ENTEROVIRUS RNA REPLICATION

肠道病毒 RNA 复制

基本信息

批准号：
2694928
负责人：
DAVID J BARTON
金额：
$ 19.79万
依托单位：
UNIVERSITY OF COLORADO DENVER
依托单位国家：
美国
项目类别：
财政年份：
1998
资助国家：
美国
起止时间：
1998-07-01 至 2003-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/2694928
关键词：
RNA binding protein RNA biosynthesis cell free system gene mutation genetic regulatory element nucleic acid sequence poliovirus recombinant DNA virus RNA virus genetics

项目摘要

DESCRIPTION: Poliovirus is the prototypic member of the Picornavirus family of positive-strand RNA viruses, a large group of related viruses that cause a diverse spectrum of human illnesses. Contrary to previous dogma, poliovirus RNA templates for negative-strand RNA synthesis require cis-active RNA elements from both the 5'- and 3'-terminal regions of the viral genome. The applicant hypothesizes that the 5'- and 3'-terminal cis-active RNA elements of poliovirus coordinately regulate viral RNA stability, RNA translation, and negative-strand RNA synthesis via temporally dynamic ribonucleoproteins. The specific aims are to: 1) Determine if cellular poly(C) binding protein bound to the 5'-terminus of poliovirus RNA mediates poliovirus RNA stability; 2) Determine how the 5'-terminal ribonucleoprotein of poliovirus participates in negative-strand RNA synthesis, a biosynthetic event initiated at the 3'-terminus of poliovirus RNA; 3) Determine why the 5'-terminal internal ribosome entry site and/or the initiation of translation is required to allow viral RNA molecules to serve as templates for negative-strand RNA synthesis; 4) Identify the RNA sequences, structures, and ribonucleoprotein complexes within the 3Dpol coding region that are required for negative-strand RNA synthesis. Technical advantages of cell-free virus replication reactions will be exploited to assay poliovirus RNA stability, RNA translation, and negative-strand RNA synthesis. Recombinant DNA technology will be used to create viral RNAs with specific mutations for structural and functional analyses. Temporally dynamic ribonucleoproteins composed of cellular proteins, viral protein, and cis-active poliovirus RNA elements will be functionally characterized to determine which step(s) of poliovirus replication they mediate and/or regulate. These studies will characterize the dynamic transformation of poliovirus mRNA into a template for negative-strand RNA synthesis, a fundamental event required for the replication of all positive-strand RNA viruses. This wok may help establish a new paradigm for RNA replication in which both 5'- and 3'-terminal cis-active RNA elements of genomic RNA coordinately regulate numerous steps of viral replication.

描述：脊髓灰质炎病毒是Picornavirus家族的原型成员阳性RNA病毒，这是一大批引起的相关病毒各种各样的人类疾病。与以前的教条相反负链RNA合成的脊髓灰质炎病毒RNA模板需要来自5'-和3末端区域的顺式主动RNA元素病毒基因组。申请人假设5'-和3末端脊髓灰质炎病毒的顺式活性RNA元素协同调节病毒RNA 稳定性，RNA翻译和负链RNA通过时间合成动态核糖核蛋白。具体目的是：1）确定是否细胞聚（C）结合蛋白与脊髓灰质炎病毒RNA的5'-末端结合介导脊髓灰质炎病毒RNA稳定性； 2）确定5'末端脊髓灰质炎病毒的核糖核蛋白参与负链RNA 合成，这是在脊髓灰质炎病毒3'-末端引发的生物合成事件 RNA； 3）确定为什么5末末端内部核糖体入口站点和/或需要翻译的启动以使病毒RNA分子达到用作负链RNA合成的模板； 4）识别RNA 3DPol中的序列，结构和核糖核蛋白复合物负链RNA合成所需的编码区域。无细胞病毒复制反应的技术优势将是利用用于测定脊髓灰质炎病毒RNA稳定性，RNA翻译和负链RNA合成。重组DNA技术将用于创建具有特定突变的病毒RNA，用于结构和功能分析。由细胞组成的时间动态核糖核蛋白蛋白质，病毒蛋白和顺式激活poliovirus RNA元素将是在功能上表征以确定脊髓灰质炎病毒的哪个步骤它们中介和/或调节的复制。这些研究将表征脊髓灰质炎病毒mRNA到模板的动态转化负链RNA合成，这是一个基本事件所有阳性RNA病毒的复制。这个锅可能有助于建立 RNA复制的新范式，其中5'-和3末端都基因组RNA的顺式活性RNA元素协调了许多步骤病毒复制。