Viral targeting of DEAD box protein 3 reveals its role in TBK1/IKKepsilon-mediated IRF activation - PubMed (original) (raw)

Viral targeting of DEAD box protein 3 reveals its role in TBK1/IKKepsilon-mediated IRF activation

Martina Schröder et al. EMBO J. 2008.

Abstract

Viruses are detected by different classes of pattern recognition receptors (PRRs), such as Toll-like receptors and RIG-like helicases. Engagement of PRRs leads to activation of interferon (IFN)-regulatory factor 3 (IRF3) and IRF7 through IKKepsilon and TBK1 and consequently IFN-beta induction. Vaccinia virus (VACV) encodes proteins that manipulate host signalling, sometimes by targeting uncharacterised proteins. Here, we describe a novel VACV protein, K7, which can inhibit PRR-induced IFN-beta induction by preventing TBK1/IKKepsilon-mediated IRF activation. We identified DEAD box protein 3 (DDX3) as a host target of K7. Expression of DDX3 enhanced Ifnb promoter induction by TBK1/IKKepsilon, whereas knockdown of DDX3 inhibited this, and virus- or dsRNA-induced IRF3 activation. Further, dominant-negative DDX3 inhibited virus-, dsRNA- and cytosolic DNA-stimulated Ccl5 promoter induction, which is also TBK1/IKKepsilon dependent. Both K7 binding and enhancement of Ifnb induction mapped to the N-terminus of DDX3. Furthermore, virus infection induced an association between DDX3 and IKKepsilon. Therefore, this study shows for the first time the involvement of a DEAD box helicase in TBK1/IKKepsilon-mediated IRF activation and Ifnb promoter induction.

PubMed Disclaimer

Figures

Figure 1

Figure 1

Identification and expression of K7. (A) Alignment of A52 (VACV_WR178) and K7 (VACV_WR039) proteins from VACV (WR strain). (B) Alignment of K7 orthologues. VACV-WR: VACV Western Reserve; VACV-AMVA: VACV-Acambis 3000 MVA; VACV-COP: VACV Copenhagen; VACV-MVA: VACV modified virus Ankara; VACV-TAN: VACV Tian Tan; RPXV-UTR: rabbitpox virus Utrecht; VARV-BSH: variola virus Bangladesh; VARV-GAR: variola virus Garcia; VARV-India, variola virus India; CMLV-CMS: camelpox virus CMS; CMLV-M96: camelpox virus M96; CPXV-BR: cowpox virus Brighton red; MPXV-ZRE: monkeypox virus Zaire. (C) VACV WR K7R was cloned into pCMV-HA. Increasing amounts of pCMV-HA-K7R DNA were transfected into HEK293T cells, and 48 h later K7 expression was analysed by immunoblotting using an anti-HA antibody. (D) HEK293 cells infected with VACV WR at 10 p.f.u. per cell were harvested at the indicated times post-infection and lysates were analysed by immunoblotting using K7-specific antiserum.

Figure 2

Figure 2

K7 inhibits TLR-induced NF-κB activation. In (A–C), HEK293 cells were transfected with pRK5-K7R or empty vector and the NF-κB luciferase reporter gene. Cells were stimulated with 20 ng/ml IL-1β for 6 h (A), transfected with 50 ng of CD4TLR4 for 24 h (B) or HEK293-TLR3 cells were stimulated with 25 μg/ml poly(I:C) for 8 h (C). Data are expressed as the mean fold induction±s.d. relative to control levels, for a representative experiment of three, each performed in triplicate. (D, E) For agonist-induced cytokines, HEK-TLR4 (D) or HEK-TLR3 (E) cells were transfected with K7R 24 h prior to stimulation with 1 μg/ml LPS or 25 μg/ml poly(I:C), respectively. After 24 h, supernatants were assayed for IL-8 (D) or RANTES (E) by ELISA. The experiments were performed four times in triplicate and data are expressed as the mean±s.d. from one representative experiment. (F) HEK293T cells were transfected with pCMV-HA-K7R and IRAK2-Myc and 48 h later, lysates were subjected to immunoprecipitation (IP) analysis with the indicated antibodies. (G) HEK293T cells were transfected with pCMV-HA-K7R and Flag–TRAF1, 2, 3, 4, 5 or 6 and 48 h later, lysates were subjected to IP analysis with the indicated antibodies. Results shown are representative of at least three experiments.

