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Argonaute (AGO) proteins bind small RNAs to silence complementary RNA transcripts and are central to RNA interference (RNAi). RNAi is critical for regulation of gene expression and antiviral defense in Aedes aegypti mosquitoes, which transmit Zika, chikungunya, dengue, and yellow fever viruses. In mosquitoes, AGO1 facilitates miRNA interactions while AGO2 mediates siRNA interactions. We applied AGO-crosslinking immunoprecipitation (AGO-CLIP) for both AGO1 and AGO2 and developed a universal software package for CLIP analysis (CLIPflexR), identifying 230 small RNAs and 5,447 small RNA targets that comprise a comprehensive RNAi network map in mosquitoes. RNAi network maps predicted expression levels of small RNA targets in specific tissues. Additionally, this resource identified unexpected, context-dependent AGO2 target preferences, including endogenous viral elements and 3′UTRs. Finally, in contrast to current thinking, mosquito AGO2 repressed imperfect targets. These findings expand our understanding of small RNA networks and have broad implications for the study of antiviral RNAi.

The phenotypic manifestations of disease induced by viruses and subviral infectious entities are the result of complex molecular interactions between host and viral factors. The viral determinants of the diseased phenotype have traditionally been sought at the level of structural or non-structural proteins. However, the discovery of RNA silencing mechanisms has led to speculations that determinants of the diseased phenotype are caused by viral nucleic acid sequences in addition to proteins. RNA silencing is a gene regulation mechanism conserved within eukaryotic kingdoms (with the exception of some yeast species), and in plants and insects it also functions as an antiviral mechanism. Non-coding RNAs of viral origin, ranging in size from 21 to 24 nucleotides (viral small interfering RNAs, vsiRNAs) accumulate in virus-infected tissues and organs, in some cases to comparable levels as the entire complement of host-encoded small interfering RNAs. Upon incorporation into RNA-induced silencing complexes, vsiRNAs can mediate cleavage or induce translational inhibition of nucleic acid targets in a sequence-specific manner. This review focuses on recent findings that suggest an increased complexity of small RNA-based interactions between virus and host. We mainly address plant viruses, but where applicable discuss insect viruses as well. Prominence is given to studies that have indisputably demonstrated that vsiRNAs determine diseased phenotype by either carrying sequence determinants or, indirectly, by altering host-gene regulatory pathways. Results from these studies suggest biotechnological applications, which are also discussed.

Interactions between the insect immune system and RNA viruses have been extensively studied in Drosophila, where RNA interference, NF-κB and JAK-STAT pathways underlie antiviral immunity. In response to RNA interference, insect viruses have convergently evolved suppressors of this pathway that act by diverse mechanisms to permit viral replication. However, interactions between the insect immune system and DNA viruses have received less attention, primarily because few Drosophila-infecting DNA virus isolates are available. Here, we use a recently-isolated DNA virus of Drosophila melanogaster, Kallithea virus (family Nudiviridae), to probe known antiviral immune responses and virus evasion tactics in the context of DNA virus infection. We find that fly mutants for RNA interference and Immune deficiency (Imd), but not Toll, pathways are more susceptible to Kallithea virus infection. We identify the Kallithea virus-encoded protein gp83 as a potent inhibitor of Toll signalling, suggesting that Toll mediates antiviral defense against Kallithea virus infection, but that it is suppressed by the virus. We find that Kallithea virus gp83 inhibits Toll signalling through the regulation of NF-κB transcription factors. Furthermore, we find that gp83 of the closely related Drosophila innubila nudivirus (DiNV) suppresses D. melanogaster Toll signalling, suggesting an evolutionary conserved function of Toll in defense against DNA viruses. Together, these results provide a broad description of known antiviral pathways in the context of DNA virus infection and identify the first Toll pathway inhibitor in a Drosophila virus, extending the known diversity of insect virus-encoded immune inhibitors.Importance Co-evolution of multicellular organisms and their natural viruses may lead to an intricate relationship in which host survival requires effective immunity, and virus survival depends on evasion of such responses. Insect antiviral immunity, and reciprocal virus immune suppression tactics, have been well-studied in Drosophila melanogaster, primarily during RNA, but not DNA, virus infection. Therefore, we describe interactions between a recently-isolated Drosophila DNA virus (Kallithea virus - KV) and immune processes known to control RNA viruses, such as RNAi and Imd pathways. We find that KV suppresses the Toll pathway, and identify gp83 as a KV-encoded protein that underlies this suppression. This immunosuppressive ability is conserved in another nudivirus, suggesting the Toll pathway has conserved antiviral activity against DNA nudiviruses, which have evolved suppressors in response. Together, these results indicate that DNA viruses induce and suppress NF-κB responses, and advance the application of KV as a model to study insect immunity.

Item does not contain fulltext RNA interference (RNAi) is a powerful host defense mechanism against viruses. As a counter-defense, many viruses encode suppressors of RNAi, which - in plants - has provoked counter-counter-defense strategies. Recently, a mechanism was proposed in Drosophila (Zhang et al.) wherein a long noncoding RNA senses a virus-encoded RNAi suppressor to activate an innate immune response.

