5' long terminal repeat (LTR)-selective methylation of latently infected HIV-1 provirus that is demethylated by reactivation signals
© Ishida et al; licensee BioMed Central Ltd. 2006
Received: 15 November 2005
Accepted: 12 October 2006
Published: 12 October 2006
We previously described selective hypermethylation of the 5'-long terminal repeat (LTR) of HTLV-1 provirus in vivo and in vitro. This prompted us to analyze CpG methylation of the two LTRs of the HIV provirus in chronically infected cell lines. The results demonstrate selective hypermethylation of the 5' LTR of the HIV provirus in ACH-2 cells. Moreover, induction of viral gene expression by TNF-α resulted in demethylation of the 5'-LTR. These results suggest that selective epigenetic modification of the 5'LTR of the HIV-1 provirus may be an important mechanism by which proviral activity is suppressed.
With the use of highly active anti-retroviral therapy (HAART) for HIV-infected individuals, greater control of viral replication is now possible. The widespread use of HAART has led to a substantial decline in the incidence of acquired immunodeficiency syndrome (AIDS) and AIDS-related mortality [1–6]. This development has led to considerable optimism , but complete eradication of HIV from an infected individual is difficult to achieve because there are latently infected resting CD4+ T cells carrying replication-competent HIV resistant to HAART [8–11]. A better understanding of mechanisms underlying latency and reactivation of HIV might yield information on how to overcome the resistance of latent HIV to treatment, and this would contribute to the goal of containment or purging of HIV.
Epigenetic control is thought to be involved in latent infection of HIV. Epigenetic mechanisms result in the heritable silencing of genes without a change in their coding sequence. Three systems, including DNA methylation, RNA-associated silencing and histone modification, are used to initiate and sustain epigenetic silencing . Histone deacetylation is important for quiescence of HIV gene expression in infected resting CD4+ T lymphocytes. Blockade of histone deacetylase (HDAC) activity can stimulate the release of virus from latently infected CD4+ T-cells in vitro and, in combination with enfuvirtde, reduces the pool of CD4+ T-cells in vivo[13–15].
CpG methylation has been implicated in silencing of the integrated provirus genome [16, 17] as well as in regulation of many imprinted genes . Demethylation induced by an inhibitor of DNA methyltransferase, 5-Azacytidine (5-AzaC), was shown to reactivate a latent provirus . In vitro studies have shown that DNA methylation suppresses the promoter activity of the HIV-1 long terminal repeat (LTR) [20–23], suggesting that CpG methylation may play an important role in viral latency in vivo.
Cytokines such as TNF-α induce HIV gene expression in chronically infected T cell lines [24, 25], as well as in latently infected lymphocytes in vivo [26, 27]. Using chronically infected T cell lines, we investigated CpG methylation of provirus LTR and its relationship to regulatory mechanisms that reactivate the latent HIV provirus. We found that CpG sites in the 5'-LTR are selectively hypermethylated, and that TNF-α-induced reactivation is associated with demethylation of the 5' LTR. Our observations provide clues to the mechanism of signal-mediated demethylation and reactivation of latent HIV.
We next asked if LTRs of integrated proviruses were methylated. Using bisulfite genomic sequencing , we analyzed methylation of each CpG site in the U3 region of the HIV-1 LTR in chronically infected cell lines. Primers used for amplification of the modified sense strand are: LTR forward primer (F-3): 5'-TTTGTTATATTTTGTGAGTTTGTAT-3' (nucleotide position: 200 to 224, 9285 to 9309), reverse primer (R-1), 5'-CAAAAAACTCCCAAACTCAAATCTA-3' (nucleotide position: 496 to 472, 9581 to 9557). Amplified products were cloned by the TA method followed by sequence analysis using an automated sequencer (Amersham Bioscience, Gene Rapid). The results showed various levels of CpG methylation in Molt 20-2, ACH-2 and U-1 cell lines, which correlated inversely with basal levels of viral gene expression (Fig. 1B and C).
Taken together, the results shown in Fig. 2 can be summarized as follows: 1) at least half of the analyzed clones were unmethylated before and after TNF-α treatment, 2) the levels of CpG methylation in the remaining clones showed a clear decrease that was related to the duration of TNF-α treatment. The primers we used can amplify both 5' and 3' LTR sequences, and methylation of only half of the amplified clones suggests that methylation may affect only the 5' or the 3' LTR, as we found previously in studies of the HTLV-1 provirus . On the other hand, the response to TNF-α provides evidence of cytokine receptor signal-mediated demethylation of the provirus LTR. The mechanism by which LTR is demethylated remains to be elucidated. However, our previous observation in HIV transgenic mice suggested a passive mechanism for demethylation of HIV LTR which depends on DNA replication as is supposed for demethylation of cellular genes.
