Figure 6.

IL6ST and ITGAV are targets of miR-142-5p and overexpression of miR-142-5p results in the reduction of ZIKV replication. (A, B) Schematic presentation of the predicted miR-142-5p binding sites within the ITGAV (A) and IL6ST (B) 3’UTR and a mutated type of ITGAV and IL6ST, as predicted by the Target Scan database. The mutated nucleotides are marked in bold. Luciferase reporter assay performed using HEK293 T cells transfected with plasmids into which the luciferase reporter gene was fused to fragment of wild type or mutant 3’UTRs of ITGAV and IL6ST. (C, D) Cells were transfected with miR-142-5p mimic or mimic negative control as indicated. 24 h later, cells were infected with ZIKV (MR766 or PRVABC59) at MOI of 1 for 24 h. (C) IL6ST, ITGAV, and ZIKV NS5 transcript levels were determined by qRT-PCR. (D) mRNA expression of ITGB1, IL-6, and TNF-α was measured by qRT-PCR. *p < 0.05, **p < 0.01, and ***p < 0.001, compared with control mimic. (E) Expression of ZIKV NS1 was examined by Western blot analysis. Quantitative densitometric analysis of Western blot analysis is presented, with normalized densitometric units plotted against treatment (shown as numbers). (F) Measurement by TCID50 assay of infectious ZIKV release into supernatant at 48 hpi is shown (MOI 1). Results represent means ± SD of two independent experiments.