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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

基本信息

批准号：
10655344
负责人：
Kristen M Ogden
金额：
$ 50.38万
依托单位：
VANDERBILT UNIVERSITY MEDICAL CENTER
依托单位国家：
美国
项目类别：
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/10655344
关键词：
Animals Antibodies Antiviral Response Bar Codes Biological Assay Cells Collection Complement Cultured Cells Cytoplasmic Inclusion Data Disease Double-Stranded RNA Electron Microscopy Elements Engineering Exclusion Family Fostering Fractionation Genes Genetic Genetic Enhancement Genetic Polymorphism Genetic Variation Genome Genomics Goals Health Human Image 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 Shapes Signal Transduction Symptoms System Testing Therapeutic Third Generation Sequencing Time Travel Vaccines Vesicle Viral Viral Genes Virulence Virus Virus Assembly Virus Replication Western Blotting Work 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

项目摘要

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，它们分类并包装 9 至 12 个正义 RNA 片段，这些片段在病毒组装到细胞质内含物中时转化为基因组双链 RNA。呼肠孤病毒科遗传多样性有助于宿主范围和疫苗反应。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排和作为集体感染单位的多个颗粒的传播。多样性的进化益处被维持介导多部分基因组包装、组装、出口和传播的相互作用的需要所抵消。因此，复制策略也可能本质上限制多样性。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，这是一种遗传上可控制的呼肠孤病毒科病毒，并已建立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很重要，但介导特定片段集合分类的 RNA 元件尚不清楚。在具体目标 1 中，我们将使用长读长和短读长方法对有缺陷的病毒基因片段进行测序，以识别最小的呼肠孤病毒 RNA 包装和分类决定因素。我们将使用反向遗传学和功能测定来确定 RNA 识别元件互换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共感染期间的片段重配，而先天细胞反应可能会通过抑制重复感染来影响重配。在具体目标 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确定感染时间对体外和体内复制和重排的影响。呼肠弧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。这项工作得出的许多原理将广泛适用于在分隔的亚细胞区域中复制、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科的预防和治疗的合理设计，并确定预测自然病毒共感染和传播结果的因素。