JOURNAL OF VIROLOGY, Feb. 2010, p. 1785–1791 Vol. 84, No. 40022-538X/10/$12.00 doi:10.1128/JVI.01362-09Copyright © 2010, American Society for Microbiology. All Rights Reserved.

Z Proteins of New World Arenaviruses Bind RIG-I andInterfere with Type I Interferon Induction�

Lina Fan, Thomas Briese, and W. Ian Lipkin*Center for Infection and Immunity, Mailman School of Public Health, Columbia University, New York, New York

Received 2 July 2009/Accepted 23 November 2009

The retinoic acid-inducible gene I product (RIG-I) is a cellular sensor of RNA virus infection that regulatesthe cellular beta interferon (IFN-�) response. The nucleoproteins (NP) of arenaviruses are reported toantagonize the IFN response by inhibiting interferon regulatory factor 3 (IRF-3). Here, we demonstrate thatthe Z proteins of four New World (NW) arenaviruses, Guanarito virus (GTOV), Junin virus (JUNV), Machupovirus (MAVC), and Sabia virus (SABV), bind to RIG-I, resulting in downregulation of the IFN-� response. Weshow that expression of the four NW arenavirus Z proteins inhibits IFN-� mRNA induction in A549 cells inresponse to RNA bearing 5� phosphates (5�pppRNA). NW arenavirus Z proteins interact with RIG-I incoimmunoprecipitation studies and colocalize with RIG-I. Furthermore, expression of Z proteins interfereswith the interaction between RIG-I and MAVS. Z expression also impedes the nuclear factor kappa light chainenhancer of activated B cells (NF-�B) and IRF-3 activation. Our results indicate that NW arenavirus Zproteins, but not Z protein of the Old World (OW) arenavirus lymphocytic choriomeningitis virus (LCMV) orLassa virus, bind to RIG-I and inhibit downstream activation of the RIG-I signaling pathway, preventing thetranscriptional induction of IFN-�.

The innate immune system recognizes virus infection and, asa first-line defense, induces antiviral responses by producingtype I interferons (alpha and beta interferon [IFN-�/�]), whichhave antiviral, antiproliferative, and immunomodulatory func-tions. Events that trigger the antiviral innate immune responseinclude (i) detection of the invading virus by immune systemreceptors and (ii) activation of protein signaling cascades thatregulate the synthesis of IFNs. The innate immune system isactivated through pattern recognition receptors (PRR) thatrecognize conserved microbial molecular structures. Toll-likereceptors (TLRs) 3, 7, 8, and 9 and retinoic acid-inducible geneI (RIG-I)-like helicases (RLHs) are the two major receptorsystems for detecting viruses. These systems localize to differ-ent compartments within the cell and recognize different li-gands; whereas TLRs recognize viral nucleic acids presenteither in the extracellular environment or in endosomes, RLHsdetect viral RNA in the cytoplasm (reviewed in reference 34).

RIG-I and another RLH, the melanoma differentiation-as-sociated gene 5 product (MDA5), are intracellular sensors ofviral RNA. RIG-I and MDA5 contain two caspase-recruitingdomains (CARD) at their N terminus, a DExD/H-box helicasedomain, and a regulatory domain (RD) at their C terminus.RNA binding requires intact helicase domains and RDs (32).After binding of RNA, the CARDs relay signals to the down-stream CARD-containing mediator MAVS (for “mitochon-drial antiviral signaling”; also known as VISA [virus-inducedsignaling adapter], IPS-1 [beta interferon promoter stimula-tor], or CARDIF [CARD adaptor inducing IFN-�]) (15, 23,35, 39). Once activated, MAVS triggers activation of two pro-

tein complexes, TBK1:IKKε (TANK-binding kinase 1:I�B ki-nase epsilon) and IKK�-IKK� (I�B kinase �-I�B kinase �),involved in the activation of IRF-3 and NF-�B transcriptionfactors, respectively. IRF-3 and NF-�B translocate into thenucleus and assemble into a stereospecific enhanceosome com-plex that binds the promoter of IFN-�, resulting in its tran-scriptional activation (reviewed in references 10 and 31).

