Reovirus Neuropathogenesis

呼肠孤病毒神经发病机制

基本信息

批准号：
10607594
负责人：
TERENCE S. DERMODY
金额：
$ 56.55万
依托单位：
UNIVERSITY OF PITTSBURGH AT PITTSBURGH
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-09-22 至 2027-07-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10607594
关键词：
3-Dimensional Adult Afferent Neurons Atomic Force Microscopy Axon Axonal Transport Binding Biophysics Brain Brain imaging CRISPR-mediated transcriptional activation Capsid Capsid Proteins Cells Cerebellum Child Clustered Regularly Interspaced Short Palindromic Repeats Cryoelectron Microscopy Cultured Cells Development Disease Double Stranded RNA Virus Dynein ATPase Electron Microscopy Encephalitis Engineering Fiber Genome Hippocampus (Brain)Human Immunocompetent Immunoglobulins In Vitro Infection Integration Host Factors Integrins Interneurons Intervention Intramuscular Knockout Mice Knowledge Malignant Neoplasms Mammals Mediating Morbidity - disease rate Motor Mus Mutagenesis Nervous system structure Neuraxis Neurons Neuropathogenesis Neurotropism Newborn Infant Oncolytic Pattern Peripheral Pharmacology Population Process Proteins Reovirus Reovirus Infections Research Resolution Role Route Sensory Receptors Series Serotyping Structure Synapses System Testing Thalamic structure Tissues Tropism Viral Viral Encephalitis Viral Pathogenesis Virulence Virus Work blind cell type clinical development experimental study fast axonal transport in vivo mortality mutant nervous system disorder neural circuit neuronal cell body neurotropic neurotropic virus neurovirulence new therapeutic target oncolytic vector particle receptor receptor function reconstruction recruit relating to nervous system reverse genetics screening targeted treatment transmission process uptake virus host interaction

项目摘要

Viral encephalitis is an important cause of morbidity and mortality in children and adults. Key knowledge gaps about the pathogenesis of viral encephalitis include how neurotropic viruses target the central nervous system (CNS), internalize into neurons, and disseminate in the brain. The proposed research uses reovirus, a genetically tractable double-stranded RNA virus that causes encephalitis and shows promise as an oncolytic agent, to dissect mechanisms of viral tropism, cell entry, and spread in the CNS. Reovirus strains display serotype-specific patterns of systemic dissemination and neurologic disease. Serotype 3 (T3) reoviruses are exquisitely neurotropic, internalize into neurons using macropinocytosis, and spread intracellularly by fast axonal transport. Reovirus uses β1 integrin to internalize into some types of cells, but its function in neural cell entry is unknown. Other previously identified reovirus receptors are dispensable for reovirus infection in the murine CNS. We recently identified a new reovirus receptor, paired immunoglobulin-like receptor B (PirB), that allows serotype-independent reovirus infection of cultured cells but is required for efficient T3 infection of neurons and full neurovirulence. Three integrated specific aims are proposed to enhance knowledge of reovirus neuropathogenesis that may have broader applications to other neurotropic viruses. In Specific Aim 1, functions of host receptors in reovirus neuropathogenesis will be determined. Biophysical interactions between PirB and reovirus will be assessed using cryo-electron microscopy, atomic-force microscopy, and structure- guided mutagenesis. The function of PirB in reovirus disease will be elucidated by comparing infection and virulence in wild-type (WT) and PirB-null mice. T3 reovirus-specific receptors that couple with PirB for neurotropism will be identified using CRISPR activation screening. In Specific Aim 2, mechanisms of reovirus entry into CNS neurons will be defined. The functions of PirB and β1 integrin in reovirus neural entry will be determined using mutant receptors and receptor-blind viruses. Receptor-dependent control of macropinocytosis will be elucidated using super-resolution, live-cell, and electron microscopy. Mechanisms of dynein motor recruitment for reovirus transport within axons will be dissected by investigating interactions between WT and mutant reovirus receptors and dynein subunits. In Specific Aim 3, the basis of reovirus dissemination in the CNS will be elucidated. The role of host receptors in reovirus neural transit will be defined using whole-brain imaging and three-dimensional reconstruction to trace reovirus infection and neural spread in WT and receptor- null mice. Mechanisms of reovirus transsynaptic transmission will be elucidated using in vitro and in vivo neural circuits. The effect of altering synaptic activity on reovirus neural transit will be tested using pharmacologic intervention and chemogenetically altered mice. Taken together, these studies will define mechanisms used by neurotropic reovirus to infect and disseminate in the brain and may promote further development of reovirus oncolytics that selectively target the nervous system.

病毒性脑炎是儿童和成人发病和死亡的重要原因关键知识差距。关于病毒性脑炎的发病机制，包括嗜神经病毒如何靶向中枢神经系统（中枢神经系统），内化到神经元中，并在大脑中传播。拟议的研究使用呼肠孤病毒，一种遗传上易于控制的双链 RNA 病毒，可引起脑炎并显示出作为溶瘤药物的前景剂，剖析病毒趋向性、细胞进入和中枢神经系统传播的机制。血清型 3 (T3) 呼肠孤病毒的全身传播和神经系统疾病具有血清型特异性模式。精致的神经营养性，通过巨胞饮作用内化到神经元中，并快速在细胞内传播轴突运输。呼肠孤病毒使用 β1 整合素内化到某些类型的细胞中，但其功能在神经细胞中。其他先前确定的呼肠孤病毒受体对于呼肠孤病毒感染来说是可有可无的。我们最近发现了一种新的呼肠孤病毒受体，配对免疫球蛋白样受体 B (PirB)，允许对培养细胞进行血清型独立的呼肠孤病毒感染，但对于有效的 T3 感染是必需的提出了三个综合的具体目标来增强呼肠孤病毒的知识。神经发病机制可能对其他嗜神经病毒有更广泛的应用。宿主受体在呼肠孤病毒神经发病机制中的功能将被确定。 PirB 和呼肠孤病毒将使用冷冻电子显微镜、原子力显微镜和结构显微镜进行评估 PirB 在呼肠孤病毒疾病中的功能将通过比较感染和诱变来阐明。野生型 (WT) 和 PirB 缺失小鼠中与 PirB 偶联的 T3 呼肠孤病毒特异性受体的毒力。在特定目标 2 中，将使用 CRISPR 激活筛选来鉴定呼肠孤病毒的机制。将定义 PirB 和 β1 整合素在呼肠孤病毒神经进入中的功能。使用突变受体和受体盲病毒的受体依赖性巨胞饮控制来确定。将使用超分辨率、活细胞和电子显微镜来阐明动力蛋白马达的机制。将通过调查 WT 和 WT 之间的相互作用来剖析呼肠孤病毒在轴突内运输的招募在特定目标 3 中，呼肠孤病毒受体和动力蛋白亚基的突变，是呼肠孤病毒在体内传播的基础。将使用全脑来阐明宿主受体在呼肠孤病毒神经传递中的作用。成像和三维重建以追踪 WT 和受体中呼肠孤病毒感染和神经传播将使用体外和体内神经来阐明呼肠孤病毒跨突触传播的机制。将使用药理学测试改变突触活性对呼肠孤病毒神经传递的影响。综合起来，这些研究将定义小鼠所使用的机制。嗜神经性呼肠孤病毒在大脑中感染和传播，并可能促进呼肠孤病毒的进一步发展选择性靶向神经系统的溶瘤药物。