, Longterm transmission of defective RNA viruses in humans and Aedes mosquitoes, Science, vol.311, pp.236-238, 2006.
, A novel system for the launch of alphavirus RNA synthesis reveals a role for the Imd pathway in arthropod antiviral response, PLoS Pathog, vol.5, p.1000582, 2009.
, Preference of RIG-I for short viral RNA molecules in infected cells revealed by next-generation sequencing, Proc. Natl. Acad. Sci. USA, vol.107, pp.16303-16308, 2010.
, The take and give between retrotransposable elements and their hosts, Annu. Rev. Genet, vol.42, pp.587-617, 2008.
, Sindbis virus expression vectors: packaging of RNA replicons by using defective helper RNAs, J. Virol, vol.67, pp.6439-6446, 1993.
, Cell Host & Microbe, vol.23, p.363, 2018.
, The RIG-I ATPase domain structure reveals insights into ATP-dependent antiviral signalling, EMBO Rep, vol.12, pp.1127-1134, 2011.
, Host alternation of chikungunya virus increases fitness while restricting population diversity and adaptability to novel selective pressures, J. Virol, vol.85, pp.1025-1035, 2011.
URL : https://hal.archives-ouvertes.fr/pasteur-00546712
, Extrachromosomal circular DNA of tandemly repeated genomic sequences in Drosophila, Genome Res, vol.13, issue.6A, pp.1133-1145, 2003.
, , 2007.
, Coupling of double-stranded RNA synthesis and siRNA generation in fission yeast RNAi, Mol. Cell, vol.27, pp.449-461
, Sequences of flavivirus-related RNA viruses persist in DNA form integrated in the genome of Aedes spp. mosquitoes, J. Gen. Virol, vol.85, 1971.
, Lentivirus envelope sequences and proviral genomes are stabilized in Escherichia coli when cloned in low-copy-number plasmid vectors, Gene, vol.124, pp.93-98, 1993.
, The DExD/ H-box helicase Dicer-2 mediates the induction of antiviral activity in drosophila, Nat. Immunol, vol.9, pp.1425-1432, 2008.
, The Jak-STAT signaling pathway is required but not sufficient for the antiviral response of drosophila, Nat. Immunol, vol.6, pp.946-953, 2005.
URL : https://hal.archives-ouvertes.fr/hal-00094350
, , 2010.
, Functional Sindbis virus replicative complexes are formed at the plasma membrane, J. Virol, vol.84, pp.11679-11695
, Essential function in vivo for Dicer-2 in host defense against RNA viruses in drosophila, Nat. Immunol, vol.7, pp.590-597, 2006.
, RNAmediated interference and reverse transcription control the persistence of RNA viruses in the insect model Drosophila, Nat. Immunol, vol.14, pp.396-403, 2013.
URL : https://hal.archives-ouvertes.fr/pasteur-01957212
, Virus-derived DNA drives mosquito vector tolerance to arboviral infection, Nat. Commun, vol.7, p.12410, 2016.
URL : https://hal.archives-ouvertes.fr/pasteur-01377742
, , 1972.
, Specific membranous structures associated with the replication of group A arboviruses, J. Virol, vol.10, pp.492-503
, Defective interfering viruses, Annu. Rev. Microbiol, vol.27, pp.101-117, 1973.
, Reverse transcription of retroviruses and LTR retrotransposons, Microbiol. Spectr, vol.3, pp.3-0027, 2015.
, Parallel ClickSeq and Nanopore sequencing elucidates the rapid evolution of defective-interfering RNAs in Flock House virus, PLoS Pathog, vol.13, p.1006365, 2017.
, Molecular characterization of Drosophila cells persistently infected with Flock House virus, Virology, vol.419, pp.43-53, 2011.
, Structural basis for the activation of innate immune pattern-recognition receptor RIG-I by viral RNA, Cell, vol.147, pp.423-435, 2011.
, , 2017.
, Sequencing the extrachromosomal circular mobilome reveals retrotransposon activity in plants, PLoS Genet, vol.13, p.1006630
, Ultrafast and memory-efficient alignment of short DNA sequences to the human genome, 2009.
, The origins of defective interfering particles of the negative-strand RNA viruses, Genome Biol, vol.10, pp.145-154, 1981.
, Distinct roles for Drosophila Dicer-1 and Dicer-2 in the siRNA/miRNA silencing pathways, Cell, vol.117, pp.69-81, 2004.
, Genetic drift, purifying selection and vector genotype shape dengue virus intra-host genetic diversity in mosquitoes, PLoS Genet, vol.12, 2016.
URL : https://hal.archives-ouvertes.fr/hal-01360140
, Induction and suppression of RNA silencing by an animal virus, Science, vol.296, pp.1319-1321, 2002.