Although MDA5 and RIG-I share similar structural archi-tectures and their signaling pathways converge at the MAVSadaptor level, gene knockout studies indicate that the twoproteins respond to distinct RNA species. RIG-I recognizes invitro-transcribed RNA and has specifically been shown to re-spond to vesicular stomatitis virus (VSV), Newcastle diseasevirus (NDV), and influenza A virus (FLUAV). In contrast,MDA5 recognizes poly(I:C), a synthetic double-stranded RNA(dsRNA) analog, and is essential for the antiviral response tothe picornavirus encephalomyocarditis virus (EMCV) (14).RIG-I, but not MDA5, recognizes RNA bearing 5� phosphates(11, 30).

Many viruses have evolved viral products that antagonize theinterferon response at different levels. The RIG-I/MAVS path-way appears to be targeted by different viruses to achieveinhibition of the IFN-� system. For example, the nonstructuralprotein 3/4a (NS3/4A) protease of hepatitis C virus (HCV)cleaves MAVS and abrogates antiviral signaling (18, 20, 23).Another example is NS1 of FLUAV, which interacts withRIG-I to inhibit the RIG-I/MAVS pathway (9, 11, 24, 25, 30).More recently, the nonstructural protein NS2 of human respi-ratory syncytial virus (RSV) has been shown to antagonize theactivation of IFN-� transcription by interacting with RIG-I(19).

Arenaviruses are enveloped single-stranded RNA viruseswith bisegmented genomes, comprising a larger (L) and asmaller (S) segment. Although classified as negative-strandRNA viruses, they employ an ambisense coding strategy. The S

* Corresponding author. Mailing address: Center for Infection andImmunity, Mailman School of Public Health, Columbia University, 722West 168th Street, New York, NY 10032. Phone: (212) 342-9033. Fax:(212) 342-9044. E-mail: wil2001@columbia.edu.

� Published ahead of print on 9 December 2009.

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segment encodes, in the opposite orientation, a nucleoprotein(NP) and a glycoprotein precursor (GPC). The L segmentencodes an RNA-dependent RNA polymerase (L) and a smallRING finger protein (Z). The Z protein functions as thearenaviral counterpart of the matrix protein found in othernegative-strand RNA viruses. Z is associated with membranes(29, 36, 37); it has also been found in the cytoplasm (33) andthe nucleus (1), interacting with cellular proteins such as ribo-somal protein P0 (2), eukaryotic translation initiation factoreIF4E (3), promyelocytic leukemia protein PML (1), and theproline-rich homeodomain protein (6).

Arenaviruses are divided into New World (NW) and OldWorld (OW) complexes based on serologic, geographic, andgenetic relationships. Some arenaviruses, including Lassa virus(LASV) and Lujo virus (LUJV) from the OW complex andGuanarito virus (GTOV), Junin virus (JUNV), Machupo virus(MAVC), Sabia virus (SABV), and Chapare virus (CHPV)from the NW complex, can cause hemorrhagic fevers in hu-mans. Evasion of the host immune response may contribute totheir pathogenicity. The NPs of the OW arenavirus lympho-cytic choriomeningitis virus (LCMV) and NW arenavirusesJUNV and MAVC but not Tacaribe virus (TCRV) have beenfound to block the innate IFN response by inhibiting IRF-3(21). To our knowledge, Z has not been implicated in immuneevasion. Here, we show that the Z proteins of GTOV, JUNV,MACV, and SABV, but not that of LCMV or Lassa virus(LASV), interact with RIG-I to curb the activation of theIFN-� response.

