Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Control of dengue virus in the midgut of Aedes aegypti by ectopic expression of the dsRNA-binding protein Loqs2

Abstract

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.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1: Production of DENV-derived siRNAs in infected mosquitoes.
Fig. 2: The antiviral siRNA pathway does not control DENV infection in the mosquito midgut but inhibits systemic dissemination and replication.
Fig. 3: The siRNA pathway triggered by endogenous and exogenous sources of dsRNA is functional in the midgut.
Fig. 4: The Aedes-specific dsRBP Loqs2 regulates the antiviral arm of the mosquito siRNA pathway.

Data availability

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.

References

  1. 1.

    Guzman, M. G., Gubler, D. J., Izquierdo, A., Martinez, E. & Halstead, S. B. Dengue infection. Nat. Rev. Dis. Primers 2, 16055 (2016).

    Article  Google Scholar 

  2. 2.

    Sanchez-Vargas, I. et al. Dengue virus type 2 infections of Aedes aegypti are modulated by the mosquito’s RNA interference pathway. PLoS Pathog. 5, e1000299 (2009).

    Article  Google Scholar 

  3. 3.

    Khoo, C. C. H., Piper, J., Sanchez-Vargas, I., Olson, K. E. & Franz, A. W. E. The RNA interference pathway affects midgut infection- and escape barriers for Sindbis virus in Aedes aegypti. BMC. Microbiol. 10, 130 (2010).

    Article  Google Scholar 

  4. 4.

    Franz, A. W. et al. Engineering RNA interference-based resistance to dengue virus type 2 in genetically modified Aedes aegypti. Proc. Natl Acad. Sci. USA 103, 4198–4203 (2006).

    CAS  Article  Google Scholar 

  5. 5.

    Campbell, C. L. et al. Aedes aegypti uses RNA interference in defense against Sindbis virus infection. BMC Microbiol. 8, 47 (2008).

    Article  Google Scholar 

  6. 6.

    Hess, A. M. et al. Small RNA profiling of dengue virus–mosquito interactions implicates the piwi RNA pathway in anti-viral defense. BMC Microbiol. 11, 45 (2011).

    CAS  Article  Google Scholar 

  7. 7.

    Miesen, P., Ivens, A., Buck, A. H. & van Rij, R. P. Small RNA profiling in dengue virus 2-infected Aedes mosquito cells reveals viral piRNAs and novel host miRNAs. PLoS Negl. Trop. Dis. 10, e0004452 (2016).

    Article  Google Scholar 

  8. 8.

    Villalon, J. M., Ghosh, A. & Jacobs-Lorena, M. The peritrophic matrix limits the rate of digestion in adult Anopheles stephensi and Aedes aegypti mosquitoes. J. Insect. Physiol. 49, 891–895 (2003).

    CAS  Article  Google Scholar 

  9. 9.

    Aguiar, E. R. G. R., Olmo, R. P. & Marques, J. T. Virus-derived small RNAs: molecular footprints of host–pathogen interactions. Wiley Interdiscip. Rev. RNA 7, 824–837 (2016).

    CAS  Article  Google Scholar 

  10. 10.

    Richardson, J., Molina-Cruz, A., Salazar, M. I. & Black, W. Quantitative analysis of dengue-2 virus RNA during the extrinsic incubation period in individual Aedes aegypti. Am. J. Trop. Med. Hyg. 74, 132–141 (2006).

    CAS  Article  Google Scholar 

  11. 11.

    Salazar, M. I., Richardson, J. H., Sánchez-Vargas, I., Olson, K. E. & Beaty, B. J. Dengue virus type 2: replication and tropisms in orally infected Aedes aegypti mosquitoes. BMC Microbiol. 7, 9 (2007).

    Article  Google Scholar 

  12. 12.

    Carthew, R. W. & Sontheimer, E. J. Origins and mechanisms of miRNAs and siRNAs. Cell 136, 642–655 (2009).

    CAS  Article  Google Scholar 

  13. 13.

    Ghildiyal, M. et al. Endogenous siRNAs derived from transposons and mRNAs in Drosophila somatic cells. Science 320, 1077–1081 (2008).

    CAS  Article  Google Scholar 

  14. 14.

    Marques, J. T. et al. Functional specialization of the small interfering RNA pathway in response to virus infection. PLoS Pathog. 9, e1003579 (2013).

    CAS  Article  Google Scholar 

  15. 15.

    Ashe, A. et al. A deletion polymorphism in the Caenorhabditis elegans RIG-I homolog disables viral RNA dicing and antiviral immunity. eLife 2, e00994 (2013).

    Article  Google Scholar 

  16. 16.

