MECHANISM OF RNA REPLICATION OF NEGATIVE-STRAND VIRUSES

横纹病毒水泡性口炎病毒(VSV)是一种模型系统 具有非节段性单链病毒群的研究 单链RNA基因组的负链意义。它包括以下内容 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，以及许多经济上感染的病毒 重要的动物。关于这些人的增长的一个中心问题 病毒是通过什么机制控制其表达的 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 将继续我们对相关蛋白质的形式和功能的研究 在新复制的核衣壳的组装中。我们将尝试 阐明N蛋白的可溶性形式之间的关系 细胞和分子的各种RNA结合活性 (基因组长度的RNA的封装，自由前导RNA的封装， 并与mRNAs结合)。其次，我们将进行分子遗传学 调控过程中的顺式作用调控序列分析 转录、复制和核衣壳组装。我们会做的 这是通过产生一个全长的基因组RNA的cdna拷贝 病毒的缺陷性干扰(DI)颗粒，并将该cDNA 在质粒PPMI中的细菌启动子的控制下。在这 通过这种方式，我们将能够产生与DI相同序列的RNA 基因组在体外。我们已经开发了一种系统来将RNA组装成 功能核衣壳，并将使用该系统在 结合对克隆的DI基因组进行定点突变 评估特定RNA序列在核衣壳组装中的作用， 转录的启动和终止，基因内的暂停 病毒粒子聚合酶，并解决与病毒作用机制有关的问题 DI颗粒对标准病毒的干扰。我们还将调查 嵌合DI核衣壳的构建及其作为载体的应用 在哺乳动物细胞中进行基因表达。

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.