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: 7661284
• 负责人: MARGARET R MACDONALD
• 金额: $ 25.29万
• 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 2011-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7661284
• 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）：本提案的目标是开发一种易于处理的模型，用于对甲病毒感染中宿主因素的作用进行遗传分析。披膜病毒科甲病毒属的成员会引起严重的人类和动物疾病，包括脑炎和死亡。该属包括 NIAID B 类优先病原体，可能用于生物恐怖主义。目前尚无可用的特异性治疗方法。虽然宿主因素无疑在甲病毒复制周期中发挥着重要作用，但它们的身份和功能仍然很大程度上未知。了解病毒复制需要哪些宿主蛋白以及病毒如何与这些宿主蛋白相互作用，可以为抗病毒疗法的开发开辟潜在的靶点。该项目将利用经过充分研究的线虫秀丽隐杆线虫作为模型生物，研究宿主因子在甲病毒复制中的作用。秀丽隐杆线虫的诱变和遗传分析技术已经得到很好的研究。选择甲病毒型辛德比斯病毒（SINV）作为模型开发的测试甲病毒。该研究的第一个目的是产生转基因动物，在特征良好的秀丽隐杆线虫启动子的控制下表达表达 GFP 的 SINV 复制子。 SINV RNA 表达将通过 GFP 表达进行监测。第二个目标是，对携带 SINV 复制子编码 DNA 的转基因动物进行化学诱变，监测 SINV 表达，并通过评估 GFP 表达来鉴定 SINV 复制减少（或增强）的突变体。将采用标准遗传方法来识别携带减少或增加 SINV 复制的突变的基因。第三个目标是，该模型将测试先前确定与病毒复制蛋白相关的宿主蛋白在病毒复制中的重要性。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，现有的突变体将被工程化（通过杂交）到 SINV 模型菌株中进行测试。或者，向 SINV 模型菌株喂食表达基因特异性双链 RNA（几乎适用于所有线虫基因）的细菌，以沉默表达。宿主蛋白水平降低对 SINV 复制的影响将通过 GFP 可视化进行评估。这些研究有可能确定甲病毒RNA复制所需的宿主因子，这可能为可能的抗病毒治疗开辟新的靶点。该模型具有扩展的潜力，可以通过使用表达 GFP 的病毒而不是复制子来检查整个甲病毒生命周期。此外，该模型还适用于其他甲病毒的研究，包括 NIAID B 类药物以及其他医学上重要的正链 RNA 病毒。公共卫生相关性：甲病毒属病毒可引起发烧、皮疹、关节炎、脑炎和死亡等疾病，但迄今为止尚无特定疗法。这项研究将开发甲病毒感染的小动物（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子。更详细地了解病毒与宿主的相互作用可能会导致特定疗法的开发。