An Accessory to the ‘Trinity’: SR-As Are Essential Pathogen Sensors of Extracellular dsRNA,Mediating Entry and Leading to Subsequent Type I IFN Responses

Extracellular RNA is becoming increasingly recognized as a signaling molecule. Virally derived double stranded (ds)RNA released into the extracellular space during virus induced cell lysis acts as a powerful inducer of classical type I interferon (IFN) responses; however, the receptor that mediates this response has not been identified. Class A scavenger receptors (SR-As) are likely candidates due to their cell surface expression and ability to bind nucleic acids. In this study, we investigated a possible role for SR-As in mediating type I IFN responses induced by extracellular dsRNA in fibroblasts, a predominant producer of IFNβ. Fibroblasts were found to express functional SR-As, even SR-A species thought to be macrophage specific. SR-A specific competitive ligands significantly blocked extracellular dsRNA binding, entry and subsequent interferon stimulated gene (ISG) induction. Candidate SR-As were systematically investigated using RNAi and the most dramatic inhibition in responses was observed when all candidate SR-As were knocked down in unison. Partial inhibition of dsRNA induced antiviral responses was observed in vivo in SR-AI/II^-/- mice compared with WT controls. The role of SR-As in mediating extracellular dsRNA entry and subsequent induced antiviral responses was observed in both murine and human fibroblasts. SR-As appear to function as ‘carriers’, facilitating dsRNA entry and delivery to the established dsRNA sensing receptors, specifically TLR3, RIGI and MDA-5. Identifying SR-As as gatekeepers of the cell, mediating innate antiviral responses, represents a novel function for this receptor family and provides insight into how cells recognize danger signals associated with lytic virus infections. Furthermore, the implications of a cell surface receptor capable of recognizing extracellular RNA may exceed beyond viral immunity to mediating other important innate immune functions.     

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.     

Cytosolic RNA:DNA hybrids activate the cGAS–STING axis

Intracellular recognition of non‐self and also self‐nucleic acids can result in the initiation of potent pro‐inflammatory and antiviral cytokine responses. Most recently, cGAS was shown to be critical for the recognition of cytoplasmic dsDNA. Binding of dsDNA to cGAS results in the synthesis of cGAMP(2′–5′), which then binds to the endoplasmic reticulum resident protein STING. This initiates a signaling cascade that triggers the induction of an antiviral immune response. While most studies on intracellular nucleic acids have focused on dsRNA or dsDNA, it has remained unexplored whether cytosolic RNA:DNA hybrids are also sensed by the innate immune system. Studying synthetic RNA:DNA hybrids, we indeed observed a strong type I interferon response upon cytosolic delivery of this class of molecule. Studies in THP‐1 knockout cells revealed that the recognition of RNA:DNA hybrids is completely attributable to the cGAS–STING pathway. Moreover, in vitro studies showed that recombinant cGAS produced cGAMP upon RNA:DNA hybrid recognition. Altogether, our results introduce RNA:DNA hybrids as a novel class of intracellular PAMP molecules and describe an alternative cGAS ligand next to dsDNA.     

The immune responses in Chinese mitten crab Eriocheir sinensis challenged with double-stranded RNA

Double-stranded RNA (dsRNA) is a virus-associated molecular pattern which induces antiviral innate immune responses and RNA interference (RNAi) in mammals. In invertebrates, RNAi phenomenon has been widely studied, but dsRNA-induced innate immune response is seldom reported. In the present study, two different dsRNAs specific for green fluorescent protein (GFP) and the putative D1 protein of photosystem II (NoPSD) from Nannochloropsis oculata, were employed to challenge Chinese mitten crab Eriocheir sinensis. The temporal changes of phenoloxidase (PO), acid phosphatase (ACP), superoxide dismutase (SOD) and malondialdehyde (MDA) content, as well as the mRNA expression of some immune-related genes were examined in order to estimate the effect of dsRNAs on the innate immunity of E. sinensis. The activities of PO, ACP and SOD significantly increased after dsRNA treatment, whereas malondialdehyde (MDA) content did not change significantly. Among the examined genes, only the mRNA expression of EsALF, an antibacterial peptide in E. sinensis, was significantly up-regulated (about 5 fold, P < 0.05) at 12 h after dsRNA treatment, while no significant expression changes were observed among the other immune genes. The increase of PO, ACP and SOD activities, and mRNA expression level of EsALF after dsRNA stimulation indicate that phenoloxidase, hydrolytic enzyme, antioxidation and EsALF were involved in dsRNA-induced innate immunity, suggesting that broad-spectrum immune responses could be induced by dsRNA in E. sinensis.     

