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: 9331794
• 负责人: Gregory Allan Smith
• 金额: $ 73.16万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE, INC
• 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/9331794
• 关键词: Address; Afferent Neurons; Axon; 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; Hereditary Disease; Herpes Labialis; Herpesviridae; Herpesvirus 1; Image; In Vitro; Individual; Kinesin; Laboratories; Mediating; Membrane; Microtubules; Modeling; Molecular; Molecular Motors; Motor; Movement; Neurons; Nuclear Export; Organelles; Pathogenesis; Pharmaceutical Preparations; Process; Production; Proteins; Public Health; Recruitment Activity; Role; Simplexvirus; 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; 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.

项目概要/摘要 嗜神经性 α 疱疹病毒（包括 1 型单纯疱疹病毒）的组装和贩运 （HSV-1）和伪狂犬病病毒（PRV）是极其复杂的过程。 DNA 包装的衣壳必须是 从受感染的细胞核输出，招募分子马达，然后沿着微管移动到达它们的位置 细胞质中包膜形成的部位。对接至反式高尔基体等细胞器表面后 网络衣壳与多种病毒结构蛋白和细胞 ESCRT 机制相互作用以进行 包围。由此产生的出口隔室，其内腔中含有成熟的包膜病毒颗粒，然后 招募分子马达并使用微管将病毒颗粒运送到细胞表面。包围 贩运对于病毒的感染性以及在细胞、组织和个体之间的传播至关重要；微管- 定向运输在神经元中尤其引人注目，其中病毒颗粒必须移动很远的距离 轴突内。 尽管它们对疾病传播很重要，但包膜组装的分子细节和 人们对运动募集知之甚少。这两个事件的核心是保守的疱疹病毒外皮蛋白 UL36p (VP1/2)。 UL36p 对于包膜至关重要，并且也被认为可以将分子马达招募到衣壳中 和被包裹的颗粒。在本提案中，我们探讨了 UL36p 在每个过程中的作用 HSV-1 和 PRV。我们的方法将还原论体外生物化学与荧光结合起来 超微结构显微镜，以及活体外植神经元中病毒组装和离体运动的成像。 研究 UL36p 在组装中的功能：我们将探讨 UL36p 在衣壳输出中的作用 从细胞核，对接至细胞器，并将细胞 ESCRT 装置募集至包膜 地点。我们还使用定量荧光显微镜，结合相关光和电子 显微镜 (CLEM)，探测 HSV-1 和 PRV 包膜细胞器的超微结构， 为了测试 UL36p 驱动蛋白结合位点在运输中的作用：我们已经确定了其中的基序 UL36p 介导与驱动蛋白 I 的附着。我们将测试这些结合位点对于运动性的重要性 衣壳和包膜病毒粒子。为了促进分子和超微结构分析，我们使用体外无细胞 该系统可在显微成像室中沿着微管重建 HSV-1 和 PRV 流量。在 与此同时，我们将想象裸露和有包膜的野生型和突变病毒在细胞体内的贩运，以及 离体培养的感觉神经元外植体的轴突。