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.     

The C proteins of human parainfluenza virus type 1 limit double-stranded RNA accumulation that would otherwise trigger activation of MDA5 and protein kinase R

Human parainfluenza virus type 1 (HPIV1) is an important respiratory pathogen in young children, the immunocompromised, and the elderly. We found that infection with wild-type (WT) HPIV1 suppressed the innate immune response in human airway epithelial cells by preventing not only phosphorylation of interferon regulatory factor 3 (IRF3) but also degradation of IκBβ, thereby inhibiting IRF3 and NF-κB activation, respectively. Both of these effects were ablated by a F170S substitution in the HPIV1 C proteins (F170S) or by silencing the C open reading frame [P(C-)], resulting in a potent beta interferon (IFN-β) response. Using murine knockout cells, we found that IFN-β induction following infection with either mutant relied mainly on melanoma-associated differentiation gene 5 (MDA5) rather than retinoic acid-inducible gene I (RIG-I). Infection with either mutant, but not WT HPIV1, induced a significant accumulation of intracellular double-stranded RNA (dsRNA). These mutant viruses directed a marked increase in the accumulation of viral genome, antigenome, and mRNA that was coincident with the accumulation of dsRNA. In addition, the amount of viral proteins was reduced compared to that of WT HPIV1. Thus, the accumulation of dsRNA might be a result of an imbalance in the N protein/genomic RNA ratio leading to incomplete encapsidation. Protein kinase R (PKR) activation and IFN-β induction followed the kinetics of dsRNA accumulation. Interestingly, the C proteins did not appear to directly inhibit intracellular signaling involved in IFN-β induction; instead, their role in preventing IFN-β induction appeared to be in suppressing the formation of dsRNA. PKR activation contributed to IFN-β induction and also was associated with the reduction in the amount of viral proteins. Thus, the HPIV1 C proteins normally limit the accumulation of dsRNA and thereby limit activation of IRF3, NF-κB, and PKR. If C protein function is compromised, as in the case of F170S HPIV1, the resulting PKR activation and reduction in viral protein levels enable the host to further reduce C protein levels and to mount a potent antiviral type I IFN response.     

Role for p53 in Gene Induction by Double-Stranded RNA

Cross talk between p53 and interferon-regulated pathways is implicated in the induction of gene expression by biologic and genotoxic stresses. We demonstrate that the interferon-stimulated gene ISG15 is induced by p53 and that p53 is required for optimal gene induction by double-stranded RNA (dsRNA), but not interferon. Interestingly, virus induces ISG15 in the absence of p53, suggesting that virus and dsRNA employ distinct signaling pathways.     

Oncogenic ras induces an inhibitor of double-stranded RNA-dependent eukaryotic initiation factor 2 alpha-kinase activation.

The interferon-inducible 68-kDa dsRNA-dependent eIF2 alpha-kinase (dsI) is a potent cellular antiviral enzyme which is activated by autophosphorylation in response to double-stranded RNA (dsRNA). Activated dsI has also been implicated as a second messenger for gene induction by platelet-derived growth factor (PDGF) and interferon (IFN). We have shown previously that introduction of a transforming ras gene into BALB/c-3T3 fibroblasts blocks induction of responsive genes by PDGF and IFN. We therefore investigated the effect of transforming ras genes on dsI activity in these cells. We report here that dsRNA-mediated activation of dsI is blocked in v-ras-containing cells in a manner specific to ras and not attributable to the transformed phenotype since: 1) a dexamethasone-inducible v-Ha-ras gene produced the effect within 18 h of induction; 2) morphologic reversion of ras-transformed cells with cAMP or the Krev-1 gene restored potential for dsI activation; and 3) transformation by v-mos or v-abl had no effect on dsI activation. Latent dsI levels were unaffected by v-ras. A heat-sensitive dsI inhibitory activity could be demonstrated in v-ras-containing cells which functioned in trans when mixed with untransformed cell extracts prior to stimulation with dsRNA. The inhibitory activity, which was destroyed by phenol-chloroform extraction, did not bind dsRNA.     

