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PubMed2005

siRNA injection induces sequence-independent protection in Penaeus monodon against white spot syndrome virus.

White spot syndrome virus (WSSV) is a major disease in crustaceans, particularly shrimp, due to the current intensity of aquaculture practices. Novel strategies including vaccination to control this virus would be highly desirable. However, invertebrates lack a true adaptive immune response system and seem to rely on various innate immune responses. An alternative and more specific approach to counteract WSSV infections in shrimp could be by the exploitation of RNA interference. As long dsRNA molecules induce a general, sequence-independent anti-viral immunity in shrimp [Robalino, J., Browdy, C.L., Prior, S., Metz, A., Parnell, P., Gross, P., Warr, G., 2004. J. Virol. 78, 10442-10448], it was investigated whether shorter 21 nt siRNAs with homology to the WSSV vp15 and vp28 genes would give a sequence-specific interference response in the shrimp Penaeus monodon. Vp28 siRNAs as well as nonspecific control gfp siRNAs were able to specifically and efficiently silence their homologous genes in a heterologous baculovirus insect cell expression system. However, in shrimps no such a specific effect was observed. Shrimp injected with vp15 or vp28 siRNAs before WSSV challenge gave a significantly lower mortality rate, but not significantly different when shrimps were injected with gfp siRNA. Thus, large dsRNA molecules as well as siRNAs induce a sequence-independent anti-viral immunity when injected in shrimp.

Animals↗
PubMed2005

Modulation of PKR activity in cells infected by bovine viral diarrhea virus.

Bovine viral diarrhea virus is an important animal pathogen. The cytopathic and noncytopathic biotypes of the virus are associated with distinct pathologic entities. A striking difference between the two biotypes is viral RNA accumulation in infected cells. Viral dsRNA is thought to activate protein kinase PKR; an important mediator of innate immunity. In this study, we investigated PKR activation and its consequences in BVDV-infected cells. Infection with cp BVDV was found to induce PKR activation, eIF2alpha phosphorylation, translation inhibition and NF-kappaB activation. In contrast, PKR activity and eIF2alpha phosphorylation were not induced during infection with the ncp BVDV. In addition, cells infected with ncp BVDV showed no PKR phosphorylation in response to infection with the unrelated poliovirus whereas uninfected ncp BVDV cells when infected with poliovirus showed high levels of phosphorylated PKR. Cells infected with ncp BVDV failed to respond to synthetic dsRNA (poly I:C) treatment with NF-kappaB activation. However, the NF-kappaB response to bacterial lipopolysaccarides (LPS) was normal in these cells, suggesting a specific suppression of antiviral response signaling in ncp BVDV infected cells. These results indicate that ncp BVDV has evolved specific mechanisms to prevent activation of PKR and its antiviral effectors, most likely to facilitate the establishment and maintenance of persistent infection.

Animals↗
PubMed2006

The reinforcement of invasion in epithelial ovarian cancer cells by 17 beta-Estradiol is associated with up-regulation of Snail.

OBJECTIVE: The transcription factor Snail, which is implicated in the triggering of epithelial-mesenchymal transitions (EMT), plays an important role in adhesion, invasion and metastasis of tumor cells. In the present study, we assessed 17beta-Estradiol (E2)'s effect on Snail, E-cadherin and MMP-2 expression of epithelial ovarian cancer cell line ES-2 and SKOV3. Then we induced Snail gene silencing by RNA interference to explore the effect of E2 on E-cadherin and MMP-2 expression when Snail gene expression was blocked. METHODS: Treated by 10(-8) M E2, Snail, E-cadherin and MMP-2 mRNA expression of the cells was measured by RT-PCR; Snail, MMP-2 protein expression was detected by IHC; and MMP-2 activity was determined by Zymography. E-cadherin protein level was measured by Western blot. We constructed the small interfering dsRNA expression vector (pRNAT-U6.1/Neo-Snail) targeting Snail gene, as well as a negative control vector (pRNAT-U6.1/Neo-Neg). Then the cells were transiently transfected with the vectors. Western blot and zymography were conducted to determine E-cadherin protein level and matrix metalloproteinase activity of the cells transfected with pRNAT-U6.1/Neo-Snail or pRNAT-U6.1/Neo-Neg after treated with E2 for 24 h. RESULTS: The expression of ER alpha mRNA and protein was negative in ES-2 cells and positive in SKOV3 cells, and ER beta expression was positive in both cell lines. 10(-8) mol/l E2 elevated expression of Snail and MMP-2 mRNA and protein in both ES-2 and SKOV3 cells, and reduced expression of E-cadherin mRNA and protein in SKOV3 cells. While in the RNAi group transfected with the small interfering dsRNA expression vector (pRNAT-U6.1/Neo-Snail) targeting Snail gene, E2 treatment did not have a significant effect on MMP-2 activity or E-cadherin protein in ES-2 and SKOV3 cells. CONCLUSIONS: 17beta-Estradiol increased Snail expression in both ER alpha-negative ES-2 cells and ER alpha-positive SKOV3 cells independent of the existence of ER alpha. The increase of MMP-2 expression in ES-2 and SKOV3 cells and decrease of E-cadherin expression in SKOV3 cells induced by E2 were associated with up-regulation of Snail.

