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

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，并在啮齿动物宿主中建立持续的终生感染。乳房病毒淋巴细胞性脉络膜脑膜炎病毒（LCMV）由自然界中的普通家鼠携带，并从母鼠垂直传播给幼鼠。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，因为病毒蛋白被幼犬正在发育的免疫系统视为自身抗原。矛盾的是，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，因此不会超越宿主。关于 LCMV 如何限制其传播的一个受欢迎的假设是通过产生有缺陷的干扰 (DI) 颗粒，这些颗粒会干扰标准传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 颗粒足以使该细胞对标准感染性病毒颗粒的后续感染产生抵抗。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繁殖和维持自身的能力。沙粒病毒 DI 颗粒干扰标准病毒传播的机制尚不清楚。对于许多 RNA 病毒来说，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。然而，尽管在 20 世纪 90 年代努力对 LCMV 基因组进行全面测序，但并未观察到此类缺失。相反，检测到 LCMV 基因组末端 3' 和 5' 非翻译区的小缺失。然而，尚不清楚这些基因组是否被包装成 DI 颗粒或是否会干扰标准病毒复制。此外，可能还存在其他候选缺陷基因组。在此应用中，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，并在功能上测试它们是否确实是 DI 粒子介导的干扰的基础。如果成功，拟议的实验将提供第一个 LCMV 基因组综合图谱，并鉴定 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是否负责阻止标准病毒传播或是否有替代机制在起作用，来回答该领域的一个重要问题。这些基本信息对于未来的研究充分定义 DI 颗粒形成和功能的机制是必要的。

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.