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CpG methylation controls reactivation of HIV from latency.

Blazkova J, Trejbalova K, Gondois-Rey F, Halfon P, Philibert P, Guiguen A, Verdin E, Olive D, Van Lint C, Hejnar J, Hirsch I - PLoS Pathog. (2009)

Bottom Line: The latency controlled solely by transcriptional interference and by chromatin-dependent mechanisms in the absence of significant promoter DNA methylation tends to be leaky and easily reactivable.In the latent reservoir of HIV-1-infected individuals without detectable plasma viremia, we found HIV-1 promoters and enhancers to be hypermethylated and resistant to reactivation, as opposed to the hypomethylated 5' LTR in viremic patients.However, even dense methylation of the HIV-1 5'LTR did not confer complete resistance to reactivation of latent HIV-1 with some histone deacetylase inhibitors, protein kinase C agonists, TNF-alpha, and their combinations with 5-aza-2deoxycytidine: the densely methylated HIV-1 promoter was most efficiently reactivated in virtual absence of T cell activation by suberoylanilide hydroxamic acid.

View Article: PubMed Central - PubMed

Affiliation: Institut National de la Santé et de la Recherche Médicale (INSERM), UMR891, Centre de Recherche en Cancérologie de Marseille; and Institut Paoli-Calmettes, Marseille, France.

ABSTRACT
DNA methylation of retroviral promoters and enhancers localized in the provirus 5' long terminal repeat (LTR) is considered to be a mechanism of transcriptional suppression that allows retroviruses to evade host immune responses and antiretroviral drugs. However, the role of DNA methylation in the control of HIV-1 latency has never been unambiguously demonstrated, in contrast to the apparent importance of transcriptional interference and chromatin structure, and has never been studied in HIV-1-infected patients. Here, we show in an in vitro model of reactivable latency and in a latent reservoir of HIV-1-infected patients that CpG methylation of the HIV-1 5' LTR is an additional epigenetic restriction mechanism, which controls resistance of latent HIV-1 to reactivation signals and thus determines the stability of the HIV-1 latency. CpG methylation acts as a late event during establishment of HIV-1 latency and is not required for the initial provirus silencing. Indeed, the latent reservoir of some aviremic patients contained high proportions of the non-methylated 5' LTR. The latency controlled solely by transcriptional interference and by chromatin-dependent mechanisms in the absence of significant promoter DNA methylation tends to be leaky and easily reactivable. In the latent reservoir of HIV-1-infected individuals without detectable plasma viremia, we found HIV-1 promoters and enhancers to be hypermethylated and resistant to reactivation, as opposed to the hypomethylated 5' LTR in viremic patients. However, even dense methylation of the HIV-1 5'LTR did not confer complete resistance to reactivation of latent HIV-1 with some histone deacetylase inhibitors, protein kinase C agonists, TNF-alpha, and their combinations with 5-aza-2deoxycytidine: the densely methylated HIV-1 promoter was most efficiently reactivated in virtual absence of T cell activation by suberoylanilide hydroxamic acid. Tight but incomplete control of HIV-1 latency by CpG methylation might have important implications for strategies aimed at eradicating HIV-1 infection.