Contains fulltext : 225985.pdf (Publisher’s version ) (Open Access) Endogenous viral elements (EVEs) are viral sequences integrated in host genomes. A large number of non-retroviral EVEs was recently detected in Aedes mosquito genomes, leading to the hypothesis that mosquito EVEs may control exogenous infections by closely related viruses. Here, we experimentally investigated the role of an EVE naturally found in Aedes aegypti populations and derived from the widespread insect-specific virus, cell-fusing agent virus (CFAV). Using CRISPR-Cas9 genome editing, we created an Ae. aegypti line lacking the CFAV EVE. Absence of the EVE resulted in increased CFAV replication in ovaries, possibly modulating vertical transmission of the virus. Viral replication was controlled by targeting of viral RNA by EVE-derived P-element-induced wimpy testis-interacting RNAs (piRNAs). Our results provide evidence that antiviral piRNAs are produced in the presence of a naturally occurring EVE and its cognate virus, demonstrating a functional link between non-retroviral EVEs and antiviral immunity in a natural insect-virus interaction.

The phenotypic manifestations of disease induced by viruses and subviral infectious entities are the result of complex molecular interactions between host and viral factors. The viral determinants of the diseased phenotype have traditionally been sought at the level of structural or non-structural proteins. However, the discovery of RNA silencing mechanisms has led to speculations that determinants of the diseased phenotype are caused by viral nucleic acid sequences in addition to proteins. RNA silencing is a gene regulation mechanism conserved within eukaryotic kingdoms (with the exception of some yeast species), and in plants and insects it also functions as an antiviral mechanism. Non-coding RNAs of viral origin, ranging in size from 21 to 24 nucleotides (viral small interfering RNAs, vsiRNAs) accumulate in virus-infected tissues and organs, in some cases to comparable levels as the entire complement of host-encoded small interfering RNAs. Upon incorporation into RNA-induced silencing complexes, vsiRNAs can mediate cleavage or induce translational inhibition of nucleic acid targets in a sequence-specific manner. This review focuses on recent findings that suggest an increased complexity of small RNA-based interactions between virus and host. We mainly address plant viruses, but where applicable discuss insect viruses as well. Prominence is given to studies that have indisputably demonstrated that vsiRNAs determine diseased phenotype by either carrying sequence determinants or, indirectly, by altering host-gene regulatory pathways. Results from these studies suggest biotechnological applications, which are also discussed.

Contains fulltext : 208771.pdf (Publisher’s version ) (Open Access) The RNA interference (RNAi) pathway is a potent antiviral defense mechanism in plants and invertebrates, in response to which viruses evolved suppressors of RNAi. In mammals, the first line of defense is mediated by the type I interferon system (IFN); however, the degree to which RNAi contributes to antiviral defense is still not completely understood. Recent work suggests that antiviral RNAi is active in undifferentiated stem cells and that antiviral RNAi can be uncovered in differentiated cells in which the IFN system is inactive or in infections with viruses lacking putative viral suppressors of RNAi. In this review, we describe the mechanism of RNAi and its antiviral functions in insects and mammals. We draw parallels and highlight differences between (antiviral) RNAi in these classes of animals and discuss open questions for future research.

Contains fulltext : 203028.pdf (Publisher’s version ) (Open Access) Small RNA mediated responses are essential for antiviral defence in mosquitoes, however, they appear to differ per virus-vector combination. To further investigate the diversity of small RNA responses against viruses in mosquitoes, we applied a small RNA deep sequencing approach on five mosquito cell lines: Culex tarsalis CT cells, Aedes albopictus U4.4 and C6/36 cells, Ae. aegypti Aag2 cells (cleared from cell fusing agent virus and Culex Y virus (CYV) by repetitive dsRNA transfections) and Ae. pseudoscutellaris AP-61 cells. De novo assembly of small RNAs revealed the presence of Phasi Charoen-like virus (PCLV), Calbertado virus, Flock House virus and a novel narnavirus in CT cells, CYV in U4.4 cells, and PCLV in Aag2 cells, whereas no insect-specific viruses (ISVs) were detected in C6/36 and AP-61 cells. Next, we investigated the small RNA responses to the identified ISVs and to acute infection with the arthropod-borne West Nile virus (WNV). We demonstrate that AP-61 and C6/36 cells do not produce siRNAs to WNV infection, suggesting that AP-61, like C6/36, are Dicer-2 deficient. CT cells produced a strong siRNA response to the persistent ISVs and acute WNV infection. Interestingly, CT cells also produced viral PIWI-interacting (pi)RNAs to PCLV, but not to WNV or any of the other ISVs. In contrast, in U4.4 and Aag2 cells, WNV siRNAs, and pi-like RNAs without typical ping-pong piRNA signature were observed, while this signature was present in PCLV piRNAs in Aag2 cells. Together, our results demonstrate that mosquito small RNA responses are strongly dependent on both the mosquito cell type and/or the mosquito species and family of the infecting virus.

