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PML/TRIM19-Dependent Inhibition of Retroviral Reverse-Transcription by Daxx.

Dutrieux J, Maarifi G, Portilho DM, Arhel NJ, Chelbi-Alix MK, Nisole S - PLoS Pathog. (2015)

Bottom Line: We demonstrate that PML does not inhibit directly retroviral infection but acts through the stabilization of one of its well-characterized partners, Daxx.In the presence of PML, cytoplasmic Daxx is found in the vicinity of incoming HIV-1 capsids and inhibits reverse-transcription.Altogether, these findings unravel a novel antiviral function for PML and PML nuclear body-associated protein Daxx.

View Article: PubMed Central - PubMed

Affiliation: Paris Descartes University, Paris, France.

ABSTRACT
PML (Promyelocytic Leukemia protein), also known as TRIM19, belongs to the family of tripartite motif (TRIM) proteins. PML is mainly expressed in the nucleus, where it forms dynamic structures known as PML nuclear bodies that recruit many other proteins, such as Sp100 and Daxx. While the role of PML/TRIM19 in antiviral defense is well documented, its effect on HIV-1 infection remains unclear. Here we show that infection by HIV-1 and other retroviruses triggers the formation of PML cytoplasmic bodies, as early as 30 minutes post-infection. Quantification of the number and size of PML cytoplasmic bodies revealed that they last approximately 8 h, with a peak at 2 h post-infection. PML re-localization is blocked by reverse-transcription inhibitors and is not observed following infection with unrelated viruses, suggesting it is specifically triggered by retroviral reverse-transcription. Furthermore, we show that PML interferes with an early step of retroviral infection since PML knockdown dramatically increases reverse-transcription efficiency. We demonstrate that PML does not inhibit directly retroviral infection but acts through the stabilization of one of its well-characterized partners, Daxx. In the presence of PML, cytoplasmic Daxx is found in the vicinity of incoming HIV-1 capsids and inhibits reverse-transcription. Interestingly, Daxx not only interferes with exogenous retroviral infections but can also inhibit retrotransposition of endogenous retroviruses, thus identifying Daxx as a broad cellular inhibitor of reverse-transcription. Altogether, these findings unravel a novel antiviral function for PML and PML nuclear body-associated protein Daxx.

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PML expression is required for Daxx stability.(A) Western-blot analysis of Daxx, Sp100 and actin expression in wt and PML KO MEFs. (B) Western-blot analysis of PML, Daxx, Sp100 and actin expression in wt HeLa cells transfected with non-targeting control (CTR), PML-specific or Daxx-specific siRNA. Intensity of individual bands was quantified using ImageJ software. (C) HeLa cells stably expressing a non-targeting control shRNA (CTR) or a PML-targeting shRNA were transfected with CTR or Daxx-specific siRNA. Expression of Daxx, PML, Sp100 and actin were evaluated by western-blot analysis. (D) Daxx mRNA relative expression in HeLa cells expressing CTR or PML-targeting shRNA, expressed as a ratio to RPL13A mRNA expression. (E) HeLa cell expressing PML-targeting shRNA were treated with MG132 for 6 h and analyzed by western-blot for Daxx and actin expression. (F) Western-blot analysis of PML, Daxx and actin expression in wt, PML KO and PML KO MEFs stably expressing each human PML isoform (PMLI to PMLVIIb). Note that endogenous PML was not detected in wt MEFs because the western-blot was revealed with anti-PML antibodies specific to human PML isoforms.
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ppat.1005280.g008: PML expression is required for Daxx stability.(A) Western-blot analysis of Daxx, Sp100 and actin expression in wt and PML KO MEFs. (B) Western-blot analysis of PML, Daxx, Sp100 and actin expression in wt HeLa cells transfected with non-targeting control (CTR), PML-specific or Daxx-specific siRNA. Intensity of individual bands was quantified using ImageJ software. (C) HeLa cells stably expressing a non-targeting control shRNA (CTR) or a PML-targeting shRNA were transfected with CTR or Daxx-specific siRNA. Expression of Daxx, PML, Sp100 and actin were evaluated by western-blot analysis. (D) Daxx mRNA relative expression in HeLa cells expressing CTR or PML-targeting shRNA, expressed as a ratio to RPL13A mRNA expression. (E) HeLa cell expressing PML-targeting shRNA were treated with MG132 for 6 h and analyzed by western-blot for Daxx and actin expression. (F) Western-blot analysis of PML, Daxx and actin expression in wt, PML KO and PML KO MEFs stably expressing each human PML isoform (PMLI to PMLVIIb). Note that endogenous PML was not detected in wt MEFs because the western-blot was revealed with anti-PML antibodies specific to human PML isoforms.

