ArticleLiterature Review

A call to arms: coevolution of animal viruses and host innate immune responses

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Abstract

Virus infection is generally disadvantageous to the host and strongly selects for host antiviral mechanisms. Therefore, viruses must develop counter-mechanisms to guarantee their survival. This arms race between pathogen and host leads to positive selection for both cellular antiviral mechanisms and viral inhibitors of such mechanisms. Here, we characterize this arms race in the context of the RNA interference (RNAi) pathway, which is used as an innate immune response against viral infection by animals. We review how RNAi is used as an antiviral strategy and the mechanisms that viruses have evolved to suppress the RNAi response.

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... # Equal contribution. genome organization, DNA editing and post-transcriptional gene silencing [13][14][15][16][17][18][19][20][21][22]. Beyond the regulation of natural biological processes, RNAi has also become a powerful tool to manipulate endogenous gene expression in research and therapeutics [7,23]. ...
... As mentioned above, long dsRNA is produced as an intermediate for viral genome replication and is processed into siRNAs by Dicer in animals or DCL proteins in plants. [21]. This mechanism of biogenesis generates antiviral siR-NAs derived from viral sequences that tend to cover the whole extent of the viral genome [116,117]. ...
Article
RNA interference (RNAi) pathways utilize small non-coding RNAs (ncRNAs) to regulate diverse biological processes in eukaryotes. Different classes of small ncRNAs function through distinct RNAi pathways and the focus of this review will be on two of these: microRNAs (miRNAs) and small interfering RNAs (siRNAs). The siRNA pathway is triggered by viral infection and repetitive elements functioning as a defense system in eukaryotes. miRNAs, on the other hand, are encoded in the genome and regulate a myriad of biological processes such as embryonic development and tissue differentiation in plants and animals. These different small RNA pathways have great potential to be used as biomarkers of the biological processes they regulate. In this review, we describe the role RNAi pathways have in diverse pathological and physiological conditions and how the detection of specific small ncRNAs can be used as biomarkers for these conditions. We discuss other areas not yet explored for the use of small ncRNAs as biomarkers and review recent patents proposing to use small RNAs as biomarkers. These patents mainly include cases where changes in expression of miRNAs, individually or in groups, have been shown to correlate with disease or disease states, most commonly cancer
... Pathogen virulence (measured as the severity of a disease) is often assumed to evolve in a strict co-evolutionary arms race between the pathogen and its host12345. The theory of virulence also commonly assumes that pathogen reproduction, and consequently the evolution of virulence, is entirely dependent on the host species [2]. ...
... Pathogen virulence (measured as the severity of a disease) is often assumed to evolve in a strict co-evolutionary arms race between the pathogen and its host [1][2][3][4][5]. The theory of virulence also commonly assumes that pathogen reproduction, and consequently the evolution of virulence, is entirely dependent on the host species [2]. ...
Article
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Pathogen virulence is usually thought to evolve in reciprocal selection with the host. While this might be true for obligate pathogens, the life histories of opportunistic pathogens typically alternate between within-host and outside-host environments during the infection-transmission cycle. As a result, opportunistic pathogens are likely to experience conflicting selection pressures across different environments, and this could affect their virulence through life-history trait correlations. We studied these correlations experimentally by exposing an opportunistic bacterial pathogen Serratia marcescens to its natural protist predator Tetrahymena thermophila for 13 weeks, after which we measured changes in bacterial traits related to both anti-predator defence and virulence. We found that anti-predator adaptation (producing predator-resistant biofilm) caused a correlative attenuation in virulence. Even though the direct mechanism was not found, reduction in virulence was most clearly connected to a predator-driven loss of a red bacterial pigment, prodigiosin. Moreover, life-history trait evolution was more divergent among replicate populations in the absence of predation, leading also to lowered virulence in some of the 'predator absent' selection lines. Together these findings suggest that the virulence of non-obligatory, opportunistic bacterial pathogens can decrease in environmental reservoirs through life history trade-offs, or random accumulation of mutations that impair virulence traits under relaxed selection.
... At the genetic level, such arms races have been described for host and virus proteins that directly interact, and particularly in those involved in host antiviral immunity and viral evasion of host immunity [2][3][4]. The interaction between the RNA interference (RNAi) antiviral immune system of many eukaryotes and viral suppressors of RNAi appears to have the potential to instigate such an arms race [5,6]. ...
... Although VSRs are predicted to be a focus of antagonistic host -virus interaction [5,6], we found little evidence for ubiquitous positive selection acting on the VSRs of plant viruses. Since our analyses are likely to have good power, we believe this is a robust result. ...
Article
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Viral suppressors of RNAi (VSRs) are proteins that actively inhibit the antiviral RNA interference (RNAi) immune response, providing an immune evasion route for viruses. It has been hypothesized that VSRs are engaged in a molecular 'arms race' with RNAi pathway genes. Two lines of evidence support this. First, VSRs from plant viruses display high sequence diversity, and are frequently gained and lost over evolutionary time scales. Second, Drosophila antiviral RNAi genes show high rates of adaptive evolution. Here, we investigate whether VSRs diversify faster than other genes and, if so, whether this is a result of positive selection, as might be expected in an arms race. By analysis of 12 plant RNA viruses, we show that the relative rate of protein evolution is higher for VSRs than for other genes, but that this is not attributable to pervasive positive selection. We argue that, because evolutionary time scales are extremely different for viruses and eukaryotes, it is improbable that viral adaptation (as measured by the ratio of non-synonymous to synonymous change) will be dominated by one-to-one coevolution with eukaryotes. Instead, for plant virus VSRs, we find strong evidence of episodic selection-diversifying selection that acts on a subset of lineages-which might be attributable to frequent shifts between different host genotypes or species.
... Thus, viral nucleic acids are a common target of several host antiviral mechanisms. [3][4][5][6] Targeting of viral RNAs often results in the generation of virus-derived small RNAs (vsRNAs) that can be detected during infection in fungi, plants, arthropods, and mammals. [7][8][9][10][11][12][13] Although vsRNAs are commonly observed in different organisms, their relative abundance can vary substantially. ...
... RNA interference (RNAi), RNA decay, and RNase L pathways are good examples of such RNA surveillance mechanisms that have been extensively reviewed elsewhere. [3][4][5][6][15][16][17][18][19][20][21][22][23][24][25] Here, we provide a few examples of how RNA degradation pathways may generate small RNA products with distinct molecular characteristics ( Figure 1). ...
... In view of the opportunistic behaviour of feline viruses, it is not difficult to imagine that the advent of domestication gave a free way to these pathogens, which readily spread, as a consequence, to every individual living in a group. As it is currently accepted that the biology of virus-host interactions is a continuous co-evolutionary process involving both host immune system and viral escape mechanisms [4], The objectives of the present study are then clearly formulated in section 2.2. Finally, although results are extensively discussed in the respective manuscripts included in this document, the overall outcome of this study is thoroughly examined in the final discussion (chapter 5), which also opens perspectives for future experiments. ...
... Due to their 78 abundance in all bacterial as well as some viral genomes, oligodeoxynucleotides (ODN) 79containing unmethylated cytosine-phosphate-guanosine (CpG) motifs are effectively 80 recognized as PAMPs by the vertebrate innate immune system[2]. Response to CpG 81 ODN stimulation is conferred through TLR9, expressed mainly in the intracellular 82 compartments of human B cells and plasmacytoid dendritic cells (pDCs)[3,4]. Alarmed 83 TLR9 is the initial instigator of gene expression profiles that strongly support antiviral 84 mechanisms: upregulation of costimulatory molecules major histocompatibility complex 85 (MHC) II, B7.1 and B7.2 on the surface of stimulated cells provides them with a stronger 86 ...
Article
Broadening the understanding of mechanisms linked to innate immunity is of primordial importance in a time of continuous emergence of rapidly spreading viral diseases. The domestic cat represents an ideal model for the study of host-virus interactions, as it is an outbred species naturally susceptible to many viruses sharing biological properties with those affecting humans. Additionally, due to their acquisition of infallible transmission strategies, rapid propagation of feline viruses within a group is particularly difficult to inhibit, reflecting the challenges linked to the prevention of pandemics. The present work was designed to gain insights on the innate immune responses of the domestic cat to viruses, and to determine whether early antiviral mechanisms can be manipulated to enhance resistance to viral infection in this species. In a first phase, real-time polymerase chain reaction (PCR) systems were developed, enabling to measure the expression of feline genes considered to be hallmarks of innate responses to viral infection, including various interferon (IFN) ? and IFN? subtypes, IFN?, intracellular antiviral Myxovirus resistance (Mx) factor, natural killer (NK) cell stimulator IL-15 and effectors perforin and granzyme B, as well as Toll-like receptors (TLRs) 3, 7, 8 and 9. These tools could then be employed to evaluate innate immune parameters in both in vitro and in vivo models of infection, conferring valuable information not only regarding strength, breadth and kinetics of antiviral defences in feline cells, but also possible biological properties of important viruses affecting the cat. In a further step, the newly developed PCR assays were utilized to assess the immunomodulatory potential of various immune response modifiers (IRMs) in feline cells in vitro. The IRMs mimicking natural viral components were selected, namely Poly IC and Resiquimod (R-848), artificial models of viral dsRNA and ssRNA, as well as dSLIM? and ODN 2216, synthetic oligonucleotides containing several unmethylated CpG motifs. Although all analysed IRMs positively modulated the innate immune state of treated peripheral mononuclear cells (PBMCs), ODN 2216 induced by far the most potent response: this molecule not only altered the gene expression profile of feline PBMCs in an antiviral orientation, but also significantly enhanced the proliferation of these immune cells and increased the presence on their surface of co-stimulatory molecules necessary for the diffusion of immunological defence signals. Moreover, when incubated in vitro with target cells of epithelial and fibroblastic origin, the supernatants of ODN 2216-stimulated PBMCs not only induced high production of intracellular antiviral proteins in these cells but also inhibited the replication of five feline viruses, namely the feline calici- (FCV), herpes- (FHV), parvo- (FPV), corona- (FCoV) and leukemia (FeLV) viruses. Altogether, this study procures a better understanding of innate antiviral mechanisms in an outbred species and highlights the promising potential of CpG-containing molecules such as ODN 2216 to protect domestic cats against a broad range of virus infections. Further in vitro and in vivo investigations will determine the feasibility of stimulation of the innate immune system by such molecules to prevent viral propagation in humans, cats and other species.
... The siRNA pathway is a major arm of the antiviral response in plants and invertebrate animals [14,15]. In Drosophila, Ago2, R2D2 and Dcr-2 mutant individuals exhibit increased sensitivity to infection by several viruses [16,17,18,19]. ...
... Obbard et al [51] showed that Ago2, R2D2 and Dcr-2 are among the fastest evolving genes in the Drosophila genome. Since many host defense and pathogen genes co-evolve in a genetic arms race, rapid evolution of Ago2, Dcr-2 and R2D2 is possibly related to their antiviral functions [14,52]. Strikingly, the loqs gene shows no sign of rapid evolution. ...
... The siRNA pathway is a major arm of the antiviral response in plants and invertebrate animals [14,15]. In Drosophila, Ago2, R2D2 and Dcr-2 mutant individuals exhibit increased sensitivity to infection by several viruses [16,17,18,19]. ...
