HIV-1 Preintegration Trafficking and Nuclear Localization

描述(申请人提供)：管理艾滋病毒/艾滋病最有效的方法是通过治疗干预。目前的抗病毒药物针对的是逆转录、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。在进入敏感的目标细胞后，病毒必须从细胞外围移动，穿过细胞质，进入细胞核，在那里它将新合成的DNA与细胞染色体重组。阻碍整合前运输的突变对病毒是致命的，但病毒如何穿过细胞质并进入细胞核的机械细节却知之甚少。这项提议将揭示艾滋病毒-1整合前贩运和核进口的基本机制细节，利用分子技术的巡回演出。蛋白质组学方法将被用来揭示基本的病毒-宿主相互作用。将利用生化分离来比较野生型和突变型复制复合体的组成，并将使用荧光共聚焦显微镜来观察复合体在细胞中移动的情况。一旦病毒核酸进入细胞核，荧光原位杂交将被用于可视化病毒核酸。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著特征。从长远来看，这一知识将为治疗相互作用确定新的目标，以开发抗击艾滋病毒/艾滋病的新的抗病毒药物。

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.