Deep sequencing the lymphocytic choriomeningitis arenavirus quasispecies to identify and functionally validate the molecular signature ofdefective interfering particles

对淋巴细胞脉络膜脑膜炎沙粒病毒准种进行深度测序，以识别和功能验证缺陷干扰颗粒的分子特征

• 批准号: 10043049
• 负责人: Jason W. Botten
• 金额: $ 24.88万
• 依托单位: UNIVERSITY OF VERMONT & ST AGRIC COLLEGE
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-06-01 至 2022-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10043049
• 关键词: 5' Untranslated Regions; Animals; Area; Arenavirus; Autoantigens; Biochemical; Bioinformatics; Cells; Complex; Defective Viruses; Disease; Environmental Risk Factor; FDA approved; Family; Future; Genome; Goals; Growth; House mice; Human; Immune response; Immune system; In Vitro; Infection; Life; Life Cycle Stages; Lymphocytic Choriomeningitis; Lymphocytic choriomeningitis virus; Maintenance; Maps; Mediating; Modeling; Molecular; Molecular Profiling; Mothers; Nature; Open Reading Frames; Pathogenesis; Pathway interactions; Positioning Attribute; Preparation; Production; Promoter Regions; RNA Viruses; Reagent; Recombinants; Refractory; Regulation; Rodent; Role; Seminal; Silent Mutation; Sorting - Cell Movement; Technology; Testing; Time; Tyrosine Phosphorylation; Untranslated Regions; Vaccines; Viral; Viral Genome; Viral Matrix Proteins; Viral Proteins; Virion; Virus; Virus Diseases; Virus Replication; Work; analysis pipeline; deep sequencing; density; experimental study; fitness; genetic signature; human pathogen; mutant; nanopore; next generation sequencing; novel; particle; preservation; pup; response; reverse genetics; ubiquitin-protein ligase

Mammarenaviruses are significant human pathogens for which FDA-approved vaccines or treatments do not exist. While these viruses cause severe disease in humans, they are completely asymptomatic in their rodent hosts, where they establish a persistent, life-long infection. The mammarenavirus lymphocytic choriomeningitis virus (LCMV) is carried by the common house mouse in nature and is transmitted vertically from mother to pup. The pups are born infected but never mount an effective immune response to clear the virus as viral proteins are seen as self-antigens by the pup’s developing immune system. Paradoxically, while LCMV can infect most cells in the host rodent, it tightly regulates its spread and therefore does not overrun its host. A favored hypothesis for how LCMV restricts its spread is through the production of defective interfering (DI) particles, which interfere with the ability of standard infectious virus particles to successfully complete the viral life cycle. A single DI particle entering a permissive host cell is sufficient to render that cell refractory to subsequent infection by a standard infectious virus particle. Thus, a virus that produces DI particles can limit its rate of spread to shield its host from the negative effects of infection while still retaining its ability to propagate and maintain itself in nature. The mechanism by which arenavirus DI particles interfere with standard virus propagation is unknown. For many RNA viruses, defective genomes containing large deletions in ORFs and/or promotor regions have been shown to be the molecular basis for interference. However, despite efforts to fully sequence the LCMV genome in the 1990s, no such deletions were observed. Instead, small deletions in the terminal 3’ and 5’ untranslated regions of the LCMV genome were detected. However, it is unknown whether these genomes are packaged into DI particles or can interfere with standard virus replication. Further, additional candidate defective genomes likely exist. In this application, we propose to apply next-generation sequencing technologies to identify candidate DI genomes in the LCMV model and functionally test whether they are indeed the basis for DI particle-mediated interference. If successful, the proposed experiments will provide the first comprehensive map of LCMV genomes and identify the molecular signature of DI particles. Further, these studies will answer a seminal question in the field by determining whether defective genomes are in fact responsible for blocking standard virus propagation or whether an alternative mechanism is at work. This fundamental information is necessary for future studies to fully define the mechanisms of DI particle formation and function.

乳房病毒是重要的人类病原体，目前尚无 FDA 批准的疫苗或治疗方法，虽然这些病毒会在人类中引起严重疾病，但它们在啮齿动物宿主中完全无症状，并会造成持续的终生感染。病毒（LCMV）由自然界中的普通家鼠携带，并从母亲垂直传播给幼崽，幼崽出生时就受到感染，但从未像以前那样产生有效的免疫反应来清除病毒。矛盾的是，幼崽正在发育的免疫系统将病毒蛋白视为自身抗原，虽然 LCMV 可以感染宿主啮齿动物的大多数细胞，但它严格调节其传播，因此不会超出宿主的范围。是通过产生有缺陷的干扰 (DI) 颗粒来干扰标准感染性病毒颗粒成功完成病毒生命周期的能力，进入允许的宿主细胞的单个 DI 颗粒足以使该细胞死亡。因此，产生 DI 颗粒的病毒可以限制其传播速度，以保护其宿主免受感染的负面影响，同时仍保留其在自然界中传播和维持自身的能力。沙粒病毒 DI 颗粒通过何种方式干扰标准病毒繁殖尚不清楚，然而，尽管努力对这些病毒进行完全测序，但 ORF 和/或启动子区域中含有大量缺失的缺陷基因组已被证明是干扰的分子基础。在 20 世纪 90 年代的 LCMV 基因组中，没有观察到此类缺失，而是在 LCMV 基因组的末端 3' 和 5' 非翻译区域检测到小缺失，但尚不清楚这些基因组是否被包装到 DI 颗粒中或会干扰。此外，在本申请中，可能存在其他候选缺陷基因组，我们建议应用下一代测序技术来识别 LCMV 模型中的候选 DI 基因组，并对其是否确实存在进行功能测试。如果成功，拟议的实验将提供第一个 LCMV 基因组图谱并鉴定 DI 粒子的分子特征。此外，这些研究将通过确定缺陷基因组是否存在来回答该领域的一个重要问题。事实上负责阻止标准病毒传播或替代机制是否在起作用。这一基本信息对于未来的研究完全定义 DI 颗粒形成和功能的机制是必要的。