Development of a genetically tractable nematode model of alphavirus infection

描述(由申请人提供)：这项建议的目标是开发一种易于处理的模型，用于对宿主因素在甲型病毒感染中的作用进行遗传分析。Togaviridae家族中的甲型病毒属成员会引起重大的人类和动物疾病，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。目前还没有特效的治疗方法。虽然宿主因素无疑在甲型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不清楚。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，可能会为抗病毒疗法的开发打开潜在的靶点。本项目将利用研究较好的线虫秀丽线虫作为模式生物，研究宿主因素在甲型病毒复制中的作用。线虫的诱变和遗传分析技术已经制定得很好了。甲型病毒辛德比斯病毒(SINV)被选为模型开发的测试甲病毒。这项研究的第一个目的是在线虫启动子的控制下产生表达GFP表达SINV复制子的转基因动物。SINV RNA的表达将通过GFP的表达进行监测。第二个目的是对携带SINV复制子编码DNA的转基因动物进行化学诱变，监测SINV的表达，并通过GFP表达的评估来鉴定SINV复制降低(或增强)的突变体。标准的遗传学方法将被用来识别含有减少或增加SINV复制的突变的基因。在第三个目标中，该模型将测试先前被确定为与病毒复制蛋白相关的宿主蛋白在病毒复制中的重要性。候选宿主蛋白将与线虫中存在的基因进行比较，如果有的话，现有的突变将被工程(通过杂交)进入SINV模式菌株进行测试。或者，SINV模型菌株将被喂养表达基因特异性双链RNA的细菌(几乎对所有线虫基因都可用)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型病毒RNA复制所需的宿主因素，这可能为可能的抗病毒治疗开辟新的靶点。该模型有可能被扩展，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的生命周期。此外，该模型还适用于其他甲型病毒的研究，包括NIAID B类药物，以及其他具有重要医学意义的正链RNA病毒。公共卫生相关性：甲型病毒属病毒可引起从发烧、皮疹和关节炎到脑炎和死亡等各种疾病，但迄今尚无特效治疗方法。这项研究将开发一种甲型病毒感染的小动物(线虫)模型，将用于采取遗传方法来确定病毒复制所需的宿主因素。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发展。

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.