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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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mTOR nuclear translocation and KAP1 activity in HCMV replicating cells.(A) Confocal microscopy coupled to immunofluorescence was performed on MRC-5, infected (TB40-E, AD169) or not (Non Infected) with HCMV, using antibodies against IE- (Alex-488, green) or mTOR (Alexa 568, red), staining DNA with Dapi (blue). White scale bar, 10 µm. (B) MRC-5 cells were transduced with a lentiviral vector expressing GFP from the TetO-PGK promoter (TetO-GFP) with or without tTR-KRAB, infected or not with HCMV and treated or not with DOX as indicated. Results are presented as % of GFP positive cells, using non-infected and Dox treated cells as a 100% reference. (n = 3, **p < 0.01, error bars as s.d.).DOI:http://dx.doi.org/10.7554/eLife.06068.016
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fig5s1: mTOR nuclear translocation and KAP1 activity in HCMV replicating cells.(A) Confocal microscopy coupled to immunofluorescence was performed on MRC-5, infected (TB40-E, AD169) or not (Non Infected) with HCMV, using antibodies against IE- (Alex-488, green) or mTOR (Alexa 568, red), staining DNA with Dapi (blue). White scale bar, 10 µm. (B) MRC-5 cells were transduced with a lentiviral vector expressing GFP from the TetO-PGK promoter (TetO-GFP) with or without tTR-KRAB, infected or not with HCMV and treated or not with DOX as indicated. Results are presented as % of GFP positive cells, using non-infected and Dox treated cells as a 100% reference. (n = 3, **p < 0.01, error bars as s.d.).DOI:http://dx.doi.org/10.7554/eLife.06068.016

Mentions: Cell fractionation analyses indicated that pS824KAP1 accumulated in the nucleus of CMV-infected cells, which also displayed increased levels of phospho-S6 ribosomal protein, a marker of mTOR activation (Figure 5A). Indirect immunofluorescence and confocal microscopy further revealed mTOR-specific foci in the nucleus of TB40- or AD169-infected but not control MRC5 cells (Figure 5B and Figure 5—figure supplement 1A). Finally, ChIP with an mTOR-specific antibody demonstrated that the kinase associated with the CMV genome at the same places regions as pS824KAP1 (Figure 5C). Consistent with a restriction of KAP1 phosphorylation and inactivation to CMV-associated molecules, the corepressor could still mediate the transcriptional silencing of an integrated TetO-PGK-GFP cassette via a Tet repressor-KRAB fusion protein (Wiznerowicz and Trono, 2003) in virus-infected cells (Figure 5—figure supplement 1B).10.7554/eLife.06068.015Figure 5.mTOR associates with the HCMV genome during 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)

mTOR nuclear translocation and KAP1 activity in HCMV replicating cells.(A) Confocal microscopy coupled to immunofluorescence was performed on MRC-5, infected (TB40-E, AD169) or not (Non Infected) with HCMV, using antibodies against IE- (Alex-488, green) or mTOR (Alexa 568, red), staining DNA with Dapi (blue). White scale bar, 10 µm. (B) MRC-5 cells were transduced with a lentiviral vector expressing GFP from the TetO-PGK promoter (TetO-GFP) with or without tTR-KRAB, infected or not with HCMV and treated or not with DOX as indicated. Results are presented as % of GFP positive cells, using non-infected and Dox treated cells as a 100% reference. (n = 3, **p < 0.01, error bars as s.d.).DOI:http://dx.doi.org/10.7554/eLife.06068.016
© Copyright Policy
Related In: Results  -  Collection

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

fig5s1: mTOR nuclear translocation and KAP1 activity in HCMV replicating cells.(A) Confocal microscopy coupled to immunofluorescence was performed on MRC-5, infected (TB40-E, AD169) or not (Non Infected) with HCMV, using antibodies against IE- (Alex-488, green) or mTOR (Alexa 568, red), staining DNA with Dapi (blue). White scale bar, 10 µm. (B) MRC-5 cells were transduced with a lentiviral vector expressing GFP from the TetO-PGK promoter (TetO-GFP) with or without tTR-KRAB, infected or not with HCMV and treated or not with DOX as indicated. Results are presented as % of GFP positive cells, using non-infected and Dox treated cells as a 100% reference. (n = 3, **p < 0.01, error bars as s.d.).DOI:http://dx.doi.org/10.7554/eLife.06068.016
Mentions: Cell fractionation analyses indicated that pS824KAP1 accumulated in the nucleus of CMV-infected cells, which also displayed increased levels of phospho-S6 ribosomal protein, a marker of mTOR activation (Figure 5A). Indirect immunofluorescence and confocal microscopy further revealed mTOR-specific foci in the nucleus of TB40- or AD169-infected but not control MRC5 cells (Figure 5B and Figure 5—figure supplement 1A). Finally, ChIP with an mTOR-specific antibody demonstrated that the kinase associated with the CMV genome at the same places regions as pS824KAP1 (Figure 5C). Consistent with a restriction of KAP1 phosphorylation and inactivation to CMV-associated molecules, the corepressor could still mediate the transcriptional silencing of an integrated TetO-PGK-GFP cassette via a Tet repressor-KRAB fusion protein (Wiznerowicz and Trono, 2003) in virus-infected cells (Figure 5—figure supplement 1B).10.7554/eLife.06068.015Figure 5.mTOR associates with the HCMV genome during 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