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At least 865 records · Page 48Linked to original sources
PubMed1990

Electrophoresis of the complementary strands of the double-stranded Kemerovo virus RNAs in agarose-urea gel.

Single-stranded (ss)RNAs derived from 10 double-stranded (ds)RNA segments of Kemerovo virus (KV) were separated into 13 RNA bands by agarose-urea gel electrophoresis. The complementary strands of the dsRNA segments 1, 9 and 10 displayed different electrophoretic mobility. An attempt was made to determine the origin of the ssRNA bands. The ssRNA bands originating from the dsRNA segments 1, 2, 3, 9 and 10 were identified unequivocally, while those originating from the dsRNA segments 4, 5, 6, 7 and 8 were characterized partially. The minus RNA strands of the dsRNA segments 9 and 10 exhibited higher electrophoretic mobilities as their complementary plus RNA strands.

Electrophoresis, Agar Gel↗
PubMed2005

A double-stranded RNA from a Phytophthora species is related to the plant endornaviruses and contains a putative UDP glycosyltransferase gene.

A new dsRNA was isolated from a Phytophthora isolate from Douglas fir. Sequence analysis showed the dsRNA to consist of 13 883 bp and to contain a single open reading frame with the potential to encode a polyprotein of 4548 aa. This polyprotein contained amino acid sequence motifs characteristic of virus RNA-dependent RNA polymerases (RdRps) in its C-terminal region and motifs characteristic of RNA helicases in its N-terminal region. These sequence motifs were related to corresponding motifs in plant viruses in the genus Endornavirus. In phylogenetic trees constructed from the RdRp and helicase motifs of a range of ssRNA and dsRNA viruses, the Phytophthora RdRp and helicase sequences clustered with those of the plant endornaviruses with good bootstrap support. The properties of the Phytophthora dsRNA are consistent with its being classified as the first non-plant member of the genus Endornavirus, for which we propose the name phytophthora endornavirus 1 (PEV1). A region between the RdRp and helicase domains of the PEV1 protein had significant amino acid sequence similarity to UDP glycosyltransferases (UGTs). Two sequence motifs were identified, one characteristic of all UGTs and the other characteristic of sterol UGTs. The PEV1 UGT would be the first for an RNA virus, although ecdysteroid UGT genes have been found in many baculoviruses. The PEV1 UGT was only distantly related to baculovirus ecdysteroid UGTs, which belong to a family distinct from the sterol UGTs.

Amino Acid Motifs↗
PubMed1999

Markers for trans-Golgi membranes and the intermediate compartment localize to induced membranes with distinct replication functions in flavivirus-infected cells.

Replication of the flavivirus Kunjin virus is associated with virus-induced membrane structures within the cytoplasm of infected cells; these membranes appear as packets of vesicles associated with the sites of viral RNA synthesis and as convoluted membranes (CM) and paracrystalline arrays (PC) containing the components of the virus-specified protease (E. G. Westaway, J. M. Mackenzie, M. T. Kenney, M. K. Jones, and A. A. Khromykh, J. Virol. 71:6650-6661, 1997). To determine the cellular origins of these membrane structures, we compared the immunolabelling patterns of several cell markers in relation to these sites by immunofluorescence and immunoelectron microscopy. A marker for the trans-Golgi membranes and the trans-Golgi network, 1,4-galactosyltransferase (GalT), was redistributed to large foci in the cytoplasm of Kunjin virus-infected cells, partially coincident with immunofluorescent foci associated with the putative sites of viral RNA synthesis. As determined by immunoelectron microscopy, the induced vesicle packets contained GalT, whereas the CM and PC contained a specific protein marker for the intermediate compartment (ERGIC53). A further indicator of the role of cellular organelles in their biogenesis was the observation that the Golgi apparatus-disrupting agent brefeldin A prevented further development of immunofluorescent foci of induced membranes if added before the end of the latent period but that once formed, these membrane foci were resistant to brefeldin A dispersion. Reticulum membranes emanating from the induced CM and PC were also labelled with the rough endoplasmic reticulum marker anti-protein disulfide isomerase and were obviously redistributed during infection. This is the first report identifying trans-Golgi membranes and the intermediate compartment as the apparent sources of the flavivirus-induced membranes involved in events of replication.

