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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–SOCS virus shows a strong latency deficit. (A) ORF73–SOCS recombinant virus exhibits normal replication in the lung. Wild-type BALB/c mice were intranasally infected with 104 p.f.u. of the indicated viruses. At the indicated days post-infection, lungs were removed and infectious viruses were titrated by plaque assay. (B) BALB/c mice were intranasally infected with 104 p.f.u. of the indicated viruses. At day 14 post-infection, latent viruses in spleens were titrated by infectious centre assay. Each point represents the titre of an individual mouse. The dashed line represents the limit of detection of the assay. (C) BALB/c mice were intranasally infected with 104 p.f.u. of the indicated viruses. At 14 days post-infection, reciprocal frequencies of viral infection in purified GC B cells (B220+/PNAhigh) were determined by limiting dilution and real-time PCR. Data were obtained from pools of five spleens per group. Bars represent the frequency of viral DNA-positive cells with 95% confidence intervals. (D) BALB/c mice were intranasally infected with 104 p.f.u. of the indicated viruses. At day 14 post-infection, spleens were removed and processed for in situ hybridisation using probes derived from viral miRNAs 1–6. Panels (a–c) show representative spleen sections from each group of viruses. All sections are magnified × 200.
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f7: ORF73–SOCS virus shows a strong latency deficit. (A) ORF73–SOCS recombinant virus exhibits normal replication in the lung. Wild-type BALB/c mice were intranasally infected with 104 p.f.u. of the indicated viruses. At the indicated days post-infection, lungs were removed and infectious viruses were titrated by plaque assay. (B) BALB/c mice were intranasally infected with 104 p.f.u. of the indicated viruses. At day 14 post-infection, latent viruses in spleens were titrated by infectious centre assay. Each point represents the titre of an individual mouse. The dashed line represents the limit of detection of the assay. (C) BALB/c mice were intranasally infected with 104 p.f.u. of the indicated viruses. At 14 days post-infection, reciprocal frequencies of viral infection in purified GC B cells (B220+/PNAhigh) were determined by limiting dilution and real-time PCR. Data were obtained from pools of five spleens per group. Bars represent the frequency of viral DNA-positive cells with 95% confidence intervals. (D) BALB/c mice were intranasally infected with 104 p.f.u. of the indicated viruses. At day 14 post-infection, spleens were removed and processed for in situ hybridisation using probes derived from viral miRNAs 1–6. Panels (a–c) show representative spleen sections from each group of viruses. All sections are magnified × 200.

Mentions: To directly investigate the biological relevance of inhibition of NF-κB signalling in gammaherpesvirus pathogenesis, we generated a recombinant MuHV-4 in which the ORF73 gene was modified to recapitulate the amino-acid substitutions of the ORF73 SOCS-box mutant, designated vSOCS. To assure that any phenotypic alteration in vSOCS was due to the engineered mutations in the SOCS-box and not from any spurious mutation introduced during mutagenesis, a second independent recombinant virus was engineered (vSOCSi). To characterise the role of the introduced mutations in a natural context of infection, we started to compare the kinetics of viral replication in vitro and during the acute phase of infection in lungs of Balb/c mice following intranasal inoculation. For comparative purposes, the viruses analysed included the vSOCS mutants alongside wild-type MuHV-4 (vWT) and a previously described (Fowler et al, 2003) ORF73 frameshift mutant (v73FS) that encodes only the 163 amino acids of the N-terminus of the protein. All these viruses were analysed for genome integrity (Supplementary Figure S1) and showed identical in vitro growth (Supplementary Figure S2), as well as normal replication in acutely infected lungs (Figure 7A). Next, we proceeded to investigate the role of the introduced mutations for the ability of MuHV-4 to induce the expansion of latency in GC B cells. To this end, we used three independent, but complementary, experimental assays: ex vivo explant co-culture assays to measure latent infection in total splenocytes, flow cytometry coupled to limiting dilution and real-time PCR to quantify the frequency of viral DNA-positive GC B cells, and in situ hybridisation analysis to identify virally infected cells within the spleen, as described earlier (Pires de Miranda et al, 2008). All three assays used were concurrent in that disruption of the SOCS-box motif in ORF73 leads to a severe latency deficit, characterised by its inability to induce the expansion of latent infection in GC B cells and persistence in the host (Figure 7B–D). This phenotype was comparable to the previously reported phenotype (Fowler et al, 2003) of a MuHV-4 lacking a functional ORF73 (v73FS in Figure 7B–D). Thus, at day 14 post-infection, the levels of the explant co-culture assay (Figure 7B) and the frequencies of infection in GC B cells (Figure 7C) obtained for the SOCS-box mutants were identical to those obtained for v73FS and significantly lower when compared with vWT. In good agreement, in situ hybridisation analysis of spleen sections from vSOCS- or v73FS-infected mice exhibited a complete lack of expansion of latently infected cells (Figure 7D, panels b and c). This was in clear contrast with vWT where large clusters of latently infected cells were observed within GCs (Figure 7D, panel a).