Adenocarcinoma, Clear Cell↗
PubMed2005

A cellular screening assay to test the ability of PKR to induce cell death in mammalian cells.

Long double-stranded RNA (>30 bp), usually expressed in cells infected with RNA viruses, triggers antiviral responses that induce apoptosis of the infected cells. PKR can be selectively activated in glioblastoma cells by in situ generation of dsRNA following introduction of antisense RNA complementary to an RNA expressed specifically in these cells. Harnessing PKR for the selective killing of cancer cells is potentially a powerful strategy for treating cancer, but we were unable to induce apoptosis by this approach in a T cell lymphoma. We therefore established a cellular screening assay to test the ability of PKR to induce death in cell lines, especially those originating from human cancers. This "PKR killing screen" is based on the infection of cells with an adenoviral vector encoding GyrB-PKR, followed by coumermycin treatment. Cancers represented by cell lines in which PKR activation leads to cell death are good candidates for the dsRNA killing approach, using antisense to RNA molecules specifically expressed in these cells. The PKR killing screen may also serve as a tool for exploring PKR signaling and other related pathways, by identifying new cases in which PKR signaling is inhibited or impaired.

Adenocarcinoma↗
PubMed2002

Involvement of TIRAP/MAL in signaling for the activation of interferon regulatory factor 3 by lipopolysaccharide.

Infections of bacteria and viruses induce host defense reactions known as innate responses that include the production of cytokines and chemokines. The production of type I interferon (IFN) is known to be induced by viral double-stranded (ds) RNA or bacterial lipopolysaccharide (LPS). Although important functions for the transcription factors NF-kappaB and interferon regulatory factor-3 (IRF-3) are indicated, the molecular signals leading to the activation of IFN genes have yet to be elucidated. We provide several lines of evidence that LPS and dsRNA trigger distinct intracellular signals upstream. Notably, our investigation revealed a critical function for TIRAP/MAL, a signaling adapter for Toll-like receptor (TLR) 4, in LPS-induced but not dsRNA-induced activation of IRF-3. These results highlight cross-talk between TLR-mediated and virus/dsRNA-induced signals resulting in activation of the IFN system.

Animals↗
PubMed2003

Genetic, morphological, and virulence characterization of the entomopathogenic fungus Verticillium lecanii.

In order to clarify relationships among genetic diversity, virulence, and other characteristics of conidia, 46 isolates of Verticillium lecanii from various hosts and geographical locations were examined. The internal transcribed spacer (ITS) and intergenic spacer (IGS) regions of ribosomal DNA (rDNA), mitochondrial small subunit rDNA (mt-SrDNA) and beta-tubulin were analyzed by PCR-RFLP. PCR-single stranded conformational polymorphism (SSCP) was performed on regions of the mitochondrial large subunit rDNA, mt-SrDNA, beta-tubulin and histone 4. There were no relationships among the results of RFLP, SSCP, isolation source, and location. However, amplified product size of IGS did have relationships with conidia size and sporulation. Six isolates with 4.0-kb IGS products had large conidia dimensions, and yielded low numbers of conidia compared with other isolates. Three out of the six isolates were high virulence (over 90%) against green peach aphids. Furthermore, double-stranded RNA (dsRNA) was detected in 22 out of 35 V. lecanii isolates and related with the amplicon sizes of IGS, though not with virulence or isolation location. Isolates containing dsRNA were divided into six distinct types based on banding pattern. These data demonstrate the level of genetic diversity of V. lecanii, and suggest relations among the genetic properties and conidial morphology.

