Arenavirus Nucleoprotein Targets Interferon Regulatory Factor

Arenavirus Nucleoprotein Targets Interferon RegulatoryFactor-Activating Kinase IKKε

Christelle Pythoud,a W. W. Shanaka I. Rodrigo,b Giulia Pasqual,a Sylvia Rothenberger,a Luis Martínez-Sobrido,b

Juan Carlos de la Torre,c and Stefan Kunza

Institute of Microbiology University Hospital Center and University of Lausanne, Lausanne, Switzerlanda; Department of Microbiology and Immunology, University ofRochester, Rochester, New York, USAb; and Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, California, USAc

Arenaviruses perturb innate antiviral defense by blocking induction of type I interferon (IFN) production. Accordingly, the are-navirus nucleoprotein (NP) was shown to block activation and nuclear translocation of interferon regulatory factor 3 (IRF3) inresponse to virus infection. Here, we sought to identify cellular factors involved in innate antiviral signaling targeted by arenavi-rus NP. Consistent with previous studies, infection with the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV)prevented phosphorylation of IRF3 in response to infection with Sendai virus, a strong inducer of the retinoic acid-induciblegene I (RIG-I)/mitochondrial antiviral signaling (MAVS) pathway of innate antiviral signaling. Using a combination of coimmu-noprecipitation and confocal microscopy, we found that LCMV NP associates with the I�B kinase (IKK)-related kinase IKK� butthat, rather unexpectedly, LCMV NP did not bind to the closely related TANK-binding kinase 1 (TBK-1). The NP-IKK� interac-tion was highly conserved among arenaviruses from different clades. In LCMV-infected cells, IKK� colocalized with NP but notwith MAVS located on the outer membrane of mitochondria. LCMV NP bound the kinase domain (KD) of IKK� (IKBKE) andblocked its autocatalytic activity and its ability to phosphorylate IRF3, without undergoing phosphorylation. Together, our dataidentify IKK� as a novel target of arenavirus NP. Engagement of NP seems to sequester IKK� in an inactive complex. Consider-ing the important functions of IKK� in innate antiviral immunity and other cellular processes, the NP-IKK� interaction likelyplays a crucial role in arenavirus-host interaction.

Arenaviruses are a large and diverse family of viruses of rele-vance as both powerful model systems for experimental virol-

ogy and clinically important human pathogens (6). The proto-typic arenavirus lymphocytic choriomeningitis virus (LCMV) hasbeen instrumental in many landmark studies that provided fun-damental concepts in molecular virology, virus-host cell interac-tion, viral pathogenesis, and viral immunology (38). On the otherhand, several arenaviruses have emerged as causative agents ofsevere hemorrhagic fevers (HF) with high morbidity and signifi-cant mortality in humans, posing serious public health problemswithin their regions of endemicity (12). The arenavirus with thehighest impact in human health is Lassa virus (LASV), whichcauses several hundred thousand infections per year in WesternAfrica, with thousands of deaths (32). There is currently no vac-cine available, and therapeutic options are limited, resulting in15% to 30% mortality in hospitalized patients. Likewise, in SouthAmerica, the arenaviruses Junin virus (JUNV), Machupo virus,Guanarito virus, and Sabia virus have emerged as causative agentsof severe HF disease (41).

Arenaviruses are enveloped viruses with a bisegmented nega-tive-strand RNA genome and a nonlytic life cycle restricted to thecell cytoplasm (9). The S RNA encodes the viral glycoprotein pre-cursor (GPC) and the nucleoprotein (NP), whereas the L RNAencodes the viral RNA-dependent RNA polymerase L and the ma-trix protein Z. The arenavirus GPC undergoes proteolytic pro-cessing by the cellular protease S1P to yield GP1, which is involvedin receptor binding, and GP2, which mediates a pH-dependentfusion event required for arenavirus cell entry (2, 23).

A hallmark of severe arenavirus infection in humans is theinability of the patient’s innate and adaptive immune systems tocontain the virus, resulting in uncontrolled virus multiplicationthat often leads to a fatal outcome (12). Thus, pathogenic arena-

viruses seem able to subvert the mechanisms of innate pathogenrecognition by the infected host (1, 13, 26). Another characteristicfeature of arenaviruses is their ability to establish persistent infec-tions in their natural rodent reservoirs and in a wide range ofmammalian cells in vitro. Arenavirus persistence is commonly as-sociated with continued replication and expression of viral pro-teins. Intriguingly, despite widespread viral replication and highviral loads in mice persistently infected with LCMV, only a modesttype I interferon (IFN) response is mounted (48), suggesting thatthe virus escapes innate detection and/or can efficiently counter-act mechanisms of innate defense (4). Accordingly, LCMV wasshown to be able to interfere with induction of type-I IFNs in thehost cell by blocking the activation of transcription factor IRF3(31), and the NP was identified as the viral component that actedas an IFN antagonist (31). This IFN-counteracting activity ishighly conserved among arenaviruses (30), and it was mapped tothe C-terminal region of NP (29), which contains a 3=-5= exonu-clease domain (15, 44). Mutations that abrogated the 3=-5= exo-nuclease domain also abolished the ability of NP to suppress theinduction of type I IFNs (15, 29, 44), indicating a link between thetwo activities.

The cytosolic pathogen recognition receptors retinoic acid-in-ducible gene I (RIG-I) and melanoma-differentiation-associatedgene 5 (MDA5) have been shown to recognize arenavirus RNA

Received 23 January 2012 Accepted 6 April 2012

Published ahead of print 24 April 2012

Address correspondence to Stefan Kunz, [email protected].

Copyright © 2012, American Society for Microbiology. All Rights Reserved.

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(14, 27, 28, 56). Upon activation, RIG-I and MDA5 associate withthe mitochondrial signaling adapter MAVS/IPS-1/Cardif/VISA(20, 34, 51, 55). MAVS consists of an amino-terminal CARD do-main, a proline-rich region (PRR) in the middle of the protein,and a C-terminal transmembrane domain that localizes the pro-tein to the outer mitochondrial membrane. Binding of active RIG-I/MDA5 induces aggregation of MAVS to activate innate antiviralresponses (18). Aggregation of MAVS leads to the generation of asignaling platform through recruitment of multiple signalingmolecules, including the classical I�B kinase (IKK) complexIKK�/IKK�/NEMO and the nonclassical IKK-related kinasesTANK-binding kinase (TBK-1) and IKKε (20, 34, 35, 51, 55). Theclassical IKK complex IKK�/IKK�/NEMO is involved in activa-tion of nuclear factor �B (NF-�B), whereas TBK-1 and IKKε ac-tivate the interferon regulatory factors (IRF) by direct phosphor-ylation of IRF3 and IRF7 (17). Upon activation, NF-�B, IRF3, andthe activator protein AP1 synergistically activate transcription ofbeta IFN (IFN-�), which acts in a autocrine and paracrine man-ner, binding to its cognate type I IFN receptor (IFNAR) and in-ducing a second wave of type I IFN expression (10). Upon recep-tor binding, type I IFNs activate the JAK/STAT signaling pathway(50) and induce the expression of hundreds of IFN-stimulatedgenes (ISGs), establishing an antiviral state within the cell (45, 47).

