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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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A49 is an NF-κB inhibitor.(A–F) Activation of firefly luciferase from an NF-κB-dependent promoter (NF-κB-Luc). Cells were transfected with NF-κB-Luc, a renilla luciferase control and an A49-expressing vector or empty vector (EV) for 24 h. When other proteins were co-expressed, these were transfected with the reporters and A49. Cells were stimulated 24 h later. NS, non-stimulated. (A) HeLa cells were stimulated with IL-1α or TNFα (100 ng/ml) for 6 h. (B–C) HEK293T cells were transfected with 50, 100 or 150 ng of pCMV-HA-A49 and then stimulated with (B) IL-1α (100 ng/ml) or (C) TNFα (250 ng/ml). (D–F) HEK293T cells were co-transfected with (D) CD4-TLR4 or TRIF, with (E) TRAF2, TRAF6, TAB3 or IKKβ, or with (F) p65 expression vectors. In all assays, data are presented as mean ± SD and show one representative experiment of at least three, each performed in triplicate. *p<0.05, **p<0.01 or ***p<0.001 comparing A49 transfected cells with EV.
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ppat-1003183-g003: A49 is an NF-κB inhibitor.(A–F) Activation of firefly luciferase from an NF-κB-dependent promoter (NF-κB-Luc). Cells were transfected with NF-κB-Luc, a renilla luciferase control and an A49-expressing vector or empty vector (EV) for 24 h. When other proteins were co-expressed, these were transfected with the reporters and A49. Cells were stimulated 24 h later. NS, non-stimulated. (A) HeLa cells were stimulated with IL-1α or TNFα (100 ng/ml) for 6 h. (B–C) HEK293T cells were transfected with 50, 100 or 150 ng of pCMV-HA-A49 and then stimulated with (B) IL-1α (100 ng/ml) or (C) TNFα (250 ng/ml). (D–F) HEK293T cells were co-transfected with (D) CD4-TLR4 or TRIF, with (E) TRAF2, TRAF6, TAB3 or IKKβ, or with (F) p65 expression vectors. In all assays, data are presented as mean ± SD and show one representative experiment of at least three, each performed in triplicate. *p<0.05, **p<0.01 or ***p<0.001 comparing A49 transfected cells with EV.

Mentions: To understand how A49 inhibited IFNβ promoter activity, additional reporter gene assays were performed. HEK293 cells were transfected with an NF-κB luciferase reporter and a plasmid expressing A49. Upon stimulation with either IL-1α or TNFα, A49 reduced NF-κB activation (Figure 3A) in a dose-dependent manner (Figure 3B, C). Moreover, A49 blocked NF-κB activation mediated by TLR signalling in HEK293T cells transfected with TLR4 fused to the CD4 dimerisation domain (CD4-TLR4) (Figure 3D). To determine where A49 was acting, NF-κB was activated by overexpression of proteins operating at different stages in the signalling cascade. A49 blocked NF-κB activation after overexpression of TRIF (Figure 3D), TRAF2, TRAF6, TGFβ-activated kinase 1 (TAK1)-binding protein 3 (TAB3), and IKKβ (Figure 3E). However, when p65 was overexpressed, A49 was not inhibitory (Figure 3F), showing that A49 suppresses NF-κB activation downstream of IKKβ and upstream of p65.


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 is an NF-κB inhibitor.(A–F) Activation of firefly luciferase from an NF-κB-dependent promoter (NF-κB-Luc). Cells were transfected with NF-κB-Luc, a renilla luciferase control and an A49-expressing vector or empty vector (EV) for 24 h. When other proteins were co-expressed, these were transfected with the reporters and A49. Cells were stimulated 24 h later. NS, non-stimulated. (A) HeLa cells were stimulated with IL-1α or TNFα (100 ng/ml) for 6 h. (B–C) HEK293T cells were transfected with 50, 100 or 150 ng of pCMV-HA-A49 and then stimulated with (B) IL-1α (100 ng/ml) or (C) TNFα (250 ng/ml). (D–F) HEK293T cells were co-transfected with (D) CD4-TLR4 or TRIF, with (E) TRAF2, TRAF6, TAB3 or IKKβ, or with (F) p65 expression vectors. In all assays, data are presented as mean ± SD and show one representative experiment of at least three, each performed in triplicate. *p<0.05, **p<0.01 or ***p<0.001 comparing A49 transfected cells with EV.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585151&req=5

ppat-1003183-g003: A49 is an NF-κB inhibitor.(A–F) Activation of firefly luciferase from an NF-κB-dependent promoter (NF-κB-Luc). Cells were transfected with NF-κB-Luc, a renilla luciferase control and an A49-expressing vector or empty vector (EV) for 24 h. When other proteins were co-expressed, these were transfected with the reporters and A49. Cells were stimulated 24 h later. NS, non-stimulated. (A) HeLa cells were stimulated with IL-1α or TNFα (100 ng/ml) for 6 h. (B–C) HEK293T cells were transfected with 50, 100 or 150 ng of pCMV-HA-A49 and then stimulated with (B) IL-1α (100 ng/ml) or (C) TNFα (250 ng/ml). (D–F) HEK293T cells were co-transfected with (D) CD4-TLR4 or TRIF, with (E) TRAF2, TRAF6, TAB3 or IKKβ, or with (F) p65 expression vectors. In all assays, data are presented as mean ± SD and show one representative experiment of at least three, each performed in triplicate. *p<0.05, **p<0.01 or ***p<0.001 comparing A49 transfected cells with EV.
Mentions: To understand how A49 inhibited IFNβ promoter activity, additional reporter gene assays were performed. HEK293 cells were transfected with an NF-κB luciferase reporter and a plasmid expressing A49. Upon stimulation with either IL-1α or TNFα, A49 reduced NF-κB activation (Figure 3A) in a dose-dependent manner (Figure 3B, C). Moreover, A49 blocked NF-κB activation mediated by TLR signalling in HEK293T cells transfected with TLR4 fused to the CD4 dimerisation domain (CD4-TLR4) (Figure 3D). To determine where A49 was acting, NF-κB was activated by overexpression of proteins operating at different stages in the signalling cascade. A49 blocked NF-κB activation after overexpression of TRIF (Figure 3D), TRAF2, TRAF6, TGFβ-activated kinase 1 (TAK1)-binding protein 3 (TAB3), and IKKβ (Figure 3E). However, when p65 was overexpressed, A49 was not inhibitory (Figure 3F), showing that A49 suppresses NF-κB activation downstream of IKKβ and upstream of p65.

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