PHARMACOLOGY OF THE HIV VIRAL DNA

人类免疫缺陷病毒(HIV)复制周期的每一步 代表了治疗干预的潜在靶点。一位批评者 步骤是通过HIV将病毒DNA整合到宿主基因组中 整合酶。我们已经建立了体外逆转录病毒DNA整合系统 使用重组HIV整合酶和对应于 HIV DNA的U5末端。整合反应可分为三步， 分析：(I)核裂解，从DNA中去除2个核苷酸 双链DNA的3‘末端(’3‘-裂解’1‘-3’-加工‘)，(Ii) DNA链转移将病毒DNA连接到 靶DNA在插入部位发生裂解 (‘整合’1‘链转移’)，和(Iii)反向反应 (‘解体’)。使用这些化验，我们已经确定了几个组 有效抑制HIV-1整合酶的药物，包括咖啡酸 苯乙酯(CAPE)。黄酮类和一些DNA嵌入剂，如 活性在微摩尔的氯喹和蒽环类药物 浓度。有趣的是，DNA拓扑异构酶I和II的抑制剂 通常是不活跃的。我们目前的努力旨在调查 黄酮类、CAPE类和木脂素类衍生物的构效关系 可用于整合酶检测的新型非DNA结合剂 体内抑制作用。 长末端重复序列(LTRs)是在两个末端重复的DNA序列 逆转录病毒DNA。除了作为HIV整合酶靶标的作用外，LTRs还包括 HIV DNA的启动子区域。它们包含定义明确的转录 对制造新的病毒RNA和 蛋白质。因为我们最近发现DNA拓扑异构酶II 人MYC原基因启动子区域内的优先位点- 癌基因(Pommier等人，癌症研究，1992；52：3125-30) 拓扑异构酶I活性似乎促进了转录，我们有 绘制了DNA拓扑异构酶与LTR军团的相互作用图 研究了不同的拓扑异构酶抑制剂对LTRs的影响。

Each step of the Human Immunodeficiency Virus (HIV) replication cycle represents a potential target for therapeutic intervention. A critical step is integration of the viral DNA into the host genome by the HIV integrase. We have set up an in vitro retroviral DNA integration system using recombinant HIV integrase and oligonucleotides which correspond to the U5 end of HIV DNA. Three steps of the integration reaction can be analyzed: (i) nucleolytic cleavage which removes 2 nucleotides from the 3'ends of the double-stranded DNA ('3'-cleavage'1'3'-processing'), (ii) DNA strand transfer which couples the Joining of the viral DNA into the target DNA with cleavage of the target DNA at the site of insertion ('integration'1 'strand transfer'), and (iii) the reverse reaction ('disintegration'). Using these assays, we have identified several groups of drugs that inhibit effectively HIV-1 integrase including caffeic acid phenethyl ester (CAPE). flavones, and some DNA intercalators such as chloroquine and anthracyclines which are active at micromolar concentrations. Interestingly, inhibitors of DNA topoisomerases I and II are generally inactive. Our current effort is aimed at investigating structure-activity of flavone, CAPE, and lignan derivatives, as well as new classes of non-DNA binders which could be tested for integrase inhibition in vivo. Long Terminal Repeats (LTRs) are repeated DNA sequences at both ends of retroviral DNA. Besides their role as HIV integrase target, the LTRs are the promoter regions of HIV DNA. They contain well defined transcription regulatory elements which are critical for making new viral RNA and proteins. Because of our recent finding that DNA topoisomerase II has preferential sites within the promoter region of the human MYC proto- oncogene (Pommier et al, Cancer Res. 1992;52:3125-30) and because topoisomerase I activity appears to facilitate transcription, we have mapped the interactions of DNA topoisomerases with the LTR legions and studied the effects of various topoisomerase inhibitors on the LTRs.