Reovirus Neuropathogenesis

病毒性脑炎是儿童和成人发病和死亡的重要原因。主要知识差距 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 (CNS)，内化到神经元中，并在脑中扩散。这项研究使用了呼肠孤病毒， 一种遗传上易处理的双链RNA病毒，可引起脑炎，并显示出作为溶瘤剂的前景 代理，剖析机制的病毒嗜性，细胞进入，并蔓延在中枢神经系统。呼肠孤病毒株展示 系统性播散和神经系统疾病的特定类型模式。血清型3（T3）呼肠孤病毒是 精细亲神经性，使用巨胞饮作用内化到神经元中，并通过快速细胞内扩散 轴突运输呼肠孤病毒利用β1整合素内化到某些类型的细胞中，但其在神经细胞中的功能 入口未知。其他先前鉴定的呼肠孤病毒受体在哺乳动物中不适合呼肠孤病毒感染。 鼠CNS。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（Pir B）， 允许培养细胞的非依赖于病毒型的呼肠孤病毒感染，但对于有效的T3感染 神经元和完整的神经毒性。提出了三个综合的具体目标，以提高对呼肠孤病毒的认识 神经致病性，可能有更广泛的应用到其他嗜神经病毒。具体目标1、 将确定宿主受体在呼肠孤病毒神经发病机制中的功能。生物物理相互作用 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-生物显微镜进行评估。 引导突变。PirB在呼肠孤病毒病中的功能将通过比较感染和 在野生型（WT）和PirB缺失小鼠中的毒力。T3呼肠孤病毒特异性受体与PirB偶联， 将使用CRISPR活化筛选来鉴定嗜神经性。在具体目标2中，呼肠孤病毒的机制 将定义进入CNS神经元。PirB和β1整联蛋白在呼肠孤病毒神经进入中的功能将被进一步研究。 使用突变受体和受体盲病毒确定。巨胞饮作用的受体依赖性调控 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 将通过研究WT和WT之间的相互作用来解剖轴突内呼肠孤病毒运输的募集。 突变呼肠孤病毒受体和动力蛋白亚基。在特定目标3中，呼肠孤病毒在非洲传播的基础 将阐明CNS。宿主受体在呼肠孤病毒神经传递中的作用将使用全脑 成像和三维重建来追踪WT和受体中的呼肠孤病毒感染和神经扩散， 无效小鼠。呼肠孤病毒跨突触传递的机制将通过体外和体内神经细胞实验来阐明。 电路.改变突触活性对呼肠孤病毒神经传递的影响将使用药理学方法进行测试。 干预和化学遗传学改变的小鼠。总之，这些研究将确定使用的机制， 嗜神经性呼肠孤病毒在脑中感染和传播，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。

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.