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At least 235 records · Page 13Linked to original sources
PubMed2003

The Orf virus E3L homologue is able to complement deletion of the vaccinia virus E3L gene in vitro but not in vivo.

Orf virus (OV), the prototypic parapoxvirus, is resistant to the effects of interferon (IFN) and this function of OV has been mapped to the OV20.0L gene. The protein product of this gene shares 31% amino acid identity to the E3L-encoded protein of vaccinia virus (VV) that is required for the broad host range and IFN-resistant phenotype of VV in cells in culture and for virulence of the virus in vivo. In this study we investigated whether the distantly related OV E3L homologue could complement the deletion of E3L in VV. The recombinant VV (VV/ORF-E3L) expressing the OV E3L homologue in place of VV E3L was indistinguishable from wt VV in its cell-culture phenotype. But VV/ORF-E3L was over a 1000-fold less pathogenic than wt VV (LD(50) > 5 x 10(6) PFU, compared to LD(50) of wtVV = 4 x 10(3) PFU) following intranasal infection of mice. While wt VV spread to the lungs and brain and replicated to high titers in the brain of infected mice, VV/ORF-E3L could not be detected in the lungs or brain following intranasal infection. VV/ORF-E3L was at least 100,000-fold less pathogenic than wt VV on intracranial injection. Domain swap experiments demonstrate that the difference in pathogenesis maps to the C-terminal domain of these proteins. This domain has been shown to be required for the dsRNA binding function of the VV E3L.

Amino Acid Sequence↗
PubMed1997

Inscuteable and Staufen mediate asymmetric localization and segregation of prospero RNA during Drosophila neuroblast cell divisions.

When neuroblasts divide, inscuteable acts to coordinate protein localization and mitotic spindle orientation, ensuring that asymmetrically localized determinants like Prospero partition into one progeny. staufen encodes a dsRNA-binding protein implicated in mRNA transport in oocytes. We demonstrate that prospero RNA is also asymmetrically localized and partitioned during neuroblast cell divisions, a process requiring both inscuteable and staufen. Inscuteable and Staufen interact and colocalize with prospero RNA on the apical cortex of interphase neuroblasts. Staufen binds prospero RNA in its 3'UTR. Our findings suggest that Inscuteable nucleates an apical complex and is required for protein localization, spindle orientation, and RNA localization. Stau, as one component of this complex, is required only for RNA localization. Hence staufen also acts zygotically, downstream of inscuteable, to effect aspects of neuroblast asymmetry.

Animals↗
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↗
PubMed1998

Orf virus encodes a homolog of the vaccinia virus interferon-resistance gene E3L.

A homolog of the vaccinia virus (VAC) interferon resistance gene E3L has been discovered in orf virus strain NZ-2, a parapoxvirus that infects sheep, goats and humans. The gene is located 20 kb from the left terminus of the orf virus genome and is transcribed towards this terminus. RNase protection studies have been used to define the limits of the gene and Northern analysis revealed that it is expressed early in infection. The predicted amino acid sequence of the orf virus protein shares 31% identity (57% similarity) with the VAC E3L protein. Four of the six residues identified as being essential to dsRNA binding in the vaccinia virus protein are conserved in the orf virus protein whilst the other two amino acid changes are conservative substitutions. The orf virus gene has been sequenced in two other orf virus strains which vary markedly in their ability to produce experimental lesions in vivo. Their predicted protein sequences vary by less than 3% from the NZ-2 protein. The recombinant orf virus protein, expressed as a fusion protein in E. coli, bound double-stranded (ds)RNA but not dsDNA, single-stranded (ss)DNA or ssRNA . This is the first demonstration of a VAC E3L-like gene encoded by a parapoxvirus.

Amino Acid Sequence↗
PubMed1998

The orf virus OV20.0L gene product is involved in interferon resistance and inhibits an interferon-inducible, double-stranded RNA-dependent kinase.

