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Hepatitis C virus protease NS34A cleavesmitochondrial antiviral signaling proteinoff the mitochondria to evade innate immunityXiao-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 reviewSeptember 29, 2005)

Hepatitis C virus (HCV) is a global epidemic manifested mainly bychronic infection. One strategy that HCV employs to establishchronic infection is to use the viral Ser protease NS34A to cleavesome unknown cellular targets involved in innate immunity. Herewe show that the target of NS34A is the mitochondrial antiviralsignaling protein, MAVS, that activates NF-B and IFN regulatoryfactor 3 to induce type-I interferons. NS34A cleaves MAVS atCys-508, resulting in the dislocation of the N-terminal fragment ofMAVS from the mitochondria. Remarkably, a point mutation ofMAVS at Cys-508 renders MAVS resistant to cleavage by NS34A,thus maintaining the ability of MAVS to induce interferons in HCVreplicon cells. NS34A binds to and colocalizes with MAVS in themitochondrial membrane, and it can cleave MAVS directly in vitro.These results provide an example of hostpathogen interaction inwhich the virus evades innate immunity by dislodging a pivotalantiviral protein from the mitochondria and suggest that blockingthe cleavageof MAVS byNS34A may be applied to thepreventionand treatment of HCV.

IFN regulatory factor 3 NF-B retinoic acid-induced gene I IB kinase

Hepatitis C virus (HCV) infects 170 million people in theworld, and 80% of the infected individuals develop persis-tent infection (1, 2). HCV is an enveloped single-strand RNA virusbelonging to the Flaviviridae family. It contains a 9.6-kb RNA

genome that encodes a large polyprotein (3,000 aa), which iscleaved into 10 structural and nonstructural (NS) proteins throughthe action of cellular peptidases as well as the viral-encodedproteases including NS34A. NS3 contains Ser protease and RNAhelicase activities that require its cofactor NS4A, which tethers theholoenzyme complex to an intracellular membrane compartment.The NS34A protease is not only essential for generating matureviralproteins required for viralreplication, butalso cansuppress thehost antiviral immune system by cleaving putative cellular targetsinvolved in the induction of type-I interferons (IFNs), such asIFN- and IFN- (3).

The induction of IFNs is regulated by the transcription factorsNF-B and IFN regulatory factor 3 (IRF3), which are activated bya signaling cascade emanating from dsRNA generated during thereplication of viral RNA (4, 5). The activation of NF-B requires

the phosphorylation of its inhibitor IB by IB kinase (IKK)complex, which can be activated by a large variety of agents,including proinflammatory cytokines and microbial pathogens (6).The phosphorylation of IB targets this inhibitor for ubiquitinationand subsequent degradation by the proteasome, thereby allowingNF-B to enter the nucleus to regulate downstream genes. Theactivation of IRF3 requires its phosphorylation by two IKK-relatedkinases, TBK1 and IKK (7, 8). After phosphorylation, IRF3 isdimerized and translocated into the nucleus where it forms anenhanceosome complex together with NF-B and other transcrip-tion factors to turn on the expression of targets genes such as IFN-(4). IFNs then induce a large array of antiviral genes through theactivation of the Janus tyrosine kinasesignal transducer and acti-vator of transcription pathway.

The receptor for intracellular viral dsRNA has recently beenidentified as retinoic acid-induced gene I (RIG-I), which containsa RNA helicase domain that binds to dsRNA (9). RIG-I alsocontains 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-terminaltransmembrane (TM) domain that targets it to the mitochondrialouter membrane (10). Importantly, the mitochondrial localizationof MAVS is essential for its signaling function, because the removalof the mitochondrial-targeting domain of MAVS abolishes itsability to induce IFNs. Epistasis experiments have shown thatMAVS functionsdownstream of RIG-I and upstream of IB kinaseand TBK1 in the viral signaling pathway.

Recent studies have shown that NS34A inhibits IFN inductionby RIG-I; however, RIG-I is not cleaved by NS34A (14, 15).NS34A also was found to cleave TIR domain-containing adapter-inducing IFN- (TRIF) (16), an adaptor protein that binds toToll-like receptors such as TLR3 and TLR4 (17). However, TRIFis not essential for IFN induction by viruses (18). Thus, the targetof NS34A in the viral pathway remains to be identified. In thiswork, we present evidence that MAVS is the proteolytic target ofNS34A. We found that NS34A cleaves MAVS at Cys-508,resulting in the dislocation of the N-terminal fragment of MAVSfrom the mitochondria,thereby suppressing the induction of IFN-.