MATERIALS AND METHODS

Cells, viruses, and antibodies. 293A and A549 cells were cultured in Dulbec-co’s modified Eagle’s medium supplemented with 10% fetal bovine serum (In-vitrogen, Carlsbad, CA) and 100 �g/ml penicillin-streptomycin (Sigma-Aldrich,St. Louis, MO). Sendai virus (SeV) strain Cantell (VR907) was purchased fromthe ATCC (Manassas, VA). Rabbit antisera against Z of arenaviruses GTOV,JUNV, MAVC and SABV were generated by immunizing rabbits with recom-binant His6-GTOV Z, His6-JUNV Z, His6-MAVC Z, and His6-SABV Z, re-spectively. The respective six-histidine (His6)-tagged proteins were expressed inprokaryotic systems (pDEST 17 Gateway vector; Invitrogen) and purified byaffinity chromatography using 1-ml HisTrap columns (GE Healthcare Life Sci-ences, Piscataway, NJ) equilibrated with denaturing binding buffer (20 mMTris-Cl, pH 8, 6 M guanidine, 0.5 M NaCl, 20 mM imidazole), washed with 5column volumes (CV) of denaturing binding buffer and 5 CV of denaturing washbuffer (20 mM Tris-Cl, pH 8, 6 M urea, 0.5 M NaCl, 20 mM imidazole), andeluted in denaturing elution buffer (20 mM Tris-Cl, pH 8, 6 M urea, 0.5 M NaCl,0.5 M imidazole) with a linear gradient over 20 CV. The following additionalantibodies were purchased: goat polyclonal antibodies against RIG-I (SC-48932),rabbit polyclonal antibodies against IRF-3 (SC-9082; Santa Cruz Biotechnology,Santa Cruz, CA), rabbit polyclonal antibodies against phospho-IRF-3 (Ser396)(4947S; Cell Signaling Technology, Beverly, MA), rabbit polyclonal antibodiesagainst green fluorescent protein (GFP) (ab290; Abcam, Cambridge, MA), rab-bit polyclonal antibodies against MAVS (A310-243A; Bethyl Laboratories,Montgomery, TX), mouse monoclonal antibody against V5 (R960-25; Invitro-gen), horseradish peroxidase (HRP)-coupled goat anti-rabbit IgG or goat anti-mouse IgG antibodies or rabbit anti-goat IgG antibodies (Bio-Rad, Hercules,CA), and Cy2-coupled goat anti-rabbit immunoglobulin (Ig) (Jackson Immu-noResearch, West Grove, PA).

Plasmids, transfections, and 5�pppRNA. pCAGGS plasmids expressing the Zproteins of GTOV, JUNV, MAVC, SABV, or LASV (pCAGGS-ZG, -ZJ -ZM,-ZS, or -ZL, respectively) were generated by insertion of the respective openreading frame (ORF) between the NotI and XhoI sites; pCAGGS-GFP wasgenerated by the insertion of the GFP ORF. pCAGGS-LCMV Z was a generousgift from Juan C. de la Torre (28). The human RIG-I ORF was cloned intopENTR 1A (Invitrogen) between the KpnI and XhoI sites. In order to generateN-terminal and C-terminal V5-tagged versions, the insert was subsequently re-combined into pcDNA3.1/nV5-DEST and pcDNA3.2/cV5-DEST vectors (In-

vitrogen) by using Gateway LR clonase enzyme mix (Invitrogen). The RIG-IORF was also cloned into pDEST 17 (Invitrogen) and His6-tagged recombinantprotein expressed and purified as described above for arenavirus Z proteins.pUNO1-RIG-I-GFP was a generous gift from Joari De Miranda (ColumbiaUniversity). pUNO1-MAVS was purchased from Invitrogen. Transfections wereperformed with either calcium phosphate or Lipofectamine 2000 (Invitrogen),according to the manufacturer’s instructions. 5�-Triphosphate RNA (5�pppRNA)was generated from an 80-bp xenopus elongation factor 1� sequence cloned intopTRI by in vitro transcription using a T7 MEGAshortscript kit and purified usinga MEGAclear kit (Ambion, Austin, TX).

qPCR. The cell monolayer was washed twice with ice-cold phosphate-bufferedsaline (PBS; Sigma-Aldrich) and RNA extracted using an RNeasy kit (Qiagen,Hilden, Germany). Total RNA was quantified by UV spectrometry. cDNA wassynthesized from 0.5 �g of RNA in a 10-�l reaction volume by using TaqManreverse transcription reagents (Applied Biosystems, Foster City, CA). HumanIFN-�-specific PCR primers and a probe labeled at the 5� end with the fluores-cent reporter dye 6-carboxyfluorescein (FAM) and at the 3� end with thequencher dye 6-carboxy-tetramethyl-rhodamine (TAMRA) were purchasedfrom Applied Biosystems. Results were normalized to �-actin gene expressionlevels in the same sample replicate by using intron/exon-spanning human �-actin-specific PCR primers and a probe labeled with a VIC reporter and a Black Holedark quencher dye (Applied Biosystems). Each 25-�l quantitative real-timereverse transcription-PCR (qPCR) amplification reaction mixture contained 5 �lof the cDNA template, 12.5 �l universal master mix (Applied Biosystems), and1.25 �l of IFN-� and �-actin primers (at 200 nM each) and probes (at 300 nM).The assay was performed with a model 7700 sequence detector system (AppliedBiosystems) with a cycling profile of 50°C for 2 min, 95°C for 10 min, and 45cycles at 95°C for 15 s, followed by 60°C for 1 min.

