HOST INTERACTIONS OF GENE PRODUCTS FROM HIV-1

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 与较大的DNA病毒相比，人类免疫缺陷病毒（HIV-1）具有相对有限的编码蛋白质库。鉴于这一事实，我们有理由预期宿主细胞构成了病毒复制所必须利用的丰富因子来源。然而，迄今为止，只有少数这样的病毒辅助宿主蛋白已被确定。在这个建议中，我们奋进确定在病毒复制过程中与HIV-1机制直接相互作用的因素，使用一个系统， 病毒已经被分子工程化以掺入有效的免疫学或生物化学标记。使用这组独立标记的复制能力衍生物，我们试图恢复宿主蛋白质，这些蛋白质在病毒的自然生命周期中与病毒特异性相互作用。由于这些工程病毒是通过基于培养物中复制能力的自我选择过程产生的，因此标记的病毒蛋白必须经历野生型病毒所遇到的相同相互作用。因此，我们相信这个系统将为我们提供一个更真实的观点，在HIV感染的正常过程中形成的短暂和稳定的分子相互作用。目前，质谱技术被用来确定通过与标记的病毒蛋白相互作用捕获的宿主蛋白和复合物的身份。这项研究的具体目标如下： I. HIV-1基因组的综合诱变和感染性、复制能力、标记病毒的选择性回收 二.利用标记的病毒定量回收复制过程中与病毒蛋白相互作用的宿主蛋白 三.相互作用蛋白质的质谱鉴定及其在HIV-1感染周期中的作用 描述这项工作的手稿正在编写中

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. 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 in preparation