, Collapse of germline piRNAs in the absence of Argonaute3 reveals somatic piRNAs in flies, Cell, vol.137, pp.509-521, 2009.
, Argonaute2 is the catalytic engine of mammalian RNAi, Science, vol.305, pp.1437-1441, 2004.
, Structural insights into RNA recognition by RIG-I, Cell, vol.147, pp.409-422, 2011.
, Duplex RNA activated ATPases (DRAs): platforms for RNA sensing, signaling and processing, RNA Biol, vol.10, pp.111-120, 2013.
, Lower survival rate, longevity and fecundity of Aedes aegypti (Diptera: Culicidae) females orally challenged with dengue virus serotype 2, Trans. R. Soc. Trop. Med. Hyg, vol.105, pp.452-458, 2011.
, Defective interfering viruses and their potential as antiviral agents, Rev. Med. Virol, vol.20, pp.51-62, 2010.
, Deduced consensus sequence of Sindbis virus strain AR339: mutations contained in laboratory strains which affect cell culture and in vivo phenotypes, 1996.
, J. Virol, vol.70, pp.1981-1989
, Flock house virus RNA replicates on outer mitochondrial membranes in Drosophila cells, J. Virol, vol.75, pp.11664-11676, 2001.
, High-throughput computing in the sciences, Methods Enzymol, vol.467, pp.197-227, 2009.
, Interferon induction by RNA viruses and antagonism by viral pathogens, Viruses, vol.6, pp.4999-5027, 2014.
, Distinct roles for Argonaute proteins in small RNA-directed RNA cleavage pathways, Genes Dev, vol.18, pp.1655-1666, 2004.
, Comparative genomics shows that viral integrations are abundant and express piRNAs in the arboviral vectors Aedes aegypti and Aedes albopictus, BMC Genomics, vol.18, p.512, 2017.
, Sensing viral RNAs by Dicer/RIG-I like ATPases across species, Curr. Opin. Immunol, vol.32, pp.106-113, 2015.
, Circular DNA throws biologists for a loop, Science, vol.356, p.996, 2017.
, Measles virus defective interfering RNAs are generated frequently and early in the absence of C protein and can be destabilized by adenosine deaminase acting on RNA-1-like hypermutations, J. Virol, vol.89, pp.7735-7747, 2015.
, Low-fidelity polymerases of alphaviruses recombine at higher rates to overproduce defective interfering particles, J. Virol, vol.90, pp.2446-2454, 2015.
, A simple method of estimating fifty per cent endpoints, Am. J. Hyg, vol.27, pp.493-497, 1938.
, Alphavirus mutator variants present host-specific defects and attenuation in mammalian and insect models, PLoS Pathog, vol.10, p.1003877, 2014.
URL : https://hal.archives-ouvertes.fr/pasteur-00974699
, Antiviral immunity in Drosophila requires systemic RNA interference spread, Nature, vol.458, pp.346-350, 2009.
URL : https://hal.archives-ouvertes.fr/pasteur-00460853
, Comparative analysis of viral RNA signatures on different RIG-I-like receptors, vol.5, p.11275, 2016.
URL : https://hal.archives-ouvertes.fr/hal-01483406
, Beyond the linear genome: comprehensive determination of the endogenous circular elements in C. elegans and human genomes via an unbiased genomic-biophysical method. bioRxiv, 2017.
, Why do RNA viruses recombine?, Nat. Rev. Microbiol, vol.9, pp.617-626, 2011.
, Drosophila dicer-2 cleavage is mediated by helicase-and dsRNA termini-dependent states that are modulated by Loquacious-PD, Mol. Cell, vol.58, pp.406-417, 2015.
, Dicer uses distinct modules for recognizing dsRNA termini, Science, vol.359, pp.329-334, 2017.
, Viral polymerase-helicase complexes regulate replication fidelity to overcome intracellular nucleotide depletion, J. Virol, vol.89, pp.11233-11244, 2015.
, PLoS Pathog, vol.11, p.1005122, 2015.
, Uncovering the Repertoire of endogenous flaviviral elements in aedes mosquito genomes, J. Virol, vol.91, pp.571-588, 2017.
URL : https://hal.archives-ouvertes.fr/hal-01636504
, Defective viral genomes arising in vivo provide critical danger signals for the triggering of lung antiviral immunity, PLoS Pathog, vol.9, pp.314-325, 2013.
, Excessive production and extreme editing of human metapneumovirus defective interfering RNA is associated with type I interferon induction, J. Gen. Virol, vol.130, pp.1625-1633, 2007.
, The RNA silencing endonuclease Argonaute 2 mediates specific antiviral immunity in Drosophila melanogaster, Genes Dev, vol.20, pp.2985-2995, 2006.
URL : https://hal.archives-ouvertes.fr/pasteur-01954329
, In silico reconstruction of viral genomes from small RNAs improves virus-derived small interfering RNA profiling, J. Virol, vol.85, pp.11016-11021, 2011.
