Affinity Gradient-Based Transport of HIV Capsid Cores through the Nuclear Pore Complex

基于亲和力梯度的HIV衣壳核心通过核孔复合体的运输： 项目摘要 慢病毒，如人类免疫缺陷病毒1型(HIV-1)，已经进化出进入细胞核的策略 并将其逆转录DNA整合到宿主基因组中。有几份报告 关于HIV-1复合体向核孔复合体(NPC)的运输和通过核孔复合体的输入 核膜通道由~30个核孔蛋白(NUP)的多个拷贝组成。最新数据 表明超分子富勒烯锥形病毒衣壳(CA)核心(~120x50 nm) 包裹的病毒基因组至少可以穿透鼻咽癌的中央通道(直径~60 nm) 部分完好无损。人们普遍认为，至少有一部分NUP与HIV-1CA蛋白相互作用 病毒核心通过其苯丙氨酸-甘氨酸(FG)基序。然而，分子机制是一种 基本完整的HIV-1衣壳通过NPC-慢病毒的一个定义特征-仍然是 已澄清。 艾滋病毒-1核进入的机制研究主要受到该系统固有复杂性的阻碍。 鼻咽癌是真核细胞中最大的组件之一(~120 mda)，而HIV-1衣壳核心是 最大的货物被送到细胞核。基于对最新发布的数据和试点的分析 结果，我们提出了一个范式转换假说，解决了HIV-1的运输机制 衣壳核心穿过鼻咽癌，挑战了关于核质运输的普遍观点。我们 认为HIV-1衣壳核心是一种复杂的转运受体，通过机会性亲和力转运 全国人大内部的渐变。亲和力梯度由特定核孔蛋白上的各种“签名”组成。 自然分布在鼻咽癌的核质轴上。这样的子集 “签名”涉及不同风格的FG主题，在NPC中被分离到不同的区域。我们将使用 我们的多学科专业知识、技术宝库和初步数据，以解决以下问题 具体目的：1.确定CA蛋白与不同FG基序的多肽形成的复合物的结构。2. 从生化和生物物理的角度研究CA与各种FG多肽之间的相互作用。3. 利用核进口区分FG基序与突变型衣壳核相互作用的不同味道 实验。科学前提是将结构、生化、生物物理和成像结合在一起 方法将提供重要的机制洞察HIV-1衣壳核心复合体与 并揭示了衣壳核心--鼻咽癌易位的结构中间体。了解以下内容 HIV-1核进口过程中的分子相互作用将揭示HIV-1部署的战略 它的基因组到细胞核；我们对鼻咽癌功能的基本了解；为研究 其他病毒的核进口；以及新的抗病毒疗法的潜在方法。

Affinity Gradient-Based Transport of HIV Capsid Cores Through the Nuclear Pore Complex: Project Summary Lentiviruses such as human immunodeficiency virus type 1 (HIV-1) have evolved strategies to enter the nucleus of non-dividing cells and integrate their reverse transcribed DNA into host genomes. There are several reports on the transport of HIV-1 complexes to, and import through, the Nuclear Pore Complex (NPC), a massive channel in the nuclear envelope composed of multiple copies of ~30 nucleoporins (NUPs). Recent data suggests that the supramolecular fullerene cone-shaped viral capsid (CA) core (~120x50 nm) that encapsulates the viral genome can penetrate the central channel (diameter of ~60 nm) of the NPC, at least partially, intact. It is widely accepted that at least a subset of NUPs interacts with the HIV-1 CA proteins of the viral core through their phenylalanine-glycine (FG) motifs. However, the molecular mechanisms by which a largely intact HIV-1 capsid passes through the NPC – a defining feature of lentiviruses – remains to be elucidated. Mechanistic studies of HIV-1 nuclear entry have been stymied primarily by the inherent complexity of the system. The NPC is one of the largest assemblies in the Eukaryotic cell (~120 MDa) and HIV-1 capsid core is one of the largest cargoes delivered to the cell nucleus. Based on analysis of state-of-the-art published data and pilot results, we propose a paradigm-shifting hypothesis that addresses the mechanism of transport of HIV-1 capsid core through the NPC and challenges the generalized view on nucleo-cytoplasmic transport. We propose that HIV-1 capsid core is a complex transport receptor that is translocated via an opportunistic affinity gradient within the NPC. The affinity gradient consists of various “signatures” on specific nucleoporins that are naturally distributed along the nucleo-cytoplasmic axis of the NPC. The subset of such “signatures” involves various flavors of FG motifs that are segregated in the NPC to distinct zones. We will use our multidisciplinary expertise, armamentarium of techniques, and preliminary data, to address the following specific aims: 1. Determine the structures of complexes of CA proteins with peptides of different FG motifs. 2. Biochemically and biophysically characterize the interactions between CA and various FG peptides. 3. Discriminate between various flavors of FG motif interactions with mutant capsid cores using nuclear import experiments. The scientific premise is that combined structural, biochemical, biophysical and imaging approaches will provide important mechanistic insight into interactions of HIV-1 capsid core complexes with NPC constituents and reveal the capsid core – NPC structural intermediates of translocation. Knowledge of the molecular interactions during HIV-1 nuclear import will shed light on the strategy HIV-1 deploys to deliver its genome to the nucleus; our fundamental understanding of NPC function; provide a framework for studies of the nuclear import of other viruses; and potential approaches to novel antiviral therapies.