In the present study, we sought to identify cellular factors tar-geted by arenavirus NP to disrupt the host innate antiviral signal-ing. Using a combination of biochemical assays and confocal mi-croscopy, we have demonstrated that the LCMV NP specificallytargets IKKε. We found that this NP-IKKε interaction is highlyconserved among arenaviruses from different clades. LCMV NPassociated with the kinase domain (KD) of IKKε and blocked itsactivity without undergoing phosphorylation. This newly uncov-ered NP-IKKε interaction may play an important role in arenavi-rus-host cell interaction during acute and persistent infection.

MATERIALS AND METHODSCells and viruses. Human embryonic kidney cells (HEK293 andHEK293T) and human lung adenocarcinoma epithelial cells (A549) weremaintained in Dulbecco’s modified Eagle medium (DMEM) (Gibco BRL)supplemented with 10% fetal calf serum (FCS) at 37°C and 5% CO2. Toinfect cells, we used lymphocytic choriomeningitis virus Armstrong 53b(LCMV) as described in reference 22 and recombinant Sendai virus-greenfluorescent protein (SeV-GFP) as described in reference 36 (kindly pro-vided by Laurent Roux, University of Geneva, Geneva, Switzerland). SeVCantell strain was grown in 10-day-old embryonated eggs, as previouslydescribed (21, 31). The viruses were diluted in 10% FCS–DMEM forLCMV or DMEM for SeV and added to the cells for 1 h at 37°C. Theinoculum was then removed and replaced by fresh cell medium. For SeV-infected cells, GFP expression was used as a control for infections.

Antibodies. Rat monoclonal antibody (MAb) antihemagglutinin(anti-HA) (high affinity) was from Roche Applied Science (Rotkreuz,Switzerland). Mouse MAb anti-�-tubulin and mouse MAb anti-FLAG(M2) were from Sigma-Aldrich (St. Louis, MO). Mouse IgG1 anti-IKKεwas a kind gift from John Hiscott, McGill University, Montreal, Canada.The nucleoprotein of LCMV (LCMV NP) was detected using guinea pigserum anti-LCMV NP or mouse MAb anti-LCMV NP (1.1.3), as previ-ously described (7). Rabbit and goat polyclonal antibodies (pAb) anti-IRF3 were from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit pAbanti-phosphoIRF3 was purchased from Cell Signaling Technology (Dan-vers, MA). Mouse MAb anti-GFP and mouse IgG2b anti-MAVS wereobtained from Clontech (Mountain View, CA) and Alexis Biochemicals(Lausen, Switzerland), respectively. Alexa Fluor 594 goat anti-mouseIgG1, Alexa Fluor 488 goat anti-mouse IgG2b, Alexa Fluor 594 F(ab=)2

fragment of goat anti-rabbit IgG, and Pacific Blue streptavidin were allpurchased from Molecular Probes (Eugene, OR), while biotinylated don-key anti-guinea pig IgG was from Jackson ImmunoResearch (Suffolk,United Kingdom). Dynabeads magnetic beads were purchased from In-vitrogen.

Plasmids. The expression plasmids for HA-tagged arenavirus (LCMV[wt], Lassa virus [LASV], Whitewater Arroyo virus [WWAV], Latino vi-rus [LATV], and Junin virus [JUNV]) NPs and HA-tagged LCMV Z weredescribed previously (30, 31). The expression plasmids for HA-taggedLCMV NP mutants (LCMV NP D382A, LCMV NP E384A, LCMV NPD459A, LCMV NP H517A, and LCMV NP D522A) were also previouslydescribed (29, 39). Expression plasmids for FLAG-tagged IKKε andTBK-1 were kindly provided by Margot Thome Miazza, University ofLausanne, Switzerland. The truncated version of IKKε (IKKε KD) encod-ing the N-terminal kinase domain (amino acids 1 to 315) was generated byintroducing a STOP codon at position 316. The forward primer was T7universal primer 5=-TAA-TAC-GAC-TCA-CTA-TAG-GG-3=, and the re-verse primer was 5=-ATA-TTC-TAG-ATT-AGG-AGA-AGA-CAT-GGA-CGA-C-3= comprising the STOP codon and XbaI restriction site, allowingfurther subcloning. Expression plasmids for FLAG-tagged MAVS andRIG-I were kind gifts of Jürg Tschopp, and an expression plasmid forTRAF3 was a kind gift from Pascal Schneider, both of the University ofLausanne, Lausanne, Switzerland. A GFP-IRF3 fusion protein-encodingplasmid and a FLAG-tagged IRF3 expression plasmid were kindly pro-vided by John Hiscott, McGill University, Canada, and Brian Seed, Har-vard Medical School, Boston, MA, respectively.

Protein expression analysis and IP. If not indicated otherwise,HEK293 cells were transfected using calcium phosphate as described pre-viously (34). For protein expression analysis, cells were lysed with sodiumdodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample-loading buffer. The samples were separated by SDS-PAGE, and proteinexpression was assessed by Western blot analysis. For immunoprecipita-tion (IP) of HA-tagged NP, cells were lysed at 48 h posttransfection withlysis buffer (50 mM Tris, 280 mM NaCl, 2 mM EGTA, 10% [wt/vol]glycerol, 0.2 mM EDTA, 50 mM NaF, 1% [wt/vol] NP-40) supplementedwith protease inhibitor cocktail (Roche, Rotkreuz, Switzerland). For IRF3immunoprecipitation, cells were lysed with lysis buffer (20 mM Tris, 137mM NaCl, 2 mM EDTA, 10% [wt/vol] glycerol, 1% [wt/vol] NP-40)complemented with protease and phosphatase inhibitor cocktail (Roche,Rotkreuz, Switzerland). Cell lysates were incubated for 2 h on a rotationwheel at 4°C with protein G-coated magnetic Dynabeads (Invitrogen,Carlsbad, CA) previously incubated with the appropriate antibody to pre-cipitate the target protein. After four wash steps with lysis buffer, magneticbeads were resuspended in SDS-PAGE sample buffer and heated to elutethe bound proteins. Eluted proteins were then separated by SDS-PAGEand analyzed by Western blotting.

IRF3 reporter assays and Western blotting. Activation of the IRF3-dependent promoter reporter plasmid (p55C1B-FF) after SeV infectionhas been previously described (31). Briefly, 2 � 105 HEK293T cells (12-well plate format, replicates) were cotransfected, in suspension, using cal-cium phosphate with 0.5 �g of p55C1B-FF and 100 ng of C-terminallyHA-tagged wild-type and 3=-5= exonuclease single-amino-acid LCMV NPpCAGGs expression plasmids, along with 50 ng of an expression plasmidencoding Renilla luciferase (RL) under the control of the simian virus 40promoter (pSV40-RL) to normalize transfection efficiencies. EmptypCAGGs plasmid was used as a negative control. Twenty-four hours post-transfection, cells were mock or SeV infected (multiplicity of infection[MOI] � 3) for 1 h at room temperature. At 16 to 18 h postinfection (p.i.),luciferase reporter activities and protein expression were analyzed usingcell lysates. Luciferase activities were determined using the Promega(Fitchburg, WA) dual-luciferase reporter assay and a Lumicount lumi-nometer. Reporter gene activation was calculated as fold induction (acti-vation) over the noninduced, empty pCAGGs multiple-cloning site(MCS)-transfected control. Protein expression was determined by West-ern blotting using anti-HA (Sigma) or anti-glyceraldehyde-3-phosphate

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dehydrogenase (anti-GAPDH) (Abcam, Cambridge, United Kingdom)polyclonal antibodies and a horseradish peroxidase (HRP)-conjugatedanti-rabbit IgG secondary antibody (GE, Amersham Biosciences, Amer-sham, United Kingdom). Protein bands were visualized using a chemilu-minescence detection kit (SprayGlo) and autoradiography films (DenvilleScientific Inc., Metuchen, NJ) according to the manufacturer’s instruc-tions.

