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

HIV衣壳核心通过核孔复合物的基于亲和力的转运： 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 并将其逆转录的DNA整合到宿主基因组中。有几份报告说 HIV-1复合物的运输，并通过核孔复合物（NPC），一个巨大的 核被膜中的一个通道，由约30个核孔蛋白（NUPs）的多个拷贝组成。最近的数据 表明超分子富勒烯锥形病毒衣壳（CA）核心（~ 120 × 50 nm）， 包封病毒基因组可以穿透NPC的中央通道（直径约60 nm），至少 部分完整广泛接受的是，NUPs的至少一个子集与HIV-1 CA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，一种新的生物学机制， 大部分完整的HIV-1衣壳通过NPC -慢病毒的一个定义特征-仍然是未知的。 阐明。 HIV-1进入核的机制研究主要受到系统固有复杂性的阻碍。 NPC是真核细胞中最大的组装体之一（~120 MDa），HIV-1衣壳核心是真核细胞中最大的组装体之一。 运送到细胞核的最大货物。根据对最新公布数据和试点的分析， 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 衣壳核心通过NPC和挑战的核质转运的广义观点。我们 我认为HIV-1衣壳核心是一个复杂的转运受体，通过机会亲和性转运 NPC内部的梯度。亲和梯度由特定核孔蛋白上的各种“签名”组成 它们沿着NPC的核质轴自然分布。这样的子集 “签名”涉及在NPC中分离到不同区域的各种风格的FG图案。我们将使用 我们的多学科专业知识，技术设备和初步数据，以解决以下问题 具体目标：1.确定CA蛋白与不同FG基序的肽的复合物的结构。2. 生物化学和生物药理学表征CA和各种FG肽之间的相互作用。3. 使用核输入区分FG基序与突变衣壳核心相互作用的各种风味 实验科学的前提是，结合结构，生物化学，生物物理和成像 这些方法将为HIV-1衣壳核心复合物与 并揭示了NPC的组分和衣壳核心- NPC结构中间体的易位.知识 HIV-1核输入过程中的分子相互作用将有助于阐明HIV-1的传播策略 它的基因组到细胞核;我们对NPC功能的基本理解;为研究提供一个框架， 其他病毒的核输入;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.