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

The FluCode project is based on two complementary methodological approaches. On the one hand, the SPLASH method is based on the crosslinking of regions of the vRNAs that are spatially close, followed by digestion of RNA by RNases and the analysis of the crosslinked regions by high throughput sequencing. This approach makes it possible to identify the regions of two different vRNAs that are close to each other in the viral particles, and therefore the contacts between vRNAs, but also the regions of the same vRNA that are spatially close due to its tridimensional structure. The identified contact areas can range from about 50 to 200 nucleotides. On the other hand, we compare the reactivity of vRNAs in intact or disassembled viral particles towards several chemical reagents. The modified RNAs are reverse transcribed under conditions inducing mutations in the cDNAs opposite the modified nucleotides. These mutations are then analyzed by high throughput sequencing. The variations in the reactivity of vRNAs when the viral particles are disassembled make it possible to identify contacts between vRNPs. In addition, the combination of several chemical reagents informs us about the nature of these contacts: RNA/RNA or RNA/NP interactions. This vRNA mapping gives information about each nucleotide but does not identify the partner regions of RNA/RNA interactions. This is why it is complementary to the SPLASH method.

The SPLASH method reveals that each vRNA makes multiple contacts with the other vRNAs, establishing a global contact network that encompasses the eight vRNAs. Mutations in the areas of contact abolish crosslinking, reinforcing the idea that the identified regions correspond not only to areas of proximity, but also to areas of interaction. Chemical probing with a SHAPE reagent reveals numerous changes in reactivity during disassembly of viral particles, including increases in reactivity over several consecutive nucleotides, which probably correspond to contacts between vRNPs. Comparison of these data with SPLAH data and bioinformatics identification of all possible stable RNA/RNA interactions within the viral genome identifies a shortlist of potential contacts between vRNAs within viral particles.

Detailed analysis of the SPLASH results on the wild virus is in progress. This methodology will now be applied to viruses carrying mutations in the NP protein or / and packaging signals that cause defects in the incorporation of vRNAs into viral particles. The SHAPE methodology will be applied to the intact and disassembled viral particles of the same mutants. In addition, we will set up chemical probing of all wild and mutant viruses with reagents that modify the RNA bases. The differences between wild and mutant viruses should highlight interactions playing a direct role in the packaging of vRNAs. Comparison of the different chemical probes will allow us to distinguish RNA / RNA interactions from RNA / NP interactions since the NP protein is expected to protect the ribose-phosphate backbone but not the RNA bases. We will then use reverse genetics to test the functional role of some of the interactions identified.

Publications are expected at the end of the project

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.