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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 binding to β-TrCP WD40 domain requires its N-terminal region.(A) Domains of β-TrCP and truncations generated. β-TrCP.ΔWD40 (1–250), β-TrCP.ΔF box (251–569). (B) HeLa cells were cotransfected with nTAP-A49 together with HA-tagged WT β-TrCP, ΔWD40 β-TrCP, ΔF-box β-TrCP or SINTBAD. After 24 h cells were lysed in IP buffer and a streptavidin pull-down was performed. WCL (2.5%) of each sample was analysed. (C) HeLa cells were cotransfected with myc-tagged β-TrCP together with nTAP-tagged WT A49, S7/12A A49, 7/11/12A A49, S7/12E A49 or C6 as a negative control. After 24 h cells were lysed in IP buffer and a streptavidin pull-down was performed. A fraction (2.5%) of each whole cell lysate (WCL) was analysed. (D) HEK293T cells were transfected with an NF-κB luciferase reporter, a renilla luciferase reporter and 100 ng of either nTAP-tagged WT, S7/12A or S7/12E A49 plasmids, or empty vector (EV). After 24 h cells were stimulated for 6 h with 20 ng/ml TNFα and luciferase activity was measured. 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 conditions pair-wise as indicated. (E) Lysates from TNFα-stimulated samples from the reporter gene assays were fractionated and immunoblotted for FLAG and tubulin. WCL (10%) of each sample was analysed. Data shown in (B–C) are one representative experiment of at least three showing indistinguishable results.
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ppat-1003183-g005: A49 binding to β-TrCP WD40 domain requires its N-terminal region.(A) Domains of β-TrCP and truncations generated. β-TrCP.ΔWD40 (1–250), β-TrCP.ΔF box (251–569). (B) HeLa cells were cotransfected with nTAP-A49 together with HA-tagged WT β-TrCP, ΔWD40 β-TrCP, ΔF-box β-TrCP or SINTBAD. After 24 h cells were lysed in IP buffer and a streptavidin pull-down was performed. WCL (2.5%) of each sample was analysed. (C) HeLa cells were cotransfected with myc-tagged β-TrCP together with nTAP-tagged WT A49, S7/12A A49, 7/11/12A A49, S7/12E A49 or C6 as a negative control. After 24 h cells were lysed in IP buffer and a streptavidin pull-down was performed. A fraction (2.5%) of each whole cell lysate (WCL) was analysed. (D) HEK293T cells were transfected with an NF-κB luciferase reporter, a renilla luciferase reporter and 100 ng of either nTAP-tagged WT, S7/12A or S7/12E A49 plasmids, or empty vector (EV). After 24 h cells were stimulated for 6 h with 20 ng/ml TNFα and luciferase activity was measured. 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 conditions pair-wise as indicated. (E) Lysates from TNFα-stimulated samples from the reporter gene assays were fractionated and immunoblotted for FLAG and tubulin. WCL (10%) of each sample was analysed. Data shown in (B–C) are one representative experiment of at least three showing indistinguishable results.

Mentions: The E3 ligase β-TrCP belongs to the F-box protein family and contains an N-terminal F-box domain and a C-terminal WD40 domain [9]. To study the A49-β-TrCP interaction, the F-box or the WD40 domains were deleted separately from β-TrCP (Figure 5A) and these truncated alleles were expressed in HeLa cells with TAP-tagged A49. After A49 pull-down, β-TrCP was co-purified only if it contained the WD40 domain (Figure 5B). This showed that A49, like IκBα, interacted with β-TrCP only if the WD40 domain was present, and that the F-box was dispensable.


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 binding to β-TrCP WD40 domain requires its N-terminal region.(A) Domains of β-TrCP and truncations generated. β-TrCP.ΔWD40 (1–250), β-TrCP.ΔF box (251–569). (B) HeLa cells were cotransfected with nTAP-A49 together with HA-tagged WT β-TrCP, ΔWD40 β-TrCP, ΔF-box β-TrCP or SINTBAD. After 24 h cells were lysed in IP buffer and a streptavidin pull-down was performed. WCL (2.5%) of each sample was analysed. (C) HeLa cells were cotransfected with myc-tagged β-TrCP together with nTAP-tagged WT A49, S7/12A A49, 7/11/12A A49, S7/12E A49 or C6 as a negative control. After 24 h cells were lysed in IP buffer and a streptavidin pull-down was performed. A fraction (2.5%) of each whole cell lysate (WCL) was analysed. (D) HEK293T cells were transfected with an NF-κB luciferase reporter, a renilla luciferase reporter and 100 ng of either nTAP-tagged WT, S7/12A or S7/12E A49 plasmids, or empty vector (EV). After 24 h cells were stimulated for 6 h with 20 ng/ml TNFα and luciferase activity was measured. 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 conditions pair-wise as indicated. (E) Lysates from TNFα-stimulated samples from the reporter gene assays were fractionated and immunoblotted for FLAG and tubulin. WCL (10%) of each sample was analysed. Data shown in (B–C) are one representative experiment of at least three showing indistinguishable results.
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Related In: Results  -  Collection

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ppat-1003183-g005: A49 binding to β-TrCP WD40 domain requires its N-terminal region.(A) Domains of β-TrCP and truncations generated. β-TrCP.ΔWD40 (1–250), β-TrCP.ΔF box (251–569). (B) HeLa cells were cotransfected with nTAP-A49 together with HA-tagged WT β-TrCP, ΔWD40 β-TrCP, ΔF-box β-TrCP or SINTBAD. After 24 h cells were lysed in IP buffer and a streptavidin pull-down was performed. WCL (2.5%) of each sample was analysed. (C) HeLa cells were cotransfected with myc-tagged β-TrCP together with nTAP-tagged WT A49, S7/12A A49, 7/11/12A A49, S7/12E A49 or C6 as a negative control. After 24 h cells were lysed in IP buffer and a streptavidin pull-down was performed. A fraction (2.5%) of each whole cell lysate (WCL) was analysed. (D) HEK293T cells were transfected with an NF-κB luciferase reporter, a renilla luciferase reporter and 100 ng of either nTAP-tagged WT, S7/12A or S7/12E A49 plasmids, or empty vector (EV). After 24 h cells were stimulated for 6 h with 20 ng/ml TNFα and luciferase activity was measured. 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 conditions pair-wise as indicated. (E) Lysates from TNFα-stimulated samples from the reporter gene assays were fractionated and immunoblotted for FLAG and tubulin. WCL (10%) of each sample was analysed. Data shown in (B–C) are one representative experiment of at least three showing indistinguishable results.
Mentions: The E3 ligase β-TrCP belongs to the F-box protein family and contains an N-terminal F-box domain and a C-terminal WD40 domain [9]. To study the A49-β-TrCP interaction, the F-box or the WD40 domains were deleted separately from β-TrCP (Figure 5A) and these truncated alleles were expressed in HeLa cells with TAP-tagged A49. After A49 pull-down, β-TrCP was co-purified only if it contained the WD40 domain (Figure 5B). This showed that A49, like IκBα, interacted with β-TrCP only if the WD40 domain was present, and that the F-box was dispensable.

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