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: 10548633
• 负责人: Dariana Torres Rivera
• 金额: $ 4.68万
• 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/10548633
• 关键词: Antiviral Agents; Appearance; Architecture; Avidity; Binding; Caliber; Capsid; Capsid Proteins; Cell Nucleus; Cells; Cellular biology; Centers for Disease Control and Prevention (U.S.); Complex; Cone; Cryoelectron Microscopy; Cyclophilin A; Cytoplasm; Data; Development; 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; Life; 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; Proteins; RNA-Directed DNA Polymerase; Research; Resistance; Resolution; Ribonucleoproteins; Role; S-nitro-N-acetylpenicillamine; Site; Structure; Techniques; Testing; Time; Training; Vaccines; Viral; Viral Reverse Transcription; Virion; Virus; Work; base; genomic RNA; insight; light microscopy; live cell imaging; mutant; novel; overexpression; protein B; protein Mx; reconstruction; response; 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.

摘要 人类免疫缺陷病毒1（HIV-1）导致终身感染，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，由 衣壳蛋白（CA）的，保护HIV-1基因组，在HIV-1感染中起主要作用，并与几个相互作用 参与病毒复合物的核输入和整合到宿主基因组中的宿主因子。粘病毒 抗性蛋白B（Mx B）被认为通过抑制脱壳和阻断核进入来限制HIV-1感染， 后者通过细胞核内的2-长末端重复序列（2-LTR）环测量。但是，MXB不 抑制感染细胞核内CA的出现，即使感染被抑制。此外，本发明还 测量细胞核中的2-LTR环，这是HIV-1细胞核输入的标志物， 生产性感染目前，对HIV-1脱壳的动力学和位点以及MxB的作用知之甚少。 来调节这个过程。HIV-1的核输入也不完全清楚。几种核孔蛋白（NUPs）， 形成核孔复合物（NPC），与CA相互作用，促进HIV-1核输入。的NPC 以前认为是一个静态结构，内径约为40 nm，太小，不允许核输入。 60 nm宽的HIV-1衣壳核心，导致假设HIV-1在进入细胞核之前必须脱壳。 然而，最近的证据表明，NPC是一个动态的结构，可能会扩大和HIV-1锥， 形核可以进入细胞核。关于NPC和MxB如何与HIV-1 CA相互作用， 分别促进或抑制HIV-1感染。因此，我假设NPC相关的MxB块 核输入抑制脱壳和核输入的HIV-1核心需要扩大 核孔复合体。在本提案的两个具体目标中，我将调查HIV-1核输入 和CA核心解体在MxB（Aim 1）的存在下，并描绘了核孔结构的变化 使用先进的显微镜技术对HIV-1（Aim 2）引起的疾病进行了研究。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-超折叠GFP（INsfGFP）的存在， 在细胞核内。我还将通过观察荧光标记的亲环素的损失来检查HIV-1的脱壳 一种以高亲合力与CA结合的衍生物。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。我会用HIV-1CA突变体感染NUP标记的细胞， 不能运输到核质，并保持与NPC相关。然后，我将想象NPC与 受激发射耗尽（STED）显微镜和随机光学重建显微镜 （风暴）。这个项目将为我提供病毒学，细胞生物学和先进显微镜的各种培训 技术，并将促进我们对HIV-1核输入机制的理解。从长远来看，我的工作 可能支持开发新的抗病毒药物，可以减轻HIV-1感染。