Double-stranded RNA induces sequence-specific antiviral silencing in addition to nonspecific immunity in a marine shrimp: convergence of RNA interference and innate immunity in the invertebrate antiviral response?

Double-stranded RNA (dsRNA) is a common by-product of viral infections and a potent inducer of innate antiviral immune responses in vertebrates. In the marine shrimp Litopenaeus vannamei, innate antiviral immunity is also induced by dsRNA in a sequence-independent manner. In this study, the hypothesis that dsRNA can evoke not only innate antiviral immunity but also a sequence-specific antiviral response in shrimp was tested. It was found that viral sequence-specific dsRNA affords potent antiviral immunity in vivo, implying the involvement of RNA interference (RNAi)-like mechanisms in the antiviral response of the shrimp. Consistent with the activation of RNAi by virus-specific dsRNA, endogenous shrimp genes could be silenced in a systemic fashion by the administration of cognate long dsRNA. While innate antiviral immunity, sequence-dependent antiviral protection, and gene silencing could all be induced by injection of long dsRNA molecules, injection of short interfering RNAs failed to induce similar responses, suggesting a size requirement for extracellular dsRNA to engage antiviral mechanisms and gene silencing. We propose a model of antiviral immunity in shrimp by which viral dsRNA engages not only innate immune pathways but also an RNAi-like mechanism to induce potent antiviral responses in vivo.     

Characterization of Aedes aegypti Innate-Immune Pathways that Limit Chikungunya Virus Replication

Replication of arboviruses in their arthropod vectors is controlled by innate immune responses. The RNA sequence-specific break down mechanism, RNA interference (RNAi), has been shown to be an important innate antiviral response in mosquitoes. In addition, immune signaling pathways have been reported to mediate arbovirus infections in mosquitoes; namely the JAK/STAT, immune deficiency (IMD) and Toll pathways. Very little is known about these pathways in response to chikungunya virus (CHIKV) infection, a mosquito-borne alphavirus (Togaviridae) transmitted by aedine species to humans resulting in a febrile and arthralgic disease. In this study, the contribution of several innate immune responses to control CHIKV replication was investigated. In vitro experiments identified the RNAi pathway as a key antiviral pathway. CHIKV was shown to repress the activity of the Toll signaling pathway in vitro but neither JAK/STAT, IMD nor Toll pathways were found to mediate antiviral activities. In vivo data further confirmed our in vitro identification of the vital role of RNAi in antiviral defence. Taken together these results indicate a complex interaction between CHIKV replication and mosquito innate immune responses and demonstrate similarities as well as differences in the control of alphaviruses and other arboviruses by mosquito immune pathways.     

Natural selection drives extremely rapid evolution in antiviral RNAi genes

RNA interference (RNAi) is perhaps best known as a laboratory tool. However, RNAi-related pathways represent an antiviral component of innate immunity in both plants and animals. Since viruses can protect themselves by suppressing RNAi, interaction between RNA viruses and host RNAi may represent an ancient coevolutionary "arms race." This could lead to strong directional selection on RNAi genes, but to date their evolution has not been studied. By comparing DNA sequences from different species of Drosophila, we show that the rate of amino acid evolution is substantially elevated in genes related to antiviral RNAi function (Dcr2, R2D2, and Ago2). They are among the fastest evolving 3% of all Drosophila genes; they evolve significantly faster than other components of innate immunity and faster than paralogous genes that mediate "housekeeping" functions. Based on DNA polymorphism data from three species of Drosophila, McDonald-Kreitman tests showed that this rapid evolution is due to strong positive selection. Furthermore, Dcr2 and Ago2 display reduced genetic diversity, indicative of a recent selective sweep in both genes. Together, these data show rapid adaptive evolution of the antiviral RNAi pathway in Drosophila. This is a signature of host-pathogen arms races and implies that the ancient battle between RNA viruses and host antiviral RNAi genes is active and significant in shaping RNAi function.     

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

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.     

Immune responses to dsRNA: implications for gene silencing technologies

Nucleic acid-induced gene silencing, such as RNA interference (RNAi), induces a multitude of responses in addition to the knockdown of a gene. This is best understood in the context of the antiviral immune response, from which the processes of RNAi are thought to be derived. Viral challenge of a vertebrate host leads to an intricate series of responses that orchestrate antiviral immunity. The success of this multifaceted system in overcoming viral encounters hinges on complex pathogen-host interactions. One aspect of these interactions, the nucleic acid-based immune response, is key to the successful resolution of a viral challenge. In particular, dsRNA, a nucleic acid associated with viral replication, is involved in numerous interactions contributing to induction, activation and regulation of antiviral mechanisms. Specifically, dsRNA is responsible for stimulating important protective responses, such as the activation of dicer-related antiviral pathways, induction of type 1 IFN, and stimulation of dsRNA-activated protein kinase and oligoadenylate synthetase. Furthermore, the modulation and shaping of this overall immune response is facilitated through nucleic acid interactions with pattern recognition receptors such as toll-like receptor 3. These diverse dsRNA-induced antiviral responses have implications for biotechnologies that use dsRNA to harness one arm of the host antiviral machinery for silencing a specific target gene. The interlinked nature of these response elements means that it may be difficult to completely isolate one element from the other arms of the antiviral response program of an organism. Thus, it is beneficial to understand all aspects of the immune response to dsRNA in order to manipulate these systems and minimize unwanted non-specific effects.     

