HIV-RT TEMPLATE PRIMER COMPLEXES AND TRANSCRIPTION FACTOR/HIV PROMOTER COMPLEXES

这一部分涉及HIV-1两个步骤的结构生物学 复制：逆转录和转录调控 从LTR中的启动子启动整合的亲病毒。 HIV逆转录酶(RT)是具有临床意义的靶标 抗逆转录病毒药物，如AZT和3TC。RT的结构有 发现了一种复杂的蛋白质，具有惊人的构象灵活性 关于机制和耐药性的主要问题没有得到回答。一个 已开发出使HIV-1RT与HIV-1RT的络合物结晶的策略 共价连接的模板引物和有序的晶体 特别是已经获得了这样的络合物。建议确定 一系列这样的络合物的结构，以便可视化 模板、底物和底物在活性部位的精确排列， 当AZTTP、ddITP和ddCTP为传入核苷酸时。的影响 关键的AZT、DDI和3TC耐药突变在RT- 模板-底物-底物复合体也将被研究 结晶学上的。结果将被用于分析药物的模式 对联合抗逆转录病毒治疗的耐药性。这个 锚链方法将扩展到RT与RNA：DNA：和 RNA：tRNA模板-引物。 HIV-1 LTR包含转录因子SP1和SP1的位点 NFkappaB.将确定NFkappaB杂二聚体的结构 (Re1同源(p50和p65的Rel同源区域)和Sp1(锌指 片段)与跨越中心NFkappaB的DNA片段结合在一起 和位于HIV-1近端增强子的SP1位点。的结构。 在串联对的两个位置结合的NFkappaB杂二聚体也将 下定决心。蛋白质：这些复合体中的蛋白质界面将是 被分析为潜在的药物靶点，并有长远的应用前景 新的方法。

This component concerns the structural biology of two steps in HIV-1 replication: reverse transcription and the regulation of transcriptional initiation from the promoter in the LTR of an integrated pro-virus. HIV reverse transcriptase (RT) is the target of clinically significant anti-retroviral drugs, such as AZT and 3TC. The structure of RT has revealed a complex protein with striking conformational flexibility but left unanswered major questions about mechanism and drug resistance. A strategy has been developed for crystallizing complexes of HIV-1 RT with covalently tethered template primers, and well-ordered crystals of a particularly such complex have been obtained. It is proposed to determine the structures of a series of such complexes, in order to visualize the precise arrangement of template, primer, and substrate in the active site, when AZTTP, ddITP, and ddCTP are the incoming nucleotides. The effects of key AZT, ddI, and 3TC resistance mutations on the conformation of the RT- template-primer-substrate complex will also be studied crystallographically. The results will be used to analyze patterns of drug resistance in response to combination anti-retroviral therapy. The tethering approach will be extended to complexes of RT with RNA:DNA: and RNA:tRNA template-primers. The HIV-1 LTR contains sites for the transcription factors SP1 and NFkappaB. The structure will be determined of the NFkappaB heterodimer (Re1 homology (Rel homology regions of p50 and p65) and Sp1 (Zn finger segment) bound together to a DNA fragment spanning the central NFkappaB and SP1 sites in the HIV-1 proximal enhancer. The structure of the NFkappaB heterodimer bound at both sites of the tandem pair will also be determined. Protein: protein interfaces in these complexes will be analyzed as potential drug targets, with a long-range view to applying novel methodologies.