CONTROL OF PARAMYXOVIRUS RNA SYNTHESIS

副粘病毒 RNA 合成的控制

• 批准号: 2700047
• 负责人: Griffith D. Parks
• 金额: $ 16.61万
• 依托单位: WAKE FOREST UNIVERSITY
• 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 2003-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2700047
• 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微生物之一 道感染，这些普遍存在的病原体是造成 全世界发病率和死亡率很高。 这些RNA病毒 已经进化出控制水平和特定的机制 感染期间合成的病毒 RNA 类型。顺式作用序列 副粘病毒基因组中包含的一个主要控制点 调节RNA合成。 拟议的工作将测试几个新的 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 原型副粘病毒SV5。 第一个目标是确定角色 影响病毒RNA复制的三个因素：（i）两个内的序列 基本和不连续的启动子元件，(ii) 这些元件的间距 沿螺旋一个面的元素和（iii）六聚体相 启动子区。 基于模型RNA的反向遗传学系统 小基因组类似物将与位点特异性诱变一起使用 确定相对贡献的体内选择方法 这些因素中的每一个都会影响启动子的活性。 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 与聚合酶结合测定一起使用以确定的基础 来自这两个启动子的病毒RNA的差异合成。 对于一些 副粘病毒，转录水平非常高 通读病毒 M 和 F 基因之间的连接处。 在 第二个目标，具有转录能力的双顺反子RNA基因组模型 用于确定转录通读的分子基础 在M-F交界处。 第三个目标是，重组病毒含有 改变的M-F基因间区域将从全长cDNA中分离出来。 这些突变病毒将被用来检验两个假设： 在感染过程中，M-F 通读转录可发挥作用 (i) 下调具有融合能力的 F 蛋白的水平或 (ii) 增加可以到达 5' 端的聚合酶分子的数量 病毒基因组。 这些实验将测试关于如何 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。 结果来自 拟议的工作将为合理的反向遗传学奠定重要基础 副流感疫苗的方法，因为有可能设计 理想的减毒重组病毒，其启动子已改变或 基因间转录活性。