Limits...
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.

Show MeSH

Related in: MedlinePlus

B14 Inhibits NF-κB ActivationHeLa cells were co-transfected with reporter plasmids for 100 ng of NF-κB (A and B), ISRE (C), AP-1 (D), and 50 ng of pSV-β-galactosidase in all cases. pCI-B14 was transfected where indicated and the amount (ng) per well is shown. The total amount of DNA applied in each reaction was adjusted to 400 ng using pCI empty vector. These transfected cells were stimulated with 100 ng/ml of IL-1β, TNFα, IFNα, or 50 ng/ml of PMA for 8 h as indicated. Luciferase activity was measured and normalized to β-galactosidase intensity in the same well in triplicate wells. (E) HEK 293 cells were co-transfected with pRL-TK and reporter plasmid for NF-κB (□), ISRE (▪), and plasmids expressing GFP, A20, and B14. These cells were stimulated with 5 μg/ml of poly(I:C) for 12 h and then were lysed to measure luciferase activity using Dual-specific luciferase assay kit (Promega). Differences in the fold activation of the indicated reporter in cells transfected with the empty vector and cells expressing B14 were analysed by Student's t-test and the p-values are indicated: * p < 0.05 or ** p < 0.01.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2233672&req=5

ppat-0040022-g001: B14 Inhibits NF-κB ActivationHeLa cells were co-transfected with reporter plasmids for 100 ng of NF-κB (A and B), ISRE (C), AP-1 (D), and 50 ng of pSV-β-galactosidase in all cases. pCI-B14 was transfected where indicated and the amount (ng) per well is shown. The total amount of DNA applied in each reaction was adjusted to 400 ng using pCI empty vector. These transfected cells were stimulated with 100 ng/ml of IL-1β, TNFα, IFNα, or 50 ng/ml of PMA for 8 h as indicated. Luciferase activity was measured and normalized to β-galactosidase intensity in the same well in triplicate wells. (E) HEK 293 cells were co-transfected with pRL-TK and reporter plasmid for NF-κB (□), ISRE (▪), and plasmids expressing GFP, A20, and B14. These cells were stimulated with 5 μg/ml of poly(I:C) for 12 h and then were lysed to measure luciferase activity using Dual-specific luciferase assay kit (Promega). Differences in the fold activation of the indicated reporter in cells transfected with the empty vector and cells expressing B14 were analysed by Student's t-test and the p-values are indicated: * p < 0.05 or ** p < 0.01.

Mentions: To investigate the effect of B14 on NF-κB activation, a plasmid containing a luciferase reporter gene linked to a NF-κB-dependent promoter was transfected into HeLa cells and these cells were stimulated with IL-1β (Figure 1A), TNFα (Figure 1A), or PMA (Figure 1B). Luciferase activity was increased greatly by addition of each stimulant but the level reached was reduced in a dose-dependent manner in the presence of B14. Similar findings were observed using HEK 293 cells (unpublished data). Moreover, B14 decreased poly (I:C)-induced NF-κB dramatically (p-value = 0.0006; 95% decrease) (Figure 1E). In contrast, B14 did not reduce luciferase activity from ISRE (Figure 1C) and AP-1 (Figure 1D) reporter genes induced by IFNα and PMA, respectively. Notably, B14 increased PMA-induced AP-1 activity slightly (p = 0.02; 1.5-fold increase; Figure 1D). We also observed a small but significant reduction in poly (I:C)-induced ISRE activity in the presence of B14 (p-value = 0.01; 29% decrease; Figure 1E). However, it is uncertain if these relatively small changes seen with these reporter assays are relevant biologically. As a control we also expressed A20, a de-ubiquitinating enzyme that downregulates NF-κB and IRF3 [39–41] and observed strong inhibition of both pathways (Figure 1E).


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 NF-κB ActivationHeLa cells were co-transfected with reporter plasmids for 100 ng of NF-κB (A and B), ISRE (C), AP-1 (D), and 50 ng of pSV-β-galactosidase in all cases. pCI-B14 was transfected where indicated and the amount (ng) per well is shown. The total amount of DNA applied in each reaction was adjusted to 400 ng using pCI empty vector. These transfected cells were stimulated with 100 ng/ml of IL-1β, TNFα, IFNα, or 50 ng/ml of PMA for 8 h as indicated. Luciferase activity was measured and normalized to β-galactosidase intensity in the same well in triplicate wells. (E) HEK 293 cells were co-transfected with pRL-TK and reporter plasmid for NF-κB (□), ISRE (▪), and plasmids expressing GFP, A20, and B14. These cells were stimulated with 5 μg/ml of poly(I:C) for 12 h and then were lysed to measure luciferase activity using Dual-specific luciferase assay kit (Promega). Differences in the fold activation of the indicated reporter in cells transfected with the empty vector and cells expressing B14 were analysed by Student's t-test and the p-values are indicated: * p < 0.05 or ** p < 0.01.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-0040022-g001: B14 Inhibits NF-κB ActivationHeLa cells were co-transfected with reporter plasmids for 100 ng of NF-κB (A and B), ISRE (C), AP-1 (D), and 50 ng of pSV-β-galactosidase in all cases. pCI-B14 was transfected where indicated and the amount (ng) per well is shown. The total amount of DNA applied in each reaction was adjusted to 400 ng using pCI empty vector. These transfected cells were stimulated with 100 ng/ml of IL-1β, TNFα, IFNα, or 50 ng/ml of PMA for 8 h as indicated. Luciferase activity was measured and normalized to β-galactosidase intensity in the same well in triplicate wells. (E) HEK 293 cells were co-transfected with pRL-TK and reporter plasmid for NF-κB (□), ISRE (▪), and plasmids expressing GFP, A20, and B14. These cells were stimulated with 5 μg/ml of poly(I:C) for 12 h and then were lysed to measure luciferase activity using Dual-specific luciferase assay kit (Promega). Differences in the fold activation of the indicated reporter in cells transfected with the empty vector and cells expressing B14 were analysed by Student's t-test and the p-values are indicated: * p < 0.05 or ** p < 0.01.
Mentions: To investigate the effect of B14 on NF-κB activation, a plasmid containing a luciferase reporter gene linked to a NF-κB-dependent promoter was transfected into HeLa cells and these cells were stimulated with IL-1β (Figure 1A), TNFα (Figure 1A), or PMA (Figure 1B). Luciferase activity was increased greatly by addition of each stimulant but the level reached was reduced in a dose-dependent manner in the presence of B14. Similar findings were observed using HEK 293 cells (unpublished data). Moreover, B14 decreased poly (I:C)-induced NF-κB dramatically (p-value = 0.0006; 95% decrease) (Figure 1E). In contrast, B14 did not reduce luciferase activity from ISRE (Figure 1C) and AP-1 (Figure 1D) reporter genes induced by IFNα and PMA, respectively. Notably, B14 increased PMA-induced AP-1 activity slightly (p = 0.02; 1.5-fold increase; Figure 1D). We also observed a small but significant reduction in poly (I:C)-induced ISRE activity in the presence of B14 (p-value = 0.01; 29% decrease; Figure 1E). However, it is uncertain if these relatively small changes seen with these reporter assays are relevant biologically. As a control we also expressed A20, a de-ubiquitinating enzyme that downregulates NF-κB and IRF3 [39–41] and observed strong inhibition of both pathways (Figure 1E).

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