HOST INTERACTIONS OF GENE PRODUCTS FROM HIV-1

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。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。在这项提案中，我们奋进确定的因素，直接与HIV-1的机器在病毒复制过程中使用的系统，其中病毒已被分子工程纳入一个有效的免疫或生化标签。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一个？自我选择？在基于培养物中的复制能力的过程中，标记的病毒蛋白必须经历与野生型病毒遇到的相同的相互作用。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。最终，质谱技术将用于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。本研究的具体目的如下：一、研究目的。HIV-1基因组的综合诱变和感染性、复制能力、标记病毒的选择性恢复II。利用标记的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III.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

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. Ultimately, mass spectrometry techniques will be 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