A point mutation at Cys-508 prevents the cleavage of MAVS byNS34A and restores IFN- induction in a HCV replicon cell line,suggesting that the cleavage of MAVS is responsible for thesuppression of IFN induction in HCV replicating cells. We alsoshow that NS34A binds to and colocalizes with MAVS in themitochondria. Finally, we show that NS34A cleaves MAVS atCys-508 in vitro, providing the direct biochemical evidence thatMAVS is the target of NS34A.

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 bysubcloning the DNA fragment encoding MAVS-HA into the XhoI

and XbaI sites of the pcDNA3-Flag vector such that the N-terminalFlag epitope was fused in frame with MAVS-HA. NS34A 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 antiviralsignaling

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

To whom correspondence should be addressed. E-mail: [email protected].

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 lineK2040 (19) and then cloned into pcDNA3 in frame with anN-terminal Flag or Myc tag. TheS139A mutant of NS34Aand theCys mutants of MAVS (C435R, C452R, and C508R) were gener-ated by site-directed mutagenesis using the QuikChange kit (Strat-agene). pET14b-NS34A was constructed by subcloning NS34Ainto the XhoI and BamH1 sites of pET14b (Novagen). All plasmidswere 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 waspurchased from NovoCastra (Newcastle, U.K.). The monoclonalAbs 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 andHeLa cells were from American Type Culture Collection. TheHuh7 and HCV replicon cell line K2040 were provided by MichaelGale (University of Texas Southwestern) (19). Transfection ofHEK293 cells was carried out by using the calcium phosphateprecipitation method. HeLa cells were transfected by using thePolyFect reagent (Qiagen, Valencia, CA). Huh7 and replicon cellswere transfected by using lipofectamine 2000 (Invitrogen) or Su-perfect (Qiagen) reagents. Luc reporter assays were performed asdescribed in ref. 10.

Subcellular Fractionation. HEK293 cells were transfected with ex-pression vectors for Flag-MAVS-HA or the C508R mutant to-gether with Myc-tagged NS34A or its inactive mutant S139A. Cellswere washed 36 h after transfection in hypotonic buffer (10 mMTrisHCl, pH 7.510 mM KCl1.5 mM MgCl2protease inhibitors)and then homogenized in the same buffer by douncing 20 times.The homogenate was centrifuged at 500 g for 5 min to removenuclei and unbroken cells. The supernatant was centrifuged againat 5,000 g for 10 min to generate membrane pellets (P; containingmostly mitochondria) and cytosolic supernatant (S). The sameprocedure was used for subcellular fractionation of Huh7 and HCVreplicon cells.

Cleavage of MAVS by NS34A Protease in Vitro. pcDNA3-Flag-NS34A or its mutant S139A was transfected into HEK293 cells toexpress the protease, which then was purified by binding to theFlag-Sepharose (M2-Sepharose, Sigma) followed by elution withtheFlagpeptide (0.2 mgml). The eluted NS34A or S139Amutantwas concentrated by using Amicon microconcentrator (Millipore,10,000 Da cut-off) and diluted in buffer A (20 mM TrisHCl150mM NaCl0.1% Triton X-1005 mM DTT, pH 7.5). This proce-dure was repeated three times to reduce the concentration of theFlag peptide. For expression of NS34A in Escherichia coli,pET14b-NS34A was transfected into the E. coli strain BL21pLysS, and the transformant was induced to express His-6-NS34Aafter the addition of IPTG. His-6-NS34A was purified by usingnickel affinity column and dialyzed against buffer B (20 mM

Tris

HCl, pH 7.5100 mM NaCl10% glycerol).Flag-MAVS or its mutant C508R was translated in vitro byusing the TNT rabbit reticulocyte lysate (Promega) supple-mented with [35S]methionine. The 35S-labeled Flag-MAVS orC508R was purified by using Flag-Sepharose as described abovefor NS34A. To measure the cleavage of MAVS by NS34A, 0.5l of35S-labeled MAVS or C508R was incubated with 0.5 l ofNS34A or S139A in a 10-l reaction containing buffer A. Afterincubation at 30C for 2 h, the reaction products were resolvedby SDSPAGE and analyzed by using PhosphorImager (Molec-ular Dynamics).

The experimental procedures for viral infection, immunoblot-ting, immunoprecipitation, and confocal microscopy are describedin ref. 10.

