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Inhibition of IkappaB kinase by vaccinia virus virulence factor B14.

Chen RA, Ryzhakov G, Cooray S, Randow F, Smith GL - PLoS Pathog. (2008)

Bottom Line: In cells infected with VACV lacking gene B14R (vDeltaB14) there was a higher level of phosphorylated IkappaBalpha but a similar level of IkappaBalpha compared to cells infected with control viruses expressing B14, suggesting B14 affects IKK activity.B14 inhibited NF-kappaB activation induced by overexpression of IKKalpha, IKKbeta, and a constitutively active mutant of IKKalpha, S176/180E, but did not inhibit a comparable mutant of IKKbeta, S177/181E.This suggested that phosphorylation of these serine residues in the activation loop of IKKbeta is targeted by B14, and this was confirmed using Ab specific for phospho-IKKbeta.

View Article: PubMed Central - PubMed

Affiliation: Department of Virology, Faculty of Medicine, Imperial College London, London, United Kingdom.

ABSTRACT
The IkappaB kinase (IKK) complex is a key regulator of signal transduction pathways leading to the induction of NF-kappaB-dependent gene expression and production of pro-inflammatory cytokines. It therefore represents a major target for the development of anti-inflammatory therapeutic drugs and may be targeted by pathogens seeking to diminish the host response to infection. Previously, the vaccinia virus (VACV) strain Western Reserve B14 protein was characterised as an intracellular virulence factor that alters the inflammatory response to infection by an unknown mechanism. Here we demonstrate that ectopic expression of B14 inhibited NF-kappaB activation in response to TNFalpha, IL-1beta, poly(I:C), and PMA. In cells infected with VACV lacking gene B14R (vDeltaB14) there was a higher level of phosphorylated IkappaBalpha but a similar level of IkappaBalpha compared to cells infected with control viruses expressing B14, suggesting B14 affects IKK activity. Direct evidence for this was obtained by showing that B14 co-purified and co-precipitated with the endogenous IKK complex from human and mouse cells and inhibited IKK complex enzymatic activity. Notably, the interaction between B14 and the IKK complex required IKKbeta but not IKKalpha, suggesting the interaction occurs via IKKbeta. B14 inhibited NF-kappaB activation induced by overexpression of IKKalpha, IKKbeta, and a constitutively active mutant of IKKalpha, S176/180E, but did not inhibit a comparable mutant of IKKbeta, S177/181E. This suggested that phosphorylation of these serine residues in the activation loop of IKKbeta is targeted by B14, and this was confirmed using Ab specific for phospho-IKKbeta.

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B14 Inhibits Phosphorylation of IKKβ on Ser 177/181HEK 293T cells were transfected with vectors expressing B14 (3, 1.2, and 0.6 μg) and HA-IKKβ (2 μg). The empty vector pCI was used to adjust the total DNA added to 5 μg per reaction per 6 cm dish. After 24 h, cell lysates were resolved by SDS-PAGE (15% gel) and analysed by immunoblotting using the Abs indicated on the right (the anti-HA mAb was used to detect total HA-IKKβ). Sizes of bands detected are indicated on the left in kDa.
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ppat-0040022-g008: B14 Inhibits Phosphorylation of IKKβ on Ser 177/181HEK 293T cells were transfected with vectors expressing B14 (3, 1.2, and 0.6 μg) and HA-IKKβ (2 μg). The empty vector pCI was used to adjust the total DNA added to 5 μg per reaction per 6 cm dish. After 24 h, cell lysates were resolved by SDS-PAGE (15% gel) and analysed by immunoblotting using the Abs indicated on the right (the anti-HA mAb was used to detect total HA-IKKβ). Sizes of bands detected are indicated on the left in kDa.

Mentions: This hypothesis was tested directly by using Ab to detect IKKβ that has been phosphorylated in the activation loop at serine 177 and 181 (Figure 8). HA-tagged IKKβ was transfected into 293 T cells either alone or together with increasing concentrations of B14. In the absence of transfected HA-IKKβ no phospho-IKKβ was detected, but after addition of HA-IKKβ, phospho-IKKβ was observed easily and was reduced in a dose-dependent manner as the concentration of B14 increased. Notably, while the amount of phospho-IKKβ decreased in the presence of B14, the amount of total HA-IKKβ remained fairly constant and blotting for tubulin confirmed equal loading of samples. Therefore, B14 inhibits NF-κB activation by preventing phosphorylation of IKKβ in the activation loop.


