Single Cell with One Particle Entry (SCOPE) for Study of HIV Infection

描述(由申请人提供) 摘要：人类免疫缺陷病毒1型(HIV-1)自1983年发现以来，通过感染CD_4阳性T淋巴细胞，已夺走了2500万人的生命。虽然已证实HIV-1通过与细胞表面的CD4和趋化因子辅助受体结合而启动T细胞感染，但HIV-1感染CD4T细胞的早期事件仍然知之甚少。多年来，人们使用了各种不同的技术来研究艾滋病毒-1进入的机制。然而，所有这些方法固有的一个技术限制是无法跟踪单个HIV-1病毒粒子从病毒进入一开始到染色体整合的命运。在所有这些实验中，培养和测量了一组病毒和细胞。由于每个进入事件都能以有限的概率导致前病毒DNA整合，因此很难在进入途径和产生性感染之间建立因果联系。该项目的目标是开发一套纳米技术和新技术，我们可以利用这些技术来确定HIV-1感染CD4T细胞的途径和相互作用。我们正在开发一种基于光学镊子的技术，它可以操纵单个HIV-1病毒粒子，将其输送到CD4T细胞，从而允许我们确定单个病毒粒子进入CD4T细胞的命运。这项技术将使我们能够明确地定义HIV-1感染的分子机制。此外，我们建议在活T细胞的背景下直接测量单个HIV-1病毒粒子和受体之间的相互作用。总的来说，这些研究将有助于明确和定量地了解艾滋病毒感染的早期事件，这可能有助于旨在阻止艾滋病毒-1进入CD4T细胞的治疗开发。本文开发的技术不仅可用于研究细胞对病毒的摄取，还可用于研究其他分子、大分子组件和纳米颗粒，并适用于细胞表面广泛的配体-受体相互作用。 公共卫生相关性：HIV感染CD4T细胞的早期事件知之甚少。这项提议旨在开发一套纳米和其他新技术来实时研究HIV对CD4T细胞的感染，一次一个病毒粒子。如果成功，这项研究的结果将首次在HIV进入途径和CD4T细胞的生产性感染之间建立起因果联系，这将有助于旨在阻止HIV-1进入CD4T细胞的治疗开发。

DESCRIPTION (Provided by the applicant) Abstract: Through infection of CD4 positive (CD4+) T lymphocytes, the Human Immunodeficiency Virus type 1 (HIV-1) has claimed twenty-five million lives since its discovery in 1983. Although it has been well established that HIV-1 initiates a T cell infection by binding to CD4 and chemokine coreceptors on the cell surface, the early events in HIV-1 infection of CD4+ T cells remain poorly understood. A variety of different techniques have been used over the years to study mechanisms of HIV-1 entry. However, one technical limitation that is inherent in all these methods is the inability to track the fate of a single HIV-1 virion from the very beginning of viral entry to chromosomal integration. In all these experiments, a population of viruses and cells were incubated and measured. Because each entry event can lead to proviral DNA integration with a finite probability, it is therefore difficult to establish a causative link between entry pathway and productive infection. The goal of this project is to develop a set of nanoscopic and novel technologies that we can harness to define the pathway and interactions by which HIV-1 infects CD4+ T cells. We are developing a technique based on optical tweezers that can manipulate a single HIV-1 virion, deliver it to CD4+ T cell and thus allows us to determine the fate of CD4+ T cell upon entry by a single virion. This technique will allow us to unambiguously define the molecular mechanisms of HIV-1 infection. Furthermore, we propose to measure directly the interactions between a single HIV-1 virion and receptors in the context of a live T cell. Collectively, these studies will contribute to a definitive and quantitative understanding of early events in HIV infection, which may help therapeutic development that is aimed to block HIV-1 entry to CD4+ T cells. The techniques developed herein can be useful for studying cellular uptake of not only viruses but other molecules, macromolecular assemblies and nanoparticles, and are applicable to a wide-range of ligand-receptor interactions on the cell surface. Public Health Relevance: The early events in HIV infection of CD4+ T cells are poorly understood. This proposal aims to develop a set of nanoscopic and other novel techniques to study HIV infection of CD4+ T cells in real time, one virion at a time. If successful, the results from this study will establish for the first time a causative link between HIV entry pathway and the productive infection of CD4+ T cells, which will help therapeutic development that is aimed to block HIV-1 entry to CD4+ T cells.