component (Figure 1). IFN-b promoter stimulator 1 (IPS-1) was identified by Kawai et al. [8] who
demonstrated that overexpression of IPS-1 activate the
IFN-a, IFN-b and NF-kB promoters, and that TBK-1 is
required for the activation of these promoters. Similar to
RIG-I, IPS-1 consists of an N-terminal CARD domain and
a C-terminal effector domain that recruits the adaptor
Fas-associated death domain protein FADD and the
kinase receptor interacting protein 1 (RIP1). The same
RIG-I adaptor molecule, named mitochondrial antiviral
signaling (MAVS), was identified by Chen et al. [9], who
showed that a C-terminal transmembrane domain, in
addition to its essential role in RIG-I-dependent signaling,
localizes MAVS to the mitochondrial membrane, thus
suggesting a novel role for mitochondrial signaling in the
cellular innate response. Furthermore, Xu et al. [10]
demonstrated that the same RIG-I adaptor molecule,
which they called virus-induced signaling adaptor (VISA),
is a crucial component of IFN-b signaling. VISA interacts
with Toll-receptor-domain-containing adaptor inducing
IFN-b(TRIF; also known as TICAM-1), tumor necrosis
factor receptor-associated factor 2 (TRAF2) and TRAF6
through a proline-rich domain, suggesting that VISA
might mediate the bifurcation of the NF-kB and IRF-3
activation pathways and have an essential role in 1471-4914/$ - see front matter Q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.molmed.2005.12.003
TRENDS in Molecular Medicine
studies had demonstrated that the HCV NS3/4A protease
interfered with NF-kB and IRF-3 induction [12] and
pointed to an unidentified component of the RIG-I
pathway as a NS3/4A target [13,14].
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Vol.12 No.2 February 2006
Crosstalk and confusion
Despite the high quality of these studies and their obvious
importance in delineating a new adaptor molecule in the
RIG-1 pathway (Figure 2), several questions remain
unresolved and will require further investigation
(Box 1). Coimmunoprecipitation experiments suggested
that VISA interacts with TBK-1 and recruits endogenous
IRF-3 in a virus-inducible manner [10]; by contrast,
Meylan et al. showed that IKKa, IKKb and IKK3, but
not TBK-1, associates with Cardif [11]. However, Kawai et
al. argued that neither TBK-1 nor IKK3 are directly
recruited by IPS-1, suggesting that other unidentified
adaptors might link the kinases to RIG-I [8].
VISA failed to activate NF-kB-dependent promoters in
the absence of TRAF6 [10]; however, Seth et al. demonstrated that endogenous activation of the gene encoding
IFN-b occurs normally in cells lacking TRAF6 [9].
Furthermore, a MAVS protein without the TRAF6binding domain can still induce IFN-b [9]. As suggested
recently, the TRAF2 adaptor might compensate for
TRENDS in Molecular Medicine
Figure 1. MAVS/IPS-1/VISA/Cardif. The MAVS/IPS-1/VISA/Cardif molecule is 540
amino acids in length and contains a CARD domain, a proline-rich (Pro) region,
which interacts with TRAF6, and a C-terminal mitochondrial membrane region
(TM). Also, the region adjacent to the TM contains Cys508 (red), which is the target
residue for the HCV NS3/4A protease.