Inhibition of IkappaB kinase by vaccinia virus virulence factor B14.

Chen RA, Ryzhakov G, Cooray S, Randow F, Smith GL - PLoS Pathog. (2008)

B14 Inhibits Phosphorylation of IKKβ on Ser 177/181HEK 293T cells were transfected with vectors expressing B14 (3, 1.2, and 0.6 μg) and HA-IKKβ (2 μg). The empty vector pCI was used to adjust the total DNA added to 5 μg per reaction per 6 cm dish. After 24 h, cell lysates were resolved by SDS-PAGE (15% gel) and analysed by immunoblotting using the Abs indicated on the right (the anti-HA mAb was used to detect total HA-IKKβ). Sizes of bands detected are indicated on the left in kDa.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2233672&req=5

ppat-0040022-g008: B14 Inhibits Phosphorylation of IKKβ on Ser 177/181HEK 293T cells were transfected with vectors expressing B14 (3, 1.2, and 0.6 μg) and HA-IKKβ (2 μg). The empty vector pCI was used to adjust the total DNA added to 5 μg per reaction per 6 cm dish. After 24 h, cell lysates were resolved by SDS-PAGE (15% gel) and analysed by immunoblotting using the Abs indicated on the right (the anti-HA mAb was used to detect total HA-IKKβ). Sizes of bands detected are indicated on the left in kDa.
Mentions: This hypothesis was tested directly by using Ab to detect IKKβ that has been phosphorylated in the activation loop at serine 177 and 181 (Figure 8). HA-tagged IKKβ was transfected into 293 T cells either alone or together with increasing concentrations of B14. In the absence of transfected HA-IKKβ no phospho-IKKβ was detected, but after addition of HA-IKKβ, phospho-IKKβ was observed easily and was reduced in a dose-dependent manner as the concentration of B14 increased. Notably, while the amount of phospho-IKKβ decreased in the presence of B14, the amount of total HA-IKKβ remained fairly constant and blotting for tubulin confirmed equal loading of samples. Therefore, B14 inhibits NF-κB activation by preventing phosphorylation of IKKβ in the activation loop.

Bottom Line: In cells infected with VACV lacking gene B14R (vDeltaB14) there was a higher level of phosphorylated IkappaBalpha but a similar level of IkappaBalpha compared to cells infected with control viruses expressing B14, suggesting B14 affects IKK activity.B14 inhibited NF-kappaB activation induced by overexpression of IKKalpha, IKKbeta, and a constitutively active mutant of IKKalpha, S176/180E, but did not inhibit a comparable mutant of IKKbeta, S177/181E.This suggested that phosphorylation of these serine residues in the activation loop of IKKbeta is targeted by B14, and this was confirmed using Ab specific for phospho-IKKbeta.

View Article: PubMed Central - PubMed

Affiliation: Department of Virology, Faculty of Medicine, Imperial College London, London, United Kingdom.

ABSTRACT
The IkappaB kinase (IKK) complex is a key regulator of signal transduction pathways leading to the induction of NF-kappaB-dependent gene expression and production of pro-inflammatory cytokines. It therefore represents a major target for the development of anti-inflammatory therapeutic drugs and may be targeted by pathogens seeking to diminish the host response to infection. Previously, the vaccinia virus (VACV) strain Western Reserve B14 protein was characterised as an intracellular virulence factor that alters the inflammatory response to infection by an unknown mechanism. Here we demonstrate that ectopic expression of B14 inhibited NF-kappaB activation in response to TNFalpha, IL-1beta, poly(I:C), and PMA. In cells infected with VACV lacking gene B14R (vDeltaB14) there was a higher level of phosphorylated IkappaBalpha but a similar level of IkappaBalpha compared to cells infected with control viruses expressing B14, suggesting B14 affects IKK activity. Direct evidence for this was obtained by showing that B14 co-purified and co-precipitated with the endogenous IKK complex from human and mouse cells and inhibited IKK complex enzymatic activity. Notably, the interaction between B14 and the IKK complex required IKKbeta but not IKKalpha, suggesting the interaction occurs via IKKbeta. B14 inhibited NF-kappaB activation induced by overexpression of IKKalpha, IKKbeta, and a constitutively active mutant of IKKalpha, S176/180E, but did not inhibit a comparable mutant of IKKbeta, S177/181E. This suggested that phosphorylation of these serine residues in the activation loop of IKKbeta is targeted by B14, and this was confirmed using Ab specific for phospho-IKKbeta.

Show MeSH
Related in: MedlinePlus