pIRS1 and pTRS1 are present in human cytomegalovirus virions.

The virus-coded proteins pIRS1 and pTRS1 were found associated with purified human cytomegalovirus virions. The proteins were not degraded when intact virions were treated with trypsin, which suggests that they are localized inside the viral particle. In transfection experiments pIRS1 and pTRS1 modestly activated expression from a reporter plasmid containing the viral major immediate-early promoter but did not influence the activity of a reporter carrying the irs1/trs1 immediate-early promoter. Both reporters were activated by the combination of pIRS1 or pTRS1 and pUL69, which is also present in virions.     

Double-stranded RNA binding by a heterodimeric complex of murine cytomegalovirus m142 and m143 proteins

In response to viral infection, cells activate a variety of antiviral responses, including several that are triggered by double-stranded (ds) RNA. Among these are the protein kinase R and oligoadenylate synthetase/RNase L pathways, both of which result in the shutoff of protein synthesis. Many viruses, including human cytomegalovirus, encode dsRNA-binding proteins that prevent the activation of these pathways and thereby enable continued protein synthesis and viral replication. We have extended these analyses to another member of the beta subfamily of herpesviruses, murine cytomegalovirus (MCMV), and now report that products of the m142 and m143 genes together bind dsRNA. Coimmunoprecipitation experiments demonstrate that these two proteins interact in infected cells, consistent with their previously reported colocalization. Jointly, but not individually, the proteins rescue replication of a vaccinia virus mutant with a deletion of the dsRNA-binding protein gene E3L (VVDeltaE3L). Like the human cytomegalovirus dsRNA-binding protein genes TRS1 and IRS1, m142 and m143 are members of the US22 gene family. We also found that two other members of the MCMV US22 family, M23 and M24, encode dsRNA-binding proteins, but they do not rescue VVDeltaE3L replication. These results reveal that MCMV, like many other viruses, encodes dsRNA-binding proteins, at least two of which can inhibit dsRNA-activated antiviral pathways. However, unlike other well-studied examples, the MCMV proteins appear to act in a heterodimeric complex.     

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     

Double-stranded RNA

High molecular weight, fully double-stranded RNA (dsRNA) has been recognized as the genetic material of many plant, animal, fungal, and bacterial viruses (Diplornaviruses): virusspecific dsRNA is also found in cells infected with single-stranded RNA viruses. DsRNA has identified in a variety of apparently normal eucaryotic cells and is associated with the "killer" character of certain strains of Saccaromyces cerevisiae.     

Double-stranded RNA bending by AU-tract sequences

Sequence-dependent structural deformations of the DNA double helix (dsDNA) have been extensively studied, where adenine tracts (A-tracts) provide a striking example for global bending in the molecule. However, in contrast to dsDNA, sequence-dependent structural features of dsRNA have received little attention. In this work, we demonstrate that the nucleotide sequence can induce a bend in a canonical Watson-Crick base-paired dsRNA helix. Using all-atom molecular dynamics simulations, we identified a sequence motif consisting of alternating adenines and uracils, or AU-tracts, that strongly bend the RNA double-helix. This finding was experimentally validated using atomic force microscopy imaging of dsRNA molecules designed to display macroscopic curvature via repetitions of phased AU-tract motifs. At the atomic level, this novel phenomenon originates from a localized compression of the dsRNA major groove and a large propeller twist at the position of the AU-tract. Moreover, the magnitude of the bending can be modulated by changing the length of the AU-tract. Altogether, our results demonstrate the possibility of modifying the dsRNA curvature by means of its nucleotide sequence, which may be exploited in the emerging field of RNA nanotechnology and might also constitute a natural mechanism for proteins to achieve recognition of specific dsRNA sequences.     

Double-stranded RNA binding may be a general plant RNA viral strategy to suppress RNA silencing

In plants, RNA silencing (RNA interference) is an efficient antiviral system, and therefore successful virus infection requires suppression of silencing. Although many viral silencing suppressors have been identified, the molecular basis of silencing suppression is poorly understood. It is proposed that various suppressors inhibit RNA silencing by targeting different steps. However, as double-stranded RNAs (dsRNAs) play key roles in silencing, it was speculated that dsRNA binding might be a general silencing suppression strategy. Indeed, it was shown that the related aureusvirus P14 and tombusvirus P19 suppressors are dsRNA-binding proteins. Interestingly, P14 is a size-independent dsRNA-binding protein, while P19 binds only 21-nucleotide ds-sRNAs (small dsRNAs having 2-nucleotide 3' overhangs), the specificity determinant of the silencing system. Much evidence supports the idea that P19 inhibits silencing by sequestering silencing-generated viral ds-sRNAs. In this study we wanted to test the hypothesis that dsRNA binding is a general silencing suppression strategy. Here we show that many plant viral silencing suppressors bind dsRNAs. Beet yellows virus Peanut P21, clump virus P15, Barley stripe mosaic virus gammaB, and Tobacco etch virus HC-Pro, like P19, bind ds-sRNAs size-selectively, while Turnip crinkle virus CP is a size-independent dsRNA-binding protein, which binds long dsRNAs as well as ds-sRNAs. We propose that size-selective ds-sRNA-binding suppressors inhibit silencing by sequestering viral ds-sRNAs, whereas size-independent dsRNA-binding suppressors inactivate silencing by sequestering long dsRNA precursors of viral sRNAs and/or by binding ds-sRNAs. The findings that many unrelated silencing suppressors bind dsRNA suggest that dsRNA binding is a general silencing suppression strategy which has evolved independently many times.     

