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Poxvirus targeting of E3 ligase β-TrCP by molecular mimicry: a mechanism to inhibit NF-κB activation and promote immune evasion and virulence.

Mansur DS, Maluquer de Motes C, Unterholzner L, Sumner RP, Ferguson BJ, Ren H, Strnadova P, Bowie AG, Smith GL - PLoS Pathog. (2013)

Bottom Line: Like IκBα, A49 binds the E3 ligase β-TrCP, thereby preventing ubiquitination and degradation of IκBα.Consequently, A49 stabilised phosphorylated IκBα (p-IκBα) and its interaction with p65, so preventing p65 nuclear translocation.Remarkably, despite encoding nine other inhibitors of NF-κB, a VACV lacking A49 showed reduced virulence in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Virology, Faculty of Medicine, Imperial College London, St Mary's Campus, London, United Kingdom.

ABSTRACT
The transcription factor NF-κB is essential for immune responses against pathogens and its activation requires the phosphorylation, ubiquitination and proteasomal degradation of IκBα. Here we describe an inhibitor of NF-κB from vaccinia virus that has a closely related counterpart in variola virus, the cause of smallpox, and mechanistic similarity with the HIV protein Vpu. Protein A49 blocks NF-κB activation by molecular mimicry and contains a motif conserved in IκBα which, in IκBα, is phosphorylated by IKKβ causing ubiquitination and degradation. Like IκBα, A49 binds the E3 ligase β-TrCP, thereby preventing ubiquitination and degradation of IκBα. Consequently, A49 stabilised phosphorylated IκBα (p-IκBα) and its interaction with p65, so preventing p65 nuclear translocation. Serine-to-alanine mutagenesis within the IκBα-like motif of A49 abolished β-TrCP binding, stabilisation of p-IκBα and inhibition of NF-κB activation. Remarkably, despite encoding nine other inhibitors of NF-κB, a VACV lacking A49 showed reduced virulence in vivo.

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Related in: MedlinePlus

A49 stabilises the IκBα/NF-κB complex.(A) HEK293T cells were transfected with 10 µg of pCI-A49 (+) or empty vector (−). Cells were stimulated with TNFα (100 ng/ml) as indicated. Whole cell lysate (4%) of each sample was separated by SDS-PAGE and analysed by immunoblotting with the indicated antibodies indicated. (B) HEK293T cells were transfected as described in (A) and after stimulation with TNFα (100 ng/ml), lysed in IP buffer and lysates immunoprecipitated with anti-p65 antibody. WCL (2.5%) of each sample was loaded. (C) HEK293T cells were transfected with Flag-tagged A49 or Flag-tagged GFP as control. After 24 h cells were treated for 4 h with MG132 (25 µM) or vehicle only, and then stimulated with TNFα (200 ng/ml) as indicated. Cells were then lysed in IP buffer and lysates immunoprecipitated with anti-IκBα antibody. WCL (2%) of each sample was analysed. (D) HeLa cells on coverslips were transfected with pCI-A49 (A49) or empty vector (EV) and 24 h later were incubated with TNFα as indicated for 30 min. Cells were then stained for Flag (green) and p65 (red) and analysed by immunofluorescence. (E) The percentage of cells with nuclear stain for p65 was calculated (n = >100 for each sample). Each condition was performed in triplicates and the assay repeated twice. **p<0.01 comparing A49 transfected cells with the EV. Data shown in (A–C) are one representative experiment of at least three.
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ppat-1003183-g006: A49 stabilises the IκBα/NF-κB complex.(A) HEK293T cells were transfected with 10 µg of pCI-A49 (+) or empty vector (−). Cells were stimulated with TNFα (100 ng/ml) as indicated. Whole cell lysate (4%) of each sample was separated by SDS-PAGE and analysed by immunoblotting with the indicated antibodies indicated. (B) HEK293T cells were transfected as described in (A) and after stimulation with TNFα (100 ng/ml), lysed in IP buffer and lysates immunoprecipitated with anti-p65 antibody. WCL (2.5%) of each sample was loaded. (C) HEK293T cells were transfected with Flag-tagged A49 or Flag-tagged GFP as control. After 24 h cells were treated for 4 h with MG132 (25 µM) or vehicle only, and then stimulated with TNFα (200 ng/ml) as indicated. Cells were then lysed in IP buffer and lysates immunoprecipitated with anti-IκBα antibody. WCL (2%) of each sample was analysed. (D) HeLa cells on coverslips were transfected with pCI-A49 (A49) or empty vector (EV) and 24 h later were incubated with TNFα as indicated for 30 min. Cells were then stained for Flag (green) and p65 (red) and analysed by immunofluorescence. (E) The percentage of cells with nuclear stain for p65 was calculated (n = >100 for each sample). Each condition was performed in triplicates and the assay repeated twice. **p<0.01 comparing A49 transfected cells with the EV. Data shown in (A–C) are one representative experiment of at least three.

