Fig. 5: Construction of the MITA and MITR modules with the CSFV IRES.

a Schematic illustration of the similar secondary structures of HCV IRES and CSFV IRES. Both HCV IRES and CSFV IRES contained targetable pseudoknot structures adjacent to the translation initiation start codon. b Reduction of CSFV IRES-mediated tagBFP translation with a 20-nt DS. c Recovery of CSFV IRES-mediated tagBFP translation with different length of RS. d Representative flow cytometry scatter plots of nIRES, dIRES, and rIRES of CSFV IRES. Each experiment was repeated three times independently with similar results. HEK293 cell line was used for the transfection experiments in panel b–d. e Construction and optimization of miR-21 responsive CSFV MITAs by variating the number of base-pairing or mismatches in designed structures. TL, SL, and AJ were all designed to sense endogenous miR-21. CSFV IRES-mediated tagBFP fluorescence intensity was measured in Huh7 (miR-21 high) and HEK293 (miR-21 low) cells. f Schematic illustration of the CSFV MITR designed by inserting miRBS fully complementary to miR-FF4. g Insertion of miRBS fully complementary to the input miRNA exhibited miRNA-responsive CSFV IRES translation repression function. HEK293 cell line was used for the transfection experiments. Data are presented as mean values with error bars representing the standard deviation of three independent biological replicates (n = 3 in each group). Statistical analysis of the results was performed by a two-tailed unpaired Welch’s t-test, assuming unequal variances. *p < 0.05. **p < 0.005. ***p < 0.001 (p = 0.0009 in Fig. 5g). arb. units, arbitrary units. Schematic illustration figures were created with BioRender.com with publication licenses. Source data are provided as a Source Data file.