Regulation Mechanisms of HIV LTR Demethylation through Reactivation Signaling

有效的抗HIV联合化疗的引入使研究者的兴趣集中在病毒潜伏的机制上，因为如果不根除储存库中的潜伏HIV，就不能实现HIV的根除。CpG甲基化作为抑制基因表达的基础之一，长期以来在病毒潜伏期中被发现。我们发现，在手性感染的细胞系中，CpG甲基化水平与病毒基因表达呈负相关。TNF-α诱导ACH-2细胞株中病毒基因表达与前病毒LTR中CpG序列的去甲基化有关，因为我们发现HTLV-1前病毒LTR中存在5 ′-LTR特异性甲基化，我们设计了亚硫酸氢盐修饰的PCR正义引物，位于5 ′-分析整合的前病毒的侧翼序列和达特编码区中的侧翼序列，并选择性地分析甲基化状态。结果显示ACH-2细胞系中5 '-LTR的选择性高甲基化和3'-LTR的低甲基化。在儿童感染的细胞系、ATL病例和HTLV-1无症状携带者中观察到相同的选择性。哺乳动物细胞可能具有使整合的逆转录病毒5 '-LTR高甲基化或使3'-LTR去甲基化的能力。

Introduction of potent anti-HIV combination chemotherapy has focused reseacher's interest on the mechanisms of viral latency, because eradication of HIV cannot be achieved without eradication of latent HIV in reservoir pool. CpG methylation has long been impricated in the viral latency as one base for repression of gene expression. We could find that the levels of CpG methylation were inversly correlated with the viral gene expression in chironically infected cell lines. Induction of viral gene expresson by TNF-α in ACH-2 cell line was shown to be associated with demethylation of CpG sequence in the provirus LTR.Because we could find that 5'-LTR specific methylation was occurred in the HTLV-1 provirus LTR, we designed the bisulfite modified PCR sense primer located in the 5'-flanking sequence of the integrated provirus and that in the tat coding region and selectively methylation status analyzed. Results revealed selective hypermethylation of 5'-LTR and hypomethylation of 3'-LTR in the ACH-2 cell line. The same selectivity was observed in the chilonically infected cell lines, ATL cases and HTLV-1 asymptomatic carriers. It was possible that mammalian cells has the ability to hypermethylate the integrated retrovirus 5'-LTR or to demethylate the 3'-LTR.