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Termination of NF-kappaB activity through a gammaherpesvirus protein that assembles an EC5S ubiquitin-ligase.

Rodrigues L, Filipe J, Seldon MP, Fonseca L, Anrather J, Soares MP, Simas JP - EMBO J. (2009)

Bottom Line: Here, we show that the ORF73 protein encoded by the murid herpesvirus-4 (MuHV-4) inhibits host nuclear factor-kappa B (NF-kappaB) transcriptional activity through poly-ubiquitination and subsequent proteasomal-dependent nuclear degradation of the NF-kappaB family member p65/RelA.The mechanism involves the assembly of an ElonginC/Cullin5/SOCS (suppressors of cytokine signalling)-like complex, mediated by an unconventional viral SOCS-box motif present in ORF73.These findings demonstrate that viral inhibition of NF-kappaB activity in latently infected GC centroblasts is critical for the establishment of a gammaherpesvirus persistent infection, underscoring the physiological importance of proteasomal degradation of RelA/NF-kappaB as a regulatory mechanism of this signalling pathway.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Microbiologia e Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Lisboa, Portugal.

ABSTRACT
Host colonisation by lymphotropic gammaherpesviruses depends critically on the expansion of viral genomes in germinal centre (GC) B cells. Yet, host and virus molecular mechanisms involved in driving such proliferation remain largely unknown. Here, we show that the ORF73 protein encoded by the murid herpesvirus-4 (MuHV-4) inhibits host nuclear factor-kappa B (NF-kappaB) transcriptional activity through poly-ubiquitination and subsequent proteasomal-dependent nuclear degradation of the NF-kappaB family member p65/RelA. The mechanism involves the assembly of an ElonginC/Cullin5/SOCS (suppressors of cytokine signalling)-like complex, mediated by an unconventional viral SOCS-box motif present in ORF73. Functional deletion of this SOCS-box motif ablated NF-kappaB inhibitory effect of ORF73, suppressed MuHV-4 expansion in GC B cells and prevented MuHV-4 persistent infection in mice. These findings demonstrate that viral inhibition of NF-kappaB activity in latently infected GC centroblasts is critical for the establishment of a gammaherpesvirus persistent infection, underscoring the physiological importance of proteasomal degradation of RelA/NF-kappaB as a regulatory mechanism of this signalling pathway.

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RelA nuclear levels are diminished in ORF73-expressing cells. (A) Reduction of NF-κB binding to κB sequences in ORF73-expressing cells correlates with diminished levels of nuclear RelA. HEK 293T cells were transiently transfected with an ORF73 expression plasmid, or control transfected. After 24 h of culture, cells were stimulated with TNF for 40 min, or left unstimulated. Nuclear extracts were prepared and subjected to electromobility shift assays (EMSAs) using the NF-κB consensus oligonucleotide from the immunoglobulin promoter region. The percentage of NF-κB binding present in each condition is shown. Nuclear extracts were analysed by immunoblotting to determine the input of RelA and ORF73 present in each binding reaction. Densitometry analysis of RelA nuclear levels present in each experimental condition, normalised to the nuclear protein LaminB, is shown. (B) ORF73 protein does not bind to κB sequences. Nuclear extracts from HEK 293T cells expressing ORF73 were subjected to a supershift assay using the specified antibodies. (C) Immunofluorescence analysis of ORF73-expressing cells. HEK 293T cells were stimulated with 50 ng/ml of TNF for the times indicated and then subjected to immunostaining with anti-RelA and anti-ORF73 antibodies. −, without; +, with; α, anti; WB, western blotting.
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f2: RelA nuclear levels are diminished in ORF73-expressing cells. (A) Reduction of NF-κB binding to κB sequences in ORF73-expressing cells correlates with diminished levels of nuclear RelA. HEK 293T cells were transiently transfected with an ORF73 expression plasmid, or control transfected. After 24 h of culture, cells were stimulated with TNF for 40 min, or left unstimulated. Nuclear extracts were prepared and subjected to electromobility shift assays (EMSAs) using the NF-κB consensus oligonucleotide from the immunoglobulin promoter region. The percentage of NF-κB binding present in each condition is shown. Nuclear extracts were analysed by immunoblotting to determine the input of RelA and ORF73 present in each binding reaction. Densitometry analysis of RelA nuclear levels present in each experimental condition, normalised to the nuclear protein LaminB, is shown. (B) ORF73 protein does not bind to κB sequences. Nuclear extracts from HEK 293T cells expressing ORF73 were subjected to a supershift assay using the specified antibodies. (C) Immunofluorescence analysis of ORF73-expressing cells. HEK 293T cells were stimulated with 50 ng/ml of TNF for the times indicated and then subjected to immunostaining with anti-RelA and anti-ORF73 antibodies. −, without; +, with; α, anti; WB, western blotting.

