Figure 3.

NSP2 decreases the silencing capacities of GIGYF2 in cellulo

(A) Artificial tethering of GIGYF2 to the 3′ UTR of a reporter mRNA. The upper panel shows a schematic of the λN/BoxB tethering assay with the RLuc-5boxB reporter construct. Recruitment of GIGYF2 to the Renilla luciferase (RLuc) mRNA was mediated by the fused λN peptide. RLuc luminescence was normalized against firefly luciferase (FLuc) level, and repression fold was calculated by dividing the relative luciferase activity of the cells transfected with the control pCI-λNV5 vector (λNV5) by the luciferase activity of λN-GIGYF2-expressing cells. The mean values (±SD) from three independent experiments are shown and the p value was determined by two-tailed Student’s t-test: (∗∗∗) p < 0.001.

(B) Artificial tethering of GIGYF2 to the 3′ UTR of a reporter mRNA which is refractory to deadenylation. The upper panel shows a schematic of the RLuc-5boxB-A₁₁₄-N₄₀-HhR reporter. HEK293T cells were co-transfected with vectors expressing either λNV5-GIGYF2, or λNV5 as a control, along with RLuc-5boxB-A₁₁₄-N₄₀-HhR and FLuc. Vectors encoding Flag-NSP2 or Flag (empty vector) were also added in the transfection mixture. RLuc luminescence was normalized against the FLuc level and analyzed as in (A). (∗∗∗) p < 0.001 (two-tailed Student’s t-test).

(C) Extracts from the HEK293T cells used in (A) and (B) were analyzed by Western blot with the indicated antibodies. GAPDH was used as a loading control.