Immunoprecipitation. Cultures of 293A cells in 6-well plates were transfectedwith various combinations of plasmids. The total DNA concentration was keptconstant by adding empty-vector DNA. Thirty-six hours after transfection, thecells were washed with PBS, trypsinized, collected by centrifugation, and thenlysed in 150 �l of nondenaturing lysis buffer (20 mM Tris-HCl [pH 8], 137 mMNaCl, 10% glycerol, 1% Nonidet P-40, 2 mM EDTA, complete protease inhib-itor cocktail [Roche Applied Science, Indianapolis, IN]). The lysates were sub-jected to centrifugation for 20 min at 10,000 � g at 4°C and the clarifiedsupernatants preadsorbed by incubation with 10 �l of protein A-Sepharose(Sigma-Aldrich) for 20 min at 4°C. For each immunoprecipitation, a 100-�laliquot of lysate was incubated with 1 �g of monoclonal antibody or 5 �l ofpolyclonal antisera overnight at 4°C on a rotator. Antibody-antigen complexeswere precipitated by adding 50 �l of a 1:1 slurry of protein A-Sepharose beadsin lysis buffer for 4 h at 4°C, followed by centrifugation for 1 min at 10,000 � g.The beads were washed four times with 1 ml of lysis buffer and then subjected toWestern blot (WB) analysis. In the case of immunoprecipitation with purifiedprotein, 200 ng of protein were combined with 5 �l polyclonal sera against Zproteins.

Native PAGE. Native polyacrylamide gel electrophoresis (PAGE) was per-formed by using ReadyGels (7.5%; Bio-Rad). The gel was prerun with 25 mMTris and 192 mM glycine, pH 8.4, with and without 1% deoxycholate (DOC) inthe cathode and anode chambers, respectively, for 30 min at 40 mA. Samples innative sample buffer (10 �g protein, 62.5 mM Tris-Cl, pH 6.8, 15% glycerol, and1% DOC) were size fractionated by electrophoresis for 60 min at 25 mA andtransferred to nitrocellulose membranes for WB analysis (12).

WB analysis. Clarified cell lysates were boiled in SDS sample buffer for 5 min(17); protein A-Sepharose beads from immunoprecipitations were boiled in 2�SDS sample buffer. The protein samples were size fractionated by SDS-PAGEand transferred to nitrocellulose membranes (Bio-Rad) (38). The membraneswere probed with the appropriate primary antibodies. After being washed withPBS containing 0.1% Tween 20, bound antibodies were probed by secondaryincubation with HRP-coupled goat anti-rabbit IgG, goat anti-mouse IgG, orrabbit anti-goat IgG antibodies. Proteins were visualized by using an ECL PlusWestern blotting detection system (GE Healthcare) and chemiluminescencescanned by a Storm phosphorimager (GE Healthcare).

ELISA. IFN-� protein was measured with an enzyme-linked immunofluores-cence assay (ELISA) (25- to 2,000-pg/ml range; PBL InterferonSource, Piscat-away, NJ).

Immunofluorescence assay and confocal microscopy. 293A cells plated on4-chamber culture slides (Becton Dickinson, Franklin Lakes, NJ) were incubatedovernight at 37°C and then transfected with vector or a Z expression plasmid. At24 h posttransfection, the cells were transfected with 5�pppRNA, and 6 h later,they were fixed with 4% paraformaldehyde in PBS for 15 min at room temper-ature (RT) and then permeabilized with 0.1% Triton X-100 in PBS for 10 min atRT. Culture slides were mounted and images taken using a BH2-RFCA micro-

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scope with a 40� oil immersion objective (Olympus, Tokyo, Japan). In colocal-ization studies, cells were cotransfected with pUNO1-RIG-I-GFP and pCAGGS-ZJ, stained with antibodies, and imaged using a scanning confocal ECLIPSETE2000-U microscope (Nikon, Tokyo, Japan).

