CONTROL OF PARAMYXOVIRUS RNA SYNTHESIS

副粘病毒 RNA 合成的控制

• 批准号: 6510756
• 负责人: Griffith D. Parks
• 金额: $ 17.37万
• 依托单位: WAKE FOREST UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-07-01 至 2003-12-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6510756
• 关键词: Paramyxovirus; RNA biosynthesis; complementary DNA; fusion gene; genetic promoter element; genetic regulatory element; genetic transcription; nucleocapsid; recombinant virus; replicase; site directed mutagenesis; virus RNA; virus genetics; virus replication

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.

副粘病毒是急性呼吸道疾病中最常见的生物之一。 肠道感染，这些无处不在的病原体导致 全世界的发病率和死亡率都很高。这些RNA病毒 已经进化出同时控制水平和特殊情况的机制 在感染过程中合成的一种病毒RNA。顺式作用序列 包含在副粘病毒基因组中的是 调节核糖核酸的合成。拟议中的工作将测试几个新的 出现的关于病毒核衣壳特征的概念 调节基因组复制和基因表达的模板 典型的副粘病毒SV5。第一个目标是确定角色 在病毒RNA复制中有三个因素：(一)序列在两个以内 基本的和不连续的启动子元件，(Ii)这些元件的间距 元素沿螺旋的一个面和(Iii)六面体相 启动子区域。一种基于模型RNA的反向遗传学系统 微基因组类似物将与定点突变和 一种确定相对贡献率的体内选择方法 这些因素中的每一个都对启动子活性有影响。嵌合RNA基因组 包含基因组和抗基因组启动子之间的交换将 与聚合酶结合分析一起用于确定 这两个启动子对病毒RNA的差异合成。对一些人来说 副粘病毒，有非常高水平的转录 在病毒M和F基因的交界处通读。在 第二个目标是，一个具有转录能力的双顺反子RNA基因组将 用于确定转录通读的分子基础 在M-F交界处。在第三个目标中，重组病毒包含 改变的M-F基因间隔区将从全长的cDNA中分离出来。 这些变异病毒将被用来检验这两个假设 在感染过程中，M-F直读转录起作用 (I)下调融合能力F蛋白的水平或(Ii) 增加可以接近5‘端的聚合酶分子的数量 病毒基因组的一部分。这些实验将测试新的概念如何 副粘病毒核衣壳相关RNA模板可调节 多功能病毒聚合酶的活性。调查结果： 拟议的工作将构成理性反向遗传学的关键基础 副流感疫苗的方法，因为有可能设计出 启动子或启动子改变的理想减毒重组病毒 基因间转录活性。