Figure 3

Figure 3

K7 inhibits IRF activation at the level of TBK1/IKKɛ. In (A–G), HEK293 cells were transfected with pRK5-K7R or pRK5-A52R, together with the indicated luciferase reporter genes for 24 h. Data are expressed as the mean fold induction±s.d. relative to control levels, for a representative experiment of at least two, each performed in triplicate. (A, B) Cells were transfected with IRF7-GAL4 (A) or IRF3-GAL4 (B) expression plasmids, and the GAL4-dependent pFR luciferase reporter gene plus 50 ng expression plasmid encoding TRIF. (C) Cells were transfected with the Ifnb promoter reporter gene prior to sendai virus infection for 16 h. (D) Cells were transfected with the ISRE reporter gene plus 50 ng MAVS expression plasmid. (E, F) Cells were transfected with IRF7-GAL4 and the pFR luciferase reporter gene, plus 50 ng of either TBK1 (E) or IKKɛ (F) expression plasmid. (G) Cells were transfected with the ISRE reporter gene plus 50 ng of either TBK1 or IRF7 expression construct for 24 h. (H) Schematic of SeV signalling pathway to Ifnb promoter induction. The deduced point of inhibition by K7 is marked with an asterisk. (I) For the IRF transactivation assay, cells were transfected as in (B), followed by stimulation with SeV for 16 h (upper panel). For immunoblot analysis, cells were transfected with K7, followed by SeV stimulation for 6 h. Western blots were probed for total IRF3 and phosphorylated IRF3 (Ser396) (lower panel).

Figure 4

Figure 4

K7 but not A52 interacts with DDX3. (A) HEK293 cell lysates were added to purified His–K7 coupled to Ni-Agarose and incubated for 2 h at 4°C. The immune complexes were precipitated, subjected to SDS–PAGE and stained with Coomassie blue. A band of approximately 70 kDa (marked with an arrow) that appeared specifically in the K7 pull-down lane was excised, prepared for MALDI-TOF analysis and shown to be DDX3. (B) HEK293T cells were transfected with HA–K7R, A52R or empty vector (EV) and 48 h later, lysates were subjected to immunoprecipitation analysis to detect endogenous DDX3. (C) HEK293 cells were transfected with Myc–DDX3 expression plasmid. After 24 h cells were mock infected or infected with WR at 10 p.f.u. per cell, and 8 h later lysates were subjected to immunoprecipitation analysis with K7- or A52-specific antiserum LC: antibody light chain. (D) HEK293 cells were mock infected or infected with WR at 10 p.f.u. per cell and 8 h later, cell lysates were subjected to immunoprecipitation with K7-specific antiserum, followed by immunoblotting with the indicated antibodies. (E) HEK293 cells were either left untreated or incubated with 25 mM leptomycin B (LMB) for 4 h. Cytoplasmic (Cy) and nuclear (N) extracts were analysed for DDX3 by immunoblotting. (F) HEK293 cells were grown on 22-mm coverslips in six-well plates and transfected with the HA–DDX3 expression plasmid for 48 h and then 25 mM LMB was added. Cells were fixed 4 h later, permeabilised and stained with anti-HA-AlexaFluor594 and the DAPI nuclear stain. The slides were examined by phase-contrast and confocal microscopy. A section of approximately 1 μm through the centre of a cell is shown. (G) HEK293 cells were grown as in (F) and transfected with HA–DDX3 and K7–EYFP expression plasmids for 48 h. LMB (25 mM) was then added and the cells were fixed 4 h later, permeabilised and stained with anti-HA-AlexaFluor594 and DAPI. The slides were examined by phase contrast and confocal microscopy. A section of approximately 1 μm through the centre of cells is shown. Results shown for (A–G) are representative of at least two experiments.

Figure 5

Figure 5

Role for the N terminus of DDX3 in TBK1/IKKɛ-induced Ifnb promoter activation. In (A–C), HEK293 cells were transfected with the indicated amount of pCMV-Myc-DDX3 (nanograms) together with the Ifnb promoter reporter gene, cells were harvested after 24 h and luciferase reporter gene activity was measured. (A) Cells were infected with sendai virus for 16 h. (B) Cells were transfected with 50 ng TBK1 expression plasmid. (C) Cells were transfected with 50 ng IKKɛ expression plasmid. (D) Cells were transfected with the Ifnb promoter reporter gene, together with 50 ng IKKɛ and the indicated amount (nanograms) of an expression plasmid encoding a mutant version of DDX3 lacking ATPase activity (K230E). (E) Cells were transfected with the Ifnb promoter reporter gene, together with 50 ng IKKɛ and 50 or 100 ng of expression vectors encoding full-length DDX3 (1–662) or the indicated N- and/or C-terminally truncated DDX3 proteins. Expression of DDX3 truncations by immunoblot is also shown. For (A–E), data are expressed as the mean fold induction±s.d. relative to control levels, for a representative experiment of at least two, each performed in triplicate. (F) Either full-length recombinant His-tagged DDX3 (1–662) or different His-tagged truncated DDX3 proteins were used in pull-down assays with cell lysates from HEK293T cells transfected with the HA–K7 expression plasmid. The precipitates were then analysed by immunoblotting with anti-HA antibody. Results are representative of three experiments. (G) Schematic of DDX3 showing in grey the positions of the nuclear export sequence (NES) and the conserved ATPase/helicase domain. The DEAD motif and the GKT motif that was mutated in the K230E point mutant are also indicated. A schematic summarising the different truncation mutants of DDX3 and their abilities to stimulate Ifnb promoter activation and to bind to K7, both of which depend on the presence of the N-terminus of DDX3 (aa 1–139), is shown below. (H) The N-terminus of DDX3 (aa 1–139) was expressed as a His-tagged recombinant protein and used in pull-down assays with cell lysates from HEK293T cells transfected with HA–K7. The precipitates were analysed by immunoblotting with anti-HA antibody. Result is representative of two experiments.

