Small RNA Profiling in Dengue Virus 2-Infected Aedes Mosquito Cells Reveals Viral piRNAs and Novel Host miRNAs

RESEARCH ARTICLE Small RNA Profiling in Dengue Virus 2Infected Aedes Mosquito Cells Reveals Viral piRNAs and Novel Host miRNAs Pascal Miesen1, Alasdair Ivens2, Amy H. Buck2, Ronald P. van Rij1* 1 Department of Medical Microbiology, Radboud University Medical Center, Radboud Institute for Molecular Life Sciences, Nijmegen, The Netherlands, 2 Centre for Immunity, Infection & Evolution, University of Edinburgh, Edinburgh, United Kingdom * Abstract OPEN ACCESS Citation: Miesen P, Ivens A, Buck AH, van Rij RP (2016) Small RNA Profiling in Dengue Virus 2Infected Aedes Mosquito Cells Reveals Viral piRNAs and Novel Host miRNAs. PLoS Negl Trop Dis 10(2): e0004452. doi:10.1371/journal.pntd.0004452 Editor: Gregory D Ebel, Colorado State University, UNITED STATES Received: October 13, 2015 Accepted: January 21, 2016 Published: February 25, 2016 Copyright: © 2016 Miesen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: Small RNA sequences have been submitted to NCBI Sequence Read Archive under accession numbers SRX1309506 to SRX1309511. All other relevant data are within the paper and its Supporting Information files. Funding: This work is financially supported by a PhD fellowship from Radboud University Medical Center (www.radboudumc.nl) to PM, a Strategic award from the Wellcome Trust to the Centre for Infection Immunity and Evolution (grant no. 095831), Welcome Trust (grant no. WT097394A1A) to AHB, an ECHO project grant from the Netherlands Organization for Scientific Research (NWO, grant no. 711.013.001) to In Aedes mosquitoes, infections with arthropod-borne viruses (arboviruses) trigger or modulate the expression of various classes of viral and host-derived small RNAs, including small interfering RNAs (siRNAs), PIWI interacting RNAs (piRNAs), and microRNAs (miRNAs). Viral siRNAs are at the core of the antiviral RNA interference machinery, one of the key pathways that limit virus replication in invertebrates. Besides siRNAs, Aedes mosquitoes and cells derived from these insects produce arbovirus-derived piRNAs, the best studied examples being viruses from the Togaviridae or Bunyaviridae families. Host miRNAs modulate the expression of a large number of genes and their levels may change in response to viral infections. In addition, some viruses, mostly with a DNA genome, express their own miRNAs to regulate host and viral gene expression. Here, we perform a comprehensive analysis of both viral and host-derived small RNAs in Aedes aegypti Aag2 cells infected with dengue virus 2 (DENV), a member of the Flaviviridae family. Aag2 cells are competent in producing all three types of small RNAs and provide a powerful tool to explore the crosstalk between arboviral infection and the distinct RNA silencing pathways. Interestingly, besides the well-characterized DENV-derived siRNAs, a specific population of viral piRNAs was identified in infected Aag2 cells. Knockdown of Piwi5, Ago3 and, to a lesser extent, Piwi6 results in reduction of vpiRNA levels, providing the first genetic evidence that Aedes PIWI proteins produce DENV-derived small RNAs. In contrast, we do not find convincing evidence for the production of virus-derived miRNAs. Neither do we find that host miRNA expression is strongly changed upon DENV2 infection. Finally, our deep-sequencing analyses detect 30 novel Aedes miRNAs, complementing the repertoire of regulatory small RNAs in this important vector species. Author Summary Mosquitoes of the Aedes family transmit many important viruses, including dengue virus, between their vertebrate hosts. In the mosquito, the growth of these viruses is limited by PLOS Neglected Tropical Diseases | DOI:10.1371/journal.pntd.0004452 February 25, 2016 1 / 22 Small RNAs in DENV2-Infected Mosquito Cells RPvR, and European Research Council Consolidator Grant under the European Union’s Seventh Framework Programme (ERC, grant no. 615680) to RPvR. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. the antiviral RNA interference pathway. Key to this pathway is a class of small non-coding RNAs known as small interfering RNAs (siRNAs). In addition, two related but distinct small RNA pathways known as the microRNA (miRNA) and the PIWI-interacting RNA (piRNA) pathway are implicated in regulating virus replication in mosquitoes. Thus, since small RNAs may critically influence the transmission of dengue virus, we set out to analyze the populations of viral and mosquito small RNAs that are produced in infected Aedes mosquito cells. We found that besides the well-known viral siRNAs, dengue virus-derived piRNAs were produced in these cells and we identified the PIWI proteins that these small RNAs rely on. In addition, we found that viral miRNAs were not expressed from the dengue virus genome and that the levels of mosquito miRNAs were barely changed upon infection. Finally, our data allowed for the identification of novel Aedes miRNAs, complementing the repertoire of these important regulatory RNAs in vector mosquitoes. Introduction Aedes mosquitoes are essential vectors for the transmission of important arthropod-borne viruses (arboviruses), including dengue virus (DENV), yellow fever virus, and chikungunya virus [1]. While several of these arboviral infections cause disease in humans, virus replication generally does not lead to severe pathology in vector mosquitoes. Infected mosquitoes thus serve as a persistent reservoir for arboviruses in the wild and they may transmit these viruses to vertebrate hosts throughout their entire lives [2]. After ingestion in a mosquito’s blood meal, arboviruses need to overcome a number of anatomical and immunological barriers to reach sufficiently high titres in the saliva. Only then can transmission to a naive vertebrate host efficiently occur. One of the most important immune responses to arboviral infection is antiviral RNA interference (RNAi) [3–5]. This pathway is triggered by the presence of double stranded RNA (dsRNA), which is produced during the replication of RNA and DNA viruses [6,7]. The dsRNA is recognized and cleaved by the RNaseIII enzyme Dicer-2 (Dcr2) into 21 nucleotide (nt) small interfering RNA duplexes (viral siRNA; vsiRNA) [8,9]. One of the siRNA strands is incorporated in Argonaute-2 (Ago2), the core protein of the RNA induced silencing complex (RISC) [10]. The siRNA-loaded RISC complex is guided to complementary viral RNA molecules and cleaves these target RNAs using the endonuclease (slicer) activity of Ago2 [11]. MicroRNAs (miRNAs) are a distinct class of small RNAs that are produced from genomeencoded stem loop-containing transcripts known as primary miRNA (p (...truncated)