Mentions: A49, like IκBα, contains a double serine motif that is needed to bind the C-terminal WD40 domain of β-TrCP. Therefore, it was possible that the ubiquitination and degradation of IκBα might be decreased in the presence of A49. As such, the IκBα/NF-κB complex would be stabilised and p65 would be retained in the cytoplasm. To address these possibilities, the effect of A49 expression on the level of total IκBα and p-IκBα (Ser 32/36) was examined by immunoblotting following TNFα stimulation (Figure 6A). In the absence of A49, p-IκBα was observed from 5 to 15 min after addition of TNFα and disappeared after 30 min, correlating with the reduction of IκBα levels at 30 mins. However, in the presence of A49, levels of both total IκBα and p-IκBα were higher when compared with cells transfected with the empty vector (Figure 6A). Conversely, A49 did not affect the phosphorylation of p65 on serine 536 by upstream kinases (Figure 6A), an event not involved in β-TrCP recognition. Furthermore, immunoprecipitation of p65 from HEK293T cells and immunoblotting for p-IκBα showed that the p-IκBα/p65 complex was de-stabilised by TNFα stimulation, but in the presence of A49 the complex remained intact (Figure 6B). To quantitate these effects, the intensity of the bands corresponding to p-IκBα and IκBα was analysed by densitometry. In all cases, the presence of A49 sustained both total and p-IκBα forms (Figure S6A, B).


Poxvirus targeting of E3 ligase β-TrCP by molecular mimicry: a mechanism to inhibit NF-κB activation and promote immune evasion and virulence.

Mansur DS, Maluquer de Motes C, Unterholzner L, Sumner RP, Ferguson BJ, Ren H, Strnadova P, Bowie AG, Smith GL - PLoS Pathog. (2013)

A49 stabilises the IκBα/NF-κB complex.(A) HEK293T cells were transfected with 10 µg of pCI-A49 (+) or empty vector (−). Cells were stimulated with TNFα (100 ng/ml) as indicated. Whole cell lysate (4%) of each sample was separated by SDS-PAGE and analysed by immunoblotting with the indicated antibodies indicated. (B) HEK293T cells were transfected as described in (A) and after stimulation with TNFα (100 ng/ml), lysed in IP buffer and lysates immunoprecipitated with anti-p65 antibody. WCL (2.5%) of each sample was loaded. (C) HEK293T cells were transfected with Flag-tagged A49 or Flag-tagged GFP as control. After 24 h cells were treated for 4 h with MG132 (25 µM) or vehicle only, and then stimulated with TNFα (200 ng/ml) as indicated. Cells were then lysed in IP buffer and lysates immunoprecipitated with anti-IκBα antibody. WCL (2%) of each sample was analysed. (D) HeLa cells on coverslips were transfected with pCI-A49 (A49) or empty vector (EV) and 24 h later were incubated with TNFα as indicated for 30 min. Cells were then stained for Flag (green) and p65 (red) and analysed by immunofluorescence. (E) The percentage of cells with nuclear stain for p65 was calculated (n = >100 for each sample). Each condition was performed in triplicates and the assay repeated twice. **p<0.01 comparing A49 transfected cells with the EV. Data shown in (A–C) are one representative experiment of at least three.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585151&req=5

