The effect of extracellular vesicle-mediated transmission on reovirus infection

细胞外囊泡介导的传播对呼肠孤病毒感染的影响

• 批准号: 10387888
• 负责人: Sydni Caet Smith
• 金额: $ 3.2万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10387888
• 关键词: Affect; Animal Model; Animals; Antibodies; Bar Codes; Capsid; Cell Culture Techniques; Cell membrane; Cells; Complement; Containment; Cultured Cells; Cytolysis; Data; Disease; Double Stranded RNA Virus; Electron Microscopy; Engineering; Family; Frequencies; Future; Genetic; Genetic Diseases; Genetic Engineering; Genome; Genotype; Goals; Human; Immune Evasion; Immune response; Infection; Kinetics; Knowledge; Lytic; Mammalian Orthoreovirus; Mediating; Medical; Membrane; Neutralizing antibody assay; Nonlytic; Oncolytic; Organism; Parents; Pathway interactions; Phenotype; Plaque Assay; Play; Population; Preparation; Property; Reoviridae; Reovirus; Reovirus Infections; Reporting; Research; Resolution; Role; Rotavirus; Site; Specific qualifier value; Structure; Testing; Viral; Viral Structural Proteins; Virion; Virulence; Virus; Virus Replication; Western Blotting; Work; cell type; extracellular vesicles; in vivo; melting; member; neutralizing antibody; novel; particle; receptor; targeted delivery; tool; transmission process; tumor; vesicular release; viral transmission

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.

项目摘要 无包膜病毒通过裂解离开细胞的传统观点受到挑战，最近的研究表明， 许多病毒家族利用细胞外囊泡（EV）进行非裂解性外出。EV介导的传播可以 使病毒免疫逃避和集体粒子传播，以潜在地增强生产性感染。的 哺乳动物正呼肠孤病毒（呼肠孤病毒）的外出机制，呼肠孤病毒科病毒家族的一个成员， 在人类和动物宿主广泛范围内的重大疾病，仍然在很大程度上研究不足。轮状病毒，另一种 呼肠孤病毒科的成员，最近报道在大型EV中从宿主细胞排出。电动汽车防护 增强轮状病毒的体内毒力。我的研究目标是了解呼肠孤病毒的机制 出口，并阐明呼肠孤病毒的传播方式如何影响呼肠孤病毒感染。使用不同类型 培养细胞和基因条形码呼肠孤病毒，我的初步工作表明i）呼肠孤病毒颗粒可以 ii）EV介导的呼肠孤病毒排出是病毒株和细胞类型依赖性的，iii）EV遏制 保护呼肠孤病毒颗粒免受抗体介导的中和，和iv）EV介导的传播增加 与游离呼肠孤病毒相比，多颗粒感染的频率。我假设EV介导的呼肠孤病毒的排出 依赖于病毒与宿主细胞的相互作用，促进多颗粒感染，并增强复制 动力学为了验证这一假设，我提出了两个具体目标。在具体目标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 群体，并确定呼肠孤病毒决定因素的细胞类型依赖性EV介导的出口。在具体目标2中， 将使用基因条形码呼肠孤病毒，空斑试验和RT-qPCR来鉴定细胞外 囊泡介导的传播对呼肠孤病毒进入、多颗粒感染和复制动力学的影响。这些发现 所提出的目标阐明的可能揭示宿主细胞辅助的非裂解性呼肠孤病毒的新机制， 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 将阐明传播模式对呼肠孤病毒毒力和宿主生物体内传播的影响。