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Ubiquitination and DegradationVirus-Induced Signaling Adaptor for The E3 Ubiquitin Ligase RNF5 Targets

and Hong-Bing ShuBo Zhong, Yu Zhang, Bo Tan, Tian-Tian Liu, Yan-Yi Wang

http://www.jimmunol.org/content/184/11/6249doi: 10.4049/jimmunol.09037482010;

2010; 184:6249-6255; Prepublished online 5 MayJ Immunol 

Referenceshttp://www.jimmunol.org/content/184/11/6249.full#ref-list-1

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The Journal of Immunology

The E3 Ubiquitin Ligase RNF5 Targets Virus-InducedSignaling Adaptor for Ubiquitination and Degradation

Bo Zhong, Yu Zhang, Bo Tan, Tian-Tian Liu, Yan-Yi Wang, and Hong-Bing Shu

Viral infection activates transcription factors, such as NF-kB and IFN regulatory factor 3, which collaborate to induce type I IFNs

and elicit innate antiviral response. Virus-induced signaling adaptor (VISA) has been identified as a critical adaptor required for

virus-triggered induction of type I IFNs. In this study, we showed that the E3 ubiquitin ligase RING-finger protein 5 (RNF5)

interacted with VISA at mitochondria in a viral infection-dependent manner. Domain mapping experiments indicated that the C-

terminal transmembrane domain of VISAwas required for its interaction with RNF5. RNF5 targeted VISA at K362 and K461 for

K48-linked ubiquitination and degradation after viral infection, whereas knockdown of RNF5 reversed virus-induced down-

regulation of VISA at the early phase. These findings suggest that RNF5-mediated ubiquitination and degradation of VISA is

one of the mechanisms of the regulation of virus-triggered induction of type I IFNs and cellular antiviral response. The Journal

of Immunology, 2010, 184: 6249–6255.

Viral infection induces host cells to express type I IFNs,including IFN-b and IFN-a family cytokines (1–4). Thesecreted IFNs bind to IFNR in both autocrine and para-

crine manners to initiate a series of signaling events leading toexpression of hundreds of downstream genes, collectively calledIFN-stimulated genes (ISGs). Proteins encoded by the ISGs col-laborate to inhibit viral replication or cause apoptosis of infectedcells, resulting in cellular antiviral response (5, 6).The mechanisms of virus-triggered induction of type I IFNs have

been extensively investigated during the past decade. Viral infectionand replication generate pathogen associated molecular patterns,such as viral RNA and DNA, which are recognized by the pattern-recognition receptors (7, 8). Among the pattern-recognition re-ceptors, the cytoplasmic RIG-I–like receptors, RIG-I and MDA5,have been demonstrated to bind to viral RNAs (1, 9, 10). Recently,it has also been shown that viral DNA is transcribed into RNA bypolymerase III and the transcribed RNA is then recognized byRIG-I (11, 12). Upon detection of viral RNA, RIG-I is associatedwith the adaptor protein virus-induced signaling adaptor (VISA)[also known as mitochondrial antiviral signaling, IFN-b promoterstimulator 1, and Cardif], which is located at the outer membrane ofthe mitochondria and acts as a central scaffold for assembling acomplex essential for virus-triggered induction of type I IFNs (13–16). VISA is associated with several downstream proteins consti-

tutively or in a viral infection-dependent manner, including TNFR-associated factor (TRAF) 3, TRAF6, and mediator of IRF3 activa-tion (MITA) (13, 17, 18). Upon viral infection, MITA recruitsTANK-binding kinase 1 to the VISA-associated complex, in whichTANK-binding kinase 1 phosphorylates IFN regulatory factor 3(18). TRAF6 in the VISA-associated complex is critical in acti-vating the inhibitor of kB kinase complex and subsequent NF-kB(13). The activated IFN regulatory factor 3 andNF-kB enter into thenucleus and collaboratively activate transcription of type I IFNgenes. It has been suggested that various proteins involved in theTNFR signaling pathway, such as TNFR-associated death domain,Fas-associated death domain, and receptor interacting protein, alsoplay roles in virus-triggered induction of type I IFNs (19, 20).Because overproduced type I IFNs cause excessive and harmful

immune responses, the production of these cytokines is tightlyregulated. For example, NLRX1 is physiologically associated withVISA to prevent its recruitment to RIG-I–like receptors, therebyblocking the production of type I IFNs in noninfected cells (21). Theubiquitin ligase RNF125 catalyzes ubiquitination and degradationof RIG-I, resulting in suppression of RIG-I–mediated signaling (22).RING-finger protein 5 (RNF5, also known as RMA1) has been

