LCMV infection blocks phosphorylation of IRF3. (A) Induction of IFN-β mRNA in cells infected with LCMV. HEK293 cells were infected with LCMV at an MOI of 10. At the indicated time points p.i., cells were lysed and total RNA was extracted and cDNA synthesized by reverse transcription. Real-time PCR was performed using a StepOne real-time PCR system and TaqMan probes specific for IFN-β and GAPDH. Gene expression levels relative to GAPDH were determined according to the 2^−ΔΔCT method (24). Data presented represent fold induction above the level seen with uninfected cells (n = 3 ± standard deviation [SD]). (B) Expression of LCMV NP. HEK293 cells were infected as described for panel A or left uninfected (u). At the indicated hour postinfection, cells were lysed. Proteins were separated in SDS-PAGE and probed in Western blot analyses performed using mouse MAb (1.1.3) to LCMV nucleoprotein (NP), combined with HRP-conjugated secondary antibodies and enhanced chemiluminescence (ECL). (C) IRF3 phosphorylation in response to LCMV infection and SeV infection. HEK293 cells were first infected with LCMV at an MOI of 0.1 or transfected with recombinant LCMV NP (4 μg). Twenty-four hours later, cells were infected with SeV at an MOI of 0.1, and 24 h later, cells lysates were prepared and subjected to immunoprecipitation using anti-IRF3 magnetic beads (IP:IRF3). Immunocomplexes were probed in Western blotting using an antibody specific to phospho-IRF3 (pIRF3) and an antibody that recognized IRF3 independently of phosphorylation (IRF3). Expression of LCMV NP (NP) in cell lysates (CL) was performed as described for panel A. The position of pIRF3 is indicated, and the asterisk designates IgG heavy chain.

LCMV NP interacts specifically with IKKε. (A) Coimmunoprecipitation (co-IP) between LCMV NP and different components of the RIG-I/MAVS signaling pathway. HEK293 cells were transiently cotransfected with an HA-tagged form of recombinant LCMV NP (4 μg) and FLAG-tagged RIG-I, MAVS, TRAF3, TBK-1, or IKKε or GFP-tagged IRF3 (4 μg each). Empty vector was used 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 anti-HA antibody to confirm the IP of LCMV NP (IB:HA). Co-IP of tagged 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 transiently transfected with recombinant HA-tagged LCMV NP (4 μg) and 24 h later infected with SeV at an MOI of 0.1. At 24 h p.i., co-IP was performed using anti-HA magnetic beads (IP:HA) as described for panel A. Immunocomplexes were probed with antibody to HA tag and a specific antibody to the endogenous form of IKKε. The presence of recombinant LCMV NP and endogenous IKKε was confirmed in the whole-cell lysate (CL). The band indicated by the asterisk corresponds to the IgG heavy chain.

The specific interaction of NP with IKKε is conserved among arenaviruses. (A) Co-IP of IKKε, but not TBK-1, with NPs derived from different arenaviruses. HEK293 cells were transiently cotransfected with HA-tagged NP of LCMV, LASV, WWAV, LATV, and JUNV together with FLAG-tagged IKKε or TBK-1 (4 μg of each plasmid). 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 to confirm IP of arenavirus NPs and antibody to FLAG to detect co-IP of IKKε or TBK-1. Expression of recombinant arenavirus NPs, IKKε, and TBK-1 was verified in whole-cell lysate (CL). IB = Western blotting; * = IgG heavy chain. (B) IKKε undergoes specific interaction with LCMV NP 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 panel A 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 heavy chain.

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 IgG2b anti-MAVS). Primary antibodies were detected with species- and isotype-specific secondary antibodies. Specimens were examined by confocal laser scanning microscopy, and image acquisition was performed with a Zeiss LSM710 Quasar confocal microscope. Images were first deconvoluted using Huygens Essential software 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 a representative 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 shown in the image (g) and scatter plot (h). Bar = 5 μm.

In HEK293 cells, TBK-1 and IKKε contribute to SeV-induced IRF3 phosphorylation and IFN-β production. (A) HEK293 cells were transfected with siRNAs 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 specific probes for IKKε and TBK-1 as described in Materials and Methods (n = 3 ± SD). (B) In HEK293 cells, induction of IFN-β mRNA in response to SeV requires both 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, mRNA levels of IFN-β were determined as described for Fig. 1A. Data represent fold induction above the level seen with uninfected cells (n = 3 ± SD). (C) IKKε is required for optimal IRF3 phosphorylation in response to SeV infection. HEK293 cells were depleted of IKKε as described for panel A. After 48 h, cells were infected 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 level by 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.

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 with IKKε. 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-IFN activity. HEK293 cells were cotransfected with 0.5 μg 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 vector to normalize transfection efficiencies. Twenty-four hours posttransfection, cells were infected with SeV (MOI = 3) and, 16 to 18 h postinfection, protein expression and reporter gene expression were analyzed. (A) Protein expression levels. Lysates (200 μg of total protein) from transfected cells were analyzed for NP 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 of LCMV-NP single amino acid substitutions on nuclear translocation of IRF3. (C) HEK293T cells cotransfected with 1 μg of pEGFP-C1-hIRF3 and 2 μg of the indicated HA-tagged LCMV wild-type (WT) NP or single amino acid substitutions in the 3′-5′ exonuclease domain were infected with SeV (MOI = 3), and subcellular 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 as described in Materials and Methods. (D) Representative images of IRF3 (green), LCMV-Z and LCMV-NP wild type (WT) and mutants (red), cellular nuclear staining (blue), and the corresponding merged images are displayed. Arrows indicate nuclear translocation of IRF3 in a cell with no detectable levels of LCMV NP. (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 NP or HA-tagged LCMV NP mutants (4 μg 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 antibodies to 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.

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) of IKKε. (B) Co-IP of IKKε KD with LCMV NP. HEK293 cells were cotransfected with 4 μg of HA-tagged LCMV NP and 4 μg 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-IP of 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.

Binding of LCMV NP affects the kinase activity of IKKε. (A) HEK293 cells were cotransfected with 4 μg of recombinant HA-tagged LCMV NP and 4 μg of FLAG-tagged IKKε or transfected with 4 μg of FLAG-tagged IKKε alone. At 48 h posttransfection, cells were subjected to IP using anti-HA magnetic beads (IP:HA) and cells transfected only with IKKε were subjected to IP using anti-FLAG magnetic beads (IP:FLAG). The presence of IKKε was assessed in co-IP with LCMV NP (NP-IKKε) and IP of IKKε-FLAG (IKKε) by Western blotting. (B) Detection of IKKε kinase activity. Normalized amounts of immunocomplexes containing IKKε alone (IKKε) or IKKε bound to LCMV NP (NP-IKKε) from the experiment described in the panel A legend were mixed with recombinant IRF3 purified from separate transfected cells, and an in vitro kinase assay was performed using [γ-³²P]ATP as described in Materials and Methods. IPs of lysates from mock-transfected cells (mock) were used as a negative control. The samples were separated by SDS-PAGE and developed by autoradiography. The presence of IRF3 in kinase assay reactions was validated by Western blotting (IB). * = IgG heavy chain.