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Methylation analysis of HIV-1 promoter in memory CD4+ T cells purified from HIV-1-infected individuals.Hypermethylation of the HIV-1 5′ LTR in HIV-1-infected long-term aviremic individuals contrasts with hypomethylation of the HIV-1 5′ LTR in viremic patients and negatively correlates with reactivation of the HIV-1 provirus. (A) CpG methylation patterns in the bisulfite-treated HIV-1 5′ LTR sequences of aviremic patients were clustered using neighbor-joining method into several groups within each patient (separated by horizontal bars) with a strong bootstrap support (>990/1000). The sequences within each group differed in addition in about 1 to 10 point-mutations. Note that the variability of bisulfite-treated sequences is underestimated due to the conversion of the majority of cytosine residues to thymine (except for cytosine in 5-methylcytosine residues). Open circles, nonmethylated CpG residues; closed circles, methylated CpG residues. (B) Percentage of methylated CpGs (mCpG) in the 5′ LTR of HIV-1 in long-term aviremic (n = 6) and viremic (n = 7) patients. p, non-parametric, two side Mann-Whitney test. (C–F) Reactivation of HIV-1 provirus in memory CD4+ T cells obtained from patients with different levels of CpG methylation. Memory CD4+ T cells cultured for three days in the presence of reactivating agents were 10-fold serially diluted in duplicate and co-cultured with PHA-activated CD4+ T cells from an allogeneic healthy donor. HIV-1 replication was followed by determination of p24 in the cell-free supernatant. Percentage of reactivable provirus is presented as a ratio of end-point dilutions of patients' CD4+ T cells producing HIV-1 virus to the number of DNA proviral copies in the cell quantity equivalent to end-point dilution and determined by quantitative PCR, as indicated in Table 1. (C) Non-stimulated CD4+ T cells. (D) CD4+ T cells stimulated with TNF-α at 10 ng/ml and 10-nM PMA. (E) CD4+ T cells stimulated with 500-nM TSA and 10-nM PMA. (F) CD4+ T cells stimulated with 5-µM 5-aza-dC. (C–F) Open symbols, viremic patients; closed symbols, aviremic patients.
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ppat-1000554-g006: Methylation analysis of HIV-1 promoter in memory CD4+ T cells purified from HIV-1-infected individuals.Hypermethylation of the HIV-1 5′ LTR in HIV-1-infected long-term aviremic individuals contrasts with hypomethylation of the HIV-1 5′ LTR in viremic patients and negatively correlates with reactivation of the HIV-1 provirus. (A) CpG methylation patterns in the bisulfite-treated HIV-1 5′ LTR sequences of aviremic patients were clustered using neighbor-joining method into several groups within each patient (separated by horizontal bars) with a strong bootstrap support (>990/1000). The sequences within each group differed in addition in about 1 to 10 point-mutations. Note that the variability of bisulfite-treated sequences is underestimated due to the conversion of the majority of cytosine residues to thymine (except for cytosine in 5-methylcytosine residues). Open circles, nonmethylated CpG residues; closed circles, methylated CpG residues. (B) Percentage of methylated CpGs (mCpG) in the 5′ LTR of HIV-1 in long-term aviremic (n = 6) and viremic (n = 7) patients. p, non-parametric, two side Mann-Whitney test. (C–F) Reactivation of HIV-1 provirus in memory CD4+ T cells obtained from patients with different levels of CpG methylation. Memory CD4+ T cells cultured for three days in the presence of reactivating agents were 10-fold serially diluted in duplicate and co-cultured with PHA-activated CD4+ T cells from an allogeneic healthy donor. HIV-1 replication was followed by determination of p24 in the cell-free supernatant. Percentage of reactivable provirus is presented as a ratio of end-point dilutions of patients' CD4+ T cells producing HIV-1 virus to the number of DNA proviral copies in the cell quantity equivalent to end-point dilution and determined by quantitative PCR, as indicated in Table 1. (C) Non-stimulated CD4+ T cells. (D) CD4+ T cells stimulated with TNF-α at 10 ng/ml and 10-nM PMA. (E) CD4+ T cells stimulated with 500-nM TSA and 10-nM PMA. (F) CD4+ T cells stimulated with 5-µM 5-aza-dC. (C–F) Open symbols, viremic patients; closed symbols, aviremic patients.

Mentions: Our next aim was to compare the methylation pattern of latent HIV-1 promoter in our in vitro model with that in infected individuals. We inspected the 5′ LTR CpG methylation in latent reservoirs of resting CD4+ T cells in patients in whom HAART resulted in long-term suppression of plasma viremia and in viremic patients (Table 1 and Table S3). The low percentage of HIV provirus-harboring cells in the latent reservoir and the low sensitivity of PCR after bisulfite treatment of DNA hampered determination of the methylation pattern in several analyzed patients. Thus, from a cohort of 18 HAART-treated patients without detectable plasma viremia, we analyzed six individuals, and from 13 viremic patients we analyzed seven individuals with ≥103 HIV-1 DNA copies per million CD4+ cells (Table 1 and Figure 6A and B).


CpG methylation controls reactivation of HIV from latency.