    Hartig, J. V. & Förstemann, K. Loqs-PD and R2D2 define independent pathways for RISC generation in Drosophila. Nucleic Acids Res. 39, 3836–3851 (2011).

    CAS  Article  Google Scholar 

  17. 17.

    Marques, J. T. et al. Loqs and R2D2 act sequentially in the siRNA pathway in Drosophila. Nat. Struct. Mol. Biol. 17, 24–30 (2010).

    CAS  Article  Google Scholar 

  18. 18.

    Tabara, H., Yigit, E., Siomi, H. & Mello, C. C. The dsRNA binding protein RDE-4 interacts with RDE-1, DCR-1, and a DExH-box helicase to direct RNAi in C. elegans. Cell 109, 861–871 (2002).

    CAS  Article  Google Scholar 

  19. 19.

    Parrish, S. & Fire, A. Distinct roles for RDE-1 and RDE-4 during RNA interference in Caenorhabditis elegans. RNA 7, 1397–1402 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. 20.

    Hartig, J. V., Esslinger, S., Böttcher, R., Saito, K. & Förstemann, K. Endo-siRNAs depend on a new isoform of loquacious and target artificially introduced, high-copy sequences. EMBO J. 28, 2932–2944 (2009).

    CAS  Article  Google Scholar 

  21. 21.

    Haac, M. E., Anderson, M. A., Eggleston, H., Myles, K. M. & Adelman, Z. N. The hub protein loquacious connects the microRNA and short interfering RNA pathways in mosquitoes. Nucleic Acids Res. 43, 3688–3700 (2015).

    CAS  Article  Google Scholar 

  22. 22.

    Edwards, M. J. et al. Characterization of a carboxypeptidase A gene from the mosquito, Aedes aegypti. Insect Mol. Biol. 9, 33–38 (2000).

    CAS  Article  Google Scholar 

  23. 23.

    Carissimo, G. et al. Antiviral immunity of Anopheles gambiae is highly compartmentalized, with distinct roles for RNA interference and gut microbiota. Proc. Natl Acad. Sci. USA 112, E176–E185 (2015).

    CAS  Article  Google Scholar 

  24. 24.

    Sarkies, P. et al. Ancient and novel small RNA pathways compensate for the loss of piRNAs in multiple independent nematode lineages. PLoS Biol. 13, e1002061 (2015).

    Article  Google Scholar 

  25. 25.

    Maillard, P. V. et al. Antiviral RNA interference in mammalian cells. Science 342, 235–238 (2013).

    CAS  Article  Google Scholar 

  26. 26.

    Li, Y., Lu, J., Han, Y., Fan, X. & Ding, S. W. RNA interference functions as an antiviral immunity mechanism in mammals. Science 342, 231–234 (2013).

    CAS  Article  Google Scholar 

  27. 27.

    Pereira, T. N., Rocha, M. N., Sucupira, P. H. F., Carvalho, F. D. & Moreira, L. A. Wolbachia significantly impacts the vector competence of Aedes aegypti for Mayaro virus. Sci. Rep. 8, 6889 (2018).

    Article  Google Scholar 

  28. 28.

    Donald, C. L. et al. Full genome sequence and sfRNA interferon antagonist activity of Zika virus from Recife, Brazil. PLoS Negl. Trop. Dis. 10, e0005048 (2016).

    Article  Google Scholar 

  29. 29.

    Sedda, L. et al. The spatial and temporal scales of local dengue virus transmission in natural settings: a retrospective analysis. Parasit. Vectors 11, 79 (2018).

    Article  Google Scholar 

  30. 30.

    Barletta, A. B. et al. Microbiota activates IMD pathway and limits Sindbis infection in Aedes aegypti. Parasit. Vectors 10, 103 (2017).

    Article  Google Scholar 

  31. 31.

    Tan, G. K. et al. A non mouse-adapted dengue virus strain as a new model of severe dengue infection in AG129 mice. PLoS Negl. Trop. Dis. 4, e672 (2010).

    Article  Google Scholar 

  32. 32.

    Lanford, R. E., Sureau, C., Jacob, J. R., White, R. & Fuerst, T. R. Demonstration of in vitro infection of chimpanzee hepatocytes with hepatitis C virus using strand-specific RT/PCR. Virology 202, 606–614 (1994).

    CAS  Article  Google Scholar 

  33. 33.

    Pfeffer, S. et al. Identification of virus-encoded microRNAs. Science 304, 734–736 (2004).

    CAS  Article  Google Scholar 

  34. 34.

