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 嗜神经性甲型疱疹病毒的组装和运输，包括1型单纯疱疹病毒 单纯疱疹病毒1型(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他们的 细胞质中形成包膜的部位。在对接到细胞器表面后，如反式高尔基体 网络衣壳与多种病毒结构蛋白和细胞ESCRT机制相互作用 包围圈。由此产生的出口隔间，在它们的管腔中包含成熟的被包裹的病毒粒子，然后 招募分子马达并使用微管将它们的病毒粒子运送到细胞表面。包络 而运输对于病毒的传染性和在细胞、组织和个人之间的传播至关重要；微管- 定向运输在神经元中尤其显著，病毒颗粒必须在那里移动非常远的距离。 在轴突内。 尽管它们对疾病的传播很重要，但包膜组装的分子细节和 人们对汽车招聘知之甚少。这两个事件的中心都是保守的疱疹病毒被膜蛋白。 UL36P(VP1/2)。UL36P对包膜是必不可少的，也被认为是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在这两个过程中的作用 HSV-1和PRV。我们的方法结合了体外还原生物化学与荧光和 超微结构显微镜，以及活体移植神经元中病毒组装和体外运动的成像。 为了研究UL36P在组装中的作用：我们将探索UL36P在衣壳出口中的作用 从细胞核到细胞器的对接和细胞ESCRT装置到包膜的募集 地点。我们还使用了定量荧光显微镜，结合相关的光和电子 显微镜(Clem)观察HSV-1和PRV囊膜细胞器的超微结构， 为了测试UL36P激动素结合位点在人口贩运中的作用：我们在 我们将测试这些结合位点对运动蛋白I运动性的重要性。 衣壳和被包裹的病毒粒子。为了便于分子和超微结构分析，我们使用了一种体外无细胞 在显微镜成像室中沿微管重建HSV-1和PRV交通的系统。在……里面 同时，我们将成像裸露和包膜的野生型和突变型病毒在细胞体和 感觉神经元外植体的轴突体外培养。