... Obbard et al [51] showed that Ago2, R2D2 and Dcr-2 are among the fastest evolving genes in the Drosophila genome. Since many host defense and pathogen genes co-evolve in a genetic arms race, rapid evolution of Ago2, Dcr-2 and R2D2 is possibly related to their antiviral functions [14,52]. Strikingly, the loqs gene shows no sign of rapid evolution. ...
Article
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In Drosophila, post-transcriptional gene silencing occurs when exogenous or endogenous double stranded RNA (dsRNA) is processed into small interfering RNAs (siRNAs) by Dicer-2 (Dcr-2) in association with a dsRNA-binding protein (dsRBP) cofactor called Loquacious (Loqs-PD). siRNAs are then loaded onto Argonaute-2 (Ago2) by the action of Dcr-2 with another dsRBP cofactor called R2D2. Loaded Ago2 executes the destruction of target RNAs that have sequence complementarity to siRNAs. Although Dcr-2, R2D2, and Ago2 are essential for innate antiviral defense, the mechanism of virus-derived siRNA (vsiRNA) biogenesis and viral target inhibition remains unclear. Here, we characterize the response mechanism mediated by siRNAs against two different RNA viruses that infect Drosophila. In both cases, we show that vsiRNAs are generated by Dcr-2 processing of dsRNA formed during viral genome replication and, to a lesser extent, viral transcription. These vsiRNAs seem to preferentially target viral polyadenylated RNA to inhibit viral replication. Loqs-PD is completely dispensable for silencing of the viruses, in contrast to its role in silencing endogenous targets. Biogenesis of vsiRNAs is independent of both Loqs-PD and R2D2. R2D2, however, is required for sorting and loading of vsiRNAs onto Ago2 and inhibition of viral RNA expression. Direct injection of viral RNA into Drosophila results in replication that is also independent of Loqs-PD. This suggests that triggering of the antiviral pathway is not related to viral mode of entry but recognition of intrinsic features of virus RNA. Our results indicate the existence of a vsiRNA pathway that is separate from the endogenous siRNA pathway and is specifically triggered by virus RNA. We speculate that this unique framework might be necessary for a prompt and efficient antiviral response.
... In contrast to AEM genes, immunity-related genes (including GO:0045087 innate immune response, GO:0006959 humoral immune response, GO:0002682 regulation of immune system process, GO:0002253 activation of immune response, GO:0006952 defense response) were generally found in bins with dN/dS Ͼ0.2 in all four lineages. This finding would be expected from pathogen-driven pressures resulting in positive selection on immunity genes in multiple evolutionary lineages (31,32). ...
... The case for adaptive evolution is supported by the finding that in all mammalian lineages examined in our study (elephant, tenrec, human, mouse, dog, and cow), immune system genes were overrepresented in the higher dN/dS bins. As noted, it is well established that pathogen-driven pressures have made immune system genes the targets of positive selection in multiple mammalian lineages (31,32). The overrepresentation of AEM genes that occurred in the higher dN/dS bins for the larger-brained and long-lived mammalian lineages further supports the case for adaptive evolution (Fig. 3, Tables S6 and S7). ...
... Viruses use different strategies to combat the host defense arsenal by producing a variety of effector molecules (Marques and Carthew, 2007). Generally, structural and regulatory viral proteins help the virus to manipulate the host system for its own advantage. ...
... The possible disadvantage for the formation of dsRNAs during (ϩ)RNA virus replication is the prompt recognition of dsRNAs by the host anti-dsRNA surveillance system, based on Dicer RNase III enzymes for gene silencing in plants and animals, dsRNA protein kinase PKR, or RIG-I and MDA5 RNA sensors in mammals (44)(45)(46)(47)(48)(49)(50)(51). The viral dsRNAs could be destroyed by cellular RNase III-like nucleases or other induced host responses, such as PKR or interferon responses (44-48, 50, 51). ...
Article
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Unlabelled: Replication of plus-strand RNA [(+)RNA] viruses of plants is a relatively simple process that involves complementary minus-strand RNA [(-)RNA] synthesis and subsequent (+)RNA synthesis. However, the actual replicative form of the (-)RNA template in the case of plant (+)RNA viruses is not yet established unambiguously. In this paper, using a cell-free replication assay supporting a full cycle of viral replication, we show that replication of Tomato bushy stunt virus (TBSV) leads to the formation of double-stranded RNA (dsRNA). Using RNase digestion, DNAzyme, and RNA mobility shift assays, we demonstrate the absence of naked (-)RNA templates during replication. Time course experiments showed the rapid appearance of dsRNA earlier than the bulk production of new (+)RNAs, suggesting an active role for dsRNA in replication. Radioactive nucleotide chase experiments showed that the mechanism of TBSV replication involves the use of dsRNA templates in strand displacement reactions, where the newly synthesized plus strand replaces the original (+)RNA in the dsRNA. We propose that the use of dsRNA as a template for (+)RNA synthesis by the viral replicase is facilitated by recruited host DEAD box helicases and the viral p33 RNA chaperone protein. Altogether, this replication strategy allows TBSV to separate minus- and plus-strand syntheses in time and regulate asymmetrical RNA replication that leads to abundant (+)RNA progeny. Importance: Positive-stranded RNA viruses of plants use their RNAs as the templates for replication. First, the minus strand is synthesized by the viral replicase complex (VRC), which then serves as a template for new plus-strand synthesis. To characterize the nature of the (-)RNA in the membrane-bound viral replicase, we performed complete RNA replication of Tomato bushy stunt virus (TBSV) in yeast cell-free extracts and in plant extracts. The experiments demonstrated that the TBSV (-)RNA is present as a double-stranded RNA that serves as the template for TBSV replication. During the production of new plus strands, the viral replicase displaces the old plus strand in the dsRNA template, leading to asymmetrical RNA synthesis. The presented data are in agreement with the model that the dsRNA is present in nuclease-resistant membranous VRCs. This strategy likely allows TBSV to protect the replicating viral RNA from degradation as well as to evade the early detection of viral dsRNAs by the host surveillance system.
... Virus infection in a plant host activates a molecular defense mechanism known as RNA silencing or RNA interference (RNAi) which causes the degradation of viral RNA. The antiviral role of RNA silencing has been extensively studied in plants (Voinnet, 2005;Ding and Voinnet, 2007;Marques and Carthew, 2007). It is induced by doublestranded viral RNA (dsRNA) or by specific single-stranded RNA (ssRNA) structures. ...
Article
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The Nicotiana tabacum genome contains four Dicer-like proteins (DCLs) and six RNA-dependent RNA polymerase (RDR) homologues involved in the RNA silencing mechanism employed against viral infection. DCL1 synthesizes 18-21 nt-long microRNA, whereas DCL2, DCL3 and DCL4 produce 22 nt, 24 nt and 21 nt-long siRNA, respectively, in the RNA silencing process. This study aimed to clarify which components among these are involved in changes in the amount of virus and the development of symptoms in Cucumber mosaic virus (CMV)-infected tobacco. Infected transgenic tobacco lines with a single down-regulation of DCL2, DCL4, RDR1 or a double down-regulation of both DCL2 and 4 were analyzed. The amounts of viral RNA in young developing leaves in transgenic tobacco lines were examined by Northern blot analysis. Most transgenic plants inoculated with CMV Pepo, a virulent strain, exhibited cyclic mosaic symptoms. The amount of viral RNA in single down-regulated lines varied based on leaf position in a similar manner to that noted in non-transgenic tobacco, while that of the double down-regulated line did not. Furthermore, the expression of RNA-silencing-related genes during high and low CMV infection did not differ among the transgenic plants. These results suggested that (i) changes in the amounts of the virus in the developing leaves of all the single down-regulated lines were associated with cyclic symptom expression in fully expanded leaves, and (ii) the lower expression of DCL2, DCL4 and RDR1 may be sufficient to establish cyclic symptom development.
... AGO2, r2d2 and Dcr-2 deficient flies are more susceptible to a range of different viruses reinforcing the idea that the siRNA pathway mediates a powerful antiviral defense [35,61,63e65]. In addition, viral suppressors of RNAi (VSRs) are commonly found in insect viruses and are capable of blocking different steps of the siRNA pathway to favor virus replication [66]. A good example is the B2 protein encoded by Flock House virus (FHV), which is able to block both the biogenesis as well as loading of siRNAs onto AGO2 [40]. ...
Article
Viral RNA is a common activator of antiviral responses. In this review, we dissect the mechanism of viral RNA recognition by the small interfering RNA pathway in Drosophila melanogaster. This antiviral response in fruit flies can help understand general principles of nucleic acid recognition.
... In response to RNAi, some viruses have been shown to express suppressors of RNA silencing (SRSs) upon infecting their plant or insect hosts (reviewed in [5]). Individual SRSs have been shown to act by sequestering dsRNA molecules, preventing siRNA-loading into RISC or through direct binding of a component of the RNAi machinery [6,7]. One of the best characterized SRSs is the B2 protein encoded by flock house virus (FHV; family Nodaviridae, genus Alphanodavirus). ...
Article
Nearly all RNA viruses produce double-stranded RNA (dsRNA) during their replication cycles-an important pathogen-associated molecular pattern recognized by the RNA interference (RNAi) pathway in invertebrates and plants. Nodamura virus (NoV) encodes a suppressor of RNA silencing termed B2, which binds to dsRNA and prevents the initiation of RNAi as well as the loading of silencing complexes. Using the published crystal structure of NoV-B2, we performed a series of molecular dynamics (MD) simulations to determine the relative electrostatic and van der Waals contributions of various residues in binding dsRNA, identifying four novel potential interactors: R56, E48, P68 and R69. Additionally, steered MD was used to simulate the binding affinity of NoV-B2 sequences bearing substitutions at positions F49, R56 or R59 to dsRNA, with F49S and R56L/R59L substitutions found to have a significant negative impact on the ability of NoV-B2 to bind dsRNA. NoV RNA1 variants were tested for self-directed replication in both vertebrate (RNAi(-)) and invertebrate (RNAi(+)) cultured cells. Consistent with a role in dsRNA binding, NoV replication in F49C and F49S variant constructs was affected negatively only in RNAi(+) cells. Thus, we used a combination of MD simulations and experimental mutagenesis to further characterize residues important for NoV-dsRNA interactions.
... Viruses are likely to be involved in host-parasite coevolution, as they are important and specialised natural pathogens but also because they are known to express RNAi suppressors (Moissiard & Voinnet 2004, Schutz & Sarnow 2006. It is also possible that these relatively high evolutionary rates are associated with the lack of pleiotropic constraints, as these genes are only involved in anti-viral defence and have no other function (Marques & Carthew 2007). ...