Stress responses are crucial processes that require activation of genetic programs that protect from the stressor. Stress responses are also energy consuming and can thus be deleterious to the organism. The mechanisms coordinating energy consumption during stress response in multicellular organisms are not well understood. Here, we show that loss of the epigenetic regulator G9a in Drosophila causes a shift in the transcriptional and metabolic responses to oxidative stress (OS) that leads to decreased survival time upon feeding the xenobiotic paraquat. During OS exposure, G9a mutants show overactivation of stress response genes, rapid depletion of glycogen, and inability to access lipid energy stores. The OS survival deficiency of G9a mutants can be rescued by a high-sugar diet. Control flies also show improved OS survival when fed a high-sugar diet, suggesting that energy availability is generally a limiting factor for OS tolerance. Directly limiting access to glycogen stores by knocking down glycogen phosphorylase recapitulates the OS-induced survival defects of G9a mutants. We propose that G9a mutants are sensitive to stress because they experience a net reduction in available energy due to (1) rapid glycogen use, (2) an inability to access lipid energy stores, and (3) an overinduced transcriptional response to stress that further exacerbates energy demands. This suggests that G9a acts as a critical regulatory hub between the transcriptional and metabolic responses to OS. Our findings, together with recent studies that established a role for G9a in hypoxia resistance in cancer cell lines, suggest that G9a is of wide importance in controlling the cellular and organismal response to multiple types of stress., Author summary Stress responses require proper activation of genetic programs to protect the organism from the stressor. However, the mechanisms controlling energy consumption during stress responses are not well understood. Here, we investigate the role of epigenetic modifier G9a in regulating metabolism and gene transcription during oxidative stress responses in Drosophila. Flies lacking G9a show a shift in the metabolic and transcriptional responses to oxidative stress, leading to decreased stress tolerance despite intact oxidative stress defense mechanisms. During oxidative stress exposure, G9a mutants show overactivation of stress response and many other genes, rapid depletion of glycogen energy stores, and an inability to access lipid energy stores. The increased susceptibility of G9a mutant flies to oxidative stress can be rescued simply by providing extra sugar. This suggests that G9a mutants are sensitive to stress because of reduced access to immediately available energy. Wild-type flies also become more tolerant to oxidative stress when they are fed extra sugar, whereas blocking energy access by genetically reducing a key metabolic enzyme leads to oxidative stress sensitivity. Though the genetic response to oxidative stress has long been appreciated, our study emphasizes the importance of energy metabolism for stress tolerance and identifies the histone methyltransferase G9a as an important player regulating both.

Coevolution of viruses and their hosts may lead to viral strategies to avoid, evade, or suppress antiviral immunity. An example is antiviral RNA interference (RNAi) in insects: the host RNAi machinery processes viral double-stranded RNA into small interfering RNAs (siRNAs) to suppress viral replication, whereas insect viruses encode suppressors of RNAi, many of which inhibit viral small interfering RNA (vsiRNA) production. Yet, many studies have analyzed viral RNAi suppressors in heterologous systems, due to the lack of experimental systems to manipulate the viral genome of interest, raising questions about in vivo functions of RNAi suppressors. To address this caveat, we generated an RNAi suppressor-defective mutant of invertebrate iridescent virus 6 (IIV6), a large DNA virus in which we previously identified the 340R protein as a suppressor of RNAi. Loss of 340R did not affect vsiRNA production, indicating that 340R binds siRNA duplexes to prevent RNA-induced silencing complex assembly. Indeed, vsiRNAs were not efficiently loaded into Argonaute 2 during wild-type IIV6 infection. Moreover, IIV6 induced a limited set of mature microRNAs in a 340R-dependent manner, most notably miR-305–3p, which we attribute to stabilization of the miR-305–5p:3p duplex by 340R. The IIV6 340R deletion mutant did not have a replication defect in cells, but was strongly attenuated in adult Drosophila . This in vivo replication defect was completely rescued in RNAi mutant flies, indicating that 340R is a bona fide RNAi suppressor, the absence of which uncovers a potent antiviral immune response that suppresses virus accumulation ∼100-fold. Together, our work indicates that viral RNAi suppressors may completely mask antiviral immunity.

Contains fulltext : 190953.pdf (Publisher’s version ) (Closed access) Insects are the most abundant and diverse group of animals on earth, but our knowledge of their viruses is biased toward insect-borne viruses that cause disease in plants, animals, or humans. Recent metagenomic studies and systematic surveys of viruses in wild-caught insects have identified an unanticipated large repertoire of novel viruses and viral sequences. These include new members of existing clades, new clades, and even entirely new virus families. These studies greatly expand the known virosphere in insects, provide opportunities to study virus-host interactions, and generate new insights into virus evolution. In this chapter, we discuss the methods used to identify novel viruses in insects and highlight some notable surprises arising from these studies.

