HIV-1 Preintegration Trafficking and Nuclear Localization

描述（由申请人提供）：管理艾滋病毒/艾滋病的最有效方法是通过治疗干预。目前的抗病毒药物靶向逆转录、病毒成熟和进入。针对尚未开发的靶点的新药是必不可少的，病毒在人类细胞中生长所必须完成的任何复制步骤都定义了一个可行的靶点。在进入易感靶细胞后，病毒必须从细胞周边移动，通过细胞质，进入细胞核，在那里它将重新组合其新合成的DNA与细胞染色体。阻碍整合前运输的突变对病毒是致命的，但对病毒如何穿过细胞质并进入细胞核的机制细节知之甚少。这项建议将揭示HIV-1融合前贩运和核进口的基本机制细节，使用一种分子技术的巡回演出。蛋白质组学的方法将被用来揭示基本的病毒-宿主相互作用。将利用生物化学分级来比较野生型和突变体复制复合物的组成，并且将使用荧光共聚焦显微镜来观察复合物在细胞中移动时的情况。一旦病毒核酸进入细胞核内，将使用荧光原位杂交来观察病毒核酸。这些实验的结果将揭示病毒如何在细胞中移动以完成其生命周期中基本的整合前任务的显着特征。从长远来看，这一知识将确定治疗相互作用的新目标，以开发防治艾滋病毒/艾滋病的新抗病毒药物。

DESCRIPTION (provided by applicant): The most effective approach for managing HIV/AIDS is through therapeutic intervention. Current anti-viral drugs target reverse transcription, virus maturation, and entry. Novel drugs against as of yet unexploited targets are essential and any replication step that the virus must accomplish to grow in human cells defines a viable target. After entering a susceptible target cell, the virus must move from the cell periphery, through the cytoplasm, and into the nucleus where it will recombine its neo- synthesized DNA with a cell chromosome. Mutations that impede preintegration trafficking are lethal to the virus, yet the mechanistic details of how the virus moves through the cytoplasm and enters the nucleus are poorly understood. This proposal will uncover essential mechanistic details of HIV-1 preintegration trafficking and nuclear import using a tour de force of molecular techniques. Proteomic approaches will be utilized to uncover essential virus-host interactions. Biochemical fractionation will be utilized to compare the composition of wild-type and mutant replication complexes, and fluorescent confocal microscopy will be used to visualize the complexes as they move through the cell. Fluorescent in situ hybridization will be used to visualize viral nucleic acids once they are inside the nucleus. The results of these experiments will reveal salient features of how the virus moves through the cell to accomplish the essential preintegration tasks in its lifecycle. This knowledge in the long run will define novel targets for therapeutic interaction to develop new anti-viral drugs in the fight against HIV/AIDS.