MasterCARD: a priceless link to innate immunity

Update

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

www.sciencedirect.com 1471-4914/$ - see front matter Q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.molmed.2005.12.003

Update

54

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].

TRAF6 10 1

77 103

CARD

177

514 535

Pro

TM 506-VP

Vol.12 No.2 February 2006

540

CHRPSP-513

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

Extracellular

Virus HCV NS3/4A

Intracellular

RIG-I TM

MAVS RNA helicase

CARDs Mitochondria

IKK

γ α

PP PP

P P

IκBα

TBK-1

IKKε

β

P

p65

P

p50

IRF-3, IRF-7 P P

TATA

NF-κB binding sites Immune regulation

NF-κB

PP PP P

P TATA

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). www.sciencedirect.com

Update

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 www.sciencedirect.com

Vol.12 No.2 February 2006

55

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. Acknowledgements 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.

References 1 Katze, M.G. et al. (2002) Viruses and interferon: a fight for supremacy. Nat. Rev. Immunol. 2, 675–687

Update

56

TRENDS in Molecular Medicine

2 Alexopoulou, L. et al. (2001) Recognition of double-stranded RNA and activation of NF-kB by Toll-like receptor 3. Nature 413, 732–738 3 Akira, S. and Takeda, K. (2004) Toll-like receptor signalling. Nat. Rev. Immunol. 4, 499–511 4 Yoneyama, M. et al. (2004) The RNA helicase RIG-I has an essential function in double-stranded RNA-induced innate antiviral responses. Nat. Immunol. 5, 730–737 5 Andrejeva, J. et al. (2004) The V proteins of paramyxoviruses bind the IFN-inducible RNA helicase, mda-5, and inhibit its activation of the IFN-b promoter. Proc. Natl. Acad. Sci. U. S. A. 101, 17264–17269 6 Kato, H. et al. (2005) Cell type-specific involvement of RIG-I in antiviral response. Immunity 23, 19–28 7 Maniatis, T. et al. (1998) Structure and function of the interferon-b enhanceosome. Cold Spring Harb. Symp. Quant. Biol. 63, 609–620 8 Kawai, T. et al. (2005) IPS-1, an adaptor triggering RIG-I- and Mda5mediated type I interferon induction. Nat. Immunol. 6, 981–988 9 Seth, R.B. et al. (2005) Identification and characterization of MAVS, a mitochondrial antiviral signaling protein that activates NF-kB and IRF 3. Cell 122, 669–682

Vol.12 No.2 February 2006

10 Xu, L.G. et al. (2005) VISA is an adapter protein required for virustriggered IFN-b signaling. Mol. Cell 19, 727–740 11 Meylan, E. et al. (2005) Cardif is an adaptor protein in the RIG-I antiviral pathway and is targeted by hepatitis C virus. Nature 437, 1167–1172 12 Gale, M., Jr. and Foy, E.M. (2005) Evasion of intracellular host defense by hepatitis C virus. Nature 436, 939–945 13 Foy, E. et al. (2005) Control of antiviral defenses through hepatitis C virus disruption of retinoic acid-inducible gene-I signaling. Proc. Natl. Acad. Sci. U. S. A. 102, 2986–2991 14 Breiman, A. et al. (2005) Inhibition of RIG-I-dependent signaling to the interferon pathway during hepatitis C virus expression and restoration of signaling by IKK3. J. Virol. 79, 3969–3978 15 McWhirter, S.M. et al. (2005) Connecting mitochondria and innate immunity. Cell 122, 645–647 16 Li, X-D. et al. Hepatitis C virus protease NS3/4A cleaves mitochondrial antiviral signaling protein off the mitochondria to evade innate immunity. Proc. Natl. Acad. Sci. U. S. A. (in press) 17 Damiano, J.S. and Reed, J.C. (2004) CARD proteins as therapeutic targets in cancer. Curr. Drug Targets 5, 367–374

Articles of interest in other Trends journals Stem cells and their applications in skin-cell therapy C. Shi, Y. Zhu, Y. Su and T. Cheng, Trends in Biotechnology, doi: 10.1016/j.tibtech.2005.11.003 How do adaptive immune systems control pathogens while avoiding autoimmunity? Carl T. Bergstrom and Rustom Antia, Trends in Ecology and Evolution, doi: 10.1016/j.tree.2005.11.008 Adipose tissue as source and target for novel therapies J. Klein, N. Perwitz, D. Kraus and M. Fasshauer, Trends in Endocrinology and Metabolism, doi: 10.1016/j.tem.2005.11.008 Tau is central in the genetic Alzheimer-frontotemporal dementia spectrum B. Dermaut, S. Kumar-Singh, R. Rademakers, J. Theuns, M. Cruts and C. Van Broeckhoven, Trends in Genetics, 21, 664-672 Triggering TLR signaling in vaccination D. van Duin, R. Medzhitov and A.C. Shaw, Trends in Immunology, doi: 10.1016/j.it.2005.11.005 The function of breast cancer resistance protein in epithelial barriers, stem cells and milk secretion drugs and xenotoxins A.E. van Herwaarden and A.H. Schinkel, Trends in Pharmacological Sciences, doi: 10.1016/j.tips.2005.11.07 www.sciencedirect.com