piRNA pathway is not required for antiviral defense in Drosophila melanogaster.

Since its discovery, RNA interference has been identified as involved in many different cellular processes, and as a natural antiviral response in plants, nematodes, and insects. In insects, the small interfering RNA (siRNA) pathway is the major antiviral response. In recent years, the Piwi-interacting RNA (piRNA) pathway also has been implicated in antiviral defense in mosquitoes infected with arboviruses. Using Drosophila melanogaster and an array of viruses that infect the fruit fly acutely or persistently or are vertically transmitted through the germ line, we investigated in detail the extent to which the piRNA pathway contributes to antiviral defense in adult flies. Following virus infection, the survival and viral titers of Piwi, Aubergine, Argonaute-3, and Zucchini mutant flies were similar to those of wild type flies. Using next-generation sequencing of small RNAs from wild type and siRNA mutant flies, we showed that no viral-derived piRNAs were produced in fruit flies during different types of viral infection. Our study provides the first evidence, to our knowledge, that the piRNA pathway does not play a major role in antiviral defense in adult Drosophila and demonstrates that viral-derived piRNA production depends on the biology of the host-virus combination rather than being part of a general antiviral process in insects.

Alphavirus mutator variants present host-specific defects and attenuation in mammalian and insect models.

Arboviruses cycle through both vertebrates and invertebrates, which requires them to adapt to disparate hosts while maintaining genetic integrity during genome replication. To study the genetic mechanisms and determinants of these processes, we use chikungunya virus (CHIKV), a re-emerging human pathogen transmitted by the Aedes mosquito. We previously isolated a high fidelity (or antimutator) polymerase variant, C483Y, which had decreased fitness in both mammalian and mosquito hosts, suggesting this residue may be a key molecular determinant. To further investigate effects of position 483 on RNA-dependent RNA-polymerase (RdRp) fidelity, we substituted every amino acid at this position. We isolated novel mutators with decreased replication fidelity and higher mutation frequencies, allowing us to examine the fitness of error-prone arbovirus variants. Although CHIKV mutators displayed no major replication defects in mammalian cell culture, they had reduced specific infectivity and were attenuated in vivo. Unexpectedly, mutator phenotypes were suppressed in mosquito cells and the variants exhibited significant defects in RNA synthesis. Consequently, these replication defects resulted in strong selection for reversion during infection of mosquitoes. Since residue 483 is conserved among alphaviruses, we examined the analogous mutations in Sindbis virus (SINV), which also reduced polymerase fidelity and generated replication defects in mosquito cells. However, replication defects were mosquito cell-specific and were not observed in Drosophila S2 cells, allowing us to evaluate the potential attenuation of mutators in insect models where pressure for reversion was absent. Indeed, the SINV mutator variant was attenuated in fruit flies. These findings confirm that residue 483 is a determinant regulating alphavirus polymerase fidelity and demonstrate proof of principle that arboviruses can be attenuated in mammalian and insect hosts by reducing fidelity.

Viral infection disrupts intestinal homeostasis via Sting-dependent NF-κB signaling in Drosophila.

Host-microbe interactions influence intestinal stem cell (ISC) activity to modulate epithelial turnover and composition. Here, we investigated the functional impacts of viral infection on intestinal homeostasis and the mechanisms by which viral infection alters ISC activity. We report that Drosophila A virus (DAV) infection disrupts intestinal homeostasis in Drosophila by inducing sustained ISC proliferation, resulting in intestinal dysplasia, loss of gut barrier function, and reduced lifespan. We found that additional viruses common in laboratory-reared Drosophila also promote ISC proliferation. The mechanism of DAV-induced ISC proliferation involves progenitor-autonomous epidermal growth factor receptor (EGFR) signaling, c-Jun N-terminal kinase (JNK) activity in enterocytes, and requires Sting-dependent nuclear factor κB (NF-κB) (Relish) activity. We further demonstrate that activating Sting-Relish signaling is sufficient to induce ISC proliferation, promote intestinal dysplasia, and reduce lifespan in the absence of infection. Our results reveal that viral infection can significantly disrupt intestinal physiology, highlight a novel role for Sting-Relish signaling, and support a role for viral infection in aging.

Innovative Toolbox for the Quantification of Drosophila C Virus, Drosophila A Virus, and Nora Virus.