Nuclear translocation of IRF3. HEK293T cells (2 � 105) werecotransfected, in suspension, by the use of calcium phosphate with 1 �g ofpEGFP-C1-hIRF3 together with 2 �g of the indicated C-terminally HA-tagged pCAGGs LCMV NP expression plasmids, as previously described(21, 31). LCMV Z-HA pCAGGs was used as a negative control. Twenty-four hours posttransfection, cells were infected with SeV (MOI � 3) for 1h at room temperature and, 16 to 18 h postinfection, cells were fixed with0.2% (vol/vol) glutaraldehyde containing 2.5% (vol/vol) formalde-hyde–1� PBS for 10 min at 4°C and permeabilized using 0.1% (vol/vol)Triton X-100 –1� PBS for 10 min at room temperature. After beingwashed with 1� PBS and blocked overnight at 4°C with 10% (wt/vol)bovine serum albumin (BSA)–1� PBS, cells were incubated with ananti-HA polyclonal antibody (Sigma) diluted in 5% (wt/vol) BSAblocking solution for 1 h at 37°C, washed, and incubated with a Rhod-amine-red-conjugated anti-rabbit IgG (H�L; Jackson ImmunoRe-search), and 4=,6=-diamidino-2-phenylindole (DAPI; Research Organics,Cleveland, OH) for cellular nucleus staining, in blocking solution for 30min at 37°C. For the quantification of the nuclear translocation of IRF3,HA-positive cells (indicative of protein expression) were considered. Tocalculate percentages of cells with nuclear translocation of GFP-IRF3, atotal of 50 cells in 3 to 7 nonoverlapping fields were counted under �20magnification, in replicate experiments.

In vitro kinase assay. HEK293 cells were seeded the day prior to trans-fection, followed by transfection using calcium phosphate. For IP ofFLAG-tagged IKKε and IRF3, lysis buffer containing the following ingre-dients was used: 50 mM Tris, 150 mM NaCl, 1 mM EGTA, 1 mM �-glyc-erophosphate, 1 mM EDTA, 1% (wt/vol) Triton X-100, and 1 mM or-thovanadate supplemented with protease inhibitor cocktail (Roche,Rotkreuz, Switzerland). For the in vitro kinase assay, the proteins wereincubated for 1 h at 30°C with 10 �Ci of [-32P]ATP per reaction in kinaseassay buffer (50 mM Tris, 12 mM MgCl2, 1 mM �-glycerophosphate, 100�M ATP, 1 mM orthovanadate) in a total volume of 50 �l. SDS-PAGEsample buffer was added to stop the reaction. Proteins were then sepa-rated by SDS-PAGE and developed by autoradiography.

Real-time quantitative PCR (qPCR). Total RNA was purified with anRNeasy minikit (Qiagen, Chatsworth, CA), and cDNA was synthesizedusing a QuantiTect reverse transcription kit (Qiagen). TaqMan probesspecific for IFN-� (Hs01077958_s1), IKKε (IKBKE; Hs01063858_m1),TBK1 (Hs00179410_m1), and GAPDH (Hs99999905_m1) were obtainedfrom Applied Biosystems (Foster City, CA). Real-time PCR (RT-PCR)was performed using a StepOne real-time PCR system (Applied Biosys-tems), and gene expression levels relative to GAPDH were determinedaccording to the 2��CT method (24).

RNAi. RNA interference (RNAi) was performed using validated smallinterfering RNAs (siRNAs) for IKKε (IKBKE; SI02655324) and TBK1(SI02224411) and scrambled siRNA (1027280) as a control from Qiagen(Basel, Switzerland). Briefly, 2 �106 HEK293 cells were reverse trans-fected with 15 nM siRNA using a10-cm-diameter dish and LipofectamineRNAiMAX (Invitrogen, Paisley, United Kingdom) according to the man-ufacturer’s recommendation. Twenty-four hours after transfection cells,were replated, and 48 h after transfection, cells were infected with SeV(MOI � 0.1) or mock infected to be assessed for IFN-� mRNA levels(RT-qPCR) or IRF3 phosphorylation (Western blotting) 24 h postinfec-tion. Depletion of IKKε and TBK1 was confirmed by RT-qPCR usingspecific probes or Western blot analysis using specific antibodies.

Colocalization analysis. To perform colocalization studies, A549 cellswere seeded on glass 8-well LabTek slides and infected with LCMV at anMOI of 1. At 48 h postinfection, cells were fixed with 2% (wt/vol) form-

aldehyde–PBS for 15 min at room temperature and washed with PBS.Cells were then permeabilized for 30 min at room temperature with 0.1%(wt/vol) saponin, 10% (vol/vol) goat serum, and 100 mM glycine–PBS.Primary and secondary antibodies were diluted in 0.1% (wt/vol) saponinand 1% (vol/vol) goat serum–PBS and incubated overnight at 4°C and 1 hat room temperature, respectively. Before acquisition, LabTek slides weremounted using Mowiol (Sigma). Image acquisition was performed with aZeiss LSM710 Quasar confocal microscope equipped with a Plan Apo-chromat lens (63�, 1.2 numerical aperture [NA] objective), 405-nm di-ode laser, argon lasers (458, 476, 488, and 514 nm), and 561-nm diode-pumped solid-state (DPSS) laser. All images for each data set wereacquired the same day with the same microscope settings. Images werefirst deconvolved using Huygens Essential software (SVI, Hilversum,Netherlands) and then analyzed for colocalization with Imaris 7.2 soft-ware (Bitplane, Zürich, Switzerland).

RESULTSArenavirus infection blocks phosphorylation of IRF3. Arenavi-rus NP can prevent the induction of type I IFNs by blocking acti-vation of IRF3 (30, 31). Specifically, expression of NP preventedthe nuclear translocation of IRF3, which is a prerequisite for acti-vation of the IFN-� promoter (30, 31). However, it was unclear atwhich level arenavirus NP interfered with IRF3 activation andconsequent IFN-� expression. To further investigate this issue, wefirst studied the effect of arenavirus infection on IRF3 phosphor-ylation. For this, HEK293 cells were infected with the prototypicarenavirus LCMV ARM 53b (henceforth referred to as LCMV),and the expression of IFN-� mRNA was monitored over time byquantitative RT-PCR (RT-qPCR). In parallel, the expression lev-els of the viral NP were detected by Western blotting. In cellsinfected at an MOI of 10, we observed a transient induction ofIFN-� mRNA at around 8 h p.i., followed by a marked decreasethereafter (Fig. 1A). The peak in IFN-� mRNA correlated with thefirst detection of NP, whereas decreased levels of IFN-� mRNA at12 h p.i. correlated with increased NP levels in infected cells (Fig.1A and B).

