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PubMed2004

Intratracheal double-stranded RNA plus interferon-gamma: a model for analysis of the acute phase response to respiratory viral infections.

Double-stranded (ds)RNA is made as a by-product of viral replication. Synthetic dsRNA induces virtually all of the same systemic symptoms as acute viral infections, such as fever and malaise. In order to develop a model of respiratory viral infections (such as influenza) suitable for use in gene knockout mice (where the deleted gene may affect viral replication), we examined C57BL/6 mouse body temperature and locomotor activity responses to the synthetic dsRNA polyriboinosinic.polyribocytidylic acid (poly[rI.rC]) introduced via the intratracheal (IT) route. We compared the IT poly[rI.rC] responses to the well-characterized intraperitoneal (IP) poly[rI.rC] responses. IT poly[rI.rC] failed to induce an acute phase response (APR) in mice, in contrast to IP poly[rI.rC]. However, addition of interferon (IFN)gamma to the IT poly[rI.rC] inoculum induced sustained hypothermia and suppressed locomotor activity responses with similar kinetics to those responses seen in acute mouse influenza. We further examined cytokine, antiviral, muscarinic M2 receptor and inducible nitric oxide synthase gene expression at 5 hr in the lungs of IT challenged mice. These studies suggested that priming the lung with IFNgamma could enhance proinflammatory (IL1beta, IL6, TNFalpha) cytokine gene expression and suppress interferon gene expression compared to IT poly[rI.rC] alone. No differences were detected for the other genes examined. While further molecular characterization of the model is required, we demonstrate that IT challenge with combined poly[rI.rC] and IFNgamma closely simulates the APR to an acute respiratory virus, and may serve as a suitable model for analyzing the molecular basis of the viral APR in gene knockout mice.

Acute-Phase Reaction↗
PubMed2004

RNA interference targeting cathepsin L and Z-like cysteine proteases of Onchocerca volvulus confirmed their essential function during L3 molting.

We describe the successful use of RNA interference (RNAi) to investigate gene function in the human filarial parasite Onchocerca volvulus third-stage larvae (L3). We targeted two specific gene products, the O. volvulus cathepsin L (Ov-CPL) and cathepsin Z-like (Ov-CPZ) cysteine proteases, which were proposed to function during O. volvulus L3 molting. We show that fluorescent-labeled Cy3-dsRNA corresponding to cpl or cpz regions encoding the mature enzymes can enter the larvae. The molting rate of larvae treated overnight with 0.5 mg ml(-1) cpl was reduced by 92% and 86% in comparison to normal control worms. It appeared that although the larvae started the molting process the last stage of molting, ecdysis was inhibited. The effect was gene specific, as larvae that did not molt in the presence of cpl or cpz dsRNA expressed the other cysteine protease, CPZ and CPL, respectively. This was confirmed by immunoelectron microscopy using antibodies directed against each enzyme. Our present study validate conclusively that both enzymes are essential for the molting of O. volvulus L3 to fourth-stage larvae. We also confirmed that the activity of the enzymes is specific to the changes that occur during the molting process on days 1-3, when the separation between the cuticles is in progress. The development of RNAi in O. volvulus L3 could further help study many of the abundant L3 and molting L3 genes identified through the filarial genome project, many of which, although have no attributed function, were identified as vaccine candidates or potential drug targets.

Animals↗
PubMed2004

RNA interference in neuroscience.

One of the most exciting findings in recent years has been the discovery of RNA interference (RNAi). RNAi is an important system that allows sequence-specific gene silencing through targeted degradation of mRNA by cognate double-stranded RNA (dsRNA). RNAi plays a role in endogenous cellular processes, such as developmental control and heterochromatin formation, and serves as an antiviral defense mechanism. Recent findings suggest that RNAi and related pathways are involved in protecting the genome against instabilities caused by transposons and repetitive sequences. Several rapidly developing RNAi methodologies provide a new approach for elucidation of gene functions. RNAi technology is also currently being evaluated as a potentially useful method to develop highly specific dsRNA-based gene-silencing therapeutics. In this paper, we review the use of RNAi in neuroscience research and as a possible therapeutic tool for treatment of neurological diseases.

Animals↗
PubMed2004

The Making of an siRNA.

An array of gene silencing pathways share a common early step: Dicer cleaves double-stranded RNA (dsRNA) into approximately 20-25 nucleotide fragments that direct the silencing machinery to specific targets. A recent report in Cell reveals how Dicer's two RNase III domains collaborate during dsRNA processing and sets the stage for a deeper understanding of Dicer's roles in later phases of silencing complex assembly.