shown to be an endoplasmic reticulum (ER)-located E3 ubiquitinligase implicated in cell motility, protein quality control in the ER,cancer and degenerative myopathy (23, 24). Recently, we dem-onstrated that RNF5 is also located at the mitochondria and in-teracts with MITA in a viral infection-dependent manner. Variousexperiments suggest that RNF5 mediates virus-triggered ubiquiti-nation and degradation of MITA and acts as a negative regulator ofvirus-triggered signaling and cellular antiviral response (25).In this study, we found that RNF5 was associated with VISA

after viral infection. RNF5 mediated K48-linked ubiquitination anddegradation of VISA. Our data collectively suggest that RNF5negatively regulates virus-triggered induction of type I IFNs andcellular antiviral response by targeting both VISA and MITA.

Materials and MethodsReagents and Abs

Mouse mAbs against Flag, hemagglutinin (HA), and b-actin (Sigma-Aldrich, St. Louis, MO) and apoptosis inducing factor and KDEL (SantaCruz Biotechnology, Santa Cruz, CA) were purchased from the indicatedmanufacturers. Sendai virus (SeV), vesicular stomatitis virus (VSV), and

College of Life Sciences, Wuhan University, Wuhan, China

Received for publication November 20, 2009. Accepted for publication March 30,2010.

This work was supported by grants from the Chinese 973 Program (2006CB504301and 2010CB911802), the National Natural Science Foundation of China (30630019and 30921001), and the Chinese Science and Technology Key Project(2008ZX10002-014).

Address correspondence and reprint requests to Prof. Hong-Bing Shu, College ofLife Sciences, Wuhan University, Wuhan 430072, China. E-mail address: shuh@whu.edu.cn

Abbreviations used in this paper: AIP4, atrophin-1 interacting protein 4; aM, anti-MITA; CARD, caspase recruitment domain; ER, endoplasmic reticulum; HA, hem-agglutinin; ISG, IFN-stimulated gene; MITA, mediator of IRF3 activation; PCBP2,poly(rC) binding protein 2; PSMA7, proteasome subunit a type-7; RING, ring-finger;RNAi, RNA interference; RNF5, RING-finger protein 5; SeV, Sendai virus; TM,transmembrane; TRAF, TNFR-associated factor; VISA, virus-induced signalingadaptor; VSV, vesicular stomatitis virus.

Copyright� 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00

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rabbit anti-VISA, mouse anti-RNF5, and mouse anti-MITA Abs werepreviously described (13, 18, 25). Goat anti-Itch was generously providedby Dr. Zhengfan Jiang (Peking University, Beijing, China).

Constructs

Mammalian expression plasmids for HA- or Flag-tagged VISA and itsmutants, MITA, and RNF5 and its mutants were previously described (13,18, 25). Mammalian expression plasmid for ankyrin repeat and deathdomain containing 1A was made in a CMV promoter-based expressionplasmid by standard molecular biology methods. Site-directed mutation ofmammalian expression plasmids for human HA- or Flag-tagged VISAwere constructed by standard molecular biology techniques. The RNF5(C42S) was a generous gift from Dr. Douglas Cyr (University of NorthCarolina, Chapel Hill, NC). HA- or Flag-tagged poly(rC) binding protein 2(PCBP2) and Itch and Itch-RNA interference (RNAi) were provided byDr. Zhengfan Jiang (Peking University) and previously described (26).

Cell fractionation assays

The cell fractionation assay was performed as previously described with fewchanges (25). The 293 cells (5 3 107) infected with SeVor left uninfectedwere washed with PBS followed by dousing 40 times in 2 ml homogeni-zation buffer (ApplyGen, Beijing, China). The homogenate was centrifugedat 5003 g for 10 min. The supernatant (S5) was centrifuged at 50003 g for10 min to precipitate mitochondria (P5K). The supernatant from this step(S5K) was further centrifuged at 50,000 3 g for 30 min to yield P50K,which contains the membrane fraction, and S50K, which mainly consistsof cytosol.