Blazkova J, Trejbalova K, Gondois-Rey F, Halfon P, Philibert P, Guiguen A, Verdin E, Olive D, Van Lint C, Hejnar J, Hirsch I - PLoS Pathog. (2009)

Methylation analysis of HIV-1 promoter in memory CD4+ T cells purified from HIV-1-infected individuals.Hypermethylation of the HIV-1 5′ LTR in HIV-1-infected long-term aviremic individuals contrasts with hypomethylation of the HIV-1 5′ LTR in viremic patients and negatively correlates with reactivation of the HIV-1 provirus. (A) CpG methylation patterns in the bisulfite-treated HIV-1 5′ LTR sequences of aviremic patients were clustered using neighbor-joining method into several groups within each patient (separated by horizontal bars) with a strong bootstrap support (>990/1000). The sequences within each group differed in addition in about 1 to 10 point-mutations. Note that the variability of bisulfite-treated sequences is underestimated due to the conversion of the majority of cytosine residues to thymine (except for cytosine in 5-methylcytosine residues). Open circles, nonmethylated CpG residues; closed circles, methylated CpG residues. (B) Percentage of methylated CpGs (mCpG) in the 5′ LTR of HIV-1 in long-term aviremic (n = 6) and viremic (n = 7) patients. p, non-parametric, two side Mann-Whitney test. (C–F) Reactivation of HIV-1 provirus in memory CD4+ T cells obtained from patients with different levels of CpG methylation. Memory CD4+ T cells cultured for three days in the presence of reactivating agents were 10-fold serially diluted in duplicate and co-cultured with PHA-activated CD4+ T cells from an allogeneic healthy donor. HIV-1 replication was followed by determination of p24 in the cell-free supernatant. Percentage of reactivable provirus is presented as a ratio of end-point dilutions of patients' CD4+ T cells producing HIV-1 virus to the number of DNA proviral copies in the cell quantity equivalent to end-point dilution and determined by quantitative PCR, as indicated in Table 1. (C) Non-stimulated CD4+ T cells. (D) CD4+ T cells stimulated with TNF-α at 10 ng/ml and 10-nM PMA. (E) CD4+ T cells stimulated with 500-nM TSA and 10-nM PMA. (F) CD4+ T cells stimulated with 5-µM 5-aza-dC. (C–F) Open symbols, viremic patients; closed symbols, aviremic patients.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2722084&req=5

ppat-1000554-g006: Methylation analysis of HIV-1 promoter in memory CD4+ T cells purified from HIV-1-infected individuals.Hypermethylation of the HIV-1 5′ LTR in HIV-1-infected long-term aviremic individuals contrasts with hypomethylation of the HIV-1 5′ LTR in viremic patients and negatively correlates with reactivation of the HIV-1 provirus. (A) CpG methylation patterns in the bisulfite-treated HIV-1 5′ LTR sequences of aviremic patients were clustered using neighbor-joining method into several groups within each patient (separated by horizontal bars) with a strong bootstrap support (>990/1000). The sequences within each group differed in addition in about 1 to 10 point-mutations. Note that the variability of bisulfite-treated sequences is underestimated due to the conversion of the majority of cytosine residues to thymine (except for cytosine in 5-methylcytosine residues). Open circles, nonmethylated CpG residues; closed circles, methylated CpG residues. (B) Percentage of methylated CpGs (mCpG) in the 5′ LTR of HIV-1 in long-term aviremic (n = 6) and viremic (n = 7) patients. p, non-parametric, two side Mann-Whitney test. (C–F) Reactivation of HIV-1 provirus in memory CD4+ T cells obtained from patients with different levels of CpG methylation. Memory CD4+ T cells cultured for three days in the presence of reactivating agents were 10-fold serially diluted in duplicate and co-cultured with PHA-activated CD4+ T cells from an allogeneic healthy donor. HIV-1 replication was followed by determination of p24 in the cell-free supernatant. Percentage of reactivable provirus is presented as a ratio of end-point dilutions of patients' CD4+ T cells producing HIV-1 virus to the number of DNA proviral copies in the cell quantity equivalent to end-point dilution and determined by quantitative PCR, as indicated in Table 1. (C) Non-stimulated CD4+ T cells. (D) CD4+ T cells stimulated with TNF-α at 10 ng/ml and 10-nM PMA. (E) CD4+ T cells stimulated with 500-nM TSA and 10-nM PMA. (F) CD4+ T cells stimulated with 5-µM 5-aza-dC. (C–F) Open symbols, viremic patients; closed symbols, aviremic patients.
Mentions: Our next aim was to compare the methylation pattern of latent HIV-1 promoter in our in vitro model with that in infected individuals. We inspected the 5′ LTR CpG methylation in latent reservoirs of resting CD4+ T cells in patients in whom HAART resulted in long-term suppression of plasma viremia and in viremic patients (Table 1 and Table S3). The low percentage of HIV provirus-harboring cells in the latent reservoir and the low sensitivity of PCR after bisulfite treatment of DNA hampered determination of the methylation pattern in several analyzed patients. Thus, from a cohort of 18 HAART-treated patients without detectable plasma viremia, we analyzed six individuals, and from 13 viremic patients we analyzed seven individuals with ≥103 HIV-1 DNA copies per million CD4+ cells (Table 1 and Figure 6A and B).

