Immunity, Vol. 11, 721–731, December, 1999, Copyright 1999 by Cell Press

JNK2 and IKKb Are Required for Activatingthe Innate Response to Viral Infection

other than viral infection, are relatively well understood(reviewed by Karin and Hunter, 1995; Verma et al., 1995),but those that control their activity as well as that of IRF

Wen-Ming Chu,* Derek Ostertag,† Zhi-Wei Li,*Lufen Chang,* Yi Chen,* Yinling Hu,*Bryan Williams,‡ Jacques Perrault,†

proteins following viral infection have yet to be eluci-and Michael Karin*§

dated (Juang et al., 1998; Wathelet et al., 1998; Yone-*Laboratory of Signal Transductionyama et al., 1998). NF-kB and AP-1 are also involved inand Gene Regulationthe induction of proinflammatory and cytotoxic cyto-Department of Pharmacologykines (Barnes and Karin, 1997; Ip and Davis, 1998).University of California, San Diego

NF-kB proteins are regulated through interaction withLa Jolla, California 92093-0636specific inhibitors, the IkBs, which retain them in the†Department of Biologycytoplasm of nonstimulated cells (Verma et al., 1995;Molecular Biology InstituteBaldwin 1996). In response to proinflammatory stimuliSan Diego State Universityincluding viral infection, the IkBs undergo rapid phos-San Diego, California 92182phorylation at conserved N-terminal sites that trigger‡Department of Cancer Biologytheir ubiquitin-dependent degradation and subsequentThe Lerner Research Institutenuclear translocation of the liberated NF-kB dimers (Del-Cleveland, Ohio 44195hase and Karin, 1999). The signal-responsive proteinkinase that phosphorylates the IkBs and thereby causesNF-kB activation was molecularly identified (DiDonatoSummaryet al., 1997; Mercurio et al., 1997; Regnier et al., 1997).This protein kinase, IKK, is composed of two catalyticViral infection or double-stranded (ds) RNA induce in-subunits, IKKa and IKKb, and one regulatory subunit,terferons (IFN) and other cytokines. Transcription fac-IKKg/NEMO (Zandi et al., 1997; 1998; Rothwarf et al.,tors mediating IFN induction are known, but the signal-1998; Yamaoka et al., 1998; Delhase and Karin, 1999).ing pathways that regulate them are less clear. WeIKKg/NEMO is required for activation of NF-kB by manynow describe two such pathways. The first pathwaystimuli, including the proinflammatory cytokines tumorleading to NF-kB depends on the dsRNA-responsivenecrosis factor a (TNFa) and IL-1, bacterial lipopolysac-protein kinase (PKR), which in turn activates IkB ki-charide (LPS), and dsRNA (Yamaoka et al., 1998). Sur-nase (IKK) through the IKKb subunit. The second viral-prisingly, however, only the IKKb catalytic subunit isand dsRNA-responsive pathway is PKR independentrequired for IKK and NF-kB activation by proinflamma-and involves Jun kinase (JNK) activation leading totory cytokines and LPS (Delhase et al., 1999; Li et al.,stimulation of AP-1. Both IKKb and JNK2 are essential1999, 1999; Tanaka et al., 1999), whereas IKKa mediatesfor efficient induction of type I IFN and other cytokinesa response to a yet-to-be identified developmental sig-in response to viral infection or dsRNA. This studynal that controls keratinocyte differentiation (Hu et al.,establishes a general role for these kinases in activa-1999; Takeda et al., 1999). Which catalytic subunit medi-tion of innate immune responses.ates the response to virus and dsRNA was heretoforeunknown.

Introduction Unlike NF-kB, c-Jun:ATF2 (and other AP-1) hetero-dimers are constitutively nuclear, but their transactivat-

Viral infection of mammalian cells results in activation ing potential is rapidly stimulated by phosphorylation ofof an innate antiviral response mediated by type I inter- their activation domains (Binetruy et al., 1991; Smeal etferons (IFNs), which impair viral gene expression and al., 1991; Gupta et al., 1995) by the Jun kinases (JNKs,replication (Sen and Lengyel, 1992; Muller et al., 1994), Hibi et al., 1993; Derijard et al., 1994; Kyriakis et al.,as well as by other cytokines, such as interleukin (IL)-6 1994; Karin, 1995). As other MAP kinases (MAPKs), the(Pulliam et al., 1995) and IL-12 (Orange and Biron, 1996), JNKs are activated via protein kinase cascades involv-which activate cell-mediated cytotoxic responses. Reg- ing two JNK kinases (JNKKs) and a much larger numberulation of IFN expression has been the subject of intense of upstream kinases (reviewed by Minden and Karin,investigation, and several transcription factors and ar- 1997). Both JNK1 and JNK2 respond to T cell activatorschitectural proteins that bind IFN gene promoters were (Su et al., 1994) and are required for induction of T cellidentified (Reis et al., 1992; Du et al., 1993; Matsuyama cytokines (Dong et al., 1998; Yang et al., 1998; Sabapa-et al., 1993; Thanos and Maniatis, 1995). One of these thy et al., 1999).proteins, HMGI(Y), facilitates assembly of an enhanceo- The signaling pathways that are activated by viral in-some that contains several signal-responsive transcrip- fection and lead to activation of NF-kB and c-Jun:ATF2tion factors, including NF-kB (p50:p65), c-Jun:ATF2 (AP-1) are rather nebulous. In the case of NF-kB, a role in theheterodimers, and members of the IRF family (Thanos viral response was ascribed to the dsRNA-dependentand Maniatis, 1995; Wathelet et al., 1998). The control of kinase PKR (Kumar et al., 1994). PKR contains two RNANF-kB and c-Jun:ATF2 activities, in response to stimuli binding regulatory domains at its N terminus and a ser-