Coimmunoprecipitation and immunoblot analysis

For transient transfection and coimmunoprecipitation experiments, 293cells (2 3 106) were transfected for 20 h. The transfected cells were lysedin 1 ml lysis buffer [20 mM Tris (pH 7.5), 150 mM NaCl, 1% Triton, 1 mMEDTA, 10 mg/ml aprotinin, 10 mg/ml leupeptin, and 1 mM PMSF]. Foreach immunoprecipitation, 0.4 ml lysate was incubated with 0.5 mg in-

dicated Ab or control IgG and 30 ml protein G-Sepharose in 20% ethanol(GE Healthcare, Piscataway, NJ) for 2 h. The Sepharose beads werewashed three times with 1 ml lysis buffer containing 500 mM NaCl. Theprecipitates were analyzed by standard immunoblot procedures.

For endogenous coimmunoprecipitation experiments, 293 cells (53 107)were infected with SeV for the indicated times or left uninfected. Thecoimmunoprecipitation and immunoblot experiments were performed asdescribed above.

Ubiquitination assays

For in vivo ubiquitination experiments, we adopted a two-step immuno-precipitation strategy (25). First-round immunoprecipitation was per-formed as described above. The immunoprecipitates were re-extracted inlysis buffer containing 1% SDS and denatured by heating for 5 min.The supernatants were diluted with regular lysis buffer until the concen-tration of SDS was decreased to 0.1%. The diluted supernatants were re-immunoprecipitated with the indicated Abs, and the immunoprecipitateswere analyzed by immunoblots with the anti-ubiquitin Ab.

For in vitro ubiquitination experiments, proteins were expressed witha TNT Quick Coupled Transcription/Translation Systems kit (Promega,Madison, WI) as the manufacturer’s instructions described. Ubiquitinationwas analyzed with an ubiquitination kit (Enzo Life Sciences, Farmingdale,NY) following the protocols recommended by the manufacturer.

ResultsDomain mapping of the association of RNF5 and VISA

Previously, we found that RNF5 interacts with VISA in a mam-malian overexpression system (25). However, the significance ofthe association between RNF5 and VISA has not been determined.We first mapped the domains of VISA that are required for its in-teraction with RNF5. As shown in Fig. 1A and 1B, full-length VISAand its deletion mutants containing the C-terminal transmembranedomain could interact with RNF5, whereas the mutants lacking the

FIGURE 1. RNF5 interacts with VISA. A, A schematic presentation of full-length VISA, its mutants, and their abilities to interact with RNF5. B, The TM

domain of VISA is required for its interaction with RNF5. The 293 cells (2 3 106) were transfected with the indicated plasmids (5 mg each). Co-

immunoprecipitations were performed with anti-Flag or control IgG. Immunoblot analysis was performed with anti-HA (upper panels). Expression levels

of the proteins were analyzed by immunoblot analysis of the lysates with anti-HA and anti-Flag (lower panels). C, A schematic presentation of full-length

RNF5, its mutants, and their abilities to interact with VISA. D, Interactions between RNF5 mutants and VISA. Experiments were performed similarly as in

B. CARD, caspase recruitment domain; TM, transmembrane.

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transmembrane domain lost their ability to associate with RNF5.These data suggest that the C-terminal transmembrane domain ofVISA is required for its interaction with RNF5.In our previous study, we also found that the transmembrane

domain of RNF5 is required for RNF5–MITA association (25). Wenext examined which domains of RNF5 are required for its in-teraction with VISA. Interestingly, deletion of either the N-terminalring-finger (RING) domain (aa 1–50) or the C-terminal trans-membrane domain (aa 169–180) of RNF5 had no marked effects onits interaction with VISA. In contrast, the truncation mutant RNF5(51–168), which lacks both the RING and the transmembranedomains, failed to interact with VISA (Fig. 1D). These resultssuggest that the intermediate domain plus either the RING or thetransmembrane domain of RNF5 are sufficient for its interactionwith VISA.We next determined whether VISA interacts with RNF5 in

untransfectedcellsandtheeffectsofviral infectionontheinteraction.Endogenous coimmunoprecipitation experiments indicated thatVISAdidnot interactwithRNF5without stimulationbutwasweaklyassociated with RNF5 at 4 h after viral infection and was stronglyassociated with RNF5 at 8 h after viral infection (Fig. 2A). In-terestingly, their association was diminished at 12 h after viral in-fection (Fig. 2A). Because VISA has been demonstrated to beexclusively localized to the outer membrane of mitochondria (14),we further determined whether their association occurs at the mi-tochondria. We isolated the mitochondria and ER fractions andperformed coimmunoprecipitation assays. As expected, althoughRNF5 distributed to both the mitochondria and the ER fractions,RNF5 interacted with VISA at the mitochondria but not the ERfraction (Fig. 2B). These results suggest thatVISA is associatedwithRNF5 at the mitochondria in a viral infection-dependent manner.

