Regulation of HIV-1 Preintegration Complexes (PICs)

描述(申请人提供)：与逆转录酶和蛋白酶不同， HIV整合酶很难作为一种分离的酶来研究，因为它的功能 在体内，在前整合复合体(PIC)的背景下，由 病毒和宿主细胞来源的成分。在静息的CD4+T细胞中，这 发现复合体以一种不活跃的形式存在，称为？前整合 复杂？或者？支持PIC，？它被假设为是 集成前延迟。需要细胞刺激才能将亲PIC转化为 整合能力强的PIC。人们对这种病毒的分子本质知之甚少。 转换过程，以及PIC的确切组成仍不清楚。这 Project将从问题的两个相反方面解决这些问题： 宿主？S一侧，在那里发生免疫过程，触发 活性PIC的形成；在病毒方面？S，分子变化的地方 将亲PIC转换为PIC。四个具体目标是：(一) 描述影响CD4+T细胞中PIC活性的细胞刺激 巨噬细胞；(2)开发原PIC和PIC的纯化方法 体外感染的细胞并用它们来证实前PIC的存在 从HIV感染者身上提取的样本中的活体PIC；(3)确定 利用DNA足迹技术对原图和原图的结构和构图， 抗体和质谱学；以及(4)使用 活性化合物寻找一类可能的整合抑制剂 阻断PIC活性而不直接抑制整合酶。加在一起， 这些研究将澄清免疫过程与 刺激艾滋病毒复制及其影响的分子变化 专业图片和图片。将获得新的工具和概念，以及精确的分子 抗艾滋病毒药物设计的目标将成为关注的焦点。

DESCRIPTION (provided by applicant): Unlike reverse transcriptase and protease, HIV integrase is difficult to study as an isolated enzyme because it functions in vivo in the context of the preintegration complex (PIC), a particle composed of both viral and host cell-derived components. In resting CD4+ T cells, this complex was found to be in an inactive form termed a ?pro-preintegration complex? or ?pro-PIC,? which is hypothesized to be the physical embodiment of preintegration latency. Cell stimulation is needed to convert the pro-PIC to an integration-competent PIC. Little is known about the molecular nature of the conversion process, and the exact composition of the PIC remains unknown. This project will address these issues from two opposite faces of the problem: on the host?s side, where immunological processes occur which trigger the formation of the active PIC; and on the virus?s side, where molecular changes convert the pro-PIC into the PIC. The four specific aims are: (1) To characterize the cellular stimuli that affect PIC activity in CD4+ T cells and macrophages; (2) To develop purification methods for pro-PICs and PICs from cells infected in vitro and to use them to establish the existence of pro-PICs and PICs in vivo in samples taken from HIV-infected individuals; (3) To define the structure and composition of pro-PICs and PICs using DNA footprinting, antibodies, and mass spectrometry; and (4) To screen PICs with a library of reactive compounds to find a putative new category of integration inhibitors which block PIC activity without directly inhibiting integrase. Taken together, these studies will clarify the linkage between the immunological processes that stimulate HIV replication and the molecular changes that they effect upon pro-PICs and PICs. New tools and concepts will be gained, and precise molecular targets for anti-HIV drug design will come into focus.