Animals↗
PubMed2004

Know-how of RNA interference and its applications in research and therapy.

Double stranded RNA (dsRNA) mediates gene silencing in a sequence specific manner. Originally recognized in plants and lower organisms, it was recently extended to higher eukaryotes and established as an important evolutionary conserved phenomenon. It has been established that the double stranded short interfering RNAs (siRNAs) originate by the activity of a dsRNA-specific endonuclease, Dicer. siRNA in conjunction with a multiple enzyme complex called RNA-induced silencing complex (RISC) locates to the specific sites on mRNA and degrades it by endonuclease and exonuclease activities. In addition to gene silencing at transcript level (degradation of messenger RNA), siRNA was also shown to reduce the expression of protein by silencing of gene promoters via de novo methylation. By virtue of their specific gene silencing activity and owing to the recent discoveries on their plasmid and virus driven expression, small dsRNAs are being widely adopted in research and therapeutics. They are rapidly replacing the conventional gene knock-out technologies. siRNA libraries are also being recruited as a new tool in genome wide functional screenings. There is no doubt that further progress in understanding the mechanism of their action as well as strategies to achieve their tightly regulated and tissue specific expression will revolutionize basic and applied biomedical research.

Animals↗
PubMed2004

Lobotomy of genes: use of RNA interference in neuroscience.

Galen of Pergamon studied nerve function by shearing nerves in various species including monkeys, dogs, bulls and even elephants (humans being off limits to researchers; Sartan, 1954). An analogous strategy to determine gene function by ablating gene expression has recently been developed. RNA interference (RNAi) is a cellular response to double-stranded RNA (dsRNA) apparently as a defense against viral or transposon activity (Denli and Hannon, 2003; Dykxhoorn et al., 2003; Plasterk, 2002; Zamore, 2002). By activating this ancient defense mechanism through the introduction of artificial dsRNA, it is now possible to inhibit expression of almost any gene in almost any cell type, among them neuronal cells. In mammalian cells the active RNAi species must be short, approximately 21 nucleotide RNAs; these 21-bp species are called short interfering RNA (siRNA; Fig 1).

Animals↗
PubMed2006

Ribonuclease revisited: structural insights into ribonuclease III family enzymes.

Ribonuclease III (RNase III) enzymes occur ubiquitously in biology and are responsible for processing RNA precursors into functional RNAs that participate in protein synthesis, RNA interference and a range of other cellular activities. Members of the RNase III enzyme family, including Escherichia coli RNase III, Rnt1, Dicer and Drosha, share the ability to recognize and cleave double-stranded RNA (dsRNA), typically at specific positions or sequences. Recent biochemical and structural data have shed new light on how RNase III enzymes catalyze dsRNA hydrolysis and how substrate specificity is achieved. A major theme emerging from these studies is that accessory domains present in different RNase III enzymes are the key determinants of substrate selectivity, which in turn dictates the specialized biological function of each type of RNase III protein.

Bacterial Proteins↗
PubMed2006

Structure of the bacteriophage phi6 nucleocapsid suggests a mechanism for sequential RNA packaging.

Bacteriophage phi6 is an enveloped dsRNA virus with a segmented genome. Phi6 specifically packages one copy of each of its three genome segments into a preassembled polymerase complex. This leads to expansion of the polymerase complex, minus and plus strand RNA synthesis, and assembly of the nucleocapsid. The phi6 in vitro assembly and packaging system is a valuable model for dsRNA virus replication. The structure of the nucleocapsid at 7.5 A resolution presented here reveals the secondary structure of the two major capsid proteins. Asymmetric P1 dimers organize as an inner T = 1 shell, and P8 trimers organize as an outer T = 13 laevo shell. The organization of the P1 molecules in the unexpanded and expanded polymerase complex suggests that the expansion is accomplished by rigid body movements of the P1 monomers. This leads to exposure of new potential RNA binding surfaces to control the sequential packaging of the genome segments.

Bacteriophage phi 6↗
PubMed2004

Cellular and molecular mechanisms for immune modulation by deoxynivalenol and other trichothecenes: unraveling a paradox.

