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.

依伐地病病毒包括重要的人类和动物病原体，各种和包装九至十二个正义的RNA片段，这些片段在细胞质包含物中的病毒组装过程中转化为基因组双链RNA。依维氏病遗传多样性有助于宿主范围和疫苗反应。为了感染和适应，雷梭式病毒具有进化的机制来促进遗传多样性和补体缺陷的颗粒。这些机制包括将多个颗粒作为集体感染单元的部分进行放置和传播。多样性的进化益处可以通过维持介导多分分组包装，组装，出口和传播的相互作用来抵消。因此，复制策略也可能固有地限制了多样性。拟议的研究的目的是了解紫ovor虫组装和传输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Reovirus，这是一种具有既定的组织培养和小鼠模型的可基因牵引性旋转病毒病毒。 Reoviridae RNA片段的末端对于包装很重要，但是介导特定片段集合的各种分类的RNA元素的定义很差。在特定的目标1中，我们将使用长阅读和短阅读方法来确定最小的异氟此里RNA包装和分类决定因素的有缺陷的病毒基因段。我们将确定RNA识别元素使用反向遗传学和功能测定的互换介导段包装和分类的能力。病毒RNA在细胞质夹杂物中的物理隔离可能会影响共同感染过程中的细分放置，而先天的细胞反应可能会通过抑制近代感染而影响重新分类。在特定的目标2中，我们将使用敏感的RNA成像探针以及感染时间对在体外和体内进行复制和在体内进行复制和重新分配的影响来确定病毒RNA的定位，并使用编码沉默的遗传多态性的病毒。在细胞外囊泡中旋转病毒病毒的传播可能会促进靶细胞同时多粒子感染。在特定的目标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。拟议的研究将洞悉病毒遗传多样性的机制，这些机制是由病毒家族中病毒的组装和传播过程介导的。从这项工作中得出的许多原则将广泛适用于在分隔的亚细胞区域复制的病毒，诱导先天抗病毒反应或在细胞外囊泡中旅行。这些发现将共同促进基于旋旋性病毒的预防剂和治疗剂的合理工程，并鉴定预测天然病毒共感染和传播结果的因素。