RESULTS

NW arenavirus Z proteins inhibit IFN-� induction by 5�pp-pRNA. During the course of building sequence libraries for adiagnostic viral microarray (13, 27), we found that Z proteinsof NW arenaviruses include a domain with a predicted second-ary structure folding pattern that resembles structures found inseveral cellular proteins implicated in innate immunity. In-deed, we found that NW arenavirus Z proteins lead to de-creased levels of IFN-� mRNA (Fig. 1). A549 cells were trans-fected with plasmids, each encoding one of the four NWarenavirus Z proteins (GTOV, JUNV, MACV, and SABV) ortwo OW arenavirus Z proteins (LCMV and LASV). All sixproteins showed comparable signals in Western blots of trans-fected cell extracts, indicating similar expression levels (notshown). Thirty-six hours after Z transfection, cells were co-transfected with 5�pppRNA for 6 h and then IFN-� levelsmeasured by qPCR. After normalization to the cellular �-actinlevel, the IFN-� expression level was calculated as fold induc-tion with respect to a baseline level measured for cells trans-fected with empty vector and not stimulated with 5�pppRNA

FIG. 1. New World arenavirus Z proteins decrease IFN-� induc-tion in A549 cells stimulated with 5�pppRNA. A549 cells (5 � 105)were transfected with 3 �g of pCAGGS-Z or empty vector by use ofcalcium phosphate. At 36 h posttransfection, cells were transfectedwith 1 �g of 5�pppRNA by use of Lipofectamine 2000. Six hours later,the cells were harvested and the IFN-� expression level was measuredby qPCR, with normalization to the �-actin expression level. Datarepresent three biological replicates. �, significant differences (P �0.05; t test).

FIG. 2. New World arenavirus Z proteins interact with RIG-I in coimmunoprecipitation experiments. (A) 293A cells (106) were cotransfectedwith 0.5 �g of pcDNA3.1-V5-RIG-I (N terminally V5 tagged [N-V5-RIG-I] or C terminally V5 tagged [C-V5-RIG-I]) and with 0.5 �g ofpCAGGS-Z (ZG, GTOV Z; ZJ, JUNV Z; ZM, MACV Z; ZS, SABV Z), pCAGGS-HA-NS1, or pCAGGS-GFP. Thirty-six hours later, cells werelysed and clarified supernatants subjected to immunoprecipitation assays using anti-V5 antibody (IP:V5). Z, NS1, and GFP were detected afterWestern blotting using anti-Z (WB:Z), anti-hemagglutinin (anti-HA) (WB:HA), and anti-GFP (WB:GFP) antibodies, respectively. “Input”represents untreated lysate prior to precipitation subjected to Western blot analysis. (B) 293A cells (106) were cotransfected with 0.5 �g ofpCAGGS-Z (ZG, GTOV Z; ZJ, JUNV Z; ZM, MACV Z; ZS, SABV Z) and 0.5 �g of pcDNA3.1/nV5-RIG-I (N terminally V5 tagged[N-V5-RIG-I]) or 0.5 �g of pcDNA3.2/cV5-RIG-I (C terminally V5 tagged [C-V5-RIG-I]) and then immunoprecipitated with antisera to therespective Z protein (IP:Z) and analyzed by Western blotting with anti-V5 antibody (WB:V5).

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(set to 1). All four NW arenavirus Z proteins reduced IFN-�mRNA induction by �60% (P � 0.05). In contrast, LCMV andLASV Z proteins had no significant effect on IFN-� mRNAinduction (Fig. 1), consistent with an earlier report on a lack ofinhibition of the type I interferon response by OW LCMV Zprotein (22). No significant effect on IFN-� mRNA levels incomparison to those for untreated cells was found in cellstransfected with vector alone or with Z expression constructs inthe absence of 5�pppRNA stimulation; comparable resultswere obtained in transfection experiments of 293A cells (notshown). A comparable �50% reduction of interferon induc-tion was also measured with all four NW arenavirus Z proteinsby ELISA (not shown). These results suggested that NWarenavirus Z proteins, but not LCMV or LASV Z protein,interfere with the transcriptional activation of IFN-�.

The Z proteins of NW arenaviruses interact with RIG-I.Since NW arenavirus Z proteins inhibited IFN-� production incells transfected with 5�pppRNA, a ligand of RIG-I, we testedwhether NW arenavirus Z proteins interact with RIG-I. 293Acells were cotransfected with a V5-tagged RIG-I expressionplasmid and one of the four arenaviral Z expression plasmids.After 36 h, interaction of RIG-I (N and C terminally V5tagged) with GTOV, JUNV, MACV, or SABV Z proteins wasassessed in coimmunoprecipitation assays using anti-V5 anti-body (Fig. 2A). FLUAV NS1 and GFP-expressing plasmidswere used as positive and negative controls, respectively, for

