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

本部分涉及HIV-1中两个步骤的结构生物学 复制：逆转录和转录调控 从整合的前病毒的LTR中的启动子起始。 HIV逆转录酶（RT）是临床上重要的 抗逆转录病毒药物，如AZT和3 TC。RT的结构具有 揭示了一种具有惊人构象灵活性的复杂蛋白质， 留下了关于机制和耐药性的未解之谜。一 已经开发了用于结晶HIV-1 RT与 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 一系列这样的复合物的结构，为了可视化， 模板、引物和底物在活性位点的精确排列， 当AZTTP、ddITP和ddCTP是进入的核苷酸时。的影响 关键的AZT，ddI和3 TC抗性突变对RT- 还将研究模板-引物-底物复合物 晶体学上。结果将用于分析药物的模式 对联合抗逆转录病毒治疗的耐药性。的 拴系方法将扩展到RT与RNA：DNA：和 RNA：tRNA模板引物。 HIV-1 LTR含有转录因子SP1和SP2的位点， NF κ B。将确定NF κ B异二聚体的结构 (Re1同源性（p50和p65的Rel同源区）和Sp1（Zn指 片段）结合在一起的DNA片段跨越中央NF κ B 和HIV-1近端增强子中的SP1位点。的结构 在串联对的两个位点结合的NF κ B异源二聚体也将是 测定蛋白质：蛋白质界面在这些复合物将 作为潜在的药物靶点进行分析，从长远来看， 新颖的方法。

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.