A Genetic/Proteomic Approach to Virus-Host Interactions

描述(申请人提供)：HIV-1感染取决于病毒和宿主因素。鉴于病毒的遗传能力有限，以及感染过程中复杂的分子事件，艾滋病毒-1必须与各种细胞因子相互作用，才能完成细胞的传递。然而，到目前为止，只鉴定了几个病毒辅助宿主蛋白。在这项提案中，我们概述了一种策略，以阐明HIV侵占的细胞结合伙伴使用一个系统感染其目标细胞，在该系统中，病毒已被分子工程设计为包含有效的免疫学或生化标签。使用这一组独立标记的(3X标志表位或生物素化信号肽，Snagg)具有复制能力的衍生物，我们寻求恢复在病毒自然生命周期中与病毒特异性相互作用的宿主蛋白。由于这些工程病毒是通过基于培养中复制能力的过程产生的，标记的整合酶蛋白可能经历与野生型蛋白相同的相互作用。我们相信，与其他传统程序相比，该系统将为我们提供更真实的视角，了解在艾滋病毒感染的正常过程中形成的瞬时和稳定的分子相互作用。最初，我们特别关注病毒整合酶蛋白--一种参与逆转录、病毒DNA的核输入以及随后整合到宿主染色体DNA中的不同角色的蛋白质。这项有针对性的蛋白质组学研究将利用亲和纯化和质谱学相结合的方法来确定通过与标记的病毒蛋白相互作用而捕获的宿主蛋白和复合体的身份。阐明艾滋病毒感染过程中细胞和病毒蛋白之间的复杂相互作用将是一项巨大的财富，不仅对于深入了解逆转录病毒生物学，而且对于它将揭示的抗病毒干预的新靶点来说也是一笔巨大的财富。人类免疫缺陷病毒(HIV-1)的病毒整合酶蛋白是病毒繁殖所必需的。除了它的其他功能外，整合酶在协调病毒的遗传信息与宿主细胞的遗传信息的融合方面发挥着关键的催化作用，从而使艾滋病毒基因组成为感染者细胞中的永久固定物。然而，我们对艾滋病毒感染中的分子事件的大多数生物学理解都是从病毒的角度出发的，而不是从宿主细胞的角度出发的，而宿主细胞必须与宿主细胞密切联系才能产生后代。因此，这个建议描述了一种方法，它努力确定病毒整合酶的细胞主角；整合酶必须依赖的因素才能发挥其不同的功能。由于病毒-宿主界面在很大程度上仍未被探索为抗逆转录病毒治疗的靶点，因此它们之间的联系的抑制剂将代表一种以前未被认识到的新的抗逆转录病毒药物。

DESCRIPTION (provided by applicant): HIV-1 infection depends on both viral and host factors. Given the limited genetic capacity of the virus and complex molecular events during its infection, HIV-1 must interact with an extensive variety of cellular factors to complete its passage through the cell. To date, however, only a few virus-assisting host proteins have been identified. In this proposal, we outline a strategy to elucidate the cellular binding partners that HIV commandeers to infect its target cells using a system in which viruses have been molecularly engineered to incorporate a potent immunological or biochemical tag. Using this panel of independently tagged (3X FLAG epitope or a biotinylation signal peptide, SNAGG), replicationcompetent derivatives, we seek to recover host proteins that interact specifically with the virus as it progresses through its natural life cycle. As these engineered viruses were generated through a process based on replication competence in culture, the tagged integrase protein likely undergo the same interactions encountered by the wild type protein. We believe that this system will afford us a more authentic view of both transient and stable molecular interactions that form during the normal course of HIV infection than other traditional procedures. Initially, we are focusing specifically on the viral integrase protein-a protein involved in such disparate roles as reverse transcription, the nuclear import of viral DNA and its subsequent integration into host chromosomal DNA. This targeted proteomic study will utilize affinity purification coupled with mass spectrometry to determine the identity of host proteins and complexes that are captured via their interaction with the tagged viral proteins. Elucidation of the complex interactions between cellular and viral proteins during HIV infection would be a great asset not only for the detailed insight into retroviral biology but also for the novel targets for antiviral interventions it would unveil. The viral integrase protein of the Human Immunodeficiency Virus (HIV-1) is absolutely required for viral propagation. In addition to its other functions, integrase is known to play a key catalytic role in coordinating the fusion of the genetic information of the virus with that of the host's cell, thereby making the HIV genome a permanent fixture in the cells of an infected individual. However, most of the biology we understand about the molecular events in HIV infection is from the perspective of the virus, and not from that of the host cell with which it must associate intimately to produce progeny. Thus, this proposal describes methodology that strives to identify the cellular protagonists of the viral integrase; the factors that integrase must rely on for its diverse functions. Because the viral-host interface remains largely unexplored as a target for antiretroviral therapy, inhibitors of their association would represent a new class of antiretroviral agents not previously appreciated.