Effects of Reoviridae assembly and transmission processes on viral genetic diversity

呼肠孤病毒科组装和传播过程对病毒遗传多样性的影响

• 批准号: 10296259
• 负责人: Kristen M Ogden
• 金额: $ 51.93万
• 依托单位: VANDERBILT UNIVERSITY MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-01 至 2026-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10296259
• 关键词: Animals; Antibodies; Antiviral Agents; Antiviral Response; Bar Codes; Biological Assay; Cells; Collection; Complement; Cultured Cells; Cytoplasmic Inclusion; Data; Disease; Double-Stranded RNA; Electron Microscopy; Elements; Engineering; Family; Fostering; Fractionation; Genes; Genetic; Genetic Enhancement; Genetic Polymorphism; Genetic Variation; Genome; Genomics; Goals; Health; Human; Image; Immunoblotting; In Vitro; Infection; Interferons; Mediating; Modeling; Mus; Oncolytic; Oncolytic viruses; Parents; Pathogenicity; Pathway interactions; Patients; Population; Predictive Factor; Preventive; Primary Infection; RNA; RNA Sequences; RNA Viruses; Recombinants; Reoviridae; Reovirus; Research; Resolution; Signal Transduction; Symptoms; System; Testing; Therapeutic; Third Generation Sequencing; Time; Travel; Vaccines; Vesicle; Viral Genes; Virulence; Virus; Virus Assembly; Work; base; cancer cell; co-infection; extracellular vesicles; human pathogen; imaging probe; improved; in vivo; insight; melting; mouse model; new technology; next generation; outcome prediction; particle; pathogen; receptor; response; reverse genetics; superinfection; tissue culture; transmission process; vaccine response; viral RNA; viral transmission; virus genetics

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.

呼肠孤病毒科病毒，包括重要的人类和动物病原体，分类和包装9至12个正义RNA片段，其在细胞质内含物中的病毒组装期间转化为基因组双链RNA。呼肠孤病毒科遗传多样性有助于宿主范围和疫苗应答。为了感染和适应，呼肠孤病毒科病毒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维持介导多组分基因组包装、组装、出口和传输的相互作用的需要所抵消。因此，复制策略也可能固有地限制多样性。拟议研究的目标是了解呼肠孤病毒科的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，一种遗传上易处理的呼肠孤病毒科病毒，具有已建立的组织培养和小鼠模型。呼肠孤病毒科RNA片段的末端对于包装是重要的，但是介导片段的特定集合的分类的RNA元件的定义很差。在具体目标1中，我们将使用长读段和短读段方法对缺陷病毒基因片段进行测序，以鉴定最小的呼肠孤病毒RNA包装和分类决定簇。我们将使用反向遗传学和功能测定来确定RNA识别元件可互换地介导片段包装和分类的能力。病毒RNA在细胞质内含物中的物理隔离可能会影响合并感染过程中的片段重配，而先天细胞反应可能会通过抑制重复感染来影响重配。在特定目标2中，我们将使用敏感的RNA成像探针确定病毒RNA在共感染过程中的定位，并使用编码沉默遗传多态性的病毒在体外和体内确定感染时机对复制和重配的影响。呼肠孤病毒科病毒在细胞外囊泡中的传播可能促进靶细胞的同时多颗粒感染。在具体目标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。拟议的研究将提供深入了解由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的机制。从这项工作中得出的许多原理将广泛应用于在亚细胞区域复制的病毒，诱导先天性抗病毒反应，或在细胞外囊泡中旅行。总之，这些发现将促进基于呼肠孤病毒的预防和治疗的合理工程，并确定预测自然病毒合并感染和传播结果的因素。