Application of fluorescence microscopy and live-cell imaging to elucidate the dynamics of viral assembly and budding

荧光标记物、荧光方法和灵敏光学检测的发展使得荧光活细胞成像成为研究细胞过程的一种强有力的方法。在这项计划中，我们将应用我们在超灵敏荧光技术和单分子研究方面的经验来初步研究HIV-1病毒粒子的萌发过程，然后将这些技术扩展到SPP 1175的其他病毒。我们的具体目标是：1)以表征Gag蛋白在胞浆中的齐聚为起点，2)通过研究Gag与Gag、脂质、包膜蛋白以及与ESCRT复合体的相互作用来研究Gag蛋白在质膜上的组装，以及3)观察和测量病毒出芽过程和病毒颗粒释放的动力学。为了以高时间和空间分辨率进行这些研究，我们将利用四种不同的荧光技术：广域成像和二维跟踪、光学切片、全内反射荧光显微镜以及荧光相关和交叉相关光谱。这些研究有望增加我们对病毒的包膜和萌发以及这些过程所需的细胞机制的了解。

Advances in the development of fluorescent markers, fluorescence methods, and sensitive optical detection have contributed to making fluorescence live-cell imaging a powerful method for investigating cellular processes. In this proposal, we will apply our experience with ultra-sensitive fluorescence techniques and single-molecule studies to initially investigate the budding process of HIV-1 virions and later on, extend these techniques to other viruses of the SPP 1175. Our specific aims are: 1) to characterize the oligomerization of Gag proteins within the cytosol as a starting point, 2) to investigate the assembly of the Gag proteins at the plasma membrane by studying the interaction of Gag with Gag, lipids, envelope proteins, and with ESCRT complexes and 3) to observe and measure the kinetics of the budding process and the release of virus particles. To perform these investigations with high temporal and spatial resolution, we will utilize four different fluorescence techniques: wide-field imaging and two-dimensional tracking, optical sectioning, total internal reflection fluorescence microscopy and fluorescence correlation and cross correlation spectroscopy. These investigations promise to increase our understanding of the envelopment and budding of viruses as well as the cellular machinery that is needed for these processes.