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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 interacts with the ubiquitin E3 ligase β-TrCP.(A) Alignment of β-TrCP recognition motif present in A49, IκBα, IκBβ, β-catenin, Emi1, HIV Vpu and p105. Phosphorylated serine residues are shown in bold and numbers indicate the first residue of the motif in each protein. (B) HeLa cells were cotransfected with myc-β-TrCP or myc-TAK1, with nTAP-A49 or nTAP-C6. Cells were lysed with IP buffer 24 h later and proteins pulled down with streptavidin beads. Whole cell lysate (WCL) (3%) of each sample was analysed. (C) HeLa cells were transfected with TAP-RIG-I or TAP-β-TrCP, and 24 h later were mock-infected or infected with vΔA49 or vA49rev at 10 PFU/cell for 6 h. Cells were lysed in IP buffer and a streptavidin pull-down was performed. D8 immunoblotting served as control for viral infection. WCL (1.5%) of each sample was analysed. (D) HeLa cells were either mock-infected or infected with vΔA49 or vA49rev with 10 PFU/cell for 6 h. After lysis with IP buffer, lysates were immunoprecipitated with a goat anti-β-TrCP antibody or an isotype control. WCL (1%) of each sample was analysed. Asterisk marks the antibody heavy chain. Data shown in (B) and (C) are one representative experiment of at least three. Data shown in (D) are one representative experiment out of two showing indistinguishable results.
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ppat-1003183-g004: A49 interacts with the ubiquitin E3 ligase β-TrCP.(A) Alignment of β-TrCP recognition motif present in A49, IκBα, IκBβ, β-catenin, Emi1, HIV Vpu and p105. Phosphorylated serine residues are shown in bold and numbers indicate the first residue of the motif in each protein. (B) HeLa cells were cotransfected with myc-β-TrCP or myc-TAK1, with nTAP-A49 or nTAP-C6. Cells were lysed with IP buffer 24 h later and proteins pulled down with streptavidin beads. Whole cell lysate (WCL) (3%) of each sample was analysed. (C) HeLa cells were transfected with TAP-RIG-I or TAP-β-TrCP, and 24 h later were mock-infected or infected with vΔA49 or vA49rev at 10 PFU/cell for 6 h. Cells were lysed in IP buffer and a streptavidin pull-down was performed. D8 immunoblotting served as control for viral infection. WCL (1.5%) of each sample was analysed. (D) HeLa cells were either mock-infected or infected with vΔA49 or vA49rev with 10 PFU/cell for 6 h. After lysis with IP buffer, lysates were immunoprecipitated with a goat anti-β-TrCP antibody or an isotype control. WCL (1%) of each sample was analysed. Asterisk marks the antibody heavy chain. Data shown in (B) and (C) are one representative experiment of at least three. Data shown in (D) are one representative experiment out of two showing indistinguishable results.

Mentions: Activation of NF-κB requires phosphorylation of IκBα on serines 32 and 36 within a short motif (DSGX2–3S) that is present in several proteins such as IκBα [8], Emi1 [52], β-catenin [12], HIV Vpu [11] and p105 [53]. Once this motif is phosphorylated, it is recognized by the E3 ligase β-TrCP [4]. Inspection of the A49 sequence identified the sequence SGNLES (aa 7–12) near the N terminus that matched the motif in β-TrCP substrates (Figure 4A). This suggested that A49 might bind β-TrCP and hence prevent β-TrCP from targeting its usual substrates. To test whether A49 interacted with β-TrCP, myc-tagged β-TrCP or TAK1 were co-expressed in HeLa cells with tandem-affinity purification (TAP)-tagged (streptavidin and FLAG) VACV proteins A49 or C6. After pull-down with streptavidin beads, an interaction between β-TrCP and A49 was seen by immunoblotting with anti-myc mAb (Figure 4B). Note that A49 did not interact with TAK1, nor did C6 with β-TrCP, confirming the specificity of the A49-β-TrCP interaction.


