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At least 1,423 records · Page 79Linked to original sources
PubMed2000

Characterization of avian reovirus non structural protein sigmaNS synthesized in Escherichia coli.

The coding region of avian reovirus S1133 genomic segment S4, encoding the non structural protein sigmaNS, was inserted into expression vector pET28a and the protein was expressed in Escherichia coli BL21(DE3) as a fusion protein containing a C-terminal peptide with six tandem histidines (His-tag). The expressed protein (esigmaNS) consistent with the expected molecular size of the avian reovirus protein sigmaNS synthesized in infected cells was readily purified by His-Bind Resin. The esigmaNS was further confirmed to be indistinguishable from viral sigmaNS by immunoblot analysis. The esigmaNS binds 32P-labeled ssRNA probe produced by run-off transcription of clone pGEM-3Zf(+)S4. The binding activity is blocked by heterologous yeast rRNA, but not by homologous avian reovirus dsRNA and heterologous infectious bursal disease virus dsRNA and salmon sperm dsDNA. Therefore, the ssRNA-binding activity of the expressed protein sigmaNS is non sequence-specific, similar to that previously described for viral sigmaNS purified from avian reovirus infected cell extracts. In addition, the recent data also show that the optimal salt (NaCl) concentration and pH for its binding are 100-150 mM and 7.0, respectively, in terms of the UV cross-linking and RNase A treatment of the reaction mixtures prior to the denaturing gel analysis.

Animals↗
PubMed2003

Construction of artificial virus-like particles exposing HIV epitopes, and the study of their immunogenic properties.

One of the major problems in the development of successful recombinant vaccines against human immunodeficiency virus (HIV) is that of correct identification of a safe and effective vaccine delivery system with which to induce protective immunity using soluble protein antigens. An original method for constructing artificial immunogens in the form of spherical particles with yeast dsRNA in the center and hybrid proteins exposing epitopes of an infectious agent on the surface is reported. The dsRNA and the proteins were linked with spermidine-polyglucin-glutathione conjugates. Particles exposing HIV-1 epitopes were constructed, and their immunogenicity tested.

AIDS Vaccines↗
PubMed2003

A double-stranded RNA mycovirus confers hypovirulence-associated traits to Botrytis cinerea.

Botrytis cinerea CCg425 contains a 33-nm isometric mycovirus whose genome is a 6.8-kb double-stranded RNA (dsRNA) molecule. Virulence bioassays, performed by direct plug mycelial inoculation on bean plant leaves, showed that B. cinerea CCg425 displays less fungal aggressivity than B. cinerea CKg54, a virulent fungal strain that is not infected by dsRNA mycoviruses. B. cinerea CCg425 also showed lower laccase activity and conidiation rate than B. cinerea CKg54. Furthermore, infection of B. cinerea CKg54 with viral particles purified from B. cinerea CCg425 resulted in diminished virulence of the infected fungus. Collectively, our results indicate that mycovirus infection confers hypovirulence to the fungal host.

Botrytis↗
PubMed2001

Cloning of a novel 2',5'-oligoadenylate synthetase-like molecule, Oasl5 in mice.

The 2',5'-oligoadenylate synthetase (2-5OAS) is a enzyme that catalyzes synthesis of 2',5'-oligoadenylates (2-5A) in a dsRNA-dependent manner, and known as a major component of the IFN-induced host defense mechanisms against microbial infections. Here, we report the presence of a novel 2-5OAS-like molecule, termed Oasl5, in mice. The size of Oasl5 cDNA was about 2 kb and encoded a protein consisting of 362 aa. The amino acid sequence showed 76% similarity to the mouse 2-5OAS, however, several motifs being important for the enzyme activity were not conserved. The Oasl5 mRNA was most significantly expressed in the brain, and relatively weak expression was found in other organs such as the spleen, kidney, ovary and testis. It was also expressed in embryonic stem (ES) cells. The Oasl5 mRNA expression in ES cells was elevated 5-fold after treatment with IFN and about 2-fold in the brain when stimulated with IFN inducer, polyinosinic-polycytidylic acid (poly[I:C]). In situ hybridization analysis revealed that Oasl5 is expressed in neurons in the central nervous system in adult mice. When Oasl5 was expressed in E. coli, it yielded 42 kDa protein that binds to dsRNA, but it did not show oligoadenylate synthetase activity. These findings suggest a novel function of Oasl5, which are independent of oligoadenylate synthetase activity, in the brain and developing embryos.

