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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 interferes with IκBα degradation during viral infection.(A) HeLa cells were infected with vA49rev or vΔA49 at 10 PFU/cell for 4 h, treated for 1 h with MG132 (20 µM) or vehicle only, and then stimulated with TNFα (200 ng/ml) as indicated. Cell extracts were separated by SDS-PAGE and analysed by immunoblotting with the antibodies indicated. N1 immunoblotting served as control for viral infection. (B) HeLa cells were infected and treated with TNFα in triplicate as in (A) and cell extracts were analysed by quantitative fluorescence immunoblotting. The amounts of p-IκBα and IκBα are shown as ratios compared with VACV protein D8. *p<0.05 or **p<0.01 comparing vA49rev with vΔA49. (C) HeLa cells were infected with vA49rev or vΔA49 at 10 PFU/cell for 6 h and then stimulated with TNFα (200 ng/ml) as indicated. Cells were then lysed in IP buffer and the lysates were immunoprecipitated with anti-IκBα antibody and immunoblotted for p65. In each assay, 2% of WCL of each sample was immunoblotted with the indicated antibodies.
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ppat-1003183-g007: A49 interferes with IκBα degradation during viral infection.(A) HeLa cells were infected with vA49rev or vΔA49 at 10 PFU/cell for 4 h, treated for 1 h with MG132 (20 µM) or vehicle only, and then stimulated with TNFα (200 ng/ml) as indicated. Cell extracts were separated by SDS-PAGE and analysed by immunoblotting with the antibodies indicated. N1 immunoblotting served as control for viral infection. (B) HeLa cells were infected and treated with TNFα in triplicate as in (A) and cell extracts were analysed by quantitative fluorescence immunoblotting. The amounts of p-IκBα and IκBα are shown as ratios compared with VACV protein D8. *p<0.05 or **p<0.01 comparing vA49rev with vΔA49. (C) HeLa cells were infected with vA49rev or vΔA49 at 10 PFU/cell for 6 h and then stimulated with TNFα (200 ng/ml) as indicated. Cells were then lysed in IP buffer and the lysates were immunoprecipitated with anti-IκBα antibody and immunoblotted for p65. In each assay, 2% of WCL of each sample was immunoblotted with the indicated antibodies.

Mentions: Next, A49 function was tested during VACV infection. HeLa cells were infected with vA49rev or vΔA49 for 4 h, treated with MG132 for 1 h, and then stimulated with TNFα for 10 or 30 min. Infection by a VACV expressing A49 prevented IκBα degradation and stabilised p-IκBα, whereas infection with vΔA49 did not (Figure 7A). Remarkably, vA49rev induced accumulation of p-IκBα even without TNF stimulation, indicating that A49 blocked NF-κB activation triggered by viral infection. In addition, failure to accumulate p-IκBα could be reversed by MG132 (both without TNF activation or 30 min post-activation), suggesting that no other VACV protein interfered with the proteasomal degradation of p-IκBα downstream of A49. To obtain a more quantitative read-out, a similar experiment in which cells were infected with vA49rev or vΔA49 and treated with TNFα for 30 mins, was performed in triplicate. The amounts of p-IκBα and total IκBα were determined by quantitative fluorescence imaging of immunoblots, and plotted as a ratio compared to the amount of viral protein D8 to account for the efficiency of infection. Infection with vA49rev sustained levels of both p-IκBα and total IκBα 30 mins post-treatment compared to infection with vΔA49, and these differences were statistically significant (Figure 7B). In the absence of TNFα, accumulation of p-IκBα and IκBα during vA49rev infection was also detected (as observed by conventional immunoblotting), but with the sample sizes tested this was not significant.


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 interferes with IκBα degradation during viral infection.(A) HeLa cells were infected with vA49rev or vΔA49 at 10 PFU/cell for 4 h, treated for 1 h with MG132 (20 µM) or vehicle only, and then stimulated with TNFα (200 ng/ml) as indicated. Cell extracts were separated by SDS-PAGE and analysed by immunoblotting with the antibodies indicated. N1 immunoblotting served as control for viral infection. (B) HeLa cells were infected and treated with TNFα in triplicate as in (A) and cell extracts were analysed by quantitative fluorescence immunoblotting. The amounts of p-IκBα and IκBα are shown as ratios compared with VACV protein D8. *p<0.05 or **p<0.01 comparing vA49rev with vΔA49. (C) HeLa cells were infected with vA49rev or vΔA49 at 10 PFU/cell for 6 h and then stimulated with TNFα (200 ng/ml) as indicated. Cells were then lysed in IP buffer and the lysates were immunoprecipitated with anti-IκBα antibody and immunoblotted for p65. In each assay, 2% of WCL of each sample was immunoblotted with the indicated antibodies.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585151&req=5

ppat-1003183-g007: A49 interferes with IκBα degradation during viral infection.(A) HeLa cells were infected with vA49rev or vΔA49 at 10 PFU/cell for 4 h, treated for 1 h with MG132 (20 µM) or vehicle only, and then stimulated with TNFα (200 ng/ml) as indicated. Cell extracts were separated by SDS-PAGE and analysed by immunoblotting with the antibodies indicated. N1 immunoblotting served as control for viral infection. (B) HeLa cells were infected and treated with TNFα in triplicate as in (A) and cell extracts were analysed by quantitative fluorescence immunoblotting. The amounts of p-IκBα and IκBα are shown as ratios compared with VACV protein D8. *p<0.05 or **p<0.01 comparing vA49rev with vΔA49. (C) HeLa cells were infected with vA49rev or vΔA49 at 10 PFU/cell for 6 h and then stimulated with TNFα (200 ng/ml) as indicated. Cells were then lysed in IP buffer and the lysates were immunoprecipitated with anti-IκBα antibody and immunoblotted for p65. In each assay, 2% of WCL of each sample was immunoblotted with the indicated antibodies.
Mentions: Next, A49 function was tested during VACV infection. HeLa cells were infected with vA49rev or vΔA49 for 4 h, treated with MG132 for 1 h, and then stimulated with TNFα for 10 or 30 min. Infection by a VACV expressing A49 prevented IκBα degradation and stabilised p-IκBα, whereas infection with vΔA49 did not (Figure 7A). Remarkably, vA49rev induced accumulation of p-IκBα even without TNF stimulation, indicating that A49 blocked NF-κB activation triggered by viral infection. In addition, failure to accumulate p-IκBα could be reversed by MG132 (both without TNF activation or 30 min post-activation), suggesting that no other VACV protein interfered with the proteasomal degradation of p-IκBα downstream of A49. To obtain a more quantitative read-out, a similar experiment in which cells were infected with vA49rev or vΔA49 and treated with TNFα for 30 mins, was performed in triplicate. The amounts of p-IκBα and total IκBα were determined by quantitative fluorescence imaging of immunoblots, and plotted as a ratio compared to the amount of viral protein D8 to account for the efficiency of infection. Infection with vA49rev sustained levels of both p-IκBα and total IκBα 30 mins post-treatment compared to infection with vΔA49, and these differences were statistically significant (Figure 7B). In the absence of TNFα, accumulation of p-IκBα and IκBα during vA49rev infection was also detected (as observed by conventional immunoblotting), but with the sample sizes tested this was not significant.

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