Hepatitis C virus protease NS3 4A cleaves mitochondrial antiviral signaling protein off the mitochondria to evade innate immunity

Xiao-Dong Li†, Lijun Sun†, Rashu B. Seth†, Gabriel Pineda, and Zhijian J. Chen‡
Howard Hughes Medical Institute, Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390-9148 Edited by Eric N. Olson, University of Texas Southwestern Medical Center, Dallas, TX, and approved October 13, 2005 (received for review September 29, 2005)

Hepatitis C virus (HCV) is a global epidemic manifested mainly by chronic infection. One strategy that HCV employs to establish chronic infection is to use the viral Ser protease NS3 4A to cleave some unknown cellular targets involved in innate immunity. Here we show that the target of NS3 4A is the mitochondrial antiviral signaling protein, MAVS, that activates NF- B and IFN regulatory factor 3 to induce type-I interferons. NS3 4A cleaves MAVS at Cys-508, resulting in the dislocation of the N-terminal fragment of MAVS from the mitochondria. Remarkably, a point mutation of MAVS at Cys-508 renders MAVS resistant to cleavage by NS3 4A, thus maintaining the ability of MAVS to induce interferons in HCV replicon cells. NS3 4A binds to and colocalizes with MAVS in the mitochondrial membrane, and it can cleave MAVS directly in vitro. These results provide an example of host–pathogen interaction in which the virus evades innate immunity by dislodging a pivotal antiviral protein from the mitochondria and suggest that blocking the cleavage of MAVS by NS3 4A may be applied to the prevention and treatment of HCV.
IFN regulatory factor 3 NF- B retinoic acid-induced gene I I B kinase

epatitis C virus (HCV) infects 170 million people in the world, and 80% of the infected individuals develop persistent infection (1, 2). HCV is an enveloped single-strand RNA virus belonging to the Flaviviridae family. It contains a 9.6-kb RNA genome that encodes a large polyprotein ( 3,000 aa), which is cleaved into 10 structural and nonstructural (NS) proteins through the action of cellular peptidases as well as the viral-encoded proteases including NS3 4A. NS3 contains Ser protease and RNA helicase activities that require its cofactor NS4A, which tethers the holoenzyme complex to an intracellular membrane compartment. The NS3 4A protease is not only essential for generating mature viral proteins required for viral replication, but also can suppress the host antiviral immune system by cleaving putative cellular targets involved in the induction of type-I interferons (IFNs), such as IFN- and IFN- (3). The induction of IFNs is regulated by the transcription factors NF- B and IFN regulatory factor 3 (IRF3), which are activated by a signaling cascade emanating from dsRNA generated during the replication of viral RNA (4, 5). The activation of NF- B requires the phosphorylation of its inhibitor I B by I B kinase (IKK) complex, which can be activated by a large variety of agents, including proinflammatory cytokines and microbial pathogens (6). The phosphorylation of I B targets this inhibitor for ubiquitination and subsequent degradation by the proteasome, thereby allowing NF- B to enter the nucleus to regulate downstream genes. The activation of IRF3 requires its phosphorylation by two IKK-related kinases, TBK1 and IKK (7, 8). After phosphorylation, IRF3 is dimerized and translocated into the nucleus where it forms an enhanceosome complex together with NF- B and other transcription factors to turn on the expression of targets genes such as IFN(4). IFNs then induce a large array of antiviral genes through the activation of the Janus tyrosine kinase signal transducer and activator of transcription pathway.
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H

The receptor for intracellular viral dsRNA has recently been identified as retinoic acid-induced gene I (RIG-I), which contains a RNA helicase domain that binds to dsRNA (9). RIG-I also contains tandem N-terminal caspase recruitment domains (CARDs) that interact with another CARD domain protein, mitochondrial antiviral signaling (MAVS; also known as IPS-1, VISA, and CARDIF) (10–13). MAVS contains a C-terminal transmembrane (TM) domain that targets it to the mitochondrial outer membrane (10). Importantly, the mitochondrial localization of MAVS is essential for its signaling function, because the removal of the mitochondrial-targeting domain of MAVS abolishes its ability to induce IFNs. Epistasis experiments have shown that MAVS functions downstream of RIG-I and upstream of I B kinase and TBK1 in the viral signaling pathway. Recent studies have shown that NS3 4A inhibits IFN induction by RIG-I; however, RIG-I is not cleaved by NS3 4A (14, 15). NS3 4A also was found to cleave TIR domain-containing adapterinducing IFN- (TRIF) (16), an adaptor protein that binds to Toll-like receptors such as TLR3 and TLR4 (17). However, TRIF is not essential for IFN induction by viruses (18). Thus, the target of NS3 4A in the viral pathway remains to be identified. In this work, we present evidence that MAVS is the proteolytic target of NS3 4A. We found that NS3 4A cleaves MAVS at Cys-508, resulting in the dislocation of the N-terminal fragment of MAVS from the mitochondria, thereby suppressing the induction of IFN- . A point mutation at Cys-508 prevents the cleavage of MAVS by NS3 4A and restores IFN- induction in a HCV replicon cell line, suggesting that the cleavage of MAVS is responsible for the suppression of IFN induction in HCV replicating cells. We also show that NS3 4A binds to and colocalizes with MAVS in the mitochondria. Finally, we show that NS3 4A cleaves MAVS at Cys-508 in vitro, providing the direct biochemical evidence that MAVS is the target of NS3 4A. Materials and Methods
Plasmids and Antibodies (Abs). Expression constructs for MAVS, MAVS (CARD-TM), TBK1, RIG-I(N), IFN- -luciferase (Luc), NF- B-Luc, UAS-Luc, and Gal4-IRF3 are described in ref. 10. pcDNA3-Flag-MAVS-hemagglutinin (HA) was constructed by subcloning the DNA fragment encoding MAVS-HA into the XhoI and XbaI sites of the pcDNA3-Flag vector such that the N-terminal Flag epitope was fused in frame with MAVS-HA. NS3 4A was
Conflict of interest statement: No conflicts declared. This paper was submitted directly (Track II) to the PNAS office. Abbreviations: CARD, caspase recruitment domain; HA, hemagglutinin; HCV, hepatitis C virus; IRF3, IFN regulatory factor 3; Luc, luciferase; MAVS, mitochondrial antiviral signaling protein; NS, nonstructural; RIG-I, retinoic acid-induced gene I; SeV, Sendai virus; TM, transmembrane; miniMAVS, truncated MAVS protein containing only the CARD and TM domains. See Commentary on page 17539.
†X.-D.L., ‡To

L.S., and R.B.S. contributed equally to this work.

whom correspondence should be addressed. E-mail: zhijian.chen@utsouthwestern.edu.

