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

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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.