Double-stranded RNA is produced by positive-strand RNA viruses and DNA viruses but not in detectable amounts by negative-strand RNA viruses

Double-stranded RNA (dsRNA) longer than 30 bp is a key activator of the innate immune response against viral infections. It is widely assumed that the generation of dsRNA during genome replication is a trait shared by all viruses. However, to our knowledge, no study exists in which the production of dsRNA by different viruses is systematically investigated. Here, we investigated the presence and localization of dsRNA in cells infected with a range of viruses, employing a dsRNA-specific antibody for immunofluorescence analysis. Our data revealed that, as predicted, significant amounts of dsRNA can be detected for viruses with a genome consisting of positive-strand RNA, dsRNA, or DNA. Surprisingly, however, no dsRNA signals were detected for negative-strand RNA viruses. Thus, dsRNA is indeed a general feature of most virus groups, but negative-strand RNA viruses appear to be an exception to that rule.     

Specific cleavage of hyper-edited dsRNAs

Extended double-stranded DNA (dsRNA) duplexes can be hyper-edited by adenosine deaminases that act on RNA (ADARs). Long uninterrupted dsRNA is relatively uncommon in cells, and is frequently associated with infection by DNA or RNA viruses. Moreover, extensive adenosine to inosine editing has been reported for various viruses. A number of cellular antiviral defence strategies are stimulated by dsRNA. An additional mechanism to remove dsRNA from cells may involve hyper-editing of dsRNA by ADARs, followed by targeted cleavage. We describe here a cytoplasmic endonuclease activity that specifically cleaves hyper-edited dsRNA. Cleavage occurs at specific sites consisting of alternating IU and UI base pairs. In contrast, unmodified dsRNA and even deaminated dsRNAs that contain four consecutive IU base pairs are not cleaved. Moreover, dsRNAs in which alternating IU and UI base pairs are replaced by isomorphic GU and UG base pairs are not cleaved. Thus, the cleavage of deaminated dsRNA appears to require an RNA structure that is unique to hyper-edited RNA, providing a molecular target for the disposal of hyper-edited viral RNA.     

A phagocytic route for uptake of double-stranded RNA in RNAi

RNA interference (RNAi) has a range of physiological functions including as a defence mechanism against viruses. To protect uninfected cells in a multicellular organism, not only a cell-autonomous RNAi response is required but also a systemic one. However, the route of RNA spread in systemic RNAi remains unclear. Here we show that phagocytosis can be a route for double-stranded RNA uptake. Double-stranded RNA expressed in Escherichia coli induces robust RNAi in Drosophila S2 cells, with effectiveness comparable to that of naked dsRNA. We could separate this phagocytic uptake route from that for RNAi induced by naked dsRNA. Therefore, phagocytic uptake of dsRNA offers a potential route for systemic spread of RNAi.     

Viral RNA Intermediates as Targets for Detection and Discovery of Novel and Emerging Mosquito-Borne Viruses

Mosquito-borne viruses encompass a range of virus families, comprising a number of significant human pathogens (e.g., dengue viruses, West Nile virus, Chikungunya virus). Virulent strains of these viruses are continually evolving and expanding their geographic range, thus rapid and sensitive screening assays are required to detect emerging viruses and monitor their prevalence and spread in mosquito populations. Double-stranded RNA (dsRNA) is produced during the replication of many of these viruses as either an intermediate in RNA replication (e.g., flaviviruses, togaviruses) or the double-stranded RNA genome (e.g., reoviruses). Detection and discovery of novel viruses from field and clinical samples usually relies on recognition of antigens or nucleotide sequences conserved within a virus genus or family. However, due to the wide antigenic and genetic variation within and between viral families, many novel or divergent species can be overlooked by these approaches. We have developed two monoclonal antibodies (mAbs) which show co-localised staining with proteins involved in viral RNA replication in immunofluorescence assay (IFA), suggesting specific reactivity to viral dsRNA. By assessing binding against a panel of synthetic dsRNA molecules, we have shown that these mAbs recognise dsRNA greater than 30 base pairs in length in a sequence-independent manner. IFA and enzyme-linked immunosorbent assay (ELISA) were employed to demonstrate detection of a panel of RNA viruses from several families, in a range of cell types. These mAbs, termed monoclonal antibodies to viral RNA intermediates in cells (MAVRIC), have now been incorporated into a high-throughput, economical ELISA-based screening system for the detection and discovery of viruses from mosquito populations. Our results have demonstrated that this simple system enables the efficient detection and isolation of a range of known and novel viruses in cells inoculated with field-caught mosquito samples, and represents a rapid, sequence-independent, and cost-effective approach to virus discovery.     