© 2005 by The National Academy of Sciences of the USA

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amplified by RT-PCR from the RNA of HCV replicon cell line K2040 (19) and then cloned into pcDNA3 in frame with an N-terminal Flag or Myc tag. The S139A mutant of NS3 4A and the Cys mutants of MAVS (C435R, C452R, and C508R) were generated by site-directed mutagenesis using the QuikChange kit (Stratagene). pET14b-NS3 4A was constructed by subcloning NS3 4A into the XhoI and BamH1 sites of pET14b (Novagen). All plasmids were verified by automatic DNA sequencing. The polyclonal Ab against MAVS was generated and purified by antigen column as described in ref. 10. The HCV NS3 Ab was purchased from NovoCastra (Newcastle, U.K.). The monoclonal Abs against Flag (M2, Sigma), Myc (9E10, Santa Cruz) and HA (HA.11, Covance) were purchased from the indicated suppliers.
Cell Culture, Transfection, and Luc Reporter Assays. HEK293 and

Results
Induction by MAVS. To determine whether NS3 4A inhibits IFN- induction by MAVS, we transfected expression vectors encoding NS3 4A and MAVS into HEK293 cells together with a Luc reporter driven by the IFN- promoter (IFN- -Luc). As controls, we infected cells with Sendai virus (SeV) or transfected cells with an expression vector encoding the Nterminal CARD domains of RIG-I [RIG-I(N)], which has previously been shown to induce IFN- (9). In each case, NS3 4A inhibited the induction of IFN- (Fig. 1A). In contrast, the induction of IFN- by TBK1 was not affected by NS3 4A, suggesting that NS3 4A inhibits the RIG-I pathway at the level of MAVS or at a step downstream of MAVS but upstream of TBK1. To determine whether the protease activity of NS3 4A was required for this inhibition, we mutated the active site Ser-139 of NS3 4A (equivalent to Ser-1165 of the HCV polyprotein) to Ala (20, 21). This mutant migrated more slowly than wild-type (WT) NS3 (Fig. 1D Bottom), because it could no longer be cleaved at the junction between NS3 and NS4A. The protease-dead mutant completely lost its ability to inhibit IFN- induction by SeV, RIG-I(N), or MAVS. Thus, it is likely that NS3 4A inhibits IFN induction by cleaving MAVS or a target downstream of MAVS. NS3 4A also prevented the MAVS-induced activation of NF- B (Fig. 1B) and IRF3 reporters (Fig. 1C), as well as the phosphorylation and dimerization of IRF3 (Fig. 1D), further supporting the notion that NS3 4A inactivates a common target required for both NF- B and IRF3 activation. NS3 4A Blocks IFNMAVS Is the Proteolytic Target of NS3 4A. To determine whether MAVS is the proteolytic target of NS3 4A, we transfected the expression vector encoding FLAG-NS3 4A or FLAG-NS3 4A (S139A) into HEK293 or Huh7 (a human hepatoma cell line). Because MAVS is a mitochondrial membrane protein, the cell lysates were separated into cytosolic fraction (S) and membrane pellets (P) by centrifugation, and the endogenous MAVS protein analyzed by immunoblotting with an affinity-purified MAVS Ab (Fig. 2A). In both cell lines, the expression of NS3 4A, but not the protease-dead mutant of NS3 4A, led to the generation of a faster-migrating fragment of MAVS that was 3–5 kDa shorter than the full-length MAVS (Fig. 2 A Upper; compare lanes 3 with 4 and 9 with 10). Interestingly, unlike the full-length MAVS that was present in the membrane pellet, the truncated fragment of MAVS was mainly present in the soluble cytosolic fraction, suggesting that the truncated fragment did not contain the C-terminal TM domain. Immunoblotting experiments also showed that the majority of NS3 is in the membrane pellet, presumably because of its association with NS4A (Fig. 2 A Lower). MAVS contains an N-terminal CARD domain, a middle Prorich domain, and a C-terminal TM domain. We have previously shown that a truncated MAVS protein containing only the CARD and TM domains (hereafter referred to as miniMAVS) is sufficient to induce IFN- (10). To further delineate the NS3 4A cleavage site of MAVS, we tested whether the miniMAVS was also sensitive to NS3 4A inhibition. As shown in Fig. 2B, IFN- induction by the miniMAVS was also inhibited by NS3 4A (Middle). Furthermore, the miniMAVS was cleaved to a shorter fragment by the WT, but not the S139A mutant, of NS3 4A (Fig. 2B Bottom). The truncated fragment was also 4 kDa shorter than the miniMAVS (Fig. 2B, compare lanes 4 and 5), suggesting that this small MAVS protein of only 140 residues in length contains a cleavage site of NS3 4A, most likely at a residue near the TM domain. NS3 4A Cleaves MAVS at Cys-508. NS3 4A is a Ser protease that cleaves at the C terminus of a Cys or Thr residue within a loosely defined consensus sequence [(E D)xxxx(C T)(S A), where x denotes any amino acid], which is found at the junction of HCV NS proteins (Fig. 2C) (22). Inspection of the miniMAVS amino acid
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HeLa cells were from American Type Culture Collection. The Huh7 and HCV replicon cell line K2040 were provided by Michael Gale (University of Texas Southwestern) (19). Transfection of HEK293 cells was carried out by using the calcium phosphate precipitation method. HeLa cells were transfected by using the PolyFect reagent (Qiagen, Valencia, CA). Huh7 and replicon cells were transfected by using lipofectamine 2000 (Invitrogen) or Superfect (Qiagen) reagents. Luc reporter assays were performed as described in ref. 10.
Subcellular Fractionation. HEK293 cells were transfected with expression vectors for Flag-MAVS-HA or the C508R mutant together with Myc-tagged NS3 4A or its inactive mutant S139A. Cells were washed 36 h after transfection in hypotonic buffer (10 mM Tris HCl, pH 7.5 10 mM KCl 1.5 mM MgCl2 protease inhibitors) and then homogenized in the same buffer by douncing 20 times. The homogenate was centrifuged at 500 g for 5 min to remove nuclei and unbroken cells. The supernatant was centrifuged again at 5,000 g for 10 min to generate membrane pellets (P; containing mostly mitochondria) and cytosolic supernatant (S). The same procedure was used for subcellular fractionation of Huh7 and HCV replicon cells. Cleavage of MAVS by NS3 4A Protease in Vitro. pcDNA3-FlagNS3 4A or its mutant S139A was transfected into HEK293 cells to express the protease, which then was purified by binding to the Flag-Sepharose (M2-Sepharose, Sigma) followed by elution with the Flag peptide (0.2 mg ml). The eluted NS3 4A or S139A mutant was concentrated by using Amicon microconcentrator (Millipore, 10,000 Da cut-off) and diluted in buffer A (20 mM Tris HCl 150 mM NaCl 0.1% Triton X-100 5 mM DTT, pH 7.5). This procedure was repeated three times to reduce the concentration of the Flag peptide. For expression of NS3 4A in Escherichia coli, pET14b-NS3 4A was transfected into the E. coli strain BL21 pLysS, and the transformant was induced to express His-6-NS3 4A after the addition of IPTG. His-6-NS3 4A was purified by using nickel affinity column and dialyzed against buffer B (20 mM Tris HCl, pH 7.5 100 mM NaCl 10% glycerol). Flag-MAVS or its mutant C508R was translated in vitro by using the TNT rabbit reticulocyte lysate (Promega) supplemented with [35S]methionine. The 35S-labeled Flag-MAVS or C508R was purified by using Flag-Sepharose as described above for NS3 4A. To measure the cleavage of MAVS by NS3 4A, 0.5 l of 35S-labeled MAVS or C508R was incubated with 0.5 l of NS3 4A or S139A in a 10- l reaction containing buffer A. After incubation at 30°C for 2 h, the reaction products were resolved by SDS PAGE and analyzed by using PhosphorImager (Molecular Dynamics). The experimental procedures for viral infection, immunoblotting, immunoprecipitation, and confocal microscopy are described in ref. 10.
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sequence revealed a potential NS3 4A cleavage sequence, 503EREVPCH509. The potential cleavage site, Cys-508, is located 6 aa from the beginning of the TM domain and 32 aa from the C terminus of MAVS. Cleavage at Cys-508 is predicted to generate an 32-residue C-terminal fragment, consistent with the size difference between the miniMAVS and the miniMAVS lacking the TM domain (Fig. 2B Bottom; compare lanes 4 and 5). Indeed, when NS3 4A was cotransfected together with an expression construct encoding a MAVS protein that is tagged with FLAG at the N terminus and HA at the C terminus, the MAVS protein was cleaved into a soluble FLAG-tagged N-terminal fragment that was smaller than the intact protein (Fig. 2D Top; compare lanes 1 and 4). Moreover, an 7-kDa C-terminal fragment was detectable in both the membrane pellet and cytosolic fractions with a HA Ab (Fig. 2D Middle; lanes 3 and 4). This cleavage product of MAVS was not detected in cells expressing the S139A mutant of NS3 4A. Although the apparent molecular mass of the C-terminal cleavage fragment was greater than the theoretical molecular mass of the fragment containing the C-terminal 32 residues of MAVS plus a HA epitope ( 5 kDa), this result is likely because of the abnormal migration of small peptides on SDS PAGE. Indeed, when we synthesized the C-terminal 32 residues of MAVS (residue 509–540) plus the HA epitope by in vitro translation, this fragment also migrated as an 7-kDa band (see Fig. 5A, which is published as supporting information on the PNAS web site), identical to the C-terminal fragment of MAVS after cleavage by NS3 4A. To demonstrate that Cys-508 is indeed the cleavage site, we mutated Cys-508 to Arg, which is predicted to disrupt NS3 4A cleavage based on the analysis of sequence requirement for HCV polyprotein cleavage (23). Remarkably, this single amino acid substitution completely blocked the cleavage of MAVS by NS3 4A (Fig. 2D, lanes 7–12). Furthermore, the protease-resistant mutant of MAVS was fully capable of inducing IFN- , and this induction was no longer inhibited by NS3 4A (Fig. 2E). In contrast to Cys-508, mutations at two adjacent Cys residues, Cys-435 and -452, did not prevent the cleavage of MAVS by NS3 4A (Fig. 5B). Together, these results indicate that NS3 4A cleaves MAVS at Cys-508 when these proteins are coexpressed in the same cells.
NS3 4A Binds to and Cleaves MAVS Directly in Vitro. To determine

