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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.

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