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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 and HCMV-associated KAP1 phosphorylation.(A–C) Confocal microscopy coupled to immunofluorescence was performed on MRC-5 (A–C), HCMV infected (TB40-E) or not (Non Infected) and treated or not (Non Treated) with mTOR inhibitors (Torin1). Staining was performed with antibodies against IE or anti-β-Tubulin (Alexa 488, green), and pS473KAP1 (Alexa 568, red), staining DNA with Dapi (blue). White scale bar, 10 µm. All pictures are representative of results obtained in three independent experiments. (D) In vitro phosphorylation of GST-KAP1 by recombinant mTOR, using Western blot and Ponseyu staining to detect pS824KAP1 and total KAP1, respectively.DOI:http://dx.doi.org/10.7554/eLife.06068.014
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fig4s2: mTOR and HCMV-associated KAP1 phosphorylation.(A–C) Confocal microscopy coupled to immunofluorescence was performed on MRC-5 (A–C), HCMV infected (TB40-E) or not (Non Infected) and treated or not (Non Treated) with mTOR inhibitors (Torin1). Staining was performed with antibodies against IE or anti-β-Tubulin (Alexa 488, green), and pS473KAP1 (Alexa 568, red), staining DNA with Dapi (blue). White scale bar, 10 µm. All pictures are representative of results obtained in three independent experiments. (D) In vitro phosphorylation of GST-KAP1 by recombinant mTOR, using Western blot and Ponseyu staining to detect pS824KAP1 and total KAP1, respectively.DOI:http://dx.doi.org/10.7554/eLife.06068.014

Mentions: We then set to identify the kinase responsible for phosphorylating KAP1 in cells productively infected with HCMV. The ATM inhibitor KU55933 did not prevent this process, suggesting that this known KAP1 kinase did not play a primary role (Figure 4—figure supplement 1B). We thus turned to the mammalian target of rapamycin (mTOR), which was previously found activated in cells replicating HCMV (Moorman and Shenk, 2010; Clippinger et al., 2011; Clippinger and Alwine, 2012; Poglitsch et al., 2012). When TB40-E infected MRC-5 cells were treated with the mTOR inhibitors rapamycin or Torin1 (Thoreen and Sabatini, 2009; Thoreen et al., 2009), the phosphoKAP1-specific immunofluorescence signal was suppressed (Figure 4C and Figure 4—figure supplement 1C), whereas total levels of KAP1 were unaffected (Figure 4—figure supplement 1D). Torin1-preventable pS824KAP1 accumulation was also documented in CD34+ HSC infected with the replicative AD169 strain (Figure 4D and Figure 4—figure supplement 1E,F). In this setting, the IE protein-specific signal was reduced by the mTOR inhibitor, consistent with some repression of viral gene expression. Of note, some KAP1 phosphorylated on serine 473 was detected in MRC5 cells, but this was independent of HCMV infection and Torin1-resistant (Figure 4—figure supplement 2A–C). In contrast, recombinant mTOR could phosphorylate GST-KAP1 on S824 in vitro (Figure 4—figure supplement 2D).


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 and HCMV-associated KAP1 phosphorylation.(A–C) Confocal microscopy coupled to immunofluorescence was performed on MRC-5 (A–C), HCMV infected (TB40-E) or not (Non Infected) and treated or not (Non Treated) with mTOR inhibitors (Torin1). Staining was performed with antibodies against IE or anti-β-Tubulin (Alexa 488, green), and pS473KAP1 (Alexa 568, red), staining DNA with Dapi (blue). White scale bar, 10 µm. All pictures are representative of results obtained in three independent experiments. (D) In vitro phosphorylation of GST-KAP1 by recombinant mTOR, using Western blot and Ponseyu staining to detect pS824KAP1 and total KAP1, respectively.DOI:http://dx.doi.org/10.7554/eLife.06068.014
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Related In: Results  -  Collection

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fig4s2: mTOR and HCMV-associated KAP1 phosphorylation.(A–C) Confocal microscopy coupled to immunofluorescence was performed on MRC-5 (A–C), HCMV infected (TB40-E) or not (Non Infected) and treated or not (Non Treated) with mTOR inhibitors (Torin1). Staining was performed with antibodies against IE or anti-β-Tubulin (Alexa 488, green), and pS473KAP1 (Alexa 568, red), staining DNA with Dapi (blue). White scale bar, 10 µm. All pictures are representative of results obtained in three independent experiments. (D) In vitro phosphorylation of GST-KAP1 by recombinant mTOR, using Western blot and Ponseyu staining to detect pS824KAP1 and total KAP1, respectively.DOI:http://dx.doi.org/10.7554/eLife.06068.014
Mentions: We then set to identify the kinase responsible for phosphorylating KAP1 in cells productively infected with HCMV. The ATM inhibitor KU55933 did not prevent this process, suggesting that this known KAP1 kinase did not play a primary role (Figure 4—figure supplement 1B). We thus turned to the mammalian target of rapamycin (mTOR), which was previously found activated in cells replicating HCMV (Moorman and Shenk, 2010; Clippinger et al., 2011; Clippinger and Alwine, 2012; Poglitsch et al., 2012). When TB40-E infected MRC-5 cells were treated with the mTOR inhibitors rapamycin or Torin1 (Thoreen and Sabatini, 2009; Thoreen et al., 2009), the phosphoKAP1-specific immunofluorescence signal was suppressed (Figure 4C and Figure 4—figure supplement 1C), whereas total levels of KAP1 were unaffected (Figure 4—figure supplement 1D). Torin1-preventable pS824KAP1 accumulation was also documented in CD34+ HSC infected with the replicative AD169 strain (Figure 4D and Figure 4—figure supplement 1E,F). In this setting, the IE protein-specific signal was reduced by the mTOR inhibitor, consistent with some repression of viral gene expression. Of note, some KAP1 phosphorylated on serine 473 was detected in MRC5 cells, but this was independent of HCMV infection and Torin1-resistant (Figure 4—figure supplement 2A–C). In contrast, recombinant mTOR could phosphorylate GST-KAP1 on S824 in vitro (Figure 4—figure supplement 2D).

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