PSiFlu

The aim of our project is to unravel the molecular mechanisms that control packaging of the genome of Influenza A viruses (IAVs). In addition to its fundamental importance, it could have enormous impact on human - and animal - health, by greatly improving the process of vaccine production and helping to predict the emergence of pandemic strains. Influenza A viruses are responsible for recurrent flu epidemics and occasional devastating pandemics. Evolution of seasonal viruses and emergence of pandemic viruses are greatly facilitated by the IAVs segmented genome consisting of 8 (-) sense RNAs (vRNAs), which allows rapid evolution by genetic reassortment but complicates its assembly, as IAVs must incorporate at least one copy of each vRNA to be infectious. The prevailing consensus is that vRNAs are selectively packaged into budding virions. This model has important implications for genetic reassortment and for the design and production of vaccines, yet little is known about the molecular mechanisms underlying specific packaging. Whilst the existence of cis-acting packaging signals has been demonstrated in all 8 vRNAs, they have only been incompletely delineated. A longstanding hypothesis is that direct vRNA/vRNA interactions within packaging signals govern selective packaging but progress in demonstrating it has been slow and limited. In an on-going collaboration to address this issue, the Marquet and Rosa-Calatrava teams combined in vitro assays with virology approaches and electron tomography to demonstrate that the 8 vRNAs of IAVs build up a single network of interactions. Importantly, they further showed that an in vitro identified vRNA/vRNA interaction is required for optimal replication of the H5N2 IAV and is involved in the selective co-packaging of the interacting vRNAs. This is the strongest evidence so far indicating that the “vRNA/vRNA interaction hypothesis” is correct. We will extensively and precisely define the vRNA primary and secondary structures of all the cis-acting IAV packaging signals. Rather than testing the effect of a limited number of mutations, usually large deletions, on viral replication, we will use a multidisciplinary approach combining molecular biology (Marquet and Rosa-Calatrava teams), virology (Marquet and Rosa-Calatrava teams) and microscopy (Rosa-Calatrava and Bron teams). First, we will apply an innovative systems biology approach (Marquet team, Smyth et al., revised manuscript under review at Nature Methods), based on the random mutation of each nucleotide of each vRNA and selection of the functional molecules by the viral machinery itself, to define, in an exhaustive and unbiased manner, the primary and secondary structure elements of each vRNA important for packaging. We will then identify and validate intermolecular vRNA/vRNA interactions and test their functional role in viral replication, co-packaging of the partner segments and viral assembly, using in vitro, in cellulo and in viro approaches. The influenza A/PuertoRico/8/34 (H1N1) strain (PR8), used for vaccine production, will be our working model. Finally, the robustness of the vRNA interaction network will be tested by electron tomography, by comparing the internal organization of PR8 virions with that of reassortant viruses where PR8 segments have been replaced by foreign segments of vaccinal interest. Our ultimate goal is to understand the rules underlying genetic reassortment of IAVs. This research will help to evaluate the likelihood of generating reassortant viruses, including those with pandemic potential. Importantly, successful completion of this project should also pave the way for improving the generation, selection and replication of reassortant viruses used for vaccine production. The strength of the proposal relies on the existing collaboration between the Marquet and Rosa-Calatrava teams and on the Bron team, with specific expertise in structural analysis of viruses.