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

乳头状病毒是重要的人类病原体，fda批准的疫苗或治疗方法尚不存在。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是完全无症状的，它们在啮齿动物宿主中建立持久的终身感染。母粒病毒淋巴细胞性脉络丛脑膜炎病毒（LCMV）在自然界中由普通家鼠携带，由母体垂直传播给幼崽。幼犬出生时就感染了病毒，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，但它严格控制其传播，因此不会超出宿主。关于LCMV如何限制其传播的一个受欢迎的假设是通过产生缺陷干扰（DI）颗粒，这些颗粒干扰标准感染性病毒颗粒成功完成病毒生命周期的能力。单个DI颗粒进入容许宿主细胞就足以使该细胞抵抗随后的标准感染性病毒颗粒的感染。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颗粒干扰标准病毒繁殖的机制尚不清楚。对于许多RNA病毒，含有orf和/或启动子区域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年代对LCMV基因组进行了全面测序，但没有观察到这种缺失。相反，在LCMV基因组的末端3 ‘和5 ’非翻译区域检测到小的缺失。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，可能存在其他候选缺陷基因组。在这项应用中，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，并对它们是否确实是DI颗粒介导干扰的基础进行功能测试。如果成功，该实验将提供首个LCMV基因组的综合图谱，并鉴定DI颗粒的分子特征。此外，这些研究将通过确定是否有缺陷的基因组实际上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开创性问题。这一基本信息对于进一步研究DI颗粒的形成机制和功能是必要的。

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.