We next examined the effect of arenavirus infection on phos-phorylation of IRF3. HEK293 cells were infected (MOI � 0.1)with LCMV or mock infected, and cells were infected 24 h laterwith Sendai virus (SeV), a strong inducer of RIG-I/MAVS-in-duced IRF3 phosphorylation and IFN expression (34), or mockinfected. Phosphorylation of IRF3 was detected by Western blot-ting with an antibody that specifically recognized the phosphory-lated form of IRF3 and compared to total IRF3 levels in the cells. Inmock-infected cells, infection with SeV induced phosphorylationof IRF3, whereas previous infection with LCMV prevented IRF3phosphorylation upon SeV infection (Fig. 1C). To address the roleof NP in the block of IRF3 phosphorylation in LCMV-infectedcells, we transfected HEK293 cells with recombinant LCMV NPusing calcium phosphate, a method that avoids induction of a typeI IFN response (31, 34). At 24 h posttransfection, cells were in-fected with SeV and IRF3 phosphorylation was assessed by West-ern blotting. SeV-induced phosphorylation of IRF3 was inhibitedin NP-transfected cells (Fig. 1C).

LCMV NP interacts specifically with IKK� but not TBK-1.Sendai virus is a strong inducer of the RIG-I/MAVS signalingpathway (34), and recent studies implicated the RIG-I/MDA5/MAVS pathway in innate detection of arenaviruses (14, 27, 28,56). In response to viral infection, activated RIG-I helicases asso-ciate with MAVS located on the outer mitochondrial membrane,followed by assembly of a signaling complex whose activation re-

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sults in IRF3 phosphorylation. The observed blockade of IRF3phosphorylation by LCMV NP (Fig. 1) raised the possibility thatNP targets one or several components of the MAVS-associatedsignaling complex upstream of IRF3. To identify potential targets,we probed possible interactions between LCMV NP and selectedcomponents of the RIG-I/MAVS signaling pathway implicated inactivation of IRF3, including IRF3 itself, RIG-I, MAVS, theadapter protein TRAF3, and the kinases TBK-1 and IKKε, bothimplicated in IRF3 phosphorylation. To this end, HEK293 cellswere cotransfected using calcium phosphate with an HA-taggedform of recombinant LCMV NP, together with FLAG-tagged re-combinant RIG-I, MAVS, TRAF3, TBK-1, and IKKε, as well asGFP-tagged IRF3. After 48 h, cell lysates were prepared and as-sayed by coimmunoprecipitation (co-IP) using anti-HA magneticbeads. Isolated immunocomplexes were subsequently probed byWestern blotting with antibodies to HA to confirm IP of LCMVNP and antibodies to FLAG tag and to GFP to detect bindingpartners. In these co-IP studies, we detected a strong and specificinteraction of LCMV NP with IKKε but not with the closely re-lated TBK-1 (Fig. 2A). IP of NP resulted sometimes in a weak

co-IP of TRAF3 (Fig. 2A), but this was not consistently found.RIG-I, MAVS, and IRF3 did not show detectable binding to NPunder our experimental conditions (Fig. 2A). To validate the de-tected interaction of LCMV NP with endogenous IKKε, HEK293cells were transfected with LCMV NP, followed by infection withSeV. At 24 h p.i., IP of the HA-tagged LCMV NP was performedand the immunocomplex probed for IKKε with a specific anti-body. We consistently detected a specific interaction between theviral NP and endogenous IKKε in SeV-infected cells (Fig. 2B). Insum, probing of a selected set of components of the RIG-I/MAVSpathway involved in IRF3 phosphorylation revealed a strong di-rect or indirect interaction of LCMV NP with IKKε but not withthe closely related TBK-1.

The interaction of NP with IKK� is conserved among arena-viruses. We next investigated if the specific interaction of NP withIKKε was conserved among arenaviruses from different phyloge-netic clades. For this purpose, we examined the interaction ofIKKε with NPs of the Old World arenavirus LASV, endemic inWestern Africa, and New World arenaviruses from clades A(White Water Arroyo virus [WWAV]), B (Junin virus [JUNV]),and C (Latino virus [LATV]). HEK293 cells were cotransfectedwith FLAG-tagged IKKε or TBK-1, together with HA-tagged ver-sions of the viral NPs. Cell lysates were precipitated with anti-HAmagnetic beads and immunocomplexes analyzed by Westernblotting as described above. All arenavirus NPs tested associatedspecifically with IKKε but not with TBK-1 (Fig. 3A). To ensurethat our co-IP assay conditions allowed detection of specific inter-actions, we included as a control an HA-tagged form of the LCMVZ protein. Only NP, and not Z, underwent co-IP with IKKε(Fig. 3B). In our experiments, we noticed apparent differences inthe binding of some NPs to IKKε. However, this has not beenconsistently observed and did not correlate with IFN antagonismof the NPs.

IKK� colocalizes with LCMV NP in infected cells. The inter-action of arenavirus NPs with IKKε may affect the stability of thekinase and thereby prevent IRF3 phosphorylation. To address thispossibility, we examined the expression of IKKε in cells infectedwith LCMV over time. HEK293 cells were either infected withLCMV or mock infected. At different time points, cells were lysedand expression of IKKε and NP was examined by Western blot-ting. Normalization of IKKε expression levels to uninfected cellsrevealed no consistent reduction of IKKε expression during 48 hof infection, despite a dramatic increase in NP expression over thecourse of infection (data not shown). This result indicated that NPexpression does not result in rapid degradation of IKKε. We there-fore explored the alternative possibility that LCMV infectionmight affect the ability of IKKε to be recruited to the MAVS sig-naling platform on mitochondria. To address this possibility, weexamined the cellular distribution of NP, IKKε, and MAVS inLCMV-infected cells by confocal microscopy. To this end, we in-fected A549 cells with LCMV and at 48 h p.i. cells were fixed andspecimens examined by confocal laser scanning microscopy. Thecolocalization of NP, IKKε, and MAVS was assessed as describedin Materials and Methods. In LCMV-infected cells, IKKε showeda strong colocalization with NP, but not MAVS, as confirmed bythe quantitative image analyses depicted in the correspondingscatter plots (Fig. 4).

TBK1 and IKK� contribute to SeV-induced IFN-� produc-tion in HEK293 cells. The specific ability of arenavirus NP totarget IKKε, but not TBK-1, was unexpected, because analysis of

FIG 1 LCMV infection blocks phosphorylation of IRF3. (A) Induction ofIFN-� mRNA in cells infected with LCMV. HEK293 cells were infected withLCMV at an MOI of 10. At the indicated time points p.i., cells were lysed andtotal RNA was extracted and cDNA synthesized by reverse transcription. Real-time PCR was performed using a StepOne real-time PCR system and TaqManprobes specific for IFN-� and GAPDH. Gene expression levels relative toGAPDH were determined according to the 2��CT method (24). Data pre-sented represent fold induction above the level seen with uninfected cells (n �3 � standard deviation [SD]). (B) Expression of LCMV NP. HEK293 cells wereinfected as described for panel A or left uninfected (u). At the indicated hourpostinfection, cells were lysed. Proteins were separated in SDS-PAGE andprobed in Western blot analyses performed using mouse MAb (1.1.3) toLCMV nucleoprotein (NP), combined with HRP-conjugated secondary anti-bodies and enhanced chemiluminescence (ECL). (C) IRF3 phosphorylation inresponse to LCMV infection and SeV infection. HEK293 cells were first in-fected with LCMV at an MOI of 0.1 or transfected with recombinant LCMVNP (4 �g). Twenty-four hours later, cells were infected with SeV at an MOI of0.1, and 24 h later, cells lysates were prepared and subjected to immunopre-cipitation using anti-IRF3 magnetic beads (IP:IRF3). Immunocomplexes wereprobed in Western blotting using an antibody specific to phospho-IRF3(pIRF3) and an antibody that recognized IRF3 independently of phosphory-lation (IRF3). Expression of LCMV NP (NP) in cell lysates (CL) was performedas described for panel A. The position of pIRF3 is indicated, and the asteriskdesignates IgG heavy chain.




