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Caspase-1 promotes Epstein-Barr virus replication by targeting the large tegument protein deneddylase to the nucleus of productively infected cells.

Gastaldello S, Chen X, Callegari S, Masucci MG - PLoS Pathog. (2013)

Bottom Line: Using as model BPLF1, the homologue encoded by Epstein-Barr virus (EBV), we found that induction of the productive virus cycle does not affect the total level of ubiquitin-conjugation but is accompanied by a BPLF1-dependent decrease of NEDD8-adducts and accumulation of free NEDD8.The inactivation of nuclear CRLs is reversed by the N-terminus of CAND1, which inhibits the binding of BPLF1 to cullins and prevents efficient viral DNA replication.Targeting of the deneddylase activity to the nucleus is dependent on processing of the catalytic N-terminus by caspase-1.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Molecular Biology, Karolinska Institutet, Stockholm, Sweden.

ABSTRACT
The large tegument proteins of herpesviruses contain N-terminal cysteine proteases with potent ubiquitin and NEDD8-specific deconjugase activities, but the function of the enzymes during virus replication remains largely unknown. Using as model BPLF1, the homologue encoded by Epstein-Barr virus (EBV), we found that induction of the productive virus cycle does not affect the total level of ubiquitin-conjugation but is accompanied by a BPLF1-dependent decrease of NEDD8-adducts and accumulation of free NEDD8. Expression of BPLF1 promotes cullin degradation and the stabilization of cullin-RING ligases (CRLs) substrates in the nucleus, while cytoplasmic CRLs and their substrates are not affected. The inactivation of nuclear CRLs is reversed by the N-terminus of CAND1, which inhibits the binding of BPLF1 to cullins and prevents efficient viral DNA replication. Targeting of the deneddylase activity to the nucleus is dependent on processing of the catalytic N-terminus by caspase-1. Inhibition of caspase-1 severely impairs viral DNA synthesis and the release of infectious virus, pointing a previously unrecognized role of the cellular response to danger signals triggered by EBV reactivation in promoting virus replication.

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BPLF1 promotes the selective degradation of nuclear cullins by the proteasome.A. Cullins are selectively degraded during EBV replication. Western blots of cell lysates from induced Akata-Bx1 were probed with the indicated antibodies. One representative experiment out of three is shown. B. The cullins are degraded by the proteasome. Ten µM of the proteasome inhibition MG132 were added to one aliquot of Akata-Bx1 cells 48 h after induction and the cells were culture overnight before western blot analysis with the indicated antibodies. One representative experiment out of three is shown. C. Representative western blots illustrating the changes in expression levels of cytoplasmic and nuclear cullins. Subcellular fractionation was performed at the indicated time after induction and the efficiency of fractionation was confirmed by probing western blots with antibodies to PARP, histone H1 and β-actin. One representative experiment out of three is shown. D. Quantification of the fold change relative to the levels of expression at time 0. The mean ± SE of three experiments are shown. Significant differences between fold changes in the cytoplasmic and nuclear fractions are indicated * = p<0.01, ** = p<0.001.
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ppat-1003664-g003: BPLF1 promotes the selective degradation of nuclear cullins by the proteasome.A. Cullins are selectively degraded during EBV replication. Western blots of cell lysates from induced Akata-Bx1 were probed with the indicated antibodies. One representative experiment out of three is shown. B. The cullins are degraded by the proteasome. Ten µM of the proteasome inhibition MG132 were added to one aliquot of Akata-Bx1 cells 48 h after induction and the cells were culture overnight before western blot analysis with the indicated antibodies. One representative experiment out of three is shown. C. Representative western blots illustrating the changes in expression levels of cytoplasmic and nuclear cullins. Subcellular fractionation was performed at the indicated time after induction and the efficiency of fractionation was confirmed by probing western blots with antibodies to PARP, histone H1 and β-actin. One representative experiment out of three is shown. D. Quantification of the fold change relative to the levels of expression at time 0. The mean ± SE of three experiments are shown. Significant differences between fold changes in the cytoplasmic and nuclear fractions are indicated * = p<0.01, ** = p<0.001.

