Vaccinia virus protein C6 is a virulence factor that binds TBK-1 adaptor proteins and inhibits activation of IRF3 and IRF7 - PubMed (original) (raw)
Vaccinia virus protein C6 is a virulence factor that binds TBK-1 adaptor proteins and inhibits activation of IRF3 and IRF7
Leonie Unterholzner et al. PLoS Pathog. 2011 Sep.
Abstract
Recognition of viruses by pattern recognition receptors (PRRs) causes interferon-β (IFN-β) induction, a key event in the anti-viral innate immune response, and also a target of viral immune evasion. Here the vaccinia virus (VACV) protein C6 is identified as an inhibitor of PRR-induced IFN-β expression by a functional screen of select VACV open reading frames expressed individually in mammalian cells. C6 is a member of a family of Bcl-2-like poxvirus proteins, many of which have been shown to inhibit innate immune signalling pathways. PRRs activate both NF-κB and IFN regulatory factors (IRFs) to activate the IFN-β promoter induction. Data presented here show that C6 inhibits IRF3 activation and translocation into the nucleus, but does not inhibit NF-κB activation. C6 inhibits IRF3 and IRF7 activation downstream of the kinases TANK binding kinase 1 (TBK1) and IκB kinase-ε (IKKε), which phosphorylate and activate these IRFs. However, C6 does not inhibit TBK1- and IKKε-independent IRF7 activation or the induction of promoters by constitutively active forms of IRF3 or IRF7, indicating that C6 acts at the level of the TBK1/IKKε complex. Consistent with this notion, C6 immunoprecipitated with the TBK1 complex scaffold proteins TANK, SINTBAD and NAP1. C6 is expressed early during infection and is present in both nucleus and cytoplasm. Mutant viruses in which the C6L gene is deleted, or mutated so that the C6 protein is not expressed, replicated normally in cell culture but were attenuated in two in vivo models of infection compared to wild type and revertant controls. Thus C6 contributes to VACV virulence and might do so via the inhibition of PRR-induced activation of IRF3 and IRF7.
Conflict of interest statement
The authors have declared that no competing interests exist.
Figures
Figure 1. C6 inhibits IFN-β and CCL5 expression.
(A–C) HEK293 cells were transfected with an IFN-β-promoter firefly luciferase reporter plasmid, a renilla luciferase transfection control, and a C6 expression plasmid (C6) or empty vector control (EV). Eight hours after transfection, cells were stimulated by transfection with 5 µg/ml poly(I∶C) (A), or 500 ng/ml poly(dA-dT) (B), with mock-transfected cells (mock) serving as control. In (C) cells were mock infected (-) or infected with Sendai virus. Firefly luciferase activity was measured 16 h after stimulation and normalized to renilla luciferase activity. (D) NIH3T3 cells were transfected with an IFN-β-promoter firefly luciferase reporter plasmid, a renilla luciferase transfection control and a C6 expression plasmid (C6) or empty vector control (EV). Cells were stimulated by transfection with 800 ng poly(I∶C) per well, and firefly and renilla luciferase activities were measured after 24 h. (E, F) HEK293 cells were transfected with empty vector (EV) or C6 expression plasmid (C6), and stimulated by infection with Sendai virus 24 h after transfection. After a further 24 h, IFN-β mRNA was measured by real-time PCR (E), or CCL5 protein secretion was measured by ELISA (F). Data are from one representative experiment of at least three, each performed in triplicate. Data are represented as mean ± SD. *p<0.05, ** p<0.01 or ***p<0.001 compared to EV.
Figure 2. C6 does not inhibit the translocation or activation of NF-κB.