Mentions: Having shown that Daxx is the mediator of retroviral restriction in PML expressing cells, we next asked why PML knockdown prevents Daxx-mediated inhibition of retroviral infections. We started by looking at Daxx expression in wt and PML KO MEFs. Unexpectedly, Daxx protein expression was dramatically reduced in PML KO compared to wt MEFs, whereas Sp100 protein level was not affected (Fig 8A). To confirm this observation in human cells, we depleted either PML or Daxx expression using siRNA in HeLa cells. Surprisingly, the siRNA directed against PML reduced the expression of PML, as anticipated, but also drastically diminished Daxx expression, as efficiently as the Daxx-targeting siRNA (Fig 8B). We performed another experiment where we silenced Daxx in HeLa cells stably expressing a non-targeting control shRNA or a shRNA targeting PML. Once again, shRNA-mediated PML knockdown dramatically reduced the expression of Daxx. Furthermore, whereas Daxx can still be detected in cells transfected with a siRNA targeting Daxx, it is completely depleted in cells expressing the shRNA targeting PML (Fig 8C).


PML/TRIM19-Dependent Inhibition of Retroviral Reverse-Transcription by Daxx.

Dutrieux J, Maarifi G, Portilho DM, Arhel NJ, Chelbi-Alix MK, Nisole S - PLoS Pathog. (2015)

PML expression is required for Daxx stability.(A) Western-blot analysis of Daxx, Sp100 and actin expression in wt and PML KO MEFs. (B) Western-blot analysis of PML, Daxx, Sp100 and actin expression in wt HeLa cells transfected with non-targeting control (CTR), PML-specific or Daxx-specific siRNA. Intensity of individual bands was quantified using ImageJ software. (C) HeLa cells stably expressing a non-targeting control shRNA (CTR) or a PML-targeting shRNA were transfected with CTR or Daxx-specific siRNA. Expression of Daxx, PML, Sp100 and actin were evaluated by western-blot analysis. (D) Daxx mRNA relative expression in HeLa cells expressing CTR or PML-targeting shRNA, expressed as a ratio to RPL13A mRNA expression. (E) HeLa cell expressing PML-targeting shRNA were treated with MG132 for 6 h and analyzed by western-blot for Daxx and actin expression. (F) Western-blot analysis of PML, Daxx and actin expression in wt, PML KO and PML KO MEFs stably expressing each human PML isoform (PMLI to PMLVIIb). Note that endogenous PML was not detected in wt MEFs because the western-blot was revealed with anti-PML antibodies specific to human PML isoforms.
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Related In: Results  -  Collection

