Figure 1

WNV replicon experiments. (A) Schematic construction of DNA based subgenomic WNV IS98 replicon models with mutations in the PBM of NS5. Rep-IS98-Gluc is a replicon containing the non-structural genes of WNV while the deleted structural genes were replaced by the non-secreted form of Gaussia luciferase (Gluc) reporter gene under the control of Sp6 promoter. The PBM sequence is indicated in green, a (−TVL) motif at the C-terminal extremity of NS5 (Rep-IS98-Gluc-wt). Three other replicons were constructed: a negative control replicon (Rep-IS98-Gluc-GVD), a replicon with a deletion of the three residues –TVL– (Rep-IS98-Gluc-∆PBM) and a mutagenized PBM in the replicon (Rep-IS98-Gluc-TVM: mPBM). (B). Replication of IS-98-Gluc replicons into BHK-21 cells. Representative replication curves after transfection into BHK-21 cells are plotted for replicons expressing different NS5 sequences: Rep-IS98-Gluc-wt (WT), Rep-IS98-Gluc-TVM (mBPM), Rep-IS98-Gluc-∆PBM (∆PBM) and a negative control replicon Rep-IS98-Gluc-GVD (GVD). The cells were lyzed for subsequent analysis post-transfection. Relative expression of luminescence was normalised to the luminescence obtained 4 h post-transfection. Average arbitrary luminescence units ± SEM is shown from 4 wells per group; experiments were done in triplicate. (C). Western blot showing NS5 protein levels in BHK-21 cells transfected with no replicon (medium only), wt replicon or mPMB replicon. Western blot of lysates from transfected BHK-21 cells at 48 h post electroporation of medium only (negative control/no replicon), mPBM replicon or wt replicon as indicated. The Western-blot is performed with the anti-NS5 antibody. Uncropped WB is shown in Supplementary Fig. 3.