Dynamics of early ESCRT recruitment during HIV budding

描述（由申请人提供）：人类免疫缺陷病毒（HIV）劫持运输所需的内体分选复合物（ESCRT），以促进从宿主质膜释放。ESCRT的组装是通过募集ALIX和TSG101启动的，它们直接结合到病毒Gag蛋白上。ALIX和TSG101招募的时空细节尚不清楚。我们最近观察到，在病毒粒子形成过程中，ALIX在Gag组装结束时被短暂招募。我们还开发了超分辨率成像方法，通过这种方法，我们将残留的ALIX定位在释放的病毒样颗粒中。我们的超分辨率图像显示ALIX不对称地位于释放的VLPs内。在这里，我们将建立ALIX招募到HIV病毒样颗粒（VLP）的事件序列，并创建工具和方法，可用于定义在HIV出芽期间如何招募其他ESCRT组件。这一信息对于理解HIV出芽机制至关重要，也将有助于我们理解膜蛋白运输和细胞分裂，因为escrt促进MVB囊泡形成和细胞质分裂的脱落阶段。通过单分子成像和光学高分辨率技术的最新技术进步，本文提出的单病毒粒子和单细胞实验成为可能。这些实验提供了一组重要的信息，补充了通过基于人群的分析容易获得的信息。总之，从传统的种群方法和从单个病毒粒子和单细胞方法收集的信息有可能彻底改变我们对病毒机器的理解。

DESCRIPTION (provided by applicant): Human immunodeficiency virus (HIV) hijacks endosomal sorting complexes required for transport (ESCRT) to facilitate release from the host plasma membrane. ESCRT assembly is initiated by recruitment of ALIX and TSG101, which bind directly to the viral Gag protein. The temporal and spatial details of ALIX and TSG101 recruitment are not yet well understood. We have recently observed that ALIX is recruited transiently at the end of Gag assembly during virion formation. We have also developed super resolution imaging approaches through which we have localized the residual ALIX within released virus like particles. Our super resolution images show ALIX asymmetrically located within released VLPs. Here we will establish the sequence of events that underlie ALIX recruitment to HIV virus like particles (VLP) and create tools and methodologies that can be used to define how other ESCRT components are recruited during HIV budding. This information is critical for understanding the mechanism of HIV budding and will also inform our understanding of membrane protein trafficking and cell division because ESCRTs facilitate MVB vesicle formation and the abscission stage of cytokinesis. The single virion and single cell experiments proposed here have been enabled through recent technological advances in single molecule imaging and optical high-resolution techniques. These experiments provide an important set of information complementary to what is readily available through population-based assays. Together, the information gathered from traditional population methods and from single virion and single cell methodologies have the potential to revolutionize our understanding of viral machines.