Retroviral Assembly

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开展研究者发起的HIV-1生物学研究。我们通过发展研究专长和促进对病毒和艾滋病的了解，为NCI的目标作出贡献。HIV-1细胞蛋白及其组装：在此期间，我们继续努力鉴定和表征HIV-1病毒粒子内的宿主蛋白及其在病毒生命周期中的相关性。细胞蛋白被整合到逆转录病毒中，可能在HIV-1的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了HIV-1在组装过程中与细胞途径之间相互作用的重要见解。与逆转录病毒发病机制实验室一起，我们开发了一种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以前在去除细胞蛋白的非病毒来源方面建立的专业知识。总之，这些技术提供了强大的工具来完全和明确地分析HIV-1病毒粒子内外的细胞蛋白质。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粒子制剂。虽然我们的大多数研究都集中在从相关转化的T细胞系产生的病毒上（Rev. Med. Virol. 12:359），但从重要的原代细胞类型（即单核巨噬细胞和T细胞）产生的HIV-1病毒粒子也在研究中。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组装和出芽中的作用[j]。化学277:38818。HIV-1出芽对蛋白酶体抑制剂敏感性的细胞基础也被研究。此外，与佛蒙特大学的马库斯·塔利的合作研究已经找到了在活细胞中动态成像HIV-1的新方法。总之，这些研究可以潜在地解释HIV-1出芽的几个未知方面。反过来，了解HIV-1的萌芽可能会确定抗逆转录病毒治疗的新目标。HIV-1组装机制：基于HIV-1 Gag多蛋白中HIV-1 NC蛋白几乎完全缺失的一系列突变体实验表明，与预期相反，该蛋白对于病毒粒子的形成是必不可少的（J. Virol. 77:5547）。其他数据表明，对HIV-1蛋白酶加工的调控是病毒组装的关键部分。这种突变体产生的颗粒没有传染性。这种突变体产生的非传染性病毒粒子使其成为生产全颗粒灭活HIV-1疫苗的另一种潜在方法。原代细胞的可逆转化。我们已经开发出一种系统，可以从人类血液中分离和永生抗原特异性T细胞系。这些细胞系由可溶性或异体抗原刺激的患者外周血单个核细胞产生，保持其抗原反应性和原代细胞特征。该系统将极大地帮助分析T细胞对病毒抗原以及其他医学相关抗原的免疫反应成分。我们还在开发一种方法，通过切除这些永生基因来逆转这些T细胞系的永生，这样这些细胞就可以用作原代T细胞，例如，在临床环境中用于过继治疗。选择性和可逆性T细胞永生化有望解剖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。

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.