The parapoxvirus orf virus was resistant to type 1 (IFN-alpha) and type 2 (IFN-gamma) interferons in cultures of ovine cells. The recently identified orf virus OV20.0L gene exhibits 31% predicted amino acid identity to the vaccinia virus E3L interferon-resistance gene, and is referred to as the (putative) orf virus interferon-resistance gene (OVIFNR). The objective of this study was to determine whether OVIFNR was involved in interferon resistance. Recombinant OVIFNR as a thioredoxin fusion protein (OVIFNR-Tx) inhibited the activation (by autophosphorylation) of an interferon-inducible, double-stranded (ds) RNA-dependent kinase (PKR) of sheep, which was shown to bind dsRNA (poly I:C). PKR in other species is involved in the inhibition of protein synthesis as part of the antiviral state in infected cells. Virus-infected cell lysates, but not control lysates, from cells grown in the presence of cytosine arabinoside also contained PKR inhibitory activity, which indicated that the inhibitory activity was associated with early viral gene expression. Significantly, the OVIFNR gene expressed in interferon-treated ovine fibroblasts protected the unrelated Semliki Forest virus from the antiviral effect of both type 1 and type 2 interferons. Taken together, the results indicate that the OVIFNR gene functions as an interferon-resistance gene, the product of which inhibits PKR in a similar way to the vaccinia virus E3L gene product.

Animals↗
PubMed1999

The expression of both domains of the 69/71 kDa 2',5' oligoadenylate synthetase generates a catalytically active enzyme and mediates an anti-viral response.

The 2',5' oligoadenylate synthetase (OAS) represents a family of interferon-induced proteins which, when activated by double-stranded (ds) RNA, polymerizes ATP into 2',5'-linked oligomers with the general formula pppA(2'p5'A)n, where n >/= 1. The 69-kDa form of human OAS has two isoforms (p69 and p71) that are identical for their first 683 amino acids and consist of two homologous and adjacent domains, each homologous to the small 40-kDa OAS. Here, we demonstrate that mRNA species specific for the isoforms p69 and p71 are enhanced in interferon-treated cells, with the p69 mRNA being more abundant than that of p71. In transfected cells, both isoforms could be expressed independently to generate enzymes with similar catalytic activity, typical of the natural 69-kDa OAS from interferon-treated cells. On the other hand, deletion mutants expressing either the N- or C-terminal domain common in p69 and p71 were greatly unstable and were found to be devoid of catalytic activity, in spite of the capacity of the C-terminal domain to bind dsRNA. Finally, we show that murine cell lines stably expressing either p69 or p71 isoforms partially resist infection by the encephalomyocarditis virus. These results indicate that both isoforms of the 69-kDa form of 2',5' OAS are expressed in interferon-treated cells, and that each isoform could be implicated in the mechanism of the anti-viral action of interferon.

2',5'-Oligoadenylate Synthetase↗
PubMed2005

Tyrosine phosphorylation acts as a molecular switch to full-scale activation of the eIF2alpha RNA-dependent protein kinase.

Phosphorylation of the alpha-subunit of translation eukaryotic initiation factor-2 (eIF2) leads to the inhibition of protein synthesis in response to diverse conditions of stress. Serine/threonine RNA-dependent protein kinase (PKR) is an eIF2alpha kinase family member induced by type I IFN and activated in response to dsRNA or virus infection. Herein, we demonstrate that human PKR is a dual specificity kinase phosphorylated at Y101, Y162 and Y293 in vitro and in vivo. Site-specific tyrosine phosphorylation is essential for efficient dsRNA-binding, dimerization, kinase activation and eIF2alpha phosphorylation of PKR. Biologically, tyrosine phosphorylation of PKR mediates the antiviral and antiproliferative properties of the kinase through its ability to control translation. Our data demonstrate an important role of tyrosine phosphorylation in biochemical and biological processes caused or mediated by the activation of the eIF2alpha kinase PKR.

Animals↗
PubMed1988

Inhibitory activity for the interferon-induced protein kinase is associated with the reovirus serotype 1 sigma 3 protein.

In this report we demonstrate that reovirus serotype 1-infected cells contain an inhibitor of the interferon-induced, double-stranded RNA (dsRNA)-dependent protein kinase. We provide evidence that suggests that the virus-encoded sigma 3 protein is likely responsible for this kinase inhibitory activity. We could not detect activation of the dsRNA-dependent protein kinase in extracts prepared from either interferon-treated or untreated reovirus serotype 1-infected mouse L cells under conditions that led to activation of the kinase in extracts prepared from either interferon-treated or untreated, uninfected cells. Extracts from reovirus-infected cells blocked activation of kinase in extracts from interferon-treated cells when the two were mixed prior to assay. The kinase inhibitory activity in extracts of reovirus-infected cells could be overcome by adding approximately 100-fold excess of dsRNA over the amount required to activate kinase in extracts of uninfected cells. Kinase inhibitory activity in extracts of interferon-treated, virus-infected cells could be overcome with somewhat less dsRNA (approximately 10-fold excess). Most of the inhibitory activity in the extracts could be removed by adsorption with immobilized anti-reovirus sigma 3 serum or immobilized dsRNA, suggesting that the dsRNA-binding sigma 3 protein is necessary for kinase inhibitory activity. Purified sigma 3 protein, when added to reaction mixtures containing partially purified kinase, inhibited enzyme activation. Control of activation of this kinase, which can modify eukaryotic protein synthesis initiation factor 2, may be relevant to the sensitivity of reovirus replication to treatment of cells with interferon and to the shutoff of host protein synthesis in reovirus-infected cells.

