Decoding the vRNP interaction network of influenza A viruses required for genome packaging

甲型流感病毒(IAV)是反复流行的流感和偶尔的破坏性大流行的罪魁祸首。它们由8个vRNA组成的分段基因组有利于进化，但需要一种复杂的包装机制，目前仍知之甚少。我们的工作和其他人的工作表明，IAV的节段基因组建立了一个由冗余的vRNA-vRNA相互作用网络连接在一起的超分子复合体，并揭示了vRNA包装信号与病毒核蛋白(NP)之间的复杂相互作用，这表明每个病毒核糖核蛋白(VRNP)都有自己的密码。我们的目标是在vRNA和NP水平上解码vRNP相互作用网络。为此，我们将把vRNPs的结构探测与几种化学探针以及野生型和已定义突变病毒颗粒内的RNA-RNA交联结合起来，并由于vRNAs或NP的突变而改变包装决定因素。我们的项目结合了三个关键方面，扩展了之前的研究。首先，通过对野生型和突变型病毒的结构分析，我们将克服识别vRNA-vRNA和vRNA-NP相互作用的困难，这些相互作用对于包装IAV基因组至关重要，而这些相互作用是固有的。其次，用完整和分解的病毒颗粒获得的RNA探测和RNA交联数据的系统比较将允许明确识别相邻vRNPs之间的相互作用。第三，通过用几种与RNA不同部分反应的试剂进行RNA探测，我们将能够区分vRNA-vRNA相互作用和vRNA-NP相互作用。总之，我们的项目应该提供原则上的证据，证明特定的RNA-RNA和/或RNA-蛋白质相互作用对于IAV基因组包装过程是必不可少的，并从结构上阐明这些相互作用的可塑性。

Influenza A viruses (IAVs) are responsible for recurrent flu epidemics and occasional devastating pandemics. Their segmented genome consisting of 8 vRNAs favors evolution but requires a complex packaging mechanism that remains poorly understood. Our work and work by others indicate that the segmented genome of IAVs build a supramolecular complex held together by a redundant vRNA-vRNA interaction network and reveals a complex interplay between the vRNA packaging signals and the viral nucleoprotein (NP), suggesting that each viral ribonucleoprotein (vRNP), formed by the association of a vRNA with a polymerase complex and multiple copies of NP, has its own code. Our aim is to decode the vRNP interaction network at the vRNA and NP levels. To that goal, we will combine structural probing of vRNPs with several chemical probes and RNA-RNA crosslinking inside particles of wild type and defined mutant viruses with altered packaging determinants due to mutations in vRNAs or NP. Our project extends previous studies by combining three crucial aspects. First, by performing our structural analysis on wild type and mutant viruses, we will overcome the difficulty of identifying vRNA-vRNA and vRNA-NP interactions crucial for packaging of the IAV genome that is inherent to the redundancy of these interactions. Second, the systematic comparison of the RNA probing and RNA crosslinking data obtained with intact and disassembled viral particles will allow the unambiguous identification of interactions between adjacent vRNPs. Third, by performing RNA probing with several reagents that react with different parts of RNA, we will be able to discriminate vRNA-vRNA interactions from vRNA-NP interactions. Altogether, the our project should provide the proof of principle that specific RNA-RNA and/or RNA-protein interactions are essential for the IAV genome packaging process and shed a structural light on the plasticity of these interactions.