ine/threonine protein kinase domain at its C terminus(reviewed by Clemens and Elia, 1997; Williams, 1997).§ To whom correspondence should be addressed (e-mail: karinoffice@

ucsd.edu). PKR expression is transcriptionally induced by IFNs,

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while its catalytic activity is stimulated by binding in either IKKa (Hu et al., 1999) or IKKb (Li et al., 1999). Weused primary embryonic fibroblasts (MEFs) from Ikka2/2dsRNAs, which are produced during viral infection. Asand Ikkb2/2 embryos to investigate the involvement ofmost cells express basal levels of PKR, its regulationthe two catalytic subunits in the response to dsRNAmay involve a feed-forward positive autoregulatory cas-and virus. No substantial differences in IKK activationcade in which dsRNA produced during viral transcriptionin response to either dsRNA (Figure 1C) or infectionor replication activates preexisting PKR, which then acti-with vesicular stomatitis virus ([VSV]; Figure 1D) werevates transcription factors that lead to IFN induction.detected between wt or IKKa-deficient MEFs. By con-Newly synthesized IFNs activate the JAK-STAT signal-trast, loss of the IKKb subunit severely impaired IKKing cascade (Darnell et al., 1994; Ihle et al., 1994), re-activation by either dsRNA (Figure 1E) or VSV (Figuresulting in induction of more PKR and other IFN-respon-1F). As a result, very little NF-kB binding activity wassive gene products. This model is supported by analysisinduced by either dsRNA or VSV in IKKb-deficient cells.of PKR-deficient mice and cells (Yang et al., 1995; KumarAs a negative control, we measured JNK activity in theet al., 1997). These studies also suggest that inductiondifferent cells and much to our surprise found it to beof IRF-1 is dependent at least in part on NF-kB (Kumarinduced by both dsRNA and VSV (Figures 1C–1F). Theet al., 1997). Although it was suggested that PKR maykinetics of JNK activation were a bit slower from thosedirectly phosphorylate IkBs (Kumar et al., 1994), NF-kBof IKK but were not affected by the loss of either IKKais activated by TNFa in PKR-deficient cells (Yang et al.,or IKKb. A similar, but even more robust, JNK activation1995; Kumar et al., 1997), whereas IKKg/NEMO-deficientresponse was detected in HEK293 cells (data notcells do not respond to dsRNA (Yamaoka et al., 1998).shown).However, it was also observed that PKR-deficient mice

Next, we examined the dependence of IKK activationand cells exhibit residual IFN induction responses toon PKR, using two immortalized mouse fibroblast cellviral infection and dsRNA, thus suggesting the existencelines derived from either wt (Pkr1/1) or PKR knockoutof additional, PKR-independent, signaling pathwaysmice (Pkr2/2) (Yang et al., 1995; Kumar et al., 1997).(Yang et al., 1995). The signaling pathways that activateWhile incubation of Pkr1/1 cells with dsRNA resulted inc-Jun:ATF2 and other forms of AP-1 in response to viralIKK activation, there was no appreciable increase in IKKinfection are largely unknown.activity during the first 4 hr of treatment with dsRNA inWe investigated signaling pathways responsive to vi-Pkr2/2 cells (Figure 2A), but after 4 hr there was a smallral infection or dsRNA that can lead to induction ofbut reproducible increase in IKK activity in Pkr2/2 cellstype I IFNs and other cytokines. We obtained evidence(data not shown). Both cell lines, however, were respon-indicating that a major pathway that leads to NF-kBsive to TNFa, an established IKK activator. It should beactivation is indeed dependent on PKR, but in this path-noted that in mouse fibroblasts, unlike HEK293 cells, theway PKR acts upstream to IKK, targeting its IKKb sub-response of IKK to dsRNA is weaker than its response tounit rather than IKKa. We demonstrate that IKKb is es-TNFa (see also Figure 1E). As previously describedsential for induction of type I IFNs and other cytokines.(Yang et al., 1995), Pkr2/2 cells exhibited a defective NF-We identified a second signaling pathway elicited bykB activation response to dsRNA but a normal responseviral infection or dsRNA that leads to JNK activation.to TNFa (Figure 2A). Surprisingly, incubation of bothThis pathway can enhance the transcriptional activity ofPkr1/1 and Pkr2/2 cells with dsRNA resulted in efficientc-Jun:ATF2 and other AP-1 dimers, and its importance isJNK activation that was similar in magnitude to the re-illustrated by JNK2-deficient cells, which exhibit a defectsponse to TNFa, one of the most potent JNK activators.in induction of type I IFNs in response to viral infectionThe defect in IKK activation by dsRNA was indeedor dsRNA.caused by the absence of functional PKR, as coex-pression of an epitope-tagged IKKb with wt PKR in

Results Pkr2/2 cells restored robust IKK activation by dsRNA(Figure 2B).