The mitochondrial antiviral signaling protein, MAVS, is cleaved during apoptosis

Apoptosis of virus-infected cells is one important host strategy used to limit viral infection. Recently a member of the innate immune signaling pathway, MAVS, was localized to mitochondria, an organelle important for apoptosis regulation. Here we investigate what role MAVS may play in apoptosis. Induction of cell death led to the rapid cleavage of MAVS, resulting in its release from the outer mitochondrial membrane. This cleavage is blocked in cells incubated with proteasome or caspase inhibitors. Transfection of synthetic viral dsRNA and dsDNA also led to cleavage of MAVS, indicating that this process may be important during infection. Preventing apoptosis by over-expression of anti-apoptotic Bcl-xL blocks MAVS cleavage, placing this process downstream of caspase activation in the apoptotic program.     

Interplay between Kaposi's sarcoma-associated herpesvirus and the innate immune system

Understanding of the innate immune response to viral infections is rapidly progressing, especially with regards to the detection of DNA viruses. Kaposi's sarcoma-associated herpesvirus (KSHV) is a large dsDNA virus that is responsible for three human diseases: Kaposi's sarcoma, primary effusion lymphoma and multicentric Castleman's disease. The major target cells of KSHV (B cells and endothelial cells) express a wide range of pattern recognition receptors (PRRs) and play a central role in mobilizing inflammatory responses. On the other hand, KSHV encodes an array of immune evasion genes, including several pirated host genes, which interfere with multiple aspects of the immune response. This review summarizes current understanding of innate immune recognition of KSHV and the role of immune evasion genes that shape the antiviral and inflammatory responses.     

Interferon-beta induction through toll-like receptor 3 depends on double-stranded RNA structure

Type I interferons (IFN-alpha/beta) play an essential role in both innate and adaptive antiviral immune responses. IFN- beta is produced by fibroblasts and myeloid dendritic cells (DCs) upon viral infection or in response to doublestranded RNA (dsRNA). Several intracellular molecules having a dsRNA-binding motif such as dsRNA-dependent protein kinase recognize dsRNA in a sequence-independent manner and induce antiviral innate responses. Toll-like receptor (TLR) 3, a member of TLR family proteins, recognizes extracellular dsRNA and activates NF- kappaB and the IFN-beta promoter leading to the induction of IFN-beta production. Here we analyzed the dsRNA structure capable of inducing TLR3-mediated IFN-beta production using various synthetic RNA duplexes. In contrast to the recognition of dsRNA by intracellular molecules, TLR3 preferentially recognizes polyriboinocinic:polyribocytidylic acid (poly(I:C)) rather than synthetic virus-derived dsRNAs. 2'-O-methyl or 2'-fluoro modification of cytidylic acid abolished the IFN-beta-inducing ability of the poly(I:C) duplex, and these modified dsRNAs inhibited poly(I:C)-induced TLR3-mediated IFN-beta production by fibroblasts and DCs. In addition, poly(dI:dC), a non-IFN inducer, also blocked poly(I:C)-induced IFN-beta induction. Since TLR3 is localized in the intracellular compartment of DCs where signaling occurs, modified dsRNAs may compete with poly(I:C) for binding to the cell-surface receptor that transfers dsRNA into TLR3-enriched vesicles. Thus, TLR3 recognizes a unique dsRNA structure that largely differs from those recognized by other dsRNA-binding proteins.     

RNAi is an antiviral immune response against a dsRNA virus in Drosophila melanogaster

Drosophila melanogaster has a robust and efficient innate immune system, which reacts to infections ranging from bacteria to fungi and, as discovered recently, viruses as well. The known Drosophila immune responses rely on humoral and cellular activities, similar to those found in the innate immune system of other animals. Recently, RNAi or 'RNA silencing' has arisen as a possible means by which Drosophila can react to a specific pathogens, transposons and retroviral elements, in a fashion similar to that of a traditional mammalian adaptive immune system instead of in a more generalized and genome encoded innate immune-based response. RNAi is a highly conserved regulation and defence mechanism, which suppresses gene expression via targeted RNA degradation directed by either exogenous dsRNA (cleaved into siRNAs) or endogenous miRNAs. In plants, RNAi has been found to act as an antiviral immune response system. Here we show that RNAi is an antiviral response used by Drosophila to combat infection by Drosophila X Virus, a birnavirus, as well. Additionally, we identify multiple core RNAi pathway genes, including piwi, vasa intronic gene (vig), aubergine (aub), armitage (armi), Rm62, r2d2 and Argonaute2 (AGO2) as having vital roles in this response in whole organisms. Our findings establish Drosophila as an ideal model for the study of antiviral RNAi responses in animals.     