RNF5 targets VISA for ubiquitination and degradation

Because RNF5 is an E3 ubiquitin ligase and associates with VISAat the mitochondria in a viral infection-dependent manner, wedetermined whether RNF5 could catalyze ubiquitination of VISA.As shown in Fig. 3A, wild-type RNF5 but not the inactive mutant(C42S) mediated ubiquitination of VISA. To exclude the possi-bility that the polyubiquitin-associated VISA is due to the asso-ciation of VISA with MITA or RNF5, we performed two lines ofexperiments. First, we examined RNF5-mediated ubiquitination ofVISA with or without RNAi-mediated knockdown of MITA andfound that knockdown of MITA had no marked effect on RNF5-mediated ubiquitination of VISA (Fig. 3B). Second, we examinedthe reimmunoprecipitation system used in our study. The resultsindicated that the denaturation and reimmunoprecipitation assaycompletely disrupted the interaction between VISA and RNF5, butthe polyubiquitin modification of VISA remained intact (Fig. 3C).These data suggest that RNF5 mediates ubiquitination of VISA.Consistent with these observations, RNF5 but not its mutant

RNF5(C42S) or MITA caused downregulation of VISA in a dose-dependent manner (Fig. 3D). Furthermore, SeVand VSV infectionresulted in ubiquitination and downregulation of endogenousVISA, and these were reversed by RNAi-mediated knockdown ofRNF5 (Fig. 3E), suggesting viral infection-caused downregulationof VISA was not viral type-specific. Kinetic experiments of VISAexpression indicated that the expression of VISA was graduallydownregulated from 6–24 h after viral infection, and knockdownof RNF5 slowed the virus-triggered downregulation of VISA (Fig.3F). In contrast, MITAwas degraded at 6–9 h after SeV infection,and knockdown of RNF5 abolished the effect. Consistently,knockdown of RNF5 maintained the VISA–MITA interaction at8 h after SeV infection (Fig. 3G). These data suggest that

RNF5 targets VISA for ubiquitination and degradation after viralinfection.

RNF5-mediated downregulation of VISA depends on theproteasome

Because proteins with the K48-linked or the K63-linked polyubi-quitin chains could be targeted for degradation in proteasome-and lysosome-dependent pathways, respectively, we next deter-mined whether RNF5 catalyzed K48- or K63-linked ubiquitinationof VISA. We previously made the ubiquitin mutant constructs inwhich all of the lysine residues except Lys48 (ubiquitin-K48) or Lys63

(ubiquitin-K63) were mutated into arginines (25). We transfectedthese ubiquitin mutants into 293 cells and examined whether theycouldmodifyVISA.RNF5catalyzedVISAubiquitinationwithwild-type ubiquitin and ubiquitin-K48 but not with ubiquitin-K63 (Fig.4A). Consistently, the proteasome inhibitor MG132 could reverseRNF5-mediated downregulation of overexpressed VISA or SeV-in-duced downregulation of endogenous VISA (Fig. 4B, 4C). Theseresults suggest that RNF5 targets VISA for K48-linked ubiquitina-tion and proteasome-dependent degradation after viral infection.

FIGURE 2. Effect of viral infection on endogenous VISA–RNF5 in-

teraction. A, Endogenous interaction between VISA and RNF5. The 293

cells (5 3 107) were left uninfected or infected with SeV for the indicated

time points. The cells were lysed, and the lysates were immunoprecipitated

with anti-RNF5 or control serum. The immunoprecipitates were analyzed

by immunoblot with anti-VISA (upper panel). The expression levels of the

endogenous proteins were detected by immunoblot analysis with anti-

MITA and anti-RNF5 (lower panels). B, RNF5 interacts with VISA at

mitochondria after viral infection. The 293 cells (1 3 108) were left un-

infected or infected with SeV for the indicated time points and fractionated

as shown in the diagram (upper schematic presentation). The fractions

were lysed, the lysates were immunoprecipitated with anti-RNF5, and the

immunoprecipitates were analyzed by immunoblot with anti-VISA (top

panel). A fraction of lysate was taken for immunoblot analysis to detect the

expression levels of the indicated proteins (lower panels).

