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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 is necessary for both establishment and maintenance of HCMV latency in HSC.HCMV kinetics analysis performed on MRC-5 fibroblasts (A) or CD34+ HSC (B). HCMV mRNA and DNA expression were quantified at day 1-3-5-7 and 10 post-infection by RT-qPCR.DOI:http://dx.doi.org/10.7554/eLife.06068.004
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fig1s1: KAP1 is necessary for both establishment and maintenance of HCMV latency in HSC.HCMV kinetics analysis performed on MRC-5 fibroblasts (A) or CD34+ HSC (B). HCMV mRNA and DNA expression were quantified at day 1-3-5-7 and 10 post-infection by RT-qPCR.DOI:http://dx.doi.org/10.7554/eLife.06068.004

Mentions: We first infected either MRC5 fibroblasts or cord blood-derived CD34+ cells with the TB40-E HCMV clinical strain. Monitoring the levels of viral DNA and various RNA transcripts during the following days confirmed that lytic replication occurred in MRC5 cells, known to be fully permissive, whereas latency was established in HSC after an initial outburst of viral gene expression (Figure 1—figure supplement 1A,B), as previously reported (Goodrum et al., 2007). To examine the possible impact of KAP1 on this process, we depleted the corepressor by lentivector-mediated RNA interference, complementing Kap1 knockdown cells by transduction with a vector expressing an shRNA-resistant Kap1 allele when relevant. We then infected these cells with TB40-E HCMV. In MRC5 cells, where KAP1 depletion was around 98% (Figure 1—figure supplement 2A), levels of viral transcripts indicative of a lytic cycle (the IE genes UL123, UL122, the E gene UL54 and the L gene UL94) were comparable in control and KAP1-depleted cells (Figure 1A) and production of viral proteins IE1 and IE2 was unchanged (Figure 1—figure supplement 2B), indicating that the regulator is not essential for productive HCMV replication. In unsorted population of HSC exposed to the knockdown lentivector, KAP1 reduction was only 85% but in sorted CD34+ GFP-positive, that is, transduced cells, it reached 95% (Figure 1—figure supplement 2C,D). In this KAP1-depleted subpopulation, expression of immediate early, early and late viral genes at 7 days post-infection was increased between 10- and 35-fold compared with control cells (Figure 1B), and massive amounts of infectious HCMV particles were released in the supernatant (Figure 1C). Furthermore, complementation of KAP1-depleted cells with an shRNA-resistant Kap1 allele restored HCMV latency (Figure 1—figure supplement 2E–I). These results thus indicated that KAP1 is necessary for the establishment of HCMV latency in HSC. We then inverted the sequence of our manipulations to induce Kap1 knockdown in CD34+ cells that had been infected 7 days earlier with the TB40-E virus (Figure 1—figure supplement 2J). 7 days after transduction, we measured the expression of the UL122, UL123, UL54 and UL94 mRNAs, which are found only during a lytic cycle (Reeves and Sinclair, 2013) (Figure 1D). All transcripts were significantly upregulated in knockdown cells, which accordingly released infectious viral particles (Figure 1E). Furthermore, the NF-κB activator TNFα increased viral gene expression and virion production from Kap1 knockdown but not from control TB40-E-infected CD34+ cells (Figure 1D,E). These data indicate that (i) KAP1 is necessary both for the establishment and for the maintenance of HCMV latency in human HSC, and (ii) the relief of KAP1-mediated repression is not in itself sufficient for full HCMV reactivation in CD34+ cells, but provides the ground for stimulation of viral gene expression by HCMV activators such as NF-κB.10.7554/eLife.06068.003Figure 1.KAP1 is required for HCMV latency.


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 is necessary for both establishment and maintenance of HCMV latency in HSC.HCMV kinetics analysis performed on MRC-5 fibroblasts (A) or CD34+ HSC (B). HCMV mRNA and DNA expression were quantified at day 1-3-5-7 and 10 post-infection by RT-qPCR.DOI:http://dx.doi.org/10.7554/eLife.06068.004
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Related In: Results  -  Collection

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fig1s1: KAP1 is necessary for both establishment and maintenance of HCMV latency in HSC.HCMV kinetics analysis performed on MRC-5 fibroblasts (A) or CD34+ HSC (B). HCMV mRNA and DNA expression were quantified at day 1-3-5-7 and 10 post-infection by RT-qPCR.DOI:http://dx.doi.org/10.7554/eLife.06068.004
Mentions: We first infected either MRC5 fibroblasts or cord blood-derived CD34+ cells with the TB40-E HCMV clinical strain. Monitoring the levels of viral DNA and various RNA transcripts during the following days confirmed that lytic replication occurred in MRC5 cells, known to be fully permissive, whereas latency was established in HSC after an initial outburst of viral gene expression (Figure 1—figure supplement 1A,B), as previously reported (Goodrum et al., 2007). To examine the possible impact of KAP1 on this process, we depleted the corepressor by lentivector-mediated RNA interference, complementing Kap1 knockdown cells by transduction with a vector expressing an shRNA-resistant Kap1 allele when relevant. We then infected these cells with TB40-E HCMV. In MRC5 cells, where KAP1 depletion was around 98% (Figure 1—figure supplement 2A), levels of viral transcripts indicative of a lytic cycle (the IE genes UL123, UL122, the E gene UL54 and the L gene UL94) were comparable in control and KAP1-depleted cells (Figure 1A) and production of viral proteins IE1 and IE2 was unchanged (Figure 1—figure supplement 2B), indicating that the regulator is not essential for productive HCMV replication. In unsorted population of HSC exposed to the knockdown lentivector, KAP1 reduction was only 85% but in sorted CD34+ GFP-positive, that is, transduced cells, it reached 95% (Figure 1—figure supplement 2C,D). In this KAP1-depleted subpopulation, expression of immediate early, early and late viral genes at 7 days post-infection was increased between 10- and 35-fold compared with control cells (Figure 1B), and massive amounts of infectious HCMV particles were released in the supernatant (Figure 1C). Furthermore, complementation of KAP1-depleted cells with an shRNA-resistant Kap1 allele restored HCMV latency (Figure 1—figure supplement 2E–I). These results thus indicated that KAP1 is necessary for the establishment of HCMV latency in HSC. We then inverted the sequence of our manipulations to induce Kap1 knockdown in CD34+ cells that had been infected 7 days earlier with the TB40-E virus (Figure 1—figure supplement 2J). 7 days after transduction, we measured the expression of the UL122, UL123, UL54 and UL94 mRNAs, which are found only during a lytic cycle (Reeves and Sinclair, 2013) (Figure 1D). All transcripts were significantly upregulated in knockdown cells, which accordingly released infectious viral particles (Figure 1E). Furthermore, the NF-κB activator TNFα increased viral gene expression and virion production from Kap1 knockdown but not from control TB40-E-infected CD34+ cells (Figure 1D,E). These data indicate that (i) KAP1 is necessary both for the establishment and for the maintenance of HCMV latency in human HSC, and (ii) the relief of KAP1-mediated repression is not in itself sufficient for full HCMV reactivation in CD34+ cells, but provides the ground for stimulation of viral gene expression by HCMV activators such as NF-κB.10.7554/eLife.06068.003Figure 1.KAP1 is required for HCMV latency.

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