Quantification of viral replication underlies investigations into host-virus interactions. In Drosophila melanogaster, persistent infections with Drosophila C virus, Drosophila A virus, and Nora virus are commonly observed in nature and in laboratory fly stocks. However, traditional endpoint dilution assays to quantify infectious titers are not compatible with persistently infecting isolates of these viruses that do not cause cytopathic effects in cell culture. Here we present a novel assay based on immunological detection of Drosophila C virus infection that allows quantification of infectious titers for a wider range of Drosophila C virus isolates. We also describe strand specific RT-qPCR assays for quantification of viral negative strand RNA produced during Drosophila C virus, Drosophila A virus, and Nora virus infection. Finally, we demonstrate the utility of these assays for quantification of viral replication during oral infections and persistent infections with each virus.

Innate immune pathways act synergistically to constrain RNA virus evolution in Drosophila melanogaster.

Host-pathogen interactions impose recurrent selective pressures that lead to constant adaptation and counter-adaptation in both competing species. Here, we sought to study this evolutionary arms-race and assessed the impact of the innate immune system on viral population diversity and evolution, using Drosophila melanogaster as model host and its natural pathogen Drosophila C virus (DCV). We isogenized eight fly genotypes generating animals defective for RNAi, Imd and Toll innate immune pathways as well as pathogen-sensing and gut renewal pathways. Wild-type or mutant flies were then orally infected with DCV and the virus was serially passaged ten times via reinfection in naive flies. Viral population diversity was studied after each viral passage by high-throughput sequencing and infection phenotypes were assessed at the beginning and at the end of the evolution experiment. We found that the absence of any of the various immune pathways studied increased viral genetic diversity while attenuating virulence. Strikingly, these effects were observed in a range of host factors described as having mainly antiviral or antibacterial functions. Together, our results indicate that the innate immune system as a whole and not specific antiviral defence pathways in isolation, generally constrains viral diversity and evolution.

Reference gene selection for gene expression studies using RT-qPCR in virus-infected planthoppers.

BACKGROUND: Planthoppers not only severely affect crops by causing mechanical damage when feeding but are also vectors of several plant virus species. The analysis of gene expression in persistently infected planthoppers might unveil the molecular basis of viral transmission. Quantitative real-time RT-PCR (RT-qPCR) is currently the most accurate and sensitive method used for quantitative gene expression analysis. In order to normalize the resulting quantitative data, reference genes with constant expression during the experimental procedures are needed. RESULTS: Partial sequences of the commonly used reference genes actin (ACT), α1-tubulin (TUB), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), elongation factor 1 alpha (EF1A), ribosomal protein S18 (RPS18) and polyubiquitin C (UBI) from Delphacodes kuscheli, a planthopper capable of persistently transmitting the plant fijivirus Mal de Río Cuarto virus (MRCV), were isolated for the first time. Specific RT-qPCR primers were designed and the expression stability of these genes was assayed in MRCV-infective and naïve planthoppers using geNorm, Normfinder and BestKeeper tools. The overall analysis showed that UBI, followed by 18S and ACT, are the most suitable genes as internal controls for quantitative gene expression studies in MRCV-infective planthoppers, while TUB and EF1A are the most variable ones. Moreover, EF1A was upregulated by MRCV infection. CONCLUSIONS: A RT-qPCR platform for gene expression analysis in the MRCV-infected planthopper vector Delphacodes kuscheli was developed. Our work is the first report on reference gene selection in virus-infected insects, and might serve as a precedent for future gene expression studies on MRCV and other virus-planthopper pathosystems.

Viral Infection and Stress Affect Protein Levels of Dicer 2 and Argonaute 2 in Drosophila melanogaster.

The small interfering RNA (siRNA) pathway of Drosophila melanogaster, mainly characterized by the activity of the enzymes Dicer 2 (Dcr-2) and Argonaute 2 (Ago-2), has been described as the major antiviral immune response. Several lines of evidence demonstrated its pivotal role in conferring resistance against viral infections at cellular and systemic level. However, only few studies have addressed the regulation and induction of this system upon infection and knowledge on stability and turnover of the siRNA pathway core components transcripts and proteins remains scarce. In the current work, we explore whether the siRNA pathway is regulated following viral infection in D. melanogaster. After infecting different fly strains with two different viruses and modes of infection, we observed changes in Dcr-2 and Ago-2 protein concentrations that were not related with changes in gene expression. This response was observed either upon viral infection or upon stress-related experimental procedure, indicating a bivalent function of the siRNA system operating as a general gene regulation rather than a specific antiviral system.