ppat-1003183-g006: A49 stabilises the IκBα/NF-κB complex.(A) HEK293T cells were transfected with 10 µg of pCI-A49 (+) or empty vector (−). Cells were stimulated with TNFα (100 ng/ml) as indicated. Whole cell lysate (4%) of each sample was separated by SDS-PAGE and analysed by immunoblotting with the indicated antibodies indicated. (B) HEK293T cells were transfected as described in (A) and after stimulation with TNFα (100 ng/ml), lysed in IP buffer and lysates immunoprecipitated with anti-p65 antibody. WCL (2.5%) of each sample was loaded. (C) HEK293T cells were transfected with Flag-tagged A49 or Flag-tagged GFP as control. After 24 h cells were treated for 4 h with MG132 (25 µM) or vehicle only, and then stimulated with TNFα (200 ng/ml) as indicated. Cells were then lysed in IP buffer and lysates immunoprecipitated with anti-IκBα antibody. WCL (2%) of each sample was analysed. (D) HeLa cells on coverslips were transfected with pCI-A49 (A49) or empty vector (EV) and 24 h later were incubated with TNFα as indicated for 30 min. Cells were then stained for Flag (green) and p65 (red) and analysed by immunofluorescence. (E) The percentage of cells with nuclear stain for p65 was calculated (n = >100 for each sample). Each condition was performed in triplicates and the assay repeated twice. **p<0.01 comparing A49 transfected cells with the EV. Data shown in (A–C) are one representative experiment of at least three.
Mentions: A49, like IκBα, contains a double serine motif that is needed to bind the C-terminal WD40 domain of β-TrCP. Therefore, it was possible that the ubiquitination and degradation of IκBα might be decreased in the presence of A49. As such, the IκBα/NF-κB complex would be stabilised and p65 would be retained in the cytoplasm. To address these possibilities, the effect of A49 expression on the level of total IκBα and p-IκBα (Ser 32/36) was examined by immunoblotting following TNFα stimulation (Figure 6A). In the absence of A49, p-IκBα was observed from 5 to 15 min after addition of TNFα and disappeared after 30 min, correlating with the reduction of IκBα levels at 30 mins. However, in the presence of A49, levels of both total IκBα and p-IκBα were higher when compared with cells transfected with the empty vector (Figure 6A). Conversely, A49 did not affect the phosphorylation of p65 on serine 536 by upstream kinases (Figure 6A), an event not involved in β-TrCP recognition. Furthermore, immunoprecipitation of p65 from HEK293T cells and immunoblotting for p-IκBα showed that the p-IκBα/p65 complex was de-stabilised by TNFα stimulation, but in the presence of A49 the complex remained intact (Figure 6B). To quantitate these effects, the intensity of the bands corresponding to p-IκBα and IκBα was analysed by densitometry. In all cases, the presence of A49 sustained both total and p-IκBα forms (Figure S6A, B).

Bottom Line: Like IκBα, A49 binds the E3 ligase β-TrCP, thereby preventing ubiquitination and degradation of IκBα.Consequently, A49 stabilised phosphorylated IκBα (p-IκBα) and its interaction with p65, so preventing p65 nuclear translocation.Remarkably, despite encoding nine other inhibitors of NF-κB, a VACV lacking A49 showed reduced virulence in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Virology, Faculty of Medicine, Imperial College London, St Mary's Campus, London, United Kingdom.

ABSTRACT
The transcription factor NF-κB is essential for immune responses against pathogens and its activation requires the phosphorylation, ubiquitination and proteasomal degradation of IκBα. Here we describe an inhibitor of NF-κB from vaccinia virus that has a closely related counterpart in variola virus, the cause of smallpox, and mechanistic similarity with the HIV protein Vpu. Protein A49 blocks NF-κB activation by molecular mimicry and contains a motif conserved in IκBα which, in IκBα, is phosphorylated by IKKβ causing ubiquitination and degradation. Like IκBα, A49 binds the E3 ligase β-TrCP, thereby preventing ubiquitination and degradation of IκBα. Consequently, A49 stabilised phosphorylated IκBα (p-IκBα) and its interaction with p65, so preventing p65 nuclear translocation. Serine-to-alanine mutagenesis within the IκBα-like motif of A49 abolished β-TrCP binding, stabilisation of p-IκBα and inhibition of NF-κB activation. Remarkably, despite encoding nine other inhibitors of NF-κB, a VACV lacking A49 showed reduced virulence in vivo.

Show MeSH
Related in: MedlinePlus