Global Landscape Of H5N1 AIV-chicken Protein Complexes

H5N1 avian influenza virus(AIV)belongs to the highly pathogenic influenza virus, posing a great threat to poultry production and public health. IAV is a single-stranded, negative-sense RNA virus, consisted of eight gene fragments such as HA, PA and NS. Influenza virus cannot replicate by itself, it depends heavily on the host cell machinery. At present, the research on protein structure and function of AIV have made great progress, but the identification and function analysis of the key host proteins in the process of AIV infection remains to be further studied.In this study, by constructing of expression vector of viral genes with the cDNA of H5N1 AIV A/Chicken/ShanXi/2/2006 strain, we captured and analyzed their host interaction proteins by affinity purification and mass spectrometry(AP-MS) and analyzed the biological characteristics of AIV–chicken interaction. The main findings are as follows:1. We constructed 8 viral protein expression vectors using pcDNA3.1- C-3×Flag- plasmid, then transfected chicken embryo fibroblast(DF1) with Lipofectamine 3000. Western blot results showed that all the expression vectors could be efficiently expressed in chicken cells.2. Viral-host protein complexes were enriched by immunoprecipitation with FLAG antibody, and significance analysis of interactome(SAINT) which was widely accepted for protein interaction scoring was used to assign confidence scores to protein-protein interaction(PPI) data generated using AP-MS. PPIs with score more than 0.9 were considered to be high confidence PPIs. Totally 1115 proteins were found to interact with H5N1 AIV. The number of host interacting proteins of PA, PB1, PB2, NP, HA, NA, NS1, M1 were: 134, 36, 595, 64, 238, 164, 145, 53. We constructed the viral-chicken interaction network based on the high confidence PPIs generated by AP-MS, and we also drawn the cellular structure pattern of the important host interacting proteins involved in the viral life process.3. Domain enrichment of viral-chicken interacting proteins was analyzed by FunRich, Our results indicated that host proteins with domains of Cation ATPase N, HELICc, DEXDc, TPR tended to have physical interactions with H5N1 AIV; GO and Pathway analysis showed that proteins which have the biological functions such as nucleic acid binding, ATP binding, protein binding, and proteins related to signaling pathways of proteasome, ribosome, cell cycle and cell apoptosis were significantly enriched in viral-host interactions, suggesting the potential roles of these proteins in with the infection of H5N1 AIV.4. 210 of 1115 host interaction proteins in this study were identical with host interacting proteins of H1N1 influenza virus, revealing the conservation of the viral-host protein interaction between different subtype of the influenza virus.5. Immunoprecipitation combined with Western Blot confirmed that chicken microtubule dynamics active protein WDR63 interacted with viral polymerase protein PB2, double-stranded RNA binding protein STAU2 interacted with viral nonstructural protein NS1, which laid a solid foundation for the mechanism analysis of functional PPIs on virus infection.