Animals↗
PubMed2002

The palm subdomain-based active site is internally permuted in viral RNA-dependent RNA polymerases of an ancient lineage.

Template-dependent polynucleotide synthesis is catalyzed by enzymes whose core component includes a ubiquitous alphabeta palm subdomain comprising A, B and C sequence motifs crucial for catalysis. Due to its unique, universal conservation in all RNA viruses, the palm subdomain of RNA-dependent RNA polymerases (RdRps) is widely used for evolutionary and taxonomic inferences. We report here the results of elaborated computer-assisted analysis of newly sequenced replicases from Thosea asigna virus (TaV) and the closely related Euprosterna elaeasa virus (EeV), insect-specific ssRNA+ viruses, which revise a capsid-based classification of these viruses with tetraviruses, an Alphavirus-like family. The replicases of TaV and EeV do not have characteristic methyltransferase and helicase domains, and include a putative RdRp with a unique C-A-B motif arrangement in the palm subdomain that is also found in two dsRNA birnaviruses. This circular motif rearrangement is a result of migration of approximately 22 amino acid (aa) residues encompassing motif C between two internal positions, separated by approximately 110 aa, in a conserved region of approximately 550 aa. Protein modeling shows that the canonical palm subdomain architecture of poliovirus (ssRNA+) RdRp could accommodate the identified sequence permutation through changes in backbone connectivity of the major structural elements in three loop regions underlying the active site. This permutation transforms the ferredoxin-like beta1alphaAbeta2beta3alphaBbeta4 fold of the palm subdomain into the beta2beta3beta1alphaAalphaBbeta4 structure and brings beta-strands carrying two principal catalytic Asp residues into sequential proximity such that unique structural properties and, ultimately, unique functionality of the permuted RdRps may result. The permuted enzymes show unprecedented interclass sequence conservation between RdRps of true ssRNA+ and dsRNA viruses and form a minor, deeply separated cluster in the RdRp tree, implying that other, as yet unidentified, viruses may employ this type of RdRp. The structural diversification of the palm subdomain might be a major event in the evolution of template-dependent polynucleotide polymerases in the RNA-protein world.

Amino Acid Motifs↗
PubMed2003

Selective regulation of gene expression by nuclear factor 110, a member of the NF90 family of double-stranded RNA-binding proteins.

Members of the nuclear factor 90 (NF90) family of double-stranded RNA (dsRNA)-binding proteins have been implicated in several biological processes including the regulation of gene expression. cDNA sequences predict that the proteins have a functional nuclear localization signal and two dsRNA-binding motifs (dsRBMs), and are identical at their N termini. Isoforms are predicted to diverge at their C termini as well as by the insertion of four amino acid residues (NVKQ) between the two dsRBMs. In this study, we verified the expression of four of the isoforms by cDNA cloning and mass spectrometric analysis of proteins isolated from human cells. Cell fractionation studies showed that NF90 and its heteromeric partner, NF45, are predominantly nuclear and largely chromatin-associated. The C-terminally extended NF90 species, NF110, are almost exclusively chromatin-bound. Both NF110 isoforms are more active than NF90 isoforms in stimulating transcription from the proliferating cell nuclear antigen reporter in a transient expression system. NF110b, which carries the NVKQ insert, was identified as the strongest activator. It stimulated transcription of some, but not all, promoters in a fashion that suggested that it functions in concert with other transcription factors. Finally, we demonstrate that NF110b associates with the dsRBM-containing transcriptional co-activator, RNA helicase A, independently of RNA binding.

Amino Acid Sequence↗
PubMed2003

PKR's protective role in viral myocarditis.

