We report here the identification of 108 human proteins that interact with flavivirus NS3 or NS5 proteins or both. Based on our Y2H screen results, we created the first flavivirus NS3 and NS5 proteins interaction network composed

of 186 interactions and involving 120 distinct human proteins. Analysis of this LY2109761 virus-host interaction network revealed the topological features of the cellular proteins targeted by the flavivirus NS3 and NS5 proteins and identified functional pathways related to flavivirus biology. Methods Plasmid DNA contructs Coding sequences for NS3 and NS5 Flaviviruses full-length proteins or NS3 helicase, NS3 protease, NS5 polymerase and NS5 methyltransferase functional domains were provided in pDONR207 entry vector (Gateway, Invitrogen) by Bruno Coutard (Architecture LY3023414 et Fonction des Macromolécules Biologiques, UMR6098, Marseille) and referenced in ViralORFeome database [17]. The viral ORFs were isolated from the following viruses: dengue virus serotype 1 (strain D1/H/IMTSSA/98/606), Alkhurma virus (strain 1176), West Nile virus (Strain paAn001), Japanese Encephalitis

virus (strain Beijing1), Kunjin virus (MRM61C) and Tick borne encephalitis virus (strain 263). Cellular ORF coding for AZI2 was purchased from Invitrogen (clone IOH41551) and coding sequences for click here NFKBIA, and TRAF4 were obtained from MYO10 the Human ORF Collection (OHS4187, Open Biosystems). Viral and cellular coding sequences were subsequently transferred by in vitro recombination from pDONR207 into different Gateway-compatible destination vectors following manufacturer’s recommendation (LR cloning reaction, Invitrogen). To perform yeast-two hybrid experiments, human prey coding sequences were recombined into pACT2 (Invitrogen) to be expressed in fusion downstream of the activation domain of Gal4 (Gal4-AD) and viral bait coding sequences into pGBKT7 to be expressed in fusion downstream of the DNA binding domain of Gal4 (Gal4-BD). In mammalian cells, GST-tag and 3xFLAG-tag fusions were achieved using pDEST27 (Invitrogen), or pCI-neo-3XFLAG (kindly

provided by Y. Jacob Institut Pasteur) vectors, respectively. Yeast two-hybrid assay Viral cDNAs cloned into bait Gal4-BD vector pGBKT7, were transformed into AH109 yeast strain (Clontech) and used to screen by mating human cDNA libraries from liver, brain, spleen and bronchial epithelia cloned in the GAL4-AD pACT2 vectors, and transformed into prey Y187 yeast strains. The mating between baits and prey yeast cells was performed on a selective medium lacking histidine and supplemented with 10 mM 3-amino-triazole (3-AT; Sigma-Aldrich). After 6 days of culture on selective medium, [His+] diploids colonies were isolated and further selected over 3 weeks by culture on selective medium to eliminate false-positives colonies.