RNA interference (RNAi) is an indispensable mechanism for antiviral defense in insects, including mosquitoes that transmit human diseases. To escape this antiviral defense system, viruses encode suppressors of RNAi that prevent elimination of viral RNAs, and thus ensure efficient virus accumulation. Although the first animal Viral Suppressor of RNAi (VSR) was identified more than a decade ago, the molecular basis of RNAi suppression by these viral proteins remains unclear. Here, we developed a single-molecule fluorescence assay to investigate how VSRs inhibit the recognition of viral RNAs by Dcr-2, a key endoribonuclease enzyme in the RNAi pathway. Using VSRs from three insect RNA viruses (Culex Y virus, Drosophila X virus and Drosophila C virus), we reveal bimodal physical interactions between RNA molecules and VSRs. During initial interactions, these VSRs rapidly discriminate short RNA substrates from long dsRNA. VSRs engage nearly irreversible binding with long dsRNAs, thereby shielding it from recognition by Dcr-2. We propose that the length-dependent switch from rapid screening to irreversible binding reflects the main mechanism by which VSRs distinguish viral dsRNA from cellular RNA species such as microRNAs.

Contains fulltext : 137035.pdf (Publisher’s version ) (Closed access) The host RNA interference (RNAi) pathway of insects senses virus infection and induces an antiviral response to restrict virus replication. Dicer-2 detects viral double-stranded RNA, produced by RNA and DNA viruses, and generates viral small interfering RNAs (vsiRNAs). Recent small RNA profiling studies provided new insights into the viral RNA substrates that trigger vsiRNA biogenesis. The importance of the antiviral RNAi pathway is underscored by the observation that viruses have evolved sophisticated mechanisms to counteract this small RNA-based immune response. More recently, it was proposed that another small RNA silencing mechanism, the piRNA pathway, also processes viral RNAs in Drosophila and mosquitoes. Here, we review recent insights into the mechanism of antiviral RNAi, viral small RNA profiles, and viral counter-defense mechanisms in insects.

Contains fulltext : 136090.pdf (Publisher’s version ) (Open Access) Invertebrate RNA viruses are targets of the host RNA interference (RNAi) pathway, which limits virus infection by degrading viral RNA substrates. Several insect RNA viruses encode suppressor proteins to counteract this antiviral response. We recently demonstrated that the dsDNA virus Invertebrate iridescent virus 6 (IIV-6) induces an RNAi response in Drosophila. Here, we show that RNAi is suppressed in IIV-6-infected cells and we mapped RNAi suppressor activity to the viral protein 340R. Using biochemical assays, we reveal that 340R binds long dsRNA and prevents Dicer-2-mediated processing of long dsRNA into small interfering RNAs (siRNAs). We demonstrate that 340R additionally binds siRNAs and inhibits siRNA loading into the RNA-induced silencing complex. Finally, we show that 340R is able to rescue a Flock House virus replicon that lacks its viral suppressor of RNAi. Together, our findings indicate that, in analogy to RNA viruses, DNA viruses antagonize the antiviral RNAi response.

Contains fulltext : 137475.pdf (Publisher’s version ) (Open Access) RNA interference (RNAi) is a crucial antiviral defense mechanism in insects, including the major mosquito species that transmit important human viruses. To counteract the potent antiviral RNAi pathway, insect viruses encode RNAi suppressors. However, whether mosquito-specific viruses suppress RNAi remains unclear. We therefore set out to study RNAi suppression by Culex Y virus (CYV), a mosquito-specific virus of the Birnaviridae family that was recently isolated from Culex pipiens mosquitoes. We found that the Culex RNAi machinery processes CYV double-stranded RNA (dsRNA) into viral small interfering RNAs (vsiRNAs). Furthermore, we show that RNAi is suppressed in CYV-infected cells and that the viral VP3 protein is responsible for RNAi antagonism. We demonstrate that VP3 can functionally replace B2, the well-characterized RNAi suppressor of Flock House virus. VP3 was found to bind long dsRNA as well as siRNAs and interfered with Dicer-2-mediated cleavage of long dsRNA into siRNAs. Slicing of target RNAs by pre-assembled RNA-induced silencing complexes was not affected by VP3. Finally, we show that the RNAi-suppressive activity of VP3 is conserved in Drosophila X virus, a birnavirus that persistently infects Drosophila cell cultures. Together, our data indicate that mosquito-specific viruses may encode RNAi antagonists to suppress antiviral RNAi.

Contains fulltext : 81506.pdf (Publisher’s version ) (Closed access) RNA interference (RNAi) - post-transcriptional gene silencing guided by small interfering RNA (siRNA) - is an important antiviral defense mechanism in insects and plants. Several recent studies in Drosophila identified endogenous siRNAs corresponding to transposons, to structured cellular transcripts and to overlapping convergent transcripts. In addition, one of these studies detected a large pool of Argonaute-2 associated siRNAs that mapped to the genome of flock house virus, a (+) RNA virus. Our bioinformatic analyses indicate that these viral siRNAs mapped in roughly equal proportions to both (+) and (-) viral RNA strands. These reports attribute an important function to RNAi in the defense against parasitic nucleic acids (viruses and transposable elements) and provide a novel mechanism for RNAi-based regulation of cellular gene expression. Furthermore, the detection of viral siRNAs of both (+) and (-) polarity implicates double-stranded RNA replication intermediates as the Dicer substrates that mediate antiviral defense.

