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At least 469 records · Page 26Linked to original sources
PubMed1989

Assignment of wound tumor virus nonstructural polypeptides to cognate dsRNA genome segments by in vitro expression of tailored full-length cDNA clones.

Presumptive full-length cDNA clones of 9 of the 12 wound tumor virus double-stranded RNA genome segments were tailored for efficient in vitro expression by a recently described strategy [Z. Xu, J.V. Anzola, and D.L. Nuss (1987) DNA6, 505-513]. In vitro synthesized polypeptides specified by synthetic transcripts corresponding to the tailored cDNAs comigrated in polyacrylamide gels with in vivo synthesized viral-specific polypeptides. This analysis confirmed the functional integrity of the tailored cDNA clones and identified cognate genome segments which encode all five viral non-structural polypeptides as well as four structural polypeptides; two which comprise the capsid, one located in the viral core and one associated with the outer protein coat.

Base Sequence↗
PubMed1991

Binding of Epstein-Barr virus small RNA EBER-1 to the double-stranded RNA-activated protein kinase DAI.

Epstein-Barr virus encodes two small RNAs, EBER-1 and -2, that are abundantly expressed in latently infected cells. Recent evidence suggests a role for EBER-1 in regulation of translation since this RNA is able to prevent the inhibition of protein synthesis by double-stranded RNA in rabbit reticulocyte lysates. We show here that EBER-1 that has been synthesized in vitro forms a complex with the dsRNA-activated inhibitor of protein synthesis DAI, a protein kinase that specifically phosphorylates polypeptide chain initiation factor eIF-2. Gel retardation assays and UV crosslinking experiments indicate that complex formation is specific for EBER-1 and requires the presence of some secondary structure in the molecule. RNA competition studies show that EBER-1-DAI complex formation is not inhibited in the presence of other small RNA species, heparin or the synthetic double-stranded RNA, poly(I).poly(C). SDS gel analysis reveals the existence of two forms of the crosslinked complex, of 64-68kDa and 46-53kDa, both of which are recognized by anti-DAI antibodies in immunoprecipitation experiments. These data suggest that EBER-1 regulates protein synthesis through its ability to interact with DAI.

Blotting, Western↗
PubMed1984

Detection of double-stranded RNA by serologically specific electron microscopy.

Details of a procedure for detecting double-stranded RNA (dsRNA) in virus and viroid infected tissue extracts using serologically specific electron microscopy are given. A method for staining dsRNA, based on in situ formation of uranyl phosphate, that consistently permits the examination of dsRNA by electron microscopy without shadowing with heavy metals, is described. The method provides for routine assays for dsRNA in crude extracts without the variable results associated with shadowing procedures. DsRNA was found to accumulate in older leaves of sorghum systemically infected with sugarcane mosaic virus. In contrast, dsRNA was not detected in older cowpea leaves systemically infected with cowpea mosaic virus but was readily found in inoculated leaves 4 days post inoculation and young systemically infected leaves.

Microscopy, Electron↗
PubMed1992

PCR amplification of a specific double-stranded RNA region of Fiji disease virus from diseased sugarcane.

A 450-bp region from one species of the segmented dsRNA genome of Fiji disease virus (FDV) was amplified from total nucleic acid extracts of diseased plants by reverse transcription with MMLV, followed by amplification with Taq DNA polymerase (RT-PCR). Other FDV-specific regions (c 150 bp and c 270 bp) were also amplified from the dsRNA template. FDV cDNA was only synthesised when the viral dsRNA template was boiled and quenched with FDV-specific or random hexamer primers. The reverse transcriptase/DNA polymerase enzyme rTth appeared to yield only the 150 bp fragment from the dsRNA template under the conditions used. The level of sensitivity of RT-PCR for purified FDV dsRNA was 100 ag, approximately 10(4)-fold more sensitive than detection with biotinylated DNA probe.

Base Sequence↗
PubMed1992

The role of double-stranded RNA in induction of the acute-phase response in an abortive influenza virus infection model.

The inducer of the acute-phase response in "flu-like" viral infections is not defined. The hypothesis that virus-associated double-stranded (ds) RNA serves this function was investigated by comparison of several acute-phase responses (fever and sleep patterns, white and nucleated red blood cell levels, serum antiviral activity and ceruloplasmin) induced by the synthetic dsRNA polyriboinosinic:polyribocytidylic acid (poly[rI:rC]) with those induced by influenza virus in rabbits. The capacity of either dsRNA or influenza virus to induce hyporesponsiveness with respect to these acute-phase parameters upon rechallenge with the same agent or cross-challenge 24 h later was also examined. Poly(rI:rC) induced only minimal hyporesponsiveness to itself but was a potent inducer of hyporesponsiveness to virus with respect to fever, sleep, leukograms, and antiviral activity. Therefore, poly(rI:rC) can substitute for virus in terms of induction of acute-phase hyporesponsiveness, suggesting that dsRNA of viral origin triggers the acute-phase response in this model of influenza.

