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. 2014 Feb 18;111(7):2746-51.
doi: 10.1073/pnas.1320123111. Epub 2014 Feb 3.

MicroRNA-like viral small RNA from Dengue virus 2 autoregulates its replication in mosquito cells

Mazhar Hussain  1 , Sassan Asgari
Affiliations

MicroRNA-like viral small RNA from Dengue virus 2 autoregulates its replication in mosquito cells

Mazhar Hussain et al. Proc Natl Acad Sci U S A. .

Abstract

MicroRNAs (miRNAs) are small regulatory RNAs that play significant roles in most cellular processes. In the seemingly endless arms race between hosts and pathogens, viruses also encode miRNAs that facilitate successful infection. In search of functional miRNAs or viral small RNAs (vsRNAs) encoded by Dengue virus (DENV), deep sequencing data of virus-infected Aedes aegypti mosquitoes were used. From six vsRNAs, with candidate stem-loop structures in the 5' and 3' untranslated regions of the viral genomic RNA, inhibition of DENV-vsRNA-5 led to significant increases in viral replication. Silencing of RNA interference (RNAi)/miRNA pathways' associated proteins showed that Argonaute 2 is mainly involved in DENV-vsRNA-5 biogenesis. Cloning of the precursor stem loop, immunoprecipitations, ectopic expression and detection in RNAi-deficient C6/36, and the mammalian Vero cell lines further confirmed DENV-vsRNA-5 production. Furthermore, significant impact of synthetic mimic and inhibitor of DENV-vsRNA-5 on DENV RNA levels revealed DENV-vsRNA-5's role in virus autoregulation by targeting the virus nonstructural protein 1 gene. Notably, DENV-vsRNA-5 homologous mimics from DENV serotypes 1 and 4, but not 3, inhibited DENV-2 replication. The results revealed that DENV is able to encode functional vsRNAs, and one of those, which resembles miRNAs, specifically targets a viral gene, opening an avenue for possible utilization of the small RNA to limit DENV replication.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Locations of DENV-2 vsRNAs in the viral genome and the effect of their inhibitors on virus replication. (A) Diagram showing the genome of DENV with locations of vsRNAs mapped to the genome. (B) RT-qPCR analysis to examine the levels of viral gRNA in Aag2 cells transfected with synthetic inhibitors of the six vsRNAs shown in A followed by DENV-2 infection at 7 dpi. ****P < 0.0001 ANOVA. (C) Virus titer determination in the medium from three replicates of the experiment described in B by using the MTT method; **P = 0.0043 ANOVA. (D) Secondary structure of the stem-loop in DENV-2 3′UTR and the location of DENV–vsRNA-5 shown with a gray shadow.
Fig. 2.
Fig. 2.
DENV–vsRNA-5 expression in Aag2 cells. (A) Northern blot analysis using a specific probe to vsRNA-5 in DENV-2–infected Aag2 cells at 3, 5, and 7 dpi; M, mock-infection. U6 RNA serves as loading control. Arrows indicate the precursor and mature vsRNA-5. (B) Northern blot analysis of vsRNA-5 in C6/36 cells infected with DENV-2 at 3, 5, and 7 dpi using the 5′ probe (Left); the same blot was used for 3′TL probe (Right). (C) Northern blot analysis for detection of vsRNA-5 in Aag2 cells transfected with 3′UTR ssRNA from DENV-2 using vsRNA-5 5′, 3′TL, and vsRNA-2 probes. SS1 and SS2 show two replicates from separate transfections. (D) miRNA RT-qPCR analysis confirming ectopic expression of vsRNA-5 in Aag2 cells transfected with pIZ/pre-5 (72 h after transfection) encoding the precursor of DENV–vsRNA-5. pIZ, empty vector. (E) miRNA RT-qPCR analysis for expression of vsRNA-5 in the mammalian Vero cells infected with DENV-2. Data were normalized against 5S rRNA.
Fig. 3.
Fig. 3.
Biogenesis of DENV–vsRNA-5 is mainly AGO2 dependent. (A) Northern blot detection of DENV sfRNA in the cytoplasmic fraction of Aag2 cells infected with DENV-2 at 7 dpi. C, cytoplasmic; M, mock infection; N, nuclear RNA fractions. To distinguish cytoplasmic and nuclear fractions, 18s rRNA and 5s rRNA are shown, respectively. (B) RT-qPCR analyses to show detection levels of vsRNA-5 in control (dsgfp), Dicer-1, Dicer-2, AGO1, and AGO2 silenced Aag2 cells infected with DENV-2 at 7 dpi. There are statistically significant differences between groups with different letters at P < 0.0001.
Fig. 4.
Fig. 4.
DENV–vsRNA-5 targets the NS1 gene and down-regulates viral gRNA levels. (A) RT-qPCR analysis of DENV gRNA levels in Aag2 cells transfected with the control mimic (Cont mim), vsRNA-5 mimic (vsRNA-5 mim), and vsRNA-6 mimic at 2 and 24 hpi. There are statistically significant differences between groups with different letters at P < 0.01. (B) Predicted target sequences of DENV–vsRNA-5 in the NS1 gene of DENV-2. (C) Cloned sequences of the NS1 target and the mutant target downstream of the GFP gene (pIZ/GFP-NS1). Complementary sequences to vsRNA-5 seed region that are highlighted gray were deleted in the mutant target construct (pIZ/GFP-∆NS1). (D) RT-qPCR analysis of RNA extracted from Aag2 cells cotransfected with the constructs in C and either vsRNA-5 or control mimic (mim) using GFP specific primers. ***P < 0.001.
Fig. 5.
Fig. 5.
Validation of DENV–vsRNA-5 and NS1 interaction. (A) Northern blot analysis of RNA from Aag2 cells cotransfected with the synthesized NS1 target ssRNA and control or vsRNA-5 mimic using a specific probe to ssRNA. aae-miR-2940 was used as loading control. (B) RT-PCR analysis of NS1 transcript levels after cotransfection of Aag2 cells with pIZ/NS1-full and control or vsRNA-5 mimics in two experimental replicates. RPS17 amplification is shown as control. (C) Western blot analysis of NS1 protein levels after cotransfection of Aag2 cells with pIZ/NS1-full and control or vsRNA-5 mimics using a monoclonal antibody to NS1. A nonspecific protein (arrowhead) bound to the antibody shows equal loading of the samples.
Fig. 6.
Fig. 6.
(A) Nucleotide sequence alignment to show conservation of DENV–vsRNA-5 among the four DENV serotypes. The accession numbers for the serotypes 1–4 are GU131786.1, M29095.1, JF808129.1, and KF041260.1, respectively. (B) RT-qPCR analysis of RNA from Aag2 cells transfected with vsRNA-5 mimics of DENV-1, -2, -3, and -4 followed by DENV-2 infection at 72 hpi. There are statistically significant differences between groups with different letters at P < 0.0001.
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