Final Report Summary - AVFLU (Interactions between avian and influenza virus proteins) Summary overview of resultsWe focused on influenza A viruses and studied host/pathogen interactions in the avian host model. Indeed, while mammals can be infected by the pathogen either sporadically or with well adapted lineages, aquatic birds are the natural reservoir of influenza A viruses. Increasing our understanding of virus/host interactions may enable us to better identify viral determinants of host range from the protein level and molecular markers of interspecies transmission. So far most of the knowledge on influenza virus and host cell interactions was gained on mammalian models and very little is known on avian cell/influenza virus interactions. We aimed at identifying avian cellular proteins that interact with influenza virus non structural 1 (NS1) proteins. The DF-1 chicken fibroblast cell line (ATCC) and the low pathogenic avian influenza virus A/turkey/Italy/977/1999(H7N1) were selected for the experiments. Co-immunoprecipitatation (co-IP) of NS1 and cellular (avian) proteins were set up and clear differences were observed on SDS page gels between infected and not infected cell lysates after IP. Mass spectrometry (nano-LC-MS/MS available at IPBS, Toulouse) analyses allowed us to identify putative interactors of NS1 in DF-1 cells. Seventeen avian proteins were overexpressed in IP of A/turkey/Italy/977/1999(H7N1) infected as compared to uninfected DF1 cells. Six proteins (with the lowest p values using the one sided paired ratios t-test) were selected. Plasmids expressing each protein of interest with a flag-tag (Genscript® Biotech Corporation) were used to transfect cells and generate cell lines overexpressing the proteins of interest. Among these, 2 cell lines were promising (with clear overexpression of the cellular proteins): SNRPA1 and CTCF expressing cell lines.Our results will allow for a better understanding of host-pathogens interactions as they complement previous reports from the literature. Qiu et al (1995) showed the interaction of U2 small nuclear ribonucleoprotein A' (U2 SNRP, encoded by the SNRPA1 gene) with influenza NS1 in human cells during mRNA splicing. CTCF is involved in cell survival and was shown to play a role in transcription and early viral genes expression and mRNA splicing in human papillomaviruses infections (Paris et al, 2015). Co-IP of NS1 and SNRPA1 as well as of NS1 and CTCF were carried out to confirm the interactions. Replicates of the experiments are still being performed to confirm the first results.The project suffered from both technical and human resources issues, which explain that we are about a year behind with our initial project objectives. We indeed started with a distinct strategy to “fish” the NS1 protein and its interactors in cells, attempting to use a flag-tagged NS1. This first approach did not work out and we then were successful at using carefully selected anti-NS1 antibodies. In addition, while we obtained funding on several other projects within the fellowship period, we were not successful at getting funds to hire a post-doctoral fellow or a PhD student on the project and thus worked with more limited human resources than anticipated. We have now secured technical expertise and reagents to continue on in the coming year.Conclusions Within the framework of the present project we set up tools to study protein-protein interactions in the virology team of IHAP. We identified 2 avian proteins that may interact with the influenza NS1 protein: SNRPA1 and CTCF. Further experiments are ongoing to confirm this hypothesis and enable disseminating the work.The CIG grant has allowed me to establish in Toulouse and combine my expertise acquired in Luxembourg (during my PhD) and in the USA (during my post-doctoral fellowship) with the host laboratory’s niche: the avian model. I have acquired the scientific knowledge and the technical know-how to successfully carry out the present proposal thanks to the financial support of the EU with this CIG grant and thanks to fruitful local and international collaborations. The first half of this CIG project period allowed me to pass my habilitation (“Habilitation à diriger des recherches”, HDR). These 4 years of CIG grants greatly helped me develop my European network and enabled me to write grants and to carry out several other research projects that led to peer-reviewed scientific articles. In addition to having a permanent position in France, I have been promoted to “Directrice de Recherche” (DR2 INRA) as of January 1st 2017.Socio-economic impacts of the projectThe benefits gained from undertaking this project at Community level are at least 3-fold:1) Fundamental: the impact of understanding the interactions between an influenza virus and an avian cell is enormous as aquatic birds are the natural reservoir of the pathogen. 2) Public health: understanding the difference between avian and mammalian protein interactions with influenza virus proteins may allow foreseeing which gene pools (or mutations) are more likely than others to contribute to a pandemic threat. 3) Animal health: avian influenza virus causes major havoc in the poultry industry with huge economic consequences. Since I joined the INRA/ENVT IHAP laboratory I have shared new techniques with the host organization. The present project requires solid skills in cellular virology and I was able to transfer knowledge in molecular biology (RNA/DNA extraction and quantification, reverse transcriptions, real-time and conventional PCRs, sequencing technique (Sanger capillary sequencer for the present project), cloning technology, reverse genetics), cell culture (with primary cells and cell lines), and virus culture (on cells and/or on embryonated chicken eggs). I also shared my knowledge in proteomics techniques: protein extraction and quantification, gel electrophoresis (SDS Page), gel extraction of proteins, basic knowledge of the use of a Maldi (Matrix Assisted Laser Desorption Ionization) Tof Tof (Tof = time of flight) for mass fingerprint analyses and sequencing of peptides. My immediate involvement, as supervisor of students represents, in the framework of this project, a first and valuable opportunity to transfer knowledge in my new hosting unit. So far during the first two years of the project I have supervised Clément Fage for his master’s degree in Microbiology that he obtained with honours in 2014. Clément Fage unfortunately failed obtaining a very competitive PhD scholarship of the graduate school that would have allowed him to continue working on the project but is now doing his PhD in Québec, Canada, still on influenza virus. Since September 2015 I also supervise Charlotte Foret within the framework of her master’s degree on the project. Charlotte is a research technician in our team and decided she would like to pursue her studies in parallel of her technician job. She therefore registered to a specific doctoral school, the “Ecole Pratique des Hautes Etudes” (EPHE) that enables her to work 50% of her time on a designated project for her master (the present project) while still working as a research technician the remaining 50% of her time. Charlotte Foret should defend her master’s degree late 2018. Finally, I transfer knowledge during IHAP Virology bi-monthly lab meetings, bi-monthly journal clubs, and IHAP monthly lab meetings. We also launched “VIROTOUL”: a network organizing seminars (2-3 per year) open to all virologists in Toulouse.
