MECHANISM OF RNA REPLICATION OF NEGATIVE-STRAND VIRUSES

负链病毒RNA复制机制

• 批准号: 3132825
• 负责人: RICHARD W PELUSO
• 金额: $ 16.25万
• 依托单位: MOUNT SINAI SCHOOL OF MEDICINE OF CUNY
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-06-01 至 1994-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3132825
• 关键词: RNA biosynthesis; Vesiculovirus; defective virus; electrofocusing; gel electrophoresis; gene expression; genetic transcription; interfering virus; messenger RNA; molecular cloning; nucleic acid sequence; nucleocapsid; plasmids; point mutation; site directed mutagenesis; tissue /cell culture; transcription factor; virus envelope; virus genetics; virus protein; virus replication

The rhabdovirus vesicular stomatitis virus (VSV) is a model system for the study of the group of viruses possessing non-segmented single- stranded RNA genomes of the negative strand sense. It includes such human pathogens as measles, mumps, rabies, respiratory syncytial virus, human parainfluenza viruses, and many viruses that infect economically important animals. A central question concerning the growth of these viruses is: by what mechanism do they control the expression of their genetic information through transcription and replication of the genome. The goals of the research in this application are two fold. First, we will continue our study of the form and function of the proteins involved in the assembly of newly replicating nucleocapsids. We will attempt to clarify the relationship between the soluble forms of the N protein in the cell and the various RNA-binding activities of the molecule (encapsidation of genome-length RNA, encapsidation of free leader RNA, and binding to mRNAs). Secondly, we will perform a molecular genetic analysis of cis-acting regulatory sequences in controlling the processes of transcription, replication, and nucleocapsid assembly. We will do this by producing a full-length cDNA copy of the genome RNA of a defective-interfering (DI) particle of the virus, and placing this cDNA under the control of a bacterial promoter in the plasmid pPMI. In this way, we will be able to produce RNA of identical sequence to the DI genome in vitro. We have developed a system to assemble the RNA into functional nucleocapsids in vitro, and will use this system in conjunction with site-directed mutagenesis of the cloned DI genome to assess the role of specific RNA sequences in nucleocapsid assembly, initiation and termination of transcription, intragenic pausing of the virion polymerase, and to address questions relating to the mechanism of interference of standard virus by DI particles. We will also investigate the possible construction and use of chimeric DI nucleocapsids as vectors for gene expression in mammalian cells.

弹状病毒水泡性口炎病毒（VSV）是一个模型系统 对具有非节段单链的病毒群的研究 负链正义链RNA基因组。 其中包括这样的 人类病原体如麻疹、腮腺炎、狂犬病、呼吸道合胞病毒、 人类副流感病毒以及许多感染经济的病毒 重要的动物。 关于这些人的成长的一个核心问题 病毒是：它们通过什么机制控制其表达 通过基因组的转录和复制获得遗传信息。 本申请的研究目标有两个。 首先，我们 我们将继续研究相关蛋白质的形式和功能 在新复制的核衣壳的组装中。 我们将尝试 阐明 N 蛋白可溶形式之间的关系 细胞和分子的各种 RNA 结合活性 （基因组长度 RNA 的衣壳化、游离前导 RNA 的衣壳化、 并与 mRNA 结合）。 其次，我们将进行分子遗传学 控制过程中顺式作用调控序列的分析 转录、复制和核衣壳组装。 我们会做 这是通过产生基因组 RNA 的全长 cDNA 副本 病毒的缺陷干扰（DI）颗粒，并将该 cDNA 置于 在质粒 pPMI 中的细菌启动子的控制下。 在这个 这样，我们就能够产生与 DI 序列相同的 RNA 体外基因组。 我们开发了一种将 RNA 组装成的系统 体外功能性核衣壳，并将在 与克隆的 DI 基因组的定点诱变相结合 评估特定 RNA 序列在核衣壳组装中的作用， 转录的起始和终止、基因内暂停 病毒体聚合酶，并解决与病毒体聚合酶机制相关的问题 DI粒子对标准病毒的干扰。 我们也会调查 嵌合 DI 核衣壳作为载体的可能构建和使用 用于哺乳动物细胞中的基因表达。