To better understand how hPIV3-C targets both the IFN-a/b and EGF signaling pathways, we characterized the functional domains of hPIV3-C that bind STAT1 and GRB2. To do so, we generated by PCR a full matrix of hPIV3-C overlapping fragments and tested their ability to interact with either STAT1 or GRB2 in the yeast two-hybrid system ( Figure 5 and 6 ). Both forward and reverse primers were designed every 75 nucleotides along hPIV3-C sequence and fused to appropriate tails to allow gap-repair recombination with linearized Gal4-DB yeast two-hybrid vector ( Figure 5A ). All possible combinations of forward and reverse primers were used to amplify hPIV3-C fragments. Finally, corresponding PCR products were transformed in a yeast strain expressing Gal4-AD fused to either STAT1 or GRB2, and growth on selective medium was used to detect potential interactions. A 124 (AA)-long peptide encompassing position 76 to 199 located in the C-terminal half of hPIV3-C was sufficient to bind STAT1 ( Figure 5B ) or GRB2 ( Figure 6A ). In an iterative process, we then generated a second, a third and a fourth set of hPIV3-C fragments corresponding to one-by-one AA deletions ( Figure 5C -E and Figure 6B -D), allowing to further reduce the STAT1 and GRB2 binding motifs to minimal peptides. A 106 AA peptide encompassing residues 90 to 195 of hPIV3-C was sufficient to observe the interaction with STAT1 ( Figure 5E ). The binding region to GRB2 was virtually the same, encompassing AA 97 to 195 ( Figure 6D ).