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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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HCMV can be forced out of latency in CD34+ cells by pharmacological manipulation.(A) Immuno-fluorescent staining was performed on CD34+ cells for total KAP1 (KAP1), using Dapi for DNA. White scale bar, 10 µm. All pictures are representative of three independent experiments. (B) Percentage of CD34+ cells after 7 days of treatment was quantified by FACS with a PE-anti-CD34 antibody. Results are presented of fold change expression vs NT of three independent experiments. HCMV DNA (C) and mRNAs (D) were quantified by qPCR in MRC-5 cells infected for 5 days with TB40-E in the presence or absence of chloroquine. Results are presented as HCMV DNA or mRNA levels normalized for GAPDH and Albumin for DNA, GAPDH and β-2M for mRNA.DOI:http://dx.doi.org/10.7554/eLife.06068.019
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fig6s2: HCMV can be forced out of latency in CD34+ cells by pharmacological manipulation.(A) Immuno-fluorescent staining was performed on CD34+ cells for total KAP1 (KAP1), using Dapi for DNA. White scale bar, 10 µm. All pictures are representative of three independent experiments. (B) Percentage of CD34+ cells after 7 days of treatment was quantified by FACS with a PE-anti-CD34 antibody. Results are presented of fold change expression vs NT of three independent experiments. HCMV DNA (C) and mRNAs (D) were quantified by qPCR in MRC-5 cells infected for 5 days with TB40-E in the presence or absence of chloroquine. Results are presented as HCMV DNA or mRNA levels normalized for GAPDH and Albumin for DNA, GAPDH and β-2M for mRNA.DOI:http://dx.doi.org/10.7554/eLife.06068.019

Mentions: These results suggested that HCMV silencing might be amenable to suppression by pharmacologically induced KAP1 phosphorylation. In the absence of strictly specific mTOR activator, we turned to ATM, a kinase previously demonstrated as capable of phosphorylating KAP1 on S824 (White et al., 2012). When HSC infected 5 days earlier with TB40-E were incubated with the ATM activator chloroquine (Bakkenist and Kastan, 2003), intracellular levels of viral RNAs and DNA increased (Figure 6A,B), and infectious viral particles were released in the supernatant (Figure 6C). Supporting the specificity of the observed phenomenon, Torin-1 did not block chloroquine-induced HCMV RNA, DNA and viral particle production, whereas the ATM inhibitor Ku55933 prevented this process (Figure 6). These results obtained in a first set of HSC obtained from three donors were confirmed in another group of three donors, where it was further observed that levels of viral transcripts increased in drug-treated cells only transiently after a single dose of drug but steadily if three doses were administered over 5 days (Figure 6—figure supplement 1A), and that single doses of the ATM activator prevented the progressive drop in viral DNA that was observed in control cells, while repeated doses triggered an augmentation in viral DNA copies, indicative of genome replication (Figure 6—figure supplement 1B). Correlating these quantitative virological data, chloroquine treatment allowed the detection by immunofluorescence of pS824KAP1-positive cells (Figure 6—figure supplement 1C). These were also systematically positive for IE antigens (Figure 6—figure supplement 1C), confirming that the virus harbored by these cells had exited latency. Finally, when the supernatant of TB40-E-infected HSC exposed to chloroquine was used to inoculate MRC5 fibroblasts, it induced the accumulation of viral DNA in these targets (Figure 6—figure supplement 1D), demonstrating that the production of replication-competent virus had been induced by ATM-activating treatment of the latently infected HSC. Noteworthy, pS824KAP1 was detected only in HCMV IE-positive cells, which strongly suggests that KAP1 became phosphorylated in these cells through the combined action of ATM and some HCMV-encoded factor. Importantly, these effects were induced without drop in KAP1 levels or loss of surface expression of the CD34 stem cell marker (Figure 6—figure supplement 2A,B). Additional evidence strongly suggested that chloroquine stimulated HCMV via KAP1 phosphorylation. First, the drug did not further boost TB40-E in MRC-5 cells, where viral replication is KAP1-insensitive (Figure 6—figure supplement 2C,D). Second, it did not further stimulate HCMV gene expression and virion release in CD34+ cells depleted for KAP1, whether or not these were complemented with a phosphorylation resistant S824A KAP1 mutant (Figure 6—figure supplement 3A,B and Figure 6—figure supplement 4AB). Third, and by contrast, chloroquine did reactivate HCMV when these knockdown cells were complemented with wild-type KAP1 (Figure 6—figure supplement 4A,B).10.7554/eLife.06068.017Figure 6.Forcing HCMV out of latency in CD34+ cells by KAP1 pharmacological manipulation.


