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PubMed1997

Involvement of dsRNA virus in the protein composition and growth kinetics of host Trichomonas vaginalis.

Trichomonas vaginalis harbors a double-stranded (ds)-RNA virus, and the presence of virus is related to upregulated expression and phenotypic variation of a prominent immunogen (Khoshnan A, Alderete JF (1994) J Virol 68: 4035-4038). To further test the influence of virus on T. vaginalis, virus-infected (V+) isolates were compared to virus-free (V-), agar-cloned progeny trichomonads derived from the parental isolates for accumulation of total proteins and cysteine proteinases. Comparative high resolution two dimensional (2D)-SDS-PAGE was performed of trichomonads grown in a chemostat under identical conditions. At least 47 proteins were identified as specifically expressed by representative V+ isolate 347, and approximately 41 spots were specific to the corresponding V- progeny, showing an association between virus and the presence and absence of parasite proteins. Qualitatively and quantitatively dissimilar cysteine proteinase patterns were detected from numerous V+ isolates and the V- progeny. A 2D analysis for isolate 347 showed the appearance of unique proteinase activities for parental parasites and presence of at least one proteinase in the V- progeny. Finally, the V+ T. vaginalis isolate 347, but not the V- isolate 347 progeny nor other V+ isolates, underwent fluctuations in density during chemostat growth allowing for purification of virus particles from the V+ isolate 347 supernatants during decreased parasite density.

Animals↗
PubMed1983

Complete nucleotide sequence of alfalfa mosaic virus RNA 1.

Double-stranded cDNA of alfalfa mosaic virus (AlMV) RNA 1 has been cloned and sequenced. From clones with overlapping inserts, and other sequence data, the complete primary sequence of the 3644 nucleotides of RNA 1 was deduced: a long open reading frame for a protein of Mr 125,685 is flanked by a 5'-terminal sequence of 100 nucleotides and a 3' noncoding region of 163 nucleotides, including the sequence of 145 nucleotides the three genomic RNAs of AlMV have in common. The two UGA-termination codons halfway RNA 1, that were postulated by Van Tol et al. (FEBS Lett. 118, 67-71, 1980) to account for partial translation of RNA 1 in vitro into Mr 58,000 and Mr 62,000 proteins, were not found in the reading frame of the Mr 125,685 protein.

Amino Acid Sequence↗
PubMed1992

Differentiation of biologically distinct cucumber mosaic virus isolates by PAGE of double-stranded RNA.

Electrophoresis on 5% polyacrylamide was used to analyze dsRNAs of 26 cucumber mosaic virus isolates propagated in Nicotiana tabacum. There was variation between isolates in the migration of each of the dsRNAs 1, 2, and 3. Comparison of the dsRNA profiles enabled each isolate to be allocated to 1 of 7 distinct dsRNA profile types. Two distinct and readily distinguishable isolates were mixed in planta and dsRNA from these infected plants compared with in vitro mixtures of them. All bands from both types were present, indicating that the differences were real and reproducible. This method is of value as a means of classifying cucumber mosaic virus isolates, as it more closely reflects a range of biological characteristics than do other methods currently used.

Electrophoresis, Polyacrylamide Gel↗
PubMed2002

Trichomonas vaginalis: observation of coexistence of multiple viruses in the same isolate.

Trichomonas vaginalis is a flagellated, parasitic protozoan that inhabits the urogenital tract of humans. Some isolates of T. vaginalis are infected with a double-stranded RNA (dsRNA) virus, which was described in the literature as homogeneous icosahedral viral particles with an isometric symmetry and 33 nm in diameter. This study examined in detail the viral particles in T. vaginalis isolate 347 and describes a heterogeneous population of viral particles. The different dsRNA viruses were only observed after a change in the technique. The sample was prepared by the negative staining carbon-film method directly onto freshly cleft mica. The detected viruses ranged in size from 33 to 200 nm. Among the shapes observed were filamentous, cylindrical, and spherical particles. These results show that T. vaginalis may be a reservoir for several different dsRNA viruses simultaneously.

Animals↗
PubMedDate not supplied

Trichomonas vaginalis: evaluating capsid proteins of dsRNA viruses and the dsRNA virus within patients attending a sexually transmitted disease clinic.

