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.
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