Fig. 1. NS3 4A blocks IFN induction by MAVS. (A) HEK293 cells were transfected with IFN- -Luc and the expression vector for either the WT or S139A mutant of NS3 4A. The next day, cells were transfected with expression vectors encoding MAVS, TBK1, or RIG-I(N), or infected with SeV. The Luc activity was measured 24 h later and normalized for transfection efficiency. The error bar represents standard deviation from the mean value of duplicated experiments. (B) Similar to A, except that NF- B Luc reporter was used in lieu of IFN- -Luc. (C) Similar to A, except that cells were transfected with the UAS-Luc reporter together with the Gal4-IRF3 expression vector. (D) HEK293 cells were transfected with the WT or S139A mutant of NS3 4A together with the expression vectors for MAVS (lanes 5–7) or TBK1 (lanes 8 –10). In lanes 2– 4, cells were infected with SeV for 24 h. Cell lysates were resolved by electrophoresis under native (Top) or denaturing condition (Middle) and then immunoblotted with an Ab against IRF3. The expression of NS3 4A and TBK1 also was analyzed by immunoblotting with a Flag Ab (Bottom).

whether NS3 4A could cleave MAVS directly in vitro, we expressed FLAG-NS3 4A or FLAG-NS3 4A (S139A) protein in HEK293 cells and immunopurified the protein using the FLAG Ab followed by elution with a FLAG peptide. We also synthesized 35S-labeled FLAG-MAVS or FLAG-MAVS-C508R protein in vitro using rabbit reticulocyte lysates and purified the proteins using the FLAG Ab. The purified MAVS proteins were incubated with NS3 4A protease or lysates containing the protease. As shown in Fig. 3A, the WT MAVS was cleaved by the active NS3 4A protease, but not the inactive NS3 4A mutant (lanes 1–5). In contrast, a point mutation at Cys-508 completely blocked the cleavage of MAVS by NS3 4A in vitro (lanes 6–10). The in vitro cleavage product represents the N-terminal fragment of MAVS, because the C-terminal 32-aa fragment does not contain any Met that can be radioactively labeled. Similar results were obtained by using NS3 4A protease expressed and purified from E. coli (see Fig. 6A, which is published as supporting information on the PNAS web site). These results indicate that NS3 4A directly cleaves MAVS at Cys-508 in vitro. The cleavage of MAVS by NS3 4A suggests that these two proteins may interact directly. To investigate this possibility, we cotransfected MAVS and NS3 4A or their mutants into HEK293 cells, immunoprecipitated MAVS with the MAVS Ab, and then determined whether NS3 4A was coprecipitated with MAVS by immunoblotting (Fig. 3B). Interestingly, only the S139A mutant, but not the WT, NS3 4A coprecipitated with MAVS (Fig. 1D Bottom, compare lanes 3 and 9), suggesting that the cleaved MAVS dissociated from the WT protease, whereas the mutant protease was able to ‘‘trap’’ its substrate. Consistent with this interpretation,
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Fig. 2. NS3 4A cleaves MAVS at Cys-508. (A) HEK293 (lanes 1– 6) or Huh7 (lanes 7–12) cells were transfected with expression vectors for the WT (lanes 3,4, 9, and 10) or S139A mutant (lanes 5, 6, 11, and 12) of NS3 4A or vector (lanes 1, 2, 7, and 8). Cell lysates were separated into membrane pellet (P) or cytosolic supernatant (S) and then immunoblotted with an Ab against MAVS or Flag. The cleavage product of MAVS was indicated as MAVS*. (B) HEK293 cells were transfected with the expression vectors for NS3 4A together with a MAVS mutant containing only the CARD and TM domains (miniMAVS; Top). The activation of IFN- by the CARD-TM fragment of MAVS in the presence of WT or S139A mutant of NS3 4A was determined by measuring the expression of the IFN- -Luc reporter (Middle). Cell lysates were analyzed by immunoblotting with the Flag Ab that detects both NS3 4A and MAVS proteins (Bottom). (C) Alignment of the junction sequences of NS proteins of HCV and the putative cleavage site at the C terminus of MAVS from different species. (D) HEK293 cells were transfected with expression vectors encoding the WT or C508R mutant of MAVS together with those encoding the WT or S139A mutant of NS3 4A. Cell lysates were separated by differential centrifugation into the membrane pellet and cytosolic supernatant, which were then resolved by 7% (Top and Bottom) or 4 –20% (Middle) SDS PAGE, followed by immunoblotting with the indicated Ab that detects the N terminus (Flag; Top) or C terminus (HA; Middle) of MAVS, or NS3 4A (Myc; Bottom). (E) HEK293 cells were transfected with MAVS and NS3 4A expression vectors as in D, except that IFN- -Luc was also cotransfected to measure the induction of IFN- by Luc assay.

C508R also bound to either WT or S139A mutant of NS3 4A, but this binding was weaker than that observed between MAVS and the S139A mutant of NS3 4A (compare lanes 6 and 12 with lane 9), suggesting that C508R mutation may partially impair its binding to the protease.
NS3 4A Colocalizes with MAVS in the Mitochondria. It has been shown

that HCV core protein and NS3 4A are localized to a mitochondrion-associated membrane structure in both cultured hepatocytes (24, 25) and liver biopsies of chronic hepatitis C patients (26).
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Because MAVS is a mitochondrial membrane protein, we examined whether MAVS and NS3 4A colocalize in the same membrane compartment. Expression vectors encoding Myc-NS3 4A and or HA-MAVS were transfected into HeLa cells, which then were stained with the corresponding Abs followed by imaging with a laser scanning confocal microscope. To stain the mitochondria, cells were incubated with Mito Tracker, a fluorescent dye taken up by the mitochondria of living cells. The staining patterns of the WT NS3 4A overlapped with that of Mito Tracker, but not the endoplasmic reticulum resident protein calnexin (Fig. 6B), suggesting
Li et al.