Article
Drosophila is involved in a wide range of interactions with parasites and pathogens (parasitoid wasps, bacteria, fungi, viruses). Drosophila hosts vary greatly at the species, population and individual level, in their response against such organisms, and much of this variation has a genetic basis. In this thesis I explored three aspects of this variation. First, using recombination mapping based on SNPs and a variation of bulk segregant analysis, I identified a QTL region on the right arm of the third chromosome of D. melanogaster associated with resistance to at least some of the parasitoid species / strains used in the experiments. The location of the QTL was further explored with deficiency complementation mapping and was narrowed down to the 96D1-97B1 region. The success of the deficiency mapping suggests that the resistant allele is not completely dominant. Second, I investigated patterns of molecular evolution in a set of immunity-related genes, using sequences from a D. melanogaster and a D. simulans population and a set of genes without known involvement in immunity for comparison. I found evidence that several of these genes have evolved under different selection pressure in each species, possibly indicating interactions with different parasites. The immunity genes tested appear to be evolving faster compared to non-immunity genes, supporting the idea that the immune system is evolving under strong selective pressure from parasites. Finally, in a D. melanogaster – sigma virus system, I measured genetic variation in the transmission of different virus genotypes, in different environments. There was poor correlation between temperatures, suggesting that environmental heterogeneity could constraint evolution of resistance (to virus transmission). The correlation between viral genotypes was also low, although relatively stronger for more closely phylogenetically related viral strains. Such interactions between host genotypes, virus genotypes and environmental conditions can maintain genetic variation in virus transmission.
... Co-evolution of virus and host fuels the arms-race between host antiviral responses and viral counter defense strategies [47]. VIFs alternately infect evolutionary distant vertebrate hosts and invertebrate vectors, which suggests that VIFs encounter distinct evolutionary pressures that together constrain their diversity [27,48]. ...
Article
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The genus Flavivirus contains pathogenic vertebrate-infecting flaviviruses (VIFs) and insect-specific flaviviruses (ISF). ISF transmission to vertebrates is inhibited at multiple stages of the cellular infection cycle, via yet to be elucidated specific antiviral responses. The zinc-finger antiviral protein (ZAP) in vertebrate cells can bind CpG dinucleotides in viral RNA, limiting virus replication. Interestingly, the genomes of ISFs contain more CpG dinucleotides compared to VIFs. In this study, we investigated whether ZAP prevents two recently discovered lineage II ISFs, Binjari (BinJV) and Hidden Valley viruses (HVV) from replicating in vertebrate cells. BinJV protein and dsRNA replication intermediates were readily observed in human ZAP knockout cells when cultured at 34 °C. In ZAP-expressing cells, inhibition of the interferon response via interferon response factors 3/7 did not improve BinJV protein expression, whereas treatment with kinase inhibitor C16, known to reduce ZAP’s antiviral function, did. Importantly, at 34 °C, both BinJV and HVV successfully completed the infection cycle in human ZAP knockout cells evident from infectious progeny virus in the cell culture supernatant. Therefore, we identify vertebrate ZAP as an important barrier that protects vertebrate cells from ISF infection. This provides new insights into flavivirus evolution and the mechanisms associated with host switching.
... A second type of siRNA arises from exogenous precursors, like viral dsRNA, and generates exo-siRNAs. Many organisms produce exo-siRNAs to mount an antiviral response (Ding and Voinnet, 2007;Marques and Carthew, 2007), including mammalian cells Maillard et al., 2013). For RNA viruses, viral replication intermediates consisting of genome-antigenome dsRNA are cleaved by Dicer to produce viral exo-siRNAs, and deepsequencing shows an enrichment of siRNAs mapping to ends of viral genomes (Aliyari et al., 2008;Li et al., 2013;Maillard et al., 2013). ...
Article
In previous studies we observed that the helicase domain of Drosophila Dicer-2 (dmDcr-2) governs substrate recognition and cleavage efficiency, and that dsRNA termini are key to this discrimination. We now provide a mechanistic basis for these observations. We show that discrimination of termini occurs during initial binding. Without ATP, dmDcr-2 binds 3' overhanging, but not blunt, termini. By contrast, with ATP, dmDcr-2 binds both types of termini, with highest-affinity binding observed with blunt dsRNA. In the presence of ATP, binding, cleavage, and ATP hydrolysis are optimal with BLT termini compared to 3'ovr termini. Limited proteolysis experiments suggest the optimal reactivity of BLT dsRNA is mediated by a conformational change that is dependent on ATP and the helicase domain. We find that dmDcr-2's partner protein, Loquacious-PD, alters termini dependence, enabling dmDcr-2 to cleave substrates normally refractory to cleavage, such as dsRNA with blocked, structured, or frayed ends. Copyright © 2015 Elsevier Inc. All rights reserved.
... Über eine Koevolution von mammalen Viren und ihren Wirten entsteht ein Gleichgewicht, in dem der Wirt keine großen Schäden im Zuge der Infektion erfährt und im Gegenzug das Virus durch die Immunantwort nur geringgradig in seiner reproduktiven Kapazität eingeschränkt wird (Marques and Carthew, 2007). Papillomaviren und ihre adaptierten Wirte haben im Laufe vieler Generationen ein derartiges Gleichgewicht erreicht (Bernard, 2005 (Burk et al., 2009). ...
Article
Das high-risk Humane Papillomavirus Typ 16 (HPV16) ist ursächlich verantwortlich für die Ausbildung von ca. 50% aller mehr als 500.000 jährlich auftretenden Zervixkarzinomfälle weltweit. Trotz der Einführung der hochwirksamen prophylaktischen HPV-Vakzine sind weiterhin Untersuchungen zum besseren Verständnis von Virus-Wirt-Interaktionen erforder-lich, die das grundlegende Verständnis für die virusinduzierte Progression einer benignen Neoplasie hin zu der Ausbildung eines Tumors erweitern. Bis heute sind die Effekte der HPV-Proteine hauptsächlich in Modellen erforscht worden, die ein oder zwei virale Onkogene unter der Kontrolle heterologer Promotoren berücksichtigen. Aus diesem experimentellen Ansatz resultiert eine Überexpression des jeweiligen Gens und die Aufklärung kooperierender Effekte mit anderen viralen Proteinen ist ausgeschlossen. Ziel dieser Arbeit war es daher, eine Analyse der biologischen Effekte der E5, E6 und E7 Onkogene von HPV16 in einem naturnahen in vitro Modell genauer zu charakterisieren. Dabei sollten die Onkoproteinfunktionen aller drei Gene im Vordergrund stehen, wenn sie unter der Kontrolle ihrer viralen Promotoren im Kontext des komplett vorliegenden Genoms exprimiert werden. Hierfür gelang es, ein transfektionsbasiertes System auf Grundlage eines loxp_HPV16_eGFP-N1-Vektoren zu etab¬lieren, das bei hoher episomaler Kopienzahl eine effiziente Manifestation von HPV16 ermög¬lichte. Benutzt wurden neben dem Wildtyp-Genom von HPV16 auch Punktmutanten aller drei Gene, die ein gezieltes „knock-out“ bewirkten. Durch eine Anpassung der Kulturbedingungen konnte die immortalisierte HaCaT-Zelllinie erstmals in organotypischer Raftkultur so kultiviert werden, dass eine Unterstützung des viralen Lebenszyklus möglich wurde. Konservierte HPV16-tragende epidermale Äquivalente bildeten dabei stark dysplastische Morphologien aus, die bisher in ähnlichen Systemen aus primären Keratinozyten bzw. bei der Verwendung der NIKS-Zelllinie nicht darstellbar waren. In Konsens mit den bekannten HPV16-Charakteris¬tiken war E7 in den vorliegenden Untersuchungen als Hauptmediator einer virusinduzierten Hyperproliferation identifizierbar. Konträr zu den allgemein anerkannten Wirkweisen von E7 und E6 resultierte hingegen die gesteigerte Proliferation nicht aus einer Degradation von pRb oder p53. Insgesamt legten sowohl die Histologie, die schwache Präsenz von E1^E4 und der ausgebliebene Nachweis einer L1-Expression den Schluss nahe, dass die erhaltenen Gewebe einer höhergradigen in vivo CIN-Läsion vergleichbar sind. Diese Ergebnisse erlauben eine teilweise neue Interpretation der Effekte der HPV-Onkogene auf die Zellproliferation und differenzierung der infizierten epithelialen Zellen. Weiterhin bietet das etablierte Modell die Möglichkeit in einem naturähnlichen System die Eigenschaften der Onkoproteine von HPV16 zu untersuchen.
... Thus, dsRNA is a common marker of viral infection and it is recognized by various mechanisms mediating an innate immune response. A role of RNA silencing in the innate immunity is supported by several lines of evidence, which were first found in plants and later also in invertebrates (reviewed in Xie and Guo 2006;Marques and Carthew 2007) (1) siRNAs derived from viral sequences were found in infected organisms (Hamilton and Baulcombe 1999), (2) inhibition of RNA silencing results in increased viral replication (Mourrain et al. 2000), and (3) some viruses produce suppressors of RNA silencing (SRS) (Voinnet et al. 1999). ...
Chapter
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Since its discovery in 1998, RNA interference has become a popular tool for experimental silencing of gene expression. On the other hand, its natural role received less attention. Recent studies in animal systems, particularly the use of the next generation sequencing and analysis of animals defective in some aspect of small RNA biogenesis, revealed novel functions of RNAi and crosstalks with other pathways. This chapter aims to provide a comprehensive view of the natural RNAi pathway in animals.
... Recognition of invading microorganisms and coordinating a defence response are critical steps for host organisms in their perpetual battle with pathogens. Viruses are generally detected initially by host sensors for foreign nucleic acids, including double-stranded RNA duplexes or viral DNA in the cytoplasm and single-or double-stranded RNA or DNA in endosomes (1,2). Reflecting a longterm, evolutionary arms race between viruses and their hosts, viruses have evolved an extensive and highly diverse range of counter-measures that block their recognition or impair the action of interferon and other effector mechanisms (3). ...
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RNA viruses infecting vertebrates differ fundamentally in their ability to establish persistent infections with markedly different patterns of transmission, disease mechanisms and evolutionary relationships with their hosts. Although interactions with host innate and adaptive responses are complex and persistence mechanisms likely multi-factorial, we previously observed associations between bioinformatically predicted RNA secondary formation in genomes of positive-stranded RNA viruses with their in vivo fitness and persistence. To analyse this interactions functionally, we transfected fibroblasts with non-replicating, non-translated RNA transcripts from RNA viral genomes with differing degrees of genome-scale ordered RNA structure (GORS). Single-stranded RNA transcripts induced interferon-β mediated though RIG-I and PKR activation, the latter associated with rapid induction of antiviral stress granules. A striking inverse correlation was observed between induction of both cellular responses with transcript RNA structure formation that was independent of both nucleotide composition and sequence length. The consistent inability of cells to recognize RNA transcripts possessing GORS extended to downstream differences from unstructured transcripts in expression of TNF-α, other interferon-stimulated genes and induction of apoptosis. This functional association provides novel insights into interactions between virus and host early after infection and provides evidence for a novel mechanism for evading intrinsic and innate immune responses.
... Defense mechanisms involving RNA interference, RNases, and endonucleases are used to combat the invading viral genome. RNA interference works by using small RNA molecules to inhibit gene transcription (a defense that can be applied to all types of virus infection regardless of the nature of their genomic material) and can cause the direct degradation of dsRNA viral genomes (Marques and Carthew, 2007). Restriction endonucleases cleave DNA at specific recognition sequence sites (often found in viral genomes, but not present or protected in host genomes) and provide general protection from DNA viruses. ...
