AN ORGANISMAL APPROACH TO VIRAL HOST FACTOR DISCOVERY

描述(申请人提供)：甲型病毒是人类和牲畜的病原体，分布在世界各地。将一些物种归类为特定病原体，并结合它们对公共卫生的影响，使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。了解病毒与之相互作用的宿主细胞成分的一个主要障碍是无法从基因上操纵宿主细胞/生物体。黑腹果蝇是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。这项拟议的研究旨在利用果蝇遗传学的力量，发现甲型病毒基因组复制所需的宿主因素。这项研究的基本概念很简单：在UAS/GAL4系统的控制下，编码报告/可选择标记的甲型病毒复制子序列将被克隆到果蝇基因组中。含有复制子序列的果蝇将与GAL4表达系杂交，以指导F1后代复制子RNA的初级转录。如果表达复制子的细胞环境允许病毒基因组复制，那么复制子的扩增和报告基因的表达就会发生。这一方法将允许(I)识别整个苍蝇体内能够支持病毒复制的组织；(Ii)对病毒基因组复制所需的宿主因子进行遗传识别。该提案中概述的研究并不复杂，只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。这种简单但强大的方法将导致识别病毒基因组复制所需的数十个(如果不是数百个)宿主因子，并显著促进我们对病毒与宿主相互作用的理解。 与公共卫生相关：确定病毒与宿主相互作用的方式对于了解疾病的根本原因和开发抗病毒药物至关重要。这项拟议的研究旨在利用模型遗传系统确定甲型病毒(蚊媒病原体)感染所需的宿主因素。

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.