Infectious Bursal Disease Virus: Ribonucleoprotein Complexes of a Double-Stranded RNA Virus

Genome-binding proteins with scaffolding and/or regulatory functions are common in living organisms and include histones in eukaryotic cells, histone-like proteins in some double-stranded DNA (dsDNA) viruses, and the nucleocapsid proteins of single-stranded RNA viruses. dsRNA viruses nevertheless lack these ribonucleoprotein (RNP) complexes and are characterized by sharing an icosahedral T = 2 core involved in the metabolism and insulation of the dsRNA genome. The birnaviruses, with a bipartite dsRNA genome, constitute a well-established exception and have a single-shelled T = 13 capsid only. Moreover, as in many negative single-stranded RNA viruses, the genomic dsRNA is bound to a nucleocapsid protein (VP3) and the RNA-dependent RNA polymerase (VPg). We used electron microscopy and functional analysis to characterize these RNP complexes of infectious bursal disease virus, the best characterized member of the Birnaviridae family. Mild disruption of viral particles revealed that VP3, the most abundant core protein, present at ∼ 450 copies per virion, is found in filamentous material tightly associated with the dsRNA. We developed a method to purify RNP and VPg-dsRNA complexes. Analysis of these complexes showed that they are linear molecules containing a constant amount of protein. Sensitivity assays to nucleases indicated that VP3 renders the genomic dsRNA less accessible for RNase III without introducing genome compaction. Additionally, we found that these RNP complexes are functionally competent for RNA synthesis in a capsid-independent manner, in contrast to most dsRNA viruses.     

Double-stranded RNA

Summary High molecular weight, fully double-stranded RNA (dsRNA) has been recognized as the genetic material of many plant, animal, fungal, and bacterial viruses (Diplomaviruses); virus-specific dsRNA is also found in cells infected with single-stranded RNA viruses.DsRNA has been identified in a variety of apparently normal eucaryotic cells and is associated with the killer character of certain strains of Saccaromyces cerevisiae.The properties and significance of these various dsRNA species are described and discussed, as well as the available information concerning the biosynthesis of such RNA in virus-infected cells, its degradation by a variety of enzymes, and some problems concerning the variables which may control this process.Finally, the biological functions of dsRNA are briefly considered, as well as the structural properties important for its activity as an inducer of interferon and an inhibitor of protein synthesis.Abbreviations dsRNA for double-stranded RNA - ssRNA for single-stranded RNA - SSC for 0.15 m sodium chloride, 0.015 m sodium citrate, pH 7 - Poly(A), poly(C), poly(U) for polyadenylate, polycytidylate and polyuridylate, respectively - Poly(A).poly(U), poly(G).poly(C), poly(I).poly(C) for double-stranded complexes formed between polyadenylate and polyuridylate, polyguanylate and polycytidylate, and polyinosinate and polycytidylate, respectively. - Poly(rA).poly(dT) for the complex formed between polyriboadenylate and polydeoxyribothymidylate - Poly(A-U), poly(G-C) for the alternating copolymers containing AMP and UMP, or GMP and CMP, respectively - Poly(rA).poly(dUz) for the complex formed between polyadenylate and poly 2-azido-2deoxyuridylate - (I)_n.(br⁵C)_n for the complex formed between polyinosinate and poly 5-bromocytidylate - (I)_n.(s²C)_n for the complex formed between polyinosinate and poly 2-thiocytidylate - (dI_n3)_n.(C)_n for the complex formed between poly 2-azido-2-deoxyinosinate and polycytidylate - MW for molecular weight     

Movement protein Pns6 of rice dwarf phytoreovirus has both ATPase and RNA binding activities

Cell-to-cell movement is essential for plant viruses to systemically infect host plants. Plant viruses encode movement proteins (MP) to facilitate such movement. Unlike the well-characterized MPs of DNA viruses and single-stranded RNA (ssRNA) viruses, knowledge of the functional mechanisms of MPs encoded by double-stranded RNA (dsRNA) viruses is very limited. In particular, many studied MPs of DNA and ssRNA viruses bind non-specifically ssRNAs, leading to models in which ribonucleoprotein complexes (RNPs) move from cell to cell. Thus, it will be of special interest to determine whether MPs of dsRNA viruses interact with genomic dsRNAs or their derivative sRNAs. To this end, we studied the biochemical functions of MP Pns6 of Rice dwarf phytoreovirus (RDV), a member of Phytoreovirus that contains a 12-segmented dsRNA genome. We report here that Pns6 binds both dsRNAs and ssRNAs. Intriguingly, Pns6 exhibits non-sequence specificity for dsRNA but shows preference for ssRNA sequences derived from the conserved genomic 5'- and 3'-terminal consensus sequences of RDV. Furthermore, Pns6 exhibits magnesium-dependent ATPase activities. Mutagenesis identified the RNA binding and ATPase activity sites of Pns6 at the N- and C-termini, respectively. Our results uncovered the novel property of a viral MP in differentially recognizing dsRNA and ssRNA and establish a biochemical basis to enable further studies on the mechanisms of dsRNA viral MP functions.     

