HIV-1 Preintegration Trafficking and Nuclear Localization

描述(由申请人提供)：HIV-1感染细胞通过一系列步骤进行，每个步骤都定义了抗病毒药物干预的潜在目标。目前的药物主要针对由蛋白酶、逆转录酶和整合酶催化的关键病毒活性，但耐药的出现要求不断开发具有新作用模式的新药。HIV-1在复制过程中依赖于与许多细胞蛋白的相互作用，马拉韦罗是一种被批准的病毒-细胞相互作用的抑制剂，这种药物可以破坏病毒包膜糖蛋白和细胞表面CCR5受体之间的相互作用，从而阻断病毒进入细胞的能力。对HIV-1复制至关重要的还有整合前复合体中间体的能力，该中间体包含整合酶和反向转录本，能够访问发生病毒DNA整合的细胞核内的染色体。像HIV-1这样的慢病毒使用能量依赖的过程通过核孔复合体运输它们的整合前复合体，这些复合体使双相核膜变得千疮百孔，尽管HIV-1核运输的分子机制尚不清楚。这项续签拨款申请支持的工作确定了许多细胞蛋白质是HIV-1复制的潜在关键辅助因素，其中相当数量的蛋白质，包括Transportin-3， 因此，核孔蛋白153和核孔蛋白358因其在细胞内的已知功能而参与整合前复合体的核输入。随后的工作确实证实了这一观点，尽管目前尚不清楚这些蛋白和其他细胞和病毒蛋白是如何准确地决定HIV-1整合前复杂核进口的。利用大量的病毒学、生化和遗传学方法，本文提出的工作将确定HIV-1核输入的机制，重点是病毒与宿主细胞成分之间的新的蛋白质相互作用，这些蛋白质对这一过程至关重要。这些发现将为开发阻断关键的艾滋病毒-宿主相互作用的抑制剂确定新的目标，预计这将在正在进行的控制艾滋病毒/艾滋病传播的战斗中增加未来抗病毒武器的广度。 与公共卫生相关：尽管HAART取得了成功，但由于对任何给定化合物建立抵抗力所需的固有病毒遗传屏障，以及复合使用的相关毒性，出现了耐药性，这严重影响了患者的依从性。这些观察结果突显了开发新的抗病毒抑制剂的持续需要，近年来，艾滋病毒-1和细胞辅助因子之间的关键相互作用已被强调为新的药物靶点。这项提案将揭示HIV-1整合前复合体(一种关键的病毒复制中间体)如何进入发生病毒整合的细胞核的新细节，这将为未来的抗病毒药物开发定义新的靶点。

DESCRIPTION (provided by applicant): Infection of a cell by HIV-1 proceeds through a series of steps, each of which defines a potential target for antiviral drug intervention. Current drugs target key viral activities catalyzed by the protease, reverse transcriptase, and integrase enzymes, but emergence of drug resistance calls for continual development of new drugs with novel modes of action. HIV-1 depends on interactions with numerous cellular proteins as it replicates and maraviroc, a drug that disrupts the interaction between the viral envelope glycoprotein and cellular surface CCR5 receptor, is an approved inhibitor of a virus-cell interaction that blocks the ability of the virus to enter the cell. Also crucial to HIV-1 replicatin is the ability for the preintegration complex intermediate that houses the integrase and reverse transcript to access the chromosomes inside the cell nucleus, where viral DNA integration takes place. Lentiviruses like HIV-1 use an energy-dependent process to transport their preintegration complexes through nuclear pore complexes that riddle the biphasic nuclear envelope, though the molecular mechanisms underlying HIV-1 nuclear transport are poorly understood. Work supported by this renewal grant application identified numerous cellular proteins as potential critical cofactors of HIV-1 replication and a significant number of these, including transportin-3, nucleoporin 153, and nucleoporin 358 were accordingly implicated in preintegration complex nuclear import due to their known functions within the cell. Subsequent work indeed confirmed this contention, though it remains unclear how these and other cellular and viral proteins precisely dictate HIV-1 preintegration complex nuclear import. Using numerous virology, biochemical, and genetic approaches, the work proposed herein will determine the mechanisms of HIV-1 nuclear import, focusing on novel protein interactions between the virus and host cellular components that are essential for the process. Such discoveries will define new targets for the development of inhibitors that block critical HIV-host interactions, which would be expected to increase the breadth of future antiviral armaments in the ongoing battle to control the spread of HIV/AIDS. PUBLIC HEALTH RELEVANCE: Despite HAART successes, drug resistance emerges due to the inherent viral genetic barrier required to build resistance to any given compound plus associated toxicity of compound use, which significantly affects patient compliance. These observations highlight the ongoing need to develop new antiviral inhibitors, and crucial interactions between HIV-1 and cellular cofactors have been highlighted in recent years as novel drug targets. This proposal will uncover novel details of how the HIV-1 preintegration complex, a critical viral replication intermediate, accesses the cellular nucleus where viral integration occurs, which will define novel targets for antiviral drug development moving forward.