Efficient Dicer processing of virus-derived double-stranded RNAs and its modulation by RIG-I-like receptor LGP2

The interferon-regulated antiviral responses are essential for the induction of both innate and adaptive immunity in mammals. Production of virus-derived small-interfering RNAs (vsiRNAs) to restrict virus infection by RNA interference (RNAi) is a recently identified mammalian immune response to several RNA viruses, which cause important human diseases such as influenza and Zika virus. However, little is known about Dicer processing of viral double-stranded RNA replicative intermediates (dsRNA-vRIs) in mammalian somatic cells. Here we show that infected somatic cells produced more influenza vsiRNAs than cellular microRNAs when both were produced by human Dicer expressed de novo, indicating that dsRNA-vRIs are not poor Dicer substrates as previously proposed according to in vitro Dicer processing of synthetic long dsRNA. We report the first evidence both for canonical vsiRNA production during wild-type Nodamura virus infection and direct vsiRNA sequestration by its RNAi suppressor protein B2 in two strains of suckling mice. Moreover, Sindbis virus (SINV) accumulation in vivo was decreased by prior production of SINV-targeting vsiRNAs triggered by infection and increased by heterologous expression of B2 in cis from SINV genome, indicating an antiviral function for the induced RNAi response. These findings reveal that unlike artificial long dsRNA, dsRNA-vRIs made during authentic infection of mature somatic cells are efficiently processed by Dicer into vsiRNAs to direct antiviral RNAi. Interestingly, Dicer processing of dsRNA-vRIs into vsiRNAs was inhibited by LGP2 (laboratory of genetics and physiology 2), which was encoded by an interferon-stimulated gene (ISG) shown recently to inhibit Dicer processing of artificial long dsRNA in cell culture. Our work thus further suggests negative modulation of antiviral RNAi by a known ISG from the interferon response.     

The Ebola virus VP35 protein is a suppressor of RNA silencing

RNA silencing or interference (RNAi) is a gene regulation mechanism in eukaryotes that controls cell differentiation and developmental processes via expression of microRNAs. RNAi also serves as an innate antiviral defence response in plants, nematodes, and insects. This antiviral response is triggered by virus-specific double-stranded RNA molecules (dsRNAs) that are produced during infection. To overcome antiviral RNAi responses, many plant and insect viruses encode RNA silencing suppressors (RSSs) that enable them to replicate at higher titers. Recently, several human viruses were shown to encode RSSs, suggesting that RNAi also serves as an innate defence response in mammals. Here, we demonstrate that the Ebola virus VP35 protein is a suppressor of RNAi in mammalian cells and that its RSS activity is functionally equivalent to that of the HIV-1 Tat protein. We show that VP35 can replace HIV-1 Tat and thereby support the replication of a Tat-minus HIV-1 variant. The VP35 dsRNA-binding domain is required for this RSS activity. Vaccinia virus E3L protein and influenza A virus NS1 protein are also capable of replacing the HIV-1 Tat RSS function. These findings support the hypothesis that RNAi is part of the innate antiviral response in mammalian cells. Moreover, the results indicate that RSSs play a critical role in mammalian virus replication.     

How to kill a mocking bug?

All metazoans have evolved means to protect themselves from threats present in the environment: injuries, viruses, fungi, bacteria and other parasites. Insect protection includes innate physical barriers and both cellular and humoral responses. The insect innate immune response, best characterized in Drosophila melanogaster, is a rapid broad response, triggered by pathogen-associated molecular patterns (PAMPs) recognition, which produces a limited range of effectors that does not alter upon continued pathogen exposure and lacks immunological memory. The Drosophila response, particularly its humoral response, has been investigated by both low and high-throughput methods. Three signalling pathways conserved between insects and mammals have been implicated in this response: Toll (equivalent to mammalian TLR), Imd (equivalent to TNFalpha) and Hop (equivalent to JAK/STAT). This review provides an entry point to the insect immune system literature outlining the main themes in D. melanogaster bacterial pathogen detection and humoral and cellular immune responses. The Drosophila immune response is compared with other insects and the mammalian immune system.     

