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

哺乳动物肾病毒是重要的人类病原体，FDA批准的疫苗或治疗不存在。虽然这些病毒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，在那里它们建立了持续的终身感染。乳病毒淋巴细胞性脉络丛脑膜炎病毒（LCMV）是由常见的家鼠自然携带，并从母亲垂直传播给幼崽。幼犬出生时就被感染，但从未产生有效的免疫反应来清除病毒，因为病毒蛋白质被幼犬发育中的免疫系统视为自身抗原。奇怪的是，虽然LCMV可以感染宿主啮齿动物中的大多数细胞，但它严格调节其传播，因此不会超越其宿主。LCMV如何限制其传播的一个有利假设是通过产生缺陷干扰（DI）颗粒，其干扰标准感染性病毒颗粒成功完成病毒生命周期的能力。进入容许宿主细胞的单个DI颗粒足以使该细胞对标准感染性病毒颗粒的后续感染不敏感。因此，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，同时仍保留其在自然界中繁殖和维持自身的能力。沙粒病毒DI颗粒干扰标准病毒繁殖的机制尚不清楚。对于许多RNA病毒，在ORF和/或启动子区域中含有大缺失的缺陷基因组已被证明是干扰的分子基础。然而，尽管在20世纪90年代努力对LCMV基因组进行完全测序，但没有观察到这样的缺失。相反，检测到LCMV基因组末端3'和5'非翻译区的小缺失。然而，目前还不清楚这些基因组是否被包装到DI颗粒中或是否会干扰标准病毒复制。此外，可能存在额外的候选缺陷基因组。在本申请中，我们建议应用下一代测序技术来鉴定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，拟议的实验将提供LCMV基因组的第一个全面图谱，并确定DI颗粒的分子特征。此外，这些研究将回答该领域的一个开创性问题，即确定缺陷基因组实际上是否负责阻止标准病毒传播，或者是否有替代机制在起作用。这一基本信息是必要的，为未来的研究，以充分定义DI颗粒的形成和功能的机制。

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.