Host determinants of alphavirus replication

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。甲病毒必须通过嗜血节肢动物媒介传播，在这种媒介中建立终身持续感染。这种感染模式意味着一种有效但不完整的免疫反应，在保持宿主健康的同时允许病毒继续复制，从而促进病毒传播。了解关键的病毒与宿主相互作用的一个主要障碍是无法从基因上操纵宿主生物。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。提出的研究旨在利用果蝇遗传学的力量来定义最近表征的甲病毒与两种节肢动物先天免疫反应途径之间的相互作用。在GAL4-UAS错表达系统的控制下，从果蝇基因组中表达编码报告基因的甲病毒复制子序列，建立了甲病毒复制分析系统。通过报告基因活性可以观察到病毒RNA的复制过程。利用该甲病毒复制子蝇系对宿主途径进行突变分析，证明其对Imd-和JAK-STAT途径具有抗病毒作用。该系统已进一步发展，以产生感染性颗粒。在果蝇中，复制子和病毒结构蛋白的共同表达导致复制子含有能够单轮感染的颗粒的产生。这些基本工具将与果蝇遗传学结合使用，以(i)确定负责激活Imd-和JAK-STAT途径的病毒成分；鉴定参与病毒诱导的先天免疫反应的宿主基因；（三）说明病毒传播对宿主的要求。这项拟议的研究将果蝇遗传学的力量与比较基因组学和分子病毒学相结合，以促进我们对甲病毒与节肢动物宿主之间相互作用的理解。

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. Alphaviruses are obligatorily transmitted by a hematophagous arthropod vector in which a lifelong persistent infection is established. This pattern of infection implies an effective but incomplete immune response that preserves host fitness while allowing virus replication to continue thus facilitating virus transmission. A major impediment to understanding the critical virus-host interactions has been the inability to genetically manipulate the host 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 to define the recently characterized interactions between alphaviruses and two arthropod innate immune response pathways. A system for the analysis of alphavirus replication has been established in which an alphavirus replicon sequence encoding a reporter gene is expressed from the genome of Drosophila under the control of the GAL4-UAS misexpression system. The resulting replication of the viral RNA is observable through reporter gene activity. Mutational analyses of host pathways using this alphavirus replicon fly line have demonstrated an antiviral role for the Imd- and JAK-STAT pathways. This system has been further developed to produce infectious particles. Co-expression in Drosophila of the replicon and viral structural proteins leads to the production of replicon containing particles capable of a single round of infection. These basic tools will be used in combination with Drosophila genetics to (i) determine the viral components responsible for activation of Imd- and JAK-STAT pathways; (ii) identify host genes involved in virus-induced innate immune response; (iii) characterize host requirements for virus spread. The proposed research combines the power of Drosophila genetics with comparative genomics and molecular virology in order to advance our understanding of the interaction between alphaviruses and an arthropod host. PUBLIC HEALTH RELEVANCE: Understanding virus-host interactions is essential to understanding the outcome of infection and determining means of interrupting transmission. The proposed research aims to examine the host response to alphaviruses using a model genetic system.