The European Science Foundation (ESF)–European Molecular Biology Organization (EMBO) Symposium on Antiviral Applications of RNA Interference took place between 5 and 10 April 2008, in Sant Feliu de Guixols, Spain, and was organized by J. Kurreck and B. Berkhout. ![][1] Small RNAs are important regulators of gene expression that operate through a range of RNA‐silencing mechanisms. Of these, RNA interference (RNAi)—gene silencing through small‐interfering RNAs (siRNAs) derived from exogenous double‐stranded RNA (dsRNA)—and the microRNA (miRNA) pathway—gene silencing guided by small RNAs encoded in the genome—are the best known. The discovery that dsRNA induces RNAi (Fire et al , 1998) and the appreciation of its antiviral activity revolutionized our view of viruses in many ways. The European Science Foundation (ESF)–European Molecular Biology Organization (EMBO) Symposium on Antiviral Applications of RNA Interference brought together about 50 scientists working at the interface between virology and RNAi. Here, I review the main issues that were discussed at the meeting. RNAi provides an antiviral defence mechanism in plants, nematodes and insects. The cleavage of viral dsRNA by the ribonuclease Dicer generates viral siRNAs (v‐siRNAs), which are incorporated into the RNA‐induced silencing complex (RISC) and direct RISC activity onto messenger RNAs (mRNAs) in a sequence‐specific manner. Recognition of a complementary sequence then triggers the endonucleic cleavage (slicer activity) or translational inhibition of the viral target RNAs by an Argonaute family member (Fig 1). Figure 1. RNA silencing in virus infections. ( A ) RNA interference (RNAi) as an antiviral defence mechanism. Viruses might produce double‐stranded RNAs (dsRNAs) that are processed by the RNAi machinery and thereby programme the RNA‐induced silencing complex (RISC) to degrade viral RNAs. ( B ) Cellular or viral microRNAs (miRNAs), or viral RNAs with structural similarity to pre‐miRNAs, regulate viral and host gene expression, and contribute to viral pathogenesis. miRISC, microRNA‐induced silencing complex; pre‐miRNA, precursor miRNA; pri‐miRNA, primary miRNA … [1]: /embed/graphic-1.gif

RNA interference or RNA silencing is a dsRNA guided mechanism that mediates sequence specific degradation of RNA. The recent demonstration that RNA interference can be used to inhibit virus replication has initiated an exciting field of research: first, as a potential novel antiviral therapeutic approach and, second, as a tool for dissecting virus-host interactions. Here we review and discuss the current data and perspectives on the use of RNA interference in the study of poliovirus as a model for positive strand RNA viruses.

Item does not contain fulltext The ESF-EMBO conference on 'Antiviral RNAi: From Molecular Biology towards Applications' took place in June 2012 in Pultusk, Poland. It brought together scientists working at the interface of RNAi and virus infections in different organisms, covering the complete range from basic mechanisms of RNA silencing to RNAi-based antiviral therapy.

Innate immunity is the first line of defence against pathogens and is essential for survival of the infected host. The fruit fly Drosophila melanogaster is an emerging model to study viral pathogenesis, yet antiviral defence responses remain poorly understood. Here, we describe the heat shock response, a cellular mechanism that prevents proteotoxicity, as a component of the antiviral immune response in Drosophila. Transcriptome analyses of Drosophila S2 cells and adult flies revealed strong induction of the heat shock response upon RNA virus infection. Dynamic induction patterns of heat shock pathway components were characterized in vitro and in vivo following infection with different classes of viruses. The heat shock transcription factor (Hsf), as well as active viral replication, were necessary for the induction of the response. Hsf-deficient adult flies were hypersensitive to virus infection, indicating a role of the heat shock response in antiviral defence. In accordance, transgenic activation of the heat shock response prolonged survival time after infection and enabled long-term control of virus replication to undetectable levels. Together, our results establish the heat shock response as an important constituent of innate antiviral immunity in Drosophila.