Mentions: Transfection of the catalytically active BPLF1 in HeLa cells promotes cullin deneddylation and their proteasomal degradation [29]. We asked therefore whether this phenotype is reproduced when the endogenous enzyme is expressed during virus replication. As illustrated by the representative western blot shown in Figure 3A, induction of the productive virus cycle was accompanied by a gradual decrease of the Cul1, Cul3, Cul4A and Cul5 specific bands in Akata-Bx1. This was not due to a global impairment of protein synthesis since neither Cul2, nor the CRL subunit RBX1 were affected. Furthermore, the decrease was rescued by treatment with MG132 confirming that cullins are degraded by the proteasome (Figure 3B). This finding is consistent with a scenario where the inactivation of CRLs by BPLF1-mediated deneddylation of cullins, and their subsequent proteasomal degradation, are key requirements for efficient virus replication. However, the failure to degrade Cul2 is surprising since the BPLF1 binding site on cullins is highly conserved [29]. To explore the possible cause of this observation, the abundance of Cul1, Cul2, Cul3, Cul4A and Cul5 was monitored over time in the nucleus and cytoplasm of the induced cells. The fractionation procedure was validated by probing of western blots with antibodies to PARP, histone H1 and β-actin. In line with their known subcellular localization, PARP and H1 were exclusively detected in the nuclear fractions, whereas β-actin was enriched in the cytoplasmic fraction (Figure 3C). Variable amounts of nuclear and cytoplasmic cullins were detected in untreated Akata-Bx1, with prevalent nuclear localization of Cul1, Cul3, Cul4A and Cul5, whereas Cul2 was detected almost exclusively in the cytoplasmic fraction (Figure 3C). Induction of the productive virus cycle was accompanied by a progressive decrease of nuclear pool of Cul1, Cul3, Cul4A and Cul5, whereas the amount of proteins detected in the cytoplasm remained unchanged throughout the observation period (Figure 3C and 3D). There was no detectable change in the expression of cytoplasmic Cul2, further supporting the conclusion that only nuclear cullins are affected.


Caspase-1 promotes Epstein-Barr virus replication by targeting the large tegument protein deneddylase to the nucleus of productively infected cells.

Gastaldello S, Chen X, Callegari S, Masucci MG - PLoS Pathog. (2013)

BPLF1 promotes the selective degradation of nuclear cullins by the proteasome.A. Cullins are selectively degraded during EBV replication. Western blots of cell lysates from induced Akata-Bx1 were probed with the indicated antibodies. One representative experiment out of three is shown. B. The cullins are degraded by the proteasome. Ten µM of the proteasome inhibition MG132 were added to one aliquot of Akata-Bx1 cells 48 h after induction and the cells were culture overnight before western blot analysis with the indicated antibodies. One representative experiment out of three is shown. C. Representative western blots illustrating the changes in expression levels of cytoplasmic and nuclear cullins. Subcellular fractionation was performed at the indicated time after induction and the efficiency of fractionation was confirmed by probing western blots with antibodies to PARP, histone H1 and β-actin. One representative experiment out of three is shown. D. Quantification of the fold change relative to the levels of expression at time 0. The mean ± SE of three experiments are shown. Significant differences between fold changes in the cytoplasmic and nuclear fractions are indicated * = p<0.01, ** = p<0.001.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3795028&req=5