Chapter
Vir-olution: Setting the Scene The Obsession with Death: Mortality from a Viral Perspective A Marriage Made in Hell The Numbers Game Fight to Death: Genes Are the Weapons The Silence of the Viruses Giving up the Viral Ghost The Makings of Virus-Host Compatibility Throwing Light on Virus-Host Evolution Sometimes it Takes More than the Odd Gene References
... TE evasion of (Obbard, et al. 2006;Kolaczkowski, et al. 2011;Obbard, et al. 2011;Bernhardt, et al. 2012;Kelleher, et al. 2012;Lee and Langley 2012;Simkin, et al. 2013). Adaptive evolution at a subset of these genes may be driven by antagonism with infectious viruses as has been seen for components of the siRNA production machinery in insects (Marques and Carthew 2007;Obbard, et al. 2009;Saleh, et al. 2009;Nayak, et al. 2010;Murray, et al. 2013;Nayak, et al. 2013;Schnettler, et al. 2014;van Mierlo, et al. 2014). ...
Article
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Transposable elements (TEs) comprise large fractions of many eukaryotic genomes and imperil host genome integrity. The host genome combats these challenges by encoding proteins that silence TE activity. Both the introduction of new TEs via horizontal transfer and TE sequence evolution requires constant innovation of host-encoded TE silencing machinery to keep pace with TEs. One form of host innovation is the adaptation of existing, single-copy host genes. Indeed, host suppressors of TE replication often harbor signatures of positive selection. Such signatures are especially evident in genes encoding the piRNA (piwi-associated RNAs) pathway of gene silencing e.g., the female germline-restricted TE silencer, HP1D/Rhino. Host genomes can also innovate via gene duplication and divergence. However, the importance of gene family expansions, contractions, and gene turnover to host genome defense has been largely unexplored. Here, we functionally characterize Oxpecker, a young, tandem duplicate gene of HP1D/rhino. We demonstrate that Oxpecker supports female fertility in Drosophila melanogaster and silences several transposable element families that are incompletely silenced by HP1D/Rhino in the female germline. We further show that, like Oxpecker, at least ten additional, structurally diverse, HP1D/rhino-derived daughter and “granddaughter” genes emerged during a short 15 million year period of Drosophila evolution. These young paralogs are transcribed primarily in germline tissues, where the genetic conflict between host genomes and TEs plays out. Our findings suggest that gene family expansion is an underappreciated yet potent evolutionary mechanism of genome defense diversification.
... On the other hand, the mosquito must evolve counter strategies to recruit highly evolvable genes to defend the virus adaptation (refractory response). This mechanism is fairly universal for evolution of most host-virus interactions (Marques and Carthew 2007). In this context, it is highly likely that our findings on role of intron on vector gene expressions corroborate to a co-evolutionary strategy between A. aegypti and dengue virus adaptability. ...
Article
Dengue virus infection causes significant morbidity and mortality in humans world-wide. The Aedes aegypti mosquito is the major vector that spreads dengue virus to humans. Interaction between dengue viruses and A. aegypti is a multi-factorial phenomena that is determined by both virus and mosquito genotypes. Although, studies have suggested significant association of mosquito vectorial capacity with population variation of dengue virus, specifications of the vector factors that may influence vector-virus compatibility are very limited in the literature. Recently, we have shown that a large number of genes are differentially expressed between MOYO-S (susceptible) and MOYO-R (refractory) A. aegypti strains upon infection with dengue virus (JAM-1409 genotype). In the current study, we show that specific intrinsic features of A. aegypti genes are significantly associated with 'responsiveness' of mosquito genes to dengue infection. Binomial logistic regression analysis further reveals differential marginal effects of these features on gene responsiveness of mosquitoes to the viral infection. Thus, our result shows that intrinsic features of genes significantly affect differential expression of A. aegypti genes to dengue infection. The information will benefit further investigations on evolution of genes among natural populations of A. aegypti conferring differential susceptibility to dengue virus.
... However, a fraction of the target insect population may survive, either because certain individuals did not consume a lethal number of virus occlusion bodies (OBs) present on the crop foliage (Evans 1999) or because the insect does not develop a lethal infection after having consumed virus inoculum (Burden et al. 2002). The later may be due to inhibition of the viral infection by the host immune system (Marques and Carthew 2007;Pascual et al. 2012), or due to the virus adopting a low virulence non-lethal infection strategy (Sorrell et al. 2009;Moreno-Garc ıa et al. 2014), or a combination of both. ...
Article
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Following the consumption of baculovirus occlusion bodies (OBs), insects may succumb to lethal disease, but the survivors can harbour sublethal covert infections and may develop, reproduce and transmit the infection to their offspring. The use of different chemical and biological stressors was examined to determine whether they could be used to activate covert infections in populations of Spodoptera exigua larvae infected by the homologous nucleopolyhedrovirus (SeMNPV). Treatment of covertly infected S. exigua second instars with Tinopal UNPA-GX, hydroxylamine, paraquat, Bacillus thuringiensis var. kurstaki crystals, spores or mixtures of crystals + spores, or a heterologous nucleopolyhedrovirus (Chrysodeixis chalcites SNPV) did not result in the activation of SeMNPV covert infections. Similarly, virus treatments involving permissive NPVs did not result in greater mortality in covertly infected insects compared with the virus-free controls. In contrast, 0.1% copper sulphate, 1% iron (II) sulphate and 1 mg/l sodium selenite treatments resulted in 12–41% lethal polyhedrosis disease in covertly infected larvae. A greenhouse trial using copper sulphate and sodium selenite as activation factors applied to covertly infected S. exigua larvae on sweet pepper plants resulted in very low levels of SeMNPV activation (<3%). These results highlight the important roles of copper, iron and selenium in insect immunity and baculovirus-induced disease. However, these substances seem unlikely to prove useful for the activation of covert SeMNPV infections in S. exigua larvae under greenhouse conditions.
... Arbovirus interactions with both the vertebrate and mosquito host have been shown to have association with coevolutionary processes that influence virus infection mechanisms and the immune systems in both hosts [109][110][111]. Using binomial logistic regression analyses to investigate known DENV responsive genes [96], we found that several intrinsic features such as gene context, intron presence, codon usage bias, paralogy, and derived versus ancestral origin of A. aegypti genes each show significant marginal effects with the observed transcriptional responses to DENV infection [112]. ...
Article
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Dengue (DENV), yellow fever, chikungunya, and Zika virus transmission to humans by a mosquito host is confounded by both intrinsic and extrinsic variables. Besides virulence factors of the individual arboviruses, likelihood of virus transmission is subject to variability in the genome of the primary mosquito vector, Aedes aegypti. The “vectorial capacity” of A. aegypti varies depending upon its density, biting rate, and survival rate, as well as its intrinsic ability to acquire, host and transmit a given arbovirus. This intrinsic ability is known as “vector competence”. Based on whole transcriptome analysis, several genes and pathways have been predicated to have an association with a susceptible or refractory response in A. aegypti to DENV infection. However, the functional genomics of vector competence of A. aegypti is not well understood, primarily due to lack of integrative approaches in genomic or transcriptomic studies. In this review, we focus on the present status of genomics studies of DENV vector competence in A. aegypti as limited information is available relative to the other arboviruses. We propose future areas of research needed to facilitate the integration of vector and virus genomics and environmental factors to work towards better understanding of vector competence and vectorial capacity in natural conditions.
... RNA interference employs robust and selective pathway for battling with various viral diseases that cause significant economic losses. Viral suppression of the immune system is a widespread phenomenon and many viruses inhibit the viRNA pathway by expressing viral suppressors of RNA interference (Marques and Carthew, 2007;Ding & Voinnet, 2007). Transgenes encoding hpRNA are greatly effective for the silencing of both endogenous genes and transgenes which are employing virus resistance in plants. ...
Article
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RNA interference studies have provided new opportunities for high-efficiency and high-throughput technology for gene suppression in plants with an aim to achieve desirable traits. RNAi which is a conserved mechanism of post transcriptional gene silencing have advantage for functional genomics. RNA construct can easily target to specific gene from any background in a dominant manner by initiating sequence specific RNA degradation pathways. Gene silencing in either way such as tissue specific silencing, inducible silencing or host delivered RNAi demonstrated to serve as a defense mechanism for an improvement of crops against nematodes, bacteria, fungi, insect, pest, parasitic weeds, and viruses. Many breeding goals have been achieved in different crops which were nightmares in the past for the researchers. But the advent of new technology has paved the way to develop resistant crops to combat biotic and abiotic stresses. It also gives a hope to attain world food security, enhances nutritional quality (in terms of bio-fortification and bio-elimination) for mankind and proving itself as an ecofriendly tool for plant protection. Especially, host gene silencing hairpin RNAi is reported as more stable gene silencing strategy in plants against different pathogens and also provide protection from invasion by foreign nucleic acid.
... There are many routes by which a pathogen may evolve host specialization (Kitano 2007 ). Although there are several experimental and evolutionary models for bacterial and viral pathogens there is a paucity of information regarding the evolution of fungal pathogens (Marques & Carthew 2007; Ochman & Moran 2001; Pallen & Wren 2007). In order to study fungal pathogen evolution, we employed a model pathogen–host system involving the serial propagation of the opportunistic fungus Aspergillus flavus through the insect host Galleria mellonella larvae (Scully & Bidochka 2005 ). A. flavus and G. mellonella are ideal species for such a model system . ...
Article
In order to study fungal pathogen evolution, we used a model system whereby the opportunistic fungus Aspergillus flavus was serially propagated through the insect (Galleria mellonella) larvae, yielding a cysteine/methionine auxotroph of A. flavus with properties of an obligate insect pathogen. The auxotroph exhibited insect host restriction but did not show any difference in virulence when compared with the wild-type (Scully LR, Bidochka MJ, 2006. Microbiology 152, 223–232). Here, we report that on 1 % insect cuticle medium and synthetic Galleria medium, the auxotroph displayed increased extracellular protease production, a virulence factor necessary for insect pathogenesis. In the wild-type strain, protease production was deregulated during carbon (glucose), nitrogen (nitrate), or sulphate deprivation. If all three were present, protease production was vastly reduced. However, in the cysteine/methionine auxotroph, protease production was deregulated in complete medium. We suggest that the deficiency in sulphate assimilation in the auxotroph resulted in deregulation of protease production. The auxotroph exhibited delayed germination and slower hyphal growth when compared to the wild-type but there were no differences in virulence or cuticle penetration, suggesting a shift in pathogenic strategy that compensated decreased growth with increased virulence factor (extracellular protease) production. We concluded that the biosynthetic deficiency that mediated insect host restriction also increased protease production in the slow-growing auxotroph, resulting in an alternate, more host-specific pathogenic strategy. However, we argue that transmission is not necessarily correlated with virulence as competition bioassays in insect larvae showed that the wild-type generally out-competed the auxotroph by producing the majority of the conidia on the sporulating cadavers. This is one of the few examples that highlight the effect of genome decay on nutrition acquisition, virulence, and transmission in fungal pathogen evolution.
