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.

项目总结/摘要 嗜神经性α疱疹病毒（包括单纯疱疹病毒1型）的组装和运输 （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 从受感染的细胞核输出，招募分子马达，然后沿着沿着微管移动，到达它们的 细胞质中包膜形成的部位。在对接到细胞器的表面，如反高尔基体， 网络衣壳与多种病毒结构蛋白和细胞ESCRT机制相互作用， - 谢谢由此产生的出口室，在其内腔中含有成熟的包膜病毒体， 招募分子发动机并使用微管将病毒体运送到细胞表面。包络 和运输是至关重要的病毒感染性和细胞之间的传播，组织和个人;微管- 定向运输在神经元中尤其引人注目，病毒颗粒必须移动很远的距离 在轴突中。 尽管它们对疾病的传播很重要，但包膜组装的分子细节和 对运动神经元的募集知之甚少。这两个事件的核心是保守的疱疹病毒被膜蛋白 UL36p（VP1/2）。UL 36 p对于衣壳是必需的，并且也被认为是将分子马达招募到衣壳中 和包膜颗粒。在本提案中，我们探讨了UL 36 p在这些过程中的作用， HSV-1和PRV。我们的方法结合了还原论体外生物化学与荧光， 超微结构显微镜和活的神经元中的病毒组装和离体运动的成像。 研究UL 36 p在组装过程中的作用：探讨UL 36 p在衣壳输出中的作用 从细胞核，对接到细胞器和细胞ESCRT装置的募集到细胞器 绝佳的价钱我们还使用定量荧光显微镜，与相关的光和电子 用CLEM显微镜观察HSV-1和PRV分泌细胞器的超微结构， 为了测试UL 36 p驱动蛋白结合位点在运输中的作用： UL 36 p介导与驱动蛋白I的连接。我们将测试这些结合位点对细胞运动性的重要性。 衣壳和有包膜的病毒体。为了便于分子和超微结构分析，我们使用体外无细胞 在显微镜成像室中重构HSV-1和PRV沿沿着微管运输的系统。在 平行地，我们将在细胞体中描绘裸的和有包膜的野生型和突变型病毒的运输， 离体培养的感觉神经元外植体的轴突。