Fig. 3. NS3 4A binds to and colocalizes with MAVS in the mitochondria, and it cleaves MAVS in vitro. (A) Flag-tagged WT or S139A mutant of NS3 4A was expressed in HEK293 cells and then purified by using Flag-Sepharose. The purified NS3 4A proteins (lanes 4, 5, 9, and 10) or lysates containing NS3 4A (lanes 1–3 and 6 – 8) were incubated with 35S-labeled MAVS or C508R mutant of MAVS, which was synthesized by in vitro translation and purified by using Flag-Sepharose. After incubation at 30°C for 2 h, proteins were separated by SDS PAGE and analyzed by PhosphorImaging (Upper) or immunoblotting (Lower). The cleavage product of MAVS is indicated as MAVS*. (B) HEK293 cells were transfected with expression vectors for Flag-NS3 4A and HA-MAVS as indicated. Cell lysates were immunoprecipitated with the MAVS Ab (lanes 3, 6, 9, and 12) or control IgG (lanes 2, 5, 8, and 11). The precipitated proteins were analyzed by immunoblotting with an Ab against HA (Upper) or Flag (Lower). (C) Expression vectors for Flag-NS3 4A and HA-MAVS or C508R mutant were transfected into HeLa cells, and the localization of these proteins was stained by the indicated Abs and visualized by confocal microscopy.

Fig. 4. MAVS is cleaved at Cys-508 in a HCV replicon cell line. (A) Cell lysates from Huh7 (lanes 1 and 2) or Replicon cells (K2040; lanes 3 and 4) were separated by centrifugation into the membrane pellet (P) and cytosolic supernatant (S), which then were analyzed by immunoblotting with an Ab against MAVS or NS3. The cleavage products of MAVS are indicated as MAVS*. MAVS- N, N-terminal truncated fragment of MAVS (see ref. 10); MAVS- N*, N-terminal truncated fragment of MAVS in which the TM domain is also cleaved by NS3 4A. (B and C) Expression vectors for MAVS, C508 mutant of MAVS, or RIG-I(N) were cotransfected with the IFN- -Luc reporter into Huh7 (B) or Replicon (C) cell lines. Twenty-four hours after transfection, cells were infected with SeV or mock infected for another 24 h before harvesting for Luc assay.

that NS3 4A is localized to the mitochondria or in a mitochondrion-associated membrane. When WT NS3 4A and MAVS were coexpressed, the majority of MAVS became cytosolic (presumably due to cleavage), as revealed by an Ab that detects the N terminus of MAVS (Fig. 3C). In sharp contrast, when the WT NS3 4A was coexpressed with MAVS-C508R, both NS3 4A and MAVS-C508R proteins colocalized in the mitochondrial membrane and showed an extensive overlapping staining pattern. These results indicate that MAVS and NS3 4A are colocalized in the mitochondrial membrane or are positioned in very close proximity, thus rendering MAVS vulnerable to cleavage by NS3 4A.
MAVS Is Cleaved in a HCV Replicon Cell Line. To determine whether

MAVS is a target of HCV during viral replication, we used the HCV replicon cell culture system in which the subgenomic RNA of HCV replicates autonomously in the human hepatoma cell line
Li et al.

Huh7 (27). The HCV subgenomic RNA encodes all of the NS proteins, including the NS3 4A protease and the NS5B RNA polymerase. It has been shown that the HCV replicon cell lines that replicate the viral RNA efficiently can suppress the host IFN induction through the proteolytic activity of NS3 4A (3). To determine whether the endogenous MAVS is cleaved in the replicon cells, we used subcellular fractionation and immunoblotting to analyze the MAVS protein from the WT Huh7 and a replicon cell line K2040 (19). As shown in Fig. 4A, the MAVS protein is present predominately in the mitochondrial membrane pellet isolated from Huh7 cells. In contrast, in the HCV replicon cells, a cleaved form of MAVS was found mostly in the soluble cytosolic fraction, whereas NS3 was detected mainly in the membrane pellets.
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Prevention of MAVS Cleavage Restores the IFN Response in HCV Replicon Cells. If the cleavage of MAVS is solely responsible for the

failure to induce IFNs in HCV replicon cells, prevention of MAVS cleavage should restore IFN induction in these cells. To test this possibility, we introduced the C508R mutant of MAVS as well as the WT MAVS into the HCV replicon cells to examine the induction of IFN- . As reported in ref. 3, SeV induced IFN- in the Huh7 cells but failed to do so in the replicon cell line (Fig. 4 B and C). Even overexpression of RIG-I(N) failed to induce IFN- in the replicon cells (Fig. 4C), presumably because the endogenous MAVS was cleaved into an inactive soluble fragment (Fig. 4A, lane 4). Overexpression of WT MAVS was able to induce partial IFN response in the replicon cells, and this response was further enhanced by SeV. This result may be due to the incomplete cleavage of the overexpressed MAVS by the NS3 4A protease in the replicon cells. Most significantly, expression of the proteaseresistant mutant of MAVS (C508R) restored IFN- induction in the replicon cells to the same level observed in Huh7 cells (Fig. 4 B and C). These results demonstrate that the evasion of innate immune response in the HCV replicon cells is due to the cleavage of MAVS by NS3 4A. Discussion We have previously shown that MAVS is an essential antiviral signaling protein and proposed that MAVS may be a prime target of viruses that have succeeded in evading the host immune system (10). In this work, we show that MAVS is indeed the proteolytic target of HCV NS3 4A protease. Specifically, we found that HCV cleaves MAVS at Cys-508, dislocating MAVS from the mitochondria, thus preventing the induction of IFN- . NS3 4A binds to and colocalizes with MAVS at the mitochondrial membrane, and it can cleave MAVS at Cys-508 directly in vitro. We also provide direct evidence that the endogenous MAVS protein is cleaved in a HCV replicon cell line that failed to elicit IFN response. Importantly, a point mutation of MAVS at Cys-508 that prevents its cleavage restores IFN induction in the HCV replicon cell line. These results provide compelling evidence that MAVS is the prime target of the HCV protease. Our present work not only provides the direct biochemical evidence that MAVS is the proteolytic target of NS3 4A (Fig. 3) but also shows that the cleavage of endogenous MAVS at Cys-508 in the HCV replicon cell line is solely responsible for the suppression of IFN induction in these cells (Fig. 4). Furthermore, our finding that NS3 4A inactivates MAVS by cleaving and dislodging it from the mitochondria underscores the
1. Lindenbach, B. D. & Rice, C. M. (2005) Nature 436, 933–938. 2. Chisari, F. V. (2005) Nature 436, 930–932. 3. Foy, E., Li, K., Wang, C., Sumpter, R., Jr., Ikeda, M., Lemon, S. M. & Gale, M., Jr. (2003) Science 300, 1145–1148. 4. Maniatis, T., Falvo, J. V., Kim, T. H., Kim, T. K., Lin, C. H., Parekh, B. S. & Wathelet, M. G. (1998) Cold Spring Harb. Symp. Quant. Biol. 63, 609–620. 5. McWhirter, S. M., Tenoever, B. R. & Maniatis, T. (2005) Cell 122, 645–647. 6. Silverman, N. & Maniatis, T. (2001) Genes Dev. 15, 2321–2342. 7. Fitzgerald, K. A., McWhirter, S. M., Faia, K. L., Rowe, D. C., Latz, E., Golenbock, D. T., Coyle, A. J., Liao, S. M. & Maniatis, T. (2003) Nat. Immunol. 4, 491–496. 8. Sharma, S., tenOever, B. R., Grandvaux, N., Zhou, G. P., Lin, R. & Hiscott, J. (2003) Science 300, 1148–1151. 9. Yoneyama, M., Kikuchi, M., Natsukawa, T., Shinobu, N., Imaizumi, T., Miyagishi, M., Taira, K., Akira, S. & Fujita, T. (2004) Nat. Immunol. 5, 730–737. 10. Seth, R. B., Sun, L., Ea, C. K. & Chen, Z. J. (2005) Cell 122, 669–682. 11. Kawai, T., Takahashi, K., Sato, S., Coban, C., Kumar, H., Kato, H., Ishii, K. J., Takeuchi, O. & Akira, S. (2005) Nat. Immunol. 6, 981–988. 12. Meylan, E., Curran, J., Hofmann, K., Moradpour, D., Binder, M., Bartenschlager, R. & Tschopp, J. (2005) Nature. 13. Xu, L. G., Wang, Y. Y., Han, K. J., Li, L. Y., Zhai, Z. & Shu, H. B. (2005) Mol. Cell 19, 727–740. 14. Breiman, A., Grandvaux, N., Lin, R., Ottone, C., Akira, S., Yoneyama, M., Fujita, T., Hiscott, J. & Meurs, E. F. (2005) J. Virol. 79, 3969–3978. 15. Foy, E., Li, K., Sumpter, R., Jr., Loo, Y. M., Johnson, C. L., Wang, C., Fish, P. M., Yoneyama, M., Fujita, T., Lemon, S. M. & Gale, M., Jr. (2005) Proc. Natl. Acad. Sci. USA 102, 2986–2991.