the antiviral response through both TLR-3 and RIG-I
virus-triggered pathways. Finally, Meylan et al. [11]
demonstrated that Cardif (the same RIG-I adaptor
molecule) interacts with RIG-I and recruits IKKa, IKKb
and IKK3 kinases through its C-terminal region. Overexpression of Cardif results in IFN-b- and NF-kBpromoter activation, and knockdown of Cardif by shortinterfering RNA (siRNA) inhibited RIG-I-dependent
antiviral responses. Importantly, this latter study also
demonstrated that Cardif is cleaved at its C-terminal end
(adjacent to the mitochondrial targeting domain) by the
NS3/4A protease of hepatitis C virus (HCV). Previous
IRF-3, IRF-7
NF-κB binding sites
Immune regulation
IRF binding sites
IFN regulation
TRENDS in Molecular Medicine
Figure 2. RIG-I–MAVS/IPS-1/VISA/Cardif signaling. RIG-I contains two N-terminal caspase recruitment domains (CARD; green) and a C-terminal RNA helicase activity that
interacts with viral RNA and is thought to recognize intracellular ribonucleoprotein complexes. A CARD-containing adaptor molecule (MAVS/IPS-1/VISA/Cardif) might be the
link between sensing the incoming virus and triggering downstream kinases. Interaction between the CARD domains of RIG-I and MAVS/IPS-1/VISA/Cardif stimulates NF-kBand IRF-dependent pathways. MAVS/IPS-1/VISA/Cardif leads to the activation of TBK-1 and IKK3 kinases and the phosphorylation of IRF-3 and IRF-7 transcription factors.
MAVS/IPS-1/VISA/Cardif can also lead to NF-kB activation via the IKKa/b/g complex, which phosphorylates the inhibitory subunit IkBa, resulting in the release of NF-kB DNAbinding subunits. MAVS/IPS-1/VISA/Cardif contains a mitochondrial transmembrane domain (TM) that localizes MAVS/IPS-1/VISA/Cardif to mitochondria, where
mitochondrial proteins might contribute to downstream signaling. NS3/4A protease activity of HCV cleaves the C-terminal domain of MAVS/IPS-1/VISA/Cardif, disrupts RIG-Imediated activation of IFN and might contribute to chronic HCV persistence. The ovals (blue) represent Bcl family members on the outer mitochondrial membrane,
suggesting their involvement in apoptotic regulation by MAVS/IPS-1/VISA/Cardif. Other adaptors linking MAVS/IPS-1/VISA/Cardif to downstream kinases remain to be
identified (dashed lines).
TRENDS in Molecular Medicine
Box 1. Outstanding questions
† Are there other adaptors in the RIG-I pathway and do they
participate in signaling events between MAVS/IPS-1/VISA/Cardif
and the TBK-1 and the IKK3 kinases? Are there other TBK-1and/or IKK3-independent mechanisms of IRF activation?
† Which mitochondrial proteins contribute to antiviral signaling or
apoptosis and how is the signaling to NF-kB and IRF coordinated
at the level of the mitochondrial membrane?
† Does MAVS/IPS-1/VISA/Cardif provide crosstalk between the
TLR-independent RIG-I pathway and other TLR-dependent pathways?
† Which roles, if any, do FADD, RIP1, TRAF2 or TRAF6 have in RIG-Imediated IRF activation?
† What is the role of the functionally related, non-redundant MDA-5
molecule in sensing virus infection relative to RIG-I?
† Will drugs that block HCV NS3/4A protease activity and its
cleavage of MAVS/IPS-1/VISA/Cardif restore innate immune
responses in vivo and influence HCV persistence?
the lack of TRAF6 because TRAF2 associates with both
RIP1 and FADD, which are additional components
implicated in virus-induced IFN-b production [15]. However, Seth et al. were unable to show an interaction
between MAVS and RIP1 or FADD but showed that RIP1
is not required for virus-induced IFN-b production [9].
Similarly, Kawai et al. showed that overexpression of
FADD and RIP1 stimulates NF-kB promoters but does not
activate IFN-b [8]. Also, overexpression of a mutant
FADD-death-effector domain blocks IPS-1-mediated activation of NF-kB but not IRF-dependent promoters,
indicating that FADD and RIP1 function exclusively
upstream of the NF-kB pathway.
