Mechanism for non-templated initiation by a paramyxovirus polymerase

描述(申请人提供)：副粘病毒科病毒家族包含许多重要的人类病原体，包括呼吸道合胞病毒(RSV)、副流感病毒、麻疹、腮腺炎和尼帕病毒。该项目将涉及RSV和副流感病毒3型(PIV-3)的工作。这些病毒共同导致了大多数儿科呼吸道疾病病例，并造成了巨大的经济和精神负担。本项目的重点是了解副粘病毒聚合酶准确启动基因组复制以避免末端核苷酸丢失的机制。副粘病毒有一个由单链负义RNA组成的基因组，它由病毒编码的RNA依赖的RNA聚合酶复制。对RSV的研究表明，复制产物的第一核苷酸是独立于模板选择的。这一发现，结合对副粘病毒启动子序列的检查，提出了一种模型，在该模型中，副粘病毒聚合酶在与模板相互作用之前预先加载了复制产物的前两个核苷酸。这个项目的目的是通过解决三个目标来检验这一假设。(1)将进行反向遗传学研究，以确定RSV复制产物的核苷酸2是否以与模板无关的方式选择，类似于核苷酸1。(2)将进行生化实验，以确定RSV聚合酶是否与核苷酸1和2特异性结合，并在没有模板的情况下产生二核苷酸引物。(3)将在PIV-3上进行实验，以确定副粘病毒科的启动机制是否保守。RSV聚合酶可以独立于模板选择起始核苷三磷酸的发现是一个非常新颖的结果。了解这种情况发生的机制将提供有关RSV复制周期中关键步骤的信息，而本研究中所做的工作将表明这一机制是否在副粘病毒家族中保守。从长远来看，这项工作可以设计出专门针对副粘病毒起始复合体的抑制剂。公共卫生相关性：副粘病毒家族包括一些重要的人类病原体。该项目的目的是了解病毒如何启动复制以产生后代基因组，这是病毒繁殖周期中的关键步骤。了解这一步骤可以提供可用于开发抗病毒药物的信息。

DESCRIPTION (provided by applicant): The virus family Paramyxoviridae contains a number of important human pathogens, including respiratory syncytial virus (RSV), parainfluenza viruses, measles, mumps, and Nipah viruses. This project will involve work on RSV and parainfluenza virus type 3 (PIV-3). Together these viruses are responsible for the majority of cases of pediatric respiratory tract disease and inflict a significant economic and emotional burden. The focus of this project is to understand the mechanism by which the paramyxovirus polymerase accurately initiates genome replication to avoid loss of terminal nucleotides. Paramyxoviruses have a genome consisting of a single strand of negative sense RNA, which is replicated by the virus encoded RNA dependent RNA polymerase. Studies with RSV have shown that the first nucleotide of the replication product is selected independently of the template. This finding, in combination with inspection of paramyxovirus promoter sequences, suggests a model in which the paramyxovirus polymerase becomes preloaded with the first two nucleotides of the replication product prior to its interaction with the template. The aim of this project is to test this hypothesis by addressing three aims. (1) Reverse-genetics studies will be performed to determine if nucleotide 2 of the RSV replication product is selected in a template independent manner, similarly to nucleotide 1. (2) Biochemical experiments will be carried out to determine if the RSV polymerase associates specifically with nucleotides 1 and 2 and generates a dinucleotide primer in the absence of a template. (3) Experiments will be carried out on PIV-3 to determine if initiation mechanisms are conserved across the Paramyxoviridae. The finding that the RSV polymerase can select the initiating nucleoside triphosphate independently of the template is a highly novel result. Understanding the mechanism by which this occurs will provide information regarding a key step in the RSV replication cycle and the work performed in this study will indicate if this mechanism is conserved across the paramyxovirus family. In the long-term, this work could allow design of inhibitors that specifically target the paramyxovirus initiation complex. PUBLIC HEALTH RELEVANCE: The paramyxovirus family of viruses includes a number of important human pathogens. The aim of this project is to understand how the virus initiates replication to produce progeny genomes, a critical step in the virus multiplication cycle. Understanding this step could provide information that could be used to develop antiviral drugs against the viruses.