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RNF5 targets K362 and K461 of VISA for ubiquitination

During our coimmunoprecipitation experiments, we routinelyobserved that RNF5 but not the C42S inactive mutant caused shiftsof VISA(361–540) to higher molecular bands (Figs. 1B, 5A). In

addition, RNF5 caused ubiquitination of wild-type VISA but not

the mutant VISA(D361–480), suggesting the possibility that the

ubiquitination sites of VISA are located in the region of aa 361–

480 of VISA (Fig. 5B). Sequence analysis identified four lysine

FIGURE 3. RNF5 targets VISA for ubiquitination and degradation. A, Overexpression of RNF5 but not the C42S inactive mutant induces ubiquitination

of VISA. The 293 cells (2 3 106) were transfected with the indicated plasmids. Twenty hours after transfection, cell lysates were immunoprecipitated with

anti-Flag or control IgG. The immunoprecipitates were denatured and reimmunoprecipitated with anti-Flag and then analyzed by immunoblot with anti-

ubiquitin (upper panel). The expression levels of the proteins were examined by immunoblots with the indicated Abs (lower panels). B, Effects of

knockdown of MITA on RNF5-mediated ubiquitination of VISA. The experiments were performed as in A. C, Denaturation and reimmunoprecipitation

treatment abolished VISA interaction with RNF5. The 293 cells (4 3 106) were transfected with the indicated plasmids (5 mg each). Twenty hours after

transfection, cell lysates were immunoprecipitated with control IgG or anti-Flag. The anti-Flag precipitants were denatured and reimmunoprecipitated with

anti-Flag or left untreated. The precipitants were analyzed by immunoblot with anti-ubiquitin, anti-HA, and anti-Flag, respectively. The expression levels of

the proteins were examined by immunoblots with the indicated Abs (lower panels). D, Overexpression of RNF5 but not RNF5(C42S) or MITA causes

downregulation of VISA but not an unrelated protein ankyrin repeat and death domain containing 1A. The 293 cells (2 3 105) were transfected with the

indicated plasmids. Twenty-four hours after transfection, cells were lysed for immunoblot analysis with anti-Flag (upper panels) or anti-HA (lower panels).

E, Effects of RNF5 knockdown on SeV- or VSV-induced ubiquitination of VISA. The 293 cells (5 3 107) were transfected with a control or the RNF5-

RNAi plasmid. Twenty-four hours after transfection, cells were infected with SeV for 8 h (left panels) or with VSV for 12 h (right panels) or left untreated.

The cell lysates were immunoprecipitated with anti-VISA. The immunoprecipitates were denatured and reimmunoprecipitated with anti-VISA and then

analyzed by immunoblot with anti-ubiquitin (upper panels). The expression of the related proteins was examined by immunoblots with the indicated Abs

(lower panels). F, Effects of RNF5 knockdown on the expression of VISA after viral infection. The 293 cells (2 3 106) were transfected with RNF5-RNAi

plasmid (8 mg). Twenty-four hours after transfection, cells were untreated or infected with SeV for the indicated time points before immunoblot analysis

was performed. G, Effects of RNF5 knockdown on VISA–MITA association. The 293 cells (2 3 107) were transfected with control or RNF5-RNAi

plasmid. Twenty-four hours after transfection, cells were untreated or infected with SeV for the indicated time points before immunoprecipitation and

immunoblot analysis was performed. aM, anti-MITA.

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residues in this region, which are K362, K371, K420, and K462.We made VISA mutants in which the lysine residues were mutatedto arginines individually or simultaneously and examined theirubiquitination by RNF5. As shown in Fig. 5C, mutation of K362or K461 to arginine reduced RNF5-mediated ubiquitination ofVISA, whereas simultaneous mutation of both K362 and K461 toarginines (K362/461R) abolished RNF5-catalyzed ubiquitination.Consistently, RNF5 caused degradation of wild-type VISA but not

VISA(K361/461R) (Fig. 5C). These data suggest that RNF5 tar-gets VISA at K362 and K461 for ubiquitination and degradation.