Systemic RNA Interference Deficiency-1 (SID-1) Extracellular Domain Selectively Binds Long Double-stranded RNA and Is Required for RNA Transport by SID-1

During systemic RNA interference (RNAi) in Caenorhabditis elegans, RNA spreads across different cells and tissues in a process that requires the systemic RNA interference deficient-1 (sid-1) gene, which encodes an integral membrane protein. SID-1 acts cell-autonomously and is required for cellular import of interfering RNAs. Heterologous expression of SID-1 in Drosophila Schneider 2 cells enables passive uptake of dsRNA and subsequent soaking RNAi. Previous studies have suggested that SID-1 may serve as an RNA channel, but its precise molecular role remains unclear. To test the hypothesis that SID-1 mediates a direct biochemical recognition of RNA molecule and subsequent permeation, we expressed the extracellular domain (ECD) of SID-1 and purified it to near homogeneity. Recombinant purified SID-1 ECD selectively binds dsRNA but not dsDNA in a length-dependent and sequence-independent manner. Genetic missense mutations in SID-1 ECD causal for deficient systemic RNAi resulted in significant reduction in its affinity for dsRNA. Furthermore, full-length proteins with these mutations decrease SID-1-mediated RNA transport efficiency, providing evidence that dsRNA binding to SID-1 ECD is related to RNA transport. To examine the functional similarity of mammalian homologs of SID-1 (SIDT1 and SIDT2), we expressed and purified mouse SIDT1 and SIDT2 ECDs. We show that they bind long dsRNA in vitro, supportive of dsRNA recognition. In summary, our study illustrates the functional importance of SID-1 ECD as a dsRNA binding domain that contributes to RNA transport.     

Double-stranded RNA analysis of strawberry plants affected by June yellows

Using an improved method for extraction of dsRNA from strawberry leaf tissue, small quantities of several dsRNA species with mol. wt greater than 1.0 × 10⁶were detected in strawberry plants free from known strawberry viruses but affected by June yellows (JY). No such dsRNA species were detected in plants of Fragaria vesca or seven strawberry cultivars known to be free from JY. Neither JY symptoms nor these dsRNA species were detected in healthy strawberry and F. vesca plants graft-inoculated with tissue from JY-affected plants. It is not known whether the JY-associated dsRNA species are those of a causal agent of JY or are a consequence of the JY condition. Nevertheless, the detection of such dsRNA species in plants affected by JY may offer a possible objective method for detecting the incipient condition in symptomless strawberry plants. However, the concentrations of dsRNA in JY-affected plants are very low and dsRNA analysis is thought not to be sufficiently reliable for routine testing of plants. The occurrence of anomalous dsRNA species in extracts from some strawberry plants was caused by dsRNA from two-spotted spider mites (Tetranychus urticae) infesting the plants.     

Double-stranded RNA deaminase ADAR1 increases host susceptibility to virus infection

The RNA-editing enzyme ADAR1 is a double-stranded RNA (dsRNA) binding protein that modifies cellular and viral RNA sequences by adenosine deamination. ADAR1 has been demonstrated to play important roles in embryonic erythropoiesis, viral response, and RNA interference. In human hepatitis virus infection, ADAR1 has been shown to target viral RNA and to suppress viral replication through dsRNA editing. It is not clear whether this antiviral effect of ADAR1 is a common mechanism in response to viral infection. Here, we report a proviral effect of ADAR1 that enhances replication of vesicular stomatitis virus (VSV) through a mechanism independent of dsRNA editing. We demonstrate that ADAR1 interacts with dsRNA-activated protein kinase PKR, inhibits its kinase activity, and suppresses the alpha subunit of eukaryotic initiation factor 2 (eIF-2alpha) phosphorylation. Consistent with the inhibitory effect on PKR activation, ADAR1 increases VSV infection in PKR+/+ mouse embryonic fibroblasts; however, no significant effect was found in PKR-/- cells. This proviral effect of ADAR1 requires the N-terminal domains but does not require the deaminase domain. These findings reveal a novel mechanism of ADAR1 that increases host susceptibility to viral infection by inhibiting PKR activation.     

