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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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Hypermethylation of 5’ LTR DNA in the HIV-1 latency model cell line 2D12 is not maintainted by DNMT3B. a DNMT3B protein expression in 2D12 cell lines that were transfected with siCTRL and with siDNMT3B at days 3 and 6 after transfection as determined by the specific antibody is depicted on the left panel (upper band). Equal protein loading was verified by anti-γ-tubulin antibodies as depicted on the left panel (lower band). The DNMT3B relative mRNA levels at days 0 (control), 3, and 6 of siCTRL or siDNMT3B transfection in the 2D12 cell line as determined by qRT-PCR are shown in the right panel. mRNA expression was normalized to the expression of the RNA polymerase II, polypeptide A (POLR2A) housekeeping gene. The data are presented as the mean ± SD of technical triplicates. Three independent experiments were performed, representative example is shown. The p values were calculated by the non-paired Student’s t test. Significance *** was assigned for p values <0.001. b Latent HIV-1 provirus reactivation levels after 6 days of DNMT3B knockdown. 2D12 cells were transfected with siCTRL or with siDNMT3B. The left column of histograms represents analyses without cellular stimulation whereas the right column represents histograms after 24 h of TNF-α and PMA stimulation. Three FACS analyses of three independent experiments are overlaid; the mean percentage of EGFP-positive cells is shown above the bar. The p values were calculated by the non-paired Student’s t test. Significance *** was assigned for p values <0.001. c CpG methylation profile of 5’ LTR in non-stimulated 2D12 cells. The 5’ LTR methylation profiles were obtained from cells transfected with siCTRL or those transfected for 3 and 6 days with siDNMT3B. 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. Mixture of genomic DNA isolated from three independent experiments was used for bisulfite analysis. The rectangle schematically represents the 5’ LTR regions U3, R, and U5 with individually analyzed CpG dinucleotides and transcription factor binding sites.
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Fig4: Hypermethylation of 5’ LTR DNA in the HIV-1 latency model cell line 2D12 is not maintainted by DNMT3B. a DNMT3B protein expression in 2D12 cell lines that were transfected with siCTRL and with siDNMT3B at days 3 and 6 after transfection as determined by the specific antibody is depicted on the left panel (upper band). Equal protein loading was verified by anti-γ-tubulin antibodies as depicted on the left panel (lower band). The DNMT3B relative mRNA levels at days 0 (control), 3, and 6 of siCTRL or siDNMT3B transfection in the 2D12 cell line as determined by qRT-PCR are shown in the right panel. mRNA expression was normalized to the expression of the RNA polymerase II, polypeptide A (POLR2A) housekeeping gene. The data are presented as the mean ± SD of technical triplicates. Three independent experiments were performed, representative example is shown. The p values were calculated by the non-paired Student’s t test. Significance *** was assigned for p values <0.001. b Latent HIV-1 provirus reactivation levels after 6 days of DNMT3B knockdown. 2D12 cells were transfected with siCTRL or with siDNMT3B. The left column of histograms represents analyses without cellular stimulation whereas the right column represents histograms after 24 h of TNF-α and PMA stimulation. Three FACS analyses of three independent experiments are overlaid; the mean percentage of EGFP-positive cells is shown above the bar. The p values were calculated by the non-paired Student’s t test. Significance *** was assigned for p values <0.001. c CpG methylation profile of 5’ LTR in non-stimulated 2D12 cells. The 5’ LTR methylation profiles were obtained from cells transfected with siCTRL or those transfected for 3 and 6 days with siDNMT3B. 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. Mixture of genomic DNA isolated from three independent experiments was used for bisulfite analysis. The rectangle schematically represents the 5’ LTR regions U3, R, and U5 with individually analyzed CpG dinucleotides and transcription factor binding sites.

Mentions: Next, we performed a knockdown of DNA methyltransferase 3B, DNMT3B, using siRNA (siDNMT3B). We verified the depletion of DNMT3B in the 2D12 cell line at both the mRNA and protein levels (Fig. 4a). The DNMT3B knockdown did not induce provirus reactivation. TNF-α and PMA stimulation slightly increased provirus reactivation in siDNMT3B-transfected 2D12 cells 6 days after transfection, by 7 % compared to siCTRL-transfected cells (Fig. 4b). We did not observe any decrease in 5’ LTR DNA methylation in the 2D12 cells with depleted DNMT3B 6 days after transfection (Fig. 4c).Fig. 4