Mentions: We proceeded to investigate whether ORF73 would be functioning at the nuclear level by impairing the binding of NF-κB to DNA κB sites. We performed electromobility shift assays (EMSA) in the presence of oligonucleotides bearing the consensus κB site from the immunoglobulin promoter region. Cells were transiently transfected with ORF73, or control transfected, and NF-κB activation was induced by TNF. As illustrated in Figure 2A, NF-κB DNA-binding levels were reduced in ORF73-expressing cells. This decrease did not reflect ORF73 interference with NF-κB DNA binding to κB sites but rather correlated with diminished levels of nuclear RelA, as assessed by immunoblotting (Figure 2A, second panel, compare lanes 2 and 4, and densitometry analysis). To assess whether ORF73 could itself bind to κB sites, a supershift assay was performed in the presence of antibodies directed against RelA (positive control), Actin (unrelated antibody) or ORF73. The addition of anti-RelA antibody to the binding reaction caused a supershift in the NF-κB/oligonucleotide complex, which indicates that these complexes contain RelA protein (Figure 2B, lane 3). In contrast, in the presence of anti-ORF73 serum no supershift was observed, as well as when an unrelated antibody was added to the binding reactions (Figure 2B, lanes 2 and 4), indicating that ORF73 does not bind to κB sites. Taken together, these data raise the hypothesis that ORF73 may be exerting its inhibitory activity towards NF-κB by promoting its degradation in the nucleus.


Termination of NF-kappaB activity through a gammaherpesvirus protein that assembles an EC5S ubiquitin-ligase.

Rodrigues L, Filipe J, Seldon MP, Fonseca L, Anrather J, Soares MP, Simas JP - EMBO J. (2009)

