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，用于宿主因子在甲病毒感染中的作用的遗传分析。毒虫病毒科甲病毒属的成员可引起严重的人类和动物疾病，包括脑炎和死亡。该属包括NIAID关注的可能用于生物恐怖主义的B类优先病原体。目前还没有专门的治疗方法。虽然宿主因子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在很大程度上仍然未知。了解哪些宿主蛋白是病毒复制所必需的，以及病毒如何与这些宿主蛋白相互作用，可以为开发抗病毒疗法开辟潜在的靶点。本项目将利用已被充分研究的秀丽隐杆线虫作为模式生物，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很成熟。选择甲型病毒Sindbis病毒（SINV）作为模型开发的测试甲型病毒。本研究的第一个目的是在线虫启动子的控制下，产生表达表达GFP的SINV复制子的转基因动物。通过GFP的表达监测SINV RNA的表达。在第二个目标中，将对携带SINV复制子编码DNA的转基因动物进行化学诱变，监测SINV的表达，并通过评估GFP表达来鉴定SINV复制减少（或增强）的突变体。标准的遗传方法将被用来识别携带减少或增加SINV复制的突变的基因。在第三个目标中，该模型将测试先前确定的与病毒复制蛋白相关的宿主蛋白在病毒复制中的意义。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，现有的突变体将被改造（通过杂交）到SINV模型菌株中进行测试。或者，将表达基因特异性双链RNA（可用于几乎所有线虫基因）的细菌喂养SINV模型菌株以沉默表达。宿主蛋白水平降低对SINV复制的影响将通过GFP可视化来评估。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型病毒生命周期。此外，该模型适用于其他甲型病毒的研究，包括NIAID B类病毒，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从发热、皮疹和关节炎到脑炎和死亡等疾病，但迄今尚无专门的治疗方法。本研究将建立一个小动物（线虫）感染甲病毒的模型，该模型将用于采用遗传方法确定病毒复制所需的宿主因子。更详细地了解病毒-宿主相互作用可能会导致特异性治疗的发展。