Figure 6

Figure 6

DDX3 is required for TBK1/IKKɛ-dependent promoters and is recruited to IKKɛ. (A) HEK293 cells were transfected with the indicated amount (nanograms) of pCMV-DDX3 and/or pCMV-DDX3(1–139), 50 ng IKKɛ expression plasmid and the Ifnb promoter reporter gene. (B) Cells were transfected with a Ccl5 (RANTES) promoter reporter gene and the indicated amount (nanograms) of either pCMV-DDX3 or pCMV-DDX3(1–139) 24 h prior to stimulation with sendai virus or transfection with either poly(I:C) (5 μg per well) or poly(dA:dT) (1 μg per well). Cells were harvested 16 h later. (C) Cells were transfected with 0 or 100 ng pCMV-DDX3(1–139) together with the IRF3 reporter genes, and either infected with sendai virus (left panel) or transfected with IKKɛ (right panel). (D) DDX3 expression was suppressed using two different RNAi oligonucleotides, DDX3-1 and DDX3-2. HEK293 cells were transfected on two consecutive days with the DDX3 RNAi oligonucleotides or a control oligonucleotide matched for GC content. At 48 h after the second transfection, the cell lysates were analysed by immunoblotting with the indicated antibodies to confirm the reduction of endogenous DDX3 protein levels (left panel). HEK293 cells were co-transfected with the Ifnb promoter reporter gene and 50 ng expression plasmid for either TBK1 (middle panel) or IKKɛ (right panel) during the second transfection with RNAi oligonucleotides. (E) Similar to (D), except cells were transfected with IRF3 reporter genes and stimulated with sendai virus (left panel) or poly(I:C) (right panel). In (A–E), reporter gene activity was measured and data are expressed as the mean fold induction±s.d. relative to control levels, for a representative experiment of at least three, each performed in triplicate. (F) HEK293T cells were transfected with Myc–DDX3 and Flag–IKKɛ for 48 h and the lysates were analysed by immunoprecipitation (IP) with the indicated antibodies. (G) Recombinant His–DDX3(1–139) was used in ‘pull-down' assays with lysates from HEK293T cells transfected with Flag–IKKɛ. The precipitates were analysed by immunoblotting with Flag Ab. (H) HEK293T cells were infected with sendai virus and samples were harvested at the indicated times after virus infection. The cell lysates were prepared, immunoprecipitated with anti-IKKɛ antibody and analysed by immunoblotting with the indicated antibodies. All results are representative of two or more experiments.

Similar articles

Cited by

References

    1. Ariumi Y, Kuroki M, Abe K, Dansako H, Ikeda M, Wakita T, Kato N (2007) DDX3 DEAD-box RNA helicase is required for hepatitis C virus RNA replication. J Virol 81: 13922–13926 - PMC - PubMed
    1. Barnes BJ, Richards J, Mancl M, Hanash S, Beretta L, Pitha PM (2004) Global and distinct targets of IRF-5 and IRF-7 during innate response to viral infection. J Biol Chem 279: 45194–45207 - PubMed
    1. Botlagunta M, Vesuna F, Mironchik Y, Raman A, Lisok A, Winnard P Jr, Mukadam S, Van Diest P, Chen JH, Farabaugh P, Patel AH, Raman V (2008) Oncogenic role of DDX3 in breast cancer biogenesis. Oncogene 27: 3912. - PMC - PubMed
    1. Bowie A, Kiss-Toth E, Symons JA, Smith GL, Dower SK, O'Neill LA (2000) A46R and A52R from vaccinia virus are antagonists of host IL-1 and Toll-like receptor signaling. Proc Natl Acad Sci USA 97: 10162–10167 - PMC - PubMed
    1. Bowie AG (2007) Translational mini-review series on Toll-like receptors: recent advances in understanding the role of Toll-like receptors in anti-viral immunity. Clin Exp Immunol 147: 217–226 - PMC - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources