HOST INTERACTIONS OF GENE PRODUCTS FROM HIV-1

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 与较大的DNA病毒相比，人类免疫缺陷病毒(HIV-1)的编码蛋白质库相对有限。鉴于这一事实，有理由认为宿主细胞构成了病毒复制所必须依赖的丰富因子来源。然而，到目前为止，只有少数这样的病毒辅助宿主蛋白被鉴定出来。在这项建议中，我们努力识别在病毒复制过程中直接与HIV-1机制相互作用的因素，在该系统中 病毒已经经过分子工程，结合了一个强大的免疫学或生化标签。使用这一组独立标记的复制能力衍生品，我们试图恢复在病毒自然生命周期中与病毒特异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自我选择过程产生的，标记的病毒蛋白必须经历与野生型病毒相同的相互作用。因此，我们相信，这个系统将为我们提供一个更真实的视角，了解在HIV感染的正常过程中形成的瞬时和稳定的分子相互作用。目前，质谱学技术正被用于确定宿主蛋白和复合体的身份，这些宿主蛋白和复合体是通过与标记的病毒蛋白相互作用而捕获的。这项研究的具体目的如下： I.HIV-1基因组的全面诱变和感染性、复制能力强的标记病毒的选择性恢复 利用标记病毒定量回收在复制过程中与病毒蛋白相互作用的宿主蛋白 质谱学鉴定相互作用蛋白及其在HIV-1感染循环中的作用 描述这项工作的手稿正在准备中

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. In comparison to larger DNA viruses, the human immunodeficiency virus (HIV-1) has a relatively limited repertoire of encoded proteins. Given this fact, it is reasonable to expect that the host cell constitutes a rich source of factors that the virus must draw upon for its replication. To date, however, only a few such virus-assisting host proteins have been identified. In this proposal, we endeavor to identify the factors that interact directly with the HIV-1 machinery during viral replication using a system in which viruses have been molecularly engineered to incorporate a potent immunological or biochemical tag. Using this panel of independently tagged replication-competent 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 self-selecting process based on replication competence in culture, the tagged viral proteins must undergo the same interactions encountered by the wild type virus. Therefore, 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. Currently, mass spectrometry techniques is being employed to determine the identity of host proteins and complexes that are captured via their interaction with the tagged viral proteins. The specific aims of this study are as follows: I. Comprehensive Mutagenesis of the HIV-1 Genome and Selective Recovery of Infectious, Replication-Competent, Tagged Viruses II. Utilization of Tagged Viruses for the Quantitative Recovery of Host Proteins that Interact with Viral Proteins during Replication III. Identification of Interacting Proteins by Mass Spectrometry and Assessment of Their Role in the HIV-1 Infectious Cycle A manuscript describing this work is under preparation