The ongoing conflict between viruses and their hosts can drive the co-evolution between host immune genes and viral suppressors of immunity. It has been suggested that an evolutionary ‘arms race’ may occur between rapidly evolving components of the antiviral RNAi pathway of Drosophila and viral genes that antagonize it. We have recently shown that viral protein 1 (VP1) of Drosophila melanogaster Nora virus (DmelNV) suppresses Argonaute-2 (AGO2)-mediated target RNA cleavage (slicer activity) to antagonize antiviral RNAi. Here we show that viral AGO2 antagonists of divergent Nora-like viruses can have host specific activities. We have identified novel Nora-like viruses in wild-caught populations of D. immigrans (DimmNV) and D. subobscura (DsubNV) that are 36% and 26% divergent from DmelNV at the amino acid level. We show that DimmNV and DsubNV VP1 are unable to suppress RNAi in D. melanogaster S2 cells, whereas DmelNV VP1 potently suppresses RNAi in this host species. Moreover, we show that the RNAi suppressor activity of DimmNV VP1 is restricted to its natural host species, D. immigrans. Specifically, we find that DimmNV VP1 interacts with D. immigrans AGO2, but not with D. melanogaster AGO2, and that it suppresses slicer activity in embryo lysates from D. immigrans, but not in lysates from D. melanogaster. This species-specific interaction is reflected in the ability of DimmNV VP1 to enhance RNA production by a recombinant Sindbis virus in a host-specific manner. Our results emphasize the importance of analyzing viral RNAi suppressor activity in the relevant host species. We suggest that rapid co-evolution between RNA viruses and their hosts may result in host species-specific activities of RNAi suppressor proteins, and therefore that viral RNAi suppressors could be host-specificity factors., Author Summary Viruses and their hosts can engage in an evolutionary arms race. Viruses may select for hosts with more effective immune responses, whereas the immune response of the host may select for viruses that evade the immune system. These viral counter-defenses may in turn drive adaptations in host immune genes. A potential outcome of this perpetual cycle is that the interaction between virus and host becomes more specific. In insects, the host antiviral RNAi machinery exerts strong evolutionary pressure that has led to the evolution of viral proteins that can antagonize the RNAi response. We have identified novel viruses that infect different fruit fly species and we show that the RNAi suppressor proteins of these viruses can be specific to their host. Furthermore, we show that these proteins can enhance virus replication in a host-specific manner. These results are in line with the hypothesis that virus-host co-evolution shapes the genomes of both virus and host. Moreover, our results suggest that RNAi suppressor proteins have the potential to determine host specificity of viruses.

Item does not contain fulltext Virus transmission and spread by arthropods is a major economic and public health concern. The ongoing dissemination of arthropod-borne viruses by blood-feeding insects is an important incentive to study antiviral immunity in these animals. RNA interference is a major mechanism for antiviral defense in insects, including the fruit fly Drosophila melanogaster and several vector mosquitoes. However, recent data suggest that the evolutionary conserved Toll, Imd and Jak-Stat signaling pathways also contribute to antiviral immunity. Moreover, symbionts, such as the intracellular bacterium Wolbachia and the gut microflora, influence the course of virus infection in insects. These results add an additional level of complexity to antiviral immunity, but also provide novel opportunities to control the spread of arboviruses. In this review, we provide an overview of the current knowledge and recent developments in antiviral immunity in Dipteran insects, with a focus on non-RNAi mediated inducible responses.

The antiviral RNA interference (RNAi) pathway processes viral double-stranded RNA (dsRNA) into viral small interfering RNAs (vsiRNA) that guide the recognition and cleavage of complementary viral target RNAs. In RNA virus infections, viral replication intermediates, dsRNA genomes or viral structured RNAs have been implicated as Dicer-2 substrates. In a recent publication, we demonstrated that a double-stranded DNA virus, Invertebrate iridescent virus 6, is a target of the Drosophila RNAi machinery, and we proposed that overlapping converging transcripts base pair to form the dsRNA substrates for vsiRNA biogenesis. Here, we discuss the role of RNAi in antiviral defense to DNA viruses in Drosophila and other invertebrate model systems.

Item does not contain fulltext MicroRNAs (miRNAs) are a ubiquitous component of gene regulatory networks that modulate the precise amounts of proteins expressed in a cell. Despite their small size, miRNA genes contain various recognition elements that enable specificity in when, where and to what extent they are expressed. The importance of precise control of miRNA expression is underscored by functional studies in model organisms and by the association between miRNA mis-expression and disease. In the last decade, identification of the pathways by which miRNAs are produced, matured and turned-over has revealed many aspects of their biogenesis that are subject to regulation. Studies in viral systems have revealed a range of mechanisms by which viruses target these pathways through viral proteins or non-coding RNAs in order to regulate cellular gene expression. In parallel, a field of study has evolved around the activation and suppression of antiviral RNA interference (RNAi) by viruses. Virus encoded suppressors of RNAi can impact miRNA biogenesis in cases where miRNA and small interfering RNA pathways converge. Here we review the literature on the mechanisms by which miRNA biogenesis and turnover are regulated in animals and the diverse strategies that viruses use to subvert or inhibit these processes.