Results

NS34A Blocks IFN- Induction by MAVS. To determine whetherNS34A inhibits IFN- induction by MAVS, we transfected ex-pression vectors encoding NS34A and MAVS into HEK293 cellstogether 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 N-terminal CARD domains of RIG-I [RIG-I(N)], which has previ-ously been shown to induce IFN- (9). In each case, NS34A

inhibited the induction of IFN- (Fig. 1A). In contrast, the induc-tion of IFN- by TBK1 was notaffected by NS34A, suggesting thatNS34A inhibits the RIG-I pathway at the level of MAVS or at astep downstream of MAVS but upstream of TBK1. To determinewhether the protease activity of NS34A was required for thisinhibition, we mutated the active site Ser-139 of NS34A (equiv-alent to Ser-1165 of the HCV polyprotein) to Ala (20, 21). Thismutant migrated more slowly than wild-type (WT) NS3 (Fig. 1DBottom), because it could no longer be cleaved at the junctionbetweenNS3 andNS4A. The protease-deadmutant completely lostits ability to inhibit IFN- induction by SeV, RIG-I(N), or MAVS.Thus, it is likely that NS34A inhibits IFN induction by cleavingMAVS or a target downstream of MAVS. NS34A also preventedthe MAVS-induced activation of NF-B (Fig. 1B) and IRF3reporters (Fig. 1C), as well as the phosphorylation and dimerization

of IRF3 (Fig. 1D), further supporting the notion that NS34Ainactivates a common target required for both NF-B and IRF3activation.

MAVS Is the Proteolytic Target of NS34A. To determine whetherMAVS is the proteolytic target of NS34A, we transfected theexpression vector encoding FLAG-NS34A or FLAG-NS34A(S139A) into HEK293 or Huh7 (a human hepatoma cell line).Because MAVS is a mitochondrial membrane protein, the celllysates were separated into cytosolic fraction (S) and membranepellets (P) by centrifugation, and the endogenous MAVS proteinanalyzed by immunoblotting with an affinity-purified MAVS Ab(Fig. 2A). In both cell lines, the expression of NS34A, but not theprotease-dead mutant of NS34A, led to the generation of a

faster-migrating fragment of MAVS that was

35 kDa shorterthan the full-length MAVS (Fig. 2 A Upper; compare lanes 3 with4 and9 with 10).Interestingly, unlikethe full-length MAVSthat waspresent in the membrane pellet, the truncated fragment of MAVSwas mainly present in the soluble cytosolic fraction, suggesting thatthe truncated fragmentdid not contain the C-terminal TM domain.Immunoblotting experiments also showed that the majority of NS3is in the membrane pellet, presumably because of its associationwith NS4A (Fig. 2 A Lower).

MAVS contains an N-terminal CARD domain, a middle Pro-rich domain, and a C-terminal TM domain. We have previouslyshown that a truncated MAVS protein containing only the CARDand TM domains (hereafter referred to as miniMAVS) is sufficientto induce IFN- (10). To further delineate the NS34A cleavagesite of MAVS, we tested whether the miniMAVS was also sensitive

to NS34A inhibition. As shown in Fig. 2B, IFN- induction by theminiMAVS was also inhibited by NS34A (Middle). Furthermore,the miniMAVS was cleaved to a shorter fragment by the WT, butnot the S139Amutant, of NS34A (Fig. 2B Bottom). The truncatedfragment was also 4 kDa shorter than the miniMAVS (Fig. 2 B,compare lanes 4 and 5), suggesting that this small MAVS proteinof only 140 residues in length contains a cleavage site of NS34A,most likely at a residue near the TM domain.

NS34A Cleaves MAVS at Cys-508. NS34A is a Ser protease thatcleaves at the C terminus of a Cys or Thr residue within a looselydefined consensus sequence [(ED)xxxx(CT)(SA), where x de-notes any amino acid], which is found at the junction of HCV NSproteins (Fig. 2C) (22). Inspection of the miniMAVS amino acid

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sequence revealed a potential NS34A cleavage sequence, 503ER-EVPCH509. The potential cleavage site, Cys-508, is located 6 aafrom the beginning of the TM domain and 32 aa from the Cterminus of MAVS. Cleavage at Cys-508 is predicted to generate an32-residue C-terminal fragment, consistent with the size differ-ence between the miniMAVS and the miniMAVS lacking the TMdomain (Fig. 2B Bottom; compare lanes 4 and 5). Indeed, whenNS34A was cotransfected together with an expression constructencoding a MAVS protein that is tagged with FLAG at the N

terminus and HA at the C terminus, theMAVS protein was cleavedinto a soluble FLAG-tagged N-terminal fragment that was smallerthan the intact protein (Fig. 2D Top; compare lanes 1 and 4).Moreover, an7-kDa C-terminal fragment was detectable in boththe membrane pellet and cytosolic fractions with a HA Ab (Fig. 2DMiddle; lanes 3 and 4). This cleavage product of MAVS was notdetected in cells expressing the S139A mutant of NS34A. Al-though the apparent molecular mass of the C-terminal cleavagefragment was greater than the theoretical molecular mass of thefragment containing the C-terminal 32 residues of MAVS plus aHA epitope (5 kDa), this result is likely because of the abnormalmigration of small peptides on SDSPAGE. Indeed, when wesynthesized the C-terminal 32 residues of MAVS (residue 509540)plus the HA epitope by in vitro translation, this fragment alsomigrated as an 7-kDa band (see Fig. 5A, which is published as

