FIG 2.

Effects of mutations introduced into CE(NiP)ΔP2-5 on viral replication and activity of P1 as a cofactor of viral RNA polymerase. (A) Growth curves of CE(NiP) and CE(NiP)ΔP2-5 in NA cells. Each virus was inoculated into NA cells at an MOI of 0.01. Viral titers in culture supernatants collected at 1, 3, and 5 dpi were determined by focus assays. (B) GFP-tagged P1s in NA cells transfected with pEGFP-P1 and pEGFP-P1ΔP2-5 together with the respective isoforms expressed in transfected cells were analyzed by Western blotting. Tubulin in each sample was also detected as a loading control. (C) Minigenome assay to compare polymerase cofactor activities of P1s of CE(NiP) and CE(NiP)ΔP2-5. NA cells were transfected with empty plasmid, pEGFP-P1, or pEGFP-P1ΔP2-5, together with plasmids expressing luciferase-based minigenome RNA and viral N and L proteins. At 48 h posttransfection, luciferase activities in cell lysates were measured. All assays were carried out in triplicate, and the values in the graph are shown as means ± standard errors of the means. ns, not significant (P ≧ 0.05).