FIG 6.

Effect of A/Viet Nam/1203/2004 H5N1 PA-X mutations in PA polymerase activity. Human HEK293T cells (96-well plate format, 1 × 10⁴ cells/well, triplicates) were transiently cotransfected with 31.25 ng of A/Viet Nam/1203/2004 H5N1 pCAGGS expression plasmids encoding the minimal components for viral genome replication and gene transcription (PB2, PB1, and PA) and NP containing a HA epitope tag, together with 62.5 ng of pPOL-I vRNA-like expression plasmids encoding Gluc or GFP under the control of the human polymerase I promoter, and 12.5 ng of the SV40-Cluc plasmid to normalize transfection efficiencies. At 24 hpt, viral replication and transcription were analyzed by GFP expression under a fluorescence microscope (A) and quantified by luminescence (B). Gluc activity was normalized to that of Cluc, and the data are represented as relative activity to that of PA WT (100%). Data represent the means and SDs of the results determined from triplicate wells. ****, P < 0.0001 (PA WT versus PA mutants), determined using one-way ANOVA. Experiment was performed three times with similar results. (C) PB2, PB1, PA, and NP protein expression levels from cell lysates were evaluated by Western blotting with a specific PAb against the HA epitope tag. A MAb against β-actin was included as a loading control. The sizes of molecular markers (kDa) are noted on the left.