Effects of Reoviridae assembly and transmission processes on viral genetic diversity

呼肠孤病毒科病毒，包括重要的人类和动物病原体，在细胞质包涵体中的病毒组装过程中，整理和包装9到12个正义RNA片段，这些片段转化为基因组双链RNA。呼肠孤病毒科的遗传多样性有助于宿主范围和疫苗反应。为了感染和适应，呼肠孤病毒科已经进化出促进遗传多样性和补充缺陷颗粒的机制。这些机制包括片段重组和多个粒子作为集体感染单位的传播。多样性的进化益处被需要维持介导多部分基因组包装、组装、输出和传播的相互作用所抵消。因此，复制策略也可能从本质上限制多样性。拟议研究的目标是了解呼肠孤病毒科组装和传播过程如何调节病毒遗传多样性的独特方面。为了实现这一目标，我们将使用呼肠孤病毒，一种遗传易感的呼肠孤病毒科病毒，建立组织培养和小鼠模型。呼肠孤病毒科RNA片段的末端对包装很重要，但介导特定片段集合分类的RNA元件定义不清。在Specific Aim 1中，我们将使用长读和短读方法对有缺陷的病毒基因片段进行测序，以确定最小的呼肠孤病毒RNA包装和分类决定因素。我们将使用反向遗传学和功能分析来确定RNA识别元件互换介导片段包装和分类的能力。在共感染过程中，细胞质内含体中病毒RNA的物理隔离可能影响片段重组，而先天细胞反应可能通过抑制重复感染来影响重组。在特异性目标2中，我们将使用敏感的RNA成像探针确定病毒RNA在共感染期间的定位，并使用编码沉默遗传多态性的病毒确定感染时间对体外和体内复制和重组的影响。呼肠孤病毒科病毒在细胞外囊泡中的传播可促进靶细胞同时发生多粒子感染。在特异性目标3中，我们将阐明囊泡介导的病毒传播对遗传多样性和毒力的贡献。拟议的研究将深入了解呼肠孤病毒科病毒的组装和传播过程介导的病毒遗传多样性机制。从这项工作中得出的许多原理将广泛适用于在分区亚细胞区域复制、诱导先天抗病毒反应或在细胞外囊泡中传播的病毒。总之，这些发现将促进基于呼肠孤病毒的预防和治疗方法的合理设计，并确定预测自然病毒合并感染和传播结果的因素。

Reoviridae viruses, which include important human and animal pathogens, assort and package nine to twelve positive-sense RNA segments that are converted to genomic double-stranded RNA during virus assembly in cytoplasmic inclusions. Reoviridae genetic diversity contributes to host range and vaccine responses. To infect and adapt, Reoviridae viruses have evolved mechanisms to promote genetic diversity and complement defective particles. These mechanisms include segment reassortment and transmission of multiple particles as collective infectious units. Evolutionary benefits of diversity are countered by a need to maintain interactions mediating multipartite genome packaging, assembly, egress, and transmission. Thus, the replication strategy also may inherently constrain diversity. The goal of the proposed research is to understand how Reoviridae assembly and transmission processes regulate unique aspects of viral genetic diversity. To accomplish this goal, we will use reovirus, a genetically tractable Reoviridae virus with established tissue culture and mouse models. The termini of Reoviridae RNA segments are important for packaging, but RNA elements that mediate assortment of a specific collection of segments are poorly defined. In Specific Aim 1, we will sequence defective viral gene segments using long-read and short-read approaches to identify minimal reovirus RNA packaging and assortment determinants. We will determine the capacity of RNA recognition elements to interchangeably mediate segment packaging and assortment using reverse genetics and functional assays. Physical sequestration of viral RNA in cytoplasmic inclusions may influence segment reassortment during coinfection, and innate cellular responses may influence reassortment by inhibiting superinfection. In Specific Aim 2, we will determine the localization of viral RNA during coinfection using sensitive RNA imaging probes and effects of infection timing on replication and reassortment in vitro and in vivo using viruses encoding silent genetic polymorphisms. Transmission of Reoviridae viruses in extracellular vesicles may promote simultaneous multi-particle infection of target cells. In Specific Aim 3, we will elucidate contributions of vesicle- mediated virus transmission to genetic diversity and virulence. The proposed studies will provide insight into mechanisms of viral genetic diversity that are mediated by the assembly and transmission processes of viruses in the Reoviridae family. Many principles derived from this work will apply broadly to viruses that replicate in compartmentalized subcellular regions, induce innate antiviral responses, or travel in extracellular vesicles. Together, these findings will promote rational engineering of Reoviridae-based preventives and therapeutics and identification of factors that predict outcomes of natural virus coinfection and transmission.