Fig. 1
: the 3′ UTR of Dengue virus (DENV) RNA contains target sequences for cellular miR-484 and miR-744. (A) Alignment of a fragment of the 3′ UTR of the four DENV serotypes. The miR-484 and miR-744 target sites common to the four serotypes are indicated; (B) the target sites for miR-484 and miR-744 fulfil the seed sequence (shaded nucleotides, first nucleotides from the 5′ end of the miRNA). Structures predicted using RNAhybrid; (C) location of the miR-484 and miR-744 target sequences in the 3′-SL, CS, and 3′UAR regions of the 3′ UTR. The secondary structure of DENV RNA was predicted using the Mfold program (Zuker 2003Zuker M. Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res. 2003; 31(13): 3406-15.). The sequences and position numbers in (B) and (C) correspond to DENV-1 RNA.
Fig. 2
: cellular miRNAs alter the expression of GFP-fused to the 3′ UTR of Dengue virus (DENV) RNA. (A) Vero cells were transfected with pGUD1, pGUD2, pGUD4, or pEGFP-C1 and the GFP expression was determined by western blotting after 24 h, using an anti-GFP antibody; (b) band intensities quantified by densitometry; (C) Vero cells were co-transfected with pGUD1, pGUD2, pGUD4, or pEGFP-C1 and pEZX-mR03-miR-484 or pEZX-mR03-miR-744, and GFP expression was then determined by western blot after 24 h using an anti-GFP antibody; (D) band intensities quantified by densitometry. Data are shown as Median and Range (two way ANOVA). Three independent replicates were performed for each experiment. (*) Statistically significant difference compared to the control (p < 0.05).
Fig. 3
: overexpression of miR-484 and miR-744 modulates Dengue virus (DENV) replication. (A) Vero cells were transfected with pEZX-mR03-miR-484, pEZX-mR03-miR-744, or the empty vector pEZX-mR03 (scramble) and after 24 h, challenged independently with each of the four DENV serotypes at an MOI of 3; (B) quantification of DENV RNA copy number in the supernatants by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). (C, D) DENV-2 NS1 expression by western blotting for each condition evaluated and the band intensities quantified by densitometry, respectively. Ponceau red was used as the loading control for western blotting, as previously reported (Romero-Calvo et al. 2010Romero-Calvo I, Ocon B, Martinez-Moya P, Suarez MD, Zarzuelo A, Martinez-Augustin O, et al. Reversible Ponceau staining as a loading control alternative to actin in Western blots. Anal Biochem. 2010; 401(2): 318-20., Gilda & Gomes 2013Gilda JE, Gomes AV. Stain-Free total protein staining is a superior loading control to b-actin for Western blots. Anal Biochem. 2013; 440(2): 186-8., Rivero-Gutiérrez et al. 2014)Rivero-Gutiérrez B, Anzola A, Martínez-Augustin O, Medina FS de. Stain-free detection as loading control alternative to Ponceau and housekeeping protein immunodetection in Western blotting. Anal Biochem. 2014; 467: 1-3.; (E) Vero cells were first challenged independently with each DENV serotype and then transfected with pEZX-mR03-miR-484, pEZX-mR03-miR-744, or the empty vector pEZX-mR03. The percentage of infected cells was evaluated at 72 hpi by flow cytometry. The data are expressed as the percentage of infected Vero cells compared with those in the scrambled infected Vero cells, defined as 100% infection. Results are shown as Median and Range (two way ANOVA, p < 0.005); (F) Quantification of DENV RNA copy number in the supernatants by RT-qPCR. (G, H) DENV-2 NS1 expression by western blotting for each condition evaluated and band intensities quantified by densitometry, respectively. Ponceau red was used as the loading control for western blotting, as previously reported (Romero-Calvo et al. 2010Romero-Calvo I, Ocon B, Martinez-Moya P, Suarez MD, Zarzuelo A, Martinez-Augustin O, et al. Reversible Ponceau staining as a loading control alternative to actin in Western blots. Anal Biochem. 2010; 401(2): 318-20., Gilda & Gomes 2013Gilda JE, Gomes AV. Stain-Free total protein staining is a superior loading control to b-actin for Western blots. Anal Biochem. 2013; 440(2): 186-8., Rivero-Gutiérrez et al. 2014)Rivero-Gutiérrez B, Anzola A, Martínez-Augustin O, Medina FS de. Stain-free detection as loading control alternative to Ponceau and housekeeping protein immunodetection in Western blotting. Anal Biochem. 2014; 467: 1-3.. The data are shown as Median and Range (two way ANOVA). Three independent replicates were performed for each experiment. (*) Statistically significant difference compared to the control (p < 0.05).
Fig. 5
: miR-484 and miR-744 expression is downregulated in Vero cells expressing the Dengue virus (DENV) 3′ UTR. Vero cells were transfected with the pGUD1, pGUD2, or pGUD4 constructs or with the empty vector pEGFP-C1 and their effect on miRNA expression was determined. The expression of miR-484 (A) and miR-744 (B) was evaluated at 12, 24, 48, and 72 hpt by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) and normalised to the untransfected control and to 18S RNA (2-ΔΔCt). Data from RT-qPCR are shown as median and bars are presented from three independent experiments. (*) Statistically significant difference compared to the control (p < 0.05).
