Defining the three-dimensional organization and dynamics of HIV-1 Envelope using superresolution microscopy

项目摘要 人类免疫缺陷病毒1（HIV-1）已经进化出复杂的策略来限制 免疫系统的病毒表位。这一点可以通过病毒能够最大限度地减少 以病毒感染性为代价暴露病毒表面的包膜蛋白。HIV-1如何调节 将病毒包膜蛋白掺入到组装颗粒中仍然非常不清楚。我们假设 在HIV-1包膜胞质结构域以及宿主细胞膜脂质中发现的特异性基序 调节Env的获得以组装病毒颗粒。在我们的第一个目标，我们将量化 HIV-1包膜和胞质结构域突变体的单分子纳米级动力学 生产细胞的表面。在我们的第二个目标中，我们将测量三维（3D）结构 HIV-1包膜在出芽病毒颗粒表面的组织和角分布。为 目的，我们建议确定关键的病毒和宿主细胞膜决定因素负责的时空 HIV-1包膜与新生病毒装配位点的关联。这两个目标都将利用数十纳米 分辨率和分子特异性的三维超分辨率显微镜，以阐明动力学 和HIV-1信封组织。我们的定量方法将提供必要的基础 构建HIV-1包膜进入病毒装配位点的精确模型的知识。我们 我相信，这些模型将有助于未来开发新的战略，旨在抑制可及性， HIV-1包膜至病毒装配位点。

Project Summary Human Immunodeficiency Virus 1 (HIV-1) has evolved sophisticated strategies to limit the presentation of viral epitopes to the immune system. This is evidenced by the ability of the virus to minimize the number of exposed Envelope proteins on the virus surface at the expense of virus infectivity. How HIV-1 regulates the incorporation of viral Envelope proteins into assembling particles is still very unclear. We hypothesize that specific motifs found within the HIV-1 Envelope cytoplasmic domain as well as host cell membrane lipids regulate the acquisition of Env into assembling virus particles. In our first aim of this proposal, we will quantify the single molecule nanoscale dynamics of HIV-1 Envelope and mutants of the cytoplasmic domain on the surface of producing cells. In our second aim, we will measure the three-dimensional (3D) structural organization and angular distribution of HIV-1 Envelope on the surface of budding virus particles. For both aims, we propose to identify key viral and host cell membrane determinants responsible for the spatiotemporal association of HIV-1 Envelope with nascent virus assembly sites. Both aims will utilize the tens of nanometer resolution and molecular specificity of three-dimensional superresolution microscopy to illuminate the dynamics and organization of HIV-1 Envelope. Our quantitative approach will provide the necessary foundational knowledge for constructing accurate models of HIV-1 Envelope acquisition into virus assembly sites. We believe that these models will aid in the future development of novel strategies aiming to inhibit accessibility of HIV-1 Envelope to virus assembly sites.