ppat-1003664-g003: BPLF1 promotes the selective degradation of nuclear cullins by the proteasome.A. Cullins are selectively degraded during EBV replication. Western blots of cell lysates from induced Akata-Bx1 were probed with the indicated antibodies. One representative experiment out of three is shown. B. The cullins are degraded by the proteasome. Ten µM of the proteasome inhibition MG132 were added to one aliquot of Akata-Bx1 cells 48 h after induction and the cells were culture overnight before western blot analysis with the indicated antibodies. One representative experiment out of three is shown. C. Representative western blots illustrating the changes in expression levels of cytoplasmic and nuclear cullins. Subcellular fractionation was performed at the indicated time after induction and the efficiency of fractionation was confirmed by probing western blots with antibodies to PARP, histone H1 and β-actin. One representative experiment out of three is shown. D. Quantification of the fold change relative to the levels of expression at time 0. The mean ± SE of three experiments are shown. Significant differences between fold changes in the cytoplasmic and nuclear fractions are indicated * = p<0.01, ** = p<0.001.
Mentions: Transfection of the catalytically active BPLF1 in HeLa cells promotes cullin deneddylation and their proteasomal degradation [29]. We asked therefore whether this phenotype is reproduced when the endogenous enzyme is expressed during virus replication. As illustrated by the representative western blot shown in Figure 3A, induction of the productive virus cycle was accompanied by a gradual decrease of the Cul1, Cul3, Cul4A and Cul5 specific bands in Akata-Bx1. This was not due to a global impairment of protein synthesis since neither Cul2, nor the CRL subunit RBX1 were affected. Furthermore, the decrease was rescued by treatment with MG132 confirming that cullins are degraded by the proteasome (Figure 3B). This finding is consistent with a scenario where the inactivation of CRLs by BPLF1-mediated deneddylation of cullins, and their subsequent proteasomal degradation, are key requirements for efficient virus replication. However, the failure to degrade Cul2 is surprising since the BPLF1 binding site on cullins is highly conserved [29]. To explore the possible cause of this observation, the abundance of Cul1, Cul2, Cul3, Cul4A and Cul5 was monitored over time in the nucleus and cytoplasm of the induced cells. The fractionation procedure was validated by probing of western blots with antibodies to PARP, histone H1 and β-actin. In line with their known subcellular localization, PARP and H1 were exclusively detected in the nuclear fractions, whereas β-actin was enriched in the cytoplasmic fraction (Figure 3C). Variable amounts of nuclear and cytoplasmic cullins were detected in untreated Akata-Bx1, with prevalent nuclear localization of Cul1, Cul3, Cul4A and Cul5, whereas Cul2 was detected almost exclusively in the cytoplasmic fraction (Figure 3C). Induction of the productive virus cycle was accompanied by a progressive decrease of nuclear pool of Cul1, Cul3, Cul4A and Cul5, whereas the amount of proteins detected in the cytoplasm remained unchanged throughout the observation period (Figure 3C and 3D). There was no detectable change in the expression of cytoplasmic Cul2, further supporting the conclusion that only nuclear cullins are affected.

Bottom Line: Using as model BPLF1, the homologue encoded by Epstein-Barr virus (EBV), we found that induction of the productive virus cycle does not affect the total level of ubiquitin-conjugation but is accompanied by a BPLF1-dependent decrease of NEDD8-adducts and accumulation of free NEDD8.The inactivation of nuclear CRLs is reversed by the N-terminus of CAND1, which inhibits the binding of BPLF1 to cullins and prevents efficient viral DNA replication.Targeting of the deneddylase activity to the nucleus is dependent on processing of the catalytic N-terminus by caspase-1.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Molecular Biology, Karolinska Institutet, Stockholm, Sweden.

ABSTRACT
The large tegument proteins of herpesviruses contain N-terminal cysteine proteases with potent ubiquitin and NEDD8-specific deconjugase activities, but the function of the enzymes during virus replication remains largely unknown. Using as model BPLF1, the homologue encoded by Epstein-Barr virus (EBV), we found that induction of the productive virus cycle does not affect the total level of ubiquitin-conjugation but is accompanied by a BPLF1-dependent decrease of NEDD8-adducts and accumulation of free NEDD8. Expression of BPLF1 promotes cullin degradation and the stabilization of cullin-RING ligases (CRLs) substrates in the nucleus, while cytoplasmic CRLs and their substrates are not affected. The inactivation of nuclear CRLs is reversed by the N-terminus of CAND1, which inhibits the binding of BPLF1 to cullins and prevents efficient viral DNA replication. Targeting of the deneddylase activity to the nucleus is dependent on processing of the catalytic N-terminus by caspase-1. Inhibition of caspase-1 severely impairs viral DNA synthesis and the release of infectious virus, pointing a previously unrecognized role of the cellular response to danger signals triggered by EBV reactivation in promoting virus replication.

Show MeSH
Related in: MedlinePlus