RNF5 and Itch independently target VISA for ubiquitination

Recently, it has been reported that the homologous to E6-AP car-boxyl terminus domain containing E3 ubiquitin ligase Itch (alsocalled atrophin-1 interacting protein 4 [AIP4]) catalyzes ubiquiti-nation of VISA and this process is mediated by PCBP2. To

FIGURE 4. RNF5-mediated downregulation of VISA depends on the proteasome. A, RNF5 catalyzes K48-linked polyubiquitination of VISA. The 293

cells (2 3 106) were transfected with the indicated plasmids. Twenty hours after transfection, immunoprecipitation, reimmunoprecipitation, and immu-

noblot analysis were performed with the indicated Abs (upper panel). The expression of the proteins was examined by immunoblots with the indicated Abs

(lower panels). B, MG132 blocks RNF5-mediated downregulation of endogenous VISA. The 293 cells (1 3 106) were transfected with the indicated

plasmids. Twenty hours after transfection, cells were treated with DMSO or MG132 (20 mM) for 6 h before immunoblot analysis was performed. C,

MG132 reverses SeV-induced downregulation of VISA. The 293 cells (13 106) were left untreated or infected with SeV. Two hours later, cells were treated

with DMSO or MG132 (20 mM) for 6 h before immunoblot analysis was performed.

FIGURE 5. Mapping of RNF5-targeted ubiquitination sites of VISA. A, Wild-type RNF5 but not its enzyme inactive mutant RNF5(C42S) causes shift of

VISA(361–540) to higher molecular bands. The 293 cells (2 3 106) were transfected with the indicated plasmids. Twenty hours later, cells were lysed, and

immunoprecipitation and immunoblot analysis were performed with the indicated Abs. B, RNF5 catalyzes ubiquitination of wild-type VISA but not VISA

(D361–480). The 293 cells (2 3 106) were transfected with the indicated plasmids. Twenty hours after transfection, immunoprecipitation, re-

immunoprecipitation, and immunoblot analysis were performed with the indicated Abs (upper panel). The expression of the proteins was examined by

immunoblots with the indicated Abs (lower panels). C, Mapping of RNF5-targeted ubiquitination sites of VISA. Experiments were performed as in A.

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determine whether RNF5 and Itch function independently or to-gether for ubiquitination of VISA, we performed two lines of ex-periments. First, we performed in vitro ubiquitination assays withrecombinant proteins. The results indicated that RNF5 directlycatalyzed ubiquitination of VISA and the E2 Ubc5 (UbcH5a,UbcH5b, or UbcH5c) was required for this process (Fig. 6A).Second, RNF5 could ubiquitinate VISA in AIP4 knockdown cells,whereas AIP4 and PCBP2 could also catalyze ubiquitination ofVISA in RNF5 knockdown cells (Fig. 6B). These data suggest thatRNF5 and Itch independently catalyze ubiquitination of VISA. Toexamine whether RNF5 and Itch function redundantly for VISAdegradation, we examined the effects of knockdown of RNF5 versusItch on the stability of VISA. As shown in Fig. 6C, SeV-induceddownregulation of VISAwas inhibited by knockdown of RNF5 at 8 hbut less at 16 h after SeV infection, whereas knockdown of Itch in-hibited degradation of VISA from 8–16 h after SeV infection. Thesedata suggest that RNF5 and Itch function in a temporal manner totarget VISA for ubiquitination after viral infection.

DiscussionOur previous studies have demonstrated that the ubiquitin ligaseRNF5 regulates antiviral response by mediating degradation of theadaptor protein MITA (25). In this report, we found that RNF5 wasassociated with VISA in a viral infection-dependant manner inuntransfected cells. Domain mapping experiments suggested thatthe transmembrane domain of VISA was required for its in-teraction with RNF5. In contrast to the observation that the trans-membrane domain of RNF5 is required for its association withMITA, we found that the intermediate domain plus either theRING or the transmembrane domain of RNF5 was sufficient forinteraction with VISA.RNF5 is an E3 ubiquitin ligase, and Cys42 of RNF5 is critical for

its ligase activity (27). Consistently, wild-type RNF5 but not theRNF5(C42S) mutant catalyzed ubiquitination and downregulationof VISA. Moreover, RNF5 also mediated virus-triggered ubiquiti-nation and degradation of VISA, as demonstrated by the observa-

tion that RNAi-mediated knockdown of RNF5 diminished theseeffects. Further studies indicated that RNF5 catalyzed K48-linkedubiquitination of VISA, and SeV infection-induced and RNF5-mediated downregulation of VISAwas reversed by treatment withthe proteasome inhibitor MG132. Site-directed mutagenesis in-dicated that RNF5 ubiquitinated VISA at K362 and K461. Con-sistently, VISA(K362/461R) was resistant to RNF5-mediateddegradation. These results suggest that RNF5 targets VISA forK48-linked ubiquitination and proteasome-dependent degradationafter viral infection.Our experiments indicated that VISA was degraded gradually