supporting information on the PNAS web site), identical to theC-terminal fragment of MAVS after cleavage by NS34A.To demonstrate that Cys-508 is indeed the cleavage site, we

mutated Cys-508 to Arg, which is predicted to disrupt NS34Acleavage based on the analysis of sequence requirement for HCVpolyprotein cleavage (23). Remarkably, this single amino acidsubstitution completely blocked the cleavage of MAVS by NS34A(Fig. 2D, lanes 712). Furthermore, the protease-resistant mutantof MAVS was fully capable of inducing IFN-, and this inductionwas no longer inhibited by NS34A (Fig. 2E). In contrast toCys-508, mutations at two adjacent Cys residues, Cys-435 and -452,did not prevent the cleavage of MAVS by NS34A (Fig. 5B).Together, these results indicate that NS34A cleaves MAVS atCys-508 when these proteins are coexpressed in the same cells.

NS34A Binds to and Cleaves MAVS Directly in Vitro. To determinewhether NS34A could cleave MAVS directly in vitro, we expressedFLAG-NS34A or FLAG-NS34A (S139A) protein in HEK293cells and immunopurified the protein using the FLAG Ab followedby elution with a FLAG peptide. We also synthesized 35S-labeledFLAG-MAVS or FLAG-MAVS-C508R protein in vitro usingrabbit reticulocytelysates and purified the proteins using the FLAGAb. The purified MAVS proteins were incubated with NS34Aprotease or lysates containing theprotease. As shownin Fig. 3A, theWT MAVS was cleaved by theactive NS34A protease, but not theinactive NS34A mutant (lanes 15). In contrast, a point mutationat Cys-508 completely blocked the cleavage of MAVS by NS34Ain vitro (lanes 610). The in vitro cleavage product represents theN-terminal fragment of MAVS, because the C-terminal 32-aafragment does not contain any Met that can be radioactively

labeled. Similar results were obtained by using NS34A proteaseexpressed and purified from E. coli (see Fig. 6A, which is publishedas supporting information on the PNAS web site). These resultsindicate that NS34A directly cleaves MAVS at Cys-508 in vitro.

The cleavage of MAVS by NS34A suggests that these twoproteins may interact directly. To investigate this possibility, wecotransfected MAVS and NS34A or their mutants into HEK293cells, immunoprecipitated MAVS with the MAVS Ab, and thendetermined whether NS34A was coprecipitated with MAVS byimmunoblotting (Fig. 3B). Interestingly, only the S139A mutant,but not the WT, NS34A coprecipitated with MAVS (Fig. 1DBottom, compare lanes 3 and 9), suggesting that the cleaved MAVSdissociated from the WT protease, whereas the mutant proteasewas able to trap its substrate. Consistent with this interpretation,

Fig. 1. NS34A blocks IFN induction by MAVS. (A) HEK293 cells were trans-fected with IFN--Luc and the expression vector for either the WT or S139A

mutant of NS34A. The next day, cells were transfected with expressionvectors 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 dupli-

cated experiments. (B) Similar to A, except that NF-B Luc reporter was used

inlieuof IFN--Luc. (C) Similar to A, except thatcells weretransfectedwith the

UAS-Luc reporter together with the Gal4-IRF3 expression vector. (D) HEK293

cells were transfected with theWT or S139Amutant of NS34A togetherwiththeexpressionvectorsforMAVS (lanes57)or TBK1 (lanes8 10).In lanes 24,

cells were infected with SeV for 24 h. Cell lysates were resolved by electro-

phoresis under native (Top) or denaturing condition (Middle) and then im-

munoblottedwith anAb against IRF3. Theexpressionof NS34AandTBK1alsowas analyzed by immunoblotting with a Flag Ab (Bottom).

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C508R also bound to either WT or S139A mutant of NS34A, butthis binding was weaker than that observed between MAVS andtheS139A mutant of NS34A (compare lanes 6 and 12 with lane 9),suggesting that C508R mutation may partially impair its binding tothe protease.

NS34A Colocalizes with MAVS in theMitochondria.It has been shownthat HCV core protein and NS34A are localized to a mitochon-drion-associated membrane structure in both cultured hepatocytes(24, 25) and liver biopsies of chronic hepatitis C patients (26).

