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Release of human cytomegalovirus from latency by a KAP1/TRIM28 phosphorylation switch.

Rauwel B, Jang SM, Cassano M, Kapopoulou A, Barde I, Trono D - Elife (2015)

Bottom Line: Here, we reveal that HCMV latency in human CD34(+) HSC reflects the recruitment on the viral genome of KAP1, a master co-repressor, together with HP1 and the SETDB1 histone methyltransferase, which results in transcriptional silencing.Correspondingly, HCMV can be forced out of latency by KAP1 knockdown or pharmacological induction of KAP1 phosphorylation, and this process can be potentiated by activating NFkB with TNF-α.These results suggest new approaches both to curtail CMV infection and to purge the virus from organ transplants.

View Article: PubMed Central - PubMed

Affiliation: School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland.

ABSTRACT
Human cytomegalovirus (HCMV) is a highly prevalent pathogen that induces life-long infections notably through the establishment of latency in hematopoietic stem cells (HSC). Bouts of reactivation are normally controlled by the immune system, but can be fatal in immuno-compromised individuals such as organ transplant recipients. Here, we reveal that HCMV latency in human CD34(+) HSC reflects the recruitment on the viral genome of KAP1, a master co-repressor, together with HP1 and the SETDB1 histone methyltransferase, which results in transcriptional silencing. During lytic infection, KAP1 is still associated with the viral genome, but its heterochromatin-inducing activity is suppressed by mTOR-mediated phosphorylation. Correspondingly, HCMV can be forced out of latency by KAP1 knockdown or pharmacological induction of KAP1 phosphorylation, and this process can be potentiated by activating NFkB with TNF-α. These results suggest new approaches both to curtail CMV infection and to purge the virus from organ transplants.

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A KAP1 phosphorylation switch distinguishes HCMV latency and lytic replication.ChIP-PCR with SETDB1-, H3K9Me3-, KAP1-, pS824KAP1-specific antibodies were performed on MRC-5 fibroblasts (A, B, F, G) or CD34+ HSC (C, D, E) infected with the HCMV TB40-E (A, B, G) or AD169 (C, D, E, F) strains, as described in Figure 3.DOI:http://dx.doi.org/10.7554/eLife.06068.011
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fig3s1: A KAP1 phosphorylation switch distinguishes HCMV latency and lytic replication.ChIP-PCR with SETDB1-, H3K9Me3-, KAP1-, pS824KAP1-specific antibodies were performed on MRC-5 fibroblasts (A, B, F, G) or CD34+ HSC (C, D, E) infected with the HCMV TB40-E (A, B, G) or AD169 (C, D, E, F) strains, as described in Figure 3.DOI:http://dx.doi.org/10.7554/eLife.06068.011

Mentions: We then examined the epigenetic status of HCMV genome when TB40-E-infected CD34+ cells were differentiated in mature dendritic cells (mDCs), a procedure previously demonstrated to result in HCMV activation. As expected, the TB40-E DNA was no longer associated with SETDB1 or HP1α and did not bear the H3K9me3 repressive mark in the CD34-derived mDCs (Figure 3A and Figure 2—figure supplement 1A). However, KAP1 was surprisingly still associated with the viral genome in these targets (Figure 3B). The same pattern was recorded in TB40-E infected MRC-5 cells, where the virus achieves a complete lytic cycle (Figure 3—figure supplement 1A,B). It suggested that HCMV reactivation and replication could occur in spite of corepressor binding. To probe this issue further, we infected CD34+ cord blood and MRC5 cells with the HCMV AD169 laboratory strain, which is incapable of inducing latency (Goodrum et al., 2007; Saffert et al., 2010). At day 7 post-infection, while the productively transcribed AD169 genome carried as expected neither SETDB1 nor H3K9Me3, it was still bound by KAP1 in both cell types as robustly as the latent TB40-E strain in HSC (Figure 3—figure supplement 1C,D). Therefore, it is not KAP1 recognition but rather the secondary recruitment of its SETDB1 effector and other KAP1-associated heterochromatin inducers such as HP1, which is responsible for HCMV latency.10.7554/eLife.06068.010Figure 3.A KAP1 phosphorylation switch governs HCMV progression from latency to lytic replication.


