Dengue virus (DENV) is an arbovirus transmitted to humans by Aedes mosquitoes1. In the insect vector, the small interfering RNA (siRNA) pathway is an important antiviral mechanism against DENV2,3,4,5. However, it remains unclear when and where the siRNA pathway acts during the virus cycle. Here, we show that the siRNA pathway fails to efficiently silence DENV in the midgut of Aedes aegypti although it is essential to restrict systemic replication. Accumulation of DENV-derived siRNAs in the midgut reveals that impaired silencing results from a defect downstream of small RNA biogenesis. Notably, silencing triggered by endogenous and exogenous dsRNAs remained effective in the midgut where known components of the siRNA pathway, including the double-stranded RNA (dsRNA)-binding proteins Loquacious and r2d2, had normal expression levels. We identified an Aedes-specific paralogue of loquacious and r2d2, hereafter named loqs2, which is not expressed in the midgut. Loqs2 interacts with Loquacious and r2d2 and is required to control systemic replication of DENV and also Zika virus. Furthermore, ectopic expression of Loqs2 in the midgut of transgenic mosquitoes is sufficient to restrict DENV replication and dissemination. Together, our data reveal a mechanism of tissue-specific regulation of the mosquito siRNA pathway controlled by Loqs2.
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Small RNA libraries from this study have been deposited in the Sequence Read Archive (SRA) at NCBI. Other publicly available RNA–seq data sets were obtained from SRA. Accession numbers and references are provided in Supplementary Table 4.
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The authors thank C.N.D. dos Santos for providing 4G2 monoclonal antibodies. This work was supported with funding from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Fundação de Amparo a Pesquisa do Estado de Minas Gerais (FAPEMIG) to J.T.M., Agence Nationale de la Recherche (ANR-11-ASV3-002) and Investissement d’Avenir Programs (ANR-10-LABX-36; ANR-11-EQPX-0022) to J.T.M and J.-L.I., and Inserm, CNRS and the University of Strasbourg to E.M. and J.-L.I. Mass spectrometry instrumentation was funded by the University of Strasbourg, IdEx Equipement mi-lourd 2015 to L.K. and P.H. National and/or international fellowships from CAPES were granted to R.P.O., T.C.I.-T., A.G.A.F., E.R.G.R.A., K.P.R.S., K.P.O. and C.D.O., and national and/or international fellowships from CNPq were granted to I.J.S.F., L.A.M. and J.T.M.
The authors declare no competing interests.
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Supplementary Figures 1–4, Supplementary Tables 2–4.
Mass spectrometry analysis of Loqs2 interacting proteins.
Plasmid sequence containing the cassette (attP loxP CP::3×FLAG-Loqs2-Sv40pA, PUb::mTurquoise2-Sv40pA loxP) flanked by piggyBac repeats.
Plasmid sequence containing the cassette (attP loxP PUb::3×FLAG-eGFPSv40pA, OpIE2::Puromicin-Sv40pA loxP) flanked by piggyBac repeats.
Plasmid sequence containing the cassette (attP loxP PUb::3×FLAG-Loqs2-Sv40pA, OpIE2::Puromicin-Sv40pA loxP) flanked by piggyBac repeats.
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Olmo, R.P., Ferreira, A.G.A., Izidoro-Toledo, T.C. et al. Control of dengue virus in the midgut of Aedes aegypti by ectopic expression of the dsRNA-binding protein Loqs2. Nat Microbiol 3, 1385–1393 (2018). https://doi.org/10.1038/s41564-018-0268-6