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ppat.1005280.g008: PML expression is required for Daxx stability.(A) Western-blot analysis of Daxx, Sp100 and actin expression in wt and PML KO MEFs. (B) Western-blot analysis of PML, Daxx, Sp100 and actin expression in wt HeLa cells transfected with non-targeting control (CTR), PML-specific or Daxx-specific siRNA. Intensity of individual bands was quantified using ImageJ software. (C) HeLa cells stably expressing a non-targeting control shRNA (CTR) or a PML-targeting shRNA were transfected with CTR or Daxx-specific siRNA. Expression of Daxx, PML, Sp100 and actin were evaluated by western-blot analysis. (D) Daxx mRNA relative expression in HeLa cells expressing CTR or PML-targeting shRNA, expressed as a ratio to RPL13A mRNA expression. (E) HeLa cell expressing PML-targeting shRNA were treated with MG132 for 6 h and analyzed by western-blot for Daxx and actin expression. (F) Western-blot analysis of PML, Daxx and actin expression in wt, PML KO and PML KO MEFs stably expressing each human PML isoform (PMLI to PMLVIIb). Note that endogenous PML was not detected in wt MEFs because the western-blot was revealed with anti-PML antibodies specific to human PML isoforms.
Mentions: Having shown that Daxx is the mediator of retroviral restriction in PML expressing cells, we next asked why PML knockdown prevents Daxx-mediated inhibition of retroviral infections. We started by looking at Daxx expression in wt and PML KO MEFs. Unexpectedly, Daxx protein expression was dramatically reduced in PML KO compared to wt MEFs, whereas Sp100 protein level was not affected (Fig 8A). To confirm this observation in human cells, we depleted either PML or Daxx expression using siRNA in HeLa cells. Surprisingly, the siRNA directed against PML reduced the expression of PML, as anticipated, but also drastically diminished Daxx expression, as efficiently as the Daxx-targeting siRNA (Fig 8B). We performed another experiment where we silenced Daxx in HeLa cells stably expressing a non-targeting control shRNA or a shRNA targeting PML. Once again, shRNA-mediated PML knockdown dramatically reduced the expression of Daxx. Furthermore, whereas Daxx can still be detected in cells transfected with a siRNA targeting Daxx, it is completely depleted in cells expressing the shRNA targeting PML (Fig 8C).

Bottom Line: We demonstrate that PML does not inhibit directly retroviral infection but acts through the stabilization of one of its well-characterized partners, Daxx.In the presence of PML, cytoplasmic Daxx is found in the vicinity of incoming HIV-1 capsids and inhibits reverse-transcription.Altogether, these findings unravel a novel antiviral function for PML and PML nuclear body-associated protein Daxx.

View Article: PubMed Central - PubMed

Affiliation: Paris Descartes University, Paris, France.

ABSTRACT
PML (Promyelocytic Leukemia protein), also known as TRIM19, belongs to the family of tripartite motif (TRIM) proteins. PML is mainly expressed in the nucleus, where it forms dynamic structures known as PML nuclear bodies that recruit many other proteins, such as Sp100 and Daxx. While the role of PML/TRIM19 in antiviral defense is well documented, its effect on HIV-1 infection remains unclear. Here we show that infection by HIV-1 and other retroviruses triggers the formation of PML cytoplasmic bodies, as early as 30 minutes post-infection. Quantification of the number and size of PML cytoplasmic bodies revealed that they last approximately 8 h, with a peak at 2 h post-infection. PML re-localization is blocked by reverse-transcription inhibitors and is not observed following infection with unrelated viruses, suggesting it is specifically triggered by retroviral reverse-transcription. Furthermore, we show that PML interferes with an early step of retroviral infection since PML knockdown dramatically increases reverse-transcription efficiency. We demonstrate that PML does not inhibit directly retroviral infection but acts through the stabilization of one of its well-characterized partners, Daxx. In the presence of PML, cytoplasmic Daxx is found in the vicinity of incoming HIV-1 capsids and inhibits reverse-transcription. Interestingly, Daxx not only interferes with exogenous retroviral infections but can also inhibit retrotransposition of endogenous retroviruses, thus identifying Daxx as a broad cellular inhibitor of reverse-transcription. Altogether, these findings unravel a novel antiviral function for PML and PML nuclear body-associated protein Daxx.

Show MeSH
Related in: MedlinePlus