importance of mitochondrial localization of MAVS in antiviral immunity. Infectious diseases are a manifestation of constant battles between the host and pathogenic microbes. This host–pathogen antagonism is now vividly demonstrated from the study of the interaction between MAVS and HCV. MAVS can orchestrate strong immune defense against HCV; however, HCV counterattacks by using the NS3 4A protease to cleave MAVS, thus crippling the immune response. Because a point mutation at Cys-508 of MAVS completely prevents its cleavage by NS3 4A and preserves its antiviral activity, it would be interesting to determine whether there are sequence variations of MAVS in human population that confer differential sensitivity to NS3 4A and, hence, differential immunity to HCV. However, the prevalence of persistent HCV infection in humans suggests that HCV might have won the battle between the virus and the host. In the next battle of ‘‘our wits versus their genes,’’ it may be possible to exploit the knowledge of MAVS–HCV interaction to fight back against HCV. For example, the cleavage of MAVS from the mitochondrial membrane may be a diagnostic marker for an established HCV infection. Furthermore, blocking the cleavage of MAVS by NS3 4A may be effective in the prevention and treatment of HCV. HCV may not be the only pathogen that targets MAVS to evade the host immune system. Although the mitochondrial localization of MAVS may allow the host immune system to detect many viruses that replicate in intracellular membrane locations proximal to the mitochondria, it also may render MAVS vulnerable to viral attack. For example, it has recently been shown that the hepatitis A virus (HAV) inhibits IFN response at a step downstream of RIG-I but upstream of TBK1 IKK (28), raising the possibility that MAVS may be a target of a HAV protein. Future studies should uncover more examples of host–pathogen interaction that revolve around the battle for MAVS to gain control of the host immune system.
Note. While this manuscript was in preparation, Meylan et al. (12) also reported the identification of MAVS CARDIF as the target of NS3 4A. We thank Dr. Michael Gale for providing the HCV replicon cell line K2040 (19). This work was supported by National Institutes of Health Grant R01-A I60919 and American Cancer Society Grant RSG0219501TBE. Confocal microscopy was supported by funding from a National Institutes of Health shared instrumentation grant 1-S10RR19406. Z.J.C. is a Burroughs Wellcome Fund Investigator in Pathogenesis of Infectious Diseases.
16. Li, K., Foy, E., Ferreon, J. C., Nakamura, M., Ferreon, A. C., Ikeda, M., Ray, S. C., Gale, M., Jr., & Lemon, S. M. (2005) Proc. Natl. Acad. Sci. USA 102, 2992–2997. 17. Akira, S. & Takeda, K. (2004) Nat. Rev. Immunol. 4, 499–511. 18. Kato, H., Sato, S., Yoneyama, M., Yamamoto, M., Uematsu, S., Matsui, K., Tsujimura, T., Takeda, K., Fujita, T., Takeuchi, O. & Akira, S. (2005) Immunity 23, 19–28. 19. Sumpter, R., Jr., Wang, C., Foy, E., Loo, Y. M. & Gale, M., Jr. (2004) J. Virol. 78, 11591–11604. 20. Kim, J. L., Morgenstern, K. A., Lin, C., Fox, T., Dwyer, M. D., Landro, J. A., Chambers, S. P., Markland, W., Lepre, C. A., O’Malley, E. T., et al. (1996) Cell 87, 343–355. 21. Love, R. A., Parge, H. E., Wickersham, J. A., Hostomsky, Z., Habuka, N., Moomaw, E. W., Adachi, T. & Hostomska, Z. (1996) Cell 87, 331–342. 22. Grakoui, A., McCourt, D. W., Wychowski, C., Feinstone, S. M. & Rice, C. M. (1993) J. Virol. 67, 2832–2843. 23. Kolykhalov, A. A., Agapov, E. V. & Rice, C. M. (1994) J. Virol. 68, 7525–7533. 24. Schwer, B., Ren, S., Pietschmann, T., Kartenbeck, J., Kaehlcke, K., Bartenschlager, R., Yen, T. S. & Ott, M. (2004) J. Virol. 78, 7958–7968. 25. Mottola, G., Cardinali, G., Ceccacci, A., Trozzi, C., Bartholomew, L., Torrisi, M. R., Pedrazzini, E., Bonatti, S. & Migliaccio, G. (2002) Virology 293, 31–43. 26. Kasprzak, A., Seidel, J., Biczysko, W., Wysocki, J., Spachacz, R. & Zabel, M. (2005) Liver Int. 25, 896–903. 27. Lohmann, V., Korner, F., Koch, J., Herian, U., Theilmann, L. & Bartenschlager, R. (1999) Science 285, 110–113. 28. Fensterl, V., Grotheer, D., Berk, I., Schlemminger, S., Vallbracht, A. & Dotzauer, A. (2005) J. Virol. 79, 10968–10977.

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REVIEW

Rashu B Seth et al. npg Cell Research (2006)16: 141-147 141 npg © 2006 IBCB, SIBS, CAS All rights reserved 1001-0602/06 $ 30.00 www.nature.com/cr

Antiviral innate immunity pathways
Rashu B Seth1, Lijun Sun1, Zhijian J Chen1
Howard Hughes Medical Institute, Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390-9148, USA
1

Recent studies have uncovered two signaling pathways that activate the host innate immunity against viral infection. One of the pathways utilizes members of the Toll-like receptor (TLR) family to detect viruses that enter the endosome through endocytosis. The TLR pathway induces interferon production through several signaling proteins that ultimately lead to the activation of the transcription factors NF-κB, IRF3 and IRF7. The other antiviral pathway uses the RNA helicase RIG-I as the receptor for intracellular viral double-stranded RNA. RIG-I activates NF-κB and IRFs through the recently identified adaptor protein MAVS, a CARD domain containing protein that resides in the mitochondrial membrane. MAVS is essential for antiviral innate immunity, but it also serves as a target of Hepatitis C virus (HCV), which employs a viral protease to cleave MAVS off the mitochondria, thereby allowing HCV to escape the host immune system. Cell Research (2006) 16:141-147. doi:10.1038/sj.cr.7310019; published online 13 February 2006 Keywords: interferon, Toll-like receptor, RIG-I, MAVS, mitochondria, NF-κB, IRF

