Computational protocol: Interferon-Induced Genes of the Expanded IFIT Family Show Conserved Antiviral Activities in Non-Mammalian Species

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Protocol publication

[…] The IFIT sequences were searched using the zebrafish genome assembly version Zv9 (www.ensembl.org/Daniorerio/), exploiting the conservation of synteny between the human and zebrafish genomes. The sequences were confirmed through PCR amplification using specific primers () to obtain the full-length open reading frame (ORF) of each gene. The PCR products were subcloned into a pCR3.1 vector (Invitrogen) and transformed into One Shot TOP10F’ competent cells (Invitrogen) for subsequent sequencing and ORF confirmation.The identity and similarity analysis between the zebrafish, human and mouse IFIT sequences was performed using MatGAT . The TPR distribution was analyzed using TPRpred (http://tprpred.tuebingen.mpg.de/) , and the theoretical isoelectric point (pI) and the calculated molecular weight were determined using ExPaSy tools (http://us.expasy.org/tools). The 3D-structure of zebrafish IFITs was predicted using the I-TASSER server , selecting the model with the best C-score, and viewed through PyMOL (http://www.pymol.org). The Template Modeling Score (TM-score), a measure of structural similarity between two proteins, was also analyzed to identify structural analogs with known crystal architecture in the Protein Data Bank (PDB; http://www.rcsb.org/pdb/). [...] IFIT-family protein sequences were retrieved from the NCBI Protein, Uniprot and Ensembl databases based on annotation. The sequences were subsequently complemented using a blastp search for homologs in different databases. The initial sequence alignment was performed using the MAFFT online server following an E-INS-i strategy . The resulting alignment was pruned using Gblocks 0.91b and subsequently analyzed using ProtTest 3.2 to determine the best-fit amino acid replacement model using the Akaike Information Criterion (AIC) , specified to estimate the maximum likelihood gene tree using PhyML 3.0 . The nodal confidence was calculated using the aLRT method . Edition and representation of the obtained tree was performed in FigTree v1.3.1 (http://tree.bio.ed.ac.uk/software/figtree/).An estimation of the rates of synonymous (silent) and nonsynonymous (amino-acid-changing) substitutions was performed to identify a positive Darwinian selection in the zebrafish IFIT family using the PAML package version 4 . The maximum-likelihood (ML) approach was implemented in the CODEML program to determine the ω value among zebrafish IFITs. […]

Pipeline specifications

Software tools MatGAT, TPRpred, I-TASSER, PyMOL, BLASTP, MAFFT, Gblocks, ProtTest, PhyML, FigTree, PAML
Databases NCBI Protein ExPASy
Applications Phylogenetics, Protein sequence analysis
Organisms Danio rerio, Homo sapiens
Diseases Virus Diseases