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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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KAP1-dependent recruitment of HP1α and SETDB1 with H3K9 trimethylation of HCMV genome in latently infected HSC.ChIP-PCR analysis of TB40-E-infected WT MRC-5 (A and B) KAP1-depleted CD34+ (A–D), WT CD34+ (A and B) or mDC (A and B) with antibodies against HP1α (A and B) SETDB1 (C) or H3K9me3 (C and D), as described in Figure 3.DOI:http://dx.doi.org/10.7554/eLife.06068.009
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fig2s1: KAP1-dependent recruitment of HP1α and SETDB1 with H3K9 trimethylation of HCMV genome in latently infected HSC.ChIP-PCR analysis of TB40-E-infected WT MRC-5 (A and B) KAP1-depleted CD34+ (A–D), WT CD34+ (A and B) or mDC (A and B) with antibodies against HP1α (A and B) SETDB1 (C) or H3K9me3 (C and D), as described in Figure 3.DOI:http://dx.doi.org/10.7554/eLife.06068.009

Mentions: Cells in which HCMV expression was induced following KAP1 depletion kept expressing the CD34 stem cell marker, indicating that viral activation was not simply due to their differentiation into normally permissive cells such as activated DCs or macrophages. Still, as KAP1 exerts pleomorphic effect on hematopoiesis (Barde et al., 2013), we could not exclude that its depletion induced HCMV lytic genes expression through indirect effects. We thus asked whether the co-repressor associates with the HCMV genome by performing KAP1-specific chromatin immunoprecipitation-deep sequencing (ChIP-seq) analyses on material isolated from human CD34+ HSC at 7 days post-TB40-E infection. Using a stringent peak-calling algorithm and validating its results by ChIP-PCR, we identified 28 major KAP1-enriched regions on the HCMV genome (Figure 2A,B and Supplementary file 1). Supporting the functional significance of KAP1 recruitment to latent HCMV genomes, ChIP-PCR analyses also detected SETDB1, H3K9me3 and HP1α at these KAP1-enriched regions (Figure 2C and Figure 2—figure supplement 1A). Furthermore, in line with our former demonstration that heterochromatin formation can spread several tens of kilobases away from primary KAP1 docking sites (Groner et al., 2010), H3K9me3 and HP1α were found at significant distances from these major KAP1 peaks, and particularly covered the major immediate early promoter (MIEP), the viral origin of lytic replication (OriLyt) and the UL112 gene. In contrast, the repressive mark was not found at genes known to be expressed during latency such as UL138 or LUNA (Reeves and Sinclair, 2013) (Figure 2D and Figure 2—figure supplement 1B). Importantly, SETDB1 and HP1α recruitment as well as H3K9 trimethylation were lost when KAP1 was depleted by RNA interference prior to HSC infection, supporting a model whereby the corepressor recruits these heterochromatin-inducing factors (Figure 2—figure supplement 1A–D).10.7554/eLife.06068.008Figure 2.KAP1, SetDB1 and H3K9Me3 are enriched on the HCMV genome in latently infected HSC.


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)

KAP1-dependent recruitment of HP1α and SETDB1 with H3K9 trimethylation of HCMV genome in latently infected HSC.ChIP-PCR analysis of TB40-E-infected WT MRC-5 (A and B) KAP1-depleted CD34+ (A–D), WT CD34+ (A and B) or mDC (A and B) with antibodies against HP1α (A and B) SETDB1 (C) or H3K9me3 (C and D), as described in Figure 3.DOI:http://dx.doi.org/10.7554/eLife.06068.009
© Copyright Policy
Related In: Results  -  Collection

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

fig2s1: KAP1-dependent recruitment of HP1α and SETDB1 with H3K9 trimethylation of HCMV genome in latently infected HSC.ChIP-PCR analysis of TB40-E-infected WT MRC-5 (A and B) KAP1-depleted CD34+ (A–D), WT CD34+ (A and B) or mDC (A and B) with antibodies against HP1α (A and B) SETDB1 (C) or H3K9me3 (C and D), as described in Figure 3.DOI:http://dx.doi.org/10.7554/eLife.06068.009
Mentions: Cells in which HCMV expression was induced following KAP1 depletion kept expressing the CD34 stem cell marker, indicating that viral activation was not simply due to their differentiation into normally permissive cells such as activated DCs or macrophages. Still, as KAP1 exerts pleomorphic effect on hematopoiesis (Barde et al., 2013), we could not exclude that its depletion induced HCMV lytic genes expression through indirect effects. We thus asked whether the co-repressor associates with the HCMV genome by performing KAP1-specific chromatin immunoprecipitation-deep sequencing (ChIP-seq) analyses on material isolated from human CD34+ HSC at 7 days post-TB40-E infection. Using a stringent peak-calling algorithm and validating its results by ChIP-PCR, we identified 28 major KAP1-enriched regions on the HCMV genome (Figure 2A,B and Supplementary file 1). Supporting the functional significance of KAP1 recruitment to latent HCMV genomes, ChIP-PCR analyses also detected SETDB1, H3K9me3 and HP1α at these KAP1-enriched regions (Figure 2C and Figure 2—figure supplement 1A). Furthermore, in line with our former demonstration that heterochromatin formation can spread several tens of kilobases away from primary KAP1 docking sites (Groner et al., 2010), H3K9me3 and HP1α were found at significant distances from these major KAP1 peaks, and particularly covered the major immediate early promoter (MIEP), the viral origin of lytic replication (OriLyt) and the UL112 gene. In contrast, the repressive mark was not found at genes known to be expressed during latency such as UL138 or LUNA (Reeves and Sinclair, 2013) (Figure 2D and Figure 2—figure supplement 1B). Importantly, SETDB1 and HP1α recruitment as well as H3K9 trimethylation were lost when KAP1 was depleted by RNA interference prior to HSC infection, supporting a model whereby the corepressor recruits these heterochromatin-inducing factors (Figure 2—figure supplement 1A–D).10.7554/eLife.06068.008Figure 2.KAP1, SetDB1 and H3K9Me3 are enriched on the HCMV genome in latently infected HSC.

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