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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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ORF73 mediates the assembly of an endogenous EC5S E3 ubiquitin-ligase to promote RelA poly-ubiquitination. (A, B) ORF73 protein co-immunoprecipitates with RelA, Cullin5 and ElonginC. HEK 293T cells were transiently transfected with the combinations of plasmids encoding the indicated proteins (top). After culture, cells were lysed and total cellular extracts were subjected to immunoprecipitation using an anti-ORF73 serum. Immunoprecipitates were analysed by immunoblotting using the indicated antibodies. In addition, representative aliquots of the total cellular lysates were used to detect the appropriate expression of RelA (A, bottom panel), Cullin5 (B, fifth panel), and ElonginC (B, bottom panel). (C, D) ElonginC and Cullin5 are required for ORF73-mediated poly-ubiquitination of RelA. (C) HEK 293T cells were transiently transfected with pools of small interfering (si) RNAs against ElonginC, Cullin5 or non-targeting oligonucleotides. After 48 h of culture, total cellular lysates were obtained and assayed for ElonginC and Cullin5 expression by immunoblotting. (D) ORF73-mediated ubiquitination of RelA is dependent on endogenous ElonginC and Cullin5 expression. HEK 293T cells were transiently transfected with the pools of siRNAs indicated above along with the indicated combinations of expression plasmids. Corresponding cellular lysates were obtained and subjected to Ni-NTA pull down as described in Figure 3A. −, without; +, with; α, anti; IP, immunoprecipitation; PD, pull down; TCL, total cellular lysates; WB, western blotting.
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f4: ORF73 mediates the assembly of an endogenous EC5S E3 ubiquitin-ligase to promote RelA poly-ubiquitination. (A, B) ORF73 protein co-immunoprecipitates with RelA, Cullin5 and ElonginC. HEK 293T cells were transiently transfected with the combinations of plasmids encoding the indicated proteins (top). After culture, cells were lysed and total cellular extracts were subjected to immunoprecipitation using an anti-ORF73 serum. Immunoprecipitates were analysed by immunoblotting using the indicated antibodies. In addition, representative aliquots of the total cellular lysates were used to detect the appropriate expression of RelA (A, bottom panel), Cullin5 (B, fifth panel), and ElonginC (B, bottom panel). (C, D) ElonginC and Cullin5 are required for ORF73-mediated poly-ubiquitination of RelA. (C) HEK 293T cells were transiently transfected with pools of small interfering (si) RNAs against ElonginC, Cullin5 or non-targeting oligonucleotides. After 48 h of culture, total cellular lysates were obtained and assayed for ElonginC and Cullin5 expression by immunoblotting. (D) ORF73-mediated ubiquitination of RelA is dependent on endogenous ElonginC and Cullin5 expression. HEK 293T cells were transiently transfected with the pools of siRNAs indicated above along with the indicated combinations of expression plasmids. Corresponding cellular lysates were obtained and subjected to Ni-NTA pull down as described in Figure 3A. −, without; +, with; α, anti; IP, immunoprecipitation; PD, pull down; TCL, total cellular lysates; WB, western blotting.

Mentions: Recently, LANA encoded by ORF73 from KSHV was also shown to possess E3 ubiquitin-ligase activity, acting as a SOCS protein responsible for substrate recognition and specificity (Cai et al, 2006). As E3 ubiquitin-ligases must interact physically with their targets to exert their function, we investigated whether ORF73 was associated with RelA in vivo. In cells overexpressing RelA and co-expressing ORF73, the latter protein was able to efficiently co-immunoprecipitate RelA (Figure 4A).


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)

ORF73 mediates the assembly of an endogenous EC5S E3 ubiquitin-ligase to promote RelA poly-ubiquitination. (A, B) ORF73 protein co-immunoprecipitates with RelA, Cullin5 and ElonginC. HEK 293T cells were transiently transfected with the combinations of plasmids encoding the indicated proteins (top). After culture, cells were lysed and total cellular extracts were subjected to immunoprecipitation using an anti-ORF73 serum. Immunoprecipitates were analysed by immunoblotting using the indicated antibodies. In addition, representative aliquots of the total cellular lysates were used to detect the appropriate expression of RelA (A, bottom panel), Cullin5 (B, fifth panel), and ElonginC (B, bottom panel). (C, D) ElonginC and Cullin5 are required for ORF73-mediated poly-ubiquitination of RelA. (C) HEK 293T cells were transiently transfected with pools of small interfering (si) RNAs against ElonginC, Cullin5 or non-targeting oligonucleotides. After 48 h of culture, total cellular lysates were obtained and assayed for ElonginC and Cullin5 expression by immunoblotting. (D) ORF73-mediated ubiquitination of RelA is dependent on endogenous ElonginC and Cullin5 expression. HEK 293T cells were transiently transfected with the pools of siRNAs indicated above along with the indicated combinations of expression plasmids. Corresponding cellular lysates were obtained and subjected to Ni-NTA pull down as described in Figure 3A. −, without; +, with; α, anti; IP, immunoprecipitation; PD, pull down; TCL, total cellular lysates; WB, western blotting.
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f4: ORF73 mediates the assembly of an endogenous EC5S E3 ubiquitin-ligase to promote RelA poly-ubiquitination. (A, B) ORF73 protein co-immunoprecipitates with RelA, Cullin5 and ElonginC. HEK 293T cells were transiently transfected with the combinations of plasmids encoding the indicated proteins (top). After culture, cells were lysed and total cellular extracts were subjected to immunoprecipitation using an anti-ORF73 serum. Immunoprecipitates were analysed by immunoblotting using the indicated antibodies. In addition, representative aliquots of the total cellular lysates were used to detect the appropriate expression of RelA (A, bottom panel), Cullin5 (B, fifth panel), and ElonginC (B, bottom panel). (C, D) ElonginC and Cullin5 are required for ORF73-mediated poly-ubiquitination of RelA. (C) HEK 293T cells were transiently transfected with pools of small interfering (si) RNAs against ElonginC, Cullin5 or non-targeting oligonucleotides. After 48 h of culture, total cellular lysates were obtained and assayed for ElonginC and Cullin5 expression by immunoblotting. (D) ORF73-mediated ubiquitination of RelA is dependent on endogenous ElonginC and Cullin5 expression. HEK 293T cells were transiently transfected with the pools of siRNAs indicated above along with the indicated combinations of expression plasmids. Corresponding cellular lysates were obtained and subjected to Ni-NTA pull down as described in Figure 3A. −, without; +, with; α, anti; IP, immunoprecipitation; PD, pull down; TCL, total cellular lysates; WB, western blotting.
Mentions: Recently, LANA encoded by ORF73 from KSHV was also shown to possess E3 ubiquitin-ligase activity, acting as a SOCS protein responsible for substrate recognition and specificity (Cai et al, 2006). As E3 ubiquitin-ligases must interact physically with their targets to exert their function, we investigated whether ORF73 was associated with RelA in vivo. In cells overexpressing RelA and co-expressing ORF73, the latter protein was able to efficiently co-immunoprecipitate RelA (Figure 4A).

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