Complex interaction between dengue virus replication and expression of miRNA-133a

Abstract

Background: Dengue virus (DENV) is the most common vector-borne viral infection worldwide with approximately 390 million cases and 25,000 reported deaths each year. MicroRNAs (miRNAs) are small non-coding RNA molecules responsible for the regulation of gene expression by repressing mRNA translation or inducing mRNA degradation. Although miRNAs possess antiviral activity against many mammalian-infecting viruses, their involvement in DENV replication is poorly understood.

Methods: Here, we explored the relationship between DENV and cellular microRNAs using bioinformatics tools. We overexpressed miRNA-133a in Vero cells to test its role in DENV replication and analyzed its expression using RT-qPCR. Furthermore, the expression of polypyrimidine tract binding protein (PTB), a protein involved in DENV replication, was analyzed by western blot. In addition, we profiled miRNA-133a expression in Vero cells challenged with DENV-2, using Taqman miRNA.

Results: Bioinformatic analysis revealed that the 3' untranslated region (3'UTR) of the DENV genome of all four DENV serotypes is targeted by several cellular miRNAs, including miRNA-133a. We found that overexpression of synthetic miRNA-133a suppressed DENV replication. Additionally, we observed that PTB transcription , a miRNA-133a target, is down-regulated during DENV infection. Based in our results we propose that 3'UTR of DENV down-regulates endogenous expression of miRNA-133a in Vero cells during the first hours of infection.

Conclusions: miRNA-133a regulates DENV replication possibly through the modulation of a host factor such as PTB. Further investigations are needed to verify whether miRNA-133a has an anti-DENV effect in vivo.

Fig. 1

Human miRNA-133a is predicted to target the 3'UTR of DENV. Using sequence alignment and Microinspector analysis, a miRNA-133a target sequence was predicted within the DENV 3'UTR sequence, which fulfills the seed sequence rule (perfect hybridization of bases 2–8 or 2–9 from the 5' end of the miRNA with the target RNA) plus binding in a 7mer-A1 fashion (a). The predicted target sequence is present in all four DENV serotypes (b). The location of the target sequence is in a loop of the 3'UTR known as the 3'SL, that contains the elements known as 3'CS and 3'UAR (c)

Fig. 2

MiRNA-133a alters the expression of GFP-fused to the DENV RNA 3'UTR. Vero cells were co-transfected with pGUD1, pGUD2, pGUD4 or synthetic miRNA-133a mimics and 24 h later, the expression of GFP was assessed by fluorescence microscopy (a) and western blot (b). Actin was used as a loading control. Semi-quantitative data of the western blots were assessed using Image J (c). A representative experiment is shown. In total three experiments were performed

Fig. 3

miRNA-133a suppresses DENV-2 replication. Vero cells were transfected with the synthetic form of miRNA-133a or with the miRNA-133a inhibitor as mimic, both at a final concentration of 50 μM. At 24 hpt, cells were challenged with DENV-2 at a MOI of 3 and infection was evaluated at 12, 24, 48 and 72 hpi. a Flow cytometry of DENV-2 challenged cells, miRNA-133a transfected and infected cells, and miRNA inhibitor transfected and infected cells. b Viral RNA copy number of DENV-2 infected cells, miRNA-133a transfected and infected cells, and miRNA inhibitor transfected and infected cells assessed by RT-PCR in culture supernatants. c Viral titer of DENV-2 infected cells, miRNA-133a transfected and infected cells, and miRNA inhibitor transfected and infected cells assessed by flow cytometry. Data are shown as median ± range from three repeated experiments. (*) Statistically significant difference compared to the control (p < 0.05)

Fig. 4

DENV up-regulates the expression of PTB through negative regulation of miRNA-133a. Vero cells were transfected with the synthetic form of miRNA-133a at a final concentration of 50 μM or 100 μM, and 24 h post-transfection PTB expression was assessed by Western blotting (a). Vero cells were challenged with DENV-2 NGC with a MOI of 3. PTB expression was measured by Western blotting at 12, 24 and 48 hpi (b). Finally, Vero cells were transfected with the synthetic form of miRNA-133a at a final concentration of 100 μM, and 24 hpt were challenged with DENV-2 at a MOI of 3. PTB expression was then measured by western blotting at 12, 24, 48 and 72 h (d). For (a, b), tubulin was used as loading control; for (d), actin was used as loading control. Semi-quantitative data of the western blot was assessed using Image J (c, e)

Fig. 5

DENV infection induces down-regulation of miRNA-133a. Vero cells were infected with DENV-2 at a MOI of 3. miRNA-133a expression was evaluated at 8, 16, 24, 32, 48, 72 hpi by RT-PCR and normalized to an uninfected control and to the 18S RNA (ΔΔ Ct). Data are shown as median ± range from three repeated experiments

Fig. 6

The expression of miRNA-133a is regulated by the 3'UTR of DENV. Vero cells were transfected with a pEGFP plasmid carrying the 3'UTR of DENV-1, −2, or −4. miRNA-133a expression was evaluated at 12, 24, 48 and 72 hpt by RT-PCR and normalized to an untransfected control and to the 18S RNA (ΔΔ Ct). Data are shown as median ± range from three repeated experiments. (*) Statistically significant difference compared to the control (p < 0.05)