Animals↗
PubMed1983

Analysis of the genomes of bluetongue virus serotype 10 vaccines and a recent BTV-10 isolate from Washington.

The 10 double-stranded RNA gene segments of 2 vaccinal strains of bluetongue virus (BTV) serotype 10 that are used in the United States (BTV CA8 California and BT-8 Colorado), and a BTV-10 isolate recently obtained from infected sheep in Washington (state) were characterized by oligonucleotide fingerprint analyses. It was determined that although the 2 BTV-10 vaccinal strains are genotypically distinct, they are closely related both to each other and to the United States prototype BTV-10 virus. The BTV-10 field isolate appears to be a naturally occurring reassortment virus with genome segments derived from both United States prototype BTV-10 and BTV-11 viruses. However, one RNA segment of the isolate was totally unlike the corresponding segments of United States prototype BTV-10, -11, -13 and -17 viruses.

Animals↗
PubMed2006

Purification and characterization of infectious myonecrosis virus of penaeid shrimp.

The causative agent of myonecrosis affecting cultured Penaeus vannamei in Brazil was demonstrated to be a virus after purification of the agent from infected shrimp tissues. Purified viral particles were injected into specific pathogen-free P. vannamei, resulting in a disease that displayed the same characteristics as those found in the original shrimp used for purification. The virus was named infectious myonecrosis virus (IMNV). The viral particles were icosahedral in shape and 40 nm in diameter, with a buoyant density of 1.366 g ml(-1) in caesium chloride. The genome consisted of a single, double-stranded (dsRNA) molecule of 7560 bp. Sequencing of the viral genome revealed two non-overlapping open reading frames (ORFs). The 5' ORF (ORF 1, nt 136-4953) encoded a putative RNA-binding protein and a capsid protein. The coding region of the RNA-binding protein was located in the first half of ORF 1 and contained a dsRNA-binding motif in the first 60 aa. The second half of ORF 1 encoded a capsid protein, as determined by amino acid sequencing, with a molecular mass of 106 kDa. The 3' ORF (ORF 2, nt 5241-7451) encoded a putative RNA-dependent RNA polymerase (RdRp) with motifs characteristic of totiviruses. Phylogenetic analysis based on the RdRp clustered IMNV with Giardia lamblia virus, a member of the family Totiviridae. Based on these findings, IMNV may be a unique member of the Totiviridae or may represent a new dsRNA virus family that infects invertebrate hosts.

Amino Acid Sequence↗
PubMed1994

A small mitochondrial double-stranded (ds) RNA element associated with a hypovirulent strain of the chestnut blight fungus and ancestrally related to yeast cytoplasmic T and W dsRNAs.

A small double-stranded (ds) RNA element was isolated from a moderately hypovirulent strain of the chestnut blight fungus Cryphonectria parasitica (Murr.) Barr. from eastern New Jersey. Virulence was somewhat lower in the dsRNA-containing strain than in a virulent dsRNA-free control strain, but colony morphology and sporulation levels were comparable. A library of cDNA clones was constructed, and overlapping clones representing the entire genome were sequenced. The 2728-bp dsRNA was considerably smaller than previously characterized C. parasitica dsRNAs, which are 12-13 kb and ancestrally related to the Potyviridae family of plant viruses. Sequence analysis revealed one large open reading frame, but only if mitochondrial codon usage (UGA = Trp) was invoked. Nuclease assays of purified mitochondria confirmed that the dsRNA was localized within mitochondria. Assuming mitochondrial translation, the deduced amino acid sequence had landmarks typical of RNA-dependent RNA polymerases. Alignments of the conserved regions indicate that this dsRNA is more closely related to yeast T and W dsRNAs and single-stranded RNA bacteriophages such as Q beta than to other hypovirulence-associated dsRNAs.

Amino Acid Sequence↗
PubMed2005

Mint virus X: a novel potexvirus associated with symptoms in 'Variegata' mint.