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embryonic fibroblasts derived from mice deficient in IKKε andTBK-1 suggested a predominant role for TBK-1, rather than IKKε,in IFN induction in response to double-stranded RNA (dsRNA)and virus infection (16, 33, 40). To address the relative contribu-tions of IKKε and TBK-1 to virus-induced IRF3 activation andIFN-� induction in our experimental system, we depleted IKKεand TBK-1 by RNA interference (RNAi) using validated siRNAs(Materials and Methods). Transfection of HEK293 cells with spe-cific siRNAs targeting IKKε or TBK-1 resulted in a marked reduc-tion in the level of the target mRNA (Fig. 5A). Next, we addressedthe roles of IKKε and TBK-1 in IFN-� induction upon infection

with SeV. For this purpose, we used siRNA treatment to depleteHEK293 cells of IKKε or TBK-1, followed by infection with SeV.We assessed levels of IFN-� mRNA by RT-qPCR as shown in Fig.1A. Depletion of IKKε or TBK-1 resulted in a significant impair-ment of IFN-� induction upon infection with SeV (Fig. 5B), sug-gesting that both kinases are required for an optimal IFN re-sponse. In contrast to other systems, such as murine embryonicfibroblasts, where TBK-1 seems to be the preeminent kinaselinked to antiviral signaling (16, 33, 40), we consistently found astronger effect of depletion of IKKε (Fig. 5B), suggesting the exis-tence of cell-type-specific differences. We further observed im-

FIG 2 LCMV NP interacts specifically with IKKε. (A) Coimmunoprecipitation (co-IP) between LCMV NP and different components of the RIG-I/MAVSsignaling pathway. HEK293 cells were transiently cotransfected with an HA-tagged form of recombinant LCMV NP (4 �g) and FLAG-tagged RIG-I, MAVS,TRAF3, TBK-1, or IKKε or GFP-tagged IRF3 (4 �g each). Empty vector was used as a negative control. At 48 h posttransfection, cells were lysed and co-IP wasperformed using anti-HA magnetic beads (IP:HA). Immunocomplexes were probed with anti-HA antibody to confirm the IP of LCMV NP (IB:HA). Co-IP oftagged proteins was detected by Western blotting with antibodies to FLAG (IB:Flag) or to GFP (IB:GFP). The presence of transfected protein in cell lysates (CL)was verified by detection with the corresponding antibodies (lower panel). (B) Co-IP of endogenous IKKε with LCMV NP. HEK293 cells were transientlytransfected with recombinant HA-tagged LCMV NP (4 �g) and 24 h later infected with SeV at an MOI of 0.1. At 24 h p.i., co-IP was performed using anti-HAmagnetic beads (IP:HA) as described for panel A. Immunocomplexes were probed with antibody to HA tag and a specific antibody to the endogenous form ofIKKε. The presence of recombinant LCMV NP and endogenous IKKε was confirmed in the whole-cell lysate (CL). The band indicated by the asterisk correspondsto the IgG heavy chain.

FIG 3 The specific interaction of NP with IKKε is conserved among arenaviruses. (A) Co-IP of IKKε, but not TBK-1, with NPs derived from differentarenaviruses. HEK293 cells were transiently cotransfected with HA-tagged NP of LCMV, LASV, WWAV, LATV, and JUNV together with FLAG-tagged IKKε orTBK-1 (4 �g of each plasmid). At 48 h posttransfection, cells were lysed and co-IP was performed using anti-HA magnetic beads (IP:HA). Immunocomplexeswere probed with antibody to HA to confirm IP of arenavirus NPs and antibody to FLAG to detect co-IP of IKKε or TBK-1. Expression of recombinant arenavirusNPs, IKKε, and TBK-1 was verified in whole-cell lysate (CL). IB � Western blotting; * � IgG heavy chain. (B) IKKε undergoes specific interaction with LCMVNP but not Z. HEK293 cells were transfected with HA-tagged LCMV NP or LCMV Z together with IKKε. After 48 h, co-IP was performed as described for panelA using anti-HA magnetic beads. IKKε and the HA-tagged viral proteins were detected in IPs and total cell lysates (CL). IB � Western blotting; * � IgG heavychain.

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portant quantitative differences in the induction of IFN-� in re-sponse to infection with SeV (Fig. 5B) and LCMV (Fig. 1A), witha circa-50-fold-stronger induction of IFN-� transcription uponinfection with SeV. We also examined the role of IKKε in virus-induced phosphorylation of IRF3. For this, we depleted HEK293cells of IKKε, followed by infection with SeV. We assessed IRF3

phosphorylation using a phosphorylation-specific antibody as de-scribed above (Fig. 1C). Depletion of IKKε at 80% at the proteinlevel resulted in a marked reduction of IRF3 phosphorylation inSeV-infected cells (Fig. 5C). Together, these data indicate that, inHEK293 cells under our experimental conditions, both TBK-1 andIKKε contributed to optimal IFN-� induction in response to SeV.

FIG 4 In infected cells, LCMV NP colocalizes with IKKε but not with MAVS. A549 cells were infected with LCMV at an MOI of 1. At 48 h p.i., cells were fixed,permeabilized, and triple stained for LCMV NP (polyclonal guinea pig IgG anti-LCMV), IKKε (mouse MAb IgG1 anti-IKKε), and MAVS (mouse MAb IgG2banti-MAVS). Primary antibodies were detected with species- and isotype-specific secondary antibodies. Specimens were examined by confocal laser scanningmicroscopy, and image acquisition was performed with a Zeiss LSM710 Quasar confocal microscope. Images were first deconvoluted using Huygens Essentialsoftware and then analyzed for colocalization with Imaris 7.2 software. (a to c) Individual staining for LCMV NP (green), IKKε (red), and MAVS (blue) in arepresentative cell. (d) Merge of the signals for LCMV NP (green), IKKε (red), and MAVS (blue). (e and f) Colocalization of LCMV NP and IKKε is shown (e)with the corresponding scatter plot (f). Colocalization appears in the diagonal of the plot. (g and h) The lack of colocalization of LCMV NP with MAVS is shownin the image (g) and scatter plot (h). Bar � 5 �m.

FIG 5 In HEK293 cells, TBK-1 and IKKε contribute to SeV-induced IRF3 phosphorylation and IFN-� production. (A) HEK293 cells were transfected withsiRNAs for IKKε or TBK-1 or control siRNA, and efficiency of depletion was assessed after 72 h by quantification of mRNA levels by RT-qPCR using specificprobes for IKKε and TBK-1 as described in Materials and Methods (n � 3 � SD). (B) In HEK293 cells, induction of IFN-� mRNA in response to SeV requiresboth IKKε and TBK-1. HEK293 cells were depleted for IKKε and TBK-1 as described for panel A and infected at 48 h with SeV at an MOI of 0.1. After 24 h, mRNAlevels of IFN-� were determined as described for Fig. 1A. Data represent fold induction above the level seen with uninfected cells (n � 3 � SD). (C) IKKε isrequired for optimal IRF3 phosphorylation in response to SeV infection. HEK293 cells were depleted of IKKε as described for panel A. After 48 h, cells wereinfected with SeV as described for panel B and phosphorylation of IRF3 was detected as described for Fig. 1C. Depletion of IKKε was verified at the protein levelby Western blotting using a specific antibody. For quantification of IRF3 phosphorylation, signals for phospho-IRF3 were normalized to signals for total IRF3(phospho-IRF3/IRF3) and cells treated with control siRNA set at 100. To quantify the IKKε knockdown, signals for IKKε were normalized to signals for tubulin(IKKε/tubulin), with cells treated with control siRNA set at 100.




