Neurotropic herpesvirus envelopment and microtubule-mediated transport

嗜神经性疱疹病毒包膜和微管介导的运输

• 批准号: 9884716
• 负责人: Gregory Allan Smith
• 金额: $ 71.53万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-03-15 至 2022-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9884716
• 关键词: Address; Afferent Neurons; Antiviral Agents; Axon; Bacterial Artificial Chromosomes; Binding; Binding Sites; Biochemistry; Biological Assay; Blindness; Capsid; Cell Nucleus; Cell surface; Cell-Free System; Cells; Cellular biology; Chickenpox; Complex; Congenital Abnormality; Cytoplasm; DNA; Development; Dissection; Docking; Electron Microscopy; Event; Fluorescence; Fluorescence Microscopy; Goals; Herpes Labialis; Herpesviridae; Herpesvirus 1; Image; In Vitro; Individual; Kinesin; Laboratories; Mediating; Membrane; Microtubules; Modeling; Molecular; Molecular Disease; Molecular Motors; Motor; Movement; Neurons; Nuclear Export; Organelles; Pathogenesis; Process; Production; Proteins; Public Health; Role; Site; Structure; Surface; Testing; Tissues; Viral; Viral Structural Proteins; Virion; Virus; Virus Assembly; base; cell motility; human disease; in vivo; light microscopy; mad itch virus; microscopic imaging; mutant; neurotropic; novel therapeutics; particle; pathogen; reconstitution; recruit; trafficking; trans-Golgi Network

Project Summary/Abstract The assembly and trafficking of neurotropic alphaherpesviruses, including herpes simplex virus type 1 (HSV-1) and pseudorabies virus (PRV), are extremely complex processes. DNA-packaged capsids must be exported from the infected cell nucleus, recruit molecular motors then move along microtubules to reach their site of envelope formation in the cytoplasm. After docking to the surface of organelles such as the trans Golgi network capsids interact with multiple viral structural proteins and the cellular ESCRT machinery to undergo envelopment. The resulting egress compartments, containing mature enveloped virions in their lumen, then recruit molecular motors and use microtubules to deliver their cargo of virions to the cell surface. Envelopment and trafficking is critical for viral infectivity and spread between cells, tissues and individuals; microtubule- directed transport is particularly dramatic in neurons, where viral particles must move very great distances within axons. Despite their importance for the spread of disease the molecular details of envelope assembly and motor recruitment are poorly understood. Central to both events is the conserved herpesvirus tegument protein UL36p (VP1/2). UL36p is essential for envelopment, and is also thought to recruit molecular motors to capsids and enveloped particles. In this proposal we explore the role of UL36p in each of these processes for both HSV-1 and PRV. Our approach combines reductionist in vitro biochemistry with fluorescence and ultrastructural microscopy, and imaging of virus assembly and movement ex vivo in living explanted neurons. To study the function of UL36p in assembly: We will explore the role of UL36p in export of capsids from the nucleus, docking to organelles and recruitment of the cellular ESCRT apparatus to the envelopment site. We also use quantitative fluorescence microscopy, in concert with correlative light and electron microscopy (CLEM), to probe the ultrastructure of HSV-1 and PRV envelopment organelles, To test the role of UL36p kinesin-binding sites in trafficking: We have identified motifs within UL36p that mediate attachment to kinesin I. We will test the importance of these binding sites for the motility of capsids and enveloped virions. To facilitate molecular and ultrastructural analysis we use an in vitro cell-free system that reconstitutes HSV-1 and PRV traffic along microtubules in a microscopic imaging chamber. In parallel we will image trafficking of naked and enveloped wild type and mutant viruses in the cell bodies and axons of sensory neuron explants cultured ex vivo.

项目摘要/摘要 神经甲状腺疱疹病毒的组装和运输，包括单纯疱疹病毒1型 （HSV-1）和伪标记病毒（PRV）是极其复杂的过程。 DNA包装的衣壳必须是 从感染的细胞核中导出，募集分子电动机然后沿着微管移动以达到其 细胞质中的包膜形成部位。停靠到细胞器的表面（例如反式高尔基） 网络衣壳与多种病毒结构蛋白和细胞质量机械相互作用以进行 信封。由此产生的出口室，其中包含成熟的包膜在其管腔中，然后 募集分子电动机并使用微管将病毒体货物运送到细胞表面。信封 贩运对于病毒感染和细胞，组织和个体之间的传播至关重要。微管 在神经元中，定向运输特别戏剧性，病毒颗粒必须移动很大的距离 轴突内。 尽管它们对疾病的传播重要性，但信封组件的分子细节和 招募运动知之甚少。这两个事件的中心是保守的疱疹病毒tegument蛋白 UL36P（VP1/2）。 UL36P对于包膜至关重要，也被认为可以募集分子电动机到Capsids 和包裹的颗粒。在此提案中，我们探讨了UL36P在每个过程中的作用 HSV-1和PRV。我们的方法结合了还原主义的体外生物化学和荧光和 超微结构显微镜，以及活体外植物神经元中病毒组装和运动的成像。 研究UL36P在组装中的功能：我们将探讨UL36P在Capsids出口中的作用 从细胞核，对接到细胞器，并募集细胞ESCRT设备再到信封 地点。我们还使用定量荧光显微镜，与相关的光和电子一起使用 显微镜（CLEM），以探测HSV-1和PRV包膜细胞器的超微结构， 测试UL36p驱动蛋白结合位点在运输中的作用：我们已经确定了主题 UL36P介导对驱动因素的附着I的UL36P。我们将测试这些结合位点的重要性 衣壳和包裹的病毒体。为了促进分子和超微结构分析，我们使用无体外细胞 在微观成像室中的微管沿着微管进行重构HSV-1和PRV流量的系统。在 平行于我们将在细胞体中对裸露和包裹的野生型和突变病毒的贩运图像 感觉神经元外植体的轴突培养了体内。