URL : https://hal.archives-ouvertes.fr/pasteur-01379334
, Virus discovery by deep sequencing and assembly of virus-derived small silencing RNAs, Proc. Natl. Acad. Sci. USA, vol.107, pp.1606-1611, 2010.
, Replication defective viral genomes exploit a cellular pro-survival mechanism to establish paramyxovirus persistence, Nat. Commun, vol.8, p.799, 2017.
, , 2006.
, A novel role for viral-defective interfering particles in enhancing dendritic cell maturation, J. Immunol, vol.177, pp.4503-4513
, MDA5 participates in the detection of paramyxovirus infection and is essential for the early activation of dendritic cells in response to Sendai Virus defective interfering particles, J. Immunol, vol.180, pp.4910-4918, 2008.
, The lymphocytic choriomeningitis virus matrix protein PPXY late domain drives the production of defective interfering particles, e1005501. detected after Sindbis-replicon injection was detected by nested PCR: a first PCR was performed using primers 91F and 1267R, vol.12, 2016.
, PCR product was amplified in a subsequent PCR using primers 414F and 913R
, days post-infection. cvDNA was isolated as described in ''Isolation of circular vDNA and detection by PCR.'' To verify the absence of viral RNA, cvDNA stock was reverse transcribed using the Maxima H Minus First Strand cDNA synthesis kit (Thermofisher Scientific) and assessed by PCR. For immunization, 4-5 days old flies were injected intrathoracically with 10 ng of cvDNA isolated from FHV-infected or non-infected S2 cells. 48 hr later, flies were challenged with 80 TCID 50 of FHV, 100 TCID 50 of DCV or control injection (Tris), Fly Injection with cvDNA and Survival S2 cells were infected with 1 MOI of FHV
, Deep-sequencing of Circular vDNA For generation of FHV cvDNA libraries, S2 cells were infected with 1 MOI of FHV, or not infected, and collected 7 days post-infection. Samples were phenol-chlorophorm purified and processed as described, 2017.
, Amplified DNA was isolated, digested with Sph1 or Mlu1, and ligated to similarly restricted pLg338-30 plasmid DNA. Ligated DNA was used to transform MAX Efficiency Stbl2 Competent Cells (Invitrogen) using procedures recommended by the manufacturer. Colonies were screened by PCR using FHV RNA 1-specific primers and positive colonies were Sanger sequenced using primers specific for FHV RNA 1, pLG338-30 and Drosophila retrotransposons (Table S1, related to Figure 4). Sequences were assembled and analyzed using MacVector. Production of the Sindbis-WT+D G and Control Stocks To produce Sindbis-WT+D G stock, BHK-21 cells were electroporated with 5 mg of Sindbis-WT RNA and 5 mg of D G RNA in an XCell Gene Pulser Biorad. After 48h, supernatant was collected and concentrated by ultracentrifugation as described. To produce the Sindbis-WT control stock, a ''fake'' in vitro transcription of D G RNA was performed, where all the reagents were present except the SP6 enzyme. BHK-21 cells were electroporated with 5 mg of Sindbis-WT RNA and the equivalent in volume of ''fake'' in vitro transcription. In this way, putative traces of D G plasmid used to produce D G RNA by in vitro trancription were present both in the Sindbis-WT and the Sindbis-WT+D G stock. Injection and Titration of Sindbis-Infected Flies Four to six-days old female flies were intrathoracically injected with 50 nL containing 400 PFU of Sindbis-WT, DNA concentration was measured with DNA PicoGreen reagent (Invitrogen) and samples were diluted to 0.2 ng/ml. One ng of DNA was used to prepare libraries with the Nextera XT library kit (Illumina). A PCR of 12 cycles was performed with index primers to amplify libraries, before DNA analysis on a high sensitivity DNA Bioanalyzer chip (Agilent Technologies, 1993.
, Feeding and Titration The insectary conditions for mosquito maintenance were 28 C, 70% relative humidity and a 12 h light: 12 h dark cycle. Adults were maintained with permanent access to 10% sucrose solution, Mosquitoes Rearing, vol.23, p.4, 2018.
