DENV 3′ UTR contains potential miR-484 and miR-744 binding sites - We were interested in determining whether any interaction occurs between DENV RNA and human host miRNAs, since it was previously reported that viral genomes can be targeted by human miRNAs. We therefore investigated whether the DENV 3′ UTR contains potential miRNA binding sites. The sequences of the 3′ UTRs plus 374 nucleotides of the coding region of NS5 of all four DENV serotypes were aligned. In total, 108 miRNAs for DENV-1, 80 for DENV-2, 94 for DENV-3, and 89 for DENV-4 were predicted to target the 3′ UTR using the MicroInspector software (data not shown). Since it is reported that the 3′ UTR sequence across all DENV serotypes is moderately conserved, and considering the hypothesis that a functional miRNA target site would be conserved across all DENV serotypes, only those target sites common to the four reference sequences were selected. Among the miRNAs that fulfilled these criteria, miR-484 and miR-744 were predicted in our analysis (Fig. 1A) using the RNAhybrid program. The StarMir program predicted the potential binding sites for miR-484 and miR-744 in the DENV 3′ UTR as the target sequences (Fig. 1B). Each of the candidate sites was assigned a logistic probability as a measure of confidence in the predicted site. Interestingly, the location of the target sequence was in a loop of the 3′ UTR known as the 3′-SL that contains the elements known as the 3′ cyclisation sequence (3’CS) and the region upstream of the AUG (3′UAR) (Fig. 1C). Secondary structure was predicted using the Mfold program (http://unafold.rna.albany.edu/?q=mfold). Using the RNAfold webserver we observed that this secondary structure is the natural-mode structure that is most stable for that region in the 3′ UTR of DENV RNA (the free energy of the thermodynamic ensemble was -140.64 kcal/mol) (http://unafold.rna.albany.edu/?q=mfold/rna-folding-form). We then wondered whether the miR-744 and miR-484 binding sites on the 3′ UTR of DENV RNA (Fig. 1C) are accessible in the presence of the SL and hairpin. Using the RNAup webserver (http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAup.cgi) we found that the total free energy of the miR-484-3′ UTR was -8.92 kcal/mol with an opening energy (accessibility) of 5.16 kcal/mol, whereas for the miR-744-3′ UTR the total energy was -7.91 kcal/mol with an opening energy of 8.13 kcal/mol, suggesting that miRNA-3′ UTR binding would proceed spontaneously.