Effect of PB1-F2 stabilization on the activity of vRdRp.

<p>(A) A minigenome reporter assay was used for the quantification of the effect of PB1-F2C-terminal K cluster mutation on the activity of vRdRp. The 293T cells were transfected with a set of expression plasmids for the vRdRp subunits pPB1, pPB2, pPA, and pNP, pRLTK (transfection efficiency normalization) and the reporter plasmid pPolPR8MLuc, encoding the firefly luciferase gene under the control of the IAV matrix protein gene segment promoter. Additionally, pPB1-F2 PR8, pPB1-F2 PR8 3KR, pPB1-F2HK97, or pPB1-F2 HK97 4KR plasmids were co-transfected. At 48 hr p.t., the samples were harvested and the activities of luciferases were quantified using a Dual Luciferase Reporter Assay System kit (Promega) on a BIOTEK Synergy HT luminometer. The samples were measured in triplicate and normalized to renilla luciferase activity (transfection efficiency) and firefly luciferase activity (vRdRp activity) in samples co-transfected with pPB1-F2 PR8. The data represent 3 independent experiments. The bars indicate the average value of the independent experiments, and the error bars indicate the intra-experiment SD values. The asterisk indicates a significant difference between the groups compared (<i>P<0</i>.<i>05</i>). (B) PB1-F2 protein and Beta-actin were detected using western blotting to compare the relative activity of vRdRp with the relative amount of the PB1-F2 in the same samples.