(A) HEK293 cells were grown on coverslips and transfected with plasmids for the expression of GFP-tagged C6 or GFP as control. Twenty-four hours later, cells were mock treated (mock), infected with Sendai virus (SeV) for 6 h, or treated with 20 ng/ml IL-1β for 15 min. Cells were fixed and stained for endogenous NF-κB p65 (red). GFP or GFP-C6 are shown in green, and nuclear DNA is stained with DAPI (blue). (B) Cells expressing GFP or GFP-C6 from (A) were observed by confocal microscopy and scored for nuclear translocation of p65. At least 100 cells were counted for each sample. Data shown are from one representative experiment of at least three. (C) HEK293 cells were transfected with a firefly luciferase reporter plasmid under the control of an NF-κB-dependent promoter, a renilla luciferase transfection control, and a C6 or B14 expression plasmid or empty vector control (EV). Twenty-four hours after transfection cells were treated with 40 ng/ml TNF-α or 20 ng/ml IL-1β for 8 h. Firefly luciferase activity was normalized to renilla luciferase activity. (D) HEK293 cells were transfected with TLR3, an NF-κB-dependent firefly luciferase reporter plasmid, a renilla luciferase transfection control, and a C6 or B14 expression plasmid or empty vector control (EV). Eight hours after transfection cells were treated with 25 µg/ml poly(I∶C) for 16 h. Firefly luciferase activity was normalized to renilla luciferase activity. Data are represented as mean ± SD from one representative of at least three experiments each performed in triplicate. ** p<0.01 or ***p<0.001 compared to EV.
Figure 3. C6 inhibits the nuclear translocation and activation of IRF3.
(A) HEK293 cells were grown on glass coverslips and transfected with plasmids for the expression of V5-tagged C6 or V5-tagged GFP as control. Twenty-four hours after transfection, cells were mock-infected (mock) or infected with Sendai virus (SeV) for 6 h. Cells were fixed and stained for endogenous IRF3 (red) and V5 (green). Nuclear DNA is stained with DAPI (blue). (B) Cells expressing V5–C6 or V5-GFP from (A) were observed by confocal microscopy and scored for nuclear translocation of IRF3. At least 100 cells were counted for each sample. Data shown are from one representative experiment of at least three. (C–E) HEK293 cells were seeded in 96-well plates, transfected with a pFR-firefly luciferase reporter plasmid under the control of the Gal4 promoter, a renilla luciferase transfection control, and an IRF3-Gal4 fusion plasmid. C6 expression vector (50, 100 or 150 ng, wedges) and empty vector (EV) were co-transfected per well. Expression plasmids for signalling factors (50 ng) were included in the transfection where indicated (D and E). In (A), cells were mock-transfected or transfected with 500 ng/ml poly(dA-dT) 8 h after the initial transfection. Cells were harvested 24 h after the first transfection, and firefly luciferase activity was measured and normalized to renilla luciferase activity. (F) HEK293 cells were transfected with a firefly luciferase reporter plasmid under the control of an ISRE element, a renilla luciferase transfection control, and a C6 expression construct or empty vector as above. The ISRE was driven by co-transfection of 2 ng of IRF3-5D. Cells were harvested 24 h after transfection, and firefly luciferase activity was measured and normalized to renilla luciferase activity. Data are represented as mean ± SD from one representative of at least three experiments each performed in triplicate. *p<0.05, ** p<0.01 or ***p<0.001 compared to EV.
Figure 4. C6 prevents IRF7 transactivation stimulated by TBK1-and IKKε-dependent pathways.
(A,E) Schematic outline of a TBK1/IKKε-dependent (A) and –independent (E) pathway resulting in the activation of IRF7. (B–D, F, G) HEK293 cells were transfected with pFR-firefly luciferase under the control of the Gal4 promoter, a renilla luciferase transfection control, and an IRF7-Gal4 fusion plasmid. C6 expression vector and empty vector (EV) were co-transfected as indicated. In (B), cells were infected with Sendai virus (SeV) for 16 h. (C, D, G) Expression plasmids (50 ng) for signalling factors were included in the initial transfection as indicated. (F) HEK293 cells expressing TLR8 were stimulated with 2.5 µg/ml CL075 or 1 µM R848 for 16 h. (H) HEK293 cells were transfected with a firefly luciferase reporter plasmid under the control of an ISRE element, a renilla luciferase transfection control, and a C6 expression plasmid or empty vector. The ISRE was driven by co-transfection of 2 ng of IRF7-4D. In all cases, cells were harvested 24 h after the first transfection, Firefly luciferase activity was measured and normalized to renilla luciferase activity. Data are represented as mean ± SD from one representative of three experiments, each performed in triplicate. ** p<0.01 or ***p<0.001 compared to EV.
