Replicon and in vitro RNA-dependent RNA polymerase (RdRp) activities of the chimeras of H5N1 Cambodia PA with PR8 and WSN. A: Replicon activity of the chimeric influenza virus ribonucleoproteins (RNPs) of WSN and PR8 with H5N1 Cambodia PA. The relative replicon activities of WSN and PR8 with H5N1 Cambodia PA in 293 T cells were compared with those of WSN and PR8. The combination of PB2, PB1, PA, and NP is indicated below the graph. W, WSN; P, PR8; C, H5N1 Cambodia. As a negative control, PR8 replicon without PA was used. B: Purified influenza virus PR8 RdRp (P) and PR8PB2-PR8PB1-C-PA RdRp (C). Each RdRp (5 pmol) was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 7.5% gels and Coomassie brilliant blue staining. The positions of PB1, 18 × His-PB2, and PA are indicated on the left. C: Western blot of the purified influenza virus chimeric RdRp. Each RdRp (5 pmol) was electro-blotted onto the nitrocellulose membrane. The PA was detected by western blotting with anti-PA antibodies. The position of PA is indicated on the left. D and E: Comparison of the chimeric RdRp activities in in vitro transcription and replication of v84 with those of PR8. The v84 model template RNA (200 nM) was transcribed by PR8RdRp (P) or PR8PB2-PR8PB1-C-PA RdRp (C) with or without (de novo) 0.1 mM ApG (ApG) or 0.5 μg globin mRNA (mRNA). F and G: Comparison of chimeric RdRp activity in in vitro replication of c84 with that of PR8. The c84 model template RNA was incubated with or without ApG (de novo). Products were analyzed by PAGE on 6% gels containing 8 M urea and the images analyzed with a Typhoon Trio plus. Examples of the images are shown in D and F. The size of the product RNA is indicated on the left. The mean and standard deviation (error bar in the graph) of the polymerase activity relative to that of PR8 RdRp were calculated from 2 independent measurements of 3 different RdRp preparations. Statistical significance was evaluated with Student’s t-test. *p < 0.05, ***p < 0.005.