Introduction
Viruses are highly infectious pathogens that depend on host cellular machinery for survival and replication. Most viral infections, like the common cold caused by Rhinoviruses, are efficiently resolved by the host innate and adaptive immune systems. The innate immune response is the first line of defense against an invading pathogen. A key aspect of the antiviral innate immune response is the synthesis and secretion of type I interferons (IFN) such as IFN-α and IFN-β, which exhibit antiviral, anti-proliferative and immunomodulatory functions [1]. Two events required to trigger an effective anti-viral innate immune response are: a) detection of the invading virus by immune system receptors; and b) initiation of protein signaling cascades that regulate the synthesis of IFNs. The cells of the innate immune system express pattern recognition receptors (PRR) that detect invariant molecular structures shared by pathogens of various origin (pathogen-associated molecular patterns, PAMP) [2]. Tolllike receptors (TLRs) 3, 7, 8 and 9 are the major PRRs that recognize distinct types of virally-derived nucleic acids and
Correspondence: Zhijian J Chen Tel: 214-648-1145, Fax: 214-648-1675; E-mail: Zhijian.Chen@UTSouthwestern.edu

activate signaling cascades that result in the induction of type I IFNs (Figure 1) [3]. Recently, retinoic acid inducible gene – I (RIG-I) has been identified as a cytosolic receptor for intracellular dsRNA [4]. RIG-I induces IFN in response to intracellular viral dsRNA in a TLR-independent manner. Thus, there are two receptor systems in place to detect the presence of virus and mount an immune response. These receptor systems localize to different compartments within a cell and recognize different ligands. Our current understanding of these receptor systems in antiviral immunity and their downstream signaling cascades is the focus of this review.

Regulation of type I IFN gene transcription
Type I IFNs include several IFN-α subtypes and a single IFN-β subtype [1]. The induction of type I IFN genes is regulated at the step of transcription and is best understood for the IFN-β promoter. A multi-protein complex called an enhanceosome is assembled at the IFN-β promoter in response to a viral challenge [5]. The enhanceosome consists of at least three classes of transcription factors – ATF-2/cJun, nuclear factor (NF)-κB and interferon regulatory factor 3 (IRF3). Of these, the activities of NF-κB and IRF3 are regulated by their subcellular localization. In the inactive state, NF-κB is held in the cytosol by inhibitory κB (IκB)

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Virus

Endosome dsRNA L TLR3 R R T I R CpG L R TLR9 R T I R RIG-I dsRNA Helicase
CARD CARD

ssRNA L TLR7/ R TLR8 R T I R

MAVS

CARD PRO

Mitochondria IRF3/7 NF-κB ATF-2/c-Jun

TRIF

MyD88

MyD88

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IFN-β Nucleus

Figure 1 TLR and RIG-I – two antiviral innate immunity pathways: Some viruses enter cells through the endocytic machinery. The genomes of such viruses are detected by members of TLR family of receptor including TLR3, TLR7, TLR8 and TLR9. These receptors have a single transmembrane domain and recognize their ligands through the leucine rich repeats (LRR) in their luminal domains. The cytoplasmic toll/IL-1 receptor (TIR) domain of these receptors enable the recruitment of adaptors such as TRIF or MyD88 that signal to downstream transcription factors ATF-2/cJun, NF-κB and IRF. RIG-I is a receptor for intracellular dsRNA. The C-terminal helicase domain of RIG-I binds dsRNA and activates the N-terminal CARD domains such that the downstream signaling cascade is initiated. MAVS is a mitochondrial protein that participates in the anti-viral signaling pathway downstream of RIG-I. Activation of either pathway leads to the induction of IFN-β.

family members [6]. In the presence of diverse stimuli, such as IL-1β, TNF-α, and viruses, the IκB kinase (IKK) is activated and it then phosphorylates IκB. Once phosphorylated, IκB is ubiquitinated and subsequently degraded by the proteasome. Free NF-κB then translocates into the nucleus and turns on its target genes. Similar to NF-κB, the inactive form of IRF3 is also cytosolic. In response to a viral challenge, IRF3 is phosphorylated by the IKK-like kinases TBK-1 and IKKe [7, 8]. Phosphorylation of IRF3 leads to its dimerization and translocation into the nucleus. Viral infection also leads to activation of stress kinases such as JNK and p38 kinase, which phosphorylate ATF2/c-Jun in the nucleus. Together with the nuclear architectural protein HMG-I (Y), NF-κB, IRF3 and ATF2/c-Jun assemble into a stereospecific enhanceosome complex that remodels the chromatin in the promoter of IFN-β, resulting in its transcriptional initiation. IFN-β binds to the IFNα/β receptor (IFNAR) in auto-

crine and paracrine manner to initiate a positive feedback loop that results in further production of type I IFNs [1]. IFNARs trigger the activation of the janus kinase (JAK) family members JAK1 and Tyk-2. These kinases in turn phosphorylate and activate the signal transducer and activator of transcription 1 (STAT1) and STAT2 proteins. These transcription factors associate with IRF9 to form a heterotrimeric complex, IFN-stimulated gene factor 3 (ISGF3). ISGF3 initiates the transcription of several interferon stimulated genes (ISGs) by binding to the IFN-stimulated response elements (ISRE) in their promoter regions. The ISGs inhibit different stages of virus replication and elicit an anti-viral state in the host. IRF7, one of the synthesized ISGs, is a member of the IRF family of transcription factors that regulates the transcription of IFN-α gene. An initial model suggested that IRF-7 is not involved in the initial phase of IFN-β induction as it is expressed at low levels in most cells in
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the absence of virus. IFN-β produced in response to a viral challenge by the IRF3 dependent pathway described above, induces transcription of IRF-7. IRF7 is then activated by phosphorylation at certain key residues by TBK-1/IKKe such that it binds and induces the promoter of IFN-α gene. A recent study using IRF7 knockout mice has demonstrated that transcription of both IFN-α and IFN-β is dependent on IRF7 [9], indicating that IRF7 is a master regulator of type I IFNs. IRF5 is another member of the IRF family that has been suggested to regulate type I IFN expression. However, recent genetic experiments using IRF5-deficient mice showed that IRF5 is not required for type I IFN induction, but is required for the induction of proinflammatory cytokines by stimulation of TLRs [10].