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Binding to IKK� involves the C-terminal domain of LCMVNP. Recent studies demonstrated that the C-terminal domain ofLCMV NP is required for its ability to act as an IFN antagonist(29). Residues D382 and E384 in the DIEGR motif (29), as well asD459, H517, and D522, which are part of a recently described3=-5= exonuclease motif highly conserved within the C terminus ofarenavirus NP, were particularly important (15, 29, 44). Whenexpressed in HEK293 cells, all NP mutants showed expressionlevels similar to the wild-type level (Fig. 6A). To confirm theirdefect in preventing type I IFN induction via IRF3, HEK293 cellswere cotransfected with wild-type NP or each of the mutant NPs,together with the reporter construct p55C1B-FF, driving expres-

sion of a Firefly luciferase reporter from an IRF3-regulated pro-moter and the pSV40-RL plasmid encoding Renilla luciferase un-der the control of the simian virus 40 promoter to normalizetransfection efficiencies. Twenty-four hours posttransfection,cells were infected with SeV, and 16 to 18 h p.i., activation of IRF3was monitored by detection of luciferase reporter activities in aluminescence assay. Consistent with previous findings (29), allmutants tested were defective in their ability to block IRF3 induc-tion by SeV infection (Fig. 6B). Results were confirmed with areporter plasmid encoding the monomeric red fluorescent pro-tein under the control of the IFN-� promoter (pIFN�-mRFP/CAT) (31) (data not shown).

FIG 6 Residues within the active site of the NP C-terminal 3=-5= exoribonuclease domain are involved in both NP anti-IFN activity and NP interaction withIKKε. Data represent the effect of single amino acid substitutions within the active site of the NP C-terminal 3=-5= exoribonuclease domain on the NP anti-IFNactivity. HEK293 cells were cotransfected with 0.5 �g of the IRF3-responsive plasmid p55C1B-FF, 100 ng of C-terminal HA-tagged LCMV-NP wild type (WT)or the mutants LCMV NP D382A, LCMV NP E384A, LCMV NP D459A, LCMV NP H517A, and LCMV NP D522A, and 50 ng of the pSV40-RL expression vectorto normalize transfection efficiencies. Twenty-four hours posttransfection, cells were infected with SeV (MOI � 3) and, 16 to 18 h postinfection, proteinexpression and reporter gene expression were analyzed. (A) Protein expression levels. Lysates (200 �g of total protein) from transfected cells were analyzed forNP expression levels by Western blotting using an anti-HA pAb. GAPDH expression levels were used to normalize total protein. (B) Reporter gene expression.IRF3-dependent activation is expressed as fold induction over the level seen with empty vector-transfected and mock-infected control cells. (C and D) Effect ofLCMV-NP single amino acid substitutions on nuclear translocation of IRF3. (C) HEK293T cells cotransfected with 1 �g of pEGFP-C1-hIRF3 and 2 �g of theindicated HA-tagged LCMV wild-type (WT) NP or single amino acid substitutions in the 3=-5= exonuclease domain were infected with SeV (MOI � 3), andsubcellular localization of GFP-IRF3 was assessed at 16 to 18 h postinfection by counting 50 cells in 3 to 7 nonoverlapping fields in replicate experiments asdescribed in Materials and Methods. (D) Representative images of IRF3 (green), LCMV-Z and LCMV-NP wild type (WT) and mutants (red), cellular nuclearstaining (blue), and the corresponding merged images are displayed. Arrows indicate nuclear translocation of IRF3 in a cell with no detectable levels of LCMVNP. (E) Co-IP of IKKε with NP mutants. HEK293 cells were transiently cotransfected with FLAG-tagged IKKε and the wild-type form of HA-tagged LCMV NPor HA-tagged LCMV NP mutants (4 �g of each plasmid). After 48 h of cotransfection, cells were lysed and co-IP was performed using anti-HA magnetic beads(IP:HA). The presence of transfected proteins in the immunocomplexes as well as in the crude cell lysate (CL) was assessed by Western blotting using antibodiesto HA and to FLAG. IB � Western blotting; * � IgG heavy chain. For quantification of the co-IP, signals for IKKε were normalized to signals for NP (IKKε/NP)and the ratio for wild-type NP was set at 100.

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In a complementary approach, we further monitored the abil-ity of our NP mutants to prevent the nuclear translocation ofIRF3. To this end, wild-type NP and mutants were cotransfectedwith a transcriptional fusion of IRF3 protein with green fluores-cent protein (GFP-IRF3). Twenty-four hours posttransfection,cells were infected with SeV as described above. At 16 to 18 hpostinfection, cells were fixed and permeabilized and intracellularstaining for NP was performed using an anti-HA polyclonal anti-body (red), along with DAPI staining to visualize nuclei (blue).Nuclear translocation of GFP-IRF3 was observed (green) in NP-expressing cells, and cells showing nuclear accumulation of GFP-IRF3 were scored. Consistent with their inability to block IRF3-dependent activation of the reporter construct p55C1B-FF, noneof the 3=-5= exonuclease mutants significantly prevented nucleartranslocation of GFP-IRF3 (Fig. 6C and D).

To assess the ability of the NP mutants to bind to IKKε, weperformed co-IP. While wild-type LCMV NP strongly interactedwith IKKε, all NP mutants tested showed consistently reducedIKKε interaction (Fig. 6E). Gross protein misfolding is highly un-likely as an explanation for the defect of these NP mutants ininteracting with IKKε, as many of these NP mutants were previ-ously shown to be active in an LCMV minigenome rescue assay(29). Our data suggest that LCMV NP binding to IKKε involvedthe C-terminal domain of NP and was affected by mutations in the3=-5= exonuclease motif.

LCMV NP binds to the kinase domain of IKK�. The IKK-related kinases IKKε and TBK-1 have a modular structure, con-sisting of a C-terminal kinase domain, a ubiquitin-like domain(UBL), and an N-terminal coiled-coil domain (17). To define thedomain(s) of IKKε involved in binding of the viral NP, we testedthe ability of the IKKε kinase domain (KD) to bind NP. FLAG-tagged full-length IKKε and IKKε KD (Fig. 7A) were cotransfectedwith LCMV NP. As a negative control, we included FLAG-taggedTBK-1. At 48 h posttransfection, cell lysates were prepared andsubjected to co-IP. Detection of FLAG-tagged proteins in total cell

lysates revealed similar expression levels for full-length IKKε andthe IKKε KD (Fig. 7B). We detected a strong association of LCMVNP with the IKKε KD, with signal intensities comparable to thoseof full-length IKKε. As expected, no interaction between NP andTBK-1 was detected.