Because MAVS is a mitochondrial membrane protein, we exam-ined whether MAVS and NS34A colocalize in the same mem-brane compartment. Expression vectors encoding Myc-NS34Aandor HA-MAVS were transfected into HeLa cells, which thenwere stained with the corresponding Abs followed by imaging witha laser scanning confocal microscope. To stain the mitochondria,cells were incubated with Mito Tracker, a fluorescent dye taken upby the mitochondria of living cells. The staining patterns of the W TNS34A overlapped with that of Mito Tracker, but not the endo-plasmic reticulum resident protein calnexin (Fig. 6B), suggesting

Fig. 2. NS34A cleaves MAVS at Cys-508. (A) HEK293 (lanes 16) or Huh7 (lanes 712) 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 cytosolicsupernatant (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 NS34A together with a MAVS mutant containing only the CARD and TM domains (miniMAVS; Top). The activationof IFN- by the CARD-TM fragment of MAVS in the presence of WT or S139A mutant of NS34A was determined by measuring the expression of the IFN--Lucreporter (Middle). Cell lysates were analyzed by immunoblotting with theFlag Ab that detects both NS34A andMAVS proteins (Bottom). (C) Alignmentof thejunction 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

withexpressionvectorsencodingthe WT orC508R mutant ofMAVS togetherwiththose encodingthe WT or S139A mutantof NS34A.Cell lysateswere separatedby differential centrifugation into the membrane pellet and cytosolic supernatant, which were then resolved by 7% ( Top and Bottom) or 420% (Middle)

SDSPAGE, followed by immunoblotting with the indicated Ab that detects the N terminus (Flag; Top) or C terminus (HA; Middle) of MAVS, or NS34A (Myc;Bottom). (E) HEK293 cells were transfected with MAVS and NS34A expression vectors as in D, except that IFN--Luc was also cotransfected to measure theinduction of IFN- by Luc assay.



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that NS34A is localized to the mitochondria or in a mitochondri-on-associated membrane. When WT NS34A and MAVS were

coexpressed, the majority of MAVS became cytosolic (presumablydue to cleavage), as revealed by an Ab that detects the N terminusof MAVS (Fig. 3C). In sharp contrast, when the WT NS34A wascoexpressedwith MAVS-C508R, bothNS34A andMAVS-C508Rproteins colocalized in the mitochondrialmembrane andshowed anextensive overlapping staining pattern. These results indicate thatMAVS and NS34A are colocalized in the mitochondrial mem-brane or are positioned in very close proximity, thus renderingMAVS vulnerable to cleavage by NS34A.

MAVS Is Cleaved in a HCV Replicon Cell Line. To determine whetherMAVS is a target of HCV during viral replication, we used theHCV replicon cell culture system in which the subgenomic RNA ofHCV replicates autonomously in the human hepatoma cell line

Huh7 (27). The HCV subgenomic RNA encodes all of the NSproteins, including the NS34A protease and the NS5B RNApolymerase. It has been shown that the HCV replicon cell lines thatreplicate the viral RNA efficiently can suppress the host IFNinduction through the proteolytic activity of NS34A (3). Todetermine whether the endogenous MAVS is cleaved in thereplicon cells, we used subcellular fractionation and immunoblot-ting to analyze the MAVS protein from the WT Huh7 and areplicon cell line K2040 (19). As shown in Fig. 4A, the MAVSprotein is present predominately in the mitochondrial membranepellet isolated from Huh7 cells. In contrast, in the HCV repliconcells, a cleaved form of MAVS was found mostly in the solublecytosolic fraction, whereas NS3 was detected mainly in the mem-brane pellets.

Fig. 3. NS34Abindsto andcolocalizes with MAVS in themitochondria, andit cleaves MAVS in vitro. (A) Flag-tagged WT or S139A mutant of NS34A wasexpressed in HEK293 cells and then purified by using Flag-Sepharose. The

purified NS34A proteins (lanes 4, 5, 9, and 10) or lysates containing NS34A

(lanes 13 and 68) were incubated with35

S-labeled MAVS or C508R mutantof MAVS, which was synthesized by in vitro translation and purified by using

Flag-Sepharose. After incubation at 30C for 2 h, proteins were separated by

SDSPAGE and analyzed by PhosphorImaging (Upper) or immunoblotting(Lower).The cleavageproduct of MAVSis indicatedas MAVS*. (B) HEK293cells

were transfected with expression vectors for Flag-NS34A and HA-MAVS asindicated. 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-NS34A and HA-MAVS or C508Rmutant weretransfectedinto HeLacells,and thelocalizationof 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 su-

pernatant (S), which then were analyzed by immunoblotting with an Ab

againstMAVS or NS3.The cleavageproducts of MAVSare indicatedas 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 NS34A. (B and C) Expression vectors for MAVS, C508 mutant ofMAVS, 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 SeVor mock infectedfor another 24 h beforeharvestingfor Luc

assay.

