Delineating HIV-1 nuclear import mechanisms through capsid interaction with MxB and the nuclear pore complex

通过衣壳与 MxB 和核孔复合体相互作用描述 HIV-1 核输入机制

• 批准号: 10753391
• 负责人: Dariana Torres Rivera
• 金额: $ 4.77万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10753391
• 关键词: Antiviral Agents; Appearance; Architecture; Avidity; Binding; Capsid; Capsid Proteins; Cell Nucleus; Cells; Cellular biology; Centers for Disease Control and Prevention (U.S.); Complex; Cone; Core Protein; Cryoelectron Microscopy; Cyclophilin A; Cytoplasm; Data; Development; Diameter; Docking; Drug Targeting; Electron Microscopy; Fluorescent Dyes; Foundations; Genome; Goals; HIV-1; Image; Imaging Techniques; Infection; Infection Control; Integrase; Integration Host Factors; Interferons; Kinetics; Label; Light; Long Terminal Repeats; Measures; Mediating; Microscopy; Modeling; Molecular; Myxovirus; Nuclear; Nuclear Import; Nuclear Pore; Nuclear Pore Complex; Nuclear Pore Complex Proteins; Nucleoplasm; Optics; Persons; Physiological; Play; Process; Productivity; Proteins; RNA-Directed DNA Polymerase; Research; Resistance; Ribonucleoproteins; Role; Shapes; Site; Structure; Techniques; Testing; Training; Vaccines; Viral; Viral Reverse Transcription; Virion; Virus; Visualization; Work; genomic RNA; insight; light microscopy; live cell imaging; mutant; novel; overexpression; protein B; protein Mx; reconstruction; response; superresolution imaging; superresolution microscopy; treatment strategy; vaccine access; viral genomics; virology

ABSTRACT Human Immunodeficiency Virus 1 (HIV-1) causes life-long infection, which requires taking antiviral drugs to control the infection. No vaccines are available against this virus. The HIV-1 cone-shaped capsid core, made up of capsid protein (CA), protects the HIV-1 genome, plays a major role in HIV-1 infection and interacts with several host factors involved in nuclear import of viral complexes and integration into the host genome. The myxovirus resistance protein B (MxB) is thought to restrict HIV-1 infection by inhibiting uncoating and blocking nuclear entry, the latter measure by 2-Long Terminal Repeats (2-LTR) circles inside the nucleus. However, MxB does not inhibit the appearance of CA inside the nucleus of infected cells, even when infection is inhibited. Additionally, measuring 2-LTR circles in the nucleus, which is a marker for HIV-1 nuclear import, does not correlate with productive infection. Currently, little is known about the kinetics and sites of HIV-1 uncoating and the role of MxB in regulating this process. HIV-1 nuclear import is also not completely understood. Several nucleoporins (NUPs), which form the nuclear pore complex (NPC), interact with CA to promote HIV-1 nuclear import. The NPC was previously thought to be a static structure with an inner diameter of ~40nm, too small to allow nuclear import of the 60nm-wide HIV-1 capsid core, leading to an assumption that HIV-1 has to uncoat before entering the nucleus. However, recent evidence suggests that NPC is a dynamic structure that may expand and that HIV-1 cone- shaped cores can enter the nucleus. Little is known about how the NPC and MxB interactions with HIV-1 CA respectively promote or inhibit HIV-1 infection. Therefore, I hypothesize that the NPC-associated MxB blocks nuclear import by inhibiting uncoating and that the nuclear import of HIV-1 core requires expansion of the nuclear pore complex. In the two Specific Aims of this proposal, I will investigate the HIV-1 nuclear import and CA core disassembly in the presence of MxB (Aim 1) and delineate the changes in nuclear pore architecture caused by HIV-1 (Aim 2) using advance microscopy techniques. In Aim 1, I will determine HIV-1 nuclear entry inhibition by MxB through visualizing the presence of fluorescently labeled integrase-superfolderGFP (INsfGFP) inside the nucleus. I will also examine HIV-1 uncoating by visualizing loss of a fluorescently labeled cyclophilin A derivative, which binds to CA with high avidity. In Aim 2, I will examine changes in the nuclear pore size upon HIV-1 infection using super-resolution microscopy. I will infect NUP-labeled cells with HIV-1 CA mutant, which fails to transport to the nucleoplasm and remains associated with NPC. I will then visualize the NPC with Stimulated Emission Depletion (STED) microscopy and Stochastic Optical Reconstruction Microscopy (STORM). This project will provide me with diverse training in virology, cell biology and advanced microscopy techniques and will advance our understanding of HIV-1 nuclear import mechanisms. In the long-term, my work may support development of novel antiviral drugs that can mitigate HIV-1 infection.

抽象的 人免疫缺陷病毒1（HIV-1）引起终生感染，这需要服用抗病毒药 控制感染。该病毒没有可用的疫苗。 HIV-1锥形的衣壳芯，组成 衣壳蛋白（CA）保护HIV-1基因组，在HIV-1感染中起主要作用，并与几种相互作用 涉及病毒复合物的核进口并融入宿主基因组的宿主因素。粘液病毒 抗性蛋白B（MXB）被认为通过抑制脱被涂层和阻断核进入来限制HIV-1感染， 后者以2长的末端重复序列（2-ltr）圆圈尺寸。但是，MXB没有 即使抑制感染，也会抑制感染细胞核内CA的外观。此外， 测量核中的2-LTR圆，这是HIV-1核进口的标记，与 生产感染。目前，关于HIV-1脱涂的动力学和位点以及MXB的作用知之甚少 在调节此过程中。 HIV-1核进口也不完全了解。几个核孔蛋白（NUP）， 形成核孔复合物（NPC）的，与CA相互作用以促进HIV-1核进口。 NPC是 以前认为是内径约40nm的静态结构，太小了，无法进口核进口 60nm宽的HIV-1衣壳核心，导致假设HIV-1在进入细胞核之前必须取消脱毛。 但是，最近的证据表明，NPC是一种动态结构，可能会扩展，HIV-1锥体 形芯可以进入细胞核。关于NPC和MXB与HIV-1 CA的相互作用知之甚少 分别促进或抑制HIV-1感染。因此，我假设与NPC相关的MXB块 核进口通过抑制脱被涂层和HIV-1核心的核进口需要扩展 核孔复合体。在该提案的两个具体目的中，我将调查HIV-1核进口 在存在MXB的情况下（AIM 1）和CA核心拆卸，并描述核孔结构的变化 由HIV-1（AIM 2）使用预先显微镜技术引起。在AIM 1中，我将确定HIV-1核进入 MXB通过可视化荧光标记的积分酶 - 苏福利克FP（INSFGFP）的抑制作用 内核内。我还将通过可视化荧光标记的环磷脂的损失来检查HIV-1的脱落 一种衍生物，该衍生物与较高的​​亲和力结合。在AIM 2中，我将检查核孔大小的变化 使用超分辨率显微镜的HIV-1感染。我将用HIV-1 CA突变体感染NUP标记的细胞， 未能运输到核质中，并保持与NPC相关。然后，我将用 刺激的发射耗竭（STED）显微镜和随机光学重建显微镜 （风暴）。该项目将为我提供病毒学，细胞生物学和高级显微镜的各种培训 技术并将提高我们对HIV-1核进口机制的理解。从长远来看，我的工作 可能支持可以减轻HIV-1感染的新型抗病毒药物的开发。