Retroviral Assembly

逆转录病毒组装

• 批准号: 7058973
• 负责人: Michael Lee
• 金额: --
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7058973
• 关键词: CD antigens; Retroviridae; T lymphocyte; gag protein; human immunodeficiency virus 1; human tissue; life cycle; mass spectrometry; proteasome; virion; virus assembly; virus replication

Our laboratory conducts investigator-initiated research into the biology of HIV-1 using molecular biology, biochemical, and cellular biological techniques. We contribute to the goals of the NCI by developing research expertise and advancing the understanding of the virus and AIDS.Cellular proteins in HIV-1 and assembly: Our efforts to identify and characterize the host proteins inside HIV-1 virions and their relevance in the viral life-cycle were continued during this period. Cellular proteins are incorporated into retroviruses and may play important roles in HIV-1 biology and pathogenesis. Additionally these cellular proteins might reveal important insights into the interactions between HIV-1 and cellular pathways during assembly. Along with the Retroviral Pathogenesis Laboratory, we have developed a CD45 immunoaffinty depletion technology that produces highly purified virus preparations. This approach complements our previously established expertise in removing non-viral sources of cellular proteins. Together, these techniques provide powerful tools to completely and unambiguously analyze the cellular proteins both inside and outside HIV-1 virions. Currently, we have begun to analyze these highly purified virion preparations using traditional biochemical analyses and state-of-the-art mass spectrometry sequencing. While most of our studies have focused on virus produced from relevant transformed T cell lines (Rev. Med. Virol. 12:359), HIV-1 virions produced from important primary cell types (i.e., monocyte-macrophages and T cells) are also being examined. From these studies, the relevance and roles of several cellular proteins in HIV-1 biology were intensively investigated. The participation of the ubiquitin-proteasome and the endosomal pathways in HIV-1 assembly and budding were studied (J. Biol. Chem. 277:38818. The cellular basis for the sensitivity of HIV-1 budding to proteasome inhibitors was also investigated. Additionally, collaborative studies with Markus Thali of the University of Vermont has lead to new ways to dynamically image HIV-1 in living cells. Together, these studies can potentially explain several unknown aspects of HIV-1 budding. In turn, understanding HIV-1 budding might identify new targets for anti-retroviral therapy. Mechanisms of HIV-1 assembly: Experiments with a series of mutants based on a nearly complete deletion of the HIV-1 NC protein from the HIV-1 Gag polyprotein demonstrated that, contrary to expectations, this protein is dispensable for virion formation (J. Virol. 77:5547. Additional data suggest that regulation of HIV-1 protease processing is a key part of viral assembly. Particles produced by this mutant are not infectious. The production of noninfectious virions by this mutant makes this another potential method to produce a whole particle inactivated HIV-1 vaccine.Reversible transformation of primary cells. We have developed a system to isolate and immortalize antigen specific T cell lines from human blood. These cell lines, generated from soluble- or allo-antigen-stimulated patient peripheral blood mononuclear cells maintain their antigen responsiveness and primary cell characteristics. This system should greatly assist the analysis of the T cell component of the immune response to viral antigens as well as other medically relevant antigens. We are also developing a method to reverse the immortalization of these T cell lines by excising the immortalizing genes so these cells could be used as primary T cells, e.g., in a clinical setting for adoptive therapy. Selective and reversible T cell immortalization promises to allow for the dissection of the host immune response to retroviruses or vaccines, the development of appropriate cell lines for vaccine production and studies, and potential applications for cancer therapy.

我们的实验室利用分子生物学、生物化学和细胞生物学技术对 HIV-1 生物学进行研究者发起的研究。我们通过发展研究专业知识和增进对病毒和艾滋病的了解，为 NCI 的目标做出贡献。 HIV-1 中的细胞蛋白和组装：我们为识别和表征 HIV-1 病毒粒子内的宿主蛋白及其在病毒中的相关性所做的努力在此期间病毒的生命周期仍在继续。细胞蛋白被整合到逆转录病毒中，可能在 HIV-1 生物学和发病机制中发挥重要作用。此外，这些细胞蛋白可能揭示组装过程中 HIV-1 和细胞通路之间相互作用的重要见解。我们与逆转录病毒发病机制实验室一起开发了 CD45 免疫亲和去除技术，可生产高度纯化的病毒制剂。这种方法补充了我们之前在去除细胞蛋白非病毒来源方面建立的专业知识。这些技术共同提供了强大的工具来完全、明确地分析 HIV-1 病毒颗粒内部和外部的细胞蛋白。目前，我们已经开始使用传统的生化分析和最先进的质谱测序来分析这些高度纯化的病毒颗粒制剂。虽然我们的大部分研究都集中在相关转化 T 细胞系产生的病毒 (Rev. Med. Virol. 12:359)，但由重要的原代细胞类型（即单核细胞-巨噬细胞和 T 细胞）产生的 HIV-1 病毒粒子也正在接受检查。从这些研究中，我们深入研究了几种细胞蛋白在 HIV-1 生物学中的相关性和作用。研究了泛素蛋白酶体和内体途径在 HIV-1 组装和出芽中的参与（J. Biol. Chem. 277:38818）。还研究了 HIV-1 出芽对蛋白酶体抑制剂敏感性的细胞基础。与佛蒙特大学的 Markus Thali 的合作研究找到了在活细胞中动态成像 HIV-1 的新方法，这些研究有可能解释 HIV-1 的几个未知方面。反过来，了解 HIV-1 的出芽可能会确定抗逆转录病毒治疗的新靶标：基于 HIV-1 NC 蛋白几乎完全缺失的一系列突变体的实验。 1 Gag 多蛋白证明，与预期相反，该蛋白对于病毒颗粒的形成是可有可无的 (J. Virol. 77:5547。其他数据表明，HIV-1 蛋白酶加工的调节是关键部分该突变体产生的病毒颗粒不具有传染性。该突变体产生的非感染性病毒颗粒使得这是生产全颗粒灭活 HIV-1 疫苗的另一种潜在方法。原代细胞的可逆转化。我们开发了一种从人类血液中分离和永生化抗原特异性 T 细胞系的系统。这些细胞系由可溶性或同种异体抗原刺激的患者外周血单核细胞产生，保持其抗原反应性和原代细胞特征。该系统应该极大地有助于分析对病毒抗原以及其他医学相关抗原的免疫反应的 T 细胞成分。我们还在开发一种方法，通过切除永生化基因来逆转这些 T 细胞系的永生化，以便这些细胞可以用作原代 T 细胞，例如在临床环境中用于过继治疗。选择性和可逆的 T 细胞永生化有望解析宿主对逆转录病毒或疫苗的免疫反应，开发用于疫苗生产和研究的适当细胞系，以及癌症治疗的潜在应用。