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)

HCMV can be forced out of latency in CD34+ cells by pharmacological manipulation.(A) Immuno-fluorescent staining was performed on CD34+ cells for total KAP1 (KAP1), using Dapi for DNA. White scale bar, 10 µm. All pictures are representative of three independent experiments. (B) Percentage of CD34+ cells after 7 days of treatment was quantified by FACS with a PE-anti-CD34 antibody. Results are presented of fold change expression vs NT of three independent experiments. HCMV DNA (C) and mRNAs (D) were quantified by qPCR in MRC-5 cells infected for 5 days with TB40-E in the presence or absence of chloroquine. Results are presented as HCMV DNA or mRNA levels normalized for GAPDH and Albumin for DNA, GAPDH and β-2M for mRNA.DOI:http://dx.doi.org/10.7554/eLife.06068.019
© Copyright Policy
Related In: Results  -  Collection

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

fig6s2: HCMV can be forced out of latency in CD34+ cells by pharmacological manipulation.(A) Immuno-fluorescent staining was performed on CD34+ cells for total KAP1 (KAP1), using Dapi for DNA. White scale bar, 10 µm. All pictures are representative of three independent experiments. (B) Percentage of CD34+ cells after 7 days of treatment was quantified by FACS with a PE-anti-CD34 antibody. Results are presented of fold change expression vs NT of three independent experiments. HCMV DNA (C) and mRNAs (D) were quantified by qPCR in MRC-5 cells infected for 5 days with TB40-E in the presence or absence of chloroquine. Results are presented as HCMV DNA or mRNA levels normalized for GAPDH and Albumin for DNA, GAPDH and β-2M for mRNA.DOI:http://dx.doi.org/10.7554/eLife.06068.019
Mentions: These results suggested that HCMV silencing might be amenable to suppression by pharmacologically induced KAP1 phosphorylation. In the absence of strictly specific mTOR activator, we turned to ATM, a kinase previously demonstrated as capable of phosphorylating KAP1 on S824 (White et al., 2012). When HSC infected 5 days earlier with TB40-E were incubated with the ATM activator chloroquine (Bakkenist and Kastan, 2003), intracellular levels of viral RNAs and DNA increased (Figure 6A,B), and infectious viral particles were released in the supernatant (Figure 6C). Supporting the specificity of the observed phenomenon, Torin-1 did not block chloroquine-induced HCMV RNA, DNA and viral particle production, whereas the ATM inhibitor Ku55933 prevented this process (Figure 6). These results obtained in a first set of HSC obtained from three donors were confirmed in another group of three donors, where it was further observed that levels of viral transcripts increased in drug-treated cells only transiently after a single dose of drug but steadily if three doses were administered over 5 days (Figure 6—figure supplement 1A), and that single doses of the ATM activator prevented the progressive drop in viral DNA that was observed in control cells, while repeated doses triggered an augmentation in viral DNA copies, indicative of genome replication (Figure 6—figure supplement 1B). Correlating these quantitative virological data, chloroquine treatment allowed the detection by immunofluorescence of pS824KAP1-positive cells (Figure 6—figure supplement 1C). These were also systematically positive for IE antigens (Figure 6—figure supplement 1C), confirming that the virus harbored by these cells had exited latency. Finally, when the supernatant of TB40-E-infected HSC exposed to chloroquine was used to inoculate MRC5 fibroblasts, it induced the accumulation of viral DNA in these targets (Figure 6—figure supplement 1D), demonstrating that the production of replication-competent virus had been induced by ATM-activating treatment of the latently infected HSC. Noteworthy, pS824KAP1 was detected only in HCMV IE-positive cells, which strongly suggests that KAP1 became phosphorylated in these cells through the combined action of ATM and some HCMV-encoded factor. Importantly, these effects were induced without drop in KAP1 levels or loss of surface expression of the CD34 stem cell marker (Figure 6—figure supplement 2A,B). Additional evidence strongly suggested that chloroquine stimulated HCMV via KAP1 phosphorylation. First, the drug did not further boost TB40-E in MRC-5 cells, where viral replication is KAP1-insensitive (Figure 6—figure supplement 2C,D). Second, it did not further stimulate HCMV gene expression and virion release in CD34+ cells depleted for KAP1, whether or not these were complemented with a phosphorylation resistant S824A KAP1 mutant (Figure 6—figure supplement 3A,B and Figure 6—figure supplement 4AB). Third, and by contrast, chloroquine did reactivate HCMV when these knockdown cells were complemented with wild-type KAP1 (Figure 6—figure supplement 4A,B).10.7554/eLife.06068.017Figure 6.Forcing HCMV out of latency in CD34+ cells by KAP1 pharmacological manipulation.

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