RNA interference (RNAi) is a major antiviral pathway that shapes evolution of RNA viruses. We show here that Nora virus, a natural Drosophila pathogen, is both a target and suppressor of RNAi. We detected viral small RNAs with a signature of Dicer-2 dependent small interfering RNAs in Nora virus infected Drosophila. Furthermore, we demonstrate that the Nora virus VP1 protein contains RNAi suppressive activity in vitro and in vivo that enhances pathogenicity of recombinant Sindbis virus in an RNAi dependent manner. Nora virus VP1 and the viral suppressor of RNAi of Cricket paralysis virus (1A) antagonized Argonaute-2 (AGO2) Slicer activity of RNA induced silencing complexes pre-loaded with a methylated single-stranded guide strand. The convergent evolution of AGO2 suppression in two unrelated insect RNA viruses highlights the importance of AGO2 in antiviral defense., Author Summary Multi-cellular organisms require a potent immune response to ensure survival under the ongoing assault by microbial pathogens. Co-evolution of virus and host shapes the genome of both pathogen and host. Using Drosophila melanogaster as a model, we study virus-host interactions in infections by Nora virus, a non-lethal natural pathogen of fruit flies. Insects depend on the RNA interference (RNAi) pathway for antiviral defense. A hallmark of the antiviral RNAi response is the production of viral small RNAs during infection. We detected Nora virus small RNAs during infection of Drosophila, demonstrating that Nora virus is a target of the antiviral RNAi pathway. Furthermore, we show that Nora virus viral protein 1 (VP1) inhibits the catalytic activity of Argonaute-2, a key protein of the RNAi pathway. The 1A protein of Cricket paralysis virus suppresses RNAi via a similar mechanism. Importantly, whereas Nora virus persistently infects Drosophila, Cricket paralysis virus induces a lethal infection. Our findings thus indicate that two distantly related viruses independently evolved an RNAi suppressor protein that targets the Argonaute-2 protein. Altogether, our results emphasize the critical role of Argonaute-2 in insect antiviral defense, both in lethal and persistent infections.

Contains fulltext : 109736.pdf (Publisher’s version ) (Open Access) The siRNA pathway is an essential antiviral mechanism in insects. Whether other RNA interference pathways are involved in antiviral defense remains unclear. Here, we report in cells derived from the two main vectors for arboviruses, Aedes albopictus and Aedes aegypti, the production of viral small RNAs that exhibit the hallmarks of ping-pong derived piwi-associated RNAs (piRNAs) after infection with positive or negative sense RNA viruses. Furthermore, these cells produce endogenous piRNAs that mapped to transposable elements. Our results show that these mosquito cells can initiate de novo piRNA production and recapitulate the ping-pong dependent piRNA pathway upon viral infection. The mechanism of viral-piRNA production is discussed.

RNA interference (RNAi) is an important pathway to combat virus infections in insects and plants. Hallmarks of antiviral RNAi in these organisms are: (1) an increase in virus replication after inactivation of major actors in the RNAi pathway, (2) production of virus-derived small interfering RNAs (v-siRNAs), and (3) suppression of RNAi by dedicated viral proteins. In this chapter, we will review the mechanism of RNAi in insects, its function as an antiviral immune system, viral small RNA profiles, and viral counterdefense strategies. We will also consider alternative, inducible antiviral immune responses.

The RNA interference (RNAi) pathway plays an important role in antiviral immunity in insects. To -counteract the RNAi-mediated immune response of their hosts, several insect viruses, such as Flock house virus, Drosophila C virus, and Cricket paralysis virus, encode potent viral suppressors of RNAi (VSRs). Because of the importance of RNAi in antiviral defense in insects, other insect viruses are likely to encode VSRs as well. In this chapter, we describe a detailed protocol for an RNAi reporter assay in Drosophila S2 cells for the identification of VSR activity.

RNA interference (RNAi) is an important pathway to combat virus infections in insects and plants. Hallmarks of antiviral RNAi in these organisms are: (1) an increase in virus replication after inactivation of major actors in the RNAi pathway, (2) production of virus-derived small interfering RNAs (v-siRNAs), and (3) suppression of RNAi by dedicated viral proteins. In this chapter, we will review the mechanism of RNAi in insects, its function as an antiviral immune system, viral small RNA profiles, and viral counterdefense strategies. We will also consider alternative, inducible antiviral immune responses.

The RNA interference (RNAi) pathway plays an important role in antiviral immunity in insects. To -counteract the RNAi-mediated immune response of their hosts, several insect viruses, such as Flock house virus, Drosophila C virus, and Cricket paralysis virus, encode potent viral suppressors of RNAi (VSRs). Because of the importance of RNAi in antiviral defense in insects, other insect viruses are likely to encode VSRs as well. In this chapter, we describe a detailed protocol for an RNAi reporter assay in Drosophila S2 cells for the identification of VSR activity.

Contains fulltext : 89222.pdf (Publisher’s version ) (Closed access) Virus-derived small interfering RNAs (siRNAs) are the hallmark of RNAi-based antiviral immunity. Wu and colleagues demonstrate how viral genomes can be assembled from these small RNA sequences. Their results provide an approach for virus discovery as well as important insights into how these siRNAs mediate antiviral defense.

Contains fulltext : 80137.pdf (Publisher’s version ) (Closed access) 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.

RNA interference (RNAi) is a mechanism for sequence-specific gene silencing triggered by double-stranded (ds) RNA. RNAi constitutes an effective antiviral defense mechanism in many organisms. Accordingly, viruses interact with the RNAi pathway at different levels. As a counter-defense, viruses have evolved suppressors of the RNAi pathway. Many DNA viruses, on the other hand, exploit the related microRNA pathway by producing microRNAs to regulate viral and host gene expression. In this chapter we summarize recent findings on these interactions and discuss how they may have shaped viral evolution.

