Zebrafish as an animal model for the antiviral RNA interference pathway
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ABSTRACT:
ORGANISM(S): Danio rerio
PROVIDER: GSE160475 | GEO | 2023/10/30
REPOSITORIES: GEO
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Project description:RNA interference (RNAi) functions as an antiviral immune response in plants and invertebrates, whereas mammalian RNAi response has been found so far only in undifferentiated cells and in differentiated cells inactive in interferon (IFN) system or in infections with viruses disabling viral suppressors of RNAi (VSRs), thereby leading to question the physiological importance of the RNAi pathway in mammals. Here, we identified that wild-type Semliki Forest virus (SFV), a prototypic alphavirus, triggered the Dicer-dependent production of abundant viral (v)siRNAs in different mammalian somatic cells in the presence of VSR. These vsiRNAs were produced from viral dsRNA replicative intermediates, almost exclusively located at the 5’ termini of the viral genome, and loaded into AGO, and they were fully active in slicing cognate viral RNAs. Besides, Sindbis virus, another alphavirus, also induced vsiRNA generation in mammalian somatic cells. AGO2 deficiency increased SFV and SINV replication, while enoxacin, a known RNAi enhancer that functions at post steps of siRNA production, efficiently reduced viral replication. The nucleotide sequence at the 5’ termini of SFV and SINV genome is conserved among the Old World alphaviruses, and mutating the conserved sequences resulted in the recombinant SFV being deficient in vsiRNA production and irresponsive to antiviral RNAi. SFV infection also enabled the production of abundant vsiRNAs and antiviral RNAi in IFN-competent adult mice, and importantly, enhanced RNAi by enoxacin protected adult mice from lethal SFV challenge and reduced the virus-induced neuropathogenesis in the central neuron system. Overall, our findings provide evidence that mammalian antiviral RNAi is active in differentiated cells and adult mice with intact IFN response even in the presence of VSR and present a therapeutic strategy against alphaviruses that include many important emerging and reemerging human pathogens.
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Project description:The function and mechanism of the interferon-regulated antiviral responses have been extensively characterized. Recent studies have demonstrated induction of antiviral RNA interference (RNAi) in somatic cells against several mammalian RNA viruses rendered incapable of RNAi suppression. However, little is known about Dicer-mediated production of virus-derived small-interfering RNAs (vsiRNAs) in these cells active in the type I interferon response. Here we show that the dsRNA precursors of influenza vsiRNAs were processed more efficiently than cellular precursor microRNA hairpins by wild-type human Dicer expressed de novo in Dicer-knockout somatic cells. We found that infection of two strains of suckling mice with wild-type Nodamura virus (NoV) was associated with production of silencing-active vsiRNAs and direct sequestration of duplex vsiRNAs by its RNAi suppressor protein B2. Our findings from in vivo infection with Sindbis virus recombinants expressing NoV B2 or carrying a vsiRNA-targeted insert provide evidence for an antiviral function of the induced RNAi response. Interestingly, NoV infection induces expression of RIG-I-like receptor LGP2 to inhibit vsiRNA biogenesis and promote virulent infection in suckling mice. Our findings together reveal efficient Dicer processing of vsiRNA precursors in interferon-competent somatic cells and suckling mice in contrast to synthetic long dsRNA examined previously by in vitro dicing.
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Project description:Small RNAs play a critical role in host-pathogen interaction. In insects, for instance, small RNA-mediated silencing or RNA interference (RNAi) represents the main antiviral defense system. However, the antiviral role of RNAi has not been clearly proven in higher vertebrates. On the contrary, it is well established that the cell response relies on the recognition of viral RNAs by host pattern recognition receptors (PRR) to trigger the activation of the interferon pathway. Based on this evidence, we wished to contribute to this research field by identifying and characterizing small non-coding RNAs produced in mammalian cells upon RNA virus infection. We focused on Sindbis virus (SINV), the prototypical arbovirus, which by definition, is able to infect both vertebrate hosts and invertebrate vectors and triggers the interferon pathway or RNAi, respectively. Taking advantage of large scale sequencing, we cloned and sequenced small RNAs from both mock and SINV-infected mammalian cells (HEK 293 and VERO). We identified a novel population of viral small RNAs (vsRNAs) that accumulate as 20 to 30 nt species during infection. We assessed that this viral small RNA population is modified in 3'end and derived from the activation of the cellular antiviral endoribonuclease RNaseL. Altogether our results indicate a potential role for the SINV-derived small RNAs in the host defense mechanism.
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Project description:Tick-borne diseases (TBDs) are the most common illnesses transmitted by ticks, and the annual number of reported TBD cases continues to increase. The Asian longhorned tick, a vector associated with at least 30 human pathogens, is native to eastern Asia and recently reached the USA as an emerging disease threat. Newly identified tick-transmitted pathogens continue to be reported, raising concerns about how TBDs occur. Interestingly, tick can harbor pathogens without being affected themselves. For viral infections, ticks have their own immune systems that protect them from infection. Meanwhile, tick-borne viruses have evolved to avoid these defenses as they establish themselves within the vector. Here, we show in detail that infecting longhorned ticks with distinct arthropod-borne RNA viruses through two approaches natural blood feeding and injection, all induce the production of vsiRNAs. Dicer2-like homolog plays a role in regulating antiviral RNAi responses as knocking down of this gene enhanced viral replication. Furthermore, we demonstrate that tick antiviral RNAi responses are inhibited through expression heterologous VSR proteins in recombinant SINV. We identify both the virus and tick factors are critical components to understanding TBDs. Importantly, our study introduces a novel, in vivo virus-vector-mouse model system for exploring TBDs in the future.
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Project description:Small RNAs play a critical role in host-pathogen interaction. In insects, for instance, small RNA-mediated silencing or RNA interference (RNAi) represents the main antiviral defense system. However, the antiviral role of RNAi has not been clearly proven in higher vertebrates. On the contrary, it is well established that the cell response relies on the recognition of viral RNAs by host pattern recognition receptors (PRR) to trigger the activation of the interferon pathway. Based on this evidence, we wished to contribute to this research field by identifying and characterizing small non-coding RNAs produced in mammalian cells upon RNA virus infection. We focused on Sindbis virus (SINV), the prototypical arbovirus, which by definition, is able to infect both vertebrate hosts and invertebrate vectors and triggers the interferon pathway or RNAi, respectively.
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Project description:Mosquito-borne flaviviruses maintain life cycles in mammals and mosquitoes. RNA interference (RNAi) has been demonstrated as an anti-flavivirus mechanism in mosquitoes; however, whether and how flavivirus induces and antagonizes RNAi-mediated antiviral immunity in mammals remains unknown. Here we showed that NS2A of Dengue virus-2 (DENV2) act as a viral suppressor of RNAi (VSR). When NS2A-mediated RNAi suppression was disabled, the resulting mutant DENV2 induced Dicer-dependent production of abundant DENV2-derived siRNAs in differentiated mammalian cells. Importantly, VSR-disabled DENV2 showed severe replication defects in mosquito and mammalian cells, and mice, which were rescued by the deficiency of RNAi. Moreover, NS2As of multiple flaviviruses act as VSRs in vitro and during viral infection in both organisms. Overall, our findings demonstrate that antiviral RNAi can be induced by flavivirus, while flavivirus uses NS2A as bona fide VSR to evade RNAi in mammals and mosquitoes, highlighting the importance of RNAi in flaviviral vector-host life cycles.
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