Mentha x gracilis 'Variegata', an ornamental mint clone first described about 200 years ago, exhibits virus-like vein banding symptoms. Double-stranded RNA and virion isolations revealed the presence of three viruses in a 'Variegata' plant. Cloning and sequencing disclosed that one of the viruses was a previously unidentified species with similarities to members of the Flexiviridae family, designated as Mint virus X (MVX). The complete nucleotide sequence of the virus was determined. Phylogenetic analysis divulged the close relationship of the virus with lily virus X and strawberry mild yellow edge virus, members of the Potexvirus genus. A reverse transcription-polymerase chain reaction protocol was developed and used for detection of MVX in other 'Variegata' plants. All clones tested, obtained from nurseries around the United States were infected with MVX, making the virus a possible causal agent of the variegated symptoms.

Genome, Viral↗
PubMed1990

Reovirus RNA is infectious.

Conditions under which reovirus RNA is infectious have been worked out. In brief, single-stranded (plus-stranded, ss) and/or double-stranded (ds) RNA of reovirus serotype 3 (ST3 virus) is lipofected into L929 mouse fibroblasts together with a rabbit reticulocyte lysate in which ss or melted dsRNA has been translated. After 8 hr the cells are then infected with a helper virus, ST2 reovirus. Virus yields are harvested 24 or 48 hr later. Under these conditions virus that forms plaques by 5 days is produced, all of which is ST3 virus; ST2 virus forms plaques only after 12 days. No reassortants are present among the progeny. The virus yields are about 0.2 PFU/cell; immunofluorescence assays show that this progeny is derived from about 4% of the cells. Double-stranded RNA is 20 times as infectious as ssRNA; ds and ssRNA together yield 10 times as much infectious virus as dsRNA alone, the reason being that dsRNA greatly increases the infectiousness of ssRNA. All species of both ss and dsRNA are required for the operation of this additive effect. The primed rabbit reticulocyte lysate is not essential, but increases virus yields by 100-fold. Its activity is proportional to the time for which translation has proceeded; however, this activity is not due solely to newly synthesized proteins because destruction of the RNA following translation abolishes activity which cannot be restored by simple addition of more RNA. Translation of all species of RNA is essential. Whereas no reassortants are formed when ss and dsRNA of different genotypes are lipofected together, mixtures of dsRNAs of different genotypes do yield reassortants. The same is true for such mixtures of ssRNA. These findings will permit the introduction of new or altered genome segments into the reovirus genome. They open the way to the identification of encapsidation and assortment signals on reovirus genome segments, the characterization of functional domains on reovirus proteins, and the development of reovirus as an expression vector.

Animals↗
PubMed2002

The role of the PKR-inhibitory genes, E3L and K3L, in determining vaccinia virus host range.

Vaccinia virus encodes two regulators of the cellular antiviral response. The E3L gene is thought to act primarily by sequestering double-stranded RNA, whereas the K3L gene is thought to act as a competitive inhibitor of the double-stranded RNA-dependent protein kinase, PKR. The broad host range associated with vaccinia virus replication appears to be related to the presence of these genes. The E3L gene is required for replication in HeLa cells, but is not required for replication in BHK cells. On the contrary, the K3L gene is required for replication in BHK cells, but is dispensable for replication in HeLa cells. Our results suggest that these cell lines varied in the expression of endogenous activatable PKR and that replication of vaccinia virus in different cell lines led to altered levels of double-stranded RNA synthesis from the virus. Vaccinia virus was able to overcome these cellular variations by regulating PKR activity through the synthesis of either E3L or K3L. The results suggest that vaccinia virus has evolved a broad host range by maintaining both the E3L and the K3L genes.

Animals↗
PubMed2000

Influenza virus NS1 protein counteracts PKR-mediated inhibition of replication.