Reovirus-induced murine myocarditis provides an excellent model for the human disease. Previously, we showed that reovirus induction of and sensitivity to interferon-beta (IFN-beta) are important determinants of protection against cardiac damage. IFN-beta induces a number of genes with antiviral activities, including the dsRNA-activated protein kinase, PKR. Once bound to viral dsRNA, PKR becomes activated and phosphorylates eukaryotic initiation factor-2 alpha (eIF2 alpha) leading to the cessation of host cell translation. Additionally, activated PKR can exert its antiviral effects by inducing phosphorylation of I kappa B, leading to the activation of the transcription factor NF kappa B and subsequent induction of IFN-beta. Thus, activated PKR can both induce and be induced by IFN-beta. Recently, numerous reports have shown PKR to be dispensable for both induction of IFN as well as protection against disease. However, both PKR's role in the heart in response to viral infection and its ability to prevent cardiac damage have gone largely unexplored. Here, we demonstrate PKR to be critical for viral induction of IFN-beta in primary cardiac myocyte cultures. Additionally, we show that loss of PKR leads to an increase in virulence for both myocarditic and nonmyocarditic reoviruses. Finally, we demonstrate PKR to be critical for protection against reovirus-induced viral myocarditis.

Animals↗
PubMed2003

RNA interference silences Microplitis demolitor bracovirus genes and implicates glc1.8 in disruption of adhesion in infected host cells.

The family Polydnaviridae consists of ds-DNA viruses that are symbiotically associated with certain parasitoid wasps. PDVs are transmitted vertically but also are injected by wasps into hosts where they cause several physiological alterations including immunosuppression. The PDV genes responsible for mediating immunosuppression and other host alterations remain poorly characterized in large measure because viral mutants cannot be produced to study gene function. Here we report the use of RNA interference (RNAi) to specifically silence the glc1.8 and egf1.0 genes from Microplitis demolitor bracovirus (MdBV) in High Five cells derived from the lepidopteran Trichoplusia ni. Dose-response studies indicated that MdBV infects High Five cells and blocks the ability of these cells to adhere to culture plates. This response was very similar to what occurs in two classes of hemocytes, granular cells, and plasmatocytes, after infection by MdBV. Screening of monoclonal antibody (mAb) markers that distinguish different classes of lepidopteran hemocytes indicated that High Five cells cross-react with three mAbs that recognize granular cells from T. ni. Double-stranded RNA (dsRNA) complementary to glc1.8 specifically silenced glc1.8 expression and rescued the adhesive phenotype of High Five cells. Reciprocally, dsRNA complementary to egf1.0 silenced egf1.0 expression but had no effect on adhesion. The simplicity and potency of RNAi could be extremely useful for analysis of other PDV genes.

Animals↗
PubMed2003

The carboxy-terminal, M3 motifs of PACT and TRBP have opposite effects on PKR activity.

PKR is an interferon(IFN)-induced, serine-threonine protein kinase, which plays a crucial role in IFN's antiviral and antiproliferative actions. The three known activators of PKR are dsRNA, heparin, and PACT. PACT activates PKR by direct protein-protein interaction in response to cellular stress. The human TAR (trans-activating region)-binding protein (TRBP), which is very homologous to PACT, also interacts with PKR, leading to an inhibition of PKR activity. Since these two highly homologous proteins have opposite effects on PKR, we examined if they interact with PKR differently by assaying their interaction with various point mutants of PKR. Our results indicate that TRBP and PACT interact with PKR through the same residues, and no differences were identified in these two interactions. Domain swap experiments between PACT and TRBP indicated that the inhibitory effects of TRBP on PKR activity are mediated through its carboxy-terminal residues, which contain TRBP's third dsRNA-binding motif.

Amino Acid Motifs↗
PubMed2003

Analysis of protein phosphatase function in Drosophila cells using RNA interference.