... For example, forest fires and volcanic eruptions are non-anthropogenic sources for particulate matter (PM) in the biosphere, thus entering the respiratory tracts of animals since they evolved [86]. Airborne viruses have challenged the immune systems of living organisms throughout their evolution, a process that is ongoing [87]. With the discovery of fire usage, humans started to burn wood to cook, to keep themselves warm, to keep away predators and more. ...
Article
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Air quality depends on the various gases and particles present in it. Both natural phenomena and human activities affect the cleanliness of air. In the last decade, many countries experienced an unprecedented industrial growth, resulting in changing air quality values, and correspondingly, affecting our life quality. Air quality can be accessed by employing microchips that qualitatively and quantitatively determine the present gases and dust particles. The so-called particular matter 2.5 (PM2.5) values are of high importance, as such small particles can penetrate the human lung barrier and enter the blood system. There are cancer cases related to many air pollutants, and especially to PM2.5, contributing to exploding costs within the healthcare system. We focus on various current and potential future air pollutants, and propose solutions on how to protect our health against such dangerous substances. Recent developments in the Organ-on-Chip (OoC) technology can be used to study air pollution as well. OoC allows determination of pollutant toxicity and speeds up the development of novel pharmaceutical drugs.
... RNA silencing is an evolutionary conserved mechanism that has an antiviral role in plants and insects (reviewed in [1,2]). Viral double-strand RNA (dsRNA) is cleaved into small interfering RNA (siRNA) by RNase III-like nucleases called Dicer or Dicer-like. ...
Article
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Virus infection in plants is limited by RNA silencing. In turn, viruses can counter RNA silencing with silencing suppressors. Viral suppressors of RNA silencing have been shown to play a role in symptom development in plants. We here study four different strategies employed by silencing suppressors: small interfering RNA (siRNA) binding, double-strand RNA (dsRNA) binding and degrading or inactivating Argonaute. We study the effect of the suppressors on viral accumulation within the cell as well as its spread on a tissue with mathematical and computational models. We find that suppressors which target Argonaute are very effective in a single cell, but that targeting dsRNA or siRNA is much more effective at the tissue level. Although targeting Argonaute can be beneficial for viral spread, it can also cause hindrance in some cases owing to raised levels of siRNAs that can spread to other cells.
... This is best studied in Drosophila [20][21][22], but is also detectable in other invertebrates [23]. It has been hypothesized that this may be a consequence of parasite-mediated 'arms-race' coevolution [21,24], either through conflict with parasite-encoded immune suppressors-as widely seen in RNA viruses [25]-or in the case of the piRNA pathway, through selection for 're-tuning' suppression mechanisms [26]. ...
Article
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Background RNA interference (RNAi) related pathways provide defense against viruses and transposable elements, and have been implicated in the suppression of meiotic drive elements. Genes in these pathways often exhibit high levels of adaptive substitution, and over longer timescales show gene duplication and loss—most likely as a consequence of their role in mediating conflict with these parasites. This is particularly striking for Argonaute 2 (Ago2), which is ancestrally the key effector of antiviral RNAi in insects, but has repeatedly formed new testis-specific duplicates in the recent history of the obscura species-group of Drosophila. Results Here we take advantage of publicly available genomic and transcriptomic data to identify six further RNAi-pathway genes that have duplicated in this clade of Drosophila, and examine their evolutionary history. As seen for Ago2, we observe high levels of adaptive amino-acid substitution and changes in sex-biased expression in many of the paralogs. However, our phylogenetic analysis suggests that co-duplications of the RNAi machinery were not synchronous, and our expression analysis fails to identify consistent male-specific expression. Conclusions These results confirm that RNAi genes, including genes of the antiviral and piRNA pathways, have undergone multiple independent duplications and that their history has been particularly labile within the obscura group. However, they also suggest that the selective pressures driving these changes have not been consistent, implying that more than one selective agent may be responsible. Electronic supplementary material The online version of this article (10.1186/s12862-019-1425-0) contains supplementary material, which is available to authorized users.
... Insects lack acquired immune system and solely dependent on the innate immune system for defending the diverse pathogens that include fungus, bacteria and viruses (Jeremy, 1997;Kingsolver et al., 2013). Among these, viruses are the most perilous parasites with very small genome controlling host machinery for their effective proliferation (Marques and Carthew, 2007). Innate immune system of insects mainly involves two types of responses, the cellular response and the humoral response. ...
Article
The domesticated silkworm, Bombyx mori, is an economically important insect that majorly contributes in financially viable sericulture industry. The baculovirus, Bombyx mori nuclear polyhedrosis virus (BmNPV) is a deadly virus that infects B. mori and inflicts heavy loss to silk production by causing high mortality of silkworms. A number of studies have been done to identify antiviral genes and pathways against BmNPV infection in B. mori. Toll-pathway is a well-known immune pathway responsible for combating bacterial and fungal infections in silkworm. However, the role of Toll-receptors in inhibiting BmNPV has not been explored. In the present study, we have attempted to examine the role of Toll-receptors in inhibiting BmNPV proliferation in B. mori larvae. The effect of Toll-receptors on viral proliferation was determined by quantifying viral DNA and counting viral occlusion bodies upon RNA interference-mediated knockdown of Toll-receptors in B. mori larvae. Knockdown of BmToll, BmToll-6, BmToll-7, BmToll-8 and BmToll-9 led to higher accumulation of BmNPV. Further, interplay between Toll-receptors and the antimicrobial proteins of B. mori, called seroins were studied using RNA interference. As a result, we found a downregulation of Seroin1 transcript level upon knockdown of BmToll, BmToll-7, BmToll-8, BmToll-10, BmToll-11 and BmToLK2, whereas Seroin2 expression was found to decrease only upon BmToll-7 knockdown. Moreover, knockdown of seroins did not result in clearly interpretable expression patterns of Toll-receptors. These results suggest the involvement of B. mori Toll-pathway in defense against BmNPV infection and provides a new insights into the antiviral defense mechanism of B. mori.
... As such, the role of the two type 145 and the four type 150 proteins already studied differs depending on the host species. The hostpathogen coevolutionary process involves reciprocal adaptive genetic changes in host and pathogen that oppose one another, such as antiviral mechanisms of the host, and inhibitors of such mechanisms in the virus and similar adaptations designed to overcome barriers to infection or replication in the host (Marques and Carthew, 2007). Hence, the host immune system and virus counter defenses evolve together and constitute a continuously evolving source of diversity (Woolhouse et al., 2002). ...
... Co-evolution of virus and host fuels the arms-race between host antiviral responses and viral counter defence strategies [47]. VIFs alternately infect evolutionary distant vertebrate hosts and invertebrate vectors, which suggests that VIFs encounter distinct evolutionary pressures that together constrain their diversity [27,48]. ...
Preprint
The genus Flavivirus contains pathogenic vertebrate-infecting flaviviruses (VIFs) and in-sect-specific flaviviruses (ISF). ISF transmission to vertebrates is inhibited at multiple stages of the cellular infection cycle, via yet to be elucidated specific antiviral responses. The Zinc-finger an-tiviral protein (ZAP) in vertebrate cells can bind CpG dinucleotides in viral RNA, limiting virus replication. Interestingly, the genomes of ISFs contain more CpG dinucleotides compared to VIFs. In this study, we investigated whether ZAP prevents two recently discovered lineage II ISFs, Binjari (BinJV) and Hidden Valley viruses (HVV) from replicating in vertebrate cells. BinJV protein and dsRNA replication intermediates were readily observed in human ZAP knockout cells when cultured at 34 ˚C. In ZAP expressing cells, inhibition of the interferon response via interferon response factors 3/7 did not improve BinJV protein expression, whereas treatment with kinase inhibitor C16, known to reduce ZAP’s antiviral function, did. Importantly, at 34 ˚C both BinJV and HVV successfully completed the infection cycle in human ZAP knockout cells evident from infectious progeny virus in the cell culture supernatant. Therefore, we identify vertebrate ZAP as an important barrier that protects vertebrate cells from ISF infection This provides new insights into flavivirus evolution and the mechanisms associated with host switching.
... It has been suggested that parasites might have lost the race to avoid direct recognition by hosts and have instead shifted their focus to interfering with the host pathways that are mediating defence (Schmid-Hempel, 2005). Indeed, there is ample empirical evidence for parasite interference with host pathways (Sacks and Sher, 2002; Bhavsar et al, 2007; Marques and Carthew, 2007). For example, parasitic protozoa Leishmania and Toxoplasma gondii are shown to inhibit the activity of specific host kinases (Olivier et al, 1992) and block nuclear localization of specific transcription factors (Denkers et al, 2004), respectively. ...
Article
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Many biological networks can maintain their function against single gene loss. However, the evolutionary mechanisms responsible for such robustness remain unclear. Here, we demonstrate that antagonistic host-parasite interactions can act as a selective pressure driving the emergence of robustness against gene loss. Using a model of host signaling networks and simulating their coevolution with parasites that interfere with network function, we find that networks evolve both redundancy and specific architectures that allow them to maintain their response despite removal of proteins. We show that when the parasite pressure is removed, subsequent evolution can lead to loss of redundancy while architecture-based robustness is retained. Contrary to intuition, increased parasite virulence hampers evolution of robustness by limiting the generation of population level diversity in the host. However, when robustness emerges under high virulence, it tends to be stronger. These findings predict an increased presence of robustness mechanisms in biological networks operating under parasite interference. Conversely, the presence of such mechanisms could indicate current or past parasite interference.
Article
The network view in systems biology, in conjunction with the continuing development of experimental technologies, is providing us with the key structural and dynamical features of both cell-wide and pathway-level regulatory, signaling and metabolic systems. These include for example modularity and presence of hub proteins at the structural level and ultrasensitivity and feedback control at the level of dynamics. The uncovering of such features, and the seeming commonality of some of them, makes many systems biologists believe that these could represent design principles that underpin cellular systems across organisms. Here, we argue that such claims on any observed feature requires an understanding of how it has emerged in evolution and how it can shape subsequent evolution. We review recent and past studies that aim to achieve such evolutionary understanding for observed features of cellular networks. We argue that this evolutionary framework could lead to deciphering evolutionary origin and relevance of proposed design principles, thereby allowing to predict their presence or absence in an organism based on its environment and biochemistry and their effect on its future evolution.
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This chapter reviews our current knowledge about mechanisms of suppression developed by pathogens to avoid host defense responses. In general, plants perceive pathogens by diverse pathogen- or microbe- or even damage-associated molecular patterns (PAMPs, MAMPs, DAMPs) and induce a variety of defense mechanisms referred to as horizontal or basal resistance, nowadays designated PAMP-triggered immunity (PTI). In addition, plants can also recognize specific pathogen-derived effectors and have derived a highly specific defense response termed effector-triggered immunity (ETI), classically called R gene-mediated, specific or vertical resistance. Both PTI and ETI are responses to potential dangers and have common components. Fungal, oomycete, and bacterial pathogens have evolved various effector-based mechanisms of suppression that interfere with such components. Plants strongly depend on RNA gene silencing to interfere with viral pathogens. Plant viruses counteract this response by encoding suppressor proteins of RNA silencing.