Acute-Phase Reaction↗
PubMed2004

Ded1p, a conserved DExD/H-box translation factor, can promote yeast L-A virus negative-strand RNA synthesis in vitro.

Viruses are intracellular parasites that must use the host machinery to multiply. Identification of the host factors that perform essential functions in viral replication is thus of crucial importance to the understanding of virus-host interactions. Here we describe Ded1p, a highly conserved DExD/H-box translation factor, as a possible host factor recruited by the yeast L-A double-stranded RNA (dsRNA) virus. We found that Ded1p interacts specifically and strongly with Gag, the L-A virus coat protein. Further analysis revealed that Ded1p interacts with the L-A virus in an RNA-independent manner and, as a result, L-A particles can be affinity purified via this interaction. The affinity-purified L-A particles are functional, as they are capable of synthesizing RNA in vitro. Critically, using purified L-A particles, we demonstrated that Ded1p specifically promotes L-A dsRNA replication by accelerating the rate of negative-strand RNA synthesis in vitro. In light of these data, we suggest that Ded1p may be a part of the long sought after activity shown to promote yeast viral dsRNA replication. This and the fact that Ded1p is also required for translating brome mosaic virus RNA2 in yeast thus raise the intriguing possibility that Ded1p is one of the key host factors favored by several evolutionarily related RNA viruses, including the human hepatitis C virus.

Cell Cycle Proteins↗
PubMed1995

The large genome segment of dsRNA bacteriophage phi6 is the key regulator in the in vitro minus and plus strand synthesis.

Bacteriophage phi6 is a double-stranded RNA (dsRNA) virus that has a genome composed of three linear dsRNA segments (L, M, S). These are encapsidated into a dodecahedral procapsid particle consisting of proteins P1, P2, P4, and P7. Empty preformed procapsids are able to package the plus-sense single-stranded RNA (ssRNA) of each genome segment, to synthesize the corresponding minus strands ("replication") to form dsRNA segments, and to continue to the plus strand synthesis ("transcription") in which the dsRNA segments are used as templates in production of plus-sense ssRNA. In this study, we have investigated the requirements for the switch-on of minus and plus strand syntheses. We show that there exists an inverse relationship between regulation of the ssRNA packaging and minus strand synthesis. The packaging of single-stranded l, which has previously been shown to be packaged as the last, is the necessary signal for the onset of the minus strand synthesis. The absolute requirement for plus strand synthesis is minus strand synthesis of l, but in addition, the minus strand synthesis of m and the packaging of s segment are needed for efficient plus strand synthesis. Furthermore, the second nucleotide at the 5'-end of each segment regulates the extent of the transcription.

Bacteriophage phi 6↗
PubMed1982

Conditions required for induction of interferon by rotaviruses and for their sensitivity to its action.

Our investigations of interferon induction by rotaviruses showed that only when cells were pretreated with interferon, i.e., primed, could infectious rotaviruses induce significant quantities of interferon. As little as 0.5 U of interferon provided sufficient priming for this induction. UV-irradiated rotaviruses induced significant levels of interferon, and priming only marginally enhanced the yields. Neither heat-inactivated virus nor serum-neutralized virus was able to induce interferon, even when cells were primed. When cells were treated with purified virus double-stranded RNA in the presence of DEAE-dextran to facilitate uptake, interferon was induced, although priming did not enhance yields. These results strongly implicate the viral double-stranded RNA as the effector for interferon induction. The insensitivity of rotaviruses to interferon in vitro was also studied. Results suggested that this lack of sensitivity was not due to any inherent resistance of the virus to the antiviral proteins, but rather to lack of activation of cellular enzymes exhibiting antiviral activity.

Animals↗
PubMed1986

A physical map of the viral genome for infectious pancreatic necrosis virus Sp: analysis of cell-free translation products derived from viral cDNA clones.

