Figure 5  Overexpressed eEF1A or/and NS5A inhibits the CSFV internal ribosome entry site (IRES) activity. (A) eEF1A inhibitory effect on the CSFV IRES activity in a dose-dependent manner. The plasmids pMyc-eEF1A (0, 0.25, 0.5 or 1 μg), pFluc/IRES/Rluc (0.75 μg) and pLXSN-T7 (0.3 μg) were cotransfected into HEK293T cells. The reporter gene activity was detected and expressed as fold induction. Protein expression of Myc-tagged eEF1A was examined by Western blotting using a rabbit anti-Myc polyclonal antibody (PAb) (1:500); (B) NS5A inhibitory effect on the CSFV IRES activity in a dose-dependent manner. The plasmids p3×Flag-NS5A (0, 0.25, 0.5 or 1 μg), pFluc/IRES/Rluc (0.75 μg) and pLXSN-T7 (0.3 μg) were cotransfected into HEK293T cells. The reporter gene activity was detected and presented as fold induction. Western blotting was performed using a mouse anti-Flag monoclonal antibody (MAb) (1:1000) to verify the expression of 3×Flag-NS5A; (C) Effects of eEF1A on the NS5A-mediated inhibition of the CSFV IRES activity. Examination of the CSFV IRES activity using a luciferase reporter assay. Luciferase reporter plasmids pFluc/IRES/Rluc (0.75 μg) and pLXSN-T7 (0.3 μg) were cotransfected into HEK293T cells with or without p3×Flag-NS5A (0.5 μg) and pMyc-eEF1A (0.5 μg). The reporter gene activity was detected and presented as fold induction. To prove the expression of 3×Flag-NS5A and Myc-eEF1A, Western blotting was performed using anti-Flag (1:1000) and anti-Myc (1:500) antibodies. GAPDH was included as an internal control. Rluc level represented the CSFV IRES activity. The Fluc gene under the control of the T7 promoter was used as an internal control. p-values were indicated above the bars. The data were averaged from six replicates of two independent experiments.
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