The availability of an influenza virus NS1 gene knockout virus (delNS1 virus) allowed us to establish the significance of the biological relationship between the influenza virus NS1 protein and double-stranded-RNA-activated protein kinase (PKR) in the life cycle and pathogenicity of influenza virus. Our results show that the lack of functional PKR permits the delNS1 virus to replicate in otherwise nonpermissive hosts, suggesting that the major function of the influenza virus NS1 protein is to counteract or prevent the PKR-mediated antiviral response.

Animals↗
PubMed1982

Influenza B virus genome: sequences and structural organization of RNA segment 8 and the mRNAs coding for the NS1 and NS2 proteins.

Double-stranded DNA derived from influenza B virus genome RNA segment 8, which codes for the NS1 and NS2 proteins, was constructed by hybridization of full-length cDNA copies of RNA segment 8 and of the NS1 mRNA. This DNA was cloned in plasmid pBR322 and sequenced. The NS1 mRNA (approximately 1,080 viral nucleotides) contains nonviral nucleotides at its 5' end and is capable of coding for a protein of 281 amino acids. Sequencing of the NS2 mRNA has shown that it contains an interrupted sequence of 655 nucleotides and is most likely synthesized by a splicing mechanism. The first approximately 75 virus-specific nucleotides at the 5' end of the NS2 mRNA are the same as are found at the 5' -end of the NS1 mRNA. This region contains the initiation codon for protein synthesis and coding information for 10 amino acids common to the two proteins. The approximately 350-nucleotide body region of the NS2 mRNA can be translated in the +1 reading frame, and the sequence indicates that the NS1 and NS2 protein-coding regions overlap by 52 amino acids translated from different reading frames. Thus, between the influenza A and B viruses, the organization of the NS1 and NS2 mRNAs and the sizes of the NS2 mRNA and protein are conserved despite the larger size of the influenza B virus RNA segment, NS1 mRNA, and NS1 protein.

Amino Acid Sequence↗
PubMed1996

A regulatory domain within the virus-response element of the interferon alpha 1 gene acts as a transcriptional repressor sequence and determinant of cell-specific gene expression.

Type-I interferons are encoded by a multigene family, the major members of which are at least 13 IFN A subtypes and a single IFN B gene. IFNs A and B are induced in response to similar stimuli, such as virus infection and double-stranded RNA, but in different cell types: the induction of IFN A is almost exclusively restricted to cells of lymphoid origin, while IFN B has been found to be induced in a variety of cell types including fibroblasts. The virus-responsive enhancer element in the promoter region of IFN A family members is largely responsible for the differential expression of individual subtypes in responsive cells. In this paper we describe experiments which address the issue of the differential expression of IFN A and IFN B in different cell types. We show that IFN-beta is induced in a variety of cells of different origin, while not all of these are able to secrete IFN-alpha. By transfection of reporter gene constructs comprising the virus-responsive enhancer from the IFN A1 and IFN B genes, we show that this differential response is mediated at the level of transcription via these control elements. More detailed analysis of the function of these regions identifies specific sequences within the IFN A1 virus response element that has an inhibitory effect on expression in cells that are normally inducible, and is also implicated in the overall suppression of IFN A induction in non-inducible cells.

Alkaline Phosphatase↗
PubMed2003

The Ebola virus VP35 protein inhibits activation of interferon regulatory factor 3.

The Ebola virus VP35 protein was previously found to act as an interferon (IFN) antagonist which could complement growth of influenza delNS1 virus, a mutant influenza virus lacking the influenza virus IFN antagonist protein, NS1. The Ebola virus VP35 could also prevent the virus- or double-stranded RNA-mediated transcriptional activation of both the beta IFN (IFN-beta) promoter and the IFN-stimulated ISG54 promoter (C. Basler et al., Proc. Natl. Acad. Sci. USA 97:12289-12294, 2000). We now show that VP35 inhibits virus infection-induced transcriptional activation of IFN regulatory factor 3 (IRF-3)-responsive mammalian promoters and that VP35 does not block signaling from the IFN-alpha/beta receptor. The ability of VP35 to inhibit this virus-induced transcription correlates with its ability to block activation of IRF-3, a cellular transcription factor of central importance in initiating the host cell IFN response. We demonstrate that VP35 blocks the Sendai virus-induced activation of two promoters which can be directly activated by IRF-3, namely, the ISG54 promoter and the ISG56 promoter. Further, expression of VP35 prevents the IRF-3-dependent activation of the IFN-alpha4 promoter in response to viral infection. The inhibition of IRF-3 appears to occur through an inhibition of IRF-3 phosphorylation. VP35 blocks virus-induced IRF-3 phosphorylation and subsequent IRF-3 dimerization and nuclear translocation. Consistent with these observations, Ebola virus infection of Vero cells activated neither transcription from the ISG54 promoter nor nuclear accumulation of IRF-3. These data suggest that in Ebola virus-infected cells, VP35 inhibits the induction of antiviral genes, including the IFN-beta gene, by blocking IRF-3 activation.

