The H5N1 NS1 L103 and I106 mutations affect binding of NS1 to human RIG- I CARD, helicase and RD domains in the bacterial reverse 2- hybrid system. a. Drop spotting of the H5N1 influenza NS1 RTHS strains on selective media. Serial dilutions (2.5 μL of each sample containing ~10n cells/mL is indicated) of the NS1 RTHS strains (and controls), were drop spotted onto selective media containing 0, 25 or 50 μM IPTG. In the absence of IPTG, there is no expression of the targeted fusion proteins; all strains therefore grow normally (top row of plates). Upon addition of IPTG (2nd and 3rd row of plates), the functional 434.P22 repressor will be reconstituted in those RTHS that contain interacting proteins (Additional file 1: Figure S1), leading to cell death. The strains containing proteins that do not interact (e.g. L103F, I106M) continue to grow normally in the presence of IPTG. b. Dose response for IPTG induction of proteins for RIGI domain binding of H5N1 and H3N2 NS1 L103 and I106 mutants as well as H1N1 and H3N2 wt NS1 proteins. RTHS constructs that possessed each of the wt H5N1 (A/HK/156/1997), H3N2 (A/HK/1/68) and H1N1 (/Brevig Mission/1/1918) or indicated 103 and 106 mutant for the H5N1 and H3N2 viruses were tested for survival under selective medium on induction with 0, 5, 10, 25 and 50 μM IPTG. The number of surviving bacterial colonies were determined by serial dilution as shown in panel A and plotted as bar graphs. NS1 proteins that do not bind the indicated RIG-I domain baits (CARD, helicase and RD) were not reduced in survival relative to the non-induced controls (0 μM IPTG).