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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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Construction of vv811 lacking A49R. (a) Schematic of the genome structure of VACV strains vv811 and Copenhagen (Cop), with the position of the known intracellular NF-κB inhibitors downstream of TNF-α or IL-1β indicated. ITR, inverted terminal repeat. (b) The A49R gene from vv811 was deleted by transient dominant selection (see Materials and Methods) to yield vv811ΔA49. A matching wild-type virus (vv811WT) was isolated from the same intermediate virus. The phenotype of the resolved viruses was analyzed by PCR from proteinase K-treated infected BSC-1 cell lysates using primers annealing to the flanking regions of A49R. The resulting PCR product sizes were compared to those obtained from the plasmid templates (pΔA49 and pA49Rev). Molecular mass markers (in kbp) are indicated on the left. (c) Expression of A49 by the recombinant vv811 viruses in lysates of BSC-1 cells mock infected or infected for 16 h with 2 PFU per cell was analyzed by immunoblotting using an anti-A49 polyclonal antiserum and anti-tubulin and anti-D8 antibodies as controls. Molecular mass markers (in kDa) are indicated on the right. (d) A549 cells were infected in duplicate with vv811 recombinant viruses or Copenhagen at 5 PFU per cell. Cells were then harvested at 0 h and 24 h, and the infectious titer of intracellular virus was determined by plaque assay on BSC-1 cells. (e) BSC-1 cells were infected with 50 PFU per well of vv811 recombinant viruses or Copenhagen, and plaques were allowed to form for 5 days. Cells were then stained with crystal violet, and the plaques were imaged and measured. Data are represented as the mean plaque radius (μm) ± SD.
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Figure 1: Construction of vv811 lacking A49R. (a) Schematic of the genome structure of VACV strains vv811 and Copenhagen (Cop), with the position of the known intracellular NF-κB inhibitors downstream of TNF-α or IL-1β indicated. ITR, inverted terminal repeat. (b) The A49R gene from vv811 was deleted by transient dominant selection (see Materials and Methods) to yield vv811ΔA49. A matching wild-type virus (vv811WT) was isolated from the same intermediate virus. The phenotype of the resolved viruses was analyzed by PCR from proteinase K-treated infected BSC-1 cell lysates using primers annealing to the flanking regions of A49R. The resulting PCR product sizes were compared to those obtained from the plasmid templates (pΔA49 and pA49Rev). Molecular mass markers (in kbp) are indicated on the left. (c) Expression of A49 by the recombinant vv811 viruses in lysates of BSC-1 cells mock infected or infected for 16 h with 2 PFU per cell was analyzed by immunoblotting using an anti-A49 polyclonal antiserum and anti-tubulin and anti-D8 antibodies as controls. Molecular mass markers (in kDa) are indicated on the right. (d) A549 cells were infected in duplicate with vv811 recombinant viruses or Copenhagen at 5 PFU per cell. Cells were then harvested at 0 h and 24 h, and the infectious titer of intracellular virus was determined by plaque assay on BSC-1 cells. (e) BSC-1 cells were infected with 50 PFU per well of vv811 recombinant viruses or Copenhagen, and plaques were allowed to form for 5 days. Cells were then stained with crystal violet, and the plaques were imaged and measured. Data are represented as the mean plaque radius (μm) ± SD.

Mentions: The deletion strain of VACV, vv811, lacks 55 ORFs in the left and right terminal regions of the genome. Fagan-Garcia and Barry (2011) demonstrated that although vv811 lacked all known TNF-α-induced NF-κB inhibitors (Fig. 1a), this virus still inhibited NF-κB activation following stimulation of cells with TNF-α or IL-1β (30). Since then, two further VACV proteins have been characterized to inhibit NF-κB activation: C4 (21) and A49 (24). Although C4L is not present in the genome of vv811, A49R is (Fig. 1a), leading us to hypothesize that this protein was the cause of the NF-κB-inhibitory activity of vv811. To investigate this possibility, a mutant of vv811 lacking the A49R gene (vv811ΔA49), as well as a matching wild-type (WT) virus that was derived from the same intermediate virus (vv811WT), was generated by transient dominant selection (31) (see Materials and Methods). The genotype of the recombinant viruses was assessed by PCR analysis using primers that annealed to the flanking regions of the A49R gene (24). As expected, a product of approximately 600 bp (corresponding to the flanking regions only) was amplified from pΔA49- and vv811ΔA49-infected cells, indicating the loss of the A49R gene (Fig. 1b).


