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Development of 5' LTR DNA methylation of latent HIV-1 provirus in cell line models and in long-term-infected individuals.

Trejbalová K, Kovářová D, Blažková J, Machala L, Jilich D, Weber J, Kučerová D, Vencálek O, Hirsch I, Hejnar J - Clin Epigenetics (2016)

Bottom Line: We detected low levels of 5' LTR DNA methylation in the resting CD4(+) T cells of the group of patients who were treated for up to 3 years.However, after long-term ART, we observed an accumulation of 5' LTR DNA methylation in the latent reservoir.Importantly, within the latent reservoir of some long-term-treated individuals, we uncovered populations of proviral molecules with a high density of 5' LTR CpG methylation.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Vídeňská 1083, CZ-14220 Prague 4, Czech Republic.

ABSTRACT

Background: Human immunodeficiency virus type 1 (HIV-1) latency represents the major barrier to virus eradication in infected individuals because cells harboring latent HIV-1 provirus are not affected by current antiretroviral therapy (ART). We previously demonstrated that DNA methylation of HIV-1 long terminal repeat (5' LTR) restricts HIV-1 reactivation and, together with chromatin conformation, represents an important mechanism of HIV-1 latency maintenance. Here, we explored the new issue of temporal development of DNA methylation in latent HIV-1 5' LTR.

Results: In the Jurkat CD4(+) T cell model of latency, we showed that the stimulation of host cells contributed to de novo DNA methylation of the latent HIV-1 5' LTR sequences. Consecutive stimulations of model CD4(+) T cell line with TNF-α and PMA or with SAHA contributed to the progressive accumulation of 5' LTR DNA methylation. Further, we showed that once established, the high DNA methylation level of the latent 5' LTR in the cell line model was a stable epigenetic mark. Finally, we explored the development of 5' LTR DNA methylation in the latent reservoir of HIV-1-infected individuals who were treated with ART. We detected low levels of 5' LTR DNA methylation in the resting CD4(+) T cells of the group of patients who were treated for up to 3 years. However, after long-term ART, we observed an accumulation of 5' LTR DNA methylation in the latent reservoir. Importantly, within the latent reservoir of some long-term-treated individuals, we uncovered populations of proviral molecules with a high density of 5' LTR CpG methylation.

Conclusions: Our data showed the presence of 5' LTR DNA methylation in the long-term reservoir of HIV-1-infected individuals and implied that the transient stimulation of cells harboring latent proviruses may contribute, at least in part, to the methylation of the HIV-1 promoter.

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5’ LTR and env methylation levels of the HIV-1 provirus in the latent reservoir of long-term treated HIV-1-infected individuals. a The levels of HIV-1 5’ LTR methylation in long-term treated patients. Each diamond represents one patient. The time (years) spent undergoing therapy is depicted on the x-axis whereas the percentage of methylated CpGs in the 5’ LTR of HIV-1 in the latent reservoir of infected patients is depicted on the y-axis. The methylation levels are presented as a mean percentage of methylated CpGs in HIV-1 promoters. b Latent HIV-1 provirus CpG methylation profiles at the 5’ LTR sequences of long-term treated patients nos. LT1 to LT10. The methylation levels are presented as a mean percentage of methylated CpGs (mCpGs) in HIV-1 promoters. An analysis of promoter molecules is shown as a linear array of open circles representing non-methylated CpG residues and closed circles representing methylated CpG residues. Each line represents one sequenced molecule of the 5’ LTR. The rectangle schematically represents the 5’ LTR region U3 with a distribution of individually analyzed CpG dinucleotides and transcription factor binding sites. c Levels of HIV-1 env methylation in selected long-term-treated individuals. The methylation levels are presented as a mean percentage of methylated CpGs mCpGs) in HIV-1 env region. An analysis of env molecules is shown as a linear array of open circles representing non-methylated CpG residues and closed circles representing methylated CpG residues. Each line represents one sequenced env molecule. The rectangle schematically represents the analyzed env region.
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Fig7: 5’ LTR and env methylation levels of the HIV-1 provirus in the latent reservoir of long-term treated HIV-1-infected individuals. a The levels of HIV-1 5’ LTR methylation in long-term treated patients. Each diamond represents one patient. The time (years) spent undergoing therapy is depicted on the x-axis whereas the percentage of methylated CpGs in the 5’ LTR of HIV-1 in the latent reservoir of infected patients is depicted on the y-axis. The methylation levels are presented as a mean percentage of methylated CpGs in HIV-1 promoters. b Latent HIV-1 provirus CpG methylation profiles at the 5’ LTR sequences of long-term treated patients nos. LT1 to LT10. The methylation levels are presented as a mean percentage of methylated CpGs (mCpGs) in HIV-1 promoters. An analysis of promoter molecules is shown as a linear array of open circles representing non-methylated CpG residues and closed circles representing methylated CpG residues. Each line represents one sequenced molecule of the 5’ LTR. The rectangle schematically represents the 5’ LTR region U3 with a distribution of individually analyzed CpG dinucleotides and transcription factor binding sites. c Levels of HIV-1 env methylation in selected long-term-treated individuals. The methylation levels are presented as a mean percentage of methylated CpGs mCpGs) in HIV-1 env region. An analysis of env molecules is shown as a linear array of open circles representing non-methylated CpG residues and closed circles representing methylated CpG residues. Each line represents one sequenced env molecule. The rectangle schematically represents the analyzed env region.