PKR Acts Upstream to IKK Pkr2/2 cells also exhibited a defect in IKK and NF-We established pools of HEK293 cells, stably expressing kB activation in response to VSV infection. While VSVeither wild-type (wt) or catalytically inactive [IKKa (KM) infection of wt fibroblasts resulted in IKK and NF-kBand IKKb (KA)] epitope-tagged forms of IKKa and IKKb. activation within 3 hr, the response of Pkr2/2 fibroblastsStable transfection of either wt IKKa or wt IKKb did to VSV infection was considerably attenuated and de-not considerably potentiate IKK or NF-kB activation by layed (Figure 2C). IKK and NF-kB activities were in-either dsRNA or TNFa (Figure 1A). However, expression creased only after 7 hr post infection in Pkr2/2 cells, andof IKKa (KM) resulted in a small decrease in IKK activity, even at that point both responses were weaker thanwhereas the IKKb (KA) mutant almost completely abol- those in Pkr1/1 cells. These results were not due toished dsRNA or TNFa induced IKK activity and markedly differences in susceptibility of the two cell lines to VSVreduced NF-kB activation. Although considerable IKK infection. For instance, the kinetics of viral RNA replica-activation is observed after a 10 min incubation with tion, following infection at an moi of 10, determined bydsRNA, kinase activity peaks only after 3 hr (Figure 1B). hybridization to the VSV L gene probe, were similar inTherefore, the response to dsRNA is considerably the two cell lines (see below). In addition, VSV infectionslower than the response to TNFa, which peaks within of both Pkr1/1 and Pkr2/2 cells resulted in similar levels15 to 20 min and decays within 30 to 60 min (Zandi et of JNK activation detected at 5–7 hr post infection (Fig-al., 1997; Delhase et al., 1999). IKK activation is maximal ure 2C).at 12.5 mg/ml of dsRNA. These experiments indicate that PKR acts upstream

to IKK in the response pathway elicited by either virusWe recently generated mutant mice that are deficient

JNK and IKK Required for Interferon Induction723

Figure 1. Involvement of IKK in NF-kB Acti-vation by dsRNA and Virus

(A) Parental HEK293 cells and pools ofHEK293 cells stably transfected with eitherwt or catalytically inactive IKK [IKKa (KM) andIKKb (KA)] expression vectors were treatedwith dsRNA (50 mg/ml) for 3 hr or TNFa (20 ng/ml) for 10 min and then lysed. Lysates wereassayed for IKK kinase activity (KA) by animmunecomplex kinase assay. Recovery ofIKK was determined by immunoblotting (IB)with anti-IKKa antibody. NF-kB DNA bindingactivity was measured by an electrophoreticmobility shift assay (EMSA).(B) IKK activity was measured at differenttime points after treatment of HEK293 cellswith 50 mg/ml dsRNA (left panel) or at 3 hrafter treatment with different concentrationsof dsRNA (right panel). For comparison, cellswere treated with TNFa (20 ng/ml) for 10 min.(C) MEFs derived from wt (Ikka1/1) andIkka2/2 embryos were treated or not withdsRNA. At the indicated time points, cellswere lysed, IKK was isolated by IKKg antibod-ies to measure IkB kinase activity (KA), andJNK was isolated with anti-JNK1 antibodiesto measure c-Jun kinase activity. Recoveryof IKK and JNK was determined by immu-noblotting (IB) with anti-IKKg and anti-JNK1,respectively.(D) Ikka1/1 and Ikka2/2 MEFs were infectedwith VSV (moi 5 10), and at the indicatedtimes IKK and JNK activities were measuredas described above.(E) MEFs derived from wt (Ikkb1/1) and Ikkb2/2

embryos were examined for activation of IKK,NF-kB, and JNK by dsRNA as describedabove.(F) Ikkb1/1 and Ikkb2/2 MEFs were examinedfor IKK, NF-kB, and JNK activation in re-sponse to VSV infection (moi 5 10).

infection or dsRNA. However, residual and delayed IKK serines were replaced by alanines was no longer respon-sive to either wt or catalytically inactive PKR, either inand NF-kB activation can occur in the absence of PKR.

Efficient IKK activation by VSV or dsRNA requires the vivo (Figure 3A) or in vitro (Figure 3B). Neither form ofPKR activated JNK1, JNK2 (Figure 3C), or p38 (data notIKKb catalytic subunit. On the other hand, PKR is not

involved to any extent in viral- or dsRNA-induced JNK shown). We also found that JNK activation by dsRNAis inhibited in cells, which stably express inactive mu-activation.tants of JNKK1 or JNKK2 (data not shown). We interpretthese results to suggest that PKR catalytic activity isDifferential Response of IKK and JNK to PKR

To further examine the relationships between PKR and mostly required for its autoactivation (Clemens and Elia1997; Williams, 1997), and once sufficient amounts ofthe IKK and JNK activation cascades, we cotransfected

expression vectors encoding epitope-tagged IKKb, JNK1, PKR are present it may activate IKK by direct protein–protein interaction.or JNK2 with either wt or catalytically inactive (a K296R

mutant) PKR expression vectors. Coexpression of HA-IKKb with wt PKR resulted in efficient dose-dependentIKK activation (Figure 3A). When expressed at low levels, JNK2 and IKKb Are Required for Optimal IFN

and Cytokine InductionPKR (K296R) led to poor IKK activation, but, surprisingly,when expressed at higher levels PKR (K296R) activated To determine whether the newly discovered PKR-inde-

pendent dsRNA-responsive signaling pathway that leadsIKK. To examine this point further, we tested whetherrecombinant wt PKR and PKR (K296R) purified from to JNK activation contributes to type I IFN gene induc-

tion, we used nonimmortalized (MEF) and immortalizedEscherichia coli can directly activate recombinant IKKbpurified from Sf9 cells. Although recombinant IKKb dis- (3T3) fibroblasts derived from wt, Jnk12/2, and Jnk22/2

