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

 描述（由申请方提供）：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发性大流行。单链负义流感病毒基因组由8个片段组成，每个片段在其末端侧接短保守元件，其形成由病毒RNA依赖性RNA聚合酶（vPOL）识别的病毒启动子。vPOL的突变和影响vPOL活性的因素可导致禽流感病毒适应人类宿主。vPOL是病毒生物发生所必需的，因为它在病毒基因组的转录和复制中起着中心作用。vPOL还严重影响宿主转录，因为它切割新生细胞RNA的加帽末端以引发病毒转录，这一过程称为Cap-snatch。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持续合成能力的细胞因子仍然在很大程度上未知。为了解决这个问题，我们开发了报告病毒，其编码标记的vPOL亚基，不会对其感染潜力产生不利影响。我们将使用这些来表征生产性感染期间的vPOL相互作用组，并表征表观遗传调节因子和新鉴定的流感毒力因子的重要性（目的1）。此外，我们将首次通过使用新开发的下一代测序方案（Aim 2）对病毒-细胞杂交mRNA进行全基因组分析来研究Cap-snatch的机制和影响。最后，我们将研究vPOL的持续合成能力，这可能为感染期间病毒基因转录的差异调节提供新的见解（目的3）。我们的提案包括在基因组，转录组和相互作用组水平上对vPOL-宿主相互作用的第一次全面和综合分析。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一瞥，而且还将导致人类体内易感性的新假设。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.