, Mosquitoes were offered the infectious or control blood meal for 30 min through a membrane feeding system (Hemotek Ltd) set at 37 C with a piece of desalted pig intestine as the membrane. Following the blood meal, fully engorged females were selected and incubated at 28 C, 70% relative humidity and under a 12 h light: 12 h dark cycle with permanent access to 10% sucrose. Individual mosquitoes were crushed in 70 mL of PBS with a Pellet Pestle, 8 days old female mosquitoes were fed with 10 5 PFU of CHIKV diluted in prewashed human blood (iCareB platform
, Sindbis vDNA Detection by qPCR vDNA detection by qPCR was performed by injecting 4 to 6-day-old female with 10 000 PFU of Sindbis virus and by freezing the flies at the indicated time point. vDNA was extracted from flies as previously described. qPCR was performed using Power SYBR Master Mix (Applied Biosystems) according to the manufacturer's instruction, and run on a StepOne Plus real-time PCR system machine (Applied Biosystems). Primers 1215F and 1267R were used for Sindbis DNA, 25% sucrose for a week or fed only with sucrose. After a week, flies were infected with 400 PFU of Sindbis or Tris-HCl 10 mM, pH 7.5 (as a control), in 50 nL using a Nanoject II injector (Drummond Scientific)
, Plot of Relative vDNA Production Over Relative Viral Load (Figure 6B) Relative vDNA Production
, Wild-type flies, or the indicated Dcr-2 mutant, where infected with 10 000 PFU of Sindbis-WT or Sindbis-highDG, and vDNA accumulation was measured by qPCR at 7 hr post-infection. Relative vDNA production is calculated by dividing the vDNA production of each fly strain by the value obtained for wild-type flies
, Relative Viral Load (x Axis)
, Wild-type or Dcr-2 mutant flies were infected with 400 PFU of Sindbis-WT or Sindbis-highDG, and titers were measured by plaque assay at 3, 5 and 7 days post-infection (n = 10 flies per condition). The percentage of Sindbis-highDG titer relative to Sindbis-WT titer was calculated for these time points. This value was averaged on 3 independent experiments. The final value was
, RNase and DNase Treatments DNA extracted from individual flies was digested with 10 mg/mL of RNase A/T1 (Thermo Fisher Scientific) 1 hr at 37 C, then phenolchlorophorm-purified. Alternatively, DNA was digested using the Turbo DNA-free kit (Life technologies) according to the manufacturers' instructions. vDNA was measured by qPCR
, Construction of Dcr-2 helicase Expression Plasmid The helicase domain of Dcr-2 was cloned as a V5/3XFLAG tagged fusion protein into pAc 5.1 V5 His(A) (Invitrogen) using the restriction free cloning method. The 3XFLAG region from p3XFLAG-CMV-9 Expression Vector (Sigma-Aldrich) was amplified by PCR using primers Flag_F and Flag_R (Table S3, related to Figure 6). This PCR product was used as a primer for a second PCR using pAc 5.1 V5 His(A) as template. During this amplification step, the His tag was removed from the original plasmid. To clone the helicase of Drosophila melanogaster Dcr-2 in pAc 5.1 V5 3XFLAG, Dcr-2 helicase was amplified by PCR from cDNA from wild-type flies (w 1118 ) with primers Dicer-2_Flag_F and Dicer-2_Flag_R (Table S3, related to Figure 6). The purified PCR product was used as primer for a PCR using pAc 5.1 V5 3XFLAG as a template. The resulting plasmid, designated pAc5.1 Dcr-2 helicase V5 3XFLAG, carries the coding region of Dcr-2 helicase downstream of the Drosophila actin promoter and two tags for protein purification in the C-terminal region, V5 and 3XFLAG. Final plasmid was sequenced to verify the presence of inserts at the desired positions without mutations, deletions or insertions. Immunoprecipitation Assays S2 cells were seeded at 1x 10 6 cells in 6 wells plate and transfected with 400 ng/well of pAc5.1 Dcr-2 helicase V5 3XFLAG using Effectene Transfection Reagent (QIAGEN) following manufacturer's indications. Controls included empty vector pAc5.1 V5 3XFLAG and pAc5.1 CG4572 V5 3XFLAG. The next day, DVGseq Pipeline Individual flies were crushed in 1 mL of TRIzol reagent (Invitrogen) using a Pellet Pestle (Sigma-Aldrich) and RNA was extracted following the manufacturer's instructions. cDNA was produced using 100 ng of RNA using the Maxima H Minus First Strand cDNA synthesis kit (Thermo Fisher Scientific) according to the manufacturer's instructions. cDNA was further amplified by PCR using Phusion polymerase using the primers 414F and 11634R for Sindbis (Table S3, related to Figure S2). PCR products were purified on NucleoSpin Gel and PCR Clean-up column (Macherey-Nagel), 2009.
, Reactions were incubated using a roller shaker for 4 h at 4 C, washed 3X in TBS buffer (50 mM Tris pH 7.5, 150 mM NaCl) supplemented with 250mM sucrose and 0.5mM DTT followed by a final wash in TBS supplemented with 250mM sucrose and 0.5mM DTT and RNase OUT. Samples were eluted either with 3X Flag Peptide (Sigma) or lysis buffer without protease inhibitors at high stringency conditions (800mM NaCl) with proteinase K treatment, Thermo Fisher Scientific) and incubated for 30 minutes at 4 C with rocking agitation