A major function of the arenavirus NP is the packaging of theviral RNA into the viral nucleocapsid, a process that is likely me-diated by charged residues at the surface of NP (15, 44). To assessa possible role of charged residues on NP in the binding to IKKε,we tested the sensitivity of the NP-IKKε interaction with respect toenhanced ionic strength. For this purpose, we performed co-IP ofLCMV NP with IKKε in the presence of increasing salt concentra-tions in the washing buffer. While the IP of NP was not affected byNaCl concentrations of up to 1,000 mM, the NP-IKKε interactionassessed by co-IP was sensitive to enhanced salt concentrations,with markedly reduced binding at 280 mM NaCl and higher (Fig.7C). The efficient dissociation of the NP-IKKε complex caused byincreased ionic strength suggests an important contribution ofionic bonds to the interaction between NP and IKKε.

Engagement of LCMV NP inhibits the kinase activity ofIKK�. The binding of LCMV NP to the kinase domain of IKKεraised the possibility that NP may affect the catalytic activity ofIKKε toward IRF3. In addition, or alternatively, the association ofNP with IKKε may result in phosphorylation of NP, which maybehave like a pseudosubstrate for the kinase. To address thesepossibilities, we sought to compare the levels of catalytic activity ofIKKε, either alone or in complex with NP. In a first step, HEK293cells were cotransfected with FLAG-tagged IKKε and HA-taggedNP or transfected with FLAG-tagged IKKε alone. At 48 h post-transfection, IKKε/NP-cotransfected cells were lysed and IP withanti-HA magnetic beads was performed. IKKε singly transfectedcells were subjected to IP with anti-FLAG magnetic beads. Immu-nocomplexes were probed for IKKε in Western blotting, revealingefficient IP of IKKε, as well as co-IP of IKKε with NP (Fig. 8A).Immunocomplexes of the IKKε IP and the IKKε/NP co-IP were

FIG 7 LCMV NP binds to the kinase domain of IKKε. (A) Schematic of full-length IKKε (wt) and the truncated protein encoding the kinase domain (KD) ofIKKε. (B) Co-IP of IKKε KD with LCMV NP. HEK293 cells were cotransfected with 4 �g of HA-tagged LCMV NP and 4 �g of FLAG-tagged full-length IKKε,IKKε KD, or TBK-1 as a negative control. At 48 h posttransfection, cells were lysed and co-IP was performed using anti-HA magnetic beads (IP:HA).Immunocomplexes were probed with antibody to HA (IB:HA) to confirm IP of LCMV NP and antibody to FLAG (IB:Flag) to identify binding partners.Expression of transfected proteins was verified in whole-cell lysate (CL). (C) Co-IP of LCMV NP with IKKε in the presence of increasing ionic strength. Co-IPof LCMV NP with IKKε was performed as described for panel B. Immunocomplexes were washed in the presence of the increasing salt concentrations [NaCl]:50 mM, 280 mM, 500 mM, and 1,000 mM.




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then mixed with recombinant IRF3 purified from separate,transiently transfected HEK293 cells. In vitro kinase assays us-ing [-32P]ATP were performed as described in Materials andMethods. The kinase reaction was stopped by adding SDS-PAGEsample buffer. Products were separated by SDS-PAGE and devel-oped by autoradiography. Consistent with published data (53),IKKε underwent apparent autophosphorylation in anti-FLAGimmunocomplexes derived from IKKε singly transfected cells(Fig. 8B). In contrast, no autophosphorylation was detected withthe IKKε present in the immunocomplex from the NP/IKKε co-IP(Fig. 8B). IKKε derived from singly transfected cells was able toefficiently phosphorylate IRF3, which was not the case for IKKε incomplex with NP (Fig. 8B). The absence of any detectable radio-labeled band in the kinase assay performed on the NP/IKKε co-IPindicates that IKKε in complex with NP was unable to phosphor-ylate itself, IRF3, or NP and that engagement of the LCMV NPinhibited the kinase activity of IKKε.

DISCUSSION

In our present study, we sought to identify cellular factors targetedby arenavirus NP to mediate suppression of innate antiviral sig-naling. The central findings derived from our studies are that (i)LCMV NP prevents activation of IRF3 by blocking phosphoryla-tion of the transcription factor; (ii) LCMV NP specifically targetsthe IKK-related kinase IKKε but not TBK-1; (iii) the specific bind-ing of NP to IKKε is conserved within the Arenaviridae; (iv)LCMV NP associates with the kinase domain of IKKε involvingNP’s C-terminal region; and (v) binding of LCMV NP inhibits thekinase activity of IKKε.

Previous studies demonstrated the ability of arenavirus NP toprevent activation and nuclear translocation of IRF3 in responseto viral infection (30, 31). Here we demonstrate that, upon infec-tion with SeV, which activates the RIG-I/MAVS pathway, LCMVNP blocks IRF3 phosphorylation required for dimerization and

subsequent nuclear translocation of the transcription factor (17).Considering the proposed role of the RIG-I/MDA5/MAVS path-way in innate detection of arenaviruses (14, 27, 28, 56), we probedthe interaction of LCMV NP with selected components of theRIG-I/MDA5/MAVS pathway implicated in IRF3 phosphoryla-tion using a co-IP approach. Although by no means comprehen-sive, our co-IP analysis revealed a strong and specific interactionof LCMV NP with IKKε but not with TBK-1, RIG-I, MAVS,TRAF3, or IRF3 under our experimental conditions. The lack ofdetectable co-IP of LCMV NP and RIG-I in our hands seems incontradiction to previous reports that demonstrated an interac-tion of LCMV NP with RIG-I. The reasons for this apparentdiscrepancy are not clear and may be due to the more stringentwashing conditions applied in our co-IP protocol. Of note, theinteraction between LCMV NP and RIG-I reported by Zhou et al.was not affected by the D382A mutation that abrogates the abilityof NP to suppress type I IFN induction (56) as well as the high-affinity interaction with IKKε (this study). Examination of NPsderived from representative members of the major arenavirusclades revealed evolutionary conservation of the specific interac-tion of NP with IKKε but not with TBK-1. Confocal microscopycombined with quantitative image analysis revealed that, inLCMV-infected cells, IKKε strongly colocalized with LCMV NPbut not with its cellular binding partner MAVS. This suggests thatNP somehow sequesters IKKε and prevents its recruitment to theMAVS signaling platform located on the outer mitochondrialmembrane. Whether this sequestration is linked to sites of virusreplication remains to be determined. Notably, when associatedwith NP, IKKε seems catalytically inactive, suggesting as ratherunlikely a role of IKKε in viral replication via phosphorylation ofcellular proteins or viral proteins or both.

The specific ability of arenavirus NP to target IKKε, but notTBK-1, was unexpected and seems different from the pattern seenwith other viruses. Targeting of IRF3 phosphorylation as a viralstrategy to subvert induction of type I IFN responses was firstdemonstrated for the phosphoprotein (P) of rabies virus, whichwas shown to interfere with phosphorylation of IRF3 by TBK-1(5). More recent studies mapped this activity to a specific domainwithin the P protein that is dispensable for replication (46). Incontrast, we found that the C-terminal region of LCMV NP,which is required for virus RNA replication and transcription,seems involved in binding to IKKε. Notably, the association of NPblocks the catalytic activity of IKKε, as evidenced by a lack ofautophosphorylation and the inability of NP-bound IKKε tophosphorylate IRF3 or NP. This is different from the mode ofaction reported for the paramyxovirus V proteins and Ebola virusVP35, which interfere with both TBK-1- and IKKε-mediated IRF3phosphorylation by acting as competing pseudosubstrates (25,43). Moreover, upon phosphorylation by TBK-1 and IKKε,paramyxovirus V proteins are degraded (25), whereas LCMV NPremains stable over time. Therefore, regarding specificity and themolecular mechanism of inhibition of IKKε and TBK-1, arenavi-rus NPs behave differently from IFN antagonist proteins of Rhab-doviridae, Paramyxoviridae, and Filoviridae.