Animals↗
PubMed2001

Dominant negative function by an alternatively spliced form of the interferon-inducible protein kinase PKR.

The double-stranded RNA (dsRNA)-activated protein kinase PKR (protein kinase dsRNA-dependent) plays an important role in the regulation of protein synthesis by phosphorylating the alpha-subunit of eukaryotic initiation factor 2. Through this activity, PKR is thought to mediate the antiviral and antiproliferative actions of interferon. Here, we show that the human T cell leukemia Jurkat cells express an alternatively spliced form of PKR with a deletion of exon 7 (PKRDeltaE7), resulting in a truncated protein that retains the two dsRNA-binding motifs. PKRDeltaE7 exhibits a dominant negative function by inhibiting both PKR autophosphorylation and eukaryotic initiation factor 2 alpha-subunit phosphorylation in vitro and in vivo. Reverse transcriptase-polymerase chain reaction assays showed that PKRDeltaE7 is expressed in a broad range of human tissues at variable levels. Interestingly, expression of PKRDeltaE7 is higher in Jurkat cells than in normal peripheral blood mononuclear cells, raising the possibility of a role in cell proliferation and/or transformation. Thus, expression of alternatively spliced forms of PKR may represent a novel mechanism of PKR autoregulation with important implications in the control of cell proliferation.

Alternative Splicing↗
PubMed2002

Genomic structure of the mouse 2',5'-oligoadenylate synthetase gene family.

2',5'-Oligoadenylate synthetase (2-5OAS) is one of the interferon (IFN)-induced proteins and mediates the antiviral action of IFN. In human, three classes of 2-5OAS genes (OAS1, OAS2, and OAS3) and one OAS-like gene (OASL) are reported. In mice, however, OAS genes corresponding to human OAS2 and OAS3 have not been identified. In this report, we identified six novel OAS family genes in mice by screening mouse genomic library and expressed sequence tag (EST) database. These genes include three homologs of the human OAS1 and each homologous gene of the human OAS2, OAS3, and OASL, respectively. Each gene displays 52%-65% amino acid identity to the corresponding human homologs. Nine 2-5OAS genes, except for two OASL genes, locate within the 210-kb genomic region and form a cluster. Each novel 2-5OAS gene showed a characteristic expression pattern among different tissues, and all of them were induced by polyinosinic-polycytidylic acid. Biochemical analyses using recombinant proteins produced in Escherichia coli showed that all the novel mouse 2-5OAS molecules have double-stranded RNA (dsRNA) binding ability, but they do not have 2-5OAS activity except for the OAS2 and OAS3 mouse homologs. These results show that there are at least 11 OAS genes, which are classified into four groups, in the mouse.

2',5'-Oligoadenylate Synthetase↗
PubMed1999

Identification of functional domains of the interferon-induced enzyme PKR in cells lacking endogenous PKR.

The interferon (IFN)-induced, double-stranded RNA (dsRNA)-activated human protein kinase (PKR) has been shown to exert antiviral and antiproliferative effects. Activation of the enzyme in mammalian cells results in protein synthesis inhibition and cell death by apoptosis. Previous studies on the structure-function relationship of PKR have been based on vectors expressing the enzyme in mammalian cells containing endogenous PKR. As exogenously expressed PKR can form heterodimers with endogenous PKR, the results obtained on the functional characterization of mutant forms of PKR have been taken with caution. To address the natural consequences of heterodimer formation between endogenous and exogenous PKR, we have analyzed the structure-function relationship of PKR ectopically expressed from vaccinia virus (VV) recombinants in cells lacking the endogenous enzyme. We demonstrate that PKR-mediated inhibition of protein synthesis and induction of apoptosis is not dependent on the presence of endogenous PKR. Further, PKR activity is independent of the presence of dsRNA binding motifs (dsRBM). Moreover, single-point mutations of the third basic domain decreased PKR activation. Our findings demonstrate that PKR can be activated in the absence of its N-terminal domain (amino acids 1-232) and that the third basic domain is important for its biologic function.