The TLR pathway of anti-viral innate immunity
The founding member of the TLRs is the Toll receptor in Drosophila, which was first found to instruct dorsalventral patterning in early embryos, and later found to also regulate anti-fungal innate immunity in adult flies. Sequence homology search in mammalian genomes has subsequently identified 11 members of TLRs [3]. These receptors contain an extracellular domain characterized by leucine-rich repeats (LRRs), a single transmembrane domain, and an intracellular signaling domain known as the Toll/IL-1R (TIR) domain. Although all TLRs share similar extracellular LRRs, they recognize very different microbial signatures. For example, TLR3 recognizes viral double-stranded RNA, TLR4 recognizes bacterial lipopolysaccharides (LPS), whereas TLR5 is a receptor for bacterial flagellin. In most cases, however, a direct binding between a TLR and a putative microbial ligand has not been demonstrated. The intracellular TIR domain recruits signaling molecules to activate downstream signaling pathways culminating in the induction of cytokines and IFNs through NF-κB and IRFs. Except for TLR3, all TLRs utilize MyD88 as an adaptor protein to recruit downstream signaling molecules including the protein kinases IRAK4 and IRAK1, and the RING domain ubiquitin ligase TRAF6. TRAF6 functions together with a dimeric ubiquitin conjugating enzyme complex Ubc13-Uev1A to catalyze the synthesis of Lys63-linked polyubiquitin chains that lead to the activation of a protein kinase complex consisting of TAK1, TAB1 and TAB2 [11, 12]. The activated TAK1 kinase phosphorylates IKKβ in the activation loop, resulting in the activation of IKK and subsequent nuclear translocation of NF-κB. The TIR domains of TLR3 and TLR4 bind to another adaptor protein TRIF, which binds directly to TRAF6 and RIP1 to activate NF-κB. TRIF can also bind to TBK1, which phosphorylates and activates
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IRF3 and IRF7. Recent studies have also shown that TRIF and MyD88 can bind to TRAF3, which activates IRFs to induce type I IFNs, but inhibits NF-κB to suppress the induction of proinflammatory cytokines [13, 14]. Among TLRs, TLR3, 7, 8 and 9 are involved in antiviral innate immune responses [3]. TLR3 recognize dsRNA viruses and may also be involved in sensing dsRNA released from dying cells. TLR7 and TLR8 are receptors for G/U-rich ssRNA associated with viruses that enter cells through endocytosis. TLR9 recognizes unmethylated CpG DNA present in DNA viruses such as herpesvirus. These receptors are localized in the endosomal membranes, with the ligand binding domain facing the lumen of the endosomes, and the TIR signaling domain positioning in the cytoplasmic side. Viral nucleic acids that arrive in this compartment through endocytosis are recognized by these receptors. The endosomal localization of TLR7, 8 and 9 is essential for signaling, as formulation of nucleic acids that allow them to retain in the endosome convert them to effective TLR ligands that induce type I IFNs [15]. This explains why plasmacytoid dendritic cells (pDC) are high producers of IFNs, as these cells effectively retain viral RNA in the endosome, whereas in conventional dendritic cells (cDC), viral RNA is rapidly transported from endosome to lysosome. The MyD88-IRAK-TRAF6 signaling module is essential for the induction of IFNs by TLR7, 8 and 9. The same signaling module is also required for the activation of NF-κB by IL-1β and other TLRs such as TLR2; however, IFNs are not induced by IL-1β or TLR2. Thus, it is likely that TLR7, 8 and 9 recruit additional components in pDCs to activate IRF7, the master regulator of IFN-α. One of such components may be TRAF3, which has recently been shown to be essential for IFN-α induction in pDCs. It has been shown that MyD88 and TRAF6 can bind to IRF7 directly, and recruit IRAK1 to phosphorylate IRF7, resulting in the nuclear translocation and activation of IRF7 [16, 17]. Indeed, IRAK1-deficient mice are defective in IFN-α production in response to stimulation of TLR7 and TLR9 [18]. Interestingly, TRAF6 appears to induce the phosphorylation of IRF7 through a mechanism that involves Ubc13-catalyzed K63 polyubiquitination [17]. It would be interesting to determine if IRAK1 or another IRF7 activating kinase is activated by TRAF6 in a ubiquitin-dependent manner. Genetic experiments clearly demonstrate that TLR7 and TLR8 are essential for interferon induction in pDCs by RNA viruses [19]. However, in many other cell types, including cDCs, macrophages and fibroblasts, deletion of both MyD88 and TRIF, which abolishes all TLR signaling, has no effect on viral induction of IFNs [20]. Furthermore, although human patients deficient in IRAK4 are more

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susceptible to bacterial infection, they have intact immune responses against viruses [21]. Therefore, there must be TLR-independent pathways that are highly effective in providing antiviral innate immunity.

The RIG-I pathway of anti-viral innate immunity
Retinoic acid inducible gene I (RIG-I) has recently been identified as an intracellular receptor for viral dsRNA [4]. RIG-I is a member of the DExD/H box-containing RNA helicase family of proteins that unwind dsRNA in an ATPase dependent manner. The helicase domain of RIG-I can bind both synthetic dsRNA [poly (I:C)] and viral dsRNA. Besides the C-terminal helicase domain, RIG-I also contains two tandem caspase recruitment domains (CARDs) at its N-terminus. Over-expression of the N-terminal region of RIG-I comprising the two CARD domains is sufficient to activate NF-κB and IRF3 in the absence of a viral challenge, whereas the full-length RIG-I is activated only in the presence of dsRNA. Thus, the binding to dsRNA to the RNA helicase domain of RIG-I likely induces a conformational change that exposes the N-terminal CARD domains to recruit downstream signaling proteins. Further structural analysis will be required to determine the exact mechanism of how RIG-I is regulated by dsRNA binding. The functional significance of RIG-I in anti-viral immunity was shown first by RNAi studies and confirmed by mouse knockout studies [4, 20]. RNAi of RIG-I in L929 cells, a mouse fibroblast cell line, inhibited not only IRF3 activation but also subsequent induction of type I IFNs in response to RNA viruses. The embryos of RIG-I knockout mice displayed severe liver degeneration and most were embryonic lethal. The mechanism underlying the lethal phenotype of RIG-I mutant mice is not understood as yet. In mouse embryonic fibroblasts (MEFs) and lung fibroblasts, it was shown that the induction of IFN-β and ISGs by several RNA viruses was abolished. Pre-treatment of the fibroblasts with IFN-β increased the resistance of the RIG-I deficient fibroblasts to VSV, indicating that RIG-I is required for the induction of IFN-β and does not affect the downstream IFN-β signaling pathway. An important question is the relative importance of signaling mediated by RIG-I vs TLRs in an in vivo system. This question has been addressed by comparing the interferon induction in fibroblasts and bone marrow derived dendritic cells from RIG-I-/- vs MyD88-/- TRIF-/- mice, which lack all TLR signaling [20]. The ability to induce IFN-β when challenged by NDV was severely compromised in the RIG-I-/-, but not MyD88-/- TRIF-/-, cDCs and fibroblasts. Opposite results were observed for the pDCs, which induce IFN-β normally in the absence of RIG-I, but not in the absence of MyD88 and TRIF. Thus, RIG-I

and TLR pathways are not redundant, but rather mediate antiviral signaling in different cell types. Besides RIG-I, MDA-5 and Lpg2 have also been identified as DExD/H box RNA helicases that function in the antiviral immune response [22, 23]. Like RIG-I, MDA-5 also contains two N-terminal CARD domains which can activate the IFN-β promoter. The importance of MDA-5 as an anti-viral protein had been suggested based on the finding that paramyxovirus V protein binds MDA-5 and inhibits its function [24]. Lpg2 lacks the CARD domain and acts as a negative regulator of the RIG-I pathway. Over-expression of Lpg2 inhibits the activation of IFN-β promoter by Sendai virus, but it does not interfere with the TLR3 signaling pathway.