FIG. 3. New World arenavirus Z proteins bind RIG-I. (A) Purified recombinant Z and RIG-I were incubated in nondenaturing lysis buffer andsubjected to immunoprecipitation assays using anti-Z antibody. RIG-I was detected by Western blotting using anti-RIG-I antibody. (B) 293A cellswere transfected with pUNO1-RIG-I-GFP (I) or pCAGGS-ZJ (II) or cotransfected with both plasmids (III to V). Proteins were visualized byconfocal immunofluorescence microscopy. (I, III) RIG-I-GFP (green). (II, IV) Staining with the ZJ antibody (anti-Z [� Junin Z]) (red).(V) Overlay (yellow).

FIG. 4. JUNV Z protein blocks complex formation betweenRIG-I and MAVS. 293A cells (1.5 � 106) were triple transfectedwith 0.5 �g of pUNO1-MAVS, 0.5 �g of pcDNA3.1nV5-RIG-I, and0.5 �g of pCAGGS-ZJ or transfected with vector without Z instead ofpCAGGS-ZJ. pCAGGS-GFP was used as a negative control. Amounts oftransfected DNA were adjusted with empty pCAGGS plasmid. At 36 hposttransfection, cells were lysed and clarified supernatants subjected toimmunoprecipitation assays using anti-MAVS antibody (IP:MAVS).RIG-I, Z, and GFP were detected by Western blotting using the respec-tive antibodies (WB:MAVS, WB:V5, WB:Z, and WB:GFP).

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RIG-I interaction. In a complementary experiment, we per-formed coimmunoprecipitation assays using four antisera spe-cific for each of the four NW arenavirus Z proteins. RIG-I wascoimmunoprecipitated with GTOV, JUNV, MACV, andSABV Z proteins (Fig. 2B). No interaction was observed be-tween OW arenavirus LCMV or LASV Z protein and RIG-I(not shown). MDA5, although structurally similar to RIG-I,did not interact with the Z proteins of GTOV, JUNV, MACV,SABV, or LCMV (not shown).

NW arenavirus Z proteins bind to and colocalize withRIG-I. In order to determine whether the interactions betweenNW arenavirus Z proteins and RIG-I are direct, we performedcoimmunoprecipitation assays using purified recombinant pro-teins. Incubation of purified recombinant His6-tagged RIG-Iwith purified recombinant His6-tagged Z proteins of GTOV,JUNV, and MACV and their cognate anti-Z antisera resulted

in coprecipitation of RIG-I (Fig. 3A). Experiments with Zprotein of SABV yielded no visible band. Whether this reflectsa difference in the avidity of the anti-Z antiserum or a bonafide failure of direct interaction between SABV Z protein andRIG-I is unknown. We also examined the cellular localizationof NW arenavirus Z proteins and RIG-I in 293A cells byconfocal immunofluorescence microscopy. Consistent withearlier work, staining of JUNV Z/RIG-I-GFP double-trans-fected cells with anti-Z antibody indicated that Z protein ismembrane bound (4, 7, 29, 36, 37) but also present in thecytoplasm (1, 2, 33), with a distribution similar to that observedfor RIG-I (Fig. 3B).

NW arenavirus Z proteins interfere with the interactionbetween RIG-I and MAVS. The finding that NW arenavirus Zproteins bind RIG-I and decrease IFN-� mRNA induction inresponse to the RIG-I ligand 5�pppRNA suggests that Z pro-

FIG. 5. JUNV Z protein interferes with NF-�B and IRF-3 activation. (A to C) 293A cells (106) were transfected with 1 �g of pCAGGS-ZJ(Junin Z) or vector. At 24 h posttransfection, cells were mock infected or infected with SeV and protein exacts subjected to SDS-PAGE (A, B)or native PAGE (C) for subsequent immunoblot analysis with anti-NF-�B p100/p52 (A), anti-phospho-IRF-3 (Ser396) (B), or anti-IRF-3(C) antibody. (D) 293A cells (2.5 � 105) were transfected with 0.25 �g of vector (I, II, V, VI) or pCAGGS-ZJ (III, IV, VII, VIII) by use ofLipofectamine 2000. At 24 h posttransfection, cells were transfected with 0.5 �g of 5�pppRNA by use of Lipofectamine 2000, and 6 h later, cellswere fixed for immunofluorescence analysis. Cells were stained with anti-IRF-3 antibody (I to IV) and DAPI (4�,6-diamidino-2-phenylindole)nuclear counterstain (V to VIII).

