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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 inhibits early innate immune signalling events.(A) HEK293T cells were transfected with an IFNβ-promoter luciferase reporter, a TK-promoter renilla luciferase transfection control, TLR3, and pCI-A49 or empty vector (EV). After 24 h cells were stimulated with 100 µg/ml of poly(I∶C) for 6 h before luciferase activity was measured. (B) RAW 264.7 cells were transfected as in (A) and stimulated with CpG or LPS for 6 h before luciferase activity was measured. (C) HEK293T cells were transfected with pCMV-HA-A49 or EV and stimulated with 500 ng/ml of poly (dA-dT) for 24 h. RNA was extracted and IFNβ mRNA measured by real-time PCR. (D) HEK293T cells were transfected as in (C) with different doses of A49 plasmid and infected 24 h later with Sendai virus (SeV) for further 24 h. CCL5 was measured by ELISA in the supernatant of infected and mock-infected cells. NI stands for non-infected, NS for non-stimulated. 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-g002: A49 inhibits early innate immune signalling events.(A) HEK293T cells were transfected with an IFNβ-promoter luciferase reporter, a TK-promoter renilla luciferase transfection control, TLR3, and pCI-A49 or empty vector (EV). After 24 h cells were stimulated with 100 µg/ml of poly(I∶C) for 6 h before luciferase activity was measured. (B) RAW 264.7 cells were transfected as in (A) and stimulated with CpG or LPS for 6 h before luciferase activity was measured. (C) HEK293T cells were transfected with pCMV-HA-A49 or EV and stimulated with 500 ng/ml of poly (dA-dT) for 24 h. RNA was extracted and IFNβ mRNA measured by real-time PCR. (D) HEK293T cells were transfected as in (C) with different doses of A49 plasmid and infected 24 h later with Sendai virus (SeV) for further 24 h. CCL5 was measured by ELISA in the supernatant of infected and mock-infected cells. NI stands for non-infected, NS for non-stimulated. 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: Next, the mechanism by which A49 inhibited activation of the IFNβ promoter was studied. The A49R ORF was amplified from VACV WR genomic DNA and cloned into a mammalian expression vector with a C-terminal Flag or an N-terminal HA tag and tested for inhibition of the IFNβ pathway. HEK293 cells were co-transfected with an IFNβ promoter-firefly luciferase reporter and an A49 expression vector or the empty vector (EV), TLR3 (to allow IFNβ induction by poly(I∶C)) and a renilla luciferase transfection control. Cells were stimulated 24 h later with poly(I∶C), poly(dA∶dT) or infected with Sendai virus (SeV) (Figure 2). A49 blocked activation of the IFNβ promoter by poly(I∶C) (Figure 2A). The same effect was seen in RAW 264.7 cells stimulated with LPS and CpG, agonists of TLR4 and TLR9, respectively (Figure 2B). A49 also diminished transcription of IFNβ mRNA in poly(dA-dT)-stimulated HEK293T cells, as shown by quantitative PCR (Figure 2C), and inhibited production of the NF-κB responsive chemokine CCL5 in SeV-infected HEK293T cells (Figure 2D).


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 inhibits early innate immune signalling events.(A) HEK293T cells were transfected with an IFNβ-promoter luciferase reporter, a TK-promoter renilla luciferase transfection control, TLR3, and pCI-A49 or empty vector (EV). After 24 h cells were stimulated with 100 µg/ml of poly(I∶C) for 6 h before luciferase activity was measured. (B) RAW 264.7 cells were transfected as in (A) and stimulated with CpG or LPS for 6 h before luciferase activity was measured. (C) HEK293T cells were transfected with pCMV-HA-A49 or EV and stimulated with 500 ng/ml of poly (dA-dT) for 24 h. RNA was extracted and IFNβ mRNA measured by real-time PCR. (D) HEK293T cells were transfected as in (C) with different doses of A49 plasmid and infected 24 h later with Sendai virus (SeV) for further 24 h. CCL5 was measured by ELISA in the supernatant of infected and mock-infected cells. NI stands for non-infected, NS for non-stimulated. 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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ppat-1003183-g002: A49 inhibits early innate immune signalling events.(A) HEK293T cells were transfected with an IFNβ-promoter luciferase reporter, a TK-promoter renilla luciferase transfection control, TLR3, and pCI-A49 or empty vector (EV). After 24 h cells were stimulated with 100 µg/ml of poly(I∶C) for 6 h before luciferase activity was measured. (B) RAW 264.7 cells were transfected as in (A) and stimulated with CpG or LPS for 6 h before luciferase activity was measured. (C) HEK293T cells were transfected with pCMV-HA-A49 or EV and stimulated with 500 ng/ml of poly (dA-dT) for 24 h. RNA was extracted and IFNβ mRNA measured by real-time PCR. (D) HEK293T cells were transfected as in (C) with different doses of A49 plasmid and infected 24 h later with Sendai virus (SeV) for further 24 h. CCL5 was measured by ELISA in the supernatant of infected and mock-infected cells. NI stands for non-infected, NS for non-stimulated. 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: Next, the mechanism by which A49 inhibited activation of the IFNβ promoter was studied. The A49R ORF was amplified from VACV WR genomic DNA and cloned into a mammalian expression vector with a C-terminal Flag or an N-terminal HA tag and tested for inhibition of the IFNβ pathway. HEK293 cells were co-transfected with an IFNβ promoter-firefly luciferase reporter and an A49 expression vector or the empty vector (EV), TLR3 (to allow IFNβ induction by poly(I∶C)) and a renilla luciferase transfection control. Cells were stimulated 24 h later with poly(I∶C), poly(dA∶dT) or infected with Sendai virus (SeV) (Figure 2). A49 blocked activation of the IFNβ promoter by poly(I∶C) (Figure 2A). The same effect was seen in RAW 264.7 cells stimulated with LPS and CpG, agonists of TLR4 and TLR9, respectively (Figure 2B). A49 also diminished transcription of IFNβ mRNA in poly(dA-dT)-stimulated HEK293T cells, as shown by quantitative PCR (Figure 2C), and inhibited production of the NF-κB responsive chemokine CCL5 in SeV-infected HEK293T cells (Figure 2D).

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