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L polymerase domains and mRNA posttranscriptional modifications in Mononegavirale

Mononegavirale 中的 L 聚合酶结构域和 mRNA 转录后修饰

基本信息

批准号：
7457099
负责人：
Valery Zurabovich Grdzelishvili
金额：
$ 21.6万
依托单位：
UNIVERSITY OF NORTH CAROLINA CHARLOTTE
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-04-01 至 2012-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7457099
关键词：
Academic Research Enhancement Awards Amino Acid Sequence Animals Biochemical Biology, Other Categories Cell Line Centers for Disease Control and Prevention (U.S.)Complex DNA-Directed RNA Polymerase Data Enzymes Family Frankfurt-Marburg Syndrome Virus Genetic Genetic Transcription Genome Genomics Human In Vitro Individual Knowledge Laboratories Lead Mediating Messenger RNA Methylation Modeling Modification Molecular Mononegavirales Paramyxovirus Pathway interactions Peptide Sequence Determination Phosphoproteins Plant Viruses Polyadenylation Polyadenylation Pathway Polymerase Protein Region Proteins Public Health Publishing RNA RNA Viruses RNA chemical synthesis RNA-Directed RNA Polymerase Rabies Range Reproduction Research Rhabdoviridae Role Sendai virus Structure System Tertiary Protein Structure Therapeutic Agents Vesicular stomatitis Indiana virus Viral Viral Physiology Virus Virus Diseases base comparative insight mRNA capping novel parainfluenza virus pathogen positional cloning protein function respiratory

项目摘要

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.

描述（由申请人提供）：该学术研究增强奖（AREA）R15申请专注于非节段负链（NNS）RNA病毒（分类顺序单负链）中病毒RNA合成和mRNA转录后修饰的分子和生化机制。该目的包括各种各样的人类、动物和植物病毒，其中RNA聚合酶复合物具有独特的特征。这项拟议的研究旨在研究病毒编码的大（L）聚合酶蛋白在病毒RNA合成和mRNA转录后修饰中的作用。该蛋白质具有在所有单负性病毒目中具有高度同源性的六个序列区（“结构域”），其已被假定构成病毒RNA聚合酶的特异性酶活性，所述病毒RNA聚合酶参与转录、mRNA加帽、54帽结构的甲基化、聚腺苷酸化和病毒基因组RNA的复制。尽管它的重要性，L蛋白没有得到很好的表征，并针对确定这些区域的特定功能的研究一直是有限的。具体目标是：1）在弹状病毒和副粘病毒中进行L蛋白帽甲基化功能的比较分析; 2）确定参与帽甲基化的两个L蛋白区域之间的关系; 3）揭示单负病毒目中mRNA帽甲基化介导的宿主限制的机制;以及4）表征在mRNA聚腺苷酸化中重要的L蛋白序列。这些研究将采用两种原型单负病毒目：水泡性口炎病毒（VSV，一种弹状病毒）和仙台病毒（SeV，一种副粘病毒），由于它们在广泛的细胞系中的稳健复制，用于RNA合成研究的成熟体外系统和可用的反向遗传学系统，它们可作为有吸引力的模型。这种双病毒方法将允许在这组病毒中鉴定通用以及病毒特异性L蛋白活性，因为VSV和SeV属于不同的家族。这些研究将为理解病毒RNA合成的分子机制和开发对抗病毒感染的新方法提供新的线索。公共卫生关系：该应用程序的重点是在非节段负链RNA病毒（分类单负链RNA病毒目）的mRNA转录后修饰的分子和生化机制。该目包含许多致命的人类病原体，包括埃博拉病毒和马尔堡病毒，它们被疾病控制和预防中心归类为A类生物武器制剂，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。单负病毒目的L蛋白被认为催化病毒RNA聚合酶的所有酶活性，所述酶活性参与转录、mRNA加帽、54帽结构的甲基化、聚腺苷酸化和病毒基因组RNA的复制。尽管它的重要性，L蛋白没有得到很好的表征，并针对确定这种蛋白质的特定功能的研究已经受到限制。拟议的研究对于确定这些病毒繁殖的基本机制非常重要，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理解，而且还将导致新的或更好的治疗药物。