Fas-associated death domain-containing protein-mediated antiviral innate immune signaling involves the regulation of Irf7

The induction of type I (alphabeta) IFN following virus infection is necessary for the stimulation of effective antiviral host defense. In fibroblasts, a subset of primary genes (including those encoding IFN-beta and IFN-alpha4) are induced directly by intracellular dsRNA generated by the virus during its replication. These primary type I IFNs induce expression of IFN regulatory factor (IRF)-7, required for production of a second cascade of IFN-alpha subtypes and the further establishment of a complete antiviral state. Previously, we had reported on a role for Fas-associated death domain-containing protein (FADD) in the control of TLR-independent innate immune responses to virus infection. Our data in this study demonstrate that FADD is not only required for efficient primary gene induction, but is also essential for induction of Irf7 and effective expression of secondary IFN-alphas and other antiviral genes. Ectopic overexpression of IRF-7 partially rescued dsRNA responsiveness and IFN-alpha production, and a constitutively active variant of IRF-7 displayed normal activity in Fadd(-/-) murine embryonic fibroblasts. MC159, a FADD-interacting viral protein encoded by the molluscum contagiosum poxvirus was found to inhibit dsRNA-activated signaling events upstream of IRF-7. These data indicate that FADD's antiviral activity involves regulation of IRF-7-dependent production of IFN-alpha subtypes and consequent induction of secondary antiviral genes.     

Mitochondria in innate immune responses

The innate immune system has a key role in the mammalian immune response. Recent research has demonstrated that mitochondria participate in a broad range of innate immune pathways, functioning as signalling platforms and contributing to effector responses. In addition to regulating antiviral signalling, mounting evidence suggests that mitochondria facilitate antibacterial immunity by generating reactive oxygen species and contribute to innate immune activation following cellular damage and stress. Therefore, in addition to their well-appreciated roles in cellular metabolism and programmed cell death, mitochondria appear to function as centrally positioned hubs in the innate immune system. Here, we review the emerging knowledge about the roles of mitochondria in innate immunity.     

Super-induction of Dicer-2 expression by alien double-stranded RNAs: an evolutionary ancient response to viral infection?

Dicer-2 is a ribonuclease involved in the insect RNAi pathway. On attempting to knockdown Dicer-2 expression in the insect Blattella germanica by RNAi, we found that treatment with Dicer-2 dsRNA upregulated the targeted mRNA. This unexpected result was also observed after treating with a nucleopolyhedrovirus dsRNA. Experiments with this alien dsRNA showed an all-or-none response with a threshold for inducing Dicer-2 upregulation between 0.4 and 0.04 μg in terms of dsRNA concentration and between 50 and 20 bp in terms of dsRNA length. The response seems specific of dsRNA given that equivalent experiments carried out with dsDNA did not affect Dicer-2 expression. In insects, Dicer-2 is postulated to be a sensor of viral infections and a key antiviral defense element. The upregulation of Dicer-2 expression after dsRNA administration fits well with this sensor role, and the occurrence of this mechanism in B. germanica, a phylogenetically basal insect, suggests that sensing alien RNAs might be an ancestral function of Dicer-2 proteins.     

Innate immune responses in human monocyte-derived dendritic cells are highly dependent on the size and the 5' phosphorylation of RNA molecules

Recognition of viral genetic material takes place via several different receptor systems, such as retinoic acid-inducible gene I-like receptors and TLRs 3, 7, 8, and 9. At present, systematic comparison of the ability of different types of RNAs to induce innate immune responses in human immune cells has been limited. In this study, we generated bacteriophage 6 and influenza A virus-specific ssRNA and dsRNA molecules ranging from 58 to 2956 nt. In human monocyte-derived dendritic cells (moDCs), short dsRNAs efficiently upregulated the expression of IFN (IFN-α, IFN-β, and IFN-λ1) and proinflammatory (TNF-α, IL-6, IL-12, and CXCL10) cytokine genes. These genes were also induced by ssRNA molecules, but size-specific differences were not as pronounced as with dsRNA molecules. Dephosphorylation of short ssRNA and dsRNA molecules led to a dramatic reduction in their ability to stimulate innate immune responses. Such a difference was not detected for long ssRNAs. RNA-induced cytokine responses correlated well with IFN regulatory factor 3 phosphorylation, suggesting that IFN regulatory factor 3 plays a major role in both ssRNA- and dsRNA-activated responses in human moDCs. We also found that IFN gene expression was efficiently stimulated following recognition of short dsRNAs by retinoic acid-inducible gene I and TLR3 in human embryonic kidney 293 cells, whereas ssRNA-induced responses were less dependent on the size of the RNA molecule. Our data suggest that human moDCs are extremely sensitive in recognizing foreign RNA, and the responses depend on RNA size, form (ssRNA versus dsRNA), and the level of 5' phosphorylation.