Figure 4.

NSP2 impairs miRNA-mediated silencing

(A) NSP2 decreases let7a-mediated silencing. The upper panel shows a schematic of the RLuc-6let7a reporter mRNA. HEK293T cells were co-transfected with RLuc or RLuc-6let7a plasmids, along with FLuc construct to account for variations in transfection efficiency. Vectors encoding Flag-NSP2 or Flag (empty vector) were also added in the transfection mixture. Repression fold was calculated by dividing the relative luciferase activity of the cells transfected with the RLuc vector by the luciferase activity of RLuc-6let7a expressing cells. Error bars indicate ±SD (n = 3). (∗∗) p < 0.01 (two-tailed Student’s t-test).

(B) Artificial tethering of GW182^SD 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. HEK293T cells were co-transfected with vectors expressing either λNV5- GW182^SD, WT, or a ΔPPGL mutant (Mut), or λNV5 as a control, along with RLuc-5boxB 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. The mean values (±SD) from three independent experiments are shown and the p value was determined by two-tailed Student’s t-test: (ns) non-significant, (∗∗∗) p < 0.001.

(C) Artificial tethering of GW182^SD 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. Transfections were performed as in (B), except that GW182^SD was tethered on the RL-5boxB-A₁₁₄-N₄₀-HhR reporter. Data are presented as mean ± SD (n = 3). (∗∗∗) p < 0.001 (two-tailed Student’s t-test).

(D) Model of NSP2-mediated negative regulation of miRNA function. In absence of NSP2 (left panel), miRISC recruits the 4EHP-GIGYF2 complex to effect translational silencing of the targeted mRNA. Following NSP2 expression (right panel), the function of 4EHP-GIGYF2 is physically targeted by NSP2 and its silencing capacity is impaired. The assembly of this complex subsequently alters the magnitude of miRNA-induced silencing.