The two segments of double-stranded RNA from infectious pancreatic necrosis virus Sp were cloned into the plasmid vector pUC8. Two sets of overlapping clones were identified by restriction enzyme and Southern blot analyses. Each of these sets was shown by Northern blot analysis to be exclusively related to either segment A or B of the genomic RNA. The entire lengths of the cloned segments were estimated to be 2.9 and 2.6 kilobases, respectively. Sequences from the two segments of viral cDNA were subcloned into the bacteriophage T7 RNA polymerase vectors pT71 and pT72. The activity of the single-stranded RNAs transcribed from these subclones in a rabbit reticulocyte lysate translation system provided information on the polarity of and the protein products coded for by each subclone. The four proteins encoded by the genome of infectious pancreatic necrosis virus were identified among the translation products of the individual cloned segments by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. By constructing plasmids containing deletions in the sequences from either the 5' or 3' end of segment A, we were able to construct a physical map for the larger segment of double-stranded RNA. The proteins derived from these plasmids indicated that the linear gene order for viral proteins encoded in segment A is beta, gamma 2, and gamma 1.

Animals↗
PubMed2000

The [KIL-d] element specifically regulates viral gene expression in yeast.

The cytoplasmically inherited [KIL-d] element epigenetically regulates killer virus gene expression in Saccharomyces cerevisiae. [KIL-d] results in variegated defects in expression of the M double-stranded RNA viral segment in haploid cells that are "healed" in diploids. We report that the [KIL-d] element is spontaneously lost with a frequency of 10(-4)-10(-5) and reappears with variegated phenotypic expression with a frequency of > or =10(-3). This high rate of loss and higher rate of reappearance is unlike any known nucleic acid replicon but resembles the behavior of yeast prions. However, [KIL-d] is distinct from the known yeast prions in its relative guanidinium hydrochloride incurability and independence of Hsp104 protein for its maintenance. Despite its transmissibility by successive cytoplasmic transfers, multiple cytoplasmic nucleic acids have been proven not to carry the [KIL-d] trait. [KIL-d] epigenetically regulates the expression of the M double-stranded RNA satellite virus genome, but fails to alter the expression of M cDNA. This specificity remained even after a cycle of mating and meiosis. Due to its unique genetic properties and viral RNA specificity, [KIL-d] represents a new type of genetic element that interacts with a viral RNA genome.

Base Sequence↗
PubMed2006

Early steps in rotavirus cell entry.

Rotaviruses, the leading cause of severe dehydrating diarrhea in infants and young children worldwide, are non-enveloped viruses formed by three concentric layers of protein that enclose a genome of double-stranded RNA. These viruses have a specific cell tropism in vivo, infecting primarily the mature enterocytes of the villi of the small intestine. It has been found that rotavirus cell entry is a complex multistep process, in which different domains of the rotavirus surface proteins interact sequentially with different cell surface molecules, which act as attachment and entry receptors. These recently described molecules include integrins (alpha2beta1, alphavbeta3, and alphaxbeta2) and a heat shock protein (hsc70), and have been found to be associated with cell membrane lipid microdomains. The requirement for several cell molecules, which might need to be present and organized in a precise fashion, could explain the cell and tissue tropism of these viruses. This review focuses on recent data describing the interactions between the virus and its receptors, the role of lipid microdomains in rotavirus infection, and the possible mechanism of rotavirus cell entry.

Animals↗
PubMedDate not supplied

The structural basis of recognition and removal of cellular mRNA 7-methyl G 'caps' by a viral capsid protein: a unique viral response to host defense.

The single segment, double-stranded RNA genome of the L-A virus (L-A) of yeast encodes two proteins: the major coat protein Gag (76 kDa) and the Gag-Pol fusion protein (180 kDa). The icosahedral L-A capsid is formed by 120 copies of Gag and has architecture similar to that seen in the reovirus, blue tongue virus and rice dwarf virus inner protein shells. Gag chemically removes the m7GMP caps from host cellular mRNAs. Previously we identified a trench on the outer surface of Gag that included His154, to which caps are covalently attached. Here we report the refined L-A coordinates at 3.4 angstroms resolution with additional structural features and the structure of L-A with bound m7GDP at 6.5 angstroms resolution, which shows the conformational change of the virus upon ligand binding. Based on site-directed mutations, residues in or adjacent to the trench that are essential (or dispensable) for the decapping reaction are described here. Along with His154, the reaction requires a cluster of positive charge adjoining the trench and residues Tyr 452, Tyr150 and either Tyr or Phe at position 538. A tentative mechanism for decapping is proposed.

Binding Sites↗
PubMed2006

Inhibition of nervous necrosis virus propagation by fish Mx proteins.

