Functional regulation of an arenavirus polymerase by the viral matrix protein and RNA ligands

沙粒病毒是地球仪上重要的人畜共患病和死亡的原因， 诱发西非的拉沙热、淋巴细胞性脉络丛脑膜炎和许多区域性出血热 南美的疾病这些病毒在没有递送大量的DNA的情况下不能感染它们的宿主细胞。 转录引发的复制和基因表达复合物，由病毒聚合酶（L）直接 与核蛋白标记的反义基因组RNA（vRNA）片段结合。沙粒病毒L 蛋白质在病毒感染周期的几乎每一个步骤中起着重要作用，包括基因表达， 基因组复制和感染性病毒核糖核蛋白的形成。一个精确的机械 由于缺乏可用的生化系统和结构， 这些多功能酶的模型。马丘波病毒（MACV）是一种沙粒病毒，负责局部 玻利维亚出血热的爆发我们的实验室率先使用MACV L作为沙粒病毒的模型 复制研究，开发多个实验系统，用于纯化和功能分析， 全长MACV L.我们实验室以前的工作已经揭示了MACV L蛋白的催化活性是 由于病毒基质蛋白的直接结合而被完全抑制（Z）。因此，L-Z复合体被锁定 在病毒RNA启动子上处于失活状态。此外，最近提出的一个模型表明， L与基因组片段（5' vRNA）的5'末端的相互作用介导RNA合成的增强 活动我提出的核心假设是RNA合成和夺帽结构域 MACV L的末端受到病毒基质蛋白（Z）和末端5'的相反调节影响， 基因组RNA序列。本项目概述的实验将解决我们在以下方面的差距 MACV系统和沙粒病毒生物学的理解：i） 沙粒病毒L RNA合成通过其蛋白质和RNA配体的调节，ii）5'端的生物化学作用， vRNA调节L活性，和iii）MACV L夺帽的催化活性和调节 该项目的发现将为沙粒病毒的未解问题提供新的见解 生物学，并将肯定适用于下游的努力，为发展有针对性的抗病毒药物 治疗策略我们的实验室最近在聚合酶结构领域取得了重大进展， 水泡性口炎病毒（VSV）L蛋白与其复合物的解析原子模型的生物学 磷蛋白辅因子此外，我们的实验室已经开发出唯一可用的体外和基于细胞的系统， 用于研究MACV L的生化功能及其独特的辅因子（Z和5' vRNA）的调节。 这些实验系统使MACV成为L结构-功能研究的理想候选者。总的来说， 这些努力将大大推进我们对沙粒病毒复制和基因表达的理解， 通过小的病毒配体调节这些活性。

Arenaviruses are responsible for significant zoonotic human illnesses and mortality across the globe, inducing Lassa fever in West Africa, lymphocytic choriomeningitis, and numerous regional hemorrhagic fever diseases in South America. These viruses are unable to infect their host cells without the delivery of a large transcriptionally primed replication and gene expression complex, consisting of the viral polymerase (L) directly bound to the nucleoprotein-encapsidated negative-sense genomic RNA (vRNA) segments. Arenavirus L proteins play essential roles in nearly every step of the virus infection cycle, including gene expression, genome replication, and formation of the infectious viral ribonucleoprotein. An accurate mechanistic understanding of L proteins has been hindered by a lack of available biochemical systems and structural models for these multifunctional enzymes. Machupo virus (MACV) is an arenavirus responsible for localized outbreaks of Bolivian hemorrhagic fever. Our lab has pioneered the use of MACV L as a model for arenavirus replication studies, developing multiple experimental systems for the purification and functional analyses of a full-length MACV L. Previous work in our lab has revealed that the catalytic activity of the MACV L protein is completely inhibited due to direct binding by the viral matrix protein (Z). As a result, the L-Z complex is locked in an inactive state on the viral RNA promoter. Moreover, a recently proposed model has suggested that the interaction of L with the 5' termini of the genome segments (5' vRNA) mediates enhanced RNA synthesis activity. The central hypothesis for my proposal is that the RNA synthesis and cap-snatching domains of MACV L are under opposing regulatory influences by the viral matrix protein (Z) and the terminal 5' genomic RNA sequences. The experiments outlined for this project will address the following gaps in our understanding of the MACV system and arenavirus biology: i) the structural mechanisms underlying the regulation of arenavirus L RNA synthesis by its protein and RNA ligands, ii) the biochemical role of the 5' vRNA in modulating L activity, and iii) the catalytic activity and regulation of the MACV L cap-snatching machinery Findings from this project will offer novel insight into the unanswered questions of arenavirus biology, and will certainly be applicable in downstream endeavors for the development of targeted antiviral therapeutic strategies. Our lab has recently made significant progress in the field of polymerase structural biology with the resolved atomic model of the vesicular stomatitis virus (VSV) L protein in complex with its phosphoprotein cofactor. Moreover, our lab has developed the only available in vitro and cell-based systems for the study of MACV L biochemical functions and regulation by its unique cofactors (Z and the 5' vRNA). These experimental systems make MACV the ideal candidate for L structure-function studies. Collectively, these efforts will significantly advance our understanding of arenavirus replication and gene expression, and the regulation of these activities via small viral ligands.