Quantitative mapping of interactions between dengue virus and its hosts

描述(申请人提供)：病毒与宿主相互作用，协调复制所需的无数生化过程。虫媒病毒如登革病毒(DENV)通过节肢动物载体传播给脊椎动物，其独特的特征是这些病毒必须在进化上相距遥远的两个宿主上保持相互作用才能成功复制。在这项建议中，比较遗传作图将用于确定对DENV复制具有重要功能的保守宿主因子：目的1-定量地定位DENV-宿主相互作用的进化限制因素两个因子之间的遗传相互作用意味着它们在功能上是相关的。单个和成对的RNAi敲除结合报告病毒系统将被用于定量绘制与DENV物理相互作用的宿主因子的遗传相互作用图。这将是第一次对基因相互作用的系统研究 寄主-病原体系统。将对目标1中产生的数据进行等级聚类，以确定具有相似遗传相互作用特征的宿主因素。先前获得的DENV-宿主和宿主-宿主蛋白质相互作用数据将被合并以定义功能模块。富集化分析将用于确定为宿主复制或限制因子而富集化的官能团和途径。AIM 1的结果将解决有关宿主对DENV复制施加的进化限制的基本问题。目的2-确定来自保守功能模块的一组宿主复制因子在病毒复制周期中的缺陷点。病毒复制将在翻译、RNA合成和退出时被RNAi从目标1所描述的保守功能模块的10个宿主复制因子敲除后在两个宿主系统中测量。本实验将为深入了解DENV在进化上相距遥远的两个宿主中复制的分子机制提供依据。最终，对DENV-宿主相互作用的进化观点将对药物开发特别有用。靶向保守或进化受限的相互作用将产生更强大的治疗方法，因为DENV在进化耐药方面的能力将受到限制。

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.