Figure 6

MiR-H3 targets the TATA box in HIV-1 5′ LTR via sequence specific manner. (A) Predicted binding site of miR-H3 in HIV-1 5′ LTR with the TATA box highlighted. The MFE of this interaction is -19.2 kcal/mol. (B) Mutations (labeled in red with underline) were introduced to the promoter, named HIV 5′ LTR mut. The effects of miR-H3 on promoter activity of wildtype or mutated promoter were determined with dual-luciferase assay. The TATA box motif was indicated with a box. (C) Top, the TATA box motif of CMV promoter was replaced by the binding site of miR-H3 on HIV-1 promoter (CMV-HIV_bs mt). The mutated nucleotides are indicated in red with underline. Bottom, the effects of miR-H3 on indicated promoter activities were determined with Dual-Luciferase assay as described above. The TATA box motif was indicated with a box. (D) ChIP assay of HEK239T cells co-transfected with pNL4-3-deltaE-EGFP and pre-miR-H3 or the empty vector with antibody against Pol II or TBP to examine the binding of these general transcription factors to the HIV-1 core promoter region. The normal IgG was used as a control. (E) The effects of miR-H3 on HIV-1 promoter activity without Tat expression. (F) Effect of miR-H3 on the activity of a TAR region deficient (deletion of 470 to 492 bp in HIV-1 5′ LTR) HIV-1 promoter. (G) The effect of miR-H3 on another retrovirus promoter. A RSV promoter-derived luciferase reporter construct was co-transfected with miR-H3 precursor or control vector, and the promoter activity was investigated by Dual-Luciferase assay. P-values were calculated using the two tailed unpaired Student’s t-test with equal variances, n = 3. *p < 0.05, ***p < 0.001.