Macrophages, T cells, and B cells of the immune system are central targets of deoxynivalenol (DON) and other trichothecenes-mycotoxins that can be immunostimulatory or immunosuppressive depending on dose, exposure frequency and timing of functional immune assay. Notably, low dose trichothecene exposure transcriptionally and post-transcriptionally upregulates expression of cytokines, chemokines and inflammatory genes with concurrent immune stimulation, whereas high dose exposure promotes leukocyte apoptosis with concomitant immune suppression. DON and other trichothecenes, via a mechanism known as the ribotoxic stress response, bind to ribosomes and rapidly activate mitogen-activated protein kinases (MAPKs). The latter are important transducers of downstream signaling events related to immune response and apoptosis. Using cloned macrophages, our laboratory has identified two critical upstream transducers of DON-induced MAPK activation. One transducer is double-stranded RNA-(dsRNA)-activated protein kinase (PKR), a widely-expressed serine/theonine protein kinase that can be activated by dsRNA, interferon, and other agents. The second transducer is hematopoetic cell kinase (Hck), a non-receptor associated Src family kinase. Inhibitors and gene silencing studies have revealed that Hck and PKR play roles in DON induced gene expression and apoptosis. Future studies should focus on the molecular linkages between these kinases and trichothecene toxicity.

Adjuvants, Immunologic↗
PubMed2005

The selective inhibition of nitric oxide production in the avian macrophage cell line HD11.

The production of reactive nitrogen, nitric oxide (NO), has previously been demonstrated to be a major mechanism by which the innate immune system defends against microbial invasion. The induction of many antimicrobial mechanisms is regulated by numerous components during the transduction of the signal from the cell surface to the cell nucleus where response genes are upregulated. Toll-like cell surface receptor activation often leads to sequential modulation of protein tyrosine kinases (PTK), mitogen activated protein kinases (MAPK), degradation of I kappa B (IkappaB) regulatory molecules which, in turn, release the nuclear factor-kappa B (NF-kappaB) family proteins for translocation into the nucleus and subsequent gene transcription. The purpose of this study was to investigate components of the upstream signal transduction pathway induced by bacterial and viral-like stimulation of NO for antimicrobial defense by the transformed chicken macrophage cell line, HD11. We quantified the production of nitrite by chicken macrophages after exposure to selective pharmacological inhibitors of specific signal transduction components prior to stimulation by polyinosinic polycytidylic acid (poly I:C), formalin-fixed Enterococcus gallinarum (EG) or formalin-fixed Klebsiella pneumoniae (KP). We found that NO production induced by dsRNA or bacteria was reduced in a dose dependent manner by specific inhibitors of PTK, p38 MAPK, IkappaB, and NF-kappaB. Inhibition efficacy varied dependent on stimulation by bacterial or viral-like ligands. In general, NO production induced by bacterial stimulation was most effectively reduced by inhibition of p38 MAPK and least effectively reduced by inhibition of IkappaB. NF-kappaB and IkappaB inhibition affected NO production induced by dsRNA more than that induced by bacterial stimulation.

Animals↗
PubMed2005

West Nile virus replication interferes with both poly(I:C)-induced interferon gene transcription and response to interferon treatment.

West Nile virus (WNV), the leading cause of viral encephalitis in the United States, is an arthropod-transmitted member of the family Flaviviridae. We have explored the interaction of this positive-strand RNA virus with signaling pathways involved in induction of the host's innate immune response. Phosphorylation of STAT-1 in response to interferon (IFN) treatment and the ability of IFN to establish an antiviral state were reduced in WNV replicon-bearing cell lines. Similarly, the activation of IRF3 and stimulation of IFN-beta transcription in response to the double-stranded RNA (dsRNA) mimetic poly(I:C) were inhibited in replicon-bearing and WNV-infected HeLa cells. In contrast, WNV replicons did not affect IRF3 activation by Sendai virus infection, suggesting that not all IRF3 activating pathways are inhibited by WNV. Taken together, these findings demonstrate that WNV replication in cultured cells interferes with both the response to IFN and synthesis of IFN-beta in response to dsRNA.

Active Transport, Cell Nucleus↗
PubMed2005

Leukemia virus long terminal repeat activates NFkappaB pathway by a TLR3-dependent mechanism.