The involvement of TBK-1 and IKK3 kinases in IRF-3
phosphorylation was questioned by Xu et al. who showed
that dominant negative mutants of TBK-1 and IKK3 do
not block VISA-mediated, IRF-mediated gene activity
[10]; nonetheless, Kawai et al. showed that IRF-3 is not
activated in TBK-1 and IKK3 knockout cells [8]. Another
unresolved question is whether MAVS also intersects with
the TLR-3-dependent pathway through recruitment of the
TIR adaptor protein TRIF. Using siRNA, Seth et al.
showed that MAVS is not required in TLR-3–TRIFmediated gene expression [9], whereas Xu et al. demonstrated a functional interaction between endogenous TRIF
and VISA and partial inhibition of TLR-3-induced
signaling in the absence of MAVS [10] (Box 1).
The mitochondrial connection
The study by Seth et al. [9] is particularly illuminating.
Confocal microscopy and biochemical fractionation
demonstrated that MAVS is present in the outer mitochondrial membrane but moves into a detergent-resistant
mitochondrial fraction upon viral infection. Deletion of the
C-terminal transmembrane domain or cleavage by the
NS3/4A protease adjacent to Cys508 cause loss of MAVSsignaling activity and relocalization of MAVS to the
cytosol [16] (Figure 2). The fact that MAVS functionality
requires mitochondrial association suggests a link
between recognition of viral infection, development of
innate immunity and mitochondrial function. In fact,
knockdown of MAVS gene expression by siRNA increases
apoptosis [9], possibly hinting at a protective role for
Vol.12 No.2 February 2006
MAVS during the early stages of viral infection. Potentially, activation of other components of the mitochondrial
membrane might also contribute to initiating the antiviral
response. In support of this idea, MAVS colocalizes with
the anti-apoptotic protein Bcl-xL in the same detergent
insoluble fraction. Among the many CARD-containing
proteins with roles in apoptosis and immunity [e.g.
apoptotic protease-activating factor 1 (APAF1), nuclear
oligomerization domain 1 (NOD1), NOD2, RIP2 and RIGI], MAVS is unique [17]. The localization of this CARDdomain-containing adaptor to the mitochondrial membrane is highly strategic and might help the host cell sense
incoming viral challenge and coordinate an immune or
apoptotic response, depending on the pathogen. Many
viruses replicate in intracellular organelles such as the
endoplasmic reticulum; a good example is HCV, which
replicates in the membranous web that connects the ER to
mitochondria. dsRNA structures, possibly within replicating ribonucleoprotein complexes, might be recognized by
RIG-I and/or mda-5, resulting in downstream signaling
through MAVS. Mitochondria might be at the center of a
delicate balancing act between the host immune response
and virus-induced apoptosis. In the case of HCV infection,
cleavage of MAVS by the NS3/4A protease seems to tip the
balance, resulting in disruption of innate immune responses
and establishment of chronic HCV persistence [12].
Concluding remarks
The identification of MAVS/IPS-1/VISA/Cardif, its role in
innate signaling, the implications of its mitochondrial
localization and its characterization as the physiologically
relevant target of the NS3/4A protease are important
landmarks in the understanding of the early host response
to viral infection (Figure 2). These studies highlight a
previously unrecognized role for mitochondria and CARDdomain-containing proteins in the coordination of the
innate immune and apoptotic responses. The implications
for the study of HCV pathogenesis are particularly
important because experimental compounds such as
BILN2061 and VX-950, which block NS3/4A protease
activity, might accomplish two goals: (i) inhibition of virus
multiplication; and (ii) processing and restoration of the
early innate immune response that is crucial to the
development of a robust adaptive response in patients.
Clearly, MAVS/IPS-1/VISA/Cardif is a leading candidate
for ‘molecule of the moment’; this important adaptor
molecule now needs a unified nomenclature. As implied
from the title of this article, with homage to a highly
successful advertising campaign, my recommendation
is evident.
This research was supported by grants from Canadian Institutes of
Health Research (J.H. and R.L.), CANVAC, 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.). R.L. is supported in part by a FRSQ Chercheur-boursier and J.H. is
supported by a CIHR Senior Investigator award.
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