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Vaccinia virus inhibits NF-κB-dependent gene expression downstream of p65 translocation.

Sumner RP, Maluquer de Motes C, Veyer DL, Smith GL - J. Virol. (2013)

Bottom Line: However, despite this translocation, vv811ΔA49 still inhibited TNF-α- and IL-1β-induced NF-κB-dependent reporter gene expression and the transcription and production of cytokines induced by these agonists.This inhibition did not require late viral gene expression.These findings indicate the presence of another inhibitor of NF-κB that is expressed early during infection and acts by a novel mechanism downstream of p65 translocation into the nucleus.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, University of Cambridge, Cambridge, United Kingdom.

ABSTRACT
The transcription factor nuclear factor kappa light-chain enhancer of activated B cells (NF-κB) plays a critical role in host defense against viral infection by inducing the production of proinflammatory mediators and type I interferon. Consequently, viruses have evolved many mechanisms to block its activation. The poxvirus vaccinia virus (VACV) encodes numerous inhibitors of NF-κB activation that target multiple points in the signaling pathway. A derivative of VACV strain Copenhagen, called vv811, lacking 55 open reading frames in the left and right terminal regions of the genome was reported to still inhibit NF-κB activation downstream of tumor necrosis factor alpha (TNF-α) and interleukin-1β (IL-1β), suggesting the presence of one or more additional inhibitors. In this study, we constructed a recombinant vv811 lacking the recently described NF-κB inhibitor A49 (vv811ΔA49), yielding a virus that lacked all currently described inhibitors downstream of TNF-α and IL-1β. Unlike vv811, vv811ΔA49 no longer inhibited degradation of the phosphorylated inhibitor of κBα and p65 translocated into the nucleus. However, despite this translocation, vv811ΔA49 still inhibited TNF-α- and IL-1β-induced NF-κB-dependent reporter gene expression and the transcription and production of cytokines induced by these agonists. This inhibition did not require late viral gene expression. These findings indicate the presence of another inhibitor of NF-κB that is expressed early during infection and acts by a novel mechanism downstream of p65 translocation into the nucleus.

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vv811ΔA49 inhibits TNF-α- and IL-1β-induced gene transcription and protein expression. A549 cells were mock infected or infected in triplicate for 6 h with 5 PFU per cell of the indicated viruses and then stimulated for 4 to 6 h with TNF-α (50 ng/ml) or IL-1β (25 ng/ml) or nonstimulated (NS) as a control by incubation with medium alone. The cell supernatant was carefully removed for ELISA, and the cells were lysed for RNA extraction. cDNA was then synthesized and used for quantitative PCR analysis. Expression of the genes for CCL-5 (a), CCL-2 (b), ICAM-1 (c), MxA (e), and IL-6 (f) was normalized to that of the gene for an internal control (GAPDH). These values were then normalized to those for the nonstimulated mock-infected cells, yielding the fold induction. The levels of the CCL-5 (d) and IL-6 (g) proteins in the cell supernatants were measured by ELISA. Data are shown as the mean ± SD and are representative of those from three experimental repeats. Significant differences between infected cells and the mock-infected control, determined using an unpaired Student's t test, are shown (*, P < 0.05; **, P < 0.01; ***, P < 0.001).
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Figure 6: vv811ΔA49 inhibits TNF-α- and IL-1β-induced gene transcription and protein expression. A549 cells were mock infected or infected in triplicate for 6 h with 5 PFU per cell of the indicated viruses and then stimulated for 4 to 6 h with TNF-α (50 ng/ml) or IL-1β (25 ng/ml) or nonstimulated (NS) as a control by incubation with medium alone. The cell supernatant was carefully removed for ELISA, and the cells were lysed for RNA extraction. cDNA was then synthesized and used for quantitative PCR analysis. Expression of the genes for CCL-5 (a), CCL-2 (b), ICAM-1 (c), MxA (e), and IL-6 (f) was normalized to that of the gene for an internal control (GAPDH). These values were then normalized to those for the nonstimulated mock-infected cells, yielding the fold induction. The levels of the CCL-5 (d) and IL-6 (g) proteins in the cell supernatants were measured by ELISA. Data are shown as the mean ± SD and are representative of those from three experimental repeats. Significant differences between infected cells and the mock-infected control, determined using an unpaired Student's t test, are shown (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