Mentions: To further explore the long-term (LT) development of 5’ LTR methylation in the latent reservoir of HIV-1-infected individuals, we enrolled another cohort of 10 patients who were treated for 3 to 22 years (median 12.5 years; Additional file 7: Table S2). The DNA methylation analysis of HIV-1 5’ LTR, which was performed in the same manner as in the first cohort, showed different levels of methylation ranging from 0 to 45 % methylated CpGs (Fig. 7a). The highest result, 45 % methylated CpGs, was found in a single patient (no. LT1); three other patients displayed 10, 15, and 19 % methylated CpGs, respectively, and six patients displayed between 0 and 3 % methylated CpGs in the 5’ LTR (Fig. 7b). In several individuals, we also analyzed the HIV-1 5’ LTR methylation levels in isolated memory CD4+ T cells. We obtained identical results for both memory and resting CD4+ T cell populations (data not shown). As in the group of patients treated for up to 3 years, the CpG methylation within the envelope coding region of four long-term-treated individuals displayed variable levels of CpG methylation ranging from 10 to 74 % (Fig. 7c). In the latent reservoir of three patients with high or moderate average 5’ LTR DNA methylation levels, we detected both heavily methylated and non-methylated 5’ LTR molecules within the same individual (patient nos. LT1, LT5, and LT10). Methylated 5’ LTR sequences of the long-term treated patients did not display large rearrangements or mutations of CpG dinucleotides (Fig. 7b). However, mutated 5’ LTR CpG dinucleotides were detected in 5’ LTR sequences with low methylation levels in patient nos. LT4 and LT9 (Fig. 7b). As for the previous cohort, our results do not correspond with the preferential methylation of defective 5’ LTRs of the HIV-1 provirus. Finally, in the highly methylated 5’ LTRs in patient nos. LT5 and LT10, we observed a lack of methylation at the CpG located within the NF-κB binding site which was identical with the hypomethylated CpG in the model cell lines. When we evaluated the 5’ LTR CpG methylation levels from all patients fused into one group, we found a statistically significant increase of the 5’ LTR DNA methylation levels (Additional file 8: Figure S6, p < 0,001). The percentage of methylated cytosines contained in the 5’ LTR arose by 7.5 % of its original value per year.Fig. 7


Development of 5' LTR DNA methylation of latent HIV-1 provirus in cell line models and in long-term-infected individuals.

Trejbalová K, Kovářová D, Blažková J, Machala L, Jilich D, Weber J, Kučerová D, Vencálek O, Hirsch I, Hejnar J - Clin Epigenetics (2016)