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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 thefailure to induce IFNs in HCV replicon cells, prevention of MAVScleavage should restore IFN induction in these cells. To test thispossibility, we introduced the C508R mutant of MAVS as well asthe WT MAVS into the HCV replicon cells to examine theinduction of IFN-. As reported in ref. 3, SeV induced IFN- in theHuh7 cells but failed to do so in the replicon cell line (Fig. 4 B andC). Even overexpression of RIG-I(N) failed to induce IFN- in the

replicon cells (Fig. 4C), presumably because the endogenousMAVS was cleaved into an inactive soluble fragment (Fig. 4 A, lane4). Overexpression of WT MAVS was able to induce partial IFNresponse in the replicon cells, and this response was furtherenhanced by SeV.This resultmay be dueto the incomplete cleavageof the overexpressed MAVS by the NS34A protease in thereplicon cells. Most significantly, expression of the protease-resistant mutant of MAVS (C508R) restored IFN- induction inthe replicon cells to the same level observed in Huh7 cells (Fig. 4B and C). These results demonstrate that the evasion of innateimmune response in the HCV replicon cells is due to the cleavageof MAVS by NS34A.

Discussion

We have previously shown that MAVS is an essential antiviral

signaling protein and proposed that MAVS may be a prime targetof viruses that have succeeded in evading the host immune system(10). In this work, we show that MAVS is indeed the proteolytictarget of HCV NS34A protease. Specifically, we found that HCVcleaves MAVS at Cys-508, dislocating MAVS from the mitochon-dria, thus preventing the induction of IFN-. NS34A binds to andcolocalizes with MAVS at the mitochondrial membrane, and it cancleave MAVS at Cys-508 directly in vitro. We also provide directevidence that the endogenous MAVS protein is cleaved in a HCVreplicon cell line that failed to elicit IFN response. Importantly, apoint mutation of MAVS at Cys-508 that prevents its cleavagerestores IFN induction in the HCV replicon cell line. These resultsprovide compelling evidence that MAVS is the prime target of theHCV protease. Our present work not only provides the directbiochemical evidence that MAVS is the proteolytic target of

NS34A (Fig. 3) but also shows that the cleavage of endogenousMAVS at Cys-508 in the HCV replicon cell line is solely responsiblefor the suppression of IFN induction in these cells (Fig. 4).Furthermore, our finding that NS34A inactivates MAVS bycleaving and dislodging it from the mitochondria underscores the

importance of mitochondrial localization of MAVS in antiviralimmunity.

Infectious diseases are a manifestation of constant battles be-tween the host and pathogenic microbes. This hostpathogenantagonism is now vividly demonstrated from the study of theinteraction between MAVS and HCV. MAVS can orchestratestrong immune defense against HCV; however, HCV counterat-tacks by using the NS34A protease to cleave MAVS, thuscripplingthe immune response. Because a point mutation at Cys-508 of

MAVS completely prevents its cleavage by NS34A and preservesits antiviral activity, it would be interesting to determine whetherthere are sequence variations of MAVS in human population thatconfer differential sensitivity to NS34A and, hence, differentialimmunity to HCV. However, the prevalence of persistent HCVinfection in humans suggests that HCV might have won the battlebetween thevirus andthe host. In thenext battle of our wits versustheir genes, it may be possible to exploit the knowledge ofMAVSHCV interaction to fight back against HCV. For example,the cleavage of MAVS from the mitochondrial membrane may bea diagnostic marker for an established HCV infection. Further-more, blocking the cleavage of MAVS by NS34A may be effectivein the prevention and treatment of HCV.

HCV may not be the only pathogen that targets MAVS to evadethe host immune system. Although the mitochondrial localizationof MAVS may allow the host immune system to detect many virusesthat replicate in intracellular membrane locations proximal to themitochondria, 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 butupstream of TBK1IKK (28), raising the possibility that MAVSmay be a target of a HAV protein. Future studies should uncovermore examples of hostpathogen interaction that revolve aroundthe battle for MAVS to gain control of the host immune system.

Note. While this manuscript was in preparation, Meylan et al. (12) alsoreported the identification of MAVSCARDIF as the target of NS34A.

We thank Dr. Michael Gale for providing the HCV replicon cell lineK2040 (19). This work was supported by National Institutes of HealthG rant R01 -AI6 09 19 and American Can cer S ociety G rantRSG0219501TBE. Confocal microscopy was supported by funding froma National Institutes of Health shared instrumentation grant 1-S10-RR19406. Z.J.C. is a Burroughs Wellcome Fund Investigator in Patho-genesis of Infectious Diseases.

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schlager, 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.