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 interacts with the ubiquitin E3 ligase β-TrCP.(A) Alignment of β-TrCP recognition motif present in A49, IκBα, IκBβ, β-catenin, Emi1, HIV Vpu and p105. Phosphorylated serine residues are shown in bold and numbers indicate the first residue of the motif in each protein. (B) HeLa cells were cotransfected with myc-β-TrCP or myc-TAK1, with nTAP-A49 or nTAP-C6. Cells were lysed with IP buffer 24 h later and proteins pulled down with streptavidin beads. Whole cell lysate (WCL) (3%) of each sample was analysed. (C) HeLa cells were transfected with TAP-RIG-I or TAP-β-TrCP, and 24 h later were mock-infected or infected with vΔA49 or vA49rev at 10 PFU/cell for 6 h. Cells were lysed in IP buffer and a streptavidin pull-down was performed. D8 immunoblotting served as control for viral infection. WCL (1.5%) of each sample was analysed. (D) HeLa cells were either mock-infected or infected with vΔA49 or vA49rev with 10 PFU/cell for 6 h. After lysis with IP buffer, lysates were immunoprecipitated with a goat anti-β-TrCP antibody or an isotype control. WCL (1%) of each sample was analysed. Asterisk marks the antibody heavy chain. Data shown in (B) and (C) are one representative experiment of at least three. Data shown in (D) are one representative experiment out of two showing indistinguishable results.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585151&req=5

ppat-1003183-g004: A49 interacts with the ubiquitin E3 ligase β-TrCP.(A) Alignment of β-TrCP recognition motif present in A49, IκBα, IκBβ, β-catenin, Emi1, HIV Vpu and p105. Phosphorylated serine residues are shown in bold and numbers indicate the first residue of the motif in each protein. (B) HeLa cells were cotransfected with myc-β-TrCP or myc-TAK1, with nTAP-A49 or nTAP-C6. Cells were lysed with IP buffer 24 h later and proteins pulled down with streptavidin beads. Whole cell lysate (WCL) (3%) of each sample was analysed. (C) HeLa cells were transfected with TAP-RIG-I or TAP-β-TrCP, and 24 h later were mock-infected or infected with vΔA49 or vA49rev at 10 PFU/cell for 6 h. Cells were lysed in IP buffer and a streptavidin pull-down was performed. D8 immunoblotting served as control for viral infection. WCL (1.5%) of each sample was analysed. (D) HeLa cells were either mock-infected or infected with vΔA49 or vA49rev with 10 PFU/cell for 6 h. After lysis with IP buffer, lysates were immunoprecipitated with a goat anti-β-TrCP antibody or an isotype control. WCL (1%) of each sample was analysed. Asterisk marks the antibody heavy chain. Data shown in (B) and (C) are one representative experiment of at least three. Data shown in (D) are one representative experiment out of two showing indistinguishable results.
Mentions: Activation of NF-κB requires phosphorylation of IκBα on serines 32 and 36 within a short motif (DSGX2–3S) that is present in several proteins such as IκBα [8], Emi1 [52], β-catenin [12], HIV Vpu [11] and p105 [53]. Once this motif is phosphorylated, it is recognized by the E3 ligase β-TrCP [4]. Inspection of the A49 sequence identified the sequence SGNLES (aa 7–12) near the N terminus that matched the motif in β-TrCP substrates (Figure 4A). This suggested that A49 might bind β-TrCP and hence prevent β-TrCP from targeting its usual substrates. To test whether A49 interacted with β-TrCP, myc-tagged β-TrCP or TAK1 were co-expressed in HeLa cells with tandem-affinity purification (TAP)-tagged (streptavidin and FLAG) VACV proteins A49 or C6. After pull-down with streptavidin beads, an interaction between β-TrCP and A49 was seen by immunoblotting with anti-myc mAb (Figure 4B). Note that A49 did not interact with TAK1, nor did C6 with β-TrCP, confirming the specificity of the A49-β-TrCP interaction.

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