Evidence For Long-Lasting Transgenerational Antiviral Immunity in Insects.

Transgenerational immune priming (TGIP) allows memory-like immune responses to be transmitted from parents to offspring in many invertebrates. Despite increasing evidence for TGIP in insects, the mechanisms involved in the transfer of information remain largely unknown. Here, we show that Drosophila melanogaster and Aedes aegypti transmit antiviral immunological memory to their progeny that lasts throughout generations. We observe that TGIP, which is virus and sequence specific but RNAi independent, is initiated by a single exposure to disparate RNA viruses and also by inoculation of a fragment of viral double-stranded RNA. The progeny, which inherit a viral DNA that is only a fragment of the viral RNA used to infect the parents, display enriched expression of genes related to chromatin and DNA binding. These findings represent a demonstration of TGIP for RNA viruses in invertebrates, broadly increasing our understanding of the immune response, host genome plasticity, and antiviral memory of the germline.

Virus infection elevates transcriptional activity of miR164a promoter in plants.

BACKGROUND: Micro RNAs (miRs) constitute a large group of endogenous small RNAs that have crucial roles in many important plant functions. Virus infection and transgenic expression of viral proteins alter accumulation and activity of miRs and so far, most of the published evidence involves post-transcriptional regulations. RESULTS: Using transgenic plants expressing a reporter gene under the promoter region of a characterized miR (P-miR164a), we monitored the reporter gene expression in different tissues and during Arabidopsis development. Strong expression was detected in both vascular tissues and hydathodes. P-miR164a activity was developmentally regulated in plants with a maximum expression at stages 1.12 to 5.1 (according to Boyes, 2001) along the transition from vegetative to reproductive growth. Upon quantification of P-miR164a-derived GUS activity after Tobacco mosaic virus Cg or Oilseed rape mosaic virus (ORMV) infection and after hormone treatments, we demonstrated that ORMV and gibberellic acid elevated P-miR164a activity. Accordingly, total mature miR164, precursor of miR164a and CUC1 mRNA (a miR164 target) levels increased after virus infection and interestingly the most severe virus (ORMV) produced the strongest promoter induction. CONCLUSION: This work shows for the first time that the alteration of miR pathways produced by viral infections possesses a transcriptional component. In addition, the degree of miR alteration correlates with virus severity since a more severe virus produces a stronger P-miR164a induction.

Mal de Río Cuarto Virus Infection Triggers the Production of Distinctive Viral-Derived siRNA Profiles in Wheat and Its Planthopper Vector.

Plant reoviruses are able to multiply in gramineae plants and delphacid vectors encountering different defense strategies with unique features. This study aims to comparatively assess alterations of small RNA (sRNA) populations in both hosts upon virus infection. For this purpose, we characterized the sRNA profiles of wheat and planthopper vectors infected by Mal de Río Cuarto virus (MRCV, Fijivirus, Reoviridae) and quantified virus genome segments by quantitative reverse transcription PCR We provide evidence that plant and insect silencing machineries differentially recognize the viral genome, thus giving rise to distinct profiles of virus-derived small interfering RNAs (vsiRNAs). In plants, most of the virus genome segments were targeted preferentially within their upstream sequences and vsiRNAs mapped with higher density to the smaller genome segments than to the medium or larger ones. This tendency, however, was not observed in insects. In both hosts, vsiRNAs were equally derived from sense and antisense RNA strands and the differences in vsiRNAs accumulation did not correlate with mRNAs accumulation. We also established that the piwi-interacting RNA (piRNA) pathway was active in the delphacid vector but, contrary to what is observed in virus-infected mosquitoes, virus-specific piRNAs were not detected. This work contributes to the understanding of the silencing response in insect and plant hosts.

Bugs Are Not to Be Silenced: Small RNA Pathways and Antiviral Responses in Insects.