Figure 5. C6 interacts with NAP1, TANK and SINTBAD.
(A) HEK293 cells were grown in 10-cm dishes and transfected with 10 µg plasmids encoding FLAG-tagged NAP1, SINTBAD, TANK or GFP as indicated. After 48 h, cells were infected with recombinant VACV expressing HA-tagged C6 (2 p.f.u. per cell) for 16 h. Cell lysates (input) were subjected to immunoprecipitation (IP) with anti-FLAG M2 agarose beads. Proteins were separated by SDS-PAGE and detected by immunoblotting as indicated on the right. (B) HEK293 cells were grown in 10-cm dishes and transfected with 5 µg plasmids encoding FLAG-tagged NAP1, SINTBAD, TANK or GFP and 5 µg TAP-tagged (consisting of FLAG and Strep epitopes) C6 as indicated. After 48 h cell lysates (input) were subjected to immunoprecipitation (IP) with Streptavidin agarose beads. Proteins were separated by SDS-PAGE and detected by immunoblotting as indicated on the right. (C–H) HEK293 cells were grown in 6-well plates and transfected with 0.5 µg luciferase-tagged TBK1 expression plasmid, 0.5 µg FLAG-tag expression plasmid and 3 µg C6 or TBD expression vector or empty vector (EV) as indicated. Cells were harvested after 24 h and subjected to immunoprecipitation with anti-FLAG antibody. Immunoprecipitated protein complexes were eluted with FLAG peptide and co-immunoprecipitated luciferase activity was measured. Data are representative of at least three experiments.
Figure 6. C6 is expressed early during infection and is present in both the cytoplasm and nucleus.
(A) BSC-1 cells were infected with VACV (5 p.f.u. per cell) in the presence or absence of cytosine arabinoside (AraC) and harvested at the indicated times post infection. Protein extracts were separated by SDS-PAGE and analysed by immunoblotting with the antibodies indicated. Molecular mass markers are indicated on the right (kDa). (B) Purified recombinant VACVs were used to infect BSC-1 cells overnight with 5 p.f.u. per cell. Protein extracts were separated by SDS-PAGE and analysed by immunoblotting with the antibodies indicated. (C) HeLa cells were infected for 16 h with 5 p.f.u. per cell. Cells were harvested and fractionated into cytoplasmic and nuclear fractions. Proteins from cell extracts were separated by SDS-PAGE and analysed by immunoblotting with the indicated antibodies. Three-fold more of the total nuclear fraction was loaded compared to the total cytoplasmic fraction. (D) HeLa cells were infected with the indicated viruses for 5 h with 5 p.f.u. per cell. Cells were then fixed and stained with a mouse anti-HA antibody. The localisation of C6 (green), nuclear and viral DNA stained with DAPI (blue), phase-contrast and merged images are shown.
Figure 7. VACV lacking C6 is attenuated in vivo.
(A, B) Virulence in a murine intranasal model of VACV infection. Female BALB/c mice (n = 10) were infected with 5×103 p.f.u. of the indicated viruses and their weights (A) and signs of illness (B) were monitored daily. Weight data (B) are expressed as the percentage ± SEM of the mean weight of the same group of animals on day 0. Signs of illness data (B) are expressed as the mean score ± SEM. The horizontal bar indicates the days on which the weight loss or signs of illness induced by vΔC6 and vC6FS were statistically different (P<0.05) from both vC6WR and vC6Rev. (C) Virulence in murine intradermal model of VACV infection. Female C57BL/6 mice (n = 10) were infected with 104 p.f.u. of the indicated viruses in both ears and the resulting lesions were measured daily. Data are expressed as the mean lesion size ± SEM. The horizontal bar indicates the days on which the lesion size caused by vΔC6 and vC6FS were statistically different (P<0.05) from both vC6WR and vC6Rev.
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