Sequestration and protection of double-stranded RNA by the betanodavirus b2 protein

Betanodavirus B2 belongs to a group of functionally related proteins from the sense-strand RNA virus family Nodaviridae that suppress cellular RNA interference. The B2 proteins of insect alphanodaviruses block RNA interference by binding to double-stranded RNA (dsRNA), thus preventing Dicer-mediated cleavage and the subsequent generation of short interfering RNAs. We show here that the fish betanodavirus B2 protein also binds dsRNA. Binding is sequence independent, and maximal binding occurs with dsRNA substrates greater than 20 bp in length. The binding of B2 to long dsRNA is sufficient to completely block Dicer cleavage of dsRNA in vitro. Protein-protein interaction studies indicated that B2 interacts with itself and with other dsRNA binding proteins, the interaction occurring through binding to shared dsRNA substrates. Induction of the dsRNA-dependent interferon response was not antagonized by B2, as the interferon-responsive Mx gene of permissive fish cells was induced by wild-type viral RNA1 but not by a B2 mutant. The induction of Mx instead relied solely on viral RNA1 accumulation, which is impaired in the B2 mutant. Hyperediting of virus dsRNA and site-specific editing of 5-HT2C mRNA were both antagonized by B2. RNA editing was not, however, observed in transfected wild-type or B2 mutant RNA1, suggesting that this pathway does not contribute to the RNA1 accumulation defect of the B2 mutant. We thus conclude that betanodavirus B2 is a dsRNA binding protein that sequesters and protects both long and short dsRNAs to protect betanodavirus from cellular RNA interference.     

Transgenic RNAi in mouse oocytes: The first decade

RNA interference (RNAi), a sequence-specific mRNA degradation induced by double-stranded RNA (dsRNA), is a common approach employed to specifically silence genes. Experimental RNAi in plant and invertebrate models is frequently induced by long dsRNA. However, in mammals, short RNA molecules are used preferentially since long dsRNA can provoke sequence-independent type I interferon response. A notable exception are mammalian oocytes where the interferon response is suppressed and long dsRNA is a potent and specific trigger of RNAi. Transgenic RNAi is an adaptation of RNAi allowing for inducing sequence-specific silencing upon expression of dsRNA. A decade ago, we have developed a vector for oocyte-specific expression of dsRNA, which has been used to study gene function in mouse oocytes on numerous occasions. This review provides an overview and discusses benefits and drawbacks encountered by us and our colleagues while working with the oocytes-specific transgenic RNAi system.     

Analysis of gene function in somatic mammalian cells using small interfering RNAs

RNA interference (RNAi) is a highly conserved gene silencing mechanism that uses double-stranded RNA (dsRNA) as a signal to trigger the degradation of homologous mRNA. The mediators of sequence-specific mRNA degradation are 21- to 23-nt small interfering RNAs (siRNAs) generated by ribonuclease III cleavage from longer dsRNAs. Twenty-one-nucleotide siRNA duplexes trigger specific gene silencing in mammalian somatic cells without activation of the unspecific interferon response. Here we provide a collection of protocols for siRNA-mediated knockdown of mammalian gene expression. Because of the robustness of the siRNA knockdown technology, genomewide analysis of human gene function in cultured cells has now become possible.     

Gene silencing in Caenorhabditis elegans by transitive RNA interference

When a cell is exposed to double-stranded RNA (dsRNA), mRNA from the homologous gene is selectively degraded by a process called RNA interference (RNAi). Here, we provide evidence that dsRNA is amplified in Caenorhabditis elegans to ensure a robust RNAi response. Our data suggest a model in which mRNA targeted by RNAi functions as a template for 5' to 3' synthesis of new dsRNA (termed transitive RNAi). Strikingly, the effect is nonautonomous: dsRNA targeted to a gene expressed in one cell type can lead to transitive RNAi-mediated silencing of a second gene expressed in a distinct cell type. These data suggest dsRNA synthesized in vivo can mediate systemic RNAi.     