Release of human cytomegalovirus from latency by a KAP1/TRIM28 phosphorylation switch.

Rauwel B, Jang SM, Cassano M, Kapopoulou A, Barde I, Trono D - Elife (2015)

A KAP1 phosphorylation switch distinguishes HCMV latency and lytic replication.ChIP-PCR with SETDB1-, H3K9Me3-, KAP1-, pS824KAP1-specific antibodies were performed on MRC-5 fibroblasts (A, B, F, G) or CD34+ HSC (C, D, E) infected with the HCMV TB40-E (A, B, G) or AD169 (C, D, E, F) strains, as described in Figure 3.DOI:http://dx.doi.org/10.7554/eLife.06068.011
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4384640&req=5

fig3s1: A KAP1 phosphorylation switch distinguishes HCMV latency and lytic replication.ChIP-PCR with SETDB1-, H3K9Me3-, KAP1-, pS824KAP1-specific antibodies were performed on MRC-5 fibroblasts (A, B, F, G) or CD34+ HSC (C, D, E) infected with the HCMV TB40-E (A, B, G) or AD169 (C, D, E, F) strains, as described in Figure 3.DOI:http://dx.doi.org/10.7554/eLife.06068.011
Mentions: We then examined the epigenetic status of HCMV genome when TB40-E-infected CD34+ cells were differentiated in mature dendritic cells (mDCs), a procedure previously demonstrated to result in HCMV activation. As expected, the TB40-E DNA was no longer associated with SETDB1 or HP1α and did not bear the H3K9me3 repressive mark in the CD34-derived mDCs (Figure 3A and Figure 2—figure supplement 1A). However, KAP1 was surprisingly still associated with the viral genome in these targets (Figure 3B). The same pattern was recorded in TB40-E infected MRC-5 cells, where the virus achieves a complete lytic cycle (Figure 3—figure supplement 1A,B). It suggested that HCMV reactivation and replication could occur in spite of corepressor binding. To probe this issue further, we infected CD34+ cord blood and MRC5 cells with the HCMV AD169 laboratory strain, which is incapable of inducing latency (Goodrum et al., 2007; Saffert et al., 2010). At day 7 post-infection, while the productively transcribed AD169 genome carried as expected neither SETDB1 nor H3K9Me3, it was still bound by KAP1 in both cell types as robustly as the latent TB40-E strain in HSC (Figure 3—figure supplement 1C,D). Therefore, it is not KAP1 recognition but rather the secondary recruitment of its SETDB1 effector and other KAP1-associated heterochromatin inducers such as HP1, which is responsible for HCMV latency.10.7554/eLife.06068.010Figure 3.A KAP1 phosphorylation switch governs HCMV progression from latency to lytic replication.

Bottom Line: Here, we reveal that HCMV latency in human CD34(+) HSC reflects the recruitment on the viral genome of KAP1, a master co-repressor, together with HP1 and the SETDB1 histone methyltransferase, which results in transcriptional silencing.Correspondingly, HCMV can be forced out of latency by KAP1 knockdown or pharmacological induction of KAP1 phosphorylation, and this process can be potentiated by activating NFkB with TNF-α.These results suggest new approaches both to curtail CMV infection and to purge the virus from organ transplants.

View Article: PubMed Central - PubMed

Affiliation: School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland.

ABSTRACT
Human cytomegalovirus (HCMV) is a highly prevalent pathogen that induces life-long infections notably through the establishment of latency in hematopoietic stem cells (HSC). Bouts of reactivation are normally controlled by the immune system, but can be fatal in immuno-compromised individuals such as organ transplant recipients. Here, we reveal that HCMV latency in human CD34(+) HSC reflects the recruitment on the viral genome of KAP1, a master co-repressor, together with HP1 and the SETDB1 histone methyltransferase, which results in transcriptional silencing. During lytic infection, KAP1 is still associated with the viral genome, but its heterochromatin-inducing activity is suppressed by mTOR-mediated phosphorylation. Correspondingly, HCMV can be forced out of latency by KAP1 knockdown or pharmacological induction of KAP1 phosphorylation, and this process can be potentiated by activating NFkB with TNF-α. These results suggest new approaches both to curtail CMV infection and to purge the virus from organ transplants.

Show MeSH
Related in: MedlinePlus