Double stranded RNA-mediated RNA interference is an effective method to downregulate the levels of protein phosphatases in Drosophila S2 cells. In many cases, nearly complete ablation of the targeted protein can be achieved. RNAi-mediated knockdown of protein phosphatases is akin to pharmacological inhibition with drugs and can be used to determine the roles of specific protein phosphatases in intact cells. RNAi can avoid the problems associated with less than adequate specificity of phosphatase inhibitors. Although information about the signaling pathways present in Drosophila S2 cells is not as well developed as many mammalian cell lines, the Drosophila system is particularly attractive for the study of oligomeric phosphatases like PP2A. Drosophila has far fewer isoforms for the phosphatases we have examined. This is especially true of the genes for PP2A regulatory subunits where over 50 isoforms are present in mammals but only four are present in Drosophila. Once hypotheses regarding phosphatase function have been generated from RNAi experiments in S2 cells, they can potentially be tested utilizing recent advances in the use of siRNAs to conduct RNAi experiments in mammalian cell lines. RNAi in Drosophila S2 cells has proven to be a powerful technique for identifying physiological functions of signaling proteins. The RNAi method is straightforward and works routinely with almost all proteins. RNAi in S2 cells can be used to assess the role of signaling proteins in specific pathways and as a screening tool to identify new roles for signaling molecules. For example, results from RNAi analysis of PP2A show that regulation of MAP kinase signaling involves the R2/B regulatory subunit and that the R5/B56 subunits play a previously unidentified role in apoptosis. While RNAi in Drosophila S2 cells is a powerful tool for analyzing protein function, the method does have limitations. Foremost, cells may exhibit an RNAi response to any nonspecific dsRNA, even in the absence of interferon. Therefore, physiological processes that respond to nonspecific dsRNA will be difficult to study. A second limitation is the need to produce antibodies that react with Drosophila isoforms. We have found that many antibodies to mammalian protein phosphatases do not cross-react with the corresponding Drosophila proteins. Finally, the physiology and signaling pathways of S2 cells have not been extensively studied. This lack of information limits the number of available readouts that can be used when assessing the effects of protein knockdowns.

Animals↗
PubMed2005

Analysis of short interfering RNA function in RNA interference by using Drosophila embryo extracts and schneider cells.

The realization that short double-stranded RNA (dsRNAs) 21-25 bp in length represent the basis for posttranscriptional gene silencing (PTGS) in plants, quelling in N. crassa, and RNA interference (RNAi) in C. elegans and Drosophila has given insight into one of the most evolutionarily conserved pathways in eukaryotes. dsRNA that arises due to viral infection, transposon mobilization, random insertion of transgenes near active promoters, transcripts from repetitive elements in the genome, or introduction of exogenous dsRNA directly is processed by one of the RNase III-related enzymes, known as the Dicers, to produce 21- to 25-bp short dsRNAs or short interfering RNAs (siRNAs) that target the degradation of the cognate RNA sequence (Denli and Hannon, 2003; Hannon, 2002; Plasterk, 2002). Proteins in the RNAi pathway and siRNA-like RNAs have also been recently demonstrated to play a role in the formation and maintenance of heterochromatin in S. pombe as well as in transgene-induced PTGS in Drosophila (Hall et al., 2002; Pal-Bhadra et al., 2004; Volpe et al., 2002). An understanding of siRNA function in these crucial regulatory pathways requires biochemical approaches to study siRNAs and their role in gene silencing as well as the formation and maintenance of heterochromatin. This chapter describes simple methods for using Drosophila embryo extracts and cultured insect cells to study siRNA function in the RNAi pathway in vivo and in vitro. We describe the most recent protocols for the preparation and use of Drosophila embryo extracts used in gene targeting studies. We present methods we have used to assay siRNA function in Drosophila embryo extracts and in cultured SL2 cells that demonstrate a combined role for siRNAs and RNA-dependent RNA polymerase (RdRp) activity in Drosophila RNAi.

Animals↗
PubMed1999

Mut-7 of C. elegans, required for transposon silencing and RNA interference, is a homolog of Werner syndrome helicase and RNaseD.

While all known natural isolates of C. elegans contain multiple copies of the Tc1 transposon, which are active in the soma, Tc1 transposition is fully silenced in the germline of many strains. We mutagenized one such silenced strain and isolated mutants in which Tc1 had been activated in the germline ("mutators"). Interestingly, many other transposons of unrelated sequence had also become active. Most of these mutants are resistant to RNA interference (RNAi). We found one of the mutated genes, mut-7, to encode a protein with homology to RNaseD. This provides support for the notion that RNAi works by dsRNA-directed, enzymatic RNA degradation. We propose a model in which MUT-7, guided by transposon-derived dsRNA, represses transposition by degrading transposon-specific messengers, thus preventing transposase production and transposition.

