Structure and Dynamics of Rev and RNA-Host Complexes

REV是一种小的HIV调节蛋白，对病毒复制至关重要，其众所周知的功能是输出 未剪接和部分剪接的病毒RNA从细胞核到细胞质。全长mRNA编码 病毒结构蛋白，并提供用于病毒包装的基因组RNA。REV以齐聚物的形式与 使用富含α螺旋精氨酸的RNA基序(ARM)的Env编码区内的RRE RNA 承认。六个Rev子单元绑定到240-NT版本的RRE，不同的子单元对接到不同的 在不同的结合模式下的RNA区域。我们最近解决了REV的高分辨率晶体结构 二聚体，与RRE RNA结合的REV二聚体的共晶结构，并组装了明确定义的核 使用CRM1/RanGTP导出复合体。这些研究表明，REV是一种高度模块化的蛋白质，它利用 用于相互作用的塑料RNA结合和寡聚化表面，并且CRM1与 REV-RRE复合体跨CRM1亚基结合。化学测绘实验进一步揭示了 全长350-nT RRE中先前未知的相互作用区，有助于形成支架以指导 REV寡聚体的结合。此外，REV似乎与更多的细胞质宿主伙伴相互作用， 我们推测这可能会重组REV-RRE齐聚物。HIV的基因组结构，其中 REV的读框与TAT和Env的读框重叠，RRE与Env也重叠的地方，施加 REV和RRE结构演化的额外约束。在此续订申请中，我们将定义 REV和RRE在结构和功能层面的动态行为：(1)确定 组装REV-RRE出口复合体中的替代RNA构象；(2)表征非出口 REV的功能及其与宿主蛋白的新相互作用；(3)REV-Env和RRE-Env的进化 重叠阅读框架；(4)通过三维作图检查活体RRE RNA结构。这些实验 将导致进一步的结构进展，旨在了解REV-RRE功能的分子基础和 确定潜在的新治疗目标。

Rev is a small HIV regulatory protein essential for viral replication whose well-known function is to export unspliced and partially spliced viral RNAs from the nucleus to the cytoplasm. The full-length mRNAs encode the viral structural proteins and supply the genomic RNA used in viral packaging. Rev binds as an oligomer to the RRE RNA within the Env coding region using an alpha-helical arginine-rich motif (ARM) for RNA recognition. Six Rev subunits bind to a 240-nt version of the RRE, with different subunits docking to different regions of the RNA in distinct binding modes. We recently solved a high-resolution crystal structure of the Rev dimer, a co-crystal structure of the Rev dimer bound to RRE RNA, and have assembled well-defined nuclear export complexes with Crm1/RanGTP. These studies revealed that Rev is a highly modular protein that utilizes plastic RNA-binding and oligomerization surfaces for interaction and that Crm1 forms a unique dimer with the Rev-RRE complex bound across the Crm1 subunits. Chemical mapping experiments further revealed a previously unknown interacting region within the full-length 350-nt RRE that helps form a scaffold to direct binding of the Rev oligomer. In addition, Rev appears to interact with additional cytoplasmic host partners, which we hypothesize may reorganize the Rev-RRE oligomer. The genomic architecture of HIV in which the reading frame of Rev overlaps with those of Tat and Env, and where the RRE also overlaps with Env, imposes extra constraints on the evolution of Rev and RRE structure. In this renewal application, we will define the dynamic behavior of Rev and the RRE at the structural and functional levels by: (1) determining the roles of alternative RNA conformations in assembling Rev-RRE export complexes; (2) characterizing non-export functions and novel host protein interactions with Rev; (3) examining the evolution of Rev-Env and RRE-Env overlapping reading frames; and (4) examining RRE RNA structure in vivo by 3D mapping. These experiments will lead to further structural advances aimed at understanding the molecular basis for Rev-RRE function and defining potential new therapeutic targets.