Some isolates of Trichomonas vaginalis, the number one, non-viral sexually transmitted disease agent, are infected with one or several distinct double stranded (ds)-RNA virus. Immune rabbit anti-capsid serum (IRS) reacted with the capsid protein of purified dsRNA virus of a subset of the virus-infected T. vaginalis isolates. A monoclonal antibody (mAb) that recognized the capsid protein reactive with the IRS was generated. Analysis of the virus capsid protein of virus-infected isolates by probing nitrocellulose blots with mAb revealed diversity among immunoreactivity and in the size of the reactive capsid protein. Despite difficulties in visualizing virus within parasites by cross-section electron microscopy, gold-conjugated mAb readily labeled the cytoplasm of virus-positive trichomonads. Finally and importantly, isolates infecting patients attending an STD clinic, 75% of which were virus-positive isolates, had capsid protein of the same size detected by mAb present in all dsRNA viruses.

Animals↗
PubMed1994

Characterization of rubella virus replication complexes using antibodies to double-stranded RNA.

A feature of the rubella virus (RV) replication cycle is the formation of cytoplasmic vesicle-containing structures known as replication complexes. Following detergent treatment of RV-infected cells, pre-embedding immunogold labeling electron microscopy using antiserum to double-stranded (ds) RNA was employed to characterize the replication complexes. Concentrations of gold particles were found associated with amorphous material located within the RV replication complex. Unlabeled long fine strands, 3-5 nm in width, were also frequently seen associated with this gold-labeled material. On some occasions gold-labeled vesicles within the replication complexes were also detected. The gold-labeled amorphous material was first detected in RV replication complexes at 12 hr postinfection, soon after the reported latent period of 8 hr. Concentrations of gold particles were not detected in mock-infected cells. The findings in this study indicate that the amorphous material is released from detergent-disrupted vesicles within the replication complex and that the vesicles contain the dsRNA. When cells were infected with the related Semliki Forest virus (SFV) and examined using the same antibody, similar gold-labeled material associated with unlabeled fine strands was also observed in SFV replication complexes. For both RV and SFV, the vesicles which line the inner membrane of the replication complexes contain the dsRNA which represent the viral replicative forms and replicative intermediates.

Animals↗
PubMed1980

Immunofluorescent detection of double-stranded RNA in cells infected with reovirus, infectious pancreatic necrosis virus, and infectious bursal disease virus.

Urea treatment of ethanol-fixed virus-infected cells exposed nucleic acid antigens for immunofluorescence. Three double-stranded (ds) RNA-containing viruses showed bright fluorescence using antibodies against dsRNA. Three single-stranded RNA-containing viruses showed less intense fluorescence with anti-dsRNA. Four out of five cell lines persistently infected with various RNA-containing viruses showed no dsRNA detectable by immunofluorescence.

Cells, Cultured↗
PubMed1995

Differentiation of cognate dsRNA genome segments of bluetongue virus reassortants by temperature gradient gel electrophoresis.

The analysis of reassortant viruses has been a valuable tool in the investigation of protein interaction and function in double-stranded (ds) RNA virus research. The differentiation of cognate dsRNA genome segments of reassortants is conventionally achieved by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). However, due to a high degree of sequence homology among different bluetongue virus (BTV) serotypes, it is not uncommon to find that certain cognate dsRNA segments cannot be differentiated by SDS-PAGE. Temperature gradient gel electrophoresis (TGGE) has been shown to be a much more sensitive method of differentiating RNA or DNA fragments of high sequence homology. Here we report the preliminary application of TGGE in analysis of genomic reassortants of two BTV serotypes, 1 and 23. While six out of ten genome segments between BTV-1 and BTV-23 could not be resolved by SDS-PAGE, all of them were differentiated by TGGE. The ability of TGGE to distinguish between dsRNA segments of high sequence homology may also make it useful in the search for BTV genes responsible for defined characteristics, such as virulence, by differentiating wild-type and mutated gene segments of viruses displaying altered phenotypes.

Bluetongue virus↗
PubMed1978

Virus-like particles and double stranded RNA from killer and non-killer strains of Saccharomyces cerevisiae.

Virus-like particles and DsRNA found in extracts of killer, non-killer and suppressive non-killer strains were co-precipitated from cell extracts using an antibody prepared against purified virus-like particles isolated from a non-killer strain having only the higher molecular weight L dsRNA. The relative amount of virus-like particles correlated roughly with the amount of dsRNA: those strains with high concentrations of dsRNA had the most particles. When a preparation of particles was subjected to sucrose gradient velocity centrifugation, particles containing the S and M dsRNA could be separated from those containing the L dsRNA. These experiments taken together suggest that the L, M and S dsRNAs are separately encapsulated by the same protein coat.

Capsid↗
PubMed1978

Semi-conservative transcription in particles of a double-stranded RNA mycovirus.