Activation of interferon regulatory factor 3 is inhibited by the influenza A virus NS1 protein

We present a novel mechanism by which viruses may inhibit the alpha/beta interferon (IFN-alpha/beta) 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-alpha/beta gene expression. IRF-3 activation and, as a consequence, IFN-beta 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 in trans, 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.     

Relationships among the positive strand and double-strand RNA viruses as viewed through their RNA-dependent RNA polymerases.

The sequences of 50 RNA-dependent RNA polymerases (RDRPs) from 43 positive strand and 7 double strand RNA (dsRNA) viruses have been compared. The alignment permitted calculation of distances among the 50 viruses and a resultant dendrogram based on every amino acid, rather than just those amino acids in the conserved motifs. Remarkably, a large subgroup of these viruses, including vertebrate, plant, and insect viruses, forms a single cluster whose only common characteristic is exploitation of insect hosts or vectors. This similarity may be due to molecular constraints associated with a present and/or past ability to infect insects and/or to common descent from insect viruses. If common descent is important, as it appears to be, all the positive strand RNA viruses of eucaryotes except for the picornaviruses may have evolved from an ancestral dsRNA virus. Viral RDRPs appear to be inherited as modules rather than as portions of single RNA segments, implying that RNA recombination has played an important role in their dissemination.     

A dsRNA-binding protein of a complex invertebrate DNA virus suppresses the Drosophila RNAi response

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

Dissection of double-stranded RNA binding protein B2 from betanodavirus

Betanodaviruses are small RNA viruses that infect teleost fish and pose a considerable threat to marine aquaculture production. These viruses possess a small protein, termed B2, which binds to and protects double-stranded RNA. This prevents cleavage of virus-derived double-stranded RNAs (dsRNAs) by Dicer and subsequent production of small interfering RNA (siRNA), which would otherwise induce an RNA-silencing response against the virus. In this work, we have performed charged-to-alanine scanning mutagenesis of the B2 protein in order to identify residues required for dsRNA binding and protection. While the majority of the 19 mutated B2 residues were required for maximal dsRNA binding and protection in vitro, residues R53 and R60 were essential for both activities. Subsequent experiments in fish cells confirmed these findings by showing that mutations in these residues abolished accumulation of both the RNA1 and RNA2 components of the viral genome, in addition to preventing any significant induction of the host interferon gene, Mx. Moreover, an obvious positive correlation was found between dsRNA binding and protection in vitro and RNA1, RNA2, and Mx accumulation in fish cells, further validating the importance of the selected amino acid residues. The same trend was also demonstrated using an RNA silencing system in HeLa cells, with residues R53 and R60 being essential for suppression of RNA silencing. Importantly, we found that siRNA-mediated knockdown of Dicer dramatically enhanced the accumulation of a B2 mutant. In addition, we found that B2 is able to induce apoptosis in fish cells but that this was not the result of dsRNA binding.     

双链RNA和植物对病毒的抗性

双链RNA能诱导转录后的基因沉默,是生物抵御病毒入侵、维持自身基因稳定的一种自我保护机制.把源自病毒的基因构建成反向重复结构转入植物体内,其转录出的RNA会通过分子内序列互补形成双链,将入侵病毒的同源序列降解,使转基因植株获得对病毒的高抗性.RNA干扰型抗病毒转基因植株中,转病毒基因的mRNA不存在或存在量很少,也不会翻译成有功能的病毒蛋白,因此不存在病毒RNA重组、异源包装及协生作用的潜在风险,具有较高的生物安全性.双链RNA抗病毒转基因正在成为一种高效、安全的植物抗病毒策略.     

DHX36 Enhances RIG-I Signaling by Facilitating PKR-Mediated Antiviral Stress Granule Formation

RIG-I is a DExD/H-box RNA helicase and functions as a critical cytoplasmic sensor for RNA viruses to initiate antiviral interferon (IFN) responses. Here we demonstrate that another DExD/H-box RNA helicase DHX36 is a key molecule for RIG-I signaling by regulating double-stranded RNA (dsRNA)-dependent protein kinase (PKR) activation, which has been shown to be essential for the formation of antiviral stress granule (avSG). We found that DHX36 and PKR form a complex in a dsRNA-dependent manner. By forming this complex, DHX36 facilitates dsRNA binding and phosphorylation of PKR through its ATPase/helicase activity. Using DHX36 KO-inducible MEF cells, we demonstrated that DHX36 deficient cells showed defect in IFN production and higher susceptibility in RNA virus infection, indicating the physiological importance of this complex in host defense. In summary, we identify a novel function of DHX36 as a critical regulator of PKR-dependent avSG to facilitate viral RNA recognition by RIG-I-like receptor (RLR).