Bottom Line: The latency controlled solely by transcriptional interference and by chromatin-dependent mechanisms in the absence of significant promoter DNA methylation tends to be leaky and easily reactivable.In the latent reservoir of HIV-1-infected individuals without detectable plasma viremia, we found HIV-1 promoters and enhancers to be hypermethylated and resistant to reactivation, as opposed to the hypomethylated 5' LTR in viremic patients.However, even dense methylation of the HIV-1 5'LTR did not confer complete resistance to reactivation of latent HIV-1 with some histone deacetylase inhibitors, protein kinase C agonists, TNF-alpha, and their combinations with 5-aza-2deoxycytidine: the densely methylated HIV-1 promoter was most efficiently reactivated in virtual absence of T cell activation by suberoylanilide hydroxamic acid.

View Article: PubMed Central - PubMed

Affiliation: Institut National de la Santé et de la Recherche Médicale (INSERM), UMR891, Centre de Recherche en Cancérologie de Marseille; and Institut Paoli-Calmettes, Marseille, France.

ABSTRACT
DNA methylation of retroviral promoters and enhancers localized in the provirus 5' long terminal repeat (LTR) is considered to be a mechanism of transcriptional suppression that allows retroviruses to evade host immune responses and antiretroviral drugs. However, the role of DNA methylation in the control of HIV-1 latency has never been unambiguously demonstrated, in contrast to the apparent importance of transcriptional interference and chromatin structure, and has never been studied in HIV-1-infected patients. Here, we show in an in vitro model of reactivable latency and in a latent reservoir of HIV-1-infected patients that CpG methylation of the HIV-1 5' LTR is an additional epigenetic restriction mechanism, which controls resistance of latent HIV-1 to reactivation signals and thus determines the stability of the HIV-1 latency. CpG methylation acts as a late event during establishment of HIV-1 latency and is not required for the initial provirus silencing. Indeed, the latent reservoir of some aviremic patients contained high proportions of the non-methylated 5' LTR. The latency controlled solely by transcriptional interference and by chromatin-dependent mechanisms in the absence of significant promoter DNA methylation tends to be leaky and easily reactivable. In the latent reservoir of HIV-1-infected individuals without detectable plasma viremia, we found HIV-1 promoters and enhancers to be hypermethylated and resistant to reactivation, as opposed to the hypomethylated 5' LTR in viremic patients. However, even dense methylation of the HIV-1 5'LTR did not confer complete resistance to reactivation of latent HIV-1 with some histone deacetylase inhibitors, protein kinase C agonists, TNF-alpha, and their combinations with 5-aza-2deoxycytidine: the densely methylated HIV-1 promoter was most efficiently reactivated in virtual absence of T cell activation by suberoylanilide hydroxamic acid. Tight but incomplete control of HIV-1 latency by CpG methylation might have important implications for strategies aimed at eradicating HIV-1 infection.

Show MeSH
Related in: MedlinePlus