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)

Construction of vv811 lacking A49R. (a) Schematic of the genome structure of VACV strains vv811 and Copenhagen (Cop), with the position of the known intracellular NF-κB inhibitors downstream of TNF-α or IL-1β indicated. ITR, inverted terminal repeat. (b) The A49R gene from vv811 was deleted by transient dominant selection (see Materials and Methods) to yield vv811ΔA49. A matching wild-type virus (vv811WT) was isolated from the same intermediate virus. The phenotype of the resolved viruses was analyzed by PCR from proteinase K-treated infected BSC-1 cell lysates using primers annealing to the flanking regions of A49R. The resulting PCR product sizes were compared to those obtained from the plasmid templates (pΔA49 and pA49Rev). Molecular mass markers (in kbp) are indicated on the left. (c) Expression of A49 by the recombinant vv811 viruses in lysates of BSC-1 cells mock infected or infected for 16 h with 2 PFU per cell was analyzed by immunoblotting using an anti-A49 polyclonal antiserum and anti-tubulin and anti-D8 antibodies as controls. Molecular mass markers (in kDa) are indicated on the right. (d) A549 cells were infected in duplicate with vv811 recombinant viruses or Copenhagen at 5 PFU per cell. Cells were then harvested at 0 h and 24 h, and the infectious titer of intracellular virus was determined by plaque assay on BSC-1 cells. (e) BSC-1 cells were infected with 50 PFU per well of vv811 recombinant viruses or Copenhagen, and plaques were allowed to form for 5 days. Cells were then stained with crystal violet, and the plaques were imaged and measured. Data are represented as the mean plaque radius (μm) ± SD.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 1: Construction of vv811 lacking A49R. (a) Schematic of the genome structure of VACV strains vv811 and Copenhagen (Cop), with the position of the known intracellular NF-κB inhibitors downstream of TNF-α or IL-1β indicated. ITR, inverted terminal repeat. (b) The A49R gene from vv811 was deleted by transient dominant selection (see Materials and Methods) to yield vv811ΔA49. A matching wild-type virus (vv811WT) was isolated from the same intermediate virus. The phenotype of the resolved viruses was analyzed by PCR from proteinase K-treated infected BSC-1 cell lysates using primers annealing to the flanking regions of A49R. The resulting PCR product sizes were compared to those obtained from the plasmid templates (pΔA49 and pA49Rev). Molecular mass markers (in kbp) are indicated on the left. (c) Expression of A49 by the recombinant vv811 viruses in lysates of BSC-1 cells mock infected or infected for 16 h with 2 PFU per cell was analyzed by immunoblotting using an anti-A49 polyclonal antiserum and anti-tubulin and anti-D8 antibodies as controls. Molecular mass markers (in kDa) are indicated on the right. (d) A549 cells were infected in duplicate with vv811 recombinant viruses or Copenhagen at 5 PFU per cell. Cells were then harvested at 0 h and 24 h, and the infectious titer of intracellular virus was determined by plaque assay on BSC-1 cells. (e) BSC-1 cells were infected with 50 PFU per well of vv811 recombinant viruses or Copenhagen, and plaques were allowed to form for 5 days. Cells were then stained with crystal violet, and the plaques were imaged and measured. Data are represented as the mean plaque radius (μm) ± SD.
Mentions: The deletion strain of VACV, vv811, lacks 55 ORFs in the left and right terminal regions of the genome. Fagan-Garcia and Barry (2011) demonstrated that although vv811 lacked all known TNF-α-induced NF-κB inhibitors (Fig. 1a), this virus still inhibited NF-κB activation following stimulation of cells with TNF-α or IL-1β (30). Since then, two further VACV proteins have been characterized to inhibit NF-κB activation: C4 (21) and A49 (24). Although C4L is not present in the genome of vv811, A49R is (Fig. 1a), leading us to hypothesize that this protein was the cause of the NF-κB-inhibitory activity of vv811. To investigate this possibility, a mutant of vv811 lacking the A49R gene (vv811ΔA49), as well as a matching wild-type (WT) virus that was derived from the same intermediate virus (vv811WT), was generated by transient dominant selection (31) (see Materials and Methods). The genotype of the recombinant viruses was assessed by PCR analysis using primers that annealed to the flanking regions of the A49R gene (24). As expected, a product of approximately 600 bp (corresponding to the flanking regions only) was amplified from pΔA49- and vv811ΔA49-infected cells, indicating the loss of the A49R gene (Fig. 1b).

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