The circular RNA circBCL2L1 regulates innate immune responses via microRNA-mediated downregulation of TRAF6 in teleost fish

Growing numbers of studies have shown that circular RNAs (circRNAs) can function as regulatory factors to regulate the innate immune response, cell proliferation, cell migration, and other important processes in mammals. However, the function and regulatory mechanism of circRNAs in lower vertebrates are still unclear. Here, we discovered a novel circRNA derived from the gene encoding Bcl-2-like protein 1 (BCL2L1) gene, named circBCL2L1, which was related to the innate immune responses in teleost fish. Results indicated that circBCL2L1 played essential roles in host antiviral immunity and antibacterial immunity. Our study also identified a microRNA, miR-30c-3-3p, which could inhibit the innate immune response by targeting inflammatory mediator TRAF6. And TRAF6 is a key signal transduction factor in innate immune response mediated by TLRs. Moreover, we also found that the antiviral and antibacterial effects inhibited by miR-30c-3-3p could be reversed with the expression of circBCL2L1. Our data revealed that circBCL2L1 functioned as a competing endogenous RNA (ceRNA) of TRAF6 by competing for binding with miR-30c-3-3p, leading to activation of the NF-κB/IRF3 inflammatory pathway and then enhancing the innate immune responses. Our results suggest that circRNAs can play an important role in the innate immune response of teleost fish.     

The Imd Pathway Is Involved in Antiviral Immune Responses in Drosophila

Cricket Paralysis virus (CrPV) is a member of the Dicistroviridae family of RNA viruses, which infect a broad range of insect hosts, including the fruit fly Drosophila melanogaster. Drosophila has emerged as an effective system for studying innate immunity because of its powerful genetic techniques and the high degree of gene and pathway conservation. Intra-abdominal injection of CrPV into adult flies causes a lethal infection that provides a robust assay for the identification of mutants with altered sensitivity to viral infection. To gain insight into the interactions between viruses and the innate immune system, we injected wild type flies with CrPV and observed that antimicrobial peptides (AMPs) were not induced and hemocytes were depleted in the course of infection. To investigate the contribution of conserved immune signaling pathways to antiviral innate immune responses, CrPV was injected into isogenic mutants of the Immune Deficiency (Imd) pathway, which resembles the mammalian Tumor Necrosis Factor Receptor (TNFR) pathway. Loss-of-function mutations in several Imd pathway genes displayed increased sensitivity to CrPV infection and higher CrPV loads. Our data show that antiviral innate immune responses in flies infected with CrPV depend upon hemocytes and signaling through the Imd pathway.     

Establishment of ex vivo systems to identify compounds acting on innate immune responses and to determine their target molecules using transgenic Drosophila

Innate immunity is an evolutionarily conserved self-defense mechanism against microbial infections. In Drosophila, induction of antimicrobial peptides is a major immune response that is regulated by two distinct signaling pathways called the IMD pathway and the Toll pathway, similar to the tumor necrosis factor-alpha signaling and Toll-like receptor/interleukin-1 signaling pathways, respectively, in mammals. In mammals, innate immunity interacts with adaptive immunity and has a key role in the regulated immune response. Therefore, innate immunity is a pharmaceutical target for the development of immune regulators. Previously, based on the striking conservation between the mechanisms that regulate Drosophila immunity and human innate immunity, we established an ex vivo culture in which compounds acting on innate immunity can be evaluated using a reporter gene that reflects activation of the IMD pathway [Yajima et al. [Yajima, M., Takada, M., Takahashi, N., Kikuchi, H., Natori, S., Oshima, Y., Kurata, S., 2003. A newly established in vitro culture using transgenic Drosophila reveals functional coupling between the phospholipase A2-generated fatty acid cascade and lipopolysaccharide-dependent activation of the immune deficiency (imd) pathway in insect immunity. The Biochemical Journal 371(Pt 1), 205-210] Biochem J 371, 205-210]. Here, we combined the ex vivo culture with a reporter gene that reflects the heat shock response and demonstrated that the resulting systems are useful for screening compounds that act specifically on innate immunity, including mammalian innate immune responses. Identification of target molecules is essential for the development of more potent medicines with fewer side effects. In this study, we also established ex vivo systems capable of identifying target molecules of the identified compounds using targeted activation of the IMD pathway.     

病毒siRNA介导的RNA干扰途径与机制

RNA干扰(RNAi)是真核生物体内重要的基因表达调控方式之一.RNAi的一种原始的作用是帮助生物体抵抗病毒,早期的研究表明无脊椎动物可以利用RNAi抵抗病毒,但是哺乳动物是否存在这一机制一直存在争议.最新的研究发现了哺乳动物RNAi抗病毒的强有力的证据,并且研究人员认为,这是一种之前被忽视的、全新的免疫途径.值得注意...     

Is innate enough? The innate immune response in Drosophila

In recent years, the innate immune system has emerged from the shadow of adaptive immune responses as a major area of research in its own right. One of the most significant model systems that has been used to investigate this phenomenon has been the fruit fly, Drosophila melanogaster. Exploration of the differential immune response presented by Drosophila led to the discovery of important signalling events and transduction pathways, which were thereafter shown to be specific for the type of infecting pathogen. These factors and pathways were subsequently found to have homologues in many other organisms, including those with adaptive immune responses. In light of the present status of studies in innate immunity, this review describes the current state of understanding of the Drosophila immune response.     

The endocytic pathway mediates cell entry of dsRNA to induce RNAi silencing

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