Mechanism of mda-5 Inhibition by Paramyxovirus V Proteins

The RNA helicases encoded by melanoma differentiation-associated gene 5 (mda-5) and retinoic acid-inducible gene I (RIG-I) detect foreign cytoplasmic RNA molecules generated during the course of a virus infection, and their activation leads to induction of type I interferon synthesis. Paramyxoviruses limit the amount of interferon produced by infected cells through the action of their V protein, which binds to and inhibits mda-5. Here we show that activation of both mda-5 and RIG-I by double-stranded RNA (dsRNA) leads to the formation of homo-oligomers through self-association of the helicase domains. We identify a region within the helicase domain of mda-5 that is targeted by all paramyxovirus V proteins and demonstrate that they inhibit activation of mda-5 by blocking dsRNA binding and consequent self-association. In addition to this commonly targeted domain, some paramyxovirus V proteins target additional regions of mda-5. In contrast, V proteins cannot bind to RIG-I and consequently have no effect on the ability of RIG-I to bind dsRNA or to form oligomers.     

Expression of double-stranded-RNA-specific RNase III of Escherichia coli is lethal to Saccharomyces cerevisiae.

The gene for the double-stranded RNA (dsRNA)-specific RNase III of Escherichia coli was expressed in Saccharomyces cerevisiae to examine the effects of this RNase activity on the yeast. Induction of the RNase III gene was found to cause abnormal cell morphology and cell death. Whereas double-stranded killer RNA is degraded by RNase III in vitro, killer RNA, rRNA, and some mRNAs were found to be stable in vivo after induction of RNase III. Variants selected for resistance to RNase III induction were isolated at a frequency of 4 X 10(-5) to 5 X 10(-5). Ten percent of these resistant strains had concomitantly lost the capacity to produce killer toxin and M dsRNA while retaining L dsRNA. The genetic alteration leading to RNase resistance was localized within the RNase III-coding region but not in the yeast chromosome. These results indicate that S. cerevisiae contains some essential RNA which is susceptible to E. coli RNase III.     

Long double-stranded RNA-mediated RNA interference and immunostimulation: long interfering double-stranded RNA as a potent anticancer therapeutics

In most applications, small interfering RNAs are designed to execute specific gene silencing via RNA interference (RNAi) without triggering nonspecific responses such as immunostimulation. However, in anticancer therapeutics, immunostimulation combined with specific oncogene silencing could be beneficial, resulting in the synergistic inhibition of cancer cell growth. In this study, we report an immunostimulatory long double-stranded RNA (dsRNA) structure with the ability to trigger RNAi-mediated specific target gene silencing, termed as long interfering dsRNA (liRNA). liRNA targeting Survivin mRNA not only efficiently and specifically triggered target gene silencing via RNAi, but also stimulated the protein kinase R pathway to induce the expression of interferon β. As a result, the ability of Survivin-targeting liRNA to inhibit cancer cell growth was superior over conventional small interfering RNA or nontargeting dsRNA structures. Our results thus provide a simple yet efficient dual function immunostimulatory RNAi-triggering structure, which is potentially applicable for the development of anticancer therapeutics.     

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.     

Sheet-like clay nanoparticles deliver RNA into developing pollen to efficiently silence a target gene

Topical application of double-stranded RNA (dsRNA) can induce RNA interference (RNAi) and modify traits in plants without genetic modification. However, delivering dsRNA into plant cells remains challenging. Using developing tomato (Solanum lycopersicum) pollen as a model plant cell system, we demonstrate that layered double hydroxide (LDH) nanoparticles up to 50 nm in diameter are readily internalized, particularly by early bicellular pollen, in both energy-dependent and energy-independent manners and without physical or chemical aids. More importantly, these LDH nanoparticles efficiently deliver dsRNA into tomato pollen within 2–4 h of incubation, resulting in an 89% decrease in transgene reporter mRNA levels in early bicellular pollen 3-d post-treatment, compared with a 37% decrease induced by the same dose of naked dsRNA. The target gene silencing is dependent on the LDH particle size, the dsRNA dose, the LDH–dsRNA complexing ratio, and the treatment time. Our findings indicate that LDH nanoparticles are an effective nonviral vector for the effective delivery of dsRNA and other biomolecules into plant cells.

Developing tomato pollen internalizes layered double hydroxide nanoparticles smaller than 50 nm that facilitate delivery of double-stranded RNA, enhancing RNA interference of a target gene.