HOST INTERACTIONS OF GENE PRODUCTS FROM HIV-1

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 与较大的 DNA 病毒相比，人类免疫缺陷病毒 (HIV-1) 的编码蛋白库相对有限。鉴于这一事实，可以合理地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，仅鉴定出少数此类病毒辅助宿主蛋白。在本提案中，我们努力使用一个系统来确定在病毒复制过程中与 HIV-1 机器直接相互作用的因素，其中 病毒经过分子工程改造，融入了有效的免疫学或生化标签。使用这组独立标记的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物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与野生型病毒相同的相互作用。因此，我们相信该系统将为我们提供关于 HIV 感染正常过程中形成的短暂和稳定的分子相互作用的更真实的视图。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。本研究的具体目的如下： I. HIV-1 基因组的全面诱变和传染性、复制能力、标记病毒的选择性恢复 二.利用标记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．通过质谱鉴定相互作用蛋白并评估其在 HIV-1 感染周期中的作用 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