from 6–24 h after viral infection. However, knockdown of RNF5reversed the degradation of VISA at 6–12 h after viral infection.Consistently, a fraction of RNF5 translocated from ER to mito-chondria and interacted with VISA at 4–8 h after viral infection,similar to its interaction with MITA (25). Previously, it has beenreported that the E3 ubiquitin ligase RNF125 is induced by viralinfection or IFN-a stimulation and catalyzes ubiquitination ofRIG-I, MDA5, and VISA (22). Recently, two studies also reportedthat Itch and proteasome subunit a type-7 (PSMA7) mediate ubiq-uitination and degradation of VISA and their expression is inducedbyviral infection (26, 28).Therefore, it is possible that viral infectionleads to translocation of RNF5 from ER to mitochondria, making itaccessible to MITA and VISA and mediating the early negativeregulation of virus-triggered induction of type I IFNs by down-regulatingVISA andMITA,whereas RNF125, Itch, and PSMA7 areinduced by viral infection and target VISA for ubiquitination at thelate phase of viral infection. In this context, we observed thatknockdown of RNF5 inhibited degradation of VISA at the earlyphase of viral infection. At the late phase of viral infection, the ex-pression of Itch (together with PCBP2) is induced, which may ac-count for the degradation of VISA. However, it should be noted thatknockdown of RNF5 or Itch did not fully restore expression ofVISAat 8 or 16 h after SeV infection, probably due to the fact thatknockdown efficiency is not 100% or alternatively other molecules,such as PSMA7 or RNF125, existing for ubiquitination and

FIGURE 6. RNF5 targets VISA for degradation independent of Itch. A, VISA is directly ubiquitinated by RNF5 in the presence of the E2 Ubc5 (UbcH5a,

UbcH5b, or UbcH5c). VISA and RNF5 were translated in vitro, and the indicated E2s were added for ubiquitination assays. Ubiquitin-conjugated VISA

was detected by immunoblot with HRP–streptavidin (upper panel). Before ubiquitination analysis, the levels of the translated proteins were detected with

the indicated Abs (lower panels). B, RNF5 catalyzes ubiquitination of VISA independent of Itch. The 293 cells (2 3 106) were transfected with the

indicated plasmids. Twenty-four hours after transfection, immunoprecipitation, reimmunoprecipitation, and immunoblot analysis were performed with the

indicated Abs (upper panel). The expression of the proteins was examined by immunoblots with the indicated Abs (lower panels). C, Effects of RNF5 or

Itch knockdown on the stability of VISA. The 293 cells (4 3 106) were transfected with the indicated plasmids (8 mg each). Twenty-four hours after

transfection, cells were infected with SeV for the indicated time points before immunoblot analysis was performed.

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degradation of VISA. Further studies are required to determine thecontributions of these molecules in mediating degradation of VISA.A recent report suggests that viral infection causes both K48- and

K63-linked ubiquitination of VISA. However, the E3 ubiquitinligases responsible for these processes were not identified (29). TheK63-linked polyubiquitin chain at K500 of VISA leads to its re-cruitment of inhibitor of kB kinase ε to the mitochondria andactivation of NF-kB and several ISGs (29). In our study, mutationof K500 to arginine had no marked effects on RNF5-mediatedubiquitination of VISA, suggesting that K500 is not a target forK48-linked ubiquitination by RNF5. Our results suggest thatRNF5 is an E3 ubiquitin ligase for K48-linked ubiquitination ofVISA. Further investigations will be needed to characterize theE3 ubiquitin ligase(s) responsible for K63-linked ubiquitinationof VISA.It has been demonstrated that VISA plays a key role in virus-

triggered induction of type I IFNs (13–16, 30, 31). Therefore, theregulation of VISA is critical for the proper control of antiviralimmune response. The identification of RNF5 as an E3 ubiquitinligase for VISA ubiquitination and degradation after viral infectionprovides a previously undiscovered strategy for the control of ex-cessive cellular antiviral response.

AcknowledgmentsWe thank Drs. Douglas Cyr and Zhengfan Jiang for reagents and members

of our laboratory for insightful discussion and technical help.

DisclosuresThe authors have no financial conflicts of interest.

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