CONTROL OF PARAMYXOVIRUS RNA SYNTHESIS

副粘病毒是急性呼吸道感染中最常见的微生物之一， 感染，这些无处不在的病原体是造成 世界范围内发病率和死亡率都很高。 这些RNA病毒 已经进化出了一种机制， 在感染过程中合成的一种病毒RNA。顺式作用序列 副粘病毒基因组中的一个主要控制点， 调节RNA合成。 这项拟议中的工作将测试几个新的 关于病毒核衣壳特征的概念 调控基因组复制和基因表达的模板， 原型副粘病毒SV 5。 第一个目标是确定角色 在病毒RNA复制的三个因素：（一）序列内的两个 必要的和不连续的启动子元件，（ii）这些元件的间隔 元件沿着一个面的螺旋和（iii）六聚体相， 启动子区 基于模型RNA的反向遗传学系统 微基因组类似物将与位点特异性诱变沿着使用， 一种体内选择方法，以确定 这些因素中的每一个都影响启动子活性。 嵌合RNA基因组 包含基因组和反基因组启动子之间的交换将 与聚合酶结合试验一起沿着使用，以确定 从这两个启动子的病毒RNA的差异合成。 对于一些 副粘病毒，有一个非常高的转录水平， 在病毒M和F基因之间的连接处的通读。 在 第二个目标，转录能力模型双顺反子RNA基因组将 用于确定转录通读的分子基础 在M-F交界处 在第三个目的中，重组病毒含有 改变的M-F基因间区将从全长cDNA中分离。 这些突变病毒将被用来测试两个假设， 在感染过程中，M-F通读转录 （i）下调有融合能力的F蛋白的水平或（ii） 增加可以进入5'端的聚合酶分子的数量 病毒基因组。 这些实验将测试新的概念， 副粘病毒核衣壳相关RNA模板可以调节 多功能病毒聚合酶的活性。 结果 拟议的工作将形成一个合理的反向遗传学的关键基础， 副流感疫苗的方法，因为它可能会设计 理想的减毒重组病毒具有改变的启动子或 基因间转录活性。

Paramyxoviruses are among the most common organisms in acute respiratory tract infections, and these ubiquitous pathogens are responsible for a high degree of morbidity and mortality worldwide. These RNA viruses have evolved mechanisms that control both the level and the particular type of viral RNA synthesized during an infection. cis-acting sequences contained within the paramyxovirus genome are a major control point for regulating RNA synthesis. The proposed work will test several new concepts that have emerged concerning features of the viral nucleocapsid template which regulate genome replication and gene expression for the prototypic paramyxovirus SV5. The first aim is to determine the role in viral RNA replication of three factors: (i) sequence within two essential and discontinuous promoter elements, (ii) the spacing of these elements along one face of a helix and (iii) the hexameric phase in the promoter region. A reverse genetics system based on model RNA minigenome analogs will be used along with site specific mutagenesis and an in vivo selection method to determine the relative contribution of each of these factors to promoter activity. Chimeric RNA genomes containing exchanges between the genomic and antigenomic promoters will be used along with polymerase binding assays to determine the basis for differential synthesis of viral RNA from these two promoters. For some paramyxoviruses, there is a very high level of transcriptional readthrough at the junction between the viral M and F genes. In the second aim, a transcription-competent model dicistronic RNA genome will be used to determine the molecular basis for transcriptional readthrough at the M-F junction. In the third aim, recombinant viruses containing altered M-F intergenic regions will be isolated from a full length cDNA. These mutant viruses will be employed to test the two hypotheses that during the course of an infection, M-F readthrough transcription serves to (i) downregulate the level of fusion-competent F protein or (ii) increase the number of polymerase molecules that can access the 5' end of the viral genome. These experiments will test new concepts on how the paramyxovirus nucleocapsid-associated RNA template can modulate the activities of the multifunctional viral polymerase. Results from the proposed work will form a critical base for a rational reverse-genetics approach to parainfluenza vaccines, since it may be possible to design ideal attenuated recombinant viruses which have altered promoter or intergenic transcription activities.