Reverse Genetics with Rotaviruses

轮状病毒的反向遗传学

基本信息

批准号：
7229820
负责人：
ROBERT Franklin RAMIG
金额：
$ 18.21万
依托单位：
BAYLOR COLLEGE OF MEDICINE
依托单位国家：
美国
项目类别：
财政年份：
2006
资助国家：
美国
起止时间：
2006-03-01 至 2009-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7229820
关键词：
Animals Antiviral Agents BCAR3 gene Cells Childhood Code Development Disease Engineering Genetic Transcription Genome Goals In Vitro Lead Left Messenger RNA Methodology Methods Modeling Modification Molecular Mutation Nucleosome Core Particle Numbers Phenotype Proteins Protocols documentation RNA RNA Viruses RNA-Directed RNA Polymerase Rotavirus Silent Mutation System Temperature Testing Time Translations Trypsin Vaccines Viral Viral Genes Viral Proteins Virion Virus Virus Diseases Virus-like particle desire gain of function genetic analysis human disease in vivo mutant particle positional cloning reconstitution replicase research study segregation vaccine development

Animals Antiviral Agents BCAR3 gene Cells Childhood Code Development Disease Engineering Genetic Transcription Genome Goals In Vitro Lead Left Messenger RNA Methodology Methods Modeling Modification Molecular Mutation Nucleosome Core Particle Numbers Phenotype Proteins Protocols documentation RNA RNA Viruses RNA-Directed RNA Polymerase Rotavirus Silent Mutation System Temperature Testing Time Translations Trypsin Vaccines Viral Viral Genes Viral Proteins Virion Virus Virus Diseases Virus-like particle desire gain of function genetic analysis human disease in vivo mutant particle positional cloning reconstitution replicase research study segregation vaccine development

展开

项目摘要

DESCRIPTION (provided by applicant): Reverse genetic systems, used to rescue manipulated viral cDNAs into infectious RNA viruses, led to rapid advances in a large number of virus systems because they allow examination of specific mutations in the context of viral infection. The rotaviruses are a group of RNA genome viruses important in human disease for which no reverse genetic system exists. The goal of the experiments proposed here is development of a reverse genetic system for rotaviruses, and its application to questions that cannot be answered without reverse genetics. We have three specific aims: (1) In vitro approaches to reverse genetics for rotaviruses. We will examine methods for "opening" core particles or virus-like particles, which result in intact particles that have replicase activity. These particles will be used to develop reverse genetic protocols. (2) In vivo approaches to reverse genetics for rotaviruses. Recently, a new model for mRNA segregation between translation and replication was presented that indicates replicated mRNAs never leave the viroplasm. We will investigate methods to introduce exogenous mRNAs into viroplasms, including (a) early introduction of exogenous RNAs into infected cells, (b) the use of tsE(1400) to manipulate the time of viroplasm formation, and (c) the isolation and manipulation of viroplasms to introduce exogenous RNA followed by reintroduction of viroplasms into cells. (3) Proof of principle application of the rotavirus reverse genetic system. We will convert a trypsin dependent virus (SA11 or RRV) to trypsin independence for plaque formation by rescuing segment 4 and VP4 from SA11-4F, a trypsin independent virus. In addition to electrophoretic mobility markers, we will introduce silent mutations into the coding region of SA11-4F VP4 for molecular confirmation of rescue. This gain of function format will allow direct selection of the desired engineered virus. These studies will make the power of reverse genetic analysis available for application to the rotaviruses for the first time. The current absence of reverse genetics renders many important problems impossible to solve. Reverse genetics will stimulate rapid advances in the rotaviruses. Rotaviruses are a significant cause of childhood disease for which no vaccine exists. Reverse genetic methods allow the examination of the effects of specific mutations in viral genes in the context of viral infection. Reverse genetic methods are not available for the rotaviruses. The ability to perform reverse genetics with rotaviruses will facilitate development of a vaccine or antivirals for this disease of childhood.

描述（由申请人提供）：用于将操纵的病毒cDNA营救成传染性RNA病毒的反向遗传系统，导致大量病毒系统的快速发展，因为它们允许在病毒感染的背景下检查特定突变。轮状病毒是一组RNA基因组病毒在人类疾病中不存在反向遗传系统的人类疾病。这里提出的实验的目的是开发用于轮状病毒的反向遗传系统，以及在没有反向遗传学的情况下无法回答的问题。我们有三个特定的目的：（1）在体外方法逆转轮式病病毒的遗传学方法。我们将研究“打开”核心颗粒或病毒样颗粒的方法，从而导致具有复制酶活性的完整颗粒。这些颗粒将用于开发反向遗传方案。（2）在体内逆转轮式病病毒的遗传学方法。最近，提出了一种新的翻译和复制之间mRNA分离模型，表明复制的mRNA永远不会离开病毒质。我们将研究将外源性mRNA引入病毒性肿瘤的方法，包括（a）早期将外源性RNA引入感染细胞中，（b）使用TSE（1400）来操纵病毒型的形成时间，以及（c）将VirOplasms的隔离和操纵以引入外源性RNA，然后将细胞引入细胞。（3）轮状病毒反向遗传系统的原理应用证明。我们将通过从SA11-4F（一种独立的胰蛋白酶独立病毒）中拯救第4和VP4，将依赖性病毒（SA11或RRV）转换为斑块形成的胰蛋白酶独立性。除了电泳迁移率标记外，我们还将在SA11-4F VP4的编码区域中引入无声突变，以进行救援的分子确认。这种功能格式的增益将允许直接选择所需的工程病毒。这些研究将使可以首次适用于轮状病毒的反向遗传分析的能力。当前缺乏反向遗传学的原因使许多重要的问题无法解决。反向遗传学将刺激轮状病毒的快速进步。轮状病毒是不存在疫苗的儿童疾病的重要原因。反向遗传学方法允许在病毒感染的情况下检查病毒基因中特异性突变的作用。反向遗传方法无法用于轮状病毒。使用轮状病毒进行反向遗传学的能力将促进这种儿童疾病的疫苗或抗病毒药。