Like every other organism on Earth, insects are infected with viruses, and they rely on RNA interference (RNAi) mechanisms to circumvent viral infections. A remarkable characteristic of RNAi is that it is both broadly acting, because it is triggered by double-stranded RNA molecules derived from virtually any virus, and extremely specific, because it targets only the particular viral sequence that initiated the process. Reviews covering the different facets of the RNAi antiviral immune response in insects have been published elsewhere. In this review, we build a framework to guide future investigation. We focus on the remaining questions and avenues of research that need to be addressed to move the field forward, including issues such as the activity of viral suppressors of RNAi, comparative genomics, the development of detailed maps of the subcellular localization of viral replication complexes with the RNAi machinery, and the regulation of the antiviral RNAi response.

Drosophila cells use nanotube-like structures to transfer dsRNA and RNAi machinery between cells.

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.

In vivo subcellular localization of Mal de Río Cuarto virus (MRCV) non-structural proteins in insect cells reveals their putative functions.

The in vivo subcellular localization of Mal de Río Cuarto virus (MRCV, Fijivirus, Reoviridae) non-structural proteins fused to GFP was analyzed by confocal microscopy. P5-1 showed a cytoplasmic vesicular-like distribution that was lost upon deleting its PDZ binding TKF motif, suggesting that P5-1 interacts with cellular PDZ proteins. P5-2 located at the nucleus and its nuclear import was affected by the deletion of its basic C-termini. P7-1 and P7-2 also entered the nucleus and therefore, along with P5-2, could function as regulators of host gene expression. P6 located in the cytoplasm and in perinuclear cloud-like inclusions, was driven to P9-1 viroplasm-like structures and co-localized with P7-2, P10 and α-tubulin, suggesting its involvement in viroplasm formation and viral intracellular movement. Finally, P9-2 was N-glycosylated and located at the plasma membrane in association with filopodia-like protrusions containing actin, suggesting a possible role in virus cell-to-cell movement and spread.

Functional and biochemical properties of Mal de Río Cuarto virus (Fijivirus, Reoviridae) P9-1 viroplasm protein show further similarities to animal reovirus counterparts.

Mal de Río Cuarto virus (MRCV) is a plant virus of the genus Fijivirus within the family Reoviridae that infects several monocotyledonous species and is transmitted by planthoppers in a persistent and propagative manner. Other members of the family replicate in viral inclusion bodies (VIBs) termed viroplasms that are formed in the cytoplasm of infected plant and insect cells. In this study, the protein coded by the first ORF of MRCV segment S9 (P9-1) was shown to establish cytoplasmic inclusion bodies resembling viroplasms after transfection of Spodoptera frugiperda insect cells. In accordance, MRCV P9-1 self-associates giving rise to high molecular weight complexes when expressed in bacteria. Strong self-interaction was also evidenced by yeast two-hybrid assays. Furthermore, biochemical characterization showed that MRCV P9-1 bound single stranded RNA and had ATPase activity. Finally, the MRCV P9-1 region required for the formation of VIB-like structures was mapped to the protein carboxy-terminal half. This extensive functional and biochemical characterization of MRCV P9-1 revealed further similarities between plant and animal reovirus viroplasm proteins.

A practical approach to the understanding and teaching of RNA silencing in plants

Gene silencing, also called RNA interference (RNAi) is a specific mechanism of RNA degradation involved in gene regulation, development and defense in eukaryotic organisms. It became an important subject in the teaching programs of molecular biology, genetics and biotechnology courses in the last years. The aim of this work is to provide simple and inexpensive assays to understand and teach gene silencing using plants as model systems. The use of transient and permanent transgenic plants for expressing reporter genes, like those derived from jellyfish green fluorescent protein (gfp) encoding gene, provides a nice, colorful and conclusive image of gene silencing. Three experimental approaches to evidence RNA silencing are depicted. In the first approach gene silencing is demonstrated after transient expression of reporter genes in non-transgenic plants. In the second, silencing is triggered against a reporter gene stably integrated into a transgenic plant. The third approach involves the triggering of RNA silencing against endogenous genes using viral vectors. In addition we illustrate systemic gene silencing showing how the silencing signal is spread over a plant and finally it is also demonstrated the suppression of gene silencing. The first group of experiments is recommended to be tough on undergraduate courses, the following two sections are recommended for graduate courses. Hopefully, it will help students to understand this important phenomenon and to unravel the importance of gene silencing as a key gene regulation mechanism and as a molecular and biotechnological tool.