REPRESSION OF HIV-1 TRANSCRIPTION BY DNA METHYLATION

DNA 甲基化抑制 HIV-1 转录

• 批准号: 3455469
• 负责人: KAREN R PRATT
• 金额: $ 10.81万
• 依托单位: UNIVERSITY OF VERMONT & ST AGRIC COLLEGE
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-11-01 至 1994-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3455469
• 关键词: DNA footprinting; DNA methylation; chloramphenicol acetyltransferase; gene induction /repression; genetic manipulation; genetic regulatory element; genetic transcription; helper T lymphocyte; human immunodeficiency virus 1; latent virus infection; methylation; molecular cloning; molecular site; nucleic acid repetitive sequence; plasmids; reporter genes; tissue /cell culture; transcription factor; transfection; virus DNA; DNA footprinting; DNA methylation; chloramphenicol acetyltransferase; gene induction /repression; genetic manipulation; genetic regulatory element; genetic transcription; helper T lymphocyte; human immunodeficiency virus 1; latent virus infection; methylation; molecular cloning; molecular site; nucleic acid repetitive sequence; plasmids; reporter genes; tissue /cell culture; transcription factor; transfection; virus DNA

DNA methylation may be important in maintaining latency of AIDS virus. It has been demonstrated that virus activation is induced in cells harboring latent virus by treatment with 5-azacytidine, a demethylating agent (Bednarik et al., J. Virol. 61, 1253-1257, 1987). Although it is well established that methylation at regulatory DNA sequences represses transcription of the associated gene (Cedar, Cell 53, 3-4, 1988), the mechanism is not known. DNA methylation may repress transcription by masking binding sites for trans-activating proteins or by binding repressor proteins. The research proposed here will systematically examine the effects on transcription of DNA methylation in the HIV-1 long terminal repeat (LTR), the segment of the AIDS virus genome which regulates transcription. Experimental plasmids will be constructed such that the HIV-1 LTR, methylated at one or more sites, will regulate transcription of a reporter gene. Control plasmids will be constructed in the same manner but without methylated sites. These will be transfected into cultured T helper cells and expression of the reporter gene will be assayed. After determining whether methylated sites block transcription, those sites responsible will be analyzed further. The protein-DNA interactions of these methylated and unmethylated sites will be compared in nuclear extracts. This research will determine which element in the LTR repress transcription when methylated and if repression involves altered binding of transcription factors to methylated sequences. Understanding the role of DNA methylation in regulating HIV-1 transcription may lead to methods to maintain viral latency in infected individuals.

DNA甲基化对于维持艾滋病病毒的潜伏期可能很重要。 它 已证明在携带的细胞中诱导病毒激活 通过5-氮杂替丁治疗的潜在病毒，一种脱甲基剂 （Bednarik等人，J。Virol。61，1253-1257，1987）。 虽然很好 确定在调节性DNA序列处的甲基化抑制 相关基因的转录（Cedar，细胞53，3-4，1988）， 机制尚不清楚。 DNA甲基化可能会通过 用于反式激活蛋白或通过结合抑制剂的掩盖结合位点 蛋白质。 这里提出的研究将系统地检查 HIV-1长末端中DNA甲基化转录的影响 重复（LTR），调节的AIDS病毒基因组段 转录。 将构建实验质粒，使得 HIV-1 LTR在一个或多个地点甲基化，将调节 记者基因。 控制质粒将以相同的方式构建 但是没有甲基化位点。 这些将被转染为培养的t 将分析辅助细胞和报告基因的表达。 后 确定甲基化位点是否阻止转录，这些位点 负责的人将进一步分析。 蛋白-DNA相互作用 这些甲基化和未甲基化位点将在核中进行比较 提取物。 这项研究将确定LTR抑制中的哪个元素 甲基化时转录，如果抑制涉及改变的结合 转录因子到甲基化序列。 了解角色 调节HIV-1转录中的DNA甲基化可能导致方法 在受感染的个体中保持病毒潜伏期。