An RNA-centric dissection of host complexes controlling flavivirus infection [LAM-PCR]

Flaviviruses including dengue virus (DENV) and Zika virus (ZIKV) cause significant human disease. Co-opting cellular factors for viral protein translation and viral genome replication at the endoplasmic reticulum (ER) is a shared replication strategy, despite different clinical outcomes. While the protein products of these viruses have been studied in depth, how the RNA genomes operate inside human cells is poorly understood. Using comprehensive identification of RNA binding proteins by mass spectrometry (ChIRP-MS), we took an RNA-centric viewpoint of flaviviral infection and identified several hundred proteins associated with both DENV and ZIKV genomic RNA inside human cells. Intersection with genome-scale knockout screens assigned putative functional relevance to the RNA-protein interactions. ER RNA binding proteins such vigilin and RRBP1 directly bound viral RNA and each operated at distinct points in the life cycle of flaviviruses. Thus, this versatile strategy can elucidate features of human biology that control pathogenesis of clinically relevant viruses. Overall design: This experiment was designed to assess, in a genome-wide manner, what human genes are required for the Zika virus to infect HAP1 cells. Genes were disrupted with lentiviral gene trap technology.黄病毒属（Flaviviruses）包含登革病毒（Dengue virus, DENV）与寨卡病毒（Zika virus, ZIKV），可引发严重的人类疾病。尽管两类病毒的临床结局存在差异，但其均采用了保守的复制策略：在内质网（endoplasmic reticulum, ER）上劫持宿主细胞因子，以完成病毒蛋白翻译与基因组复制。目前学界已对这两类病毒的蛋白产物开展了深入研究，但对于其RNA基因组在人类细胞内的运作机制仍知之甚少。 本研究借助基于质谱的RNA结合蛋白综合鉴定技术（comprehensive identification of RNA binding proteins by mass spectrometry, ChIRP-MS），以病毒RNA为核心视角开展黄病毒感染研究，在人类细胞中鉴定出了数百种可与登革病毒、寨卡病毒基因组RNA结合的宿主蛋白。通过与全基因组敲除筛选结果进行交集分析，我们为这些RNA-蛋白质相互作用赋予了推定的功能相关性。内质网RNA结合蛋白如vigilin与RRBP1可直接结合病毒RNA，并分别在黄病毒生命周期的不同阶段发挥调控作用。综上，本研究所采用的通用研究策略可阐明调控临床相关病毒致病机制的人类生物学特征。 整体实验设计：本实验以全基因组规模评估寨卡病毒感染HAP1细胞所必需的人类基因，采用慢病毒基因陷阱技术对宿主基因进行敲除干扰。