Mentions: The inhibitory activity of the vv811 recombinant viruses was tested further by real-time PCR and ELISA analyses on infected and TNF-α/IL-1β-stimulated A549 cells. Consistent with reporter gene assay data, both vv811WT and vv811ΔA49 inhibited the transcription of the NF-κB-responsive genes for CCL-5 (Fig. 6a), CCL-2 (Fig. 6b), and intercellular adhesion molecule 1 (ICAM-1) (Fig. 6c) in response to both TNF-α and IL-1β. Furthermore, the level of CCL-5 protein production by these cells was inhibited by both viruses (Fig. 6d). Transcription of the viral DNA polymerase in these samples was also measured by real-time PCR and was found to be similar for the two viruses, indicating that the cells were infected with equivalent titers (data not shown). Upon further analysis of other NF-κB-responsive genes, it was noted that although vv811WT inhibited MxA (Fig. 6e) and IL-6 (Fig. 6f) gene transcription, vv811ΔA49 failed to do so, suggesting that some genes are more susceptible to the NF-κB-inhibitory activity of this virus. These results for IL-6 were confirmed at the protein level by ELISA (Fig. 6g). Importantly, these data demonstrate that the inhibition of NF-κB-dependent gene expression and protein production by vv811ΔA49 is not due to a general translational shutoff by the virus, because production of IL-6 in vv811ΔA49-infected cells was equivalent to that in mock-infected cells. On the other hand, production of IL-6 was inhibited strongly by Copenhagen infection (Fig. 6g).


Vaccinia virus inhibits NF-κB-dependent gene expression downstream of p65 translocation.

Sumner RP, Maluquer de Motes C, Veyer DL, Smith GL - J. Virol. (2013)

