ABSTRACT
The RNAi pathway confers antiviral immunity in insects. Virus-specific siRNA responses are amplified via the reverse transcription of viral RNA to viral DNA (vDNA). The nature, biogenesis, and regulation of vDNA are unclear. We find that vDNA produced during RNA virus infection of Drosophila and mosquitoes is present in both linear and circular forms. Circular vDNA (cvDNA) is sufficient to produce siRNAs that confer partially protective immunity when challenged with a cognate virus. cvDNAs bear homology to defective viral genomes (DVGs), and DVGs serve as templates for vDNA and cvDNA synthesis. Accordingly, DVGs promote the amplification of vDNA-mediated antiviral RNAi responses in infected Drosophila. Furthermore, vDNA synthesis is regulated by the DExD/H helicase domain of Dicer-2 in a mechanism distinct from its role in siRNA generation. We suggest that, analogous to mammalian RIG-I-like receptors, Dicer-2 functions like a pattern recognition receptor for DVGs to modulate antiviral immunity in insects.
Subject(s)
Antiviral Agents/immunology , DNA, Viral/metabolism , Drosophila Proteins/metabolism , Drosophila/immunology , RNA Helicases/metabolism , RNA Viruses/immunology , Ribonuclease III/metabolism , Animals , Arboviruses/immunology , Arboviruses/pathogenicity , Culicidae/immunology , DEAD-box RNA Helicases/metabolism , Drosophila Proteins/genetics , Genes, Viral/genetics , Genome, Viral , Host-Pathogen Interactions/genetics , Host-Pathogen Interactions/immunology , Point Mutation , RNA Helicases/genetics , RNA Interference/immunology , RNA Virus Infections , RNA Viruses/genetics , RNA Viruses/pathogenicity , RNA, Small Interfering/genetics , RNA, Viral/metabolism , Ribonuclease III/genetics , Viral Load , Virus ReplicationABSTRACT
Mosquitoes develop long-lasting viral infections without substantial deleterious effects, despite high viral loads. This makes mosquitoes efficient vectors for emerging viral diseases with enormous burden on public health. How mosquitoes resist and/or tolerate these viruses is poorly understood. Here we show that two species of Aedes mosquitoes infected with two arboviruses from distinct families (dengue or chikungunya) generate a viral-derived DNA (vDNA) that is essential for mosquito survival and viral tolerance. Inhibition of vDNA formation leads to extreme susceptibility to viral infections, reduction of viral small RNAs due to an impaired immune response, and loss of viral tolerance. Our results highlight an essential role of vDNA in viral tolerance that allows mosquito survival and thus may be important for arbovirus dissemination and transmission. Elucidating the mechanisms of mosquito tolerance to arbovirus infection paves the way to conceptualize new antivectorial strategies to selectively eliminate arbovirus-infected mosquitoes.
Subject(s)
Aedes/virology , Arboviruses/genetics , Chikungunya virus/genetics , DNA, Viral/genetics , Dengue Virus/genetics , Mosquito Vectors/virology , Animals , Cell Line , Chikungunya Fever/transmission , Chikungunya Fever/virology , Chikungunya virus/growth & development , Chlorocebus aethiops , Cricetinae , DNA, Viral/biosynthesis , Dengue/transmission , Dengue/virology , Dengue Virus/growth & development , RNA Interference , RNA, Viral/biosynthesis , RNA, Viral/genetics , Vero Cells , Viral LoadABSTRACT
Tunnelling nanotubes and cytonemes function as highways for the transport of organelles, cytosolic and membrane-bound molecules, and pathogens between cells. During viral infection in the model organism Drosophila melanogaster, a systemic RNAi antiviral response is established presumably through the transport of a silencing signal from one cell to another via an unknown mechanism. Because of their role in cell-cell communication, we investigated whether nanotube-like structures could be a mediator of the silencing signal. Here, we describe for the first time in the context of a viral infection the presence of nanotube-like structures in different Drosophila cell types. These tubules, made of actin and tubulin, were associated with components of the RNAi machinery, including Argonaute 2, double-stranded RNA, and CG4572. Moreover, they were more abundant during viral, but not bacterial, infection. Super resolution structured illumination microscopy showed that Argonaute 2 and tubulin reside inside the tubules. We propose that nanotube-like structures are one of the mechanisms by which Argonaute 2, as part of the antiviral RNAi machinery, is transported between infected and non-infected cells to trigger systemic antiviral immunity in Drosophila.