The long terminal repeat (LTR) region of leukemia viruses plays a critical role in tissue tropism and pathogenic potential of the viruses. We have previously reported that U3-LTR from Moloney murine and feline leukemia viruses (Mo-MuLV and FeLV) upregulates specific cellular genes in trans in an integration-independent way. The U3-LTR region necessary for this action does not encode a protein but instead makes a specific RNA transcript. Because several cellular genes transactivated by the U3-LTR can also be activated by NFkappaB, and because the antiapoptotic and growth promoting activities of NFkappaB have been implicated in leukemogenesis, we investigated whether FeLV U3-LTR can activate NFkappaB signaling. Here, we demonstrate that FeLV U3-LTR indeed upregulates the NFkappaB signaling pathway via activation of Ras-Raf-IkappaB kinase (IKK) and degradation of IkappaB. LTR-mediated transcriptional activation of genes did not require new protein synthesis suggesting an active role of the LTR transcript in the process. Using Toll-like receptor (TLR) deficient HEK293 cells and PKR(-/-) mouse embryo fibroblasts, we further demonstrate that although dsRNA-activated protein kinase R (PKR) is not necessary, TLR3 is required for the activation of NFkappaB by the LTR. Our study thus demonstrates involvement of a TLR3-dependent but PKR-independent dsRNA-mediated signaling pathway for NFkappaB activation and thus provides a new mechanistic explanation of LTR-mediated cellular gene transactivation.

3T3 Cells↗
PubMed2005

Variation in the ability of human influenza A viruses to induce and inhibit the IFN-beta pathway.

We investigated the ability of a selection of human influenza A viruses, including recent clinical isolates, to induce IFN-beta production in cultured cell lines. In contrast to the well-characterized laboratory strain A/PR/8/34, several, but not all, recent isolates of H3N2 viruses resulted in moderate IFN-beta stimulation. Through the generation of recombinant viruses, we were able to show that this is not due to a loss of the ability of the NS1 genes to suppress IFN-beta induction; indeed, the NS1 genes behaved similarly with respect to their abilities to block dsRNA signaling. Interestingly, replication of A/Sydney/5/97 virus was less susceptible to pre-treatment with IFN-alpha than the other viruses. In contrast to the universal effect on dsRNA signaling, we noted differences in the effect of NS1 proteins on expression of interferon stimulated genes and also genes induced by a distinct pathway. The majority of NS1 proteins blocked expression from both IFN-dependent and TNF-dependent promoters by an apparent post-transcriptional mechanism. The NS1 gene of A/PR/8/34 NS1 did not confer these blocks. We noted striking differences in the cellular localization of different influenza A virus NS1 proteins during infection, which might explain differences in biological activity.

Animals↗
PubMed2006

Binding of the influenza A virus NS1 protein to PKR mediates the inhibition of its activation by either PACT or double-stranded RNA.

A major component of the cellular antiviral system is the latent protein kinase PKR, which is activated by binding to either double-stranded RNA (dsRNA) or the cellular PACT protein. Activated PKR phosphorylates the translation initiation factor eIF2, thereby inhibiting viral and cellular protein synthesis and virus replication. To evade the antiviral effects of PKR, many viruses, including influenza A virus, have evolved multiple mechanisms. For influenza A virus, the non-structural (NS1A) protein plays a major role in blocking activation of PKR during virus infection. The mechanism by which the NS1A protein inhibits PKR activation in infected cells has not been established. In the present study, we first carried out a series of in vitro experiments to determine whether the NS1A protein could utilize a common mechanism to inhibit PKR activation by both PACT and dsRNA, despite their different modes of activation. We demonstrated that the direct binding of the NS1A protein to the N-terminal 230 amino acid region of PKR can serve as such a common mechanism and that this binding does not require the RNA-binding activity of the NS1A protein. The lack of requirement for NS1A RNA-binding activity for the inhibition of PKR activation in vivo was established by two approaches. First, we showed that an NS1A protein lacking RNA-binding activity, like the wild-type (wt) protein, blocked PKR activation by PACT in vivo, as well as the downstream effects of PKR activation in cells, namely, eIF2 phosphorylation and apoptosis. In addition, we demonstrated that PKR activation is inhibited in cells infected with a recombinant influenza A virus expressing NS1A mutant protein that cannot bind RNA, as is the case in cells infected with wild-type influenza A virus.

Apoptosis↗
PubMed2006

siRNA targeting vaccinia virus double-stranded RNA binding protein [E3L] exerts potent antiviral effects.