Sequence-specific single-strand RNA binding protein encoded by the human LINE-1 retrotransposon.

Previous experiments using human teratocarcinoma cells indicated that p40, the protein encoded by the first open reading frame (ORF) of the human LINE-1 (L1Hs) retrotransposon, occurs in a large cytoplasmic ribonucleoprotein complex in direct association with L1Hs RNA(s), the p40 RNP complex. We have now investigated the interaction between partially purified p40 and L1Hs RNA in vitro using an RNA binding assay dependent on co-immunoprecipitation of p40 and bound RNA. These experiments identified two p40 binding sites on the full-length sense strand L1Hs RNA. Both sites are in the second ORF of the 6000 nt RNA: site A between residues 1999 and 2039 and site B between residues 4839 and 4875. The two RNA segments share homologous regions. Experiments involving UV cross-linking followed by immunoprecipitation indicate that p40 in the in vitro complex is directly associated with L1Hs RNA, as it is in the p40 RNP complex found in teratocarcinoma cells. Binding and competition experiments demonstrate that p40 binds to single-stranded RNA containing a p40 binding site, but not to single-stranded or double-stranded DNA, double-stranded RNA or a DNA-RNA hybrid containing a binding site sequence. Thus, p40 appears to be a sequence-specific, single-strand RNA binding protein.     

细菌介导的发夹结构双链RNA对秀丽小杆线虫中par-1基因的RNA干扰

秀丽小杆线虫(Caenorhabditis elegans)是一种重要的模式生物,目前在C.elegans中发现的许多基因在序列和功能上与哺乳动物的基因有很高的同源性,对其基因功能的研究有助于阐明哺乳动物的基因功能。为检测细菌介导的通过发夹结构表达双链RNA(dsRNA)的RNA干扰(RNAi)在C.elegans中的作用效果,我们设计并构建了可以转录表达母源极性基因par-1的发夹dsRNA的RNAi载体,并转入E.coli HT115中,经过IPTG诱导后浓缩。浓缩菌液在25℃下喂食C.elegans48h后,观察C.elegans胚胎发育情况,同时以含有空载体的菌液作为对照,并与par-1突变体及野生型比较。结果显示:发夹结构表达的dsRNA对par-1基因进行RNAi后,虫体内par-1 mRNA几乎消失,C.elegans早期胚胎分裂不对称性丧失,par-1(RNAi)干扰率在95%以上。     

Double-stranded RNA regulates IL-4 expression

dsRNA, as genomic fragment, replicative intermediate, or stem and loop structure in cells infected by viruses, can act to signal the immune system of the presence of viral infections. Although most viral infections are associated with strong Th1 immune responses, Th2-type responses have also been observed. In this study, we characterize the effects of dsRNA on the induction of Th2 responses in human lymphocytes. We report that in addition to the well-known Th1-inducing capabilities of dsRNA, treatment of human lymphocytes with low concentrations of dsRNA (0.1-1 microg/ml) leads to the expression of the prototypic Th2 cytokine IL-4. This induction was accompanied with the concentration-dependent activation of NF-kappaB and NF-AT2 but not NF-AT1. In addition, dsRNA can directly activate an IL-4 promoter-driven chloramphenicol acetyltransferase reporter gene in transiently transfected Jurkat cells. These results are the first demonstration of a non-TCR-associated activator of NF-AT in human cells and suggest that dsRNA directly influences IL-4 gene expression through its effect on NF-AT activation. Our data provide support for the idea that dsRNA at low concentrations in vivo may induce a Th2-dominant response that is not optimal for protective immunity to the virus.     

Double-stranded RNA mycovirus from Fusarium graminearum

Double-stranded RNA (dsRNA) viruses in some fungi are associated with hypovirulence and have been used or proposed as biological control agents. We isolated 7.5-kb dsRNAs from 13 of 286 field strains of Fusarium graminearum isolated from maize in Korea. One of these strains, DK21, was examined in more detail. This strain had pronounced morphological changes, including reduction in mycelial growth, increased pigmentation, reduced virulence towards wheat, and decreased (60-fold) production of trichothecene mycotoxins. The presence or absence of the 7.5-kb dsRNA was correlated with the changes in pathogenicity and morphology. The dsRNA could be transferred to virus-free strains by hyphal fusion, and the recipient strain acquired the virus-associated phenotype of the donor strain. The dsRNA was transmitted to approximately 50% of the conidia, and only colonies resulting from conidia carrying the mycovirus had the virus-associated phenotype. Partial nucleotide sequences of the purified dsRNA identify an RNA-dependent RNA polymerase sequence and an ATP-dependent helicase that are closely related to those of Cryphonectria hypovirus and Barley yellow mosaic virus. Collectively, these results suggest that this dsRNA isolated from F. graminearum encodes traits for hypovirulence.