Subject(s)
Argonaute Proteins/genetics , Drosophila Proteins/genetics , Drosophila melanogaster/metabolism , Organelles/metabolism , RNA, Double-Stranded/genetics , Viral Proteins/antagonists & inhibitors , Actins/genetics , Actins/metabolism , Animals , Argonaute Proteins/metabolism , Biological Transport , Cell Communication , Cell Line , Dicistroviridae/genetics , Dicistroviridae/growth & development , Drosophila Proteins/metabolism , Drosophila melanogaster/microbiology , Drosophila melanogaster/ultrastructure , Drosophila melanogaster/virology , Gene Expression Regulation , Genes, Reporter , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Organelles/microbiology , Organelles/ultrastructure , Organelles/virology , Pectobacterium carotovorum/genetics , Pectobacterium carotovorum/growth & development , RNA Interference , RNA, Double-Stranded/metabolism , Tubulin/genetics , Tubulin/metabolism , Viral Proteins/genetics , Viral Proteins/metabolism , rab GTP-Binding Proteins/genetics , rab GTP-Binding Proteins/metabolism , rab7 GTP-Binding ProteinsABSTRACT
RNA interference (RNAi) controls gene expression in eukaryotic cells and thus, cellular homeostasis. In addition, in plants, nematodes and arthropods it is a central antiviral effector mechanism. Antiviral RNAi has been well described as a cell autonomous response, which is triggered by double-stranded RNA (dsRNA) molecules. This dsRNA is the precursor for the silencing of viral RNA in a sequence-specific manner. In plants, systemic antiviral immunity has been demonstrated, however much less is known in animals. Recently, some evidence for a systemic antiviral response in arthropods has come to light. Cell autonomous RNAi may not be sufficient to reach an efficient antiviral response, and the organism might rely on the spread and uptake of an RNAi signal of unknown origin. In this review, we offer a perspective on how RNAi-mediated antiviral immunity could confer systemic protection in insects and we propose directions for future research to understand the mechanism of RNAi-immune signal sorting, spreading and amplification.
Subject(s)
Arbovirus Infections/immunology , Arboviruses/immunology , Clathrin-Coated Vesicles/metabolism , Drosophila melanogaster/immunology , RNA Interference , Animals , Antigens, Viral/immunology , Argonaute Proteins/metabolism , Clathrin-Coated Vesicles/immunology , Drosophila Proteins/metabolism , Drosophila melanogaster/virology , Endocytosis/immunology , Humans , Immunity, Innate/genetics , RNA Helicases/metabolism , RNA Interference/immunology , RNA, Double-Stranded/immunology , RNA, Viral/immunology , Ribonuclease III/metabolismABSTRACT
How persistent viral infections are established and maintained is widely debated and remains poorly understood. We found here that the persistence of RNA viruses in Drosophila melanogaster was achieved through the combined action of cellular reverse-transcriptase activity and the RNA-mediated interference (RNAi) pathway. Fragments of diverse RNA viruses were reverse-transcribed early during infection, which resulted in DNA forms embedded in retrotransposon sequences. Those virus-retrotransposon DNA chimeras produced transcripts processed by the RNAi machinery, which in turn inhibited viral replication. Conversely, inhibition of reverse transcription hindered the appearance of chimeric DNA and prevented persistence. Our results identify a cooperative function for retrotransposons and antiviral RNAi in the control of lethal acute infection for the establishment of viral persistence.
Subject(s)
Drosophila melanogaster/genetics , Drosophila melanogaster/virology , RNA Interference , RNA Virus Infections/virology , RNA Viruses/genetics , Reverse Transcription , Animals , Base Sequence , Cell Line , DNA Viruses/chemistry , DNA Viruses/genetics , DNA Viruses/metabolism , Disease Models, Animal , Female , Gene Order , Models, Biological , Molecular Sequence Data , RNA Viruses/chemistry , RNA Viruses/metabolism , RNA, Small Interfering/genetics , Retroelements , Viral Load , Virus Replication/geneticsABSTRACT
Persistent infection is a situation of metastability in which the pathogen and the host coexist. A common outcome for viral infections, persistence is a widespread phenomenon through all kingdoms. With a clear benefit for the virus and/or the host at the population level, persistent infections act as modulators of the ecosystem. The origin of persistence being long time elusive, here we explore the concept of 'endogenization' of viral sequences with concomitant activation of the host immune pathways, as a main way to establish and maintain viral persistent infections. Current concepts on viral persistence mechanisms and biological role are discussed.
