AN ORGANISMAL APPROACH TO VIRAL HOST FACTOR DISCOVERY

描述（由申请人提供）：甲病毒是人类和牲畜的病原体，分布在世界各地。鉴于某些物种对公共卫生的影响，将它们分类为选定物剂，因此了解它们与宿主的相互作用和制定干预措施成为高度优先事项。了解与病毒相互作用的宿主细胞成分的主要障碍是无法从基因上操纵宿主细胞/生物体。黑腹果蝇代表了一个很好的实验系统，用于阐明许多生理和发育过程背后的遗传、分子和生化机制。拟议的研究旨在利用果蝇遗传学的力量来发现甲病毒基因组复制所需的宿主因子。这项研究的基本概念很简单；在UAS/GAL4系统的控制下，将编码报告/选择标记的甲病毒复制子序列克隆到果蝇基因组中。将含有复制子序列的果蝇与GAL4表达系杂交，在F1后代中指导复制子RNA的初级转录。如果复制子表达的细胞环境是允许病毒基因组复制的，那么就会发生复制子扩增和报告基因表达。这种方法将允许(i)识别整个苍蝇能够支持病毒复制的组织；（ii）病毒基因组复制所需宿主因子的遗传鉴定。提案中概述的研究并不复杂，涉及对果蝇和甲型病毒基因组的相对简单的操作；然而，潜在的回报是巨大的。这种简单但功能强大的方法将导致识别数十甚至数百个病毒基因组复制所需的宿主因子，并显著提高我们对病毒-宿主相互作用的理解。

DESCRIPTION (provided by applicant): Alphaviruses are pathogens of humans and livestock with worldwide distribution. The classification of some species as select agents in conjunction with their impact on public health makes understanding their interaction with the host and development of interventions a high priority. A major impediment to understanding the host cell components with which the virus interacts has been the inability to genetically manipulate the host cell/organism. Drosophila melanogaster represents an excellent experimental system for elucidating the genetic, molecular, and biochemical mechanisms underlying numerous physiological and developmental processes. The proposed research aims to exploit the power of Drosophila genetics for the discovery of host factors required for alphavirus genome replication. The basic concept for the research is straightforward; an alphavirus replicon sequence encoding a reporter/selectable marker will be cloned into the Drosophila genome under the control of the UAS/GAL4 system. Flies containing the replicon sequence will be crossed with a GAL4 expressing line to direct primary transcription of the replicon RNA in F1 progeny. If the cellular environment in which the replicon is expressed is permissive for virus genome replication then replicon amplification and reporter gene expression will occur. This approach will allow (i) the identification of tissues throughout the fly capable of supporting virus replication; (ii) genetic identification of host factors required for viral genome replication. The research outlined in the proposal is not complex, involving relatively simple manipulations of the Drosophila and alphaviral genomes; however the potential payoff is enormous. This simple but powerful approach will lead to the identification of dozens if not hundreds of host factors required for viral genome replication, and significantly advance our understanding of the virus-host interaction. PUBLIC HEALTH RELEVANCE: Identifying the ways in which a virus interacts with it's host is crucial to understanding the underlying cause of disease and developing antiviral drugs. The proposed research aims to determine host factors required for alphavirus (mosquito-borne pathogens) infection using a model genetic system.