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)

Hypermethylation of 5’ LTR DNA in the HIV-1 latency model cell line 2D12 is not maintainted by DNMT3B. a DNMT3B protein expression in 2D12 cell lines that were transfected with siCTRL and with siDNMT3B at days 3 and 6 after transfection as determined by the specific antibody is depicted on the left panel (upper band). Equal protein loading was verified by anti-γ-tubulin antibodies as depicted on the left panel (lower band). The DNMT3B relative mRNA levels at days 0 (control), 3, and 6 of siCTRL or siDNMT3B transfection in the 2D12 cell line as determined by qRT-PCR are shown in the right panel. mRNA expression was normalized to the expression of the RNA polymerase II, polypeptide A (POLR2A) housekeeping gene. The data are presented as the mean ± SD of technical triplicates. Three independent experiments were performed, representative example is shown. The p values were calculated by the non-paired Student’s t test. Significance *** was assigned for p values <0.001. b Latent HIV-1 provirus reactivation levels after 6 days of DNMT3B knockdown. 2D12 cells were transfected with siCTRL or with siDNMT3B. The left column of histograms represents analyses without cellular stimulation whereas the right column represents histograms after 24 h of TNF-α and PMA stimulation. Three FACS analyses of three independent experiments are overlaid; the mean percentage of EGFP-positive cells is shown above the bar. The p values were calculated by the non-paired Student’s t test. Significance *** was assigned for p values <0.001. c CpG methylation profile of 5’ LTR in non-stimulated 2D12 cells. The 5’ LTR methylation profiles were obtained from cells transfected with siCTRL or those transfected for 3 and 6 days with siDNMT3B. 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. Mixture of genomic DNA isolated from three independent experiments was used for bisulfite analysis. The rectangle schematically represents the 5’ LTR regions U3, R, and U5 with individually analyzed CpG dinucleotides and transcription factor binding sites.
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Fig4: Hypermethylation of 5’ LTR DNA in the HIV-1 latency model cell line 2D12 is not maintainted by DNMT3B. a DNMT3B protein expression in 2D12 cell lines that were transfected with siCTRL and with siDNMT3B at days 3 and 6 after transfection as determined by the specific antibody is depicted on the left panel (upper band). Equal protein loading was verified by anti-γ-tubulin antibodies as depicted on the left panel (lower band). The DNMT3B relative mRNA levels at days 0 (control), 3, and 6 of siCTRL or siDNMT3B transfection in the 2D12 cell line as determined by qRT-PCR are shown in the right panel. mRNA expression was normalized to the expression of the RNA polymerase II, polypeptide A (POLR2A) housekeeping gene. The data are presented as the mean ± SD of technical triplicates. Three independent experiments were performed, representative example is shown. The p values were calculated by the non-paired Student’s t test. Significance *** was assigned for p values <0.001. b Latent HIV-1 provirus reactivation levels after 6 days of DNMT3B knockdown. 2D12 cells were transfected with siCTRL or with siDNMT3B. The left column of histograms represents analyses without cellular stimulation whereas the right column represents histograms after 24 h of TNF-α and PMA stimulation. Three FACS analyses of three independent experiments are overlaid; the mean percentage of EGFP-positive cells is shown above the bar. The p values were calculated by the non-paired Student’s t test. Significance *** was assigned for p values <0.001. c CpG methylation profile of 5’ LTR in non-stimulated 2D12 cells. The 5’ LTR methylation profiles were obtained from cells transfected with siCTRL or those transfected for 3 and 6 days with siDNMT3B. 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. Mixture of genomic DNA isolated from three independent experiments was used for bisulfite analysis. The rectangle schematically represents the 5’ LTR regions U3, R, and U5 with individually analyzed CpG dinucleotides and transcription factor binding sites.
Mentions: Next, we performed a knockdown of DNA methyltransferase 3B, DNMT3B, using siRNA (siDNMT3B). We verified the depletion of DNMT3B in the 2D12 cell line at both the mRNA and protein levels (Fig. 4a). The DNMT3B knockdown did not induce provirus reactivation. TNF-α and PMA stimulation slightly increased provirus reactivation in siDNMT3B-transfected 2D12 cells 6 days after transfection, by 7 % compared to siCTRL-transfected cells (Fig. 4b). We did not observe any decrease in 5’ LTR DNA methylation in the 2D12 cells with depleted DNMT3B 6 days after transfection (Fig. 4c).Fig. 4

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