Mx proteins are interferon induced, antiviral proteins, expressed in response to treatment with double stranded RNA or virus infection. Here we report the cloning, sequencing, and antiviral property of three forms of Mx genes, MxI, MxII, and MxIII from grouper (Epinephelus coioides). Multiple comparison of grouper Mx amino acid sequences revealed the conservation of Mx putative GTP-binding domain, dynamin family signature, and leucine zipper motif. We have established a new cell line, grouper brain 3 (GB3), and prepared stable clones expressing Flag-epitope tagged grouper MxI, MxII, and MxIII. Immunostaining shows that all the three grouper Mx proteins are localized in the cytoplasm. To examine the antiviral activity of grouper Mx proteins, these stable clones were infected by a nodavirus, yellow grouper nervous necrosis virus (YGNNV), and the results showed that all the three Mx isoforms have the efficiency of reducing the titre of virus from 10- to 100-fold. Moreover, through immunocytochemistry we demonstrated that Mx protein can inhibit the YGNNV propagation in GB3 cells. Taken together, this study demonstrates that grouper Mx proteins have efficient inhibitory activity against nodavirus, the most endangered virus of fish, and this information would be helpful to design effective DNA vaccines that can confer an early non-specific antiviral protection.

Amino Acid Sequence↗
PubMed2002

Virus in Trichomonas--an ultrastructural study.

Trichomonas vaginalis is a flagellated, parasitic protozoan that inhabits the urogenital tract of humans. Approximately one-half of isolates of T. vaginalis are infected with a double-stranded (ds) RNA virus, which was described in the literature as a homogeneous population of icosahedral virus with isometric symmetry and 33 nm in diameter. The present study describes the heterogeneous virus population found in T. vaginalis isolate 347. This population comprises different virus sizes (33-200 nm) and shape (filamentous, cylindrical, and spherical particles). These observations were made in CsCl-purified virus fractions as well as the thin sections of parasites. Some viruses were only observed after slight changes in the technique where the sample was prepared by the negative staining carbon-film method directly onto freshly cleft mica. The VLPs were found in the cytoplasm closely associated with the Golgi complex, with some VLPs budding from the Golgi, and other VLPs were detected adjacent to the plasma membrane. Unidentified cytoplasmic inclusions were observed in the region close to the VLPs and Golgi. These results indicate that T. vaginalis organisms may be infected with different dsRNA viruses simultaneously and suggest that T. vaginalis may be a reservoir for several viruses. We also showed some steps in the route of T. vaginalis virus and some aspects of the cytopathology of this infection. Purified VLPs were transfected to virus-free T. vaginalis isolates. Our results demonstrate that TVV attach and penetrate into trichomonads through endocytic coated pits and are maintained within vacuoles during batch culture for several daily passages. Immediately after virus transfection, many cells were lysed, whereas some intact reminiscent cells were recruited forming large clusters. Virus particles were found outside the cells, and in coated pits, within vacuoles in the cytoplasm, and infrequently within the nucleus. The Golgi complex showed changes in its electron density and in the cisternae structure. In lysed cells, virus particles were clearly seen over the residual membranes.

Animals↗
PubMed1986

Molecular basis of rotavirus virulence: role of gene segment 4.

Bovine rotavirus NCDV and simian rotavirus SA-11 exhibited markedly different patterns of gastrointestinal tract disease when inoculated orally into newborn mice. A genetic approach was used to define the molecular basis of these differences. The SA-11 strain of rotavirus was more virulent than the NCDV strain when inoculated orally into newborn mice; the dose of SA-11 required to cause diarrhea in 50% of infant mice was 50-fold less than that required for NCDV. Nineteen reassortant viruses were derived by coinfection of MA-104 cells in vitro with the SA-11 and NCDV strains. The parental origin of reassortant virus double-stranded RNA segments was determined by gene segment migration differences in polyacrylamide gels and hybridization with radioactively labeled parental viral transcripts. The neutralization antigen phenotype of reassortant viruses was determined by plaque reduction neutralization. We found that the dose of SA-11 and NCDV rotavirus required to induce gastroenteritis in newborn mice was determined by gene segment 4. The results suggest that rotavirus virulence may be manipulated by modification or reassortment of gene segment 4.

Antigens, Viral↗
PubMed1993

Genetics of heat-curability of killer virus of yeast.

The cytoplasmically inherited M double-stranded (ds) RNA genome segment of killer virus of Saccharomyces cerevisiae is heat-curable in some yeast strains but not in others. Temperature sensitivity is conferred on both M1 and M2 dsRNA satellite virus segments by the L-A-HN allele of the killer helper virus genome, but not by the L-A-H allele. Both diploidy and mating type heterozygosity of the host cell are also correlated with increased virus curability.

Base Sequence↗