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. 2009 Mar 25;83(11):5525–5534. doi: 10.1128/JVI.02289-08

FIG. 5.

FIG. 5.

Transcription activity of the N mutants. (A) Schematic of the plasmid carrying a negative-sense VSV minigenome with eGFP. The trailer (tr) and leader (le) regions of VSV along with the eGFP sequence in the negative-sense orientation flanked by the hammerhead ribozyme (HH) and the hepatitis delta virus ribozyme (δ) are shown. The bent arrows indicate the cleavage sites for the two ribozymes. The T7 RNA polymerase promoter (φ10) and the terminator (Tφ) are also shown in the plasmid. (B) Transcription of a negative-sense minireplicon of VSV expressing eGFP as a reporter for transcription activity. BHK-21 cells were transfected with pP, pL, and pVSVmg-eGFP and with (panel ii) or without (panel i) the plasmid encoding the wt N protein. At 24 hpt, the cells were directly observed for expression of eGFP. (C) Western blot analysis for detection of eGFP as a function of transcription activity of the minireplicon template. The cell lysates from the above-mentioned experiment were subjected to Western blot analysis by using anti-GFP antibody which detects the eGFP in the cells transfected with the plasmids expressing the P protein, the L protein, and the VSVmg-eGFP minireplicon along with the plasmid expressing (lane 2) or not expressing (lane 1) the N protein. (D) Representative fluorescent images of cells transfected with the plasmids expressing the P protein, the L protein, and the VSVmg-eGFP minireplicon along with the plasmid expressing the wt or mutant N proteins. Results for a control experiment in which wt N was not transfected (−N) are also shown. The panel numbers represent the alanine-scanning mutants corresponding to those positions in the N protein. (E) Western blot analysis for detection of eGFP as a function of transcription activity of the mutant N-RNA templates. The cell lysates from the experiment described for panel D were subjected to Western blot analysis. The upper panel shows the levels of eGFP; the lower panel shows the levels of actin determined using antiactin antibody in the Western blot. (F) Transcription activities of the N mutants per unit of the template. The ratios of average transcription activity to average replication activity (as shown in Fig. 2B) are shown for the respective N mutants. The ratio for the wt N protein is set at 1.