Amino Acid Sequence↗
PubMed2002

RNA synthesis in a cage--structural studies of reovirus polymerase lambda3.

The reovirus polymerase and those of other dsRNA viruses function within the confines of a protein capsid to transcribe the tightly packed dsRNA genome segments. The crystal structure of the reovirus polymerase, lambda3, determined at 2.5 A resolution, shows a fingers-palm-thumb core, similar to those of other viral polymerases, surrounded by major N- and C-terminal elaborations, which create a cage-like structure, with four channels leading to the catalytic site. This "caged" polymerase has allowed us to visualize the results of several rounds of RNA polymerization directly in the crystals. A 5' cap binding site on the surface of lambda3 suggests a template retention mechanism by which attachment of the 5' end of the plus-sense strand facilitates insertion of the 3' end of the minus-sense strand into the template channel.

Amino Acid Sequence↗
PubMed1998

Efficient cloning of cDNA from grapevine leafroll-associated virus 4 and demonstration of probe specificity by the viral antibody.

Using a random-PCR method, a cDNA clone (LR4) was constructed from the replicative form dsRNA of grapevine leafroll-associated virus 4 (GLRaV-4). Northern blot analysis showed hybridization of LR4 to dsRNA in an extract of a Thompson Seedless grapevine clone from which GLRaV-4 was isolated originally by Hu et al. (1990). The cDNA clone was sequenced and shown to be specific to GLRaV-4 by reverse-transcription-PCR using GLRaV-4 particles enriched by the virus antibody coupled to magnetic beads. Reverse-transcription-PCR was used successfully to screen different varieties of grapevines for the virus. Western blot analysis of GLRaV-4 extracts from different varieties of infected grapevines revealed two distinct species of capsid protein with estimated Mr of either 35500 or 38000 depending on the variety used. Both proteins reacted with polyclonal as well as monoclonal antibodies.

Antibodies, Viral↗
PubMed1998

Rapid isolation of both double-stranded RNA and PCR-suitable DNA from the obligate biotrophic phytopathogenic fungus Uncinula necator using a commercially available reagent.

A method for rapid extraction of both double-stranded RNA (dsRNA) and DNA from an obligate biotrophic phytopathogenic fungus is described. Lyophilised fungal material is incubated in a commercial guanidium thiocyanate reagent. Proteins and cell debris are centrifuged by chloroform precipitation. After precipitation in isopropanol and washing in 75% ethanol, nucleic acids are resuspended in water (10 microl/mg fungal dry weight). DsRNA is directly visualised by agarose gel electrophoresis. DNA contained in 10-fold dilutions of the samples proved to be suitable for PCR-based experiments.

Ascomycota↗
PubMed2000

Double-stranded RNA interference in Trypanosoma brucei using head-to-head promoters.

The discovery of double-stranded RNA interference (dsRNAi) in Trypanosoma brucei provides a convenient method to generate knockout phenotypes in this protozoan parasite [Ngo H, Tschudi C, Gull K, Ullu E. Double-stranded RNA induces mRNA degradation in Trypanosoma brucei. Proc Natl Acad Sci USA 1998;95:14687-14692]. The presence of double-stranded RNA (dsRNA) dominantly silences gene expression in a sequence-specific manner by causing the corresponding endogenous RNA to be degraded. To simplify the generation of knockout phenotypes in T. brucei via dsRNAi, we used two promoters arranged as an inverted repeat on a plasmid. This promoter arrangement generates transcripts of both strands of DNA inserted between the promoters, which then form dsRNA. We have used plasmids encoding either two T. brucei ribosomal RNA promoters or two bacteriophage T7 promoters to interfere with expression of alpha-tubulin (TUB), green fluorescent protein (GFP), paraflagellar rod protein A (PFRA), flagellum-adhesion glycoprotein 1 (FLA1), and histone 2B (H2B) in T. brucei. We show here that FLA1 is required for flagellar attachment in T. brucei and that H2B is required for parasite growth. Thus, the two-promoter approach efficiently generates dsRNAi in T. brucei and can be used to produce both specific and random knockout phenotypes in T. brucei. This approach should be useful in generating knockout phenotypes in other kinetoplastid parasites including Trypanosoma cruzi and Leishmania.

Animals↗