In DNA from unstimulated ACH-2 cells, among 10 clones derived from the 5' LTR, six showed methylation of all 9 CpG sites, and two had only two unmethylated CpGs, although two clones showed complete hypomethylation of the 9 CpG sites. In contrast, all 10 clones derived from the 3' LTR were hypomethylated, with only one methylated site in one clone. The 3' LTR did not show any changes in methylation on TNF-α stimulation (Fig. 4). TNF-α stimulation resulted in increase in unmethylated CpGs in the 5' LTR. After 24 hours of TNF-α stimulation, only one clone out of 10 remained completely methylated, and most clones had one to five unmethylated sites, while one was completely unmethylated. After 48 hours of stimulation, none of the sequenced clones were completely methylated (Fig. 4). Demethylated CpGs appeared to cluster in the first 5 CpG sites (reading 5' to 3'), with higher frequency at the 5th site. Three of 10 clones from unstimulated ACH-2 cells had an unmethylated 5th CpG site, and 6 clones each had unmethylated 5th CpG site after 24 and 48 hours of stimulation. Furthermore, the number of unmethylated CpGs in the first 5 sites increased with the time of TNF-α stimulation. Before stimulation, 12 (24%) of total 50 sites were unmethylated, whereas after stimulation 22 (44%) and 26 (52%) sites were unmethylated at 24 and 48 hours respectively. On the other hand, in the cluster of 4 sites at the 3 end of the cluster, among a total of 40 sites analyzed 8 (20%) were unmethylated in unstimulated cells, and after TNF-α stimulation 6 (15%) and 17 (42.5%) sites were unmethylated at 24 and 48 hours respectively. Taken together, demethylation induced by TNF-α showed a tendency to cluster in the CpG sites located at the 5' end. Furthermore, the 5th CpG site may be a hot spot for demethylation in the TNF-α stimulated cells.
Nevertheless, our results demonstrate that in the setting of full reactivation of viral gene expression by TNF-α stimulation, the provirus LTR showed only a partial demethylation at scattered sites (Fig. 4). This observation is inconsistent with the widely accepted idea that promoter activity is regulated by the density of CpG methylation [22, 32, 33]. Our results may provide support for the idea that demethylation of a specific CpG site plays an important role in promoter activity of the HIV LTR, which we have previously reported using HIV transgenic mice . It is also consistent with the notion that CpG methylation of specific sites plays an important role in controlling the promoter activities of EBV and imprinted genes [34–38].
Differential methylation of two LTRs located within 10 kbp of each other in the provirus genome, which we previously reported in the human retrovirus HTLV-1 , may suggest the presence of an unknown mechanism of methylation targeting that discriminates the 5'from the 3' LTR. The difference may depend on chemical modification of histone H3, such as lysine 9 (K9) methylation, since repression of gene expression mediated by histone H3 K9 methylation is thought to be stabilized by DNA methylation.
Previous studies in vitro suggested CpG methylation of the HIV-1 LTR as a mechanism to maintain HIV-1 latency [20–23, 40, 41]. However, no information is available as to the CpG methylation status of the HIV provirus in the reservoir pool in vivo, because extremely low copy numbers of HIV provirus make it infeasible to directly analyze CpG methylation with bisulfite genomic sequencing. In spite of the widely accepted idea that CpG methylation is involved in suppression of HIV gene expression and latency, a recent report suggested that proviral DNA methylation may not be involved in transcriptional suppression of integrated HIV provirus . However, this report used an artificial system in which provirus methylation was analyzed on a defective HIV genome or a vector having only HIV LTR as the promoter. Because we lack information on the state of the latent HIV provirus in vivo, the notion remains to be examined.
Decipherment of the mechanisms for reactivation of latently infected HIV in the reservoir pool will provide the basis for designing treatment strategies for containment or purging of HIV. Thus our observations of 5' LTR selective methylation in ACH-2 cells, and signal-induced demethylation of HIV provirus in the transgenic mice model and latently infected cell lines , provide information that will be useful in future investigations.
human immunodeficiency virus
long terminal repeat
highly active anti-retrovirus therapy
inverse polymerase chain reaction
We thank Drs. David IK Martin and K Suzuki for comments on the manuscript and Ms. S Aizawa and Ms. Maruyama-Nagai for technical assistance. This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan to T Ishida and T Watanabe, and by a Grant from Japanese Foundation for AIDS Prevention to T Ishida and T Watanabe.
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