Multi-level analysis of influenza virus polymerase and its role in pathogenesis

 描述(申请人提供)：甲型流感病毒是一种主要的人类呼吸道病原体，可引起季节性流行和偶尔的大流行。单链负义流感病毒基因组由8个片段组成，每个片段末端都有短的保守元件，形成病毒启动子，由病毒RNA依赖的RNA聚合酶(VPOL)识别。VPOL基因突变和影响vPOL活性的因素可导致禽类病毒适应人类宿主。VPOL对病毒的生物发生是必不可少的，因为它在病毒基因组的转录和复制中起着核心作用。VPOL还严重影响宿主转录，因为它切割新生细胞RNA的顶端以启动病毒转录，这一过程被称为帽抓取。这些不同的vPOL活性的基础，以及可以影响其在宿主细胞中的处理能力的细胞因素，在很大程度上仍不清楚。为了解决这个问题，我们开发了报告病毒，编码标记的vPOL亚基，不会对其感染潜力产生不利影响。我们将利用这些来描述vPOL在生产性感染过程中的相互作用组，并表征表观遗传调节因子和新发现的流感毒力因子的重要性(目标1)。此外，我们将首次通过使用新开发的下一代测序协议(AIM 2)在全基因组范围内对病毒-细胞杂交mRNAs进行剖析来研究Cap-Setch的机制和影响。最后，我们将检查vPOL的处理能力，这可能为感染过程中病毒基因转录的差异调控提供新的见解(目标3)。我们的建议包括首次在基因组、转录组和互作组水平上全面和集成地分析vPOL与宿主的相互作用。了解人类系统中的这些病毒和细胞方面不仅将提供对早期感染事件的第一次窥探，而且还将导致关于人类体内易感性的新假说。值得注意的是，我们为这项提议开发的新方法将广泛适用于研究其他类型的病毒。

 DESCRIPTION (provided by applicant): Influenza A virus is a major human respiratory pathogen that causes seasonal epidemics and occasional pandemics. The single-stranded negative-sense influenza virus genome consists of 8 segments, each flanked by short conserved elements at their termini, which form the viral promoter that is recognized by the viral RNA-dependent RNA polymerase (vPOL). Mutations in vPOL and factors that affect vPOL activity can cause adaptation of avian viruses to the human host. vPOL is essential for viral biogenesis because of its central role in transcription and replication of the viral genome. vPOL also severely impacts host transcription as it cleaves the capped ends of nascent cellular RNAs to prime viral transcription, a process known as Cap-snatch. The basis for these distinct vPOL activities, as well as the cellular factors that can affect its processivity in host cells still reain largely unknown. To address this, we developed reporter viruses that encode tagged vPOL subunits that do not adversely affect their infection potential. We will use these to characterize the vPOL interactome during productive infection, and to characterize the importance of epigenetic regulators and newly identified factors for influenza virulence (Aim 1). Moreover we will, for the first time, study the mechanism and impact of Cap-snatch by genome-wide profiling of the viral-cellular hybrid mRNAs using newly developed next-generation sequencing protocols (Aim 2). Finally, we will examine vPOL processivity, which may provide new insights into differential regulation of viral gene transcription during infection (Aim 3). Our proposal comprise the first comprehensive and integrated analysis of vPOL-host interactions at the genome, transcriptome and interactome level. Understanding these viral and cellular aspects in a human system will not only provide the first glimpse into early infection events, but also lead to new hypotheses for in vivo susceptibility in humans. Notably, the novel methodologies we developed for this proposal will be widely applicable to studying other types of viruses.