5’ LTR and env methylation levels of the HIV-1 provirus in the latent reservoir of long-term treated HIV-1-infected individuals. a The levels of HIV-1 5’ LTR methylation in long-term treated patients. Each diamond represents one patient. The time (years) spent undergoing therapy is depicted on the x-axis whereas the percentage of methylated CpGs in the 5’ LTR of HIV-1 in the latent reservoir of infected patients is depicted on the y-axis. The methylation levels are presented as a mean percentage of methylated CpGs in HIV-1 promoters. b Latent HIV-1 provirus CpG methylation profiles at the 5’ LTR sequences of long-term treated patients nos. LT1 to LT10. The methylation levels are presented as a mean percentage of methylated CpGs (mCpGs) in HIV-1 promoters. An analysis of promoter molecules is shown as a linear array of open circles representing non-methylated CpG residues and closed circles representing methylated CpG residues. Each line represents one sequenced molecule of the 5’ LTR. The rectangle schematically represents the 5’ LTR region U3 with a distribution of individually analyzed CpG dinucleotides and transcription factor binding sites. c Levels of HIV-1 env methylation in selected long-term-treated individuals. The methylation levels are presented as a mean percentage of methylated CpGs mCpGs) in HIV-1 env region. An analysis of env molecules is shown as a linear array of open circles representing non-methylated CpG residues and closed circles representing methylated CpG residues. Each line represents one sequenced env molecule. The rectangle schematically represents the analyzed env region.
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Fig7: 5’ LTR and env methylation levels of the HIV-1 provirus in the latent reservoir of long-term treated HIV-1-infected individuals. a The levels of HIV-1 5’ LTR methylation in long-term treated patients. Each diamond represents one patient. The time (years) spent undergoing therapy is depicted on the x-axis whereas the percentage of methylated CpGs in the 5’ LTR of HIV-1 in the latent reservoir of infected patients is depicted on the y-axis. The methylation levels are presented as a mean percentage of methylated CpGs in HIV-1 promoters. b Latent HIV-1 provirus CpG methylation profiles at the 5’ LTR sequences of long-term treated patients nos. LT1 to LT10. The methylation levels are presented as a mean percentage of methylated CpGs (mCpGs) in HIV-1 promoters. An analysis of promoter molecules is shown as a linear array of open circles representing non-methylated CpG residues and closed circles representing methylated CpG residues. Each line represents one sequenced molecule of the 5’ LTR. The rectangle schematically represents the 5’ LTR region U3 with a distribution of individually analyzed CpG dinucleotides and transcription factor binding sites. c Levels of HIV-1 env methylation in selected long-term-treated individuals. The methylation levels are presented as a mean percentage of methylated CpGs mCpGs) in HIV-1 env region. An analysis of env molecules is shown as a linear array of open circles representing non-methylated CpG residues and closed circles representing methylated CpG residues. Each line represents one sequenced env molecule. The rectangle schematically represents the analyzed env region.
Mentions: To further explore the long-term (LT) development of 5’ LTR methylation in the latent reservoir of HIV-1-infected individuals, we enrolled another cohort of 10 patients who were treated for 3 to 22 years (median 12.5 years; Additional file 7: Table S2). The DNA methylation analysis of HIV-1 5’ LTR, which was performed in the same manner as in the first cohort, showed different levels of methylation ranging from 0 to 45 % methylated CpGs (Fig. 7a). The highest result, 45 % methylated CpGs, was found in a single patient (no. LT1); three other patients displayed 10, 15, and 19 % methylated CpGs, respectively, and six patients displayed between 0 and 3 % methylated CpGs in the 5’ LTR (Fig. 7b). In several individuals, we also analyzed the HIV-1 5’ LTR methylation levels in isolated memory CD4+ T cells. We obtained identical results for both memory and resting CD4+ T cell populations (data not shown). As in the group of patients treated for up to 3 years, the CpG methylation within the envelope coding region of four long-term-treated individuals displayed variable levels of CpG methylation ranging from 10 to 74 % (Fig. 7c). In the latent reservoir of three patients with high or moderate average 5’ LTR DNA methylation levels, we detected both heavily methylated and non-methylated 5’ LTR molecules within the same individual (patient nos. LT1, LT5, and LT10). Methylated 5’ LTR sequences of the long-term treated patients did not display large rearrangements or mutations of CpG dinucleotides (Fig. 7b). However, mutated 5’ LTR CpG dinucleotides were detected in 5’ LTR sequences with low methylation levels in patient nos. LT4 and LT9 (Fig. 7b). As for the previous cohort, our results do not correspond with the preferential methylation of defective 5’ LTRs of the HIV-1 provirus. Finally, in the highly methylated 5’ LTRs in patient nos. LT5 and LT10, we observed a lack of methylation at the CpG located within the NF-κB binding site which was identical with the hypomethylated CpG in the model cell lines. When we evaluated the 5’ LTR CpG methylation levels from all patients fused into one group, we found a statistically significant increase of the 5’ LTR DNA methylation levels (Additional file 8: Figure S6, p < 0,001). The percentage of methylated cytosines contained in the 5’ LTR arose by 7.5 % of its original value per year.Fig. 7

Bottom Line: We detected low levels of 5' LTR DNA methylation in the resting CD4(+) T cells of the group of patients who were treated for up to 3 years.However, after long-term ART, we observed an accumulation of 5' LTR DNA methylation in the latent reservoir.Importantly, within the latent reservoir of some long-term-treated individuals, we uncovered populations of proviral molecules with a high density of 5' LTR CpG methylation.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Vídeňská 1083, CZ-14220 Prague 4, Czech Republic.

ABSTRACT

Background: Human immunodeficiency virus type 1 (HIV-1) latency represents the major barrier to virus eradication in infected individuals because cells harboring latent HIV-1 provirus are not affected by current antiretroviral therapy (ART). We previously demonstrated that DNA methylation of HIV-1 long terminal repeat (5' LTR) restricts HIV-1 reactivation and, together with chromatin conformation, represents an important mechanism of HIV-1 latency maintenance. Here, we explored the new issue of temporal development of DNA methylation in latent HIV-1 5' LTR.

Results: In the Jurkat CD4(+) T cell model of latency, we showed that the stimulation of host cells contributed to de novo DNA methylation of the latent HIV-1 5' LTR sequences. Consecutive stimulations of model CD4(+) T cell line with TNF-α and PMA or with SAHA contributed to the progressive accumulation of 5' LTR DNA methylation. Further, we showed that once established, the high DNA methylation level of the latent 5' LTR in the cell line model was a stable epigenetic mark. Finally, we explored the development of 5' LTR DNA methylation in the latent reservoir of HIV-1-infected individuals who were treated with ART. We detected low levels of 5' LTR DNA methylation in the resting CD4(+) T cells of the group of patients who were treated for up to 3 years. However, after long-term ART, we observed an accumulation of 5' LTR DNA methylation in the latent reservoir. Importantly, within the latent reservoir of some long-term-treated individuals, we uncovered populations of proviral molecules with a high density of 5' LTR CpG methylation.

Conclusions: Our data showed the presence of 5' LTR DNA methylation in the long-term reservoir of HIV-1-infected individuals and implied that the transient stimulation of cells harboring latent proviruses may contribute, at least in part, to the methylation of the HIV-1 promoter.

Show MeSH
Related in: MedlinePlus