Journal of General Virology

Fig. 3. Effect of NS1, NS1 DT and NS1 DOT on reporter gene expression driven by either the P38 promoter (A, B) or the CMV early promoter (C, D). (A, B) Cultures of 1.5x10⁵ A9 cells were co-transfected with 50 ng of the reporter plasmid P38-Luc and indicated amounts of effector plasmid pX-NS1wt, pX-DT-NLS or pX-DOT-NLS. Effector plasmids were given either separately (A) or in combination (B). The DNA inoculum was adjusted to 400 ng with the control plasmid pX. Transfected cultures were incubated for 48h and assayed for luciferase activity. The relative light units (RLU) measured for the P38 promoter-driven reporter plasmid co-transfected with each effector are shown (A) and the fold induction of the P38 promoter is shown for the different effectors (B). (C, D) The CMV early promoter-driven reporter construct pCMV SEAP was used to measure the transinhibiting effects of wt or mutant NS1-expressing plasmids that were inoculated either separately (C) or in combination (D). All transfection mixtures were adjusted to 600 ng of effector plasmid with the empty vector pX. SEAP activity was measured with the Phospha-Light kit (Tropix), according to the manufacturer's instructions. The RLU measured for the CMV promoter-driven reporter plasmid co-transfected with each effector are shown (C) and the fold inhibition of the CMV promoter is shown for the different effectors (D). Data are average values from three independent transfections, each carried out in triplicate.

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