3T3 Cells↗
PubMed1996

Regulated expression of the interferon-induced protein kinase p68 (PKR) by vaccinia virus recombinants inhibits the replication of vesicular stomatitis virus but not that of poliovirus.

A direct antiviral role of the interferon-induced human protein kinase p68 has been shown only against encephalomyocarditis virus (EMCV) and vaccinia virus (VV). To determine if p68 kinase (PKR) has a broad antiviral effect, we have used coinfections between VV recombinants expressing p68 kinase under regulation of the lac I operator/repressor elements of Escherichia coli and two RNA viruses, vesicular stomatitis virus (VSV) and poliovirus. In cells coinfected with VV recombinants and VSV, induction with isopropyl-B-D-thiogalactoside (IPTG) of wild-type p68 kinase or a mutant lacking the dsRNA binding domain resulted in inhibition of both VV and VSV protein synthesis. This inhibition is not observed in cells infected with a catalytically inactive point mutant lys-arg296 of p68 kinase. When cells are coinfected with VV recombinants and poliovirus, induction of active p68 kinase resulted in a decrease in VV proteins but not in poliovirus proteins or poliovirus yields. Immunoblot analysis revealed that p68 kinase was expressed during mixed infections. Our results demonstrate a differential effect of p68 kinase on the replication of VV, VSV, and poliovirus. We suggest that in a particular virus-cell system, the different sensitivity of a virus to p68 kinase is probably due to levels of active enzyme.

Animals↗
PubMed1998

Role of the vaccinia virus E3L and K3L gene products in rescue of VSV and EMCV from the effects of IFN-alpha.

Vaccinia virus (VV) has been shown to be relatively resistant to the antiviral effects of interferon-alpha (IFN-alpha) and to rescue replication of IFN-sensitive viruses, such as encephalomyocarditis virus (EMCV) and vesicular stomatitis virus (VSV), from the antiviral effects of IFN. The E3L and K3L gene products have been implicated in the IFN resistance of VV. We have investigated the role that these VV-encoded functions play in the rescue of VSV and EMCV from the effects of IFN. Transient expression of the E3L open reading frame (ORF) was sufficient to rescue VSV but not EMCV from the IFN-induced antiviral state. Rescue of VSV by mutants of E3L correlated with the ability of the mutated E3L gene products to bind dsRNA. Conversely, transient expression of the K3L ORF was sufficient to partially rescue EMCV but not VSV from the effects of IFN. Results with VV deleted of either the K3L or E3L ORFs were consistent with results obtained by transient expression of these genes. These results demonstrate that the VV E3L gene products are likely responsible for the VV-mediated rescue of VSV from the effects of IFN and the K3L gene product is likely at least partly responsible for rescue of EMCV.

Antiviral Agents↗
PubMed2006

Characterization of DGCR8/Pasha, the essential cofactor for Drosha in primary miRNA processing.

DGCR8/Pasha is an essential cofactor for Drosha, a nuclear RNase III that cleaves the local hairpin structures embedded in long primary microRNA transcripts (pri-miRNAs) in eukaryotes. Although our knowledge of pri-miRNA processing has significantly advanced in recent years, the precise role of DGCR8 in this pathway remains unclear. In our present study, we dissect the domains in DGCR8 that contribute to the processing of pri-miRNAs and the subcellular localization of DGCR8. Drosha is stabilized through an interaction between its middle domain and the conserved C-terminal domain of DGCR8. Furthermore, DGCR8, but not Drosha, can directly and stably interact with pri-miRNAs, and the tandem dsRNA-binding domains (dsRBDs) in DGCR8 are responsible for this recognition. Moreover, the DGCR8 N-terminal region upstream of its dsRBDs is unnecessary for pri-miRNA processing but is critical for nuclear localization. Our study thus provides further insights into the mechanism of action of the Drosha-DGCR8 complex in pri-miRNA processing.

Amino Acid Sequence↗
PubMed1998

Comparative sequence analysis of American, European and Asian isolates of viruses in the genus Coltivirus.