The Vaccinia virus gene, E3L, encodes a double-stranded RNA [dsRNA]-binding protein. We hypothesized that, owing to the critical nature of dsRNA in triggering host innate antiviral responses, E3L-specific small-interfering RNAs [siRNAs] should be effective antiviral agents against pox viruses, for which Vaccinia virus is an appropriate surrogate. In this study, we have utilized two human cell types, namely, HeLa and 293T, one which responds to interferon [IFN]-beta and the other produces and responds to IFN-beta, respectively. The antiviral effects were equally robust in HeLa and 293T cells. However, in the case of 293T cells, several distinct features were observed, when IFN-beta is activated in these cells. Vaccinia virus replication was inhibited by 97% and 98% as compared to control infection in HeLa and 293T cells transfected with E3L-specific siRNAs, respectively. These studies demonstrate the utility of E3L-specific siRNAs as potent antiviral agents for small pox and related pox viruses.

Antiviral Agents↗
PubMed2004

RNA silencing of rotavirus gene expression.

RNA interference (RNAi) is a double-stranded RNA (dsRNA)-triggered mechanism for suppressing gene expression, which is conserved in evolution and has emerged as a powerful tool to study gene function. Rotaviruses, the leading cause of severe diarrhea in young children, are formed by three concentric layers of protein, and a genome composed of 11 segments of dsRNA. Here, we show that the RNAi machinery can be triggered to silence rotavirus gene expression by sequence-specific short interfering RNAs (siRNAs). RNAi is also useful for the study of the virus-cell interactions, through the silencing of cellular genes that are potentially important for the replication of the virus. Interestingly, while the translation of mRNAs is readily stopped by the RNAi machinery, the viral transcripts involved in virus genome replication do not seem to be susceptible to RNAi. Since gene silencing by RNAi is very efficient and specific, this system could become a novel therapeutic approach for rotavirus and other virus infections, once efficient methods for in vivo delivery of siRNAs are developed. Although the use of RNAi as an antiviral therapeutic tool remains to be demonstrated, there is no doubt that this technology will influence drastically the way postgenomic virus research is conducted.

Gene Expression Regulation, Viral↗
PubMed2004

Plant viral suppressors of RNA silencing.

RNA silencing is an ancient eukaryotic pathway in which double stranded RNA (dsRNA) triggers destruction of related RNAs in the cell. Early studies in plants pointed to a role for this pathway as a defense against viruses. Most known plant viruses have RNA genomes and replicate via dsRNA intermediates, thereby serving as potent inducers of RNA silencing early in replication and as silencing targets later in infection. Because RNA silencing is an antiviral mechanism, it is not surprising that many plant viruses encode suppressors of RNA silencing. This review focuses on the currently known plant virus encoded suppressors of silencing and the functional assays used to identify these proteins. Because they interfere with silencing at different points in the pathway, these viral suppressors are powerful tools to help unravel the mechanism of RNA silencing in plants.

Cysteine Endopeptidases↗
PubMed2005

Three unrelated viruses occur in a single isolate of Gremmeniella abietina var. abietina type A.

Five enclosed double-stranded RNA (dsRNA) bands in electrophoresis, probably of viral origin, were found from a single isolate (SurS4) of Gremmeniella abietina var. abietina type A. Analysis of the dsRNAs revealed that they represented three different viruses, named as Gremmeniella abietina mitochondrial RNA virus S2 (GaMRV-S2), Gremmeniella abietina RNA virus MS2 (GaRV-MS2) and Gremmeniella abietina RNA virus L2 (GaRV-L2). The genome of GaMRV-S2 was 2587 base pairs (bp) long and had a very low GC content (31%). Sequence variations occurred at both ends. The genome coded for a putative RNA-dependent RNA polymerase (RdRp) under a mitochondrial translation code. The GaRV-MS2 genome was composed of three dsRNA molecules (1781 bp, 1586 bp and 1186 bp). They coded for a putative RdRp, a coat protein (CP) and a protein with an unknown function, respectively. The GaRV-L2 genome was 5129 bp long and contained two ORFs. The 5'-proximal ORF coded for a putative CP, whereas the 3'-proximal ORF encoded for a putative RdRp. The buoyant density of GaRV-MS2 and GaRV-L2 were 1.37 and 1.42 g/ml, respectively. GaMRV-S2, GaRV-MS2 and GaRV-L2 were closely related to the previously described viruses GaMRV-S1, GaRV-MS1 and GaRV-L1, respectively, and are putative members of the genera Mitovirus, Partitivirus and Totivirus, respectively. This is the first report on the occurrence of viruses of all these different genera in a single fungal isolate.

Amino Acid Sequence↗