E12 mouse embryos. We compared the induction ofplays considerable basal activity (Zandi et al., 1998), itsincubation with either wt PKR or PKR (K296R) further IFNa1 and IFNb mRNA in these cells to their induction

in Pkr2/2 fibroblasts and IKK-deficient cells. As pre-increased its IkB kinase activity (Figure 3B). This activa-tion required phosphorylation at Ser-177 and Ser-181 viously described (Yang et al., 1995), little induction of

IFNa1 or IFNb mRNA was observed in Pkr2/2 fibroblasts(Delhase et al., 1999), because a mutant in which these

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Figure 3. PKR Activates IKK but Not JNKFigure 2. Role of PKR in IKK and JNK Activation by dsRNA or Virus (A) Wt 3T3 fibroblasts were transfected with HA-IKKb, HA-IKKb (AA),(A) Immortalized fibroblasts (3T3) derived from wt (Pkr1/1) or Pkr2/2 or HA-IKKb (KA) expression vectors (200 ng/plate) along with wtembryos (Kumar et al., 1997) were treated with dsRNA (100 mg/ml) PKR or catalytically inactive PKR (K296R) expression vectors (200for the indicated periods or with TNFa for 10 min, after which IKK ng/plate in lanes 2 and 5, 400 ng/plate in lanes 3 and 6, or 800 ng/and JNK activities were measured by immunecomplex kinase plate in lanes 4, 7, 10, and 13). Where indicated, instead of a PKRassays (KA). Recoveries of IKK (IKKa) and JNK1 were determined expression vector, the cells were transfected with an empty expres-by immunoblotting (IB). NF-kB and NF-1 DNA binding activities were sion vector (400–600 ng/plate, lanes 1, 5, 8, 9, 11, and 14) anddetermined by EMSA. were either left untreated or treated with TNFa for 10 min prior to(B) Exogenous PKR restores IKK activation in Pkr2/2 cells. Pkr2/2 harvesting. After 36 hr, the cells were lysed, HA-IKKb containing IKKfibroblasts were cotransfected with an HA-IKKb expression vector complexes were isolated by immunoprecipitation, and their kinasealong with either “empty” or PKR expression vectors. After 36 hr, activity and recoveries determined by immunecomplex kinase assaythe cells were treated with dsRNA, and HA-IKKb associated kinase (KA) and immunoblotting (IB), respectively.activity and expression were determined as above. (B) In vitro activation. Wt IKKb (1 ng) or IKKb (AA) (10 ng) purified(C) Pkr1/1 and Pkr2/2 cells were infected with VSV (moi 5 10). At from baculovirus infected Sf9 cells were incubated with either wtthe indicated times post infection, cell lysates were prepared and PKR or PKR (K296R) (12.5 ng in lanes 2 and 6; 25 ng in lanes 3, 7,used to measure IKK and JNK activities and expression and NF-kB 11, and 13, or 50 ng in lanes 4, 5, 8, 9, 12, and 14) for 30 min, afterand NF-1 DNA binding activities. which the IkB kinase activities of the IKKb proteins were examined.

Wt PKR or PKR (K296R) were also tested directly for IkBa (1–54)phosphorylation (lanes 5 and 9, respectively).upon treatment with dsRNA (Figure 4A). A similar reduc-(C) Wt 3T3 cells were transfected with either HA-JNK1 or HA-JNK2

tion in IFN mRNA induction was observed in both nonim- expression vectors (0.5 mg/plate) along with empty expression vec-mortalized and immortalized fibroblasts from Jnk22/2

tor or a PKR expression vector (0.4 mg/plate). Where indicated, cellswere treated with TNFa for 10 min before harvesting. After 24 hr,mice (Figure 4B). A more modest reduction in IFN mRNAcell lysates were prepared and HA-JNK immunoprecipitated, andinduction was observed in Jnk12/2 fibroblasts (data nottheir kinase activity and recoveries were determined by an im-shown). Consistent with the effects on IKK activationmunecomplex kinase assay (KA) and immunoblotting (IB), respec-(Figure 1C), little effect on IFN mRNA induction wastively.

observed in Ikka2/2 MEFs (Figure 4C). However, a largedecrease in induction of IFNa1 and IFNb mRNAs bydsRNA was observed in Ikkb2/2 MEFs (Figure 4D).

JNK and IKK Required for Interferon Induction725

Figure 4. Defective IFN mRNA Induction bydsRNA in Cells Lacking PKR, JNK2, or IKKb

(A) Wt (Pkr1/1) and Pkr2/2 3T3s were left un-treated or incubated with dsRNA (100 mg/ml)for 1 hr. After washing with PBS, the cellswere incubated in complete medium for 3 hr,after which total RNA was extracted. IFNa1,IFNb, and GAPDH mRNA levels were deter-mined by Northern blot analysis.(B) Wt (Jnk21/1) and Jnk22/2 MEFs and 3T3fibroblasts were left untreated or treated withdsRNA as above. IFNa1, IFNb, and GAPDHmRNA levels were determined as describedabove.(C) Ikka1/1 and Ikka2/2 MEFs or (D) Ikkb1/1

and Ikkb2/2 MEFs were treated as above andanalyzed for induction of IFN mRNA.