Arenavirus NP is a versatile protein that fulfills a plethora offunctions in virus replication, virion assembly, and interactionwith host cell factors. The examination of a set of previously de-scribed and well-characterized NP mutants (29) revealed a role ofthe C-terminal domain of NP in binding to IKKε. The C-terminaldomain of NP is involved in binding to the viral matrix Z protein

FIG 8 Binding of LCMV NP affects the kinase activity of IKKε. (A) HEK293cells were cotransfected with 4 �g of recombinant HA-tagged LCMV NP and 4�g of FLAG-tagged IKKε or transfected with 4 �g of FLAG-tagged IKKε alone.At 48 h posttransfection, cells were subjected to IP using anti-HA magneticbeads (IP:HA) and cells transfected only with IKKε were subjected to IP usinganti-FLAG magnetic beads (IP:FLAG). The presence of IKKε was assessed inco-IP with LCMV NP (NP-IKKε) and IP of IKKε-FLAG (IKKε) by Westernblotting. (B) Detection of IKKε kinase activity. Normalized amounts of im-munocomplexes containing IKKε alone (IKKε) or IKKε bound to LCMV NP(NP-IKKε) from the experiment described in the panel A legend were mixedwith recombinant IRF3 purified from separate transfected cells, and an in vitrokinase assay was performed using [-32P]ATP as described in Materials andMethods. IPs of lysates from mock-transfected cells (mock) were used as anegative control. The samples were separated by SDS-PAGE and developed byautoradiography. The presence of IRF3 in kinase assay reactions was validatedby Western blotting (IB). * � IgG heavy chain.

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(39) and contains a 3=-5= exonuclease domain, whose activity waslinked to the anti-IFN activity of NP (15, 29, 44). The interactionwith Z and the suppression of IFN activation map to partiallyoverlapping regions of NP but also involve distinct functional do-mains (39). Our present study was limited to NP mutations caus-ing impairment in the 3=-5= exonuclease function and IFN sup-pression. All mutants tested showed significantly weaker bindingto IKKε, suggesting that overlapping domains of NP are involvedin 3=-5= exonuclease function and IKKε binding. Future compre-hensive mutation-function studies would be required to structur-ally separate the two functions and to distinguish their individualcontributions to virus-induced suppression of the induction oftype I IFN.

The specific ability of arenavirus NP to target IKKε, but notTBK-1, was unexpected, because existing evidence indicates amore important role for TBK-1 in the induction of type I IFNs inresponse to virus infection (16, 33, 40). Analysis of murine em-bryonic fibroblasts derived from mice deficient in IKKε or TBK-1suggested a predominant role for TBK-1, rather than IKKε, in IFNinduction in response to dsRNA and SeV infection (16, 33, 40).However, in macrophages, the IFN response to SeV was normal inTBK-1-deficient cells (40), indicating cell-type-specific differ-ences. To address the relative contributions of TBK-1 and IKKε toIRF3 phosphorylation and IFN-� induction in response to SeV inour system, we performed RNAi. In HEK293 cells under our ex-perimental conditions, we found that both TBK-1 and IKKε wererequired for optimal induction of IFN-� upon SeV infection. Theapparently nonredundant roles of IKKε and TBK-1 suggest thatSeV-induced IRF3 phosphorylation in HEK293 cells involves acomplex requiring both kinases. It is conceivable that NP targetingIKKε may also affect the function of TBK-1, resulting in a block inIRF3 phosphorylation. However, several lines of evidence suggestthat TBK-1 can function in the absence of IKKε (16, 33, 40). In-nate detection of RNA viruses, e.g., by Toll-like receptor 3 (TLR-3), results in specific activation of TBK-1 but not IKKε via thesignaling adapter TRIF (49). The absence of a direct interactionbetween arenavirus NP and TBK-1 found here indicates that are-naviruses may use other strategies to evade TBK-1-dependentpathways. One possible strategy is illustrated by the degradation ofviral RNA by the 3=-5= exonuclease activity of LCMV NP that islinked to its function as a type I IFN antagonist (15, 29, 44). Bydegradation of viral RNA, the 3=-5= exonuclease of NP may elim-inate the “danger signal” preventing recognition by RIG-I heli-cases. The specificity of NP for IKKε, but not TBK-1, would allowpersistent arenaviruses to leave major TBK-1-dependent path-ways of innate immunity intact. This strategy may contribute toprotecting the natural reservoir host against infection by otherpathogens, including DNA viruses, bacteria, fungi, and parasites.

The remarkably conserved specificity of arenavirus NPs forIKKε suggests other nonredundant roles for IKKε in arenavirus-host cell interaction. IKKε has also been implicated in activationof NF-�B in response to phorbol myristate acetate (PMA) and Tcell receptor activation but not tumor necrosis factor alpha(TNF-�) and interleukin-1 (IL-1) (42). In breast cancer, IKKε isknown to be an oncogene involved in uncontrolled NF-�B acti-vation (3, 11). Interestingly, as noted in the accompanying article,Rodrigo et al. have reported that LCMV NP is able to specificallyinhibit activation of NF-�B in response to virus infection withonly a mild effect on NF-�B induction by TNF-� (46a). In addi-tion to its role in activation of IRF3 and NF-�B, IKKε but not

TBK-1 affects IFN-regulated gene expression by phosphorylationof STAT1 on serine 708 (54). IKKε-mediated phosphorylation ofSTAT1 affects the quality of the gene expression profile in re-sponse to type I and type II IFNs (37, 54). Apart from antiviralsignaling, IKKε has been implicated in the regulation of energymetabolism in models of obesity (8) and cell transformation incancer cells (19). Since arenaviruses are carried in nature by per-sistent infection of their reservoir rodent species, a major thrust ofour current research is to study the role of the arenavirus NP-IKKεinteraction in the context of viral persistence. The specificity of NPfor IKKε may allow using the virus as a molecular probe to dissectnonredundant functions of IKKε and to elucidate their role forarenavirus-host interaction.

ACKNOWLEDGMENTS

We are especially indebted to the late Jürg Tschopp for stimulating dis-cussions, tools, and reagents. Without his crucial input, ideas, and sup-port, this study would not have been possible. We thank John Hiscott(McGill University Montreal), Laurent Roux (University of Geneva),Brian Seed (Harvard Medical School), and Margot Thome Miazza andPascal Schneider (both University of Lausanne) for valuable reagents.

This research was supported by a grant from the VonTobel Founda-tion (to S.K.) and funds from the University of Lausanne (to S.K.). Re-search at the J.C.D.L.T. laboratory was supported by grants RO1AI047140, RO1 AI077719, and RO1 AI079665 from NIH/NIAID. Re-search in the L.M.-S. laboratory was partially funded by NIH grants RO1AI077719 and HHSN272201000055C.

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Arenavirus Nucleoprotein Targets Interferon Regulatory Factor