L polymerase domains and mRNA posttranscriptional modifications in Mononegavirale

描述(由申请人提供)：这项学术研究增强奖(AREA)R15的申请集中在病毒RNA合成和非片段负链(NNS)RNA病毒(分类目Mononegavirales)的RNA转录后修饰的分子和生化机制上。该目包括各种各样的人类、动物和植物病毒，其中RNA聚合酶复合体具有独特的特征。这项拟议的研究旨在研究病毒编码的大分子(L)聚合酶蛋白在病毒核糖核酸合成和mRNA转录后修饰中的作用。该蛋白有6个序列区域(“结构域”)，在所有单核病毒科中具有高度同源性，它们被认为构成了病毒RNA聚合酶的特异性酶活性，参与了病毒基因组RNA的转录、mRNA封端、54个帽结构的甲基化、多聚腺苷化和复制。尽管L蛋白很重要，但它的特性还不是很好，针对这些区域特定功能的研究也受到了限制。其具体目的是：1)比较分析L蛋白帽甲基化在横纹肌病毒和副粘病毒中的作用；2)确定参与帽甲基化的两个L蛋白区域之间的关系；3)揭示帽甲基化介导的宿主限制机制；以及4)确定在mR NA多腺化过程中起重要作用的L蛋白序列。这项研究将采用两种典型的单粒病毒：水疱性口炎病毒(VSV，一种横纹病毒)和仙台病毒(SeV，一种副粘病毒)，这两种病毒因其在广泛的细胞系中的强劲复制、成熟的RNA合成研究的体外系统以及可用的反向遗传学系统而成为有吸引力的模型。这种双病毒方法将允许在这组病毒中鉴定普遍的和病毒特异性的L蛋白活性，因为病毒和轮状病毒属于不同的家族。这些研究应该为理解病毒RNA合成的分子机制和开发新的抗击病毒感染的方法提供新的线索。公共卫生相关性：这一应用侧重于非节段性负链RNA病毒(分类目单病毒目)中mRNA转录后修饰的分子和生化机制。这一顺序包含许多致命的人类病原体，包括被疾病控制和预防中心列为A类生物武器制剂的埃博拉和马尔堡病毒，以及高度流行的人类病原体，如呼吸道合胞病毒和副流感病毒。病毒L蛋白催化病毒核糖核酸聚合酶的所有酶活性，包括转录、核糖核酸封端、54个帽结构的甲基化、多聚腺苷化和病毒基因组核糖核酸的复制。尽管L蛋白很重要，但它的特性还不是很好，针对该蛋白特定功能的研究也受到了限制。这些研究对确定这些病毒复制的基本机制很重要，最终希望这些知识不仅有助于对RNA大分子生物合成途径的基本了解，而且还将导致新的或更好的治疗药物。

DESCRIPTION (provided by applicant): This Academic Research Enhancement Award (AREA) R15 application is focused on the molecular and biochemical mechanisms of viral RNA synthesis and mRNA posttranscriptional modifications in nonsegmented negative-strand (NNS) RNA viruses (taxonomic order Mononegavirales). This order includes a wide variety of human, animal and plant viruses, in which the RNA polymerase complex possesses unique features. The proposed research aims to examine the role of the virus-encoded large (L) polymerase protein in viral RNA synthesis and mRNA posttranscriptional modifications. This protein has six sequence regions ("domains") with a high degree of homology among all Mononegavirales, which have been postulated to constitute specific enzymatic activities of the viral RNA polymerase involved in transcription, mRNA capping, methylation of 54 cap structures, polyadenylation, and replication of viral genomic RNA. Despite its importance, the L protein is not well characterized, and studies directed at identifying specific functions of these regions have been limited. The specific aims are: 1) Performing a comparative analysis of the L protein cap methylation function in rhabdo- and paramyxoviruses; 2) Defining the relationship between the two L protein regions involved in cap methylation; 3) Revealing the mechanism of mRNA cap methylation- mediated host restriction in Mononegavirales; and 4) Characterizing the L protein sequences important in mRNA polyadenylation. The studies will employ two prototypic Mononegavirales: vesicular stomatitis virus (VSV, a rhabdovirus) and Sendai virus (SeV, a paramyxovirus), which serve as attractive models due to their robust replication in a wide range of cell lines, well-established in vitro systems for the study of RNA synthesis, and available reverse genetics systems. This two-virus approach will allow for the identification of universal as well as virus-specific L protein activities within this group of viruses, because VSV and SeV belong to different families. These studies should provide new clues for understanding the molecular mechanisms of viral RNA synthesis and for developing new ways to combat viral infections. PUBLIC HEALTH RELEVANCE: This application is focused on the molecular and biochemical mechanisms of mRNA posttranscriptional modifications in nonsegmented negative-strand RNA viruses (taxonomic order Mononegavirales). This order contains many lethal human pathogens, including the Ebola and Marburg viruses which are classified as category A bioweapon agents by the Centers for Disease Control and Prevention, and highly prevalent human pathogens, such as the respiratory syncytial and parainfluenza viruses. The L protein of Mononegavirales has been postulated to catalyze all enzymatic activities of the viral RNA polymerase involved in transcription, mRNA capping, methylation of 54 cap structures, polyadenylation, and replication of viral genomic RNA. Despite its importance, the L protein is not well characterized, and studies directed at identifying specific functions of this protein have been limited. The proposed studies are important to define the basic mechanisms of reproduction of these viruses, with the ultimate hope that this knowledge will contribute not only to the basic understanding of RNA macromolecular biosynthetic pathways, but will also lead to new or better therapeutic agents.