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Control of Paramyxovirus RNA Synthesis

副粘病毒 RNA 合成的控制

基本信息

批准号：
7319657
负责人：
Griffith D. Parks
金额：
$ 23.36万
依托单位：
WAKE FOREST UNIVERSITY HEALTH SCIENCES
依托单位国家：
美国
项目类别：
财政年份：
1998
资助国家：
美国
起止时间：
1998-07-01 至 2010-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7319657
关键词：
Address Affect Antiviral Response Apoptosis Apoptotic Attenuated Biochemical Biological Assay Canis familiaris Cell Communication Cell Line Cells Defect Engineering Failure Gene Expression Genes Genetic Transcription Genome Glycine decarboxylase Goals Growth Human Immune response Infection Interferon Activation Interferon Inducers Interferon Type I Interferons Kinetics Mediating Messenger RNA Modeling Mutation Paramyxovirus Pathway interactions Phenotype Polymerase Polymerase Chain Reaction Polymerase Gene Property Protein Overexpression Proteins RNA chemical synthesis Recombinants Safety Signal Pathway Signal Transduction Simian virus 5 Testing Time Vaccines Variant Viral Viral Proteins Viral Vector Virus Work base cell killing design improved mutant novel novel strategies parainfluenza virus prototype response role model vector vaccine viral RNA

项目摘要

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（由申请人提供）：我们最近的工作导致了以下总体假设：感染后，副粘病毒延迟大量RNA合成，为病毒介导的宿主抗病毒反应抑制提供时间。这一假设出现在我们的结果与原型副粘病毒猴病毒5（SV 5）的变种，加速或延迟RNA合成的动力学。这项工作的一个显著结果是，我们发现P/V基因中加速病毒RNA合成的突变也将SV 5从非细胞病变病毒转化为激活I型干扰素（IFN）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的病毒：宿主细胞相互作用。我们已经分离出一种天然存在的SV 5变体（WF-CPIV），与WT SV 5相比，该变体显示出病毒基因表达的显著延迟。在目标1中，我们将确定WF-CPIV基因表达延迟的基础。将使用实时PCR测定来检验延迟的WF-CPIV基因表达是由于初级或次级转录缺陷所致的假设。含有聚合酶相关基因交换的重组SV 5病毒将鉴定导致延迟基因表达的WF-CPIV组分。目的2阐述了我们的发现，即在P/V基因中具有取代的rSV 5比WT rSV 5更早地表达病毒mRNA和蛋白质，并且表达水平更高。将产生新的rSV 5病毒，其从不同的转录单位表达WT或突变体P和V蛋白。将使用生物化学和微型基因组复制测定来检验P/V置换降低V抑制SV 5基因组复制的能力或赋予P蛋白更高的RNA合成活性的假设。在目的3中，我们将检验以下假设：含有P/V置换的致细胞病变rSV 5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。具有WT和突变P和V蛋白的诱导表达的细胞系以及新的rSV 5 P/V突变体将用于测试加速和延迟的SV 5 RNA合成在IFN诱导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起始时间，以避免宿主的抗病毒反应，并且加速或显著延迟RNA合成的变化可以激活IFN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，并提供新的方法来提高疫苗载体的安全性和效力。