2',5'-Oligoadenylate Synthetase↗
PubMedDate not supplied

Identification of viruses with bi- and trisegmented double-stranded RNA genome in faeces of children with gastroenteritis.

Polyacrylamide gel electrophoresis of nucleic acid extracted from faecal samples of diarrhoeic children revealed the presence of group A rotavirus in 50 (23.4%) samples and group C rotavirus in 1 (0.5%) sample out of 214 tested. One other sample showed the presence of three bands (with apparent length of 2.92, 2.37 and 1.32 kbp) which by enzymatic digestion analysis, were shown to consist of double-stranded RNA (dsRNA). The sample was shown by electron microscopy to contain virus particles with a diameter of 32-34 nm. On the basis of morphology and genomic characteristics, this virus closely resembles a virus hitherto described only in chickens by Leite et al. in 1990 and tentatively named "picotrirnavirus". From the same group of 214, one sample containing a "picobirnavirus" was also identified. Thus, small icosahedral viruses with either a bior trisegmented dsRNA genome appear to infect humans. However, their pathogenic potential remains to be established.

Child↗
PubMedDate not supplied

Characterization and structural localization of the reovirus lambda 3 protein.

The putative reovirus RNA polymerase, protein lambda 3, was characterized using antiserum prepared against a TrpE-lambda 3 fusion protein synthesized in Escherichia coli. Immunofluorescence microscopy showed that lambda 3 accumulated in perinuclear inclusion bodies in reovirus-infected cells. Analysis of lambda 3 accumulation in infected cells indicates that, once synthesized, lambda 3 is quite stable throughout the course of infection. Anti-lambda 3 serum did not immunoprecipitate virions, core particles or iodinated surface proteins of either virions or cores. These results indicate that lambda 3 is located in the inner part of the core. Experiments involving urea denaturation of purified reovirus cores indicate that lambda 3 cannot be selectively removed from the core without total denaturation of the core structure. When the dsRNA genome was eliminated from the core, lambda 3 remained associated with the other viral proteins in the core. Thus, lambda 3 appears to be a stable, structural component of the reovirus core, not bound to genomic dsRNA or free in soluble form inside the core.

Animals↗
PubMed2003

Rhinovirus increases human beta-defensin-2 and -3 mRNA expression in cultured bronchial epithelial cells.

Human beta-defensins (hBDs) are antimicrobial peptides that play important roles in host defense against infection, inflammation and immunity. Previous studies showed that micro-organisms and proinflammatory mediators regulate the expression of these peptides in airway epithelial cells. The aim of the present study was to investigate the modulation of expression of hBDs in cultured primary bronchial epithelial cells (PBEC) by rhinovirus-16 (RV16), a respiratory virus responsible for the common cold and associated with asthma exacerbations. RV16 was found to induce expression of hBD-2 and -3 mRNA in PBEC, but did not affect hBD-1 mRNA. Viral replication appeared essential for rhinovirus-induced beta-defensin mRNA expression, since UV-inactivated rhinovirus did not increase expression of hBD-2 and hBD-3 mRNA. Exposure to synthetic double-stranded RNA (dsRNA) molecule polyinosinic:polycytidylic acid had a similar effect as RV16 on mRNA expression of these peptides in PBEC. In line with this, PBEC were found to express TLR3, a Toll-like receptor involved in recognition of dsRNA. This study shows that rhinovirus infection of PBEC leads to increased hBD-2 and hBD-3 mRNA expression, which may play a role in both the uncomplicated common cold and in virus-associated exacerbations of asthma.

Bronchi↗
PubMed2000

RNAi of the receptor tyrosine phosphatase HmLAR2 in a single cell of an intact leech embryo leads to growth-cone collapse.