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Ebola virus (EBOV) causes a lethal hemorrhagic fever for which there is no approved effective treatment or prevention strategy. EBOV VP35 is a virulence factor that blocks innate antiviral host responses, including the induction of and response to alpha/beta interferon. VP35 is also an RNA silencing suppressor (RSS). By inhibiting microRNA-directed silencing, mammalian virus RSSs have the capacity to alter the cellular environment to benefit replication. A reporter gene containing specific microRNA target sequences was used to demonstrate that prior expression of wild-type VP35 was able to block establishment of microRNA silencing in mammalian cells. In addition, wild-type VP35 C-terminal domain (CTD) protein fusions were shown to bind small interfering RNA (siRNA). Analysis of mutant proteins demonstrated that reporter activity in RSS assays did not correlate with their ability to antagonize double-stranded RNA (dsRNA)-activated protein kinase R (PKR) or bind siRNA. The results suggest that enhanced reporter activity in the presence of VP35 is a composite of nonspecific translational enhancement and silencing suppression. Moreover, most of the specific RSS activity in mammalian cells is RNA binding independent, consistent with VP35's proposed role in sequestering one or more silencing complex proteins. To examine RSS activity in a system without interferon, VP35 was tested in well-characterized plant silencing suppression assays. VP35 was shown to possess potent plant RSS activity, and the activities of mutant proteins correlated strongly, but not exclusively, with RNA binding ability. The results suggest the importance of VP35-protein interactions in blocking silencing in a system (mammalian) that cannot amplify dsRNA.
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Shrimp production, using a small‐scale enclosed pond system, is a rapidly growing aquaculture sector, which is valued around USD 18.30 billion in 2020. Intensified shrimp culture leads to the outbreak of transmissible diseases to eventually cause a huge loss in the production process and thus the economy. Studies on microRNA (miRNA) reveal that miRNA has an influential role in the host‐pathogen interaction during an infection. Recently, shrimp miRNA has been shown to help pathogen‐like viruses for their replication and infection. Several shrimp miRNAs were reported to be involved in enhancing host immunity against viral infection, especially white spot syndrome virus (WSSV) infection and Vibrio infection caused by bacterial species, whereas some shrimp miRNAs were reported to be hijacked by WSSV and to enhance the viral replication and establish the infection in shrimp. This gives an insight into the double‐edged sword role played by shrimp miRNA during host‐pathogen interaction. In future, this role could be employed against the virus to strengthen the shrimp culture. In this review, we discuss the role of shrimp miRNA and their mechanism(s) associated with the establishment of host‐pathogen interaction during infection, which will reveal the complexity associated with shrimp infection.
Conference Paper
The "11K" gene family is notable for having homologs in both baculoviruses and entomopoxviruses and are classified as either type 145 or type 150, according to their similarity with the ac145 or ac150 genes of Autographa californica multiple nucleopolyhedrovirus (AcMNPV). One homolog of ac145 (sf138) and two homologs of ac150 (sf68 and sf95) are present in Spodoptera frugiperda multiple nucleopolyhedrovirus (SfMNPV). Recombinant bacmids lacking sf68, sf95 or sf138 (Sf68null, Sf95null and Sf138null, respectively) and the respective repair bacmids were generated from a bacmid comprising the complete virus genome. Occlusion bodies (OBs) of the Sf138null virus were ∼15-fold less orally infective to insects, which was attributed to a 100-fold reduction in ODV infectious titer. Inoculation of insects with Sf138null OBs in mixtures with an optical brightener failed to restore the pathogenicity of Sf138null OBs to that of the parental virus, indicating that the effects of sf138 deletion on OB pathogenicity were unlikely to involve an interaction with the gut peritrophic matrix. In contrast, deletion of sf68 and sf95 resulted in a slower speed-of-kill by 9 hours, and a concurrent increase in the yield of OBs. Phylogenetic analysis indicated that sf68 and sf95 were not generated after a duplication event of an ancestral gene homologous to the ac150 gene. We conclude that type 145 genes modulate the primary infection process of the virus, whereas type 150 genes appear to have a role in spreading systemic infection within the insect. Copyright © 2015. Published by Elsevier Inc.
Article
The fruit fly Drosophila melanogaster is a powerful model to study host‐pathogen interactions. Most studies so far have focused on extracellular pathogens such as bacteria and fungi. More recently, viruses have come to the front, and RNA interference was shown to play a critical role in the control of viral infections in drosophila. We review here our current knowledge on drosophila viruses. A diverse set of RNA viruses belonging to several families (Rhabdoviridae, Dicistroviridae, Birnaviridae, Reoviridae, Errantiviridae) has been reported in D. melanogaster. By contrast, no DNA virus has been recovered up to now. The drosophila viruses represent powerful tools to study virus‐cell interactions in vivo. Analysis of the literature however reveals that for many of them, important gaps exist in our understanding of their replication cycle, genome organization, morphology or pathogenesis. The data obtained in the past few years on antiviral defense mechanisms in drosophila, which point to evolutionary conserved pathways, highlight the potential of the D. melanogaster model to study antiviral innate immunity and to better understand the complex interaction between arthropod‐borne viruses and their insect vectors.
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Evidence is accumulating that small RNAs processed from both host genomic sources and exogenous, viral, double stranded RNAs are biologically active in pathways that downregulate translation and increase degradation of specific transcripts in eukaryotic cells. Here, the mechanisms of small RNA production and processing in Drosophila melanogaster are described, and recent evidence that these pathways are involved in a pathogen-specific, systemic innate immune response is summarised. Small interfering RNAs (siRNAs) processed from viral dsRNAs are presented in the cell as templates to enable sequence-specific silencing of complementary viral transcripts in infected insect cells. Further, there is evidence that, as part of the ongoing immunological arms race between host and pathogen, insect viruses have evolved means to disrupt or subvert the host siRNA pathway.
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Virus surveillance in vector insects is potentially of great benefit to public health. Large-scale sequencing of small and long RNAs has previously been used to detect viruses, but without any formal comparison of different strategies. Furthermore, the identification of viral sequences largely depends on similarity searches against reference databases. Here, we developed a sequence-independent strategy based on virus-derived small RNAs produced by the host response, such as the RNA interference pathway. In insects, we compared sequences of small and long RNAs, demonstrating that viral sequences are enriched in the small RNA fraction. We also noted that the small RNA size profile is a unique signature for each virus and can be used to identify novel viral sequences without known relatives in reference databases. Using this strategy, we characterized six novel viruses in the viromes of laboratory fruit flies and wild populations of two insect vectors: mosquitoes and sandflies. We also show that the small RNA profile could be used to infer viral tropism for ovaries among other aspects of virus biology. Additionally, our results suggest that virus detection utilizing small RNAs can also be applied to vertebrates, although not as efficiently as to plants and insects.
Chapter
The phenotypic effects of drugs of abuse are partially mediated by transcriptional and epigenetic regulatory mechanisms. This chapter will provide a brief overview of substance abuse and then focus on the roles of three epigenetic regulatory mechanisms in addictive processes: histone modifications, DNA modifications, and noncoding RNAs. This chapter will conclude with a focus on three other important areas: (1) the potential for long-lasting epigenetic effects due to drugs of abuse, (2) obstacles and opportunities in this scientific area as they pertain to addiction biology, and (3) the potential for translating epigenomic and noncoding RNA discoveries into improvements in human health and the treatment of substance use disorders.
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Argonaute2 (Ago2) is a rapidly evolving nuclease in the Drosophila melanogaster RNA interference (RNAi) pathway that targets viruses and transposable elements in somatic tissues. Here we reconstruct the history of Ago2 duplications across the Drosophila obscura group, and use patterns of gene expression to infer new functional specialization. We show that some duplications are old, shared by the entire species group, and that losses may be common, including previously undetected losses in the lineage leading to D. pseudoobscura We find that while the original (syntenic) gene copy has generally retained the ancestral ubiquitous expression pattern, most of the novel Ago2 paralogues have independently specialized to testis-specific expression. Using population genetic analyses, we show that most testis-specific paralogues have significantly lower genetic diversity than the genome-wide average. This suggests recent positive selection in three different species, and model-based analyses provide strong evidence of recent hard selective sweeps in or near four of the six D. pseudoobscura Ago2 paralogues. We speculate that the repeated evolution of testis-specificity in obscura group Ago2 genes, combined with their dynamic turnover and strong signatures of adaptive evolution, may be associated with highly derived roles in the suppression of transposable elements or meiotic drive. Our study highlights the lability of RNAi pathways, even within well-studied groups such as Drosophila, and suggests that strong selection may act quickly after duplication in RNAi pathways, potentially giving rise to new and unknown RNAi functions in non-model species.
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This report is the outcome of an EFSA procurement aiming at investigating and summarising the state of knowledge on (I) the mode-of-action of dsRNA and miRNA pathways, (II) the potential for nontarget gene regulation by dsRNA-derived siRNAs or miRNAs, (III) the determination of siRNA pools in plant tissues and the importance of individual siRNAs for silencing. The report is based on a comprehensive and systematic literature search, starting with the identification and retrieval of~190,000 publications related to the research area and further filtered down with keywords to produce focused collections used for subsequent screening of titles and abstracts. The report is comprised of an (I) Introduction to the field of small RNAs, (II) a Data and Methodologies section containing strategies used for literature search and study selection, and (III) the Results of the literature review organized according to the three main procurement tasks. The outcome of the first task reviews dsRNA and miRNA pathways in mammals (including humans), birds, fish, arthropods, annelids, molluscs, nematodes, and plants. Eight taxon-dedicated chapters are based on ~1,400 cumulative references chosen from ~10,000 inspected titles and abstracts. We review conserved and divergent aspects of small RNA pathways and dsRNA responses in animals and plants including structure and function of key proteins as well as four basic mechanisms: genome-encoded posttranscriptional regulations (miRNA), degradation of RNAs by short interfering RNA pools generated from long dsRNA (RNAi), transcriptional silencing, and sequence-independent responses to dsRNA. The outcome of the second task focuses on base pairing between small RNAs and their target RNAs and predictability of biological effects of small RNAs in animals and plants. The outcome of the last task reviews methodology, siRNA pools, and movement of small RNAs in plants. Potential transfer of small RNAs between species and circulating miRNAs in mammals is described in the final chapter. full text: http://onlinelibrary.wiley.com/doi/10.2903/sp.efsa.2017.EN-1246/pdf
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We present a novel mechanism by which viruses may inhibit the alpha/beta interferon (IFN-α/β) cascade. The double-stranded RNA (dsRNA) binding protein NS1 of influenza virus is shown to prevent the potent antiviral interferon response by inhibiting the activation of interferon regulatory factor 3 (IRF-3), a key regulator of IFN-α/β gene expression. IRF-3 activation and, as a consequence, IFN-β mRNA induction are inhibited in wild-type (PR8) influenza virus-infected cells but not in cells infected with an isogenic virus lacking the NS1 gene (delNS1 virus). Furthermore, NS1 is shown to be a general inhibitor of the interferon signaling pathway. Inhibition of IRF-3 activation can be achieved by the expression of wild-type NS1 intrans, not only in delNS1 virus-infected cells but also in cells infected with a heterologous RNA virus (Newcastle disease virus). We propose that inhibition of IRF-3 activation by a dsRNA binding protein significantly contributes to the virulence of influenza A viruses and possibly to that of other viruses.
