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

基本信息

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
项目类别：
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

项目摘要

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.

抽象的人类免疫缺陷病毒1（HIV-1）会导致终生感染，需要服用抗病毒药物控制感染。没有针对这种病毒的疫苗。 HIV-1 锥形衣壳核心，组成衣壳蛋白 (CA)，保护 HIV-1 基因组，在 HIV-1 感染中发挥重要作用，并与多种相互作用参与病毒复合物核输入和整合到宿主基因组中的宿主因子。粘病毒耐药蛋白 B (MxB) 被认为可以通过抑制脱壳和阻止核进入来限制 HIV-1 感染，后者通过细胞核内的 2 长末端重复 (2-LTR) 环进行测量。然而，MxB 并不即使感染被抑制，也能抑制受感染细胞核内 CA 的出现。此外，测量细胞核中的 2-LTR 环（这是 HIV-1 核输入的标记）与生产性感染。目前，人们对 HIV-1 脱壳的动力学和位点以及 MxB 的作用知之甚少。在调节这个过程中。 HIV-1 的核输入也尚未完全明了。几种核孔蛋白（NUP），形成核孔复合体（NPC），与CA相互作用促进HIV-1核输入。全国人大是以前认为是一个内径约为 40nm 的静态结构，太小而不允许核导入 60nm 宽的 HIV-1 衣壳核心，导致 HIV-1 在进入细胞核之前必须脱去外壳的假设。然而，最近的证据表明，NPC 是一个可能扩展的动态结构，并且 HIV-1 异形核心可以进入细胞核。关于 NPC 和 MxB 如何与 HIV-1 CA 相互作用知之甚少分别促进或抑制HIV-1感染。因此，我假设 NPC 相关的 MxB 会阻止通过抑制脱壳来进行核输入，并且 HIV-1 核心的核输入需要扩展核孔复合体。在该提案的两个具体目标中，我将调查 HIV-1 核输入和 CA 核心在 MxB 存在下的分解（目标 1）并描绘核孔结构的变化使用先进的显微镜技术检测由 HIV-1（目标 2）引起的病毒。在目标 1 中，我将确定 HIV-1 进入核通过观察荧光标记的整合酶超级文件夹 GFP (INsfGFP) 的存在来抑制 MxB 核内。我还将通过可视化荧光标记的亲环蛋白的损失来检查 HIV-1 脱壳一种衍生物，以高亲和力与 CA 结合。在目标 2 中，我将检查核孔径的变化使用超分辨率显微镜观察 HIV-1 感染。我将用 HIV-1 CA 突变体感染 NUP 标记的细胞，这无法转运至核质并仍与 NPC 相关。然后我将用以下方式可视化 NPC 受激发射损耗 (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学方面的多样化培训技术并将增进我们对 HIV-1 核输入机制的理解。从长远来看，我的工作可能支持开发可以减轻 HIV-1 感染的新型抗病毒药物。