We also compared induction of IFN mRNA in response or replication, because VSV genomic RNA accumulatedwith slightly faster kinetics in both Ikkb2/2 and Jnk22/2to VSV infection of the different fibroblast cultures. In-

duction of IFNa1 and IFNb mRNA was normal in Ikka2/2 cells than in wt cells. A considerable reduction in IFNgene induction in response to VSV infection also wascells (Figure 5A). However, a considerable reduction in

IFN gene induction was observed in Ikkb2/2 cells (Figure observed in Pkr2/2 cells (Figure 5D).In addition to type I IFNs, treatment of cells with5B). A partial decrease in both IFNa1 and IFNb mRNA

induction was also observed in Jnk22/2 fibroblasts (Fig- dsRNA results in induction of cytokines such as TNFa,IL-1, IL-6, and IL-12 (Gendelman et al., 1990; Verdijk eture 5C). The reduction in IFN induction in both Ikkb2/2

and Jnk22/2 cells was not due to a defect in viral infection al., 1999). We found that JNK2- and IKKb-deficient cells

Figure 5. Defective IFN mRNA Induction inResponse to VSV Infection in PKR-, IKK-, andJNK-Deficient Cells

(A) Ikka1/1 and Ikka2/2 MEFs were infectedwith VSV at moi 5 10 or mock infected. Atthe indicated times post infection (7 hr formock infected cells), total RNA was extractedand analyzed for expression of IFNa1, IFNb,and GAPDH mRNAs. VSV replication (Rep)was monitored by hybridization to a probefor the L gene.(B) Ikkb1/1 and Ikkb2/2 MEFs, (C) Jnk21/1 andJnk22/2 MEFs; and (D) Pkr1/1 and Pkr2/2 3T3swere infected and analyzed as describedabove.

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and 7E). While both promoters were highly responsiveto VSV in wt cells, very little activation of either promoterwas observed in either Jnk22/2 or Ikkb2/2 cells, thusconfirming the results of the RNA analysis.

Discussion

Role of JNK in the Antiviral ResponseViral infection or dsRNA treatment results in inductionof type I IFNs (IFNa1 and IFNb), which in turn confer anantiviral state (Sen and Lengyel, 1992). Initially, it wasassumed that the only signaling molecule activated inresponse to virus or synthetic dsRNA, a mimic of earlyproducts of viral infections, is the dsRNA-dependentprotein kinase PKR. Not only is PKR activity and expres-sion induced by dsRNA and viral infections (Clemensand Elia, 1997; Williams, 1997), but its overexpressionprotects cultured cells from vaccinia virus (Lee and Es-teban, 1993) and encephalomyocarditis virus but notfrom VSV (Meurs et al., 1992). In addition, several viruseshave evolved different strategies to inhibit PKR activityand thereby became IFN resistant (Katze, 1992; Tayloret al., 1999). The use of certain protein kinase inhibitors,which are not particularly specific to PKR, suggestedthat PKR is essential for IFN induction (Zinn et al., 1988).It was therefore surprising that PKR-deficient mice gen-Figure 6. Defective Cytokine Induction in JNK2- and IKKb-Deficienterated by gene targeting exhibited nearly normal induc-Fibroblaststion of type I IFNs in response to viral infection or dsRNAMEFs from wild-type, Jnk22/2, and Ikkb2/2 mice were treated with

either IL-1 (20 ng/ml), LPS (10 mg/ml), or dsRNA (100 mg/ml) or left (Yang et al., 1995) and normal antiviral responses (Yanguntreated. After 4 hr, total RNA was isolated and expression of IL-6, et al., 1995; Abraham et al., 1999). The results, however,IL-12, IFNb, and GAPDH determined by Northern blot hybridization. turn out to depend on the type of virus used, and PKR-

deficient mice were recently found to be highly sensitiveto VSV infection (N. Sonnenberg, personal communica-

were also defective in induction of IL-6 and IL-12 by tion). MEFs derived from PKR-deficient mice exhibit de-dsRNA, IL-1, or LPS (Figure 6). As expected, IL-1 and fective IFN gene induction, in response to either virusLPS did not induce INFb expression in these cells. or dsRNA. This defect can be rescued by “priming” with

low levels of type I IFN (Yang et al., 1995). It was thereforesuggested that the response of Pkr2/2 mice to dsRNA isJNK2 Is Required for IFNa and IFNb

Promoter Activation caused by low-level IFN production due to backgroundexposure to environmental pathogens. Nevertheless, itTo determine whether the defect in IFN mRNA induc-

tion in Jnk22/2 cells occurs at the level of IFN gene tran- was also suggested that there must be another, PKR-independent activation pathway involved in IFN genescription, we transfected established wt, Jnk12/2, and