Subject(s)
DNA Virus Infections/immunology , RNA Virus Infections/immunology , Virus Latency , Virus Physiological Phenomena , Animals , DNA Virus Infections/virology , Genome, Viral , Host-Pathogen Interactions , Humans , RNA Virus Infections/virology , Viruses/genetics , Viruses/immunologyABSTRACT
RNA interference (RNAi) is the essential component of antiviral immunity in invertebrates and plants. One of the landmarks of the antiviral RNAi response is the production of virus-derived small interfering RNA (vsiRNA) from viral double-stranded RNA (dsRNA). vsiRNAs constitute a fragmented image of the viral genome sequence that results from Dicer cleavage. vsiRNA sequence profiling is used extensively as a surrogate to study the antiviral RNAi response by determining the nature of the viral dsRNA molecules exposed to and processed by the RNAi machinery. The accuracy of these profiles depends on the actual viral genome sequence used as a reference to align vsiRNA reads, and the interpretation of inaccurate profiles can be misleading. Using Flock house virus and Drosophila melanogaster as a model RNAi-competent organism, we show accurate reconstruction of full-length virus reference sequence from vsiRNAs and prediction of the structure of defective interfering particles (DIs). We developed a Perl script, named Paparazzi, that reconstitutes viral genomes through an iterative alignment/consensus call procedure using a related reference sequence as scaffold. As prevalent DI-derived reads introduce artifacts during reconstruction, Paparazzi eliminates DI-specific reads to improve the quality of the reconstructed genome. Paparazzi constitutes a promising alternative to Sanger sequencing in this context and an effective tool to study antiviral RNAi mechanisms by accurately quantifying vsiRNA along the replicating viral genome. We further discuss Paparazzi as a companion tool for virus discovery as it provides full-length genome sequences and corrects for potential artifacts of assembly.
Subject(s)
Drosophila melanogaster/virology , Genome, Viral , Molecular Biology/methods , Nodaviridae/genetics , RNA Interference , RNA, Small Interfering/genetics , RNA, Viral/genetics , Animals , Computational Biology/methods , Drosophila melanogaster/immunology , Molecular Sequence Data , Nodaviridae/immunology , Sequence Analysis, DNAABSTRACT
Multicellular organisms evolved sophisticated defence systems to confer protection against pathogens. An important characteristic of these immune systems is their ability to act both locally at the site of infection and at distal uninfected locations. In insects, such as Drosophila melanogaster, RNA interference (RNAi) mediates antiviral immunity. However, the antiviral RNAi defence in flies seems to be a local, cell-autonomous process, as flies are thought to be unable to generate a systemic RNAi response. Here we show that a recently defined double-stranded RNA (dsRNA) uptake pathway is essential for effective antiviral RNAi immunity in adult flies. Mutant flies defective in this dsRNA uptake pathway were hypersensitive to infection with Drosophila C virus and Sindbis virus. Mortality in dsRNA-uptake-defective flies was accompanied by 100-to 10(5)-fold increases in viral titres and higher levels of viral RNA. Furthermore, inoculating naked dsRNA into flies elicited a sequence-specific antiviral immune response that required an intact dsRNA uptake pathway. These findings suggest that spread of dsRNA to uninfected sites is essential for effective antiviral immunity. Notably, infection with green fluorescent protein (GFP)-tagged Sindbis virus suppressed expression of host-encoded GFP at a distal site. Thus, similar to protein-based immunity in vertebrates, the antiviral RNAi response in flies also relies on the systemic spread of a virus-specific immunity signal.
Subject(s)
Drosophila melanogaster/immunology , Drosophila melanogaster/virology , RNA Interference/immunology , RNA Viruses/immunology , Animals , Cell Line , Drosophila melanogaster/genetics , Drosophila melanogaster/microbiology , Micrococcus luteus/immunology , Pectobacterium carotovorum/immunology , RNA Viruses/physiology , RNA, Double-Stranded/genetics , RNA, Double-Stranded/immunology , RNA, Double-Stranded/metabolism , Sindbis Virus/genetics , Sindbis Virus/growth & development , Sindbis Virus/immunology , Substrate SpecificityABSTRACT
The infectious salmon anemia virus (ISAV), which belongs to the new genus Isavirus of the Orthomyxoviridae family, is an important pathogen of the salmon farming industry. Indirect immunofluorescence assays carried out with monoclonal antibodies specific for the nucleoprotein (NP) reveal differential staining of sub-cellular compartments in infected cells. Particularly interesting was the staining of the nucleolus, which showed co-localization with nucleolin in CHSE-214, EPC and SHK-1 cells infected with ISAV. These results were confirmed by co-immunoprecipitation studies showing an interaction between NP and nucleolin. In addition, in situ hybridization carried out with probes specific for each of the 8 RNA segments of ISAV showed that the genomic as well as the anti-genomic strands were also localized in the nucleolus. These results suggest a role of the nucleolus in the replication and/or in the packaging of the ISAV genome.