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REVIEW

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

npg

Antiviral innate immunity pathways

Rashu B Seth1, Lijun Sun1, Zhijian J Chen1

1Howard Hughes Medical Institute, Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas,

TX 75390-9148, USA

Correspondence: Zhijian J Chen

Tel: 214-648-1145, Fax: 214-648-1675;

E-mail: [email protected]

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-B, 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-B and IRFs through the

recently identied 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-B, 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 Rhino-

viruses, are efciently resolved by the host innate and

adaptive immune systems. The innate immune response

is the rst 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- and IFN-, 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 invad-

ing 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]. Toll-

like receptors (TLRs) 3, 7, 8 and 9 are the major PRRs that

recognize distinct types of virally-derived nucleic acids and

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 identied 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 under-

standing 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- subtypes and a single

IFN- subtype[1]. The induction of type I IFN genes is

regulated at the step of transcription and is best understood

for the IFN- promoter. A multi-protein complex called an

enhanceosome is assembled at the IFN- promoter in re-

sponse to a viral challenge[5]. The enhanceosome consists

of at least three classes of transcription factors ATF-2/c-

Jun, nuclear factor (NF)-B and interferon regulatory factor

3 (IRF3). Of these, the activities of NF-B and IRF3 are

regulated by their subcellular localization. In the inactive

state, NF-B is held in the cytosol by inhibitory B (IB)


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family members [6]. In the presence of diverse stimuli,

such as IL-1, TNF-, and viruses, the IB kinase (IKK)

is activated and it then phosphorylates IB. Once phos-

phorylated, IB is ubiquitinated and subsequently degraded

by the proteasome. Free NF-B then translocates into the

nucleus and turns on its target genes. Similar to NF-B,

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 IRF3leads 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-B, IRF3 and ATF2/c-Jun assemble into

a stereospecic enhanceosome complex that remodels the

chromatin in the promoter of IFN-, resulting in its tran-

scriptional initiation.

IFN- binds to the IFN/ 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 activa-

tor of transcription 1 (STAT1) and STAT2 proteins. These

transcription factors associate with IRF9 to form a hetero-

trimeric complex, IFN-stimulated gene factor 3 (ISGF3).

ISGF3 initiates the transcription of several interferonstimulated 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- gene. An initial model suggested

that IRF-7 is not involved in the initial phase of IFN-

induction as it is expressed at low levels in most cells in

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-B 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 ofeither pathway leads to the induction of IFN-.

dsRNA

Endosome

ssRNA

TLR3TLR7/TLR8

TLR9

CpG

L

R

R

L

R

R

L

R

R

TI

R

TI

R

TI

R

TRIFMyD88

MyD88IRF3/7

NF-B

ATF-2/c-Jun

Cytoplasm

Nucleus

IFN-

Mitochondria

MAVS

dsRNA

RIG-I

CARDCARD

Helicase

CARDPRO

Virus


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the absence of virus. IFN- 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- gene.

A recent study using IRF7 knockout mice has demonstrated

that transcription of both IFN- and IFN- is dependent

on IRF7 [9], indicating that IRF7 is a master regulator oftype I IFNs.

IRF5 is another member of the IRF family that has been

suggested to regulate type I IFN expression. However, re-

cent genetic experiments using IRF5-decient mice showed

that IRF5 is not required for type I IFN induction, but is

required for the induction of proinammatory 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 rst found to instruct dorsal-ventral patterning in early embryos, and later found to

also regulate anti-fungal innate immunity in adult ies.

Sequence homology search in mammalian genomes has

subsequently identied 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 differ-

ent microbial signatures. For example, TLR3 recognizes

viral double-stranded RNA, TLR4 recognizes bacterial

lipopolysaccharides (LPS), whereas TLR5 is a receptor

for bacterial agellin. 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 sig-

naling pathways culminating in the induction of cytokines

and IFNs through NF-B and IRFs. Except for TLR3,

all TLRs utilize MyD88 as an adaptor protein to recruit

downstream signaling molecules including the protein ki-

nases 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 con-

sisting of TAK1, TAB1 and TAB2 [11, 12]. The activated

TAK1 kinase phosphorylates IKK in the activation loop,

resulting in the activation of IKK and subsequent nuclear

translocation of NF-B. The TIR domains of TLR3 and

TLR4 bind to another adaptor protein TRIF, which binds

directly to TRAF6 and RIP1 to activate NF-B. TRIF can

also bind to TBK1, which phosphorylates and activates

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-B to suppress the

induction of proinammatory cytokines [13, 14].

Among TLRs, TLR3, 7, 8 and 9 are involved in antivi-

ral innate immune responses [3]. TLR3 recognize dsRNA

viruses and may also be involved in sensing dsRNA re-

leased from dying cells. TLR7 and TLR8 are receptors forG/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 endo-

some to lysosome.

