Figure 5

Alanine substitutions in the putative diacidic motif in ORF6 exert an effect on impedance of nsp8 expression. (A) A schematic of the ORF6 amino acid sequence showing the position of two putative motifs, a lysosomal targeting motif and a diacidic motif. (B) Vero E6 cells were transfected with 1 μg of pXJ40myc-nsp8 and either 0 μg, 1 μg, 2 μg of either pXJ3'-ORF6 (left panel), pXJ3'-ORF6A49-52 (centre panel) or pXJ3'-ORF6A53-56 (right panel). 16 hours post transfection, cells were harvested, lysed and subjected to Western Blotting using an antibody directed against either ORF6 or myc-nsp8. Actin was used as a loading control. Similar results were obtained in three independent experiments, and a representative set of data is presented. (C) Vero E6 cells were similarly transfected as in (A) and total RNA was harvested 16 hours later, reverse transcribed and subjected to SYBR-Green QPCR using specific primers for nsp8, with GAPDH as a housekeeping control. Relative quotients were calculated and plotted from the Ct values obtained, using the ΔΔCt method. The average values with standard deviations are plotted and the standard deviations are also shown in parentheses in the x-axis labels. Students' T-test was performed on selected results and the p-values are indicated. P < 0.05 was considered statistically significant. Similar results were obtained in five independent experiments, and a representative set of data is presented.