Animals↗
PubMed1998

The 2-5A system: modulation of viral and cellular processes through acceleration of RNA degradation.

The 2-5A system is an RNA degradation pathway that can be induced by the interferons (IFNs). Treatment of cells with IFN activates genes encoding several double-stranded RNA (dsRNA)-dependent synthetases. These enzymes generate 5'-triphosphorylated, 2',5'-phosphodiester-linked oligoadenylates (2-5A) from ATP. The effects of 2-5A in cells are transient since 2-5A is unstable in cells due to the activities of phosphodiesterase and phosphatase. 2-5A activates the endoribonuclease 2-5A-dependent RNase L, causing degradation of single-stranded RNA with moderate specificity. The human 2-5A-dependent RNase is an 83.5 kDa polypeptide that has little, if any, RNase activity, unless 2-5A is present. 2-5A binding to RNase L switches the enzyme from its off-state to its on-state. At least three 2',5'-linked oligoadenylates and a single 5'-phosphoryl group are required for maximal activation of the RNase. Even though the constitutive presence of 2-5A-dependent RNase is observed in nearly all mammalian cell types, cellular amounts of 2-5A-dependent mRNA and activity can increase after IFN treatment. One well-established role of the 2-5A system is as a host defense against some types of viruses. Since virus infection of cells results in the production and secretion of IFNs, and since dsRNA is both a frequent product of virus infection and an activator of 2-5A synthesis, the replication of encephalomyocarditis virus, which produces dsRNA during its life cycle, is greatly suppressed in IFN-treated cells as a direct result of RNA decay by the activated 2-5A-dependent RNase. This review covers the organic chemistry, enzymology, and molecular biology of 2-5A and its associated enzymes. Additional possible biological roles of the 2-5A system, such as in cell growth and differentiation, human immunodeficiency virus replication, heat shock, atherosclerotic plaque, pathogenesis of Type I diabetes, and apoptosis, are presented.

Adenine Nucleotides↗
PubMed2002

Inhibition of viral gene expression and replication in mosquito cells by dsRNA-triggered RNA interference.

Mosquitoes transmit numerous viral pathogens to humans including dengue virus which affects approximately 50 million individuals per year. Inhibition of viral gene expression within an insect host could be used to block virus replication and subsequent transmission of the pathogen to humans. A naturally occurring gene silencing mechanism triggered by double-stranded RNA (dsRNA), RNA interference (RNAi), has recently been described in a number of species including Drosophila. To ascertain if dsRNA-triggered RNAi is present in mosquito cells, we used Aedes albopictus C6/36 cells, and to investigate the feasibility of blocking viral gene expression and replication, we used two mosquito-borne viruses, Semliki Forest virus (SFV) and the serotype 1 dengue virus (DEN1). We demonstrate that dsRNA can specifically inhibit transgene expression in C6/36 cells from both plasmid and SFV replicons and can significantly modify the kinetics of DEN1 RNA and virus replication. The inhibition mediated by dsRNA was sequence-specific and either equal or superior to that induced by antisense single-stranded RNA (ssRNA). This study demonstrates dsRNA-triggered inhibition of gene expression and virus replication in mosquito cells and suggests that this mechanism could be used to block pathogen replication within an insect host and, thus, block disease transmission.

Animals↗