Receptor protein tyrosine phosphatases (RPTPs) are important for growth-cone migration [1-5], but their specific roles have yet to be defined. Previously, we showed that the growth cones of the Comb cell, an embryonic cell in the leech, express high levels of an RPTP called HmLAR2 [6,7]. Here, we report the use of RNA interference (RNAi) to block expression of HmLAR2 in individual Comb cells in the developing embryo. HmLAR2 mRNA levels were reduced in the soma, processes and growth cones of Comb cells injected with double-stranded RNA (dsRNA) for HmLAR2, but no decrease was detected when control dsRNAs were injected. Consistent with this observation, the level of phosphotyrosine increased significantly in the growth cones of Comb cells injected with HmLAR2 dsRNA. Within 24 hours, the growth cones of treated cells showed a distinct collapsed phenotype, with sharp reductions in lamellipodial surface area and in numbers of filopodia. These experiments indicate a key role for LAR-like RPTPs in maintaining the integrity of the growth cone.

Animals↗
PubMed2002

PPW-1, a PAZ/PIWI protein required for efficient germline RNAi, is defective in a natural isolate of C. elegans.

One of the remarkable aspects about RNA interference (RNAi) in Caenorhabditis elegans is that the trigger molecules, dsRNA, can be administered via the animal's food. We assayed whether this feature is a universal property of the species by testing numerous strains that have been isolated from different parts of the globe. We found that one isolate from Hawaii had a defect in RNAi that was specific to the germline and was a result of multiple mutations in a PAZ/PIWI domain-containing protein, which we named PPW-1. Deleting ppw-1 in the canonical C. elegans strain Bristol N2 makes it resistant to feeding of dsRNA directed against germline-expressed genes. PPW-1 belongs to the Argonaute family of proteins, which act in posttranscriptional gene silencing and development, and is homologous to the RNAi gene rde-1. Our data indicate that at least two members of this family are required for complete and effective RNAi in C. elegans.

Amino Acid Sequence↗
PubMed1995

PKR: a new name and new roles.

The double-stranded RNA (dsRNA)-activated protein kinase, now called PKR, was first discovered by virtue of its ability to phosphorylate translation initiation factor eIF-2 and inhibit its activity. Recent studies have shown that expression of inactive mutants of PKR in cultured cells causes them to acquire characteristics typical of transformed cells. These and other findings indicate that PKR plays a role in the normal control of cell growth and differentiation. It seems likely that, in addition to eIF-2, PKR has other substrates including the protein I-kappa B, which regulates the transcription of certain genes. Indeed, it now seems likely that PKR mediates the regulation of selected genes by dsRNA.

Enzyme Activation↗
PubMed2000

Methyl-RNA: an evolutionary bridge between RNA and DNA?

Given the apparent limitation of double-stranded RNA (dsRNA) genomes to about 30 kb, together with the complexity of DNA synthesis, it appears difficult for a dsRNA genome to encode all the information required before the transition from an RNA to a DNA genome. Ribonucleotide reductase itself, which synthesizes deoxyribonucleotides from ribonucleotides, requires complex protein radical chemistry, and RNA world genomes may have reached their limits of coding capacity well before such complex enzymes had evolved. The transition from RNA to DNA thus appears to require intermediate steps, and we suggest that the naturally occurring 2'-O-methylated RNA, with chemical properties intermediate between RNA and DNA, is a suitable candidate.

DNA↗
PubMed2001

Self-assembly of a viral molecular machine from purified protein and RNA constituents.

We present the assembly of the polymerase complex (procapsid) of a dsRNA virus from purified recombinant proteins. This molecular machine packages and replicates viral ssRNA genomic precursors in vitro. After addition of an external protein shell, these in vitro self-assembled viral core particles can penetrate the host plasma membrane and initiate a productive infection. Thus, a viral procapsid has been assembled and rendered infectious using purified components. Using this system, we have studied the mechanism of assembly of the common dsRNA virus shell and the incorporation of a symmetry mismatch within an icosahedral capsid. Our work demonstrates that this molecular machine, self-assembled under defined conditions in vitro, can function in its natural environment, the cell cytoplasm.

Capsid↗
PubMed2000

dADAR, a Drosophila double-stranded RNA-specific adenosine deaminase is highly developmentally regulated and is itself a target for RNA editing.

