Identifying determinants of HIV-1 responsible for the nanoscale distribution and dynamics of virus assembly

项目摘要。 人类免疫缺陷病毒1型(HIV-1)组装机制，即获得包膜 糖蛋白(Env)进入组装的病毒晶格，仍然不是很清楚。持续创新 用来研究这些分子和纳米级组装过程的工具正在开始揭示一些 基本原则。本建议书旨在以这些创新为基础，量化环境需求 通过询问调节细胞内运输的病毒和宿主因子决定因素，表面展示， 病毒的整合和细胞间的传播。为了实现这些目标，我们将采用目前的超分辨率 和我们实验室开发的单分子跟踪方法来理解三维 环境蛋白在装配阶段和细胞间的结构组织和时空动力学 散开。接下来，我们将解剖Env的细胞质长尾，以了解哪些残基/区域驱动Env 成立为法团。最后，我们建议利用基因敲除来理解已识别的宿主的作用 调节环境表面水平的贩运因素。我们的初步数据支持我们的 通过展示我们提取有关纳米组织的定量信息的能力来实现这一方法 和三维环境中的单分子动力学。这些集体方法将为我们提供新的线索 HIV-1组装和细胞间病毒转移，提供了针对这些病毒的新模型和方法 以治疗的方式进行。

Project Summary. Human Immunodeficiency Virus Type-1 (HIV-1) assembly mechanisms, namely the acquisition of the envelope glycoprotein (Env) into the assembling viral lattice, remains inadequately understood. The continued innovation of tools to interrogate these molecular and nanoscale assembly processes is beginning to disclose some of the basic principles. This proposal aims to build upon these innovations and quantify the requirements for Env assembly by interrogating viral and host factor determinants regulating intracellular trafficking, surface display, virus incorporation, and cell-to-cell spread. To accomplish these aims, we will adapt current superresolution and single-molecule tracking approaches developed by our laboratory to understand the three-dimensional structural organization and spatiotemporal dynamics of Env throughout assembly stages and cell-to-cell spread. Next, we will dissect the long Env cytoplasmic tail to understand which residues/regions drive Env incorporation. Finally, we propose to utilize genetic knock-out to understand the role of identified host trafficking factors in regulating the surface levels of Env. Our preliminary data supports the feasibility of our approach by demonstrating our ability to extract quantitative information regarding the nanoscale organization and single-molecule dynamics of Env in three-dimensions. These collective approaches will shed new light on HIV-1 assembly and cell-to-cell virus transfer, informing new models and approaches aimed at targeting these processes therapeutically.