Quantitative mapping of interactions between dengue virus and its hosts

描述（由申请人提供）：病毒与宿主相互作用，以协调复制所需的无数生化过程。虫媒病毒如登革病毒（DENV）通过节肢动物传播给脊椎动物的一个独特特征是，这些病毒必须在两个进化上遥远的宿主中保持相互作用才能成功复制。在该提议中，比较遗传相互作用作图将用于鉴定对DENV复制功能重要的保守宿主因子：目的1-DENV-宿主相互作用的定量图谱进化约束两个因子之间的遗传相互作用暗示它们在功能上相关。将使用与报告病毒系统偶联的单个和成对RNAi敲低来定量绘制与DENV物理相互作用的宿主因子的遗传相互作用。这将是第一次系统地研究遗传相互作用， 寄主-病原体系统将对目标1中生成的数据进行分层聚类，以确定具有相似遗传相互作用特征的宿主因子。先前获得的DENV-宿主和宿主-宿主蛋白相互作用数据将被并入以定义功能模块。富集分析将用于鉴定富集宿主复制或限制因子的官能团和途径。目标1的结果将解决有关宿主对DENV复制施加的进化约束的基本问题。目的2-鉴定来自保守功能模块的宿主复制因子子集在病毒复制周期中的缺陷点。在通过RNAi敲低来自目的1中表征的保守功能模块的10种宿主复制因子后，在两种宿主系统中的翻译、RNA合成和退出点测量病毒复制。在这个目标的实验将提供深入了解登革病毒复制的分子机制在两个进化上遥远的主机。最终，DENV-宿主相互作用的进化观点将对药物开发特别有用。靶向保守的或进化上受约束的相互作用将产生更强大的疗法，因为DENV在其进化抗性的能力方面将受到限制。

DESCRIPTION (provided by applicant): Viruses interact with the host to orchestrate myriad biochemical processes required for replication. A unique characteristic of arboviruses such as dengue virus (DENV), which are transmitted to vertebrates by arthropod vectors, is that these viruses must maintain interactions in two evolutionarily distant hosts to successfully replicate. I this proposal, comparative genetic interaction mapping will be used to identify conserved host factors that are functionally important for DENV replication: Aim 1-Quantitatively map evolutionary constraints of DENV-host interactions A genetic interaction between two factors implies that they are functionally related. Single and pairwise RNAi knockdown coupled with a reporter virus system will be used to quantitatively map genetic interactions of host factors that physically interact with DENV. This will be the first systematic study of genetic interactions in a host-pathogen system. Hierarchical clustering will be applied to the data generated in Aim 1 to identify host factors with similar genetic interaction profiles. Previously acquired DENV-host and host-host protein interaction data will be incorporated to define functional modules. Enrichment analysis will be used to identify functional groups and pathways that are enriched for host replication or restriction factors. The results from Aim 1 will address fundamental questions regarding the evolutionary constraints imposed by hosts on DENV replication. Aim 2-Identify the point of defect in the viral replication cycle for a subset of host replication factors from conserved functional modules Viral replication will be measured at the point of translation, RNA synthesis and exit in both host systems following knockdown by RNAi for 10 host replication factors from conserved functional modules characterized in Aim 1. The experiments in this aim will provide insight into the molecular mechanism of DENV replication in two evolutionarily distant hosts. Ultimately, an evolutionary perspective on DENV-host interactions will be particularly useful for drug development. Targeting conserved or evolutionarily constrained interactions will yield more robust therapies, as DENV will be limited in its ability to evolve resistance.