The effect of extracellular vesicle-mediated transmission on reovirus infection

项目总结 无包膜病毒通过裂解退出细胞的传统观念正受到挑战，最近的研究表明 许多病毒家族利用细胞外小泡(EVS)进行非裂解排泄。EV介导的传播可以 使病毒免疫逃避和集体颗粒传播潜在地增强生产性感染。这个 哺乳动物呼肠孤病毒(呼肠孤病毒)的出口机制，呼肠孤病毒是呼肠孤病毒科病毒家族的成员，可引起 在广泛的人类和动物宿主中的重大疾病，在很大程度上仍然没有得到充分的研究。轮状病毒，另一种 呼肠孤病毒科的成员，最近被报道在大型EV中从宿主细胞中排出。电动汽车围堵 体内轮状病毒毒力增强。我提出的研究的目的是了解呼肠孤病毒的机制。 并阐明呼肠孤病毒的传播方式如何影响呼肠孤病毒感染。使用不同的类型 在培养细胞和基因条形码呼肠孤病毒中，我的初步工作表明：i)呼肠孤病毒颗粒可以 大EV中的出口，ii)EV介导的呼肠孤病毒出口取决于病毒株和细胞类型，III)EV遏制 保护呼肠孤病毒颗粒免受抗体介导的中和，以及iv)EV介导的传播增加 与游离呼肠孤病毒相比，多粒子感染的频率。我推测EV介导的呼肠孤病毒出口 依赖于病毒与宿主细胞的相互作用，促进多粒子感染，并增强复制 运动学。为了检验这一假设，我提出了两个具体目标。在特定的目标1中，我将使用免疫印迹， 电子显微镜和基因工程呼肠孤病毒以确定呼肠孤病毒相关EV的特性 并确定依赖细胞类型的EV介导的出口的呼肠孤病毒决定因素。在具体目标2中，我 将使用基因条形码呼肠孤病毒、空斑分析和RT-qPCR来确定细胞外的影响 囊泡介导的呼肠孤病毒进入、多粒子感染和复制动力学的传播。调查结果 被提议的AIMS阐明将可能揭示宿主细胞辅助的非裂解性呼肠孤病毒的新机制。 出口，这可能适用于呼肠孤病毒科的其他病毒。在这些发现的基础上继续进行研究 将阐明传播方式对呼肠孤病毒毒力和在宿主体内传播的影响。

PROJECT SUMMARY The traditional idea that non-enveloped viruses exit cells via lysis is being challenged with recent studies showing that many virus families utilize extracellular vesicles (EVs) for non-lytic egress. EV-mediated transmission can enable viral immune evasion and collective particle transmission to potentially enhance productive infection. The egress mechanisms of mammalian orthoreovirus (reovirus), a member of the Reoviridae virus family that causes significant disease in a broad range of human and animal hosts, remain largely understudied. Rotavirus, another member of the Reoviridae family, was recently reported to egress from host cells in large EVs. EV containment enhanced rotavirus virulence in vivo. The goal of my proposed research is to understand mechanisms of reovirus egress and to elucidate how the mode of reovirus transmission affects reovirus infection. Using different types of cultured cells and genetically barcoded reovirus, my preliminary work indicates that i) reovirus particles can egress in large EVs, ii) EV-mediated reovirus egress is virus strain- and cell type-dependent, iii) EV containment protects reovirus particles from antibody-mediated neutralization, and iv) EV-mediated transmission increases the frequency of multiparticle infection compared to free reovirus. I hypothesize that EV-mediated reovirus egress is dependent on viral interaction with the host cell, promotes multiparticle infection, and enhances replication kinetics. To test this hypothesis, I propose two specific aims. In Specific Aim 1, I will use immunoblotting, electron microscopy, and genetically engineered reovirus to define the properties of reovirus-associated EV populations and identify reovirus determinants of cell type-dependent EV-mediated egress. In Specific Aim 2, I will use genetically barcoded reovirus, plaque assays, and RT-qPCR to identify the effects of extracellular vesicle-mediated transmission on reovirus entry, multiparticle infection, and replication kinetics. The findings elucidated by the proposed aims will likely reveal a novel mechanism of host cell-assisted, nonlytic reovirus egress, which may apply to other viruses of the Reoviridae family. Continued studies building on these findings will illuminate the impact of transmission mode on reovirus virulence and dissemination within a host organism.