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MicroRNAs (miRNAs), which function as regulators of gene expression in eukaryotes, are processed from larger transcripts by sequential action of nuclear and cytoplasmic ribonuclease III–like endonucleases. We show that Exportin-5 (Exp5) mediates efficient nuclear export of short miRNA precursors (pre-miRNAs) and that its depletion by RNA interference results in reduced miRNA levels. Exp5 binds correctly processed pre-miRNAs directly and specifically, in a Ran guanosine triphosphate–dependent manner, but interacts only weakly with extended pre-miRNAs that yield incorrect miRNAs when processed by Dicer in vitro. Thus, Exp5 is key to miRNA biogenesis and may help coordinate nuclear and cytoplasmic processing steps.
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Homology-dependent RNA silencing occurs in many eukaryotic cells. We reported recently that nodaviral infection triggers an RNA silencing-based antiviral response (RSAR) in Drosophila, which is capable of a rapid virus clearance in the absence of expression of a virus-encoded suppressor. Here, we present further evidence to show that the Drosophila RSAR is mediated by the RNA interference (RNAi) pathway, as the viral suppressor of RSAR inhibits experimental RNAi initiated by exogenous double-stranded RNA and RSAR requires the RNAi machinery. We demonstrate that RNAi also functions as a natural antiviral immunity in mosquito cells. We further show that vaccinia virus and human influenza A, B, and C viruses each encode an essential protein that suppresses RSAR in Drosophila. The vaccinia and influenza viral suppressors, E3L and NS1, are distinct double-stranded RNA-binding proteins and essential for pathogenesis by inhibiting the mammalian IFN-regulated innate antiviral response. We found that the double-stranded RNA-binding domain of NS1, implicated in innate immunity suppression, is both essential and sufficient for RSAR suppression. These findings provide evidence that mammalian virus proteins can inhibit RNA silencing, implicating this mechanism as a nucleic acid-based antiviral immunity in mammalian cells.
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RNA silencing processes are guided by small RNAs that are derived from double-stranded RNA. To probe for function of RNA silencing during infection of human cells by a DNA virus, we recorded the small RNA profile of cells infected by Epstein-Barr virus (EBV). We show that EBV expresses several microRNA (miRNA) genes. Given that miRNAs function in RNA silencing pathways either by targeting messenger RNAs for degradation or by repressing translation, we identified viral regulators of host and/or viral gene expression.
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Gene silencing through RNA interference (RNAi) is carried out by RISC, the RNA-induced silencing complex. RISC contains two signature components, small interfering RNAs (siRNAs) and Argonaute family proteins. Here, we show that the multiple Argonaute proteins present in mammals are both biologically and biochemically distinct, with a single mammalian family member, Argonaute2, being responsible for messenger RNA cleavage activity. This protein is essential for mouse development, and cells lacking Argonaute2 are unable to mount an experimental response to siRNAs. Mutations within a cryptic ribonuclease H domain within Argonaute2, as identified by comparison with the structure of an archeal Argonaute protein, inactivate RISC. Thus, our evidence supports a model in which Argonaute contributes “Slicer” activity to RISC, providing the catalytic engine for RNAi.
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The biogenesis and function of mature human microRNAs is dependent on the nuclear export of pre-microRNA precursors by Exportin 5 (Exp5). The precursor for the human miR-30 microRNA, which is a 63 nt long RNA hairpin bearing a 2 nt 3′ overhang, forms a specific complex with Exp5 and the Ran-GTP cofactor. Here, we have examined the structural requirements for pre-microRNA binding by Exp5. Our data indicate that pre-miR-30 binding requires an RNA stem of >16 bp and is facilitated by a 3′ overhang. Although a blunt-ended derivative of the pre-miR-30 stem–loop remained capable of binding Exp5, 5′ overhangs were inhibitory. miR-30 variants that had lost the ability to bind Exp5 effectively were not efficiently exported from the nucleus and were also expressed at reduced levels. Furthermore, formation of a pre-microRNA/Exp5/Ran-GTP complex inhibited exonucleolytic digestion of the pre-miRNA in vitro. Together, these data demonstrate that pre-microRNA binding by Exp5 involves recognition of almost all of the RNA hairpin, with the exception of the terminal loop. Moreover, these results argue that Exp5 binding not only mediates pre-microRNA nuclear export but also prevents nuclear pre-microRNA degradation.
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MicroRNAs (miRNAs) constitute a large family of noncoding RNAs that function as guide molecules in diverse gene silencing pathways. Current efforts are focused on the regulatory function of miRNAs, while little is known about how these unusual genes themselves are regulated. Here we present the first direct evidence that miRNA genes are transcribed by RNA polymerase II (pol II). The primary miRNA transcripts (pri-miRNAs) contain cap structures as well as poly(A) tails, which are the unique properties of class II gene transcripts. The treatment of human cells with alpha-amanitin decreased the level of pri-miRNAs at a concentration that selectively inhibits pol II activity. Furthermore, chromatin immunoprecipitation analyses show that pol II is physically associated with a miRNA promoter. We also describe, for the first time, the detailed structure of a miRNA gene by determining the promoter and the terminator of mir-23a approximately 27a approximately 24-2. These data indicate that pol II is the main, if not the only, RNA polymerase for miRNA gene transcription. Our study offers a basis for understanding the structure and regulation of miRNA genes.
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The factors regulating the expression of microRNAs (miRNAs), a ubiquitous family of approximately 22-nt noncoding regulatory RNAs, remain undefined. However, it is known that miRNAs are first transcribed as a largely unstructured precursor, termed a primary miRNA (pri-miRNA), which is sequentially processed in the nucleus, to give the approximately 65-nt pre-miRNA hairpin intermediate, and then in the cytoplasm, to give the mature miRNA. Here we have sought to identify the RNA polymerase responsible for miRNA transcription and to define the structure of a full-length human miRNA. We show that the pri-miRNA precursors for nine human miRNAs are both capped and polyadenylated and report the sequence of the full-length, approximately 3433-nt pri-miR-21 RNA. This pri-miR-21 gene sequence is flanked 5' by a promoter element able to transcribe heterologous mRNAs and 3' by a consensus polyadenylation sequence. Nuclear processing of pri-miRNAs was found to be efficient, thus largely preventing the nuclear export of full-length pri-miRNAs. Nevertheless, an intact miRNA stem-loop precursor located in the 3' UTR of a protein coding gene only moderately inhibited expression of the linked open reading frame, probably because the 3' truncated mRNA could still be exported and expressed. Together, these data show that human pri-miRNAs are not only structurally similar to mRNAs but can, in fact, function both as pri-miRNAs and mRNAs.
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Although inhibition of RNA interference (RNAi) by plant virus proteins has been shown to enhance viral replication and pathogenesis in plants, no viral gene product has as yet been shown to inhibit RNAi in vertebrate cells. Here, we present evidence demonstrating that the highly structured ∼160-nucleotide adenoviral VA1 noncoding RNA can inhibit RNAi at physiological levels of expression. VA1, which is expressed at very high levels in adenovirus-infected cells, potently inhibited RNAi induced by short hairpin RNAs (shRNAs) or human microRNA precursors but did not affect RNAi induced by artificial short interfering RNA duplexes. Inhibition appeared to be due both to inhibition of nuclear export of shRNA or premicro-RNA precursors, competition for the Exportin 5 nuclear export factor, and inhibition of Dicer function by direct binding of Dicer. Together, these data argue that adenovirus infection can result in inhibition of RNAi and identify VA1 RNA as the first viral gene product able to inhibit RNAi in human cells.
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RNA interference (RNAi) is triggered in eukaryotic organisms by double-stranded RNA (dsRNA), and it destroys any mRNA that has sequence identity with the dsRNA trigger. The RNAi pathway in Anopheles gambiae can be silenced by transfecting cells with dsRNA derived from exon sequence of the A. gambiae Argonaute2 (AgAgo2) gene. We hypothesized that RNAi may also act as an antagonist to alphavirus replication in A. gambiae because RNA viruses form dsRNA during replication. Silencing AgAgo2 expression would make A. gambiae mosquitoes more permissive to virus infection. To determine whether RNAi conditions the vector competence of A. gambiae for O'nyong-nyong virus (ONNV), we engineered a genetically modified ONNV that expresses enhanced GFP (eGFP) as a marker. After intrathoracic injection, ONNV-eGFP slowly spread to other A. gambiae tissues over a 9-day incubation period. Mosquitoes were then coinjected with virus and either control β-galactosidase dsRNA (dsβgal; note that “ds” is used as a prefix to indicate the dsRNA derived from a given gene throughout) or ONNV dsnsP3. Treatment with dsnsP3 inhibited virus spread significantly, as determined by eGFP expression patterns. ONNV-eGFP titers from mosquitoes coinjected with dsnsP3 were significantly lower at 3 and 6 days after injection than in mosquitoes coinjected with dsβgal. Mosquitoes were then coinjected with ONNV-eGFP and dsAgAgo2. Mosquitoes coinjected with virus and AgAgo2 dsRNA displayed widespread eGFP expression and virus titers 16-fold higher than dsβgal controls after 3 or 6 days after injection. These observations provide direct evidence that RNAi is an antagonist of ONNV replication in A. gambiae, and they suggest that the innate immune response conditions vector competence. • innate immunity • mosquito • vector competence
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In eukaryotes, small RNA molecules engage in sequence-specific interactions to inhibit gene expression by RNA silencing. This process fulfils fundamental regulatory roles, as well as antiviral functions, through the activities of microRNAs and small interfering RNAs. As a counter-defence mechanism, viruses have evolved various anti-silencing strategies that are being progressively unravelled. These studies have not only highlighted our basic understanding of host-parasite interactions, but also provide key insights into the diversity, regulation and evolution of RNA-silencing pathways.
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Epstein-Barr virus (EBV or HHV4), a member of the human herpesvirus (HHV) family, has recently been shown to encode microRNAs (miRNAs). In contrast to most eukaryotic miRNAs, these viral miRNAs do not have close homologs in other viral genomes or in the genome of the human host. To identify other miRNA genes in pathogenic viruses, we combined a new miRNA gene prediction method with small-RNA cloning from several virus-infected cell types. We cloned ten miRNAs in the Kaposi sarcoma-associated virus (KSHV or HHV8), nine miRNAs in the mouse gammaherpesvirus 68 (MHV68) and nine miRNAs in the human cytomegalovirus (HCMV or HHV5). These miRNA genes are expressed individually or in clusters from either polymerase (pol) II or pol III promoters, and share no substantial sequence homology with one another or with the known human miRNAs. Generally, we predicted miRNAs in several large DNA viruses, and we could neither predict nor experimentally identify miRNAs in the genomes of small RNA viruses or retroviruses.
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In eukaryotes, 21- to 24-nucleotide-long RNAs engage in sequence-specific interactions that inhibit gene expression by RNA silencing. This process has regulatory roles involving microRNAs and, in plants and insects, it also forms the basis of a defense mechanism directed by small interfering RNAs that derive from replicative or integrated viral genomes. We show that a cellular microRNA effectively restricts the accumulation of the retrovirus primate foamy virus type 1 (PFV-1) in human cells. PFV-1 also encodes a protein, Tas, that suppresses microRNA-directed functions in mammalian cells and displays cross-kingdom antisilencing activities. Therefore, through fortuitous recognition of foreign nucleic acids, cellular microRNAs have direct antiviral effects in addition to their regulatory functions.
