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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 is a virulence factor.Groups of 5 BALB/c mice were infected intranasally with 5×103 PFU of the indicated viruses and their weights (A) and signs of illness (B) were monitored daily. Weights are expressed as the percentage ± SEM of the mean weight of the same group of animals on day 0. Signs of illness (B) are expressed as the mean score ± SEM. (C) At day 2, 5 and 7 pi, lungs from infected animals were extracted and virus titres were assessed by plaque assay. Statistical significance is indicated by horizontal bars after analysis with one-way ANOVA with Friedman and Dunn's multiple comparison test (A), or unpaired t-test comparing WT with vΔA49 (B–C). Data correspond to one representative experiment out of two showing indistinguishable results.
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ppat-1003183-g001: A49 is a virulence factor.Groups of 5 BALB/c mice were infected intranasally with 5×103 PFU of the indicated viruses and their weights (A) and signs of illness (B) were monitored daily. Weights are expressed as the percentage ± SEM of the mean weight of the same group of animals on day 0. Signs of illness (B) are expressed as the mean score ± SEM. (C) At day 2, 5 and 7 pi, lungs from infected animals were extracted and virus titres were assessed by plaque assay. Statistical significance is indicated by horizontal bars after analysis with one-way ANOVA with Friedman and Dunn's multiple comparison test (A), or unpaired t-test comparing WT with vΔA49 (B–C). Data correspond to one representative experiment out of two showing indistinguishable results.

Mentions: The contribution of A49 to virulence was tested by infecting groups of BALB/c mice intranasally and measuring weight loss and signs of illness [43]. Animals infected with vΔA49 lost less weight and recovered more quickly than controls (Figure 1A) and showed fewer signs of illness on days 4 to 10 post infection (pi) (Figure 1B). Measurement of infectious virus in lungs showed that all viruses replicated to similar titres by day 2 pi, but on days 5 and 7 pi mice infected with vΔA49 had significantly lower titres, showing more rapid clearance of virus (Figure 1C). Collectively, these data indicate that A49 is non-essential for replication, but is a virulence factor. The degree of attenuation seen by deletion of the A49 gene is similar to that deriving from deletion of many other VACV immunomodulators in this model [16], [17], [22], [23], [35], [37], [43]–[50].


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 a virulence factor.Groups of 5 BALB/c mice were infected intranasally with 5×103 PFU of the indicated viruses and their weights (A) and signs of illness (B) were monitored daily. Weights are expressed as the percentage ± SEM of the mean weight of the same group of animals on day 0. Signs of illness (B) are expressed as the mean score ± SEM. (C) At day 2, 5 and 7 pi, lungs from infected animals were extracted and virus titres were assessed by plaque assay. Statistical significance is indicated by horizontal bars after analysis with one-way ANOVA with Friedman and Dunn's multiple comparison test (A), or unpaired t-test comparing WT with vΔA49 (B–C). Data correspond to one representative experiment out of two showing indistinguishable results.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3585151&req=5

ppat-1003183-g001: A49 is a virulence factor.Groups of 5 BALB/c mice were infected intranasally with 5×103 PFU of the indicated viruses and their weights (A) and signs of illness (B) were monitored daily. Weights are expressed as the percentage ± SEM of the mean weight of the same group of animals on day 0. Signs of illness (B) are expressed as the mean score ± SEM. (C) At day 2, 5 and 7 pi, lungs from infected animals were extracted and virus titres were assessed by plaque assay. Statistical significance is indicated by horizontal bars after analysis with one-way ANOVA with Friedman and Dunn's multiple comparison test (A), or unpaired t-test comparing WT with vΔA49 (B–C). Data correspond to one representative experiment out of two showing indistinguishable results.
Mentions: The contribution of A49 to virulence was tested by infecting groups of BALB/c mice intranasally and measuring weight loss and signs of illness [43]. Animals infected with vΔA49 lost less weight and recovered more quickly than controls (Figure 1A) and showed fewer signs of illness on days 4 to 10 post infection (pi) (Figure 1B). Measurement of infectious virus in lungs showed that all viruses replicated to similar titres by day 2 pi, but on days 5 and 7 pi mice infected with vΔA49 had significantly lower titres, showing more rapid clearance of virus (Figure 1C). Collectively, these data indicate that A49 is non-essential for replication, but is a virulence factor. The degree of attenuation seen by deletion of the A49 gene is similar to that deriving from deletion of many other VACV immunomodulators in this model [16], [17], [22], [23], [35], [37], [43]–[50].

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