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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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SOCS-box disruption reverts ORF73 inhibition of NF-κB transcriptional activity. (A) ORF73–SOCS mutant is unable to promote RelA poly-ubiquitination. HEK 293T cells were transfected with the different combinations of plasmids (top). After culture, poly-ubiquitination of RelA was assessed as described in Figure 3A. −, without; +, with; α, anti; PD, pull down; TCL, total cellular lysates; WB, western blotting. (B) ORF73–SOCS mutant is unable to inhibit TNF-driven NF-κB transcriptional activity. HEK 293T cells were transfected with the NF-κB luciferase reporter together with the plasmids allowing the expression of the indicated proteins (bottom). Transfected cells were either stimulated with (open bars) or without (filled bars) 50 ng/ml of TNF. NF-κB activity associated with each condition was assayed using a luminometer. Error bars represent the standard deviations of the mean in three independent experiments.
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f6: SOCS-box disruption reverts ORF73 inhibition of NF-κB transcriptional activity. (A) ORF73–SOCS mutant is unable to promote RelA poly-ubiquitination. HEK 293T cells were transfected with the different combinations of plasmids (top). After culture, poly-ubiquitination of RelA was assessed as described in Figure 3A. −, without; +, with; α, anti; PD, pull down; TCL, total cellular lysates; WB, western blotting. (B) ORF73–SOCS mutant is unable to inhibit TNF-driven NF-κB transcriptional activity. HEK 293T cells were transfected with the NF-κB luciferase reporter together with the plasmids allowing the expression of the indicated proteins (bottom). Transfected cells were either stimulated with (open bars) or without (filled bars) 50 ng/ml of TNF. NF-κB activity associated with each condition was assayed using a luminometer. Error bars represent the standard deviations of the mean in three independent experiments.

Mentions: Further experiments showed that the ORF73–SOCS mutant was unable to potentiate RelA poly-ubiquitination (Figure 6A), confirming that the amino-acid substitutions introduced disrupted the ORF73 SOCS-box domain, and that the presence of this motif is essential for ORF73 to function as a mediator of RelA poly-ubiquitination. To investigate whether ORF73–SOCS failure to promote RelA poly-ubiquitination would be reflected by its inability to suppress NF-κB transcriptional activity, we performed gene reporter assays in ORF73–SOCS-expressing cells. The effect of the expression of ORF73–SOCS on NF-κB transcriptional activity, in conditions of TNF stimulation, was assessed by quantifying the luciferase activity present in each experimental condition (Figure 6B). We observed that disruption of the ORF73–SOCS-box motif impaired ORF73 ability to inhibit NF-κB-mediated signalling.


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)

SOCS-box disruption reverts ORF73 inhibition of NF-κB transcriptional activity. (A) ORF73–SOCS mutant is unable to promote RelA poly-ubiquitination. HEK 293T cells were transfected with the different combinations of plasmids (top). After culture, poly-ubiquitination of RelA was assessed as described in Figure 3A. −, without; +, with; α, anti; PD, pull down; TCL, total cellular lysates; WB, western blotting. (B) ORF73–SOCS mutant is unable to inhibit TNF-driven NF-κB transcriptional activity. HEK 293T cells were transfected with the NF-κB luciferase reporter together with the plasmids allowing the expression of the indicated proteins (bottom). Transfected cells were either stimulated with (open bars) or without (filled bars) 50 ng/ml of TNF. NF-κB activity associated with each condition was assayed using a luminometer. Error bars represent the standard deviations of the mean in three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2664658&req=5

f6: SOCS-box disruption reverts ORF73 inhibition of NF-κB transcriptional activity. (A) ORF73–SOCS mutant is unable to promote RelA poly-ubiquitination. HEK 293T cells were transfected with the different combinations of plasmids (top). After culture, poly-ubiquitination of RelA was assessed as described in Figure 3A. −, without; +, with; α, anti; PD, pull down; TCL, total cellular lysates; WB, western blotting. (B) ORF73–SOCS mutant is unable to inhibit TNF-driven NF-κB transcriptional activity. HEK 293T cells were transfected with the NF-κB luciferase reporter together with the plasmids allowing the expression of the indicated proteins (bottom). Transfected cells were either stimulated with (open bars) or without (filled bars) 50 ng/ml of TNF. NF-κB activity associated with each condition was assayed using a luminometer. Error bars represent the standard deviations of the mean in three independent experiments.
Mentions: Further experiments showed that the ORF73–SOCS mutant was unable to potentiate RelA poly-ubiquitination (Figure 6A), confirming that the amino-acid substitutions introduced disrupted the ORF73 SOCS-box domain, and that the presence of this motif is essential for ORF73 to function as a mediator of RelA poly-ubiquitination. To investigate whether ORF73–SOCS failure to promote RelA poly-ubiquitination would be reflected by its inability to suppress NF-κB transcriptional activity, we performed gene reporter assays in ORF73–SOCS-expressing cells. The effect of the expression of ORF73–SOCS on NF-κB transcriptional activity, in conditions of TNF stimulation, was assessed by quantifying the luciferase activity present in each experimental condition (Figure 6B). We observed that disruption of the ORF73–SOCS-box motif impaired ORF73 ability to inhibit NF-κB-mediated signalling.

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