PMC:4576194 / 14578-18263 JSONTXT

{"project":"2_test","denotations":[{"id":"26266418-19264615-144197540","span":{"begin":167,"end":169},"obj":"19264615"}],"text":"2.5. eEF1A Reduces the Translation Efficiency of CSFV IRES \nA previous study showed that NS5A decreases the CSFV IRES-mediated translation in a dose-dependent manner [13]. To examine the effects of eEF1A on the NS5A-mediated inhibition of the CSFV IRES activity, the luciferase reporter assay was used in this study. The results showed that eEF1A (Figure 5A), as well as NS5A (Figure 5B), inhibited the CSFV IRES activity in a dose-dependent manner. To further investigate the inhibitory effects of eEF1A and NS5A coexpression, eEF1A- and NS5A-expressing plasmids were cotransfected with the luciferase reporter plasmids into HEK293T cells. The results indicated that the eEF1A did not antagonize the inhibition of the IRES activity by NS5A when NS5A and eEF1A were coexpressed (Figure 5C).\nFigure 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.\nFigure 6 eEF1A binds to the CSFV internal ribosome entry site (IRES). (A) eEF1A as well as NS5A interacts with the CSFV IRES. HEK293T cells were transfected with pMyc-eEF1A or pMyc-NS5A. The cell lysate was incubated with the biotinylated CSFV IRES or porcine reproductive and respiratory syndrome virus (PRRSV) 3ʹ-UTR, followed by incubation with streptavidin beads. The bound proteins were analyzed by immunoblotting using the anti-Myc monoclonal antibody (MAb) (1:1000); (B) eEF1A interacts with the CSFV IRES in a dose-dependent manner. HEK293T cells grown in the 6-well plate were transfected with an increased amount of pMyc-eEF1A (0, 1, 2 and 4 μg), followed by streptavidin pulldown assay as described above; (C) Competitive RNA pulldown assay. HEK293T cells grown in the 6-well plate were transfected with the indicated pMyc-eEF1A (4 μg). The cell lysate was incubated with an increased amount of the IRES RNA (0, 0.75, 1.5 and 3.0 μg) for 1 h at 4 °C and incubated with the biotinylated IRES RNA and streptavidin beads. The bound proteins were subjected to immunoblotting using the anti-Myc MAb.\n\n2"}