Jnk22/2 fibroblasts with a luciferase (LUC) reporter induction (Yang et al., 1995; Abraham et al., 1999). Ourresults identify one such signaling pathway as the onedriven by the IFNb promoter (truncated at 2125; Yone-

yama et al., 1998). After normalization for transfection that leads to JNK activation. We demonstrate that JNKactivity is potently stimulated by dsRNA or infectionefficiency (which was quite similar in the different cell

lines), the IFNb promoter was approximately 4-fold less with the RNA virus VSV, in a manner that is entirelyindependent of PKR. Furthermore, JNK activity is notactive in Jnk22/2 cells, although a residual response to

dsRNA was still observed (Figure 7A). In Jnk12/2 cells, responsive to overexpression of PKR. Using MEFs andimmortalized (3T3) fibroblast lines derived from JNK2-the response was similar to that in wt fibroblasts. Similar

results were obtained using an IFNb-CAT reporter (trun- deficient mice (Sabapathy et al., 1999), we provide ge-netic evidence that JNK activation is required for optimalcated at 2110; Figure 7B). Induction of the IFNb pro-

moter by dsRNA in Jnk22/2 cells, and to a lesser extent induction of IFNa1 and IFNb transcription.The discovery of this physiological function for thebasal promoter activity, were potentiated by cotransfec-

tion of a JNK2 vector (Figure 7C). By contrast, cotrans- JNK pathway fits nicely with previous analysis of theIFNb promoter, which contains four positive regulatoryfection of a nonactivatable JNK2 mutant, JNK2 (APF)

(Kallunki et al., 1994), reduced basal IFNb promoter ac- domains (PRDs) required for its induction by virus ordsRNA (Du et al., 1993; Thanos and Maniatis, 1995).tivity and completely prevented its induction. This mu-

tant most likely acts as a dominant-negative inhibitor of PRD IV is recognized by a heterodimer of c-Jun andATF2, as well as the structural protein HMGI(Y) (Du et al.,the residual JNK activity present in Jnk22/2 cells.

We also compared the activation of the IFNa1 (trun- 1993). c-Jun and ATF2, which belong to the AP-1/ATFfamily of transcription factors, are the most extensivelycated at -140; Yoneyama et al., 1998) and IFNb promot-

ers by VSV in wt, Jnk22/2, and Ikkb2/2 cells (Figures 7D characterized JNK substrates (Karin, 1995). In both

JNK and IKK Required for Interferon Induction727

Figure 7. JNK2 Is Involved in Control of IFNb

Gene Transcription

(A) Wt, Jnk12/2, and Jnk22/2 3T3 cells weretransiently transfected with an 2125IFNb-Luc reporter (125-Luc; Yoneyama et al.,1998). After 36 hr, the cells were left untreatedor incubated with dsRNA (100 mg/ml) for 4hr. The cells were collected and lysed, andluciferase activity was determined. The re-sults shown are averages of two independentexperiments normalized for transfection effi-ciency determined by cotransfection of aSV40-LacZ reporter and measurement ofb-galactosidase activity.(B) Wt and Jnk22/2 3T3 cells were cotrans-fected with -110IFNb-CAT (Wathelet et al., 1998)and SV40-LacZ reporters. After 48 hr, the cellswere treated or not with dsRNA for 4 hr. Thecells were collected and lysed, and CAT andb-galactosidase activities were determined.Shown are the results of a typical CAT assayand the relative fold-induction of CAT activitynormalized to b-galactosidase expression.(C) Jnk22/2 3T3 cells were cotransfected with2125IFNb-Luc and SV40-LacZ reporters andeither empty, HA-JNK2, or HA-JNK2 (APF) ex-pression vectors. After 24 hr, the cells weretreated or not with dsRNA, and cell lysateswere prepared 4 hr later. Luciferase andb-galactosidase activities were determined.Shown are average luciferase activities fromtwo independent experiments normalized tob-galactosidase levels.

(D) Wt, Ikkb2/2, and Jnk22/2 cells were transfected with 2140IFNa1-Luc (La-Luc; Yoneyama et al. 1998) and SV40-LacZ reporters. After 46hr, the cells were either mock infected or infected with VSV at m.o.i 5 10. After 9 more hr, lysates were prepared and luciferase andb-galactosidase activities determined as above.(E) Similar experiment to the one above except that an 2125IFNb-Luc reporter was used.

cases, JNK-mediated phosphorylation of specific sites points. After several hours, residual NF-kB activationcan be observed in Pkr2/2 cells. This delayed responsewithin their activation domains enhances their ability to

activate transcription (Smeal et al., 1991; Hibi et al., could, in principle, be due to the priming effect dis-cussed above or to activation of a PKR-independent1993; Derijard et al., 1994; Gupta et al., 1995). Our pres-

ent results explain how viral infection results in stimula- pathway, possibly the one that leads to JNK activation.As NF-kB binds to PRDII of the IFNb promoter (Lenardotion of c-Jun:ATF2 activity.

The inability of Pkr2/2 MEFs to respond to dsRNA with et al., 1989; Visvanathan and Goodbourn, 1989), its de-fective activation can explain the defect in IFNb induc-IFN gene induction can be remediated by priming with

small amounts of IFN (Yang et al., 1995). We therefore tion in Pkr2/2 cells. However, Pkr2/2 cells also exhibit adefect in IFNa1 induction (Yang et al., 1995; Figures 4examined whether JNK activation by dsRNA or virus

may be a secondary response that depends on low-level and 5), despite the absence of NF-kB binding sites inthe proximal IFNa1 promoter (MacDonald et al., 1990).IFN induction, which may occur even in the absence

of PKR. However, JNK activation was not inhibited by The similar reduction in both IFNa1 and IFNb mRNAinduction and promoter activation in Ikkb2/2 MEFspretreatment with the protein synthesis inhibitor cyclo-

heximide and treatment of 3T3 or HEK293 cells with strongly suggests that NF-kB is, nonetheless, involvedIFNa or IFNg did not activate JNK (W.-M. C. and M. K., in IFNa1 induction. Although the IFNa1 promoter mayunpublished data). In addition, the loss of IKKb, which lack NF-kB binding sites, NF-kB may still be recruited tocauses a substantial reduction in IFN induction (Figures this promoter via an interaction with other transcription4 and 5), has no effect on JNK activation (Figure 1). factors, such as AP-1 (Stein et al., 1993).Thus, it is unlikely that JNK activation is a consequence The defect in NF-kB activation in PKR-deficient cells isof IFN induction. due to their inability to rapidly activate the IKK complex.