We have identified a homolog of the ADAR (adenosine deaminases that act on RNA) class of RNA editases from Drosophila, dADAR. The dADAR locus has been localized to the 2B6-7 region of the X chromosome and the complete genomic sequence organization is reported here. dADAR is most homologous to the mammalian RNA editing enzyme ADAR2, the enzyme that specifically edits the Q/R site in the pre-mRNA encoding the glutamate receptor subunit GluR-B. Partially purified dADAR expressed in Pichia pastoris has robust nonspecific A-to-I deaminase activity on synthetic dsRNA substrates. Transcripts of the dADAR locus originate from two regulated promoters. In addition, alternative splicing generates at least four major dADAR isoforms that differ at their amino-termini as well as altering the spacing between their dsRNA binding motifs. dADAR is expressed in the developing nervous system, making it a candidate for the editase that acts on para voltage-gated Na+ channel transcripts in the central nervous system. Surprisingly, dADAR itself undergoes developmentally regulated RNA editing that changes a conserved residue in the catalytic domain. Taken together, these findings show that both transcription and processing of dADAR transcripts are under strict developmental control and suggest that the process of RNA editing in Drosophila is dynamically regulated.

Adenosine Deaminase↗
PubMed2002

Functional siRNA expression from transfected PCR products.

RNA interference (RNAi) is a process in which double-stranded RNA (dsRNA) induces the postranscriptional degradation of homologous transcripts. RNAi can be initiated by exposing cells to dsRNA either via transfection or endogenous expression. In mammalian systems, the sequence-specific RNAi effect has been observed by expression of 21-23 base transcripts capable of forming duplexes, or via expression of short hairpin RNAs. We describe here a facile PCR based strategy for rapid synthesis of siRNA expression units and their testing in mammalian cells. The siRNA expression constructs are constructed by PCR, and the PCR products are directly transfected into mammalian cells resulting in functional expression of siRNAs. This approach should prove useful for identification of optimal siRNA-target combinations and for multiplexing siRNA expression in mammalian cells.

Blotting, Northern↗
PubMed2000

Double-stranded RNA adenosine deaminases ADAR1 and ADAR2 have overlapping specificities.

Adenosine deaminases that act on RNA (ADARs) deaminate adenosines to produce inosines within RNAs that are largely double-stranded (ds). Like most dsRNA binding proteins, the enzymes will bind to any dsRNA without apparent sequence specificity. However, once bound, ADARs deaminate certain adenosines more efficiently than others. Most of what is known about the intrinsic deamination specificity of ADARs derives from analyses of Xenopus ADAR1. In addition to ADAR1, mammalian cells have a second ADAR, named ADAR2; the deamination specificity of this enzyme has not been rigorously studied. Here we directly compare the specificity of human ADAR1 and ADAR2. We find that, like ADAR1, ADAR2 has a 5' neighbor preference (A approximately U > C = G), but, unlike ADAR1, also has a 3' neighbor preference (U = G > C = A). Simultaneous analysis of both neighbor preferences reveals that ADAR2 prefers certain trinucleotide sequences (UAU, AAG, UAG, AAU). In addition to characterizing ADAR2 preferences, we analyzed the fraction of adenosines deaminated in a given RNA at complete reaction, or the enzyme's selectivity. We find that ADAR1 and ADAR2 deaminate a given RNA with the same selectivity, and this appears to be dictated by features of the RNA substrate. Finally, we observed that Xenopus and human ADAR1 deaminate the same adenosines on all RNAs tested, emphasizing the similarity of ADAR1 in these two species. Our data add substantially to the understanding of ADAR2 specificity, and aid in efforts to predict which ADAR deaminates a given editing site adenosine in vivo.

Adenosine Deaminase↗
PubMed2001

Selective binding by the RNA binding domain of PKR revealed by affinity cleavage.