vv811ΔA49 inhibits TNF-α- and IL-1β-induced gene transcription and protein expression. A549 cells were mock infected or infected in triplicate for 6 h with 5 PFU per cell of the indicated viruses and then stimulated for 4 to 6 h with TNF-α (50 ng/ml) or IL-1β (25 ng/ml) or nonstimulated (NS) as a control by incubation with medium alone. The cell supernatant was carefully removed for ELISA, and the cells were lysed for RNA extraction. cDNA was then synthesized and used for quantitative PCR analysis. Expression of the genes for CCL-5 (a), CCL-2 (b), ICAM-1 (c), MxA (e), and IL-6 (f) was normalized to that of the gene for an internal control (GAPDH). These values were then normalized to those for the nonstimulated mock-infected cells, yielding the fold induction. The levels of the CCL-5 (d) and IL-6 (g) proteins in the cell supernatants were measured by ELISA. Data are shown as the mean ± SD and are representative of those from three experimental repeats. Significant differences between infected cells and the mock-infected control, determined using an unpaired Student's t test, are shown (*, P < 0.05; **, P < 0.01; ***, P < 0.001).
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Figure 6: vv811ΔA49 inhibits TNF-α- and IL-1β-induced gene transcription and protein expression. A549 cells were mock infected or infected in triplicate for 6 h with 5 PFU per cell of the indicated viruses and then stimulated for 4 to 6 h with TNF-α (50 ng/ml) or IL-1β (25 ng/ml) or nonstimulated (NS) as a control by incubation with medium alone. The cell supernatant was carefully removed for ELISA, and the cells were lysed for RNA extraction. cDNA was then synthesized and used for quantitative PCR analysis. Expression of the genes for CCL-5 (a), CCL-2 (b), ICAM-1 (c), MxA (e), and IL-6 (f) was normalized to that of the gene for an internal control (GAPDH). These values were then normalized to those for the nonstimulated mock-infected cells, yielding the fold induction. The levels of the CCL-5 (d) and IL-6 (g) proteins in the cell supernatants were measured by ELISA. Data are shown as the mean ± SD and are representative of those from three experimental repeats. Significant differences between infected cells and the mock-infected control, determined using an unpaired Student's t test, are shown (*, P < 0.05; **, P < 0.01; ***, P < 0.001).
Mentions: The inhibitory activity of the vv811 recombinant viruses was tested further by real-time PCR and ELISA analyses on infected and TNF-α/IL-1β-stimulated A549 cells. Consistent with reporter gene assay data, both vv811WT and vv811ΔA49 inhibited the transcription of the NF-κB-responsive genes for CCL-5 (Fig. 6a), CCL-2 (Fig. 6b), and intercellular adhesion molecule 1 (ICAM-1) (Fig. 6c) in response to both TNF-α and IL-1β. Furthermore, the level of CCL-5 protein production by these cells was inhibited by both viruses (Fig. 6d). Transcription of the viral DNA polymerase in these samples was also measured by real-time PCR and was found to be similar for the two viruses, indicating that the cells were infected with equivalent titers (data not shown). Upon further analysis of other NF-κB-responsive genes, it was noted that although vv811WT inhibited MxA (Fig. 6e) and IL-6 (Fig. 6f) gene transcription, vv811ΔA49 failed to do so, suggesting that some genes are more susceptible to the NF-κB-inhibitory activity of this virus. These results for IL-6 were confirmed at the protein level by ELISA (Fig. 6g). Importantly, these data demonstrate that the inhibition of NF-κB-dependent gene expression and protein production by vv811ΔA49 is not due to a general translational shutoff by the virus, because production of IL-6 in vv811ΔA49-infected cells was equivalent to that in mock-infected cells. On the other hand, production of IL-6 was inhibited strongly by Copenhagen infection (Fig. 6g).

Bottom Line: However, despite this translocation, vv811ΔA49 still inhibited TNF-α- and IL-1β-induced NF-κB-dependent reporter gene expression and the transcription and production of cytokines induced by these agonists.This inhibition did not require late viral gene expression.These findings indicate the presence of another inhibitor of NF-κB that is expressed early during infection and acts by a novel mechanism downstream of p65 translocation into the nucleus.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, University of Cambridge, Cambridge, United Kingdom.

ABSTRACT
The transcription factor nuclear factor kappa light-chain enhancer of activated B cells (NF-κB) plays a critical role in host defense against viral infection by inducing the production of proinflammatory mediators and type I interferon. Consequently, viruses have evolved many mechanisms to block its activation. The poxvirus vaccinia virus (VACV) encodes numerous inhibitors of NF-κB activation that target multiple points in the signaling pathway. A derivative of VACV strain Copenhagen, called vv811, lacking 55 open reading frames in the left and right terminal regions of the genome was reported to still inhibit NF-κB activation downstream of tumor necrosis factor alpha (TNF-α) and interleukin-1β (IL-1β), suggesting the presence of one or more additional inhibitors. In this study, we constructed a recombinant vv811 lacking the recently described NF-κB inhibitor A49 (vv811ΔA49), yielding a virus that lacked all currently described inhibitors downstream of TNF-α and IL-1β. Unlike vv811, vv811ΔA49 no longer inhibited degradation of the phosphorylated inhibitor of κBα and p65 translocated into the nucleus. However, despite this translocation, vv811ΔA49 still inhibited TNF-α- and IL-1β-induced NF-κB-dependent reporter gene expression and the transcription and production of cytokines induced by these agonists. This inhibition did not require late viral gene expression. These findings indicate the presence of another inhibitor of NF-κB that is expressed early during infection and acts by a novel mechanism downstream of p65 translocation into the nucleus.

Show MeSH
Related in: MedlinePlus