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teins may deregulate RIG-I signaling to downstream adapters.MAVS acts downstream of RIG-I in signal transmissionthrough complex formation with activated RIG-I. To investi-gate whether Z proteins abrogate the interaction betweenRIG-I and MAVS, we conducted coimmunoprecipitation anal-yses. 293A cells were cotransfected with MAVS and N-termi-nally V5-tagged RIG-I expression vector as well as with aJUNV Z expression vector or vector without Z (control).Whereas MAVS-binding to RIG-I was readily demonstrated inthe absence of Z protein, no interaction was seen in the pres-ence of JUNV Z protein (Fig. 4). These results are consistentwith JUNV Z protein preventing the RIG-I/MAVS complexformation.

New World arenavirus Z proteins impede NF-�B and IRF-3activity. RIG-I signaling activates transcription factors NF-�Band IRF-3 via MAVS binding. We examined the activity ofendogenous NF-�B and IRF-3 in 293A cells expressing JUNVZ. Twenty-four hours after transfection, cells were mock in-fected or infected with SeV for 16 h. Cell lysates were sub-jected to SDS-PAGE or native PAGE. The presence of JUNVZ protein blocked the activation of NF-�B. The active form ofNF-�B2, p52, was not detected in lysates from SeV-infectedcells expressing JUNV Z protein (Fig. 5A). Phosphorylation ofIRF-3 at Ser396 was observed only in SeV-infected cells whenJUNV Z protein was absent; uninfected cells and infected cellsthat express JUNV Z protein showed no IRF-3 phosphoryla-tion (Fig. 5B). IRF-3 dimer formation is a consequence ofIRF-3 phosphorylation and enables nuclear translocation. Asanticipated, IRF-3 dimerization was reduced in SeV-infectedcells expressing JUNV Z protein (Fig. 5C). Similarly, nucleartranslocation of IRF-3 was blocked by the expression of JUNVZ protein (Fig. 5D).

DISCUSSION

Given the central role of RIG-I in virus sensing and IFNinduction, both RNA and DNA viruses have devised geneproducts that target the RIG-I signaling pathway to inhibit IFNproduction by infected cells. Inhibition of RIG-I signaling isachieved by targeting different steps of the pathway. In manyinstances, viruses express redundant IFN-antagonizing activi-ties. For example, the severe acute respiratory syndrome coro-navirus (SARS-CoV) encodes three proteins with IFN antag-onism activity, the N, ORF 3b, and ORF 6 products (16). Inarenaviruses, the NP antagonizes IFN by inhibiting IRF-3 (21).Our data reveal an additional strategy whereby NW arenavi-ruses evade the host innate immune response. In a mechanismthat may be specific to NW arenaviruses, Z proteins inhibithost cell IFN mRNA induction via direct binding of Z toRIG-I. This interaction disrupts the necessary complex forma-tion between RIG-I and MAVS, impeding downstream IRF-3and NF-�B activation and resulting in decreased IFN-� induc-tion.

The precise mechanism by which NW arenavirus Z proteinsinhibit RIG-I activity remains to be determined. However,several recent studies provided more-detailed insights into thepotential mechanism: (i) studies by Cui et al. (5) show that theC-terminal RD of RIG-I binds viral RNA in a 5�-triphosphate-dependent manner and activates the RIG-I ATPase throughRNA-dependent dimerization, (ii) RIG-I requires ubiquitina-

tion by TRIM25 at its N-terminal CARD to interact withMAVS (8), and (iii) studies by Oshiumi et al. (26) show thatRiplet/RNF135 binds and ubiquitinates the C-terminal regionof RIG-I to promote RIG-I-mediated IFN-� promoter activa-tion. Interaction of NW arenavirus Z proteins with RIG-I maydisrupt RIG-I interaction with any of the aforementioned pro-teins or even the viral RNA itself. Future work will focus onfine mapping the interaction between RIG-I and NW arena-virus Z proteins in an effort to understand the immunosup-pression observed in NW arenavirus infections.

ACKNOWLEDGMENTS

We thank Brent Williams, Kavitha Yaddanapudi, Joari De Miranda,and Gustavo Palacios for helpful discussions and critical comments.

This work was supported by the Department of Defense and byNational Institutes of Health award AI57158 (Northeast BiodefenseCenter—Lipkin).

REFERENCES

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