The 103L and 106I residues in the H5N1- NS gene antagonize poly I: C induced IFN- β and IRF -3 promoter transcriptional activation but differ in CPSF- 30 F2F3 binding. a. H5N1-NS1 proteins (wt and mutants) as well as CPSF30-F2F3-FLAG were expressed by coupled in vitro transcription and translation of expression plasmids or empty vector in the presence of 35S labeled methionine and cysteine with detection by autoradiography. Radiolabeled NS1 proteins or empty vector were mixed with radiolabeled CPSF30- F2F3-FLAG and subject to pull-down using α-FLAG M2 antibody and detected by SDS-PAGE and autoradiography. b. Representative pull-down of 2 independent replicates showing that the I106 mutation abrogated binding but L103 maintained binding. c. IFN-β promoter activation was measured in human 293-T cells in triplicate biological replicates for the H5N1-NS-wt and the three NS mutant (L103F, I106M and L103F+I106M) expression plasmids using a dual-luciferase reporter assay. Each of the NS expression plasmids along with the firefly luciferase p125 under the control of the human IFN-β promoter as well as the renilla luciferase control pRL-SV40 reporter plasmids were transfected in 293T cells. Sixteen hours following transfection, IFN-β promoter activation was stimulated by poly I:C transfection. The luciferase activities were measured following a 24h incubation period and are shown as the ratio relative to the internal renilla luciferase control. d. The same experiment was repeated using the firefly luviferase reporter plasmid under the control of the human IRF-3 promoter. Data represent the means of n = 3 values ± SD (*p<0.05, **p<0.01, *** p<0.001; two-tailed student’s t-test) of firefly luciferase activity relative to renilla.