A proteome-wide mapping of interactions between hepatitis C virus (HCV) and

A proteome-wide mapping of interactions between hepatitis C virus (HCV) and human proteins was performed to provide a comprehensive view of the cellular infection. appeared as a major perturbator of this network. Focal adhesion was identified as a new function affected by HCV, mainly by NS3 and NS5A proteins. (2007). Degree, betweenness and shortest path followed the same tendency with HEBV proteins (Supplementary Table SV and Supplementary Figure S2) and were in good agreement with a previous report (Calderwood and centrality measures (and recombination sites fused to forward and reverse primers, then cloned into pDONR223 (Rual (X-Gal colorimetric assay) and (growth assay on 5-FOA supplemented medium). Positive clones that displayed at least two out of three positive phenotypes were retested in fresh yeasts: bait vectors were retransformed into MAV203 and each prey cDNA (obtained by colony PCR, see below) were transformed in combination with linearized prey vector (gap repair; Walhout and Vidal, 2001). Clones that did not retest were discarded. AD-cDNA were PCR-amplified and inserts were sequenced to identify interactors. IMAP2 screens were performed by yeast mating, using AH109 and Y187 yeast strains (Clontech; Albers et al, 2005). Bait vectors were transformed into AH109 (bait strain), and human spleen and fetal brain AD-cDNA libraries (Invitrogen) SRC were transformed into Y187 (prey strain). Single bait 2831-75-6 strains were mated with prey strain, then diploids were plated on SD?W?L?H+3?AT medium. Positive clones were maintained onto this selective medium for 15 days to eliminate any contaminant AD-cDNA plasmid (Vidalain et al, 2004). AD-cDNAs were PCR-amplified and inserts were sequenced. Text-mining of interactions 2831-75-6 between HCV and human proteins Literature-curated interactions (LCI), describing binary interactions between cellular and HCV proteins, were extracted from BIND database and PubMed (publications before August 2007) by using an automatic text-mining pipeline completed by expert curation process. For the text-mining approach, all abstracts related to HCV’ and protein interactions’ keywords were retrieved, subjected to a sentencizer (sentence partition) and a part-of-speech tagger for gene name (based on NCBI gene name and aliases) and interaction verbs (Rebholz-Schuhmann et al, 2008) (interact, bind, attach and so on). Sentences presenting co-occurrences of at least one human gene name, one viral gene name and one interaction term were prioritized to curation by human expert. Validation by co-affinity purification Cellular ORFs (interacting domains found in Y2H screens) were cloned by recombinational cloning from a pool of human cDNA library or the MGC cDNA plasmids using KOD polymerase (Toyobo) into pDONR207 (Invitrogen). After validation by sequencing, these ORFs were transferred into pCi-neo-3 FLAG gateway-converted. HCV ORFs were transferred into pDEST27 (GST fusion in N-term). A total of 4 105 HEK-293T cells were then co-transfected (6 l JetPei, Polyplus) with 1.5 g 2831-75-6 of each pair of plasmid. Controls are GST-alone against 3 FLAG-tagged prey. Two days after transfection, cells were harvested and lysed (0.5% NP-40, 20 mM TrisCHCl (pH 8.0), 180 mM NaCl, 1 mM EDTA and Roche complete protease inhibitor cocktail). Cell lysates were cleared by centrifugation for 20 min at 13 000 r.p.m. at 4C and soluble protein complexes were purified by incubating 300 g of cleared cell lysate with 40 l glutathione sepharose 4B beads (GE Healthcare). Beads were then washed extensively with lysis buffer and proteins were separated on SDSCPAGE and transferred to nitrocellulose membrane. A total of 50 g of cleared cell lysate was analysed by western blot to check the amount of 3 FLAG-tagged cell protein. GST-tagged viral proteins and 3 FLAG-tagged cellular proteins were detected using standard immunoblotting techniques using anti-GST (Covance) and anti-FLAG M2 (Sigma) monoclonal antibodies. Integrated human interactome network (HCH network) Only physical and direct binary protein-protein interactions were retrieved from BIND (Bader et al, 2003), BioGRID (Stark et al, 2006), DIP (Xenarios et al, 2002), GeneRIF (Lu et al, 2007), HPRD (Peri et al, 2004), IntAct (Kerrien et al, 2007), MINT (Chatr-aryamontri et al, 2007) and Reactome (Vastrik et al, 2007). NCBI official 2831-75-6 gene names were used to unify protein ACC, protein ID, gene name, symbol or alias defined in different genome reference databases (i.e ENSEMBL, UNIPROT, NCBI, INTACT, HPRD and so on) and to eliminate interaction redundancy due to the existence of different protein isoforms for a single gene. Thus, the gene name was used in the text to identify the proteins. Finally, only non-redundant proteinCprotein interactions were retained 2831-75-6 for building the human interactome data set. Topological analysis The R (http://www.r-project.org/) statistical environment was used to perform statistical analysis and the igraph R package (http://cneurocvs.rmki.kfki.hu/igraph/) to compute network connected components, centrality (degree, betweenness) and shortest path measures. The WilcoxonCMannCWhitney rank sum test (the U-test) was chosen to statistically challenge observed differences. The U-test is a non-parametric alternative to the paired Student’s t-test for the case of two related samples or repeated measurements on a single sample. The generalized linear model and.