Previous work has demonstrated that the IKKg/NEMOregulatory subunit is required for NF-kB activation byRole of IKK and Other Signaling PathwaysdsRNA (Yamaoka et al., 1998). Here, we show that IKKIn addition to the defect in IFN gene induction, Pkr2/2

catalytic activity is stimulated by either dsRNA or viralMEFs can not mount a normal NF-kB activation re-infection and that this response is largely dependent onsponse to dsRNA (Yang et al., 1995). We show that thisPKR. As previously shown for TNFa and IL-1 (Delhasedefect can be extended to VSV as an inducer. However,et al., 1999; Li et al., 1999, 1999; Tanaka et al., 1999),with both dsRNA and VSV, the defect in NF-kB activation

is not absolute and is mostly restricted to early time the stimulation of IKK activity by dsRNA or VSV and

Immunity728

consequently activation of NF-kB are dependent on AP-1 and NF-kB transcription factors in vertebrates (Ipand Davis, 1998; Hoffmann et al., 1999). The role of NF-IKKb, but not IKKa. In cotransfection experiments, we

find that PKR efficiently stimulates phosphorylation of kB in the control of innate immune and inflammatoryresponses in mammals is well recognized (Baldwin,IKKb at the same activating phosphoacceptor sites used

by the TNFa pathway (W.-M. C. and M. K., unpublished 1996; May and Ghosh, 1998). Recent results have estab-lished the role of the JNK pathway in induction of severaldata). As high levels of catalytically inactive PKR can

result in IKK activation, both in vivo and in vitro, we important cytokines, including IL-2, IL-4, and IFNg, inresponse to T cell activation (Dong et al., 1998; Yang etsurmise that PKR activates IKK via protein–protein inter-

action that stimulates the autophosphorylation of IKKb al., 1998; Sabapathy et al., 1999). It is also well estab-lished that JNK and IKK are activated by LPS or the(Delhase and Karin, 1999), rather than by direct phos-

phorylation of IKKb. In support of this interpretation of proinflammatory cytokines. The results presented heredemonstrate that JNK and IKK are also activated inthe results shown in Figure 3, we obtained evidence

suggesting a physical interaction between PKR and IKK response to viral infection and dsRNA. Furthermore,JNK2 and IKKb are essential elements of the innate(W.-M. C. and M. K., unpublished data). Most likely,

the catalytic activity of PKR is required for its initial response to viral infection or dsRNA, required for induc-tion of type I IFNs and other cytokines, including IL-6activation that eventually results in its transcription in-

duction and elevated expression (Clemens and Elia, and IL-12. Whereas IFNs inhibit virus replication, IL-6and IL-12, which are also induced by bacterial infec-1997; Williams, 1997).

Another viral- and dsRNA-responsive transcription tions, elicit cytotoxic responses needed for eliminationof intracellular pathogens. Thus, JNK and IKK serve asfactor involved in type I IFN gene induction is IRF3 or

IRF7 (Juang et al., 1998; Schafer et al., 1998; Wathelet universal activators of innate immune responses to bac-teria and viruses.et al., 1998; Yoneyama et al., 1998). These closely related

members of the IRF family bind to PRDI and PRDIII of Our results may also explain how dsRNA can inducecostimulation of T cells (Hoffmann et al., 1999) andthe IFNb promoter and to related elements in the IFNa1

promoter. In response to viral infection, IRF3 is phos- upregulate expression of numerous cytokines (Gendel-man et al., 1990; Verdijk et al., 1999). Thus, JNK andphorylated at a cluster of serines near its C terminus,

and this modification induces its nuclear entry and inter- IKK, being responsive to LPS and dsRNA, provide animportant link between innate and adaptive immunity. Inaction with the coactivator CBP/p300 (Juang et al., 1998;

Yoneyama et al., 1998). The protein kinase responsible this context, it is appealing to draw an analogy betweendsRNA and LPS. While the first serves as a genericfor IRF3 phosphorylation has not been identified, but

examination of the identified C-terminal phosphoaccep- signal for viral infection, the second serves as a genericsignal indicating infection by Gram-negative bacteria.tor sites suggests that they are not a target for PKR,

JNK, or IKK. Indeed, we have not been able to detect LPS and other byproducts of bacterial infections arerecognized by the Toll family of pattern recognition re-efficient phosphorylation of the C-terminal portion of

IRF3 by JNK or IKK (W.-M. C. and M. K., unpublished ceptors (Medzhitov and Janeway, 1997; Hoffmann etal., 1999). It is possible that dsRNA, whose chemicaldata).

Induction of type I IFN in most cell types is a specific properties resemble those of LPS (being a negativelycharged polysaccharide), may also activate membersresponse to viral infection that does not occur following

cell stimulation with TNFa (Thanos and Maniatis, 1995), of this rapidly growing family.IL-1, or LPS (Figure 6). However, TNFa, IL-1, and LPS

Experimental Proceduresare potent activators of the IKK and JNK pathways,which lead to stimulation of NF-kB and AP-1 activities.