Most organisms have evolved defense mechanisms to protect themselves from viruses and other pathogens. Arthropods lack the protein-based adaptive immune response found in vertebrates. Here we show that the central catalytic component of the RNA-induced silencing complex (RISC), the nuclease Argonaute 2 (Ago-2), is essential for antiviral defense in adult Drosophila melanogaster. Ago-2-defective flies are hypersensitive to infection with a major fruit fly pathogen, Drosophila C virus (DCV), and with Cricket Paralysis virus (CrPV). Increased mortality in ago-2 mutant flies was associated with a dramatic increase in viral RNA accumulation and virus titers. The physiological significance of this antiviral mechanism is underscored by our finding that DCV encodes a potent suppressor of RNA interference (RNAi). This suppressor binds long double-stranded RNA (dsRNA) and inhibits Dicer-2-mediated processing of dsRNA into short interfering RNA (siRNA), but does not bind short siRNAs or disrupt the microRNA (miRNA) pathway. Based on these results we propose that RNAi is a major antiviral immune defense mechanism in Drosophila.

Many metazoan cells can take up exogenous double-stranded (ds) RNA and use it to initiate an RNA silencing response, however, the mechanism for this uptake is ill-defined. Here, we identify the pathway for dsRNA uptake in Drosophila melanogaster S2 cells. Biochemical and cell biological analyses, and a genome-wide screen for components of the dsRNA-uptake machinery, indicated that dsRNA is taken up by an active process involving receptor-mediated endocytosis. Pharmacological inhibition of endocytic pathways disrupted exogenous dsRNA entry and the induction of gene silencing. This dsRNA uptake mechanism seems to be evolutionarily conserved, as knockdown of orthologues in Caenorhabditis elegans inactivated the RNA interference response in worms. Thus, this entry pathway is required for systemic RNA silencing in whole organisms. In Drosophila cells, pharmacological evidence suggests that dsRNA entry is mediated by pattern-recognition receptors. The possible role of these receptors in dsRNA entry may link RNA interference (RNAi) silencing to other innate immune responses.

RNA interference (RNAi) is a mechanism for sequence-specific gene silencing guided by double-stranded RNA. In plants and insects it is well established that RNAi is instrumental in the response to viral infections; whether RNAi has a similar function in mammals is under intense investigation. Recent studies to address this question have identified some unanticipated interactions between the RNAi machinery and mammalian viruses. Furthermore, introduction of virus-specific small interfering RNAs (siRNAs) into cells, thus programming the RNAi machinery to target viruses, is an effective therapeutic approach to inhibit virus replication in vitro and in animal models. Although several issues remain to be addressed, such as delivery and viral escape, these findings hold tremendous potential for the development of RNAi-based antiviral therapeutics.

Little is known about the tolerance mechanisms that reduce the negative effects of microbial infection on host fitness. Here, we demonstrate that the histone H3 lysine 9 methyltransferase G9a regulates tolerance to virus infection by shaping the response of the evolutionary conserved Jak-Stat pathway in Drosophila. G9a-deficient mutants are more sensitive to RNA virus infection and succumb faster to infection than wild-type controls, which was associated with strongly increased Jak-Stat dependent responses, but not with major differences in viral load. Genetic experiments indicate that hyperactivated Jak-Stat responses are associated with early lethality in virus-infected flies. Our results identify an essential epigenetic mechanism underlying tolerance to virus infection., Author Summary Multicellular organisms deploy various strategies to fight microbial infections. Invading pathogens may be eradicated directly by antimicrobial effectors of the immune system. Another strategy consists of increasing the tolerance of the host to infection, for example, by limiting the adverse effects of the immune response. The molecular mechanisms underlying this novel concept remain largely uncharacterized. Here, we demonstrate that the epigenetic regulator G9a mediates tolerance to virus infection in Drosophila. We found that G9a-deficient flies succumb faster than control flies to infection with RNA viruses, but that the viral burden did not significantly differ. Unexpectedly, mutant flies express higher levels of genes that are regulated by the Jak-Stat signaling pathway, which in other studies was found to be important for antiviral defense. Exploiting the genetic toolbox in Drosophila, we demonstrate that Jak-Stat hyperactivation induces early mortality after virus infection. Precise control of immune pathways is essential to ensure efficient immunity, while preventing damage due to excessive immune responses. Our results indicate that G9a, an epigenetic modifier, dampens Jak-Stat responses to prevent immunopathology. Therefore, we propose epigenetic regulation of immunity as a new paradigm for disease tolerance.

RNAi A Guide to Gene Silencing. Gregory J. Hannon, Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2003. 444 pp. $130, £95. ISBN 0-87969-641-9. The contributors, who include many pioneers in the field, present the principles behind RNA interference and a series of protocols for the laboratory use of RNAi in a variety of plant and animal systems.