MAVS signaling in the RIG-I pathway
Recent studies have identified a CARD domain containing protein that acts downstream of RIG-I. This protein, independently identified by four different groups, has been called mitochondrial anti-viral signaling protein (MAVS) [25], IFN-β promoter stimulator 1 (IPS-1) [26], virus-induced signaling adaptor (VISA) [27] and CARD adaptor inducing IFN-β (CARDIF) [28]. Based on its biological function as an antiviral protein and the importance of mitochondrial localization for the function of this protein (discussed below), we will refer to this protein as MAVS in this review. Several lines of evidence demonstrate an essential role for MAVS in the antiviral signaling pathway. First, overexpression of MAVS leads to the activation of NF-κB and IRF3, and therefore type I IFN production. Second, knockdown of MAVS expression by RNAi abolishes the induction of IFNs by viruses as well as by RIG-I over-expression. Third, the activation of kinases responsible for NF-κB and IRF3 activation is abrogated in the absence of MAVS expression. Fourth, over-expression of MAVS protects cells from the cytopathic effects of VSV, whereas RNAi of MAVS renders the cells more susceptible to killing by the virus. Further epistasis studies show that MAVS functions downstream of RIG-I and upstream of IKK and TBK1 (Figure 2). Besides the N-terminal CARD domain, MAVS also contains a proline-rich (PRO) region and a C-terminal hydrophobic transmembrane (TM) region [25]. Deletion analyses have shown that the CARD domain and the TM domain are essential for the function of MAVS. Unlike the RIG-I CARD domains, overexpression of MAVS CARD domain is not sufficient to induce IFN-β. But when the CARD domain is fused with the C-terminal TM domain, this truncated ‘mini-MAVS’ protein, which represents just one-fourth of the total length of MAVS, is sufficient
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Virus

Intracellualar dsRNA Helicase CARD CARD HCV NS3/4A

RIG-I

MAVS

CARD PRO Mitochondria

IKKγ Ub IKKα Ub Ub Ub IKKβ P

TBK-1/ IKKe

Proteasome Cytoplasm

IκBα

NF-κB

IRF3

P

Nucleus

NF-κB

IRF3

P

IFN-β

Figure 2 The RIG-I – MAVS signaling pathway: RIG-I is a receptor for intracellular dsRNA. It contains a C-terminal RNA helicase domain that binds to viral dsRNA, and two tandem CARD domains at the N-terminus. The binding of dsRNA to the helicase domain presumably induces a conformational change that exposes the CARD domains to initiate a signaling cascade. MAVS is a CARD domain containing mitochondrial protein that functions downstream of RIG-I. MAVS can signal to both NF-κB and IRF3 signaling pathways by activating the IKK and TBK-1/IKKe kinase complexes. Once activated, NF-κB and IRF3 translocate into the nucleus and turn on the IFN-β gene promoter. The mechanism by which MAVS activates downstream kinase pathways is not clear, although it has been shown that the mitochondrial membrane localization of MAVS is essential for its signaling function. The importance of the mitochondrial localization of MAVS is underscored by the recent discovery that the hepatitis C virus protease NS3/4A cleaves MAVS off the mitochondria to evade the host innate immune system.

to activate the downstream pathway. The TM sequence of MAVS resembles the mitochondrial targeting sequences of several C-tail anchored mitochondrial membrane proteins, including the cell survival proteins Bcl-2 and Bcl-xL. Indeed, biochemical and microscopic imaging experiments show that the C-terminal TM domain of MAVS targets the protein to the mitochondrial outer membrane. Importantly, the mitochondrial localization of MAVS is essential for its activity because the deletion of the TM domain, which mislocalizes the protein to the cytosol, abolishes the signalwww.cell-research.com | Cell Research

ing function of MAVS. When the TM domain of MAVS was replaced with the mitochondrial membrane targeting domain of Bcl-2 or Bcl-xL, the function of MAVS was fully restored, indicating that it is the mitochondrial localization but not the sequence of the TM domain that is essential for MAVS activity. This conclusion is further supported by the experiments showing that mislocalization of MAVS to other membrane compartments such as plasma membrane and endoplasmic reticulum greatly impairs the ability of MAVS to induce IFNs.

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The mechanisms by which MAVS is regulated by RIG-I and how MAVS signals to downstream kinases remain to be further investigated. Although MAVS has been shown to interact with RIG-I in over-expression experiments by several groups, it has not been clearly demonstrated that endogenous MAVS and RIG-I can interact in a virus-dependent manner. The signaling mechanism of MAVS is a subject of debate at present. Two groups show that MAVS can interact with TRAF6 and both identified TRAF6 binding sites within MAVS [25, 27]. However, Seth et al presented evidence that the MAVS mutant (mini-MAVS) lacking all TRAF-binding sites is still capable of inducing IFN-β. Furthermore, TRAF6-deficient cells have normal induction of IFN-β following viral infection [25, 29]. Kawai et al showed that MAVS interacted with RIP-1 and FADD, and proposed that these molecules linked MAVS to IKK activation [26]. However, RIP1-deficient MEF cells are also fully capable of inducing IFN-β following viral challenges [25, 30]. Meylan et al reported that MAVS bound to IKKα and IKKe directly, but such interaction was not found by the other groups. Thus, there is no consensus mechanism that emerges from these independent studies. Further studies are clearly required to elucidate the mechanism of MAVS signaling. The discovery of MAVS has also provided a breakthrough in the field of hepatitis C virus (HCV) research. HCV infects more than 170 million people worldwide, and approximately 80% of the infected individuals develop persistent infection [31]. The persistence of HCV infection is caused in part by the suppression of the host immune system by HCV viral proteins, such as the NS3/4A serine protease. Previous studies have shown that NS3/4A inhibits the induction of IFN-β by the RIG-I signaling pathway [32-34], however, the target of NS3/4A was not known previously. Two groups have now shown that MAVS is the long-sought target of NS3/4A [28, 35]. NS3/4A cleaves MAVS at Cys-508, which is located only a few residues before the mitochondrial targeting domain of MAVS. As a result of the proteolytic cleavage, MAVS is dislodged from the mitochondria and becomes an inactive cytosolic fragement. Li et al also showed that NS3/4A binds to and co-localizes with MAVS in the mitochondrial membrane, and it can cleave MAVS directly in vitro. Using a replicon cell culture system in which the RNA genome of HCV replicates autonomously, Li et al demonstrated that endogenous MAVS is indeed cleaved by NS3/4A, and that a mutation at C508 of MAVS that prevents its cleavage restores IFN induction in the replicon cells. Meylan et al also showed that transfected MAVS is cleaved in a liver cell line infected with the recently developed HCV virus. Taken together, these results show that HCV paralyzes the host immune system by cleaving MAVS off the mitochondria,

further underscoring the importance of the mitochondrial localization of MAVS in its antiviral signaling.

Conclusions and perspectives
Research in the past few years has uncovered two antiviral innate immunity pathways leading to the induction of interferons. The TLR pathway operates mainly in pDCs to detect viral RNA and DNA associated with endocytosed viral particles. In most other cell types, the RIG-I pathway is essential for innate immune responses against intracellular viral replication. While the receptors for both antiviral pathways have now been identified, the signaling pathways downstream of both receptors remain to be fully elucidated. In the TLR pathway, although it is clear that MyD88, IRAK, TRAF6 and TRAF3 are essential for the induction of IFN-α in pDCs, how these proteins lead to the phosphorylation and activation of IRF7 is not understood. Similarly, in the RIG-I pathway, although MAVS is clearly an essential adaptor molecule that links RIG-I to IKK and TBK1 activation, how MAVS is regulated by RIG-I and how it activates downstream kinases remains largely unknown. While the mitochondrial localization of MAVS is essential for its signaling function, how mitochondria play a role in activating IKK and TBK1 is still a mystery. Future studies should also uncover more examples of host-virus interaction, as illustrated from the proteolytic cleavage of MAVS by the HCV protease. It is quite possible that other viruses may have also evolved to develop novel strategies to target pivotal host immune response proteins such as MAVS. Measures to counter the viral suppression of the host immune system may prove effective in the prevention and treatment of viral diseases.

Acknowledgments
Research in the Chen laboratory is supported by grants from NIH (RO1-AI60919 and RO1-GM63692), American Cancer Society (RSG0219501TBE), and the Welch Foundation (I-1389). Zhijian J Chen is an Investigator of Howard Hughes Medical Institute and a Burroughs Wellcome Fund Investigator in Pathogenesis of Infectious Diseases.

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