During transcription in vitro catalysed by the virion RNA polymerase of Aspergillus foetidus virus AfV-S in the presence of tritiated UTP, the virus double-stranded RNA becomes labelled in one strand, which has the same sequence as the single-stranded RNA transcripts produced. Most of the label incorporated into double-stranded RNA could be chased into single stranded RNA by further reaction with excess unlabelled nucleoside triphosphates. In reactions containing tritiated UTP the single-stranded RNA transcripts released after the first round of transcription were unlabelled. It is concluded that transcription in virions of AfV-S occurs by displacement of one of the strands of double-stranded RNA by the RNA strand being newly synthesised i.e. the reaction is semi-conservative with respect to double-stranded RNA.

Aspergillus↗
PubMed1996

The Ustilago maydis virally encoded KP1 killer toxin.

Some strains of the plant-pathogenic fungus Ustilago maydis secrete toxins (killer toxins) that are lethal to susceptible strains of the same fungus. There are three well-characterized killer toxins in U. maydis-KP1, KP4, and KP6-which are secreted by the P1, P4, and P6 subtypes, respectively. These killer toxins are small polypeptides encoded by segments of an endogenous, persistent double-stranded RNA (dsRNA) virus in each U. maydis subtype. In P4 and P6, the M2 dsRNA segment encodes the toxin. In this work, the KP1 killer toxin was purified for internal amino acid sequence analysis, and P1M2 was identified as the KP1 toxin-encoding segment by sequence analysis of cDNA clones. The KP1 toxin is a monomer with a predicted molecular weight of 13.4kDa and does not have extensive sequence similarity with other viral anti-fungal toxins. The P1M2 segment is different from the P4 and P6 toxin-encoding dsRNA segments in that the 3' non-coding region of its plus strand has no sequence homology to the 3' ends of the plus strands of P1M1, P4M2, or P6M2.

Cloning, Molecular↗
PubMed2001

The H1 double-stranded RNA genome of Ustilago maydis virus-H1 encodes a polyprotein that contains structural motifs for capsid polypeptide, papain-like protease, and RNA-dependent RNA polymerase.

The Ustilago maydis viral (UmV) genome consists of three distinct size groups of double-stranded RNA (dsRNA) segments: H (heavy), M (medium), and L (light). The H segments have been suggested to encode all essential viral proteins, but without any molecular evidences. As a preliminary step to understand viral genomic organization and the molecular mechanism governing gene expression in UmV, we determined the complete nucleotide sequence of the H1 dsRNA genome in P1 viral killer subtype. The H1 dsRNA genome (designated UmV-H1) contained a single open reading frame that encodes a polyprotein of 1820 residues, which is predicted to be autocatalytically processed by a viral papain-like protease to generate viral proteins. The amino-terminal region is the capsid polypeptide with a predicted molecular mass of 79.9 kDa. The carboxy-terminal region is the RNA-dependent RNA polymerase (RDRP) that has a high sequence homology to those of the totiviruses. The H2 dsRNA also encodes a distinct RDRP, suggesting that UmV is a complex virus system like the Saccharomyces cerevisiae viruses ScV-L1 and -La.

Amino Acid Sequence↗
PubMed1997

Characterization of an RNA-dependent RNA polymerase activity associated with La France isometric virus.

Purified preparations of La France isometric virus (LIV), an unclassified, double-stranded RNA (dsRNA) virus of Agaricus bisporus, were associated with an RNA-dependent RNA polymerase (RDRP) activity. RDRP activity cosedimented with the 36-nm isometric particles and genomic dsRNAs of LIV during rate-zonal centrifugation in sucrose density gradients, suggesting that the enzyme is a constituent of the virion. Enzyme activity was maximal in the presence of all four nucleotides, a reducing agent (dithiothreitol or beta-mercaptoethanol), and Mg2+ and was resistant to inhibitors of DNA-dependent RNA polymerases (actinomycin D, alpha-amanitin, and rifampin). The radiolabeled enzyme reaction products were predominantly (95%) single-stranded RNA (ssRNA) as determined by cellulose column chromatography and ionic-strength-dependent sensitivity to hydrolysis by RNase A. Three major size classes of ssRNA transcripts of 0.95, 1.3, and 1.8 kb were detected by agarose gel electrophoresis, although the transcripts hybridized to all nine of the virion-associated dsRNAs. The RNA products synthesized in vitro appeared to be of a single polarity, as they hybridized to an ssDNA corresponding to one strand of a genomic dsRNA and not to the complementary strand. Similarly, reverse transcription-PCR with total cellular ssRNA as a template and strand-specific primers targeting a genomic dsRNA during synthesis of cDNA suggested that only the coding strand was transcribed in vivo. Our data indicate that the RDRP activity associated with virions of LIV is probably a transcriptase engaged in the synthesis of ssRNA transcripts corresponding to each of the virion-associated dsRNAs.