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–SOCS virus shows a strong latency deficit. (A) ORF73–SOCS recombinant virus exhibits normal replication in the lung. Wild-type BALB/c mice were intranasally infected with 104 p.f.u. of the indicated viruses. At the indicated days post-infection, lungs were removed and infectious viruses were titrated by plaque assay. (B) BALB/c mice were intranasally infected with 104 p.f.u. of the indicated viruses. At day 14 post-infection, latent viruses in spleens were titrated by infectious centre assay. Each point represents the titre of an individual mouse. The dashed line represents the limit of detection of the assay. (C) BALB/c mice were intranasally infected with 104 p.f.u. of the indicated viruses. At 14 days post-infection, reciprocal frequencies of viral infection in purified GC B cells (B220+/PNAhigh) were determined by limiting dilution and real-time PCR. Data were obtained from pools of five spleens per group. Bars represent the frequency of viral DNA-positive cells with 95% confidence intervals. (D) BALB/c mice were intranasally infected with 104 p.f.u. of the indicated viruses. At day 14 post-infection, spleens were removed and processed for in situ hybridisation using probes derived from viral miRNAs 1–6. Panels (a–c) show representative spleen sections from each group of viruses. All sections are magnified × 200.
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f7: ORF73–SOCS virus shows a strong latency deficit. (A) ORF73–SOCS recombinant virus exhibits normal replication in the lung. Wild-type BALB/c mice were intranasally infected with 104 p.f.u. of the indicated viruses. At the indicated days post-infection, lungs were removed and infectious viruses were titrated by plaque assay. (B) BALB/c mice were intranasally infected with 104 p.f.u. of the indicated viruses. At day 14 post-infection, latent viruses in spleens were titrated by infectious centre assay. Each point represents the titre of an individual mouse. The dashed line represents the limit of detection of the assay. (C) BALB/c mice were intranasally infected with 104 p.f.u. of the indicated viruses. At 14 days post-infection, reciprocal frequencies of viral infection in purified GC B cells (B220+/PNAhigh) were determined by limiting dilution and real-time PCR. Data were obtained from pools of five spleens per group. Bars represent the frequency of viral DNA-positive cells with 95% confidence intervals. (D) BALB/c mice were intranasally infected with 104 p.f.u. of the indicated viruses. At day 14 post-infection, spleens were removed and processed for in situ hybridisation using probes derived from viral miRNAs 1–6. Panels (a–c) show representative spleen sections from each group of viruses. All sections are magnified × 200.
Mentions: To directly investigate the biological relevance of inhibition of NF-κB signalling in gammaherpesvirus pathogenesis, we generated a recombinant MuHV-4 in which the ORF73 gene was modified to recapitulate the amino-acid substitutions of the ORF73 SOCS-box mutant, designated vSOCS. To assure that any phenotypic alteration in vSOCS was due to the engineered mutations in the SOCS-box and not from any spurious mutation introduced during mutagenesis, a second independent recombinant virus was engineered (vSOCSi). To characterise the role of the introduced mutations in a natural context of infection, we started to compare the kinetics of viral replication in vitro and during the acute phase of infection in lungs of Balb/c mice following intranasal inoculation. For comparative purposes, the viruses analysed included the vSOCS mutants alongside wild-type MuHV-4 (vWT) and a previously described (Fowler et al, 2003) ORF73 frameshift mutant (v73FS) that encodes only the 163 amino acids of the N-terminus of the protein. All these viruses were analysed for genome integrity (Supplementary Figure S1) and showed identical in vitro growth (Supplementary Figure S2), as well as normal replication in acutely infected lungs (Figure 7A). Next, we proceeded to investigate the role of the introduced mutations for the ability of MuHV-4 to induce the expansion of latency in GC B cells. To this end, we used three independent, but complementary, experimental assays: ex vivo explant co-culture assays to measure latent infection in total splenocytes, flow cytometry coupled to limiting dilution and real-time PCR to quantify the frequency of viral DNA-positive GC B cells, and in situ hybridisation analysis to identify virally infected cells within the spleen, as described earlier (Pires de Miranda et al, 2008). All three assays used were concurrent in that disruption of the SOCS-box motif in ORF73 leads to a severe latency deficit, characterised by its inability to induce the expansion of latent infection in GC B cells and persistence in the host (Figure 7B–D). This phenotype was comparable to the previously reported phenotype (Fowler et al, 2003) of a MuHV-4 lacking a functional ORF73 (v73FS in Figure 7B–D). Thus, at day 14 post-infection, the levels of the explant co-culture assay (Figure 7B) and the frequencies of infection in GC B cells (Figure 7C) obtained for the SOCS-box mutants were identical to those obtained for v73FS and significantly lower when compared with vWT. In good agreement, in situ hybridisation analysis of spleen sections from vSOCS- or v73FS-infected mice exhibited a complete lack of expansion of latently infected cells (Figure 7D, panels b and c). This was in clear contrast with vWT where large clusters of latently infected cells were observed within GCs (Figure 7D, panel a).

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