In this study, the basis for the classification of virus isolates grouped within the genus Coltivirus, family Reoviridae, is discussed. Sequences of dsRNA segments from American (segments 9-12), European (segment 12) and Asian (segments 7-12) isolates were characterized and polythetic criteria were defined for their taxonomic classification. These criteria (including sequence analysis) permitted the different species to be distinguished and classified into two groups. In both groups, subgroups were defined according to the degree of homology between the genomic sequences. American and European isolates are classified within group A, which includes subgroups A1 (Colorado tick fever virus species) and A2 (Eyach virus species). Asian isolates are classified in group B, which includes subgroups B1 (JKT-7075 virus species) and B2 (JKT-6423 virus species). The proteins encoded by the sequenced genomic segments were analysed. This allowed the identification of dsRNA binding domains in the proteins encoded by segment 8 of subgroup B1 isolates and segment 12 of subgroup B2 isolates. A conserved pattern of amino acids in segment 7 of group B isolates matched sequences found in the catalytic domains of protein kinases.

Amino Acid Sequence↗
PubMed2000

Complete sequence determination and genetic analysis of Banna virus and Kadipiro virus: proposal for assignment to a new genus (Seadornavirus) within the family Reoviridae.

Arboviruses with genomes composed of 12 segments of double-stranded (ds) RNA have previously been classified as members or probable members of the genus Coltivirus within the family REOVIRIDAE: A number of these viruses have been isolated in North America and Europe and are serologically and genetically related to Colorado tick fever virus, the Coltivirus type species. These isolates constitute subgroup A of the coltiviruses. The complete genome sequences are now presented of two Asian arboviruses, Kadipiro virus (KDV) and Banna virus (BAV), which are currently classified as subgroup B coltiviruses. Analysis of the viral protein sequences shows that all of the BAV genome segments have cognate genes in KDV. The functions of several of these proteins were also indicated by this analysis. Proteins with dsRNA-binding domains or with significant similarities to polymerases, methyltransferases, NTPases or protein kinases were identified. Comparisons of amino acid sequences of the conserved polymerase protein have shown that BAV and KDV are only very distantly related to the subgroup A coltiviruses. These data demonstrate a requirement for the subgroup B viruses to be reassigned to a separate new genus, for which the name Seadornavirus is proposed.

Animals↗
PubMed1998

Miranda mediates asymmetric protein and RNA localization in the developing nervous system.

Neuroblasts undergo asymmetric stem cell divisions to generate a series of ganglion mother cells (GMCs). During these divisions, the cell fate determinant Prospero is asymmetrically partitioned to the GMC by Miranda protein, which tethers it to the basal cortex of the dividing neuroblast. Interestingly, prospero mRNA is similarly segregated by the dsRNA binding protein, Staufen. Here we show that Staufen interacts in vivo with a segment of the prospero 3' UTR. Staufen protein and prospero RNA colocalize to the apical side of the neuroblast at interphase, but move to the basal side during prophase. Both the apical and basal localization of Staufen are abolished by the removal of a conserved domain from the carboxyl terminus of the protein, which interacts in a yeast two-hybrid screen with Miranda protein. Furthermore, Miranda colocalizes with Staufen protein and prospero mRNA during neuroblast divisions, and neither Staufen nor prospero RNA are localized in miranda mutants. Thus Miranda, which localizes Prospero protein, also localizes prospero RNA through its interaction with Staufen protein.

Animals↗
PubMed2004

The Drosha-DGCR8 complex in primary microRNA processing.

RNase III proteins play key roles in microRNA (miRNA) biogenesis. The nuclear RNase III Drosha cleaves primary miRNAs (pri-miRNAs) to release hairpin-shaped pre-miRNAs that are subsequently cut by the cytoplasmic RNase III Dicer to generate mature miRNAs. While Dicer (class III) and other simple RNase III proteins (class I) have been studied intensively, the class II enzyme Drosha remains to be characterized. Here we dissected the action mechanism of human Drosha by generating mutants and by characterizing its new interacting partner, DGCR8. The basic action mechanism of Drosha was found to be similar to that of human Dicer; the RNase III domains A and B form an intramolecular dimer and cleave the 3' and 5' strands of the stem, respectively. Human Drosha fractionates at approximately 650 kDa, indicating that Drosha functions as a large complex. In this complex, Drosha interacts with DGCR8, which contains two double-stranded RNA (dsRNA)-binding domains. By RNAi and biochemical reconstitution, we show that DGCR8 may be an essential component of the pri-miRNA processing complex, along with Drosha. Based on these results, we propose a model for the action mechanism of class II RNase III proteins.

Blotting, Northern↗