The RNA-dependent protein kinase (PKR) is regulated by the binding of double-stranded RNA (dsRNA) or single-stranded RNAs with extensive duplex secondary structure. PKR has an RNA binding domain (RBD) composed of two copies of the dsRNA binding motif (dsRBM). The dsRBM is an alpha-beta-beta-beta-alpha structure present in a number of proteins that bind RNA, and the selectivity demonstrated by these proteins is currently not well understood. We have used affinity cleavage to study the binding of PKR's RBD to RNA. In this study, we site-specifically modified the first dsRBM of PKR's RBD at two different amino acid positions with the hydroxyl radical generator EDTA.Fe. Cleavage by these proteins of a synthetic stem-loop ligand of PKR indicates that PKR's dsRBMI binds the RNA in a preferred orientation, placing the loop between strands beta1 and beta2 near the single-stranded RNA loop. Additional cleavage experiments demonstrated that defects in the RNA stem, such as an A bulge and two GA mismatches, do not dictate dsRBMI's binding orientation preference. Cleavage of VA(I) RNA, an adenoviral RNA inhibitor of PKR, indicates that dsRBMI is bound near the loop of the apical stem of this RNA in the same orientation as observed with the synthetic stem-loop RNA ligands. This work, along with an NMR study of the binding of a dsRBM derived from the Drosophila protein Staufen, indicates that dsRBMs can bind stem-loop RNAs in distinct ways. In addition, the successful application of the affinity cleavage technique to localizing dsRBMI of PKR on stem-loop RNAs and defining its orientation suggests this approach could be applied to dsRBMs found in other proteins.

Adenoviridae↗
PubMed2002

Characterization of subunit-specific interactions in a double-stranded RNA virus: Raman difference spectroscopy of the phi6 procapsid.

The icosahedral core of a double-stranded (ds) RNA virus hosts RNA-dependent polymerase activity and provides the molecular machinery for RNA packaging. The stringent requirements of dsRNA metabolism may explain the similarities observed in core architecture among a broad spectrum of dsRNA viruses, from the mammalian rotaviruses to the Pseudomonas bacteriophage phi6. Although the structure of the assembled core has been described in atomic detail for Reoviridae (blue tongue virus and reovirus), the molecular mechanism of assembly has not been characterized in terms of conformational changes and key interactions of protein constituents. In the present study, we address such questions through the application of Raman spectroscopy to an in vitro core assembly system--the procapsid of phi6. The phi6 procapsid, which comprises multiple copies of viral proteins P1 (copy number 120), P2 (12), P4 (72), and P7 (60), represents a precursor of the core that is devoid of RNA. Raman signatures of the procapsid, its purified recombinant core protein components, and purified sub-assemblies lacking either one or two of the protein components have been obtained and interpreted. The major procapsid protein (P1), which forms the skeletal frame of the core, is an elongated and monomeric molecule of high alpha-helical content. The fold of the core RNA polymerase (P2) is also mostly alpha-helical. On the other hand, the folds of both the procapsid accessory protein (P7) and RNA-packaging ATPase (P4) are of the alpha/beta type. Raman difference spectra show that conformational changes occur upon interaction of P1 with either P4 or P7 in the procapsid. These changes involve substantial ordering of the polypeptide backbone. Conversely, conformations of procapsid subunits are not significantly affected by interactions with P2. An assembly model is proposed in which P1 induces alpha-helix in P4 during formation of the nucleation complex. Subsequently, the partially disordered P7 subunit is folded within the context of the procapsid shell.

Bacteriophage phi 6↗
PubMed2005

The structure of a mixed LNA/DNA:RNA duplex is driven by conformational coupling between LNA and deoxyribose residues as determined from 13C relaxation measurements.

A study of the internal dynamics of an LNA/DNA:RNA duplex has been performed to further characterize the conformational changes associated with the incorporation of locked nucleic acid (LNA) nucleotides in a DNA:RNA duplex. In general, it was demonstrated that the LNA/DNA:RNA duplex has a very high degree of order compared to dsDNA and dsRNA duplexes. The order parameters of the aromatic carbon atoms in the LNA/DNA strand are uniformly high, whereas a sharp drop in the degree of order was seen in the RNA strand in the beginning of the AUAU stretch in the middle of the strand. This can be related to a return to normal dsRNA dynamics for the central A:U base pair. The high order of the heteroduplex is consistent with preorganization of the chimera strand for an A-form duplex conformation. These results partly explain the dramatic increase in T(m) of the chimeric heteroduplex over dsDNA and DNA:RNA hybrids of the same sequence.

Base Sequence↗