The MyD88-IRAK-TRAF6 signaling module is es-

sential for the induction of IFNs by TLR7, 8 and 9. The

same signaling module is also required for the activation

of NF-B by IL-1 and other TLRs such as TLR2; how-

ever, IFNs are not induced by IL-1 or TLR2. Thus, it is

likely that TLR7, 8 and 9 recruit additional components in

pDCs to activate IRF7, the master regulator of IFN-. One

of such components may be TRAF3, which has recently

been shown to be essential for IFN- 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-decient mice are defective

in IFN- 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 ubiqui-

tin-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 broblasts, deletion of

both MyD88 and TRIF, which abolishes all TLR signaling,

has no effect on viral induction of IFNs [20]. Furthermore,

although human patients decient 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 beenidentied as an intracellular receptor for viral dsRNA [4].

RIG-I is a member of the DExD/H box-containing RNA he-

licase 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. Be-

sides 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 sufcient to

activate NF-B and IRF3 in the absence of a viral chal-

lenge, whereas the full-length RIG-I is activated only in

the presence of dsRNA. Thus, the binding to dsRNA to theRNA helicase domain of RIG-I likely induces a conforma-

tional 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 signicance of RIG-I in anti-viral im-

munity was shown rst by RNAi studies and conrmed by

mouse knockout studies [4, 20]. RNAi of RIG-I in L929

cells, a mouse broblast 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 broblasts (MEFs) and lung bro-

blasts, it was shown that the induction of IFN- and ISGs

by several RNA viruses was abolished. Pre-treatment of

the broblasts with IFN- increased the resistance of the

RIG-I decient broblasts to VSV, indicating that RIG-I

is required for the induction of IFN- and does not affect

the downstream IFN- 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 inter-

feron induction in broblasts and bone marrow derived

dendritic cells from RIG-I-/- vs MyD88-/- TRIF-/- mice,

which lack all TLR signaling [20]. The ability to induce

IFN- when challenged by NDV was severely compro-

mised in the RIG-I-/-, but not MyD88-/- TRIF-/-, cDCs and

broblasts. Opposite results were observed for the pDCs,

which induce IFN- 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 iden-

tied 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- promoter. The importance of MDA-5

as an anti-viral protein had been suggested based on thending 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-

promoter by Sendai virus, but it does not interfere with the

TLR3 signaling pathway.

MAVS signaling in the RIG-I pathway

Recent studies have identied a CARD domain contain-

ing protein that acts downstream of RIG-I. This protein,

independently identied by four different groups, has beencalled mitochondrial anti-viral signaling protein (MAVS)

[25], IFN- promoter stimulator 1 (IPS-1) [26], virus-in-

duced signaling adaptor (VISA) [27] and CARD adaptor

inducing IFN- (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, over-

expression of MAVS leads to the activation of NF-B

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-ex-

pression. Third, the activation of kinases responsible for

NF-B 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 kill-

ing 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 sufcient to induce IFN-. 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 sufcient


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to activate the downstream pathway. The TM sequence ofMAVS resembles the mitochondrial targeting sequences of

several C-tail anchored mitochondrial membrane proteins,

including the cell survival proteins Bcl-2 and Bcl-xL. In-

deed, 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 signal-

ing function of MAVS. When the TM domain of MAVSwas 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.

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-B and IRF3 signaling

pathways by activating the IKK and TBK-1/IKKe kinase complexes. Once activated, NF-B and IRF3 translocate into the nucleus

and turn on the IFN- 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.

Virus

RIG-IHelicase

CARDCARD

Intracellualar

dsRNA

MAVS CARDPRO

HCV

NS3/4A

Mitochondria

TBK-1/IKKe

IRF3P

IKK

IKK IKKUb

UbUb

Ub PIB

NF-B

Proteasome

Cytoplasm

NF-B IRF3P

IFN-

Nucleus


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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-de-

pendent manner. The signaling mechanism of MAVS is a

subject of debate at present. Two groups show that MAVScan 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-. Furthermore, TRAF6-decient cells have normal

induction of IFN- following viral infection [25, 29]. Kawai

et alshowed that MAVS interacted with RIP-1 and FADD,

and proposed that these molecules linked MAVS to IKK

activation [26]. However, RIP1-decient MEF cells are also

fully capable of inducing IFN- following viral challenges

[25, 30]. Meylan et alreported that MAVS bound to IKK

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 break-

through in the eld 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- 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 alalso showed that NS3/4A binds to and

co-localizes with MAVS in the mitochondrial membrane,

and it can cleave MAVS directlyin vitro. Using a replicon

cell culture system in which the RNA genome of HCV

replicates autonomously, Li et al demonstrated that en-

dogenous 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 an-

tiviral innate immunity pathways leading to the induction

of interferons. The TLR pathway operates mainly in pDCs

to detect viral RNA and DNA associated with endocy-

tosed 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 identied, 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- 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 un-

known. 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 thehost 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 Foun-

dation (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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