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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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Reverse-transcription is the step affected by PML expression and the required event triggering PML CB formation.(A) Wt or PML KO MEFs were transduced for different times at MOI 10 with VSV-G pseudotyped HIV-1 derived vector, DNA was extracted and early and late reverse transcript were quantified by qPCR. (B) Wt or PML KO MEFs were treated for 30 min with nevirapine (NVP) before a 2 h transduction at MOI 10 with HIV-1 vector. DNA was extracted and early and late RT products were quantified by qPCR (left panel). PML (in green) and p24 (in red) localization was determined by immunofluorescence (middle panel). For each condition, the number of PML CBs was quantified in 50 cells from 3 independent fields (right panel). Student t-test was performed to determine P values (**P<0.01). (C) The amount of SUMOylated proteins in wt MEFs treated with GA for 24 h was evaluated by western-blot (left panel). In parallel, wt MEFs pretreated with GA for 4 h were transduced for 2 h at MOI 10 with HIV-1 vector and PML (in green) and HIV-1 p24 (in red) localization was determined by immunofluorescence (right panel).
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ppat.1005280.g003: Reverse-transcription is the step affected by PML expression and the required event triggering PML CB formation.(A) Wt or PML KO MEFs were transduced for different times at MOI 10 with VSV-G pseudotyped HIV-1 derived vector, DNA was extracted and early and late reverse transcript were quantified by qPCR. (B) Wt or PML KO MEFs were treated for 30 min with nevirapine (NVP) before a 2 h transduction at MOI 10 with HIV-1 vector. DNA was extracted and early and late RT products were quantified by qPCR (left panel). PML (in green) and p24 (in red) localization was determined by immunofluorescence (middle panel). For each condition, the number of PML CBs was quantified in 50 cells from 3 independent fields (right panel). Student t-test was performed to determine P values (**P<0.01). (C) The amount of SUMOylated proteins in wt MEFs treated with GA for 24 h was evaluated by western-blot (left panel). In parallel, wt MEFs pretreated with GA for 4 h were transduced for 2 h at MOI 10 with HIV-1 vector and PML (in green) and HIV-1 p24 (in red) localization was determined by immunofluorescence (right panel).

Mentions: Next, we investigated the precise step of HIV replicative cycle that is enhanced following PML knockdown in murine cells. To do this, we performed a kinetic analysis of HIV transduction in wt and PML KO MEFs and determined the amount of early and late RT products by quantitative PCR. As shown in Fig 3A, the amount of RT products was much higher in PML KO compared to wt MEFs, suggesting that PML expression prevents the accumulation of RT products. We therefore asked whether RT triggers PML re-localization to the cytoplasm. We treated wt MEFs with nevirapine (NVP), a non-nucleoside RT inhibitor and transduced them with HIV-1. As expected, NVP treatment led to a dramatic reduction of both early and late RT products, quantified by quantitative PCR (Fig 3B, left panel). Interestingly, whereas PML bodies can be detected in the cytoplasm of untreated cells 2 h post-transduction, no PML CBs could be detected in NVP-treated cells (Fig 3A, right panel), indicating that RT is a prerequisite for the formation of PML CBs.


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)

Reverse-transcription is the step affected by PML expression and the required event triggering PML CB formation.(A) Wt or PML KO MEFs were transduced for different times at MOI 10 with VSV-G pseudotyped HIV-1 derived vector, DNA was extracted and early and late reverse transcript were quantified by qPCR. (B) Wt or PML KO MEFs were treated for 30 min with nevirapine (NVP) before a 2 h transduction at MOI 10 with HIV-1 vector. DNA was extracted and early and late RT products were quantified by qPCR (left panel). PML (in green) and p24 (in red) localization was determined by immunofluorescence (middle panel). For each condition, the number of PML CBs was quantified in 50 cells from 3 independent fields (right panel). Student t-test was performed to determine P values (**P<0.01). (C) The amount of SUMOylated proteins in wt MEFs treated with GA for 24 h was evaluated by western-blot (left panel). In parallel, wt MEFs pretreated with GA for 4 h were transduced for 2 h at MOI 10 with HIV-1 vector and PML (in green) and HIV-1 p24 (in red) localization was determined by immunofluorescence (right panel).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4643962&req=5

ppat.1005280.g003: Reverse-transcription is the step affected by PML expression and the required event triggering PML CB formation.(A) Wt or PML KO MEFs were transduced for different times at MOI 10 with VSV-G pseudotyped HIV-1 derived vector, DNA was extracted and early and late reverse transcript were quantified by qPCR. (B) Wt or PML KO MEFs were treated for 30 min with nevirapine (NVP) before a 2 h transduction at MOI 10 with HIV-1 vector. DNA was extracted and early and late RT products were quantified by qPCR (left panel). PML (in green) and p24 (in red) localization was determined by immunofluorescence (middle panel). For each condition, the number of PML CBs was quantified in 50 cells from 3 independent fields (right panel). Student t-test was performed to determine P values (**P<0.01). (C) The amount of SUMOylated proteins in wt MEFs treated with GA for 24 h was evaluated by western-blot (left panel). In parallel, wt MEFs pretreated with GA for 4 h were transduced for 2 h at MOI 10 with HIV-1 vector and PML (in green) and HIV-1 p24 (in red) localization was determined by immunofluorescence (right panel).
Mentions: Next, we investigated the precise step of HIV replicative cycle that is enhanced following PML knockdown in murine cells. To do this, we performed a kinetic analysis of HIV transduction in wt and PML KO MEFs and determined the amount of early and late RT products by quantitative PCR. As shown in Fig 3A, the amount of RT products was much higher in PML KO compared to wt MEFs, suggesting that PML expression prevents the accumulation of RT products. We therefore asked whether RT triggers PML re-localization to the cytoplasm. We treated wt MEFs with nevirapine (NVP), a non-nucleoside RT inhibitor and transduced them with HIV-1. As expected, NVP treatment led to a dramatic reduction of both early and late RT products, quantified by quantitative PCR (Fig 3B, left panel). Interestingly, whereas PML bodies can be detected in the cytoplasm of untreated cells 2 h post-transduction, no PML CBs could be detected in NVP-treated cells (Fig 3A, right panel), indicating that RT is a prerequisite for the formation of PML CBs.

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