Basidiomycota↗
PubMed2002

Double-stranded nef RNA interferes with human immunodeficiency virus type 1 replication.

RNA interference (RNAi) has been reported to be post-transcriptional gene silencing (PTGS) by approximately 500 nucleotide-(nt)-long double-stranded (ds) RNA that specifically targets homologous sequences of messenger RNA. In this report, we describe inhibition of HIV-1 transcription by synthetic dsRNAs constructed with mutated nef genes (nef dsRNAs) derived from long-term non-progressors (LTNPs) using cotransfection of the target gene-expressing plasmid and dsRNA. The effects of nef dsRNAs were examined with luciferase (Luc) reporter which is combined with the HIV-1 (SF2) LTR in persistently HIV-1-infected T cell and macrophage cell lines. At 48 hr, a defective nef dsRNA (556 nt) suppressed Luc activity more potently than did SF2 full-length nef dsRNA (744 nt), suggesting that approximately 500 nt-long nef dsRNA could interfere with the HIV-1 transcription.

Gene Products, nef↗
PubMed2002

Transmission of double-stranded RNA in Heterobasidion annosum.

Transmission of dsRNA viruses between homo- and heterokaryotic mycelia paired on agar plates and into conidia has been studied in Heterobasidion annosum. Horizontal transmission of dsRNA occurred between both homo- and heterokaryotic isolates, as well as between isolates belonging to different intersterility groups. The proportions of vertical transmission into conidia were 3% and 55%, respectively, for the two isolates included in the study. RT-PCR of dsRNA and PCR-RFLP of mitochondrial markers were used to confirm transmission of dsRNA between the cytoplasms of different mycelia. The identity of nuclei and nuclear migration during experiments were verified using PCR-RFLP of several nuclear markers.

Basidiomycota↗
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↗
PubMed1996

RNA-dependent RNA polymerase activity related to the 20S RNA replicon of Saccharomyces cerevisiae.

Saccharomyces cerevisiae contains two double-stranded RNA (dsRNA) viruses (L-A and L-BC) and two different single-stranded (ssRNA) replicons (20S RNA and 23S RNA). Replicase (dsRNA synthesis on a ssRNA template) and transcriptase (ssRNA synthesis on a dsRNA template) activities have been described for L-A and L-BC viruses, but not for 20S or 23S RNA. We report the characterization of a new in vitro RNA replicase activity in S. cerevisiae. This activity is detected after partial purification of a particulate fraction in CsCl gradients where it migrates at the density of free protein. The activity does not require the presence of L-A or L-BC viruses or 23S RNA, and its presence or absence is correlated with the presence or absence of the 20S RNA replicon. Strains lacking both this RNA polymerase activity and 20S RNA acquire this activity when they acquire 20S RNA by cytoduction (cytoplasmic mixing). This polymerase activity converts added ssRNA to dsRNA by synthesis of the complementary strand, but has no specificity for the 3' end or internal template sequence. Although it replicates all tested RNA templates, it has a template size requirement, being unable to replicate templates larger than 1 kb. The replicase makes dsRNA from a ssRNA template, but many single-stranded products due to a terminal transferase activity are also formed. These results suggest that, in contrast to the L-A and L-BC RNA polymerases, dissociation of 20S RNA polymerase from its RNA (or perhaps some cellular factor) makes the enzyme change its specificity.

Nucleotides↗
PubMed1993

Temperature-sensitive mutants in the vaccinia virus A18R gene increase double-stranded RNA synthesis as a result of aberrant viral transcription.

Mutations in the vaccinia gene A18R cause activation of the cellular ribonucleolytic 2-5A pathway. To determine the mechanism of 2-5A pathway activation, mutant infections were analyzed for synthesis of double-stranded RNA and for transcription of individual virus genes. At late times postinfection, A18R mutant-infected cells contained an increased amount of complementary RNA and a higher steady state level of RNA from regions of the genome transcribed normally only early in the infection. The phenotype of A18R ts mutants is indistinguishable from that of wild-type infections done in the presence of isatin-beta-thiosemicarbazone (IBT). Actinomycin D is a potent inhibitor of activation of the 2-5A pathway in IBT-treated wt infections. Based on these observations, we conclude that the phenotype induced by A18R mutants or by IBT treatment of wt infections is caused by a loss of control of late viral transcription.

2',5'-Oligoadenylate Synthetase↗