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MicroRNAs (miRNAs) are generated by a two-step processing pathway to yield RNA molecules of approximately 22 nucleotides that negatively regulate target gene expression at the post-transcriptional level. Primary miRNAs are processed to precursor miRNAs (pre-miRNAs) by the Microprocessor complex. These pre-miRNAs are cleaved by the RNase III Dicer to generate mature miRNAs that direct the RNA-induced silencing complex (RISC) to messenger RNAs with complementary sequence. Here we show that TRBP (the human immunodeficiency virus transactivating response RNA-binding protein), which contains three double-stranded, RNA-binding domains, is an integral component of a Dicer-containing complex. Biochemical analysis of TRBP-containing complexes revealed the association of Dicer-TRBP with Argonaute 2 (Ago2), the catalytic engine of RISC. The physical association of Dicer-TRBP and Ago2 was confirmed after the isolation of the ternary complex using Flag-tagged Ago2 cell lines. In vitro reconstitution assays demonstrated that TRBP is required for the recruitment of Ago2 to the small interfering RNA (siRNA) bound by Dicer. Knockdown of TRBP results in destabilization of Dicer and a consequent loss of miRNA biogenesis. Finally, depletion of the Dicer-TRBP complex via exogenously introduced siRNAs diminished RISC-mediated reporter gene silencing. These results support a role of the Dicer-TRBP complex not only in miRNA processing but also as a platform for RISC assembly.
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We show that human adenovirus inhibits RNA interference (RNAi) at late times of infection by suppressing the activity of two key enzyme systems involved, Dicer and RNA-induced silencing complex (RISC). To define the mechanisms by which adenovirus blocks RNAi, we used a panel of mutant adenoviruses defective in virus-associated (VA) RNA expression. The results show that the virus-associated RNAs, VA RNAI and VA RNAII, function as suppressors of RNAi by interfering with the activity of Dicer. The VA RNAs bind Dicer and function as competitive substrates squelching Dicer. Further, we show that VA RNAI and VA RNAII are processed by Dicer, both in vitro and during a lytic infection, and that the resulting short interfering RNAs (siRNAs) are incorporated into active RISC. Dicer cleaves the terminal stem of both VA RNAI and VA RNAII. However, whereas both strands of the VA RNAI-specific siRNA are incorporated into RISC, the 3′ strand of the VA RNAII-specific siRNA is selectively incorporated during a lytic infection. In summary, our work shows that adenovirus suppresses RNAi during a lytic infection and gives insight into the mechanisms of RNAi suppression by VA RNA.
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p53 has been well characterized as a tumor suppressor gene, but its role in antiviral defense remains unclear. A recent report has demonstrated that p53 can be induced by interferons and is activated after vesicular stomatitis virus (VSV) infection. We observed that different nononcogenic viruses, including encephalomyocarditis virus (EMCV) and human parainfluenza virus type 3 (HPIV3), induced down-regulation of p53 in infected cells. Double-stranded RNA (dsRNA) and a mutant vaccinia virus lacking the dsRNA binding protein E3L can also induce this effect, indicating that dsRNA formed during viral infection is likely the trigger for down-regulation of p53. The mechanism of down-regulation of p53 by dsRNA relies on translation inhibition mediated by the PKR and RNase L pathways. In the absence of p53, the replication of both EMCV and HPIV3 was retarded, whereas, conversely, VSV replication was enhanced. Cell cycle analysis indicated that wild-type (WT) but not p53 knockout (KO) fibroblasts undergo an early-G1 arrest following dsRNA treatment. Moreover, in WT cells the onset of dsRNA-induced apoptosis begins after p53 levels are down-regulated, whereas p53 KO cells, which lack the early-G1 arrest, rapidly undergo apoptosis. Hence, our data suggest that the down-regulation of p53 facilitates apoptosis, thereby limiting viral replication.
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RNA interference (RNAi) is an evolutionarily conserved sequence-specific post-transcriptional gene silencing mechanism that is well defined genetically in Caenorhabditis elegans. RNAi has been postulated to function as an adaptive antiviral immune mechanism in the worm, but there is no experimental evidence for this. Part of the limitation is that there are no known natural viral pathogens of C. elegans. Here we describe an infection model in C. elegans using the mammalian pathogen vesicular stomatitis virus (VSV) to study the role of RNAi in antiviral immunity. VSV infection is potentiated in cells derived from RNAi-defective worm mutants (rde-1; rde-4), leading to the production of infectious progeny virus, and is inhibited in mutants with an enhanced RNAi response (rrf-3; eri-1). Because the RNAi response occurs in the absence of exogenously added VSV small interfering RNAs, these results show that RNAi is activated during VSV infection and that RNAi is a genuine antiviral immune defence mechanism in the worm.
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The worm Caenorhabditis elegans is a model system for studying many aspects of biology, including host responses to bacterial pathogens, but it is not known to support replication of any virus. Plants and insects encode multiple Dicer enzymes that recognize distinct precursors of small RNAs and may act cooperatively. However, it is not known whether the single Dicer of worms and mammals is able to initiate the small RNA-guided RNA interference (RNAi) antiviral immunity as occurs in plants and insects. Here we show complete replication of the Flock house virus (FHV) bipartite, plus-strand RNA genome in C. elegans. We show that FHV replication in C. elegans triggers potent antiviral silencing that requires RDE-1, an Argonaute protein essential for RNAi mediated by small interfering RNAs (siRNAs) but not by microRNAs. This immunity system is capable of rapid virus clearance in the absence of FHV B2 protein, which acts as a broad-spectrum RNAi inhibitor upstream of rde-1 by targeting the siRNA precursor. This work establishes a C. elegans model for genetic studies of animal virus-host interactions and indicates that mammals might use a siRNA pathway as an antiviral response.
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Antisense transcription (transcription from the opposite strand to a protein-coding or sense strand) has been ascribed roles in gene regulation involving degradation of the corresponding sense transcripts (RNA interference), as well as gene silencing at the chromatin level. Global transcriptome analysis provides evidence that a large proportion of the genome can produce transcripts from both strands, and that antisense transcripts commonly link neighboring “genes” in complex loci into chains of linked transcriptional units. Expression profiling reveals frequent concordant regulation of sense/antisense pairs. We present experimental evidence that perturbation of an antisense RNA can alter the expression of sense messenger RNAs, suggesting that antisense transcription contributes to control of transcriptional outputs in mammals.
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Dicer is a key enzyme involved in RNA interference (RNAi) and microRNA (miRNA) pathways. It is required for biogenesis of miRNAs and small interfering RNAs (siRNAs), and also has a role in the effector steps of RNA silencing. Apart from Argonautes, no proteins are known to associate with Dicer in mammalian cells. In this work, we describe the identification of TRBP (human immunodeficiency virus (HIV-1) transactivating response (TAR) RNA-binding protein) as a protein partner of human Dicer. We show that TRBP is required for optimal RNA silencing mediated by siRNAs and endogenous miRNAs, and that it facilitates cleavage of pre-miRNA in vitro. TRBP had previously been assigned several functions, including inhibition of the interferon-induced double-stranded RNA-regulated protein kinase PKR and modulation of HIV-1 gene expression by association with TAR. The TRBP-Dicer interaction shown raises interesting questions about the potential interplay between RNAi and interferon-PKR pathways.
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As a counter-defense against antiviral RNA silencing during infection, the insect Flock House virus (FHV) expresses the silencing suppressor protein B2. Biochemical experiments show that B2 binds to double-stranded RNA (dsRNA) without regard to length and inhibits cleavage of dsRNA by Dicer in vitro. A cocrystal structure reveals that a B2 dimer forms a four-helix bundle that binds to one face of an A-form RNA duplex independently of sequence. These results suggest that B2 blocks both cleavage of the FHV genome by Dicer and incorporation of FHV small interfering RNAs into the RNA-induced silencing complex.
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RNA interference (RNAi) is a sequence-specific gene-silencing mechanism triggered by exogenous dsRNA. In plants an RNAi-like mechanism defends against viruses, but the hypothesis that animals possess a similar natural antiviral mechanism related to RNAi remains relatively untested. To test whether genes needed for RNAi defend animal cells against virus infection, we infected wild-type and RNAi-defective cells of the nematode C. elegans with vesicular stomatitis virus engineered to encode a GFP fusion protein. We show that upon infection, cells lacking components of the RNAi apparatus produce more GFP and infective particles than wild-type cells. Furthermore, we show that mutant cells with enhanced RNAi produce less GFP. Our observation that multiple genes required for RNAi are also required for resistance to vesicular stomatitis virus suggests that the RNAi machinery functions in resistance to viruses in nature. • vesicular stomatitis virus • virus
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A vaccinia virus-encoded double-stranded RNA-binding protein, p25, has been previously implicated in inhibition of the interferon-induced, double-stranded RNA-activated protein kinase. In this study, we have identified the vaccinia viral gene (WR strain) that encodes p25. Amino acid sequence analysis of a chymotryptic fragment of p25 revealed a close match to the vaccinia virus (Copenhagen strain) E3L gene. The WR strain E3L gene was cloned and expressed either in COS-1 cells or in rabbit reticulocyte lysates in vitro. A M(r) 25,000 polypeptide that could bind to poly(rI).poly(rC)-agarose and that reacted with p25-specific antiserum was produced in each case. In addition, COS cells expressing E3L gene products inhibited activation of the double-stranded RNA-activated protein kinase in extracts from interferon-treated cells. Removal of E3L-encoded products by adsorption with anti-p25 antiserum resulted in loss of kinase inhibitory activity. These results demonstrate that the vaccinia virus E3L gene encodes p25 and that the products of the E3L gene have kinase inhibitory activity. Comparison of the deduced amino acid sequence of the E3L gene products with the protein sequence data base revealed a region closely related to the human interferon-induced, double-stranded RNA-activated protein kinase.
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A phylogenetic analysis of mammalian type I interferon (IFN) genes showed: (1) that the three main subfamilies of these genes in mammals (IFN-beta, IFN-alpha, and IFN-omega) diverged after the divergence of birds and mammals but before radiation of the eutherian orders and (2) that IFN-beta diverged first. Although apparent cases of interlocus recombination among mouse IFN-alpha genes were identified, the hypothesis that coding regions of IFN-alpha genes have been homogenized within species by interlocus recombination was not supported. Flanking regions as well as coding regions of IFN-alpha were more similar within human and mouse than between these species; and reconstruction of the pattern of nucleotide substitution in IFN-alpha coding regions of four mammalian species by the maximum parsimony method suggested that parallel substitutions have occurred far more frequently between species than within species. Therefore, it seems likely that IFN-alpha genes have duplicated independently within different eutherian orders. In general, type I IFN genes are subject to purifying selection, which in the case of IFN-alpha and IFN-beta is strongest in the putative receptor-binding domains. However, analysis of the pattern of nucleotide substitution among IFN-omega genes suggested that positive Darwinian selection may have acted in some cases to diversify members of this subfamily at the amino acid level.
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