Imaging HIV-1 Egress by Evanescent Field Microscopy

描述(申请人提供)：最近的生化和遗传学研究表明，哺乳动物空泡蛋白分选途径的组成部分与各种RNA病毒的释放有关，包括灵长类慢病毒。目前尚不清楚这些通常由内切溶酶体系统托管的成分是如何被招募到激活的淋巴细胞等细胞类型的质膜上的，在这些细胞类型中，HIV-1主要通过从细胞表面出芽退出。在这里，我们建议研究使用消失场荧光显微镜(EF-FM)，也被称为全内反射荧光显微镜(TIR-FM)，实时研究HIV-1芽形成的质膜微室的可行性。EF-FM/TIR-FM允许对质膜和底层约100 nm的细胞质进行选择性照射。荧光蛋白标记的病毒和细胞蛋白将被用来确定作为HIV-1萌芽平台的质膜微域是在病毒结构蛋白到达时同时组装的，还是它们是预先存在的位点。我们预计EF-FM/TIR-FM将成为一个重要的工具，以高空间和时间分辨率阐明最终导致新形成的病毒颗粒释放的事件序列。

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.