PlasmidsTherefore, other transcription factors, most likely IRF3Expression vectors encoding HA-tagged wt and mutant IKKa and

and/or IRF7, that interact with IFN gene promoters, must IKKb, HA-tagged JNK1 and JNK2, wt PKR, and catalytically inactivebe responsible for this response specificity. By contrast, PKR (296R) were previously described (Kallunki et al., 1994; Kumar

et al., 1997; Zandi et al., 1997). Reporter plasmids containing IFNbactivation of AP-1 and NF-kB may be sufficient for induc-and IFNa1 promoter were kindly provided by Drs. T. Maniatis (-110tion of other cytokines, such as IL-6 and IL-12, whoseIFN-CAT; Thanos and Maniatis, 1995) and T. Fujita (-125-Luc-; -140promoters contain binding sites for these factorsLa-Luc; Yoneyama et al., 1998). IFNa1 and IFNb cDNA plasmids(Sanceau et al., 1995).were kindly provided by Dr. Y.-L. Yang.

Cell Culture, Transfection, dsRNA Treatment,The Role of JNK and IKK in Innateand Viral InfectionImmune ResponsesCells were cultured in DMEM supplemented with 10% FBS andInfection with bacterial, fungal, and viral pathogens re-antibiotics. Cells were transfected using Lipofectamine (Gibco) orsults in activation of innate immune responses, whichSuperfect (Qiagen), according to manufacturers’ recommendations.

provide the first line of defense against infection and Cells were washed twice with PBS before incubating with dsRNAalert the adaptive immune system to the presence of in serum-free medium containing dextran sulfate (5 mg/ml). VSV

strain T1026R1 (obtained from Dr. J. Lucas-Lenard, U. of Connecti-pathogens (Medzhitov and Janeway, 1997). Innate im-cut, Storrs) was used in all experiments, as it is a good IFN inducermunity has appeared early in evolution, preceding the(Boulares et al., 1996). High-titer virus stocks were prepared in BHKappearance of adaptive immunity, which is unique tocells from a single plaque isolate. Mouse cell monolayers grown tovertebrates (Hoffmann et al., 1999). Analysis of innate80%–90% confluency on 6 cm dishes were infected at an moi of

immune responses to bacteria and fungi in Drosophila 10 (based on pfu titer determined on BHK cells). Virus was absorbedhave implicated the involvement of signaling pathways at room temperature for 30 min in 0.5 ml of medium. Incubation at

378C was initiated concurrent with addition of 3.5 ml of mediumthat are highly similar to those that lead to activation of

JNK and IKK Required for Interferon Induction729

(time 0). In some experiments, resulting virus titers were determined Boulares, A.H., Ferran, M.C., and Lucaslenard, J. (1996). NF-kappa-Bactivation is delayed in mouse L929 cells infected with interferonat about 24 hr post infection (p.i.) by plaque assay on BHK cells.suppressing, but not inducing, vesicular stomatitis strains. Virology218, 71–80.Immunoprecipitation, Kinase, Electrophoretic Mobility,Clemens, M.J., and Elia, A. (1997). The double-stranded RNA-depen-and Reporter Assaysdent protein kinase PKR: structure and function. J. Interferon Cyto-Lysates were prepared and incubated with antibody at 48C over-kine Res. 17, 503–524.night. Immunoprecipitates were washed and used either for kinase

assays or for immunoblotting. Immunecomplex kinase assays, im- Darnell, J.E., Kerr, I.M., and Stark, G.R. (1994). Jak-STAT pathwaysmunoblot analysis, and electrophoretic mobility shift assay (EMSA) and transcriptional activation in response to IFNs and other extracel-were carried out as described (Zandi et al., 1997; Rothwarf et al., lular signaling proteins. Science 264, 1415–1421.1998). Wt and catalytically inactive PKR were expressed and purified Delhase, M., and Karin, M. (1999). The IkB kinase: a master regulatorfrom E. coli, while IKKb was expressed and purified from Sf9 cells of NF-kB, innate immunity and epidermal differentiation. Cold Spring(Zandi et al., 1998). The proteins were coincubated in kinase buffer Harb. Symp. Quant. Biol. 64, in press.in the presence of ATP and then the IkB kinase activity of IKKb was

Delhase, M., Hayakawa, M., Chen, Y., and Karin, M. (1999). Positiveassayed by standard procedures. CAT and Luciferase assays wereand negative regulation of IkB kinase activity through IKKb subunit

performed as described (Wathelet et al., 1998).phosphorylation. Science 284, 309–313.

Derijard, B., Hibi, M., Wu, I.-H., Barrett, T., Su, B., Deng, T., Karin,RNA Analysis

M., and Davis, R.J. (1994). JNK1: a protein kinase stimulated by UVFor Northern blot analysis, 10 mg of total RNA per lane was fraction-

light and Ha-Ras that binds and phosphorylates the c-Jun activationated on a 1% denaturing agarose gel (Yang et al., 1995). Northern

domain. Cell 76, 1025–1037.blots were hybridized with random-primed 32P-labeled probes for

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Hibi, M., Lin, A., Smeal, T., Minden, A., and Karin, M. (1993). Identifi-Viral genome replication was probed with 32P-labeled T7 transcriptcation of an oncoprotein-responsive and UV-responsive protein ki-containing nucleotides 286 to 2559 of L gene (plus strand).nase that binds and potentiates the c-Jun activation domain. GenesDev. 7, 2135–2148.

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