Cloning PDZ Domains and NS5 Proteins

All PDZ domains (257/266 PDZ) and ORFs containing PDZ domains (91 full-length proteins out of the 152 human proteins) were introduced into Gateway destination vector pYN2H-LEU-N1^{45 (link)} whereas NS5 and NS5∆PBM (NS5 with a deletion of the three residues –TVL–) were introduced into Gateway destination vector pYN2H-TRP-N2, via LR clonase-mediated Gateway reaction (Life Technologies), following the protocol previously described^{46 (link)}. Briefly, LR reaction products were subsequently transformed into DH5α competent bacterial cells and grown for 24 h at 37 °C at 900 rpm in carbenicillin-containing TB liquid medium. Plasmid DNA was extracted using a NucleoSpin 96 Plasmid kit from Macherey–Nagel, following manufacturer's instructions. All DNA were sequenced using plasmid-specific primers.