RelA nuclear levels are diminished in ORF73-expressing cells. (A) Reduction of NF-κB binding to κB sequences in ORF73-expressing cells correlates with diminished levels of nuclear RelA. HEK 293T cells were transiently transfected with an ORF73 expression plasmid, or control transfected. After 24 h of culture, cells were stimulated with TNF for 40 min, or left unstimulated. Nuclear extracts were prepared and subjected to electromobility shift assays (EMSAs) using the NF-κB consensus oligonucleotide from the immunoglobulin promoter region. The percentage of NF-κB binding present in each condition is shown. Nuclear extracts were analysed by immunoblotting to determine the input of RelA and ORF73 present in each binding reaction. Densitometry analysis of RelA nuclear levels present in each experimental condition, normalised to the nuclear protein LaminB, is shown. (B) ORF73 protein does not bind to κB sequences. Nuclear extracts from HEK 293T cells expressing ORF73 were subjected to a supershift assay using the specified antibodies. (C) Immunofluorescence analysis of ORF73-expressing cells. HEK 293T cells were stimulated with 50 ng/ml of TNF for the times indicated and then subjected to immunostaining with anti-RelA and anti-ORF73 antibodies. −, without; +, with; α, anti; WB, western blotting.
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f2: RelA nuclear levels are diminished in ORF73-expressing cells. (A) Reduction of NF-κB binding to κB sequences in ORF73-expressing cells correlates with diminished levels of nuclear RelA. HEK 293T cells were transiently transfected with an ORF73 expression plasmid, or control transfected. After 24 h of culture, cells were stimulated with TNF for 40 min, or left unstimulated. Nuclear extracts were prepared and subjected to electromobility shift assays (EMSAs) using the NF-κB consensus oligonucleotide from the immunoglobulin promoter region. The percentage of NF-κB binding present in each condition is shown. Nuclear extracts were analysed by immunoblotting to determine the input of RelA and ORF73 present in each binding reaction. Densitometry analysis of RelA nuclear levels present in each experimental condition, normalised to the nuclear protein LaminB, is shown. (B) ORF73 protein does not bind to κB sequences. Nuclear extracts from HEK 293T cells expressing ORF73 were subjected to a supershift assay using the specified antibodies. (C) Immunofluorescence analysis of ORF73-expressing cells. HEK 293T cells were stimulated with 50 ng/ml of TNF for the times indicated and then subjected to immunostaining with anti-RelA and anti-ORF73 antibodies. −, without; +, with; α, anti; WB, western blotting.
Mentions: We proceeded to investigate whether ORF73 would be functioning at the nuclear level by impairing the binding of NF-κB to DNA κB sites. We performed electromobility shift assays (EMSA) in the presence of oligonucleotides bearing the consensus κB site from the immunoglobulin promoter region. Cells were transiently transfected with ORF73, or control transfected, and NF-κB activation was induced by TNF. As illustrated in Figure 2A, NF-κB DNA-binding levels were reduced in ORF73-expressing cells. This decrease did not reflect ORF73 interference with NF-κB DNA binding to κB sites but rather correlated with diminished levels of nuclear RelA, as assessed by immunoblotting (Figure 2A, second panel, compare lanes 2 and 4, and densitometry analysis). To assess whether ORF73 could itself bind to κB sites, a supershift assay was performed in the presence of antibodies directed against RelA (positive control), Actin (unrelated antibody) or ORF73. The addition of anti-RelA antibody to the binding reaction caused a supershift in the NF-κB/oligonucleotide complex, which indicates that these complexes contain RelA protein (Figure 2B, lane 3). In contrast, in the presence of anti-ORF73 serum no supershift was observed, as well as when an unrelated antibody was added to the binding reactions (Figure 2B, lanes 2 and 4), indicating that ORF73 does not bind to κB sites. Taken together, these data raise the hypothesis that ORF73 may be exerting its inhibitory activity towards NF-κB by promoting its degradation in the nucleus.

Bottom Line: Here, we show that the ORF73 protein encoded by the murid herpesvirus-4 (MuHV-4) inhibits host nuclear factor-kappa B (NF-kappaB) transcriptional activity through poly-ubiquitination and subsequent proteasomal-dependent nuclear degradation of the NF-kappaB family member p65/RelA.The mechanism involves the assembly of an ElonginC/Cullin5/SOCS (suppressors of cytokine signalling)-like complex, mediated by an unconventional viral SOCS-box motif present in ORF73.These findings demonstrate that viral inhibition of NF-kappaB activity in latently infected GC centroblasts is critical for the establishment of a gammaherpesvirus persistent infection, underscoring the physiological importance of proteasomal degradation of RelA/NF-kappaB as a regulatory mechanism of this signalling pathway.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Microbiologia e Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Lisboa, Portugal.

ABSTRACT
Host colonisation by lymphotropic gammaherpesviruses depends critically on the expansion of viral genomes in germinal centre (GC) B cells. Yet, host and virus molecular mechanisms involved in driving such proliferation remain largely unknown. Here, we show that the ORF73 protein encoded by the murid herpesvirus-4 (MuHV-4) inhibits host nuclear factor-kappa B (NF-kappaB) transcriptional activity through poly-ubiquitination and subsequent proteasomal-dependent nuclear degradation of the NF-kappaB family member p65/RelA. The mechanism involves the assembly of an ElonginC/Cullin5/SOCS (suppressors of cytokine signalling)-like complex, mediated by an unconventional viral SOCS-box motif present in ORF73. Functional deletion of this SOCS-box motif ablated NF-kappaB inhibitory effect of ORF73, suppressed MuHV-4 expansion in GC B cells and prevented MuHV-4 persistent infection in mice. These findings demonstrate that viral inhibition of NF-kappaB activity in latently infected GC centroblasts is critical for the establishment of a gammaherpesvirus persistent infection, underscoring the physiological importance of proteasomal degradation of RelA/NF-kappaB as a regulatory mechanism of this signalling pathway.

Show MeSH
Related in: MedlinePlus