Development of a genetically tractable nematode model of alphavirus infection

甲病毒感染的遗传易驯化线虫模型的开发

• 批准号: 7764768
• 负责人: MARGARET R MACDONALD
• 金额: $ 20.91万
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-02-05 至 2012-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7764768
• 关键词: Alphavirus; Alphavirus Infections; Animal Diseases; Animal Feed; Animal Model; Animals; Antiviral Agents; Antiviral Therapy; Arthritis; Bacteria; Bioterrorism; C. elegans genome; Caenorhabditis elegans; Categories; Cessation of life; Chemicals; Complex; DNA; Development; Disease; Double-Stranded RNA; Encephalitis; Engineering; Epidemic; Equus caballus; Exanthema; Failure; Family; Fever; Future; Genes; Genetic; Genetic Models; Genetic Transcription; Genome; Goals; Green Fluorescent Proteins; Heating; Homologous Protein; Human; Imagery; Insect Proteins; Integration Host Factors; Intervention; Lead; Life Cycle Stages; Modeling; Monitor; Mutagenesis; Mutation; National Institute of Allergy and Infectious Disease; Nematoda; Play; Proteins; Proteomics; RNA; RNA Viruses; RNA replication; Replicon; Research Design; Role; Sindbis Virus; Symptoms; System; Techniques; Testing; Togaviridae; Transgenic Animals; Transgenic Organisms; Viral; Virus; Virus Replication; Work; chikungunya; combat; feeding; genetic analysis; member; model development; mutant; novel; pathogen; promoter; protein expression; public health relevance; replicase; tool; viral RNA; virus host interaction

DESCRIPTION (provided by applicant): The goal of this proposal is to develop a tractable model for genetic analyses of the role of host factors in Alphavirus infection. Members of the Alphavirus genus in the Togaviridae family cause significant human and animal disease, including encephalitis and death. The genus includes NIAID Category B priority pathogens of concern for possible bioterrorism use. Currently there is no specific therapy available. While host factors undoubtedly play important roles in the Alphavirus replication cycle, their identities and functions remain largely unknown. Understanding which host proteins are required for virus replication and how the virus interacts with these host proteins could open up potential targets for the development of antiviral therapies. This project will utilize the well-studied nematode Caenorhabditis elegans as a model organism for studying the role of host factors in Alphavirus replication. Techniques for mutagenesis and genetic analysis of C. elegans are well worked out. The type Alphavirus, Sindbis virus (SINV), was chosen as a test Alphavirus for model development. The first aim of the study is to generate transgenic animals expressing a GFP- expressing SINV replicon under the control of a well-characterized C. elegans promoter. SINV RNA expression will be monitored by GFP expression. In the second aim, the transgenic animals harboring the SINV replicon-encoding DNA will be subjected to chemical mutagenesis, SINV expression will be monitored, and mutants with decreased (or enhanced) SINV replication will be identified by assessment of GFP expression. Standard genetic approaches will be employed to identify the genes harboring mutations that decrease or increase SINV replication. In the third aim, the model will test for the significance in viral replication of host proteins previously identified as associating with viral replication proteins. Candidate host proteins will be compared to the genes present in C. elegans, and if available, existing mutants will be engineered (by crossing) into the SINV model strain for testing. Alternatively, the SINV model strain will be fed bacteria expressing gene-specific double stranded RNA (available for virtually all the nematode genes) to silence expression. The effect of reduced levels of the host protein on SINV replication will be assessed by GFP visualization. These studies have the potential to identify host factors necessary for Alphavirus RNA replication, which may open up new targets for possible antiviral treatment. The model has the potential to be expanded to allow examination of the entire Alphavirus life cycle through use of GFP expressing virus, rather than a replicon. In addition, the model is amenable to the study of other Alphaviruses, including NIAID Category B agents, as well as other medically important positive strand RNA viruses. PUBLIC HEALTH RELEVANCE: Viruses in the Alphavirus genus cause diseases ranging from fever, rash and arthritis, to encephalitis and death, yet to date no specific therapies exist. This study will develop a small animal (nematode) model of Alphavirus infection, which will be used to take a genetic approach to identify host factors that are required for viral replication. Understanding the virus-host interaction in greater detail may lead to the development of specific therapies.

描述（由申请人提供）：该提案的目的是开发一种可进行的模型，用于遗传分析宿主因子在α病毒感染中的作用。 Togaviridae家族中α病毒属的成员引起了重大的人类和动物疾病，包括脑炎和死亡。该属包括对可能使用生物恐怖的关注的NIAID类别的优先病原体。目前尚无特定疗法。尽管宿主因素无疑在α病毒复制周期中起着重要作用，但其身份和功能在很大程度上仍然未知。了解病毒复制需要哪种宿主蛋白​​以及病毒与这些宿主蛋白相互作用的方式可以为开发抗病毒药疗法的潜在靶标。该项目将利用秀丽隐杆线虫的良好线虫作为模型生物体，用于研究宿主因子在α病毒复制中的作用。秀丽隐杆线虫的诱变和遗传分析的技术已经很好地解决了。选择α病毒（SINV）的类型α病毒作为模型开发的测试α病毒。该研究的第一个目的是产生在特征良好的秀丽隐杆线虫启动子的控制下表达GFP表达SINV复制子的转基因动物。 SINV RNA表达将通过GFP表达来监测。在第二个目标中，携带SINV复制子编码DNA的转基因动物将受到化学诱变，将监测SINV表达，并通过评估GFP表达来鉴定（或增强）SINV复制的突变体。将采用标准遗传方法来识别携带突变的基因，这些突变减少或增加了SINV复制。在第三个目标中，该模型将测试先前鉴定为与病毒复制蛋白相关的宿主蛋白的病毒复制的显着性。候选宿主蛋白将与秀丽隐杆线虫中存在的基因进行比较，如果有的话，将通过（通过越过）将现有突变体设计到SINV模型菌株中进行测试。或者，SINV模型应被喂食的细菌表达基因特异性双链RNA（几乎可用于所有线虫基因）以静音表达。宿主蛋白水平降低对SINV复制的影响将通过GFP可视化评估。这些研究有可能识别α病毒RNA复制所需的宿主因素，这可能为可能的抗病毒药治疗打开了新的靶标。该模型具有扩展的潜力，可以通过使用GFP表达病毒而不是复制子来检查整个α病毒生命周期。此外，该模型适合研究其他α类病毒，包括NIAID类别B类药物，以及其他重要的医学上重要的阳性链RNA病毒。公共卫生相关性：α病毒属的病毒引起的疾病范围从发烧，皮疹和关节炎，到脑炎和死亡，但迄今为止尚无特定疗法。这项研究将开发出α病毒感染的小动物（线虫）模型，该模型将用于采用遗传方法来识别病毒复制所需的宿主因素。更详细地了解病毒宿主相互作用可能会导致特定疗法的发展。