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.

毛甘脉病毒是不存在FDA批准的疫苗或治疗的重要人类病原体。尽管这些病毒在人类中引起严重疾病，但它们在啮齿动物的宿主中完全不对称，在那里它们建立了持久的，终生的感染。 Mammermanavirus淋巴细胞性绒毛膜炎病毒（LCMV）是由自然界中的普通室小鼠携带的，并从母亲到幼犬垂直传播。这些幼崽天生被感染，但永远不会产生有效的免疫响应以清除病毒，因为幼犬的发育中的免疫系统将病毒蛋白视为自我抗原。矛盾的是，虽然LCMV会感染宿主啮齿动物中的大多数细胞，但它会严格调节其扩散，因此不会超越其宿主。 LCMV如何通过产生有缺陷的干扰（DI）颗粒来限制其扩散的偏爱假设，该颗粒干扰了标准感染性病毒颗粒颗粒的能力，即进入允许宿主细胞的单个DI粒子，足以使该细胞使该细胞对后续的标准感染粒子的感染使细胞折磨。这样，产生DI颗粒的病毒可以限制其传播速率以保护其宿主免受感染的负面影响，同时仍保持其在自然界中传播和维持自身的能力。尚不清楚竞技病毒DI颗粒干扰标准病毒传播的机制。对于许多RNA病毒，已证明在ORF和/或启动子区域中包含大量缺失的缺陷基因组被证明是干扰的分子基础。但是，尚不清楚这些基因组是将其包装到DI颗粒中还是会干扰标准病毒复制。此外，可能存在其他候选缺陷的基因组。在此应用中，我们建议应用下一代测序技术以识别LCMV模型中的候选DI基因组，并在功能上测试它们是否确实是DI颗粒介导的干扰的基础。如果成功，则提出的实验将提供LCMV基因组的第一个综合图，并确定DI颗粒的分子特征。此外，这些研究将通过确定有缺陷的基因组实际上负责阻止标准病毒传播或替代机制是否在起作用来回答该领域的第二个问题。将来的研究完全定义了DI颗粒形成和功能的机制，因此这种基本信息是必要的。