Control of Paramyxovirus RNA Synthesis

副粘病毒 RNA 合成的控制

• 批准号: 6720400
• 负责人: Griffith D. Parks
• 金额: $ 25.72万
• 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 2008-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6720400
• 关键词: Paramyxovirus; RNA biosynthesis; apoptosis; biochemical evolution; biochemistry; biotechnology; cell line; gene expression; gene expression profiling; gene mutation; genetic transcription; host organism interaction; interferons; microarray technology; polymerase chain reaction; recombinant virus; simian virus; transfection /expression vector; vector vaccine; virus genetics; virus infection mechanism; virus replication

DESCRIPTION (provided by applicant): Our recent work has led to the overall hypothesis that following infection, paramyxoviruses delay abundant RNA synthesis to provide time for virus-mediated suppression of host anti-virus responses. This hypothesis emerged from our results with variants of the prototype paramyxovirus Simian Virus 5 (SV5) that have either accelerated or delayed kinetics of RNA synthesis. A remarkable result from this work was our finding that mutations in the P/V gene that accelerate viral RNA synthesis also convert SV5 from a noncytopathic virus into a virus that activates type I interferon (IFN) and kills cells by apoptosis. Thus, the overall goal of our work is to understand mechanisms that can accelerate or delay paramyxovirus RNA synthesis and how changes in the timing of viral gene expression affect key virus:host cell interactions. We have isolated a naturally-occurring SV5 variant (WF-CPIV) that shows a remarkable delay in viral gene expression compared to WT SV5. In Aim 1, we will determine the basis for the delay in WF-CPIV gene expression. Real-time PCR assays will be used to test the hypotheses that delayed WF-CPIV gene expression is due to a defect in either primary or secondary transcription. Recombinant SV5 viruses containing exchanges of polymerase-associate genes will identify WF-CPIV component(s) responsible for delayed gene expression. Aim 2 addresses our finding that an rSV5 with substitutions in the P/V gene expresses viral mRNA and proteins earlier and to higher levels than WT rSV5. Novel rSV5 viruses will be generated that express WT or mutant P and V proteins from separate transcription units. Biochemical and minigenome replication assays will be used to test the hypotheses that P/V substitutions either decrease the ability of V to inhibit SV5 genome replication or confer higher RNA synthesis activity on the P protein. In Aim 3, we will test the hypothesis that the cytopathic rSV5 variant containing P/V substitutions induces interferon (IFN) synthesis and apoptosis due to premature expression of viral gene products. Cell lines with inducible expression of WT and mutant P and V proteins, and new rSV5 P/V mutants will be used to test a two-step model for the role of accelerated and delayed SV5 RNA synthesis in the induction of IFN and apoptosis. We hypothesize that paramyxoviruses have evolved to optimize the timing of the onset of viral gene expression to avoid host antiviral responses, and changes that accelerate or significantly delay RNA synthesis can activate IFN and/or apoptotic pathways. Our work will increase our understanding of important virus-host cell interactions, as well as provide new approaches to improve the safety and potency of vaccine vectors.

描述(申请人提供)：我们最近的工作导致了一个总体假设，即感染后，副粘病毒延迟了丰富的RNA合成，为病毒介导的宿主抗病毒反应的抑制提供了时间。这一假说来自我们对副粘病毒原型猿猴病毒5(SV5)的变种的结果，这些变种加速或延迟了RNA合成的动力学。这项工作的一个显着结果是我们发现P/V基因的突变加速了病毒RNA的合成，也将SV5从非细胞病变病毒转化为激活I型干扰素(干扰素)并通过凋亡杀死细胞的病毒。因此，我们工作的总体目标是了解可以加速或延迟副粘病毒RNA合成的机制，以及病毒基因表达时间的变化如何影响关键病毒：宿主细胞相互作用。 我们已经分离出一种自然产生的SV5变异株(WF-CPIV)，与WTSV5相比，该变异株在病毒基因表达方面表现出明显的延迟。在目标1中，我们将确定WF-CPIV基因表达延迟的基础。实时聚合酶链式反应分析将被用来检验WF-CPIV基因延迟表达是由于初级或次级转录缺陷的假说。含有聚合酶相关基因交换的重组SV5病毒将识别导致基因表达延迟的WF-CPIV成分(S)。目的2解决我们的发现，在P/V基因有替换的rSV5比WT rSV5更早和更高水平表达病毒的mRNA和蛋白质。将产生新的rSV5病毒，表达来自不同转录单位的WT或突变的P和V蛋白。生化和微型基因组复制分析将被用来检验P/V替换要么降低V抑制SV5基因组复制的能力，要么赋予P蛋白更高的RNA合成活性的假设。在目标3中，我们将验证一种假设，即含有P/V替换的细胞性rSV5变体由于病毒基因产物的过早表达而诱导干扰素(IFN)合成和细胞凋亡。可诱导表达WT和突变的P和V蛋白的细胞系以及新的rSV5P/V突变株将用于测试加速和延迟SV5RNA合成在诱导干扰素和细胞凋亡中的作用的两步模型。 我们假设副粘病毒已经进化到优化病毒基因表达的开始时间以避免宿主抗病毒反应，并且加速或显著延迟RNA合成的变化可以激活干扰素和/或凋亡途径。我们的工作将增加我们对重要的病毒-宿主细胞相互作用的理解，并为提高疫苗载体的安全性和有效性提供新的方法。