Imaging HIV-1 Egress by Evanescent Field Microscopy

描述（由申请人提供）：最近的生物化学和遗传学研究表明，哺乳动物液泡蛋白分选途径的组分参与了各种RNA病毒（包括灵长类慢病毒）的释放。目前还不清楚这些通常由内-溶酶体系统宿主的组分如何被募集到细胞类型（如活化的淋巴细胞）的质膜上，其中HIV-1主要通过从细胞表面出芽而离开。在这里，我们建议研究的可行性，使用倏逝场荧光显微镜（EF-FM），也称为全内反射荧光显微镜（TIR-FM），调查在真实的时间，如何质膜微区室从HIV-1芽形成。EF-FM/TIR-FM允许选择性照射质膜和下面约100 nm的细胞质。将利用荧光蛋白标记的病毒和细胞蛋白来确定作为HIV-1出芽平台的质膜微区是否与病毒结构蛋白的到达同时组装，或者它们是否是预先存在的基因座。我们预计，EF-FM/TIR-FM将成为一个重要的工具来阐明，具有高的空间和时间分辨率，最终在新形成的病毒颗粒的释放事件的序列。

DESCRIPTION (provided by applicant): Recent biochemical and genetic studies have implicated components of the mammalian vacuolar protein sorting pathway in the release of various RNA viruses, including the primate lentiviruses. It is unclear how these components, which are normally hosted by the endo-lysosomal system, are recruited to the plasma membrane in cell types such as activated lymphocytes, where HIV-1 exits primarily by budding from the cell surface. Here we propose to study the feasibility of using evanescent field fluorescence microscopy (EF-FM), also called total internal reflection fluorescence microscopy (TIR-FM), to investigate in real time, how the plasma membrane microcompartments from which HIV-1 buds are formed. EF-FM/TIR-FM allows selective illumination of the plasma membrane and the underlying approximately 100 nm of cytoplasm. Fluorescent protein-tagged viral and cellular proteins will be utilized to determine if the plasma membrane microdomains, which serve as budding platforms for HIV-1 are assembled simultaneously with the arrival of the viral structural proteins or if they are pre-existing loci. We anticipate that EF-FM/TIR-FM will become an important tool to elucidate, with high spatial and temporal resolution, the sequence of events culminating in the release of newly formed viral particles.