    Jayaprakash, A. D., Jabado, O., Brown, B. D. & Sachidanandam, R. Identification and remediation of biases in the activity of RNA ligases in small-RNA deep sequencing. Nucleic Acids Res. 39, e141 (2011).

    CAS  Article  Google Scholar 

  35. 35.

    Martin, M. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet J. 17, 10–12 (2011).

    Article  Google Scholar 

  36. 36.

    Langmead, B., Trapnell, C., Pop, M. & Salzberg, S. L. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 10, R25 (2009).

    Article  Google Scholar 

  37. 37.

    Dobin, A. et al. STAR: ultrafast universal RNA–seq aligner. Bioinformatics 29, 15–21 (2013).

    CAS  Article  Google Scholar 

  38. 38.

    Li, B. & Dewey, C. N. RSEM: accurate transcript quantification from RNA–seq data with or without a reference genome. BMC Bioinformatics 12, 323 (2011).

    CAS  Article  Google Scholar 

  39. 39.

    Robinson, M. D., McCarthy, D. J. & Smyth, G. K. edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26, 139–140 (2010).

    CAS  Article  Google Scholar 

  40. 40.

    Sievers, F. et al. Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol. Syst. Biol. 7, 539 (2011).

    Article  Google Scholar 

  41. 41.

    Engler, C. & Marillonnet, S. Combinatorial DNA assembly using Golden Gate cloning. Methods Mol. Biol. 1073, 141–156 (2013).

    CAS  Article  Google Scholar 

  42. 42.

    Geissler, R. et al. Transcriptional activators of human genes with programmable DNA-specificity. PLoS ONE 6, e19509 (2011).

    CAS  Article  Google Scholar 

  43. 43.

    Volohonsky, G. et al. Tools for Anopheles gambiae transgenesis. G3 (Bethesda) 5, 1151–1163 (2015).

    CAS  Article  Google Scholar 

  44. 44.

    Jasinskiene, N., Juhn, J. & James, A. A. Microinjection of A. aegypti embryos to obtain transgenic mosquitoes. J. Vis. Exp. 2007, 219 (2007).

    Google Scholar 

  45. 45.

    Morris, A. C., Eggleston, P. & Crampton, J. M. Genetic transformation of the mosquito Aedes aegypti by micro-injection of DNA. Med. Vet. Entomol. 3, 1–7 (1989).

    CAS  Article  Google Scholar 

  46. 46.

    Stoetzel, C. et al. A mutation in VPS15 (PIK3R4) causes a ciliopathy and affects IFT20 release from the cis-Golgi. Nat. Commun. 7, 13586 (2016).

    CAS  Article  Google Scholar 

  47. 47.

    Chicher, J. et al. Purification of mRNA-programmed translation initiation complexes suitable for mass spectrometry analysis. Proteomics 15, 2417–2425 (2015).

    CAS  Article  Google Scholar 

  48. 48.

    Perkins, D. N., Pappin, D. J. C., Creasy, D. M. & Cottrell, J. S. Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis 20, 3551–3567 (1999).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

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.

Author information

Affiliations

Authors

Contributions

R.P.O. and J.T.M. designed the project. R.P.O., A.G.A.F., T.C.I.-T., I.J.S.F., K.P.R.S., K.P.O., P.H., E.G.A., Y.M.T, M.N.R., T.H.J.F.L., S.C.G.A., J.N.A. and S.P. performed experiments. R.P.O., E.R.G.R.A. and L.K. performed bioinformatics analysis. C.D.O., F.D.C., L.A.M and E.M. contributed to mosquito experiments. R.P.O., E.R.G.R.A., J.-L.I. and J.T.M. analysed the data. R.P.O., J.-L.I. and J.T.M. wrote the paper. All authors discussed the results and commented on the manuscript.

Corresponding author

Correspondence to João T. Marques.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Supplementary Figures 1–4, Supplementary Tables 2–4.

Reporting Summary

Supplementary Table 1

Mass spectrometry analysis of Loqs2 interacting proteins.

Supplementary File 1

Plasmid sequence containing the cassette (attP loxP CP::3×FLAG-Loqs2-Sv40pA, PUb::mTurquoise2-Sv40pA loxP) flanked by piggyBac repeats.

Supplementary File 2

Plasmid sequence containing the cassette (attP loxP PUb::3×FLAG-eGFPSv40pA, OpIE2::Puromicin-Sv40pA loxP) flanked by piggyBac repeats.

Supplementary File 3

Plasmid sequence containing the cassette (attP loxP PUb::3×FLAG-Loqs2-Sv40pA, OpIE2::Puromicin-Sv40pA loxP) flanked by piggyBac repeats.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

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

Download citation

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing