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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↗
PubMed1999

A comparison of six cypovirus isolates by cross-hybridisation of their dsRNA genome segments.

Genetic relationships between the genome segments of six cypovirus (CPV) isolates were analysed by RNA cross-hybridisation. These included three type 1 viruses and single isolates of types 2, 5 and 12, which collectively are identical to those previously compared by serology and electrophoresis [Mertens et al. (1989), J Gen Virol 70: 173-185]. Since only genome segment 10 of three cypovirus types and segments 8 and 9 of a single virus strain (of type 1) have currently been sequenced, this initial study provides some additional information on sequence variation/similarity in each of the ten genome segments. The RNA of the type 1 viruses showed high levels of cross-hybridisation. Significant but much lower levels of cross-hybridisation were detected between type 1 and the related type 12 CPV. However, only very low levels of cross-hybridisation were detected between the other pairs of viruses. Apart from evidence of a slightly higher level of sequence similarity between the largest segments, the RNA sequence appeared to vary uniformly across the whole genome. There was no evidence for any type specific RNA sequences restricted to individual genome segment(s). The sequence variation, reflected in the levels of RNA sequence similarity and cross hybridisation, correlates well with serological data, showing large differences between CPV types and supports the continued use of electropherotype as one of the 'species parameters' for the classification of cypoviruses.

Animals↗
PubMed2006

Transgenes consisting of a dsRNA of an RNAi suppressor plus the 3' UTR provide resistance to Citrus tristeza virus sequences in Nicotiana benthamiana but not in citrus.

In an attempt to utilize post-transcriptional gene silencing (PTGS) as a means to impart resistance against Citrus tristeza virus (CTV) into citrus plants, the p23 + 3'UTR sequence (p23U) of the VT strain of CTV was engineered to fold into a double-stranded (ds) RNA structure. The resulting construct (p23UI) was introduced into Nicotiana benthamiana and Alemow (Citrus macrophylla) plants by Agrobacterium-mediated transformation. Transgenic p23UI- N. benthamiana were resistant to infection with a viral vector made of Grapevine virus A (GVA) + p23U (GVA-p23U), as indicated by the absence of the chimeric virus from inoculated plants. Inoculation of transgenic p23UI Alemow plants with CTV resulted in delayed appearance of symptoms in 9 out of the 70 transgenic plants. However, none of the plants showed durable resistance, as indicated by the obtaining of similar Northern hybridization signals from both transgenic and non-transgenic citrus plants. The possible causes for the failure of transgenic citrus plants to confer durable resistance to CTV are discussed.

3' Untranslated Regions↗
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↗
PubMed1978

Interferon-associated, dsRNA-dependent enzyme activities in a mutant 3T6 cell engaged in the semiconstitutive synthesis of interferon.

Cytoplasmic extracts of untreated cultures of a virus-resistant mutant of mouse 3T6 cells, designated 3T6-VrB2, contain two double-stranded, RNA-activated enzyme activities associated with interferon action. These are the synthesis of a low molecular weight oligonucleotide inhibitor of cell-free protein synthesis from ATP, and the phosphorylation of a 67,000 dalton polypeptide by transfer of the gamma phosphate of ATP. Basal levels of both enzyme activities are detectable in extracts of untreated parental 3T6 cells, and are greatly enhanced upon interferon pretreatment. A procedure was developed, using a nonionic detergent to effect cell lysis, which allowed the analysis of the protein kinase activity from as few as 2 x 10(7) cells. Using this procedure, direct proportionalities were demonstrated between the concentration of interferon to which 3T6 cells were exposed, and both the level of protein kinase activity and the magnitude of the antiviral state were established in these cells. Furthermore, untreated cultures of 3T6-VrB2 exhibited both an antiviral state and an intracellular protein kinase activity equal to that of cultures of the parental 3T6 cells pretreated with a single concentration of mouse interferon.

Cell Line↗
PubMed1982

Yeast L dsRNA consists of at least three distinct RNAs; evidence that the non-Mendelian genes [HOK], [NEX] and [EXL] are on one of these dsRNAs.

[HOK], [NEX] and [EXL] are non-Mendelian genes affecting the K1 and K2 killer systems of Saccharomyces cerevisiae. T1 fingerprints of L double-stranded RNA from [HOK]-, [NEX]- and [EXL]-containing cells and their heat-cured derivatives indicate that: there are three distinct double-stranded RNAs, L-A, L-B and L-C; [HOK], [NEX] and [EXL] are all located on L-A; there are three functional variants of L-A that produce the [HOK] [NEX], [HOK] [EXL] or [EXL] phenotypes; L-A is compatible with L-B or L-C; and there are additional sequences present in lower copy number. Although their fingerprint patterns are unrelated, solution hybridization shows that L-B and L-C share sequence homology. Strains carrying L-A as the major double-stranded RNA or only L-B or only L-C all have similarly sedimenting (160S) virus-like particles with RNA polymerase activity. Virus-like particles from strains with L-A all have proteins of 81,000 and 180,000 daltons that are absent from isogenic strains cured of L-A. Virus-like particles from strains with only L-B or only L-C both have major proteins of 77,000 and 73,000 daltons.

DNA, Fungal↗
PubMedDate not supplied

Mismatched dsRNA (ampligen) induces protection against genomic variants of the human immunodeficiency virus type 1 (HIV-1) in a multiplicity of target cells.

Mismatched double-stranded RNA of the form r(I)n.r(C12-U)n (Ampligen) has been shown to be active against human immunodeficiency virus type 1 (HIV-1) using CEM and C3 cells as targets for infection by the highly similar HIV-1 isolates HTLV-IIIB and LAV (Montefiori, D.C. and Mitchell, W.M., 1987, Proc. Natl. Acad. Sci. U.S.A., 84, 2985-2989). The scope of Ampligen's anti-HIV-1 activity was examined in this study using the genetically divergent HIV-1 isolate HTLV-IIIRF, two additional target T-cell lines, H9 and MT-2, and a monocyte/macrophage cell line, U937. As judged by indirect immunofluorescence, reverse transcriptase activity and vital dye uptake, Ampligen was active against HTLV-IIIRF in H9, MT-2, C3 and U937 cells in addition to being active against HTLV-IIIB in U937 cells. A minimum of 1 h preincubation of cells (MT-2) with Ampligen was required for maximum activity. These results suggest that Ampligen's potential clinical efficacy may not be limited by either the highly variable nature or host cell range of HIV-1.

Antiviral Agents↗
PubMed2005

Novel splice variants of human ADAR2 mRNA: skipping of the exon encoding the dsRNA-binding domains, and multiple C-terminal splice sites.

We report here two previously unknown alternative splice sites in the mRNA of human adenosine deaminase acting on RNA type 2 (ADAR2), an RNA editing enzyme. One splices out the whole of exon 2, which encodes two double-stranded RNA-binding domains (dsRBDs), resulting in a frameshift that introduces a stop codon in the downstream exon. This variant accounts for between 13% and 20% of the total ADAR2 mRNA in each developmental stage and brain region examined, even though its translated product is not expressed at levels that are detectable by Western blot analysis. The other new splice site is located in exon 9, 83 nucleotides downstream of the stop codon for the long C-terminus, resulting in a new 3' untranslated region (UTR) that is about 80 bp longer than the previously reported short C-terminus. The variant produced by this splice site has a stop codon at the same site as that in ADAR2 mRNA containing canonical exons 9 and 10, and is predicted to be translated as an enzymatically active ADAR2 protein. With these two additional splice sites, a total of 48 mRNA variants are theoretically possible, because each splicing event occurs independently. Among them, variants containing the long C-terminus are translated in human brains in situ, implying that alternative splicing in the 3' UTR of ADAR2 might regulate translational efficiency and mRNA stability in vivo.

3' Untranslated Regions↗
PubMed2006

Increased IL-1beta production from dsRNA-stimulated leukocytes in febrile seizures.

This study examined the possibility that children with and without a history of febrile seizures might mount different immune responses to double-stranded ribonucleic acid, which is a common viral factor that induces host cell immune responses, and is recognized by Toll-like receptor 3. The production of interleukin-1beta and interferon-alpha from double-stranded ribonucleic acid-stimulated leukocytes was examined in 27 children (age 3.6+/-0.3 years) with a history of febrile seizures and in 18 children (age 3.4+/-0.2 years) without a history of febrile seizures. Significantly (P=0.0007) increased interleukin-1beta production was observed in children with a history of febrile seizures, compared with control subjects. When patients with a single prior episode of febrile seizures (n=9) and those with multiple prior episodes of febrile seizures (n=18) were compared, a significant difference in interleukin-1beta production was not observed. Genotyping of interleukin-1beta(-511), Toll-like receptor 3, Toll-IL-1 receptor domain-containing adapter inducing interferon-beta, and interleukin-1 receptor antagonist polymorphisms revealed no significant differences in allelic distribution among febrile seizure patients and control subjects. Interleukin-1beta production was not significantly influenced by genotype. Viral infection results in increased interleukin-1beta production in febrile seizure patients, and this may play a role in febrile seizures.

Adaptor Proteins, Vesicular Transport↗
PubMed2005

Evaluation of rotavirus dsRNA load in specimens and body fluids from experimentally infected juvenile macaques by real-time PCR.

We recently established a non-human primate model of rotavirus infection that is characterized by consistent and high levels of virus antigen shedding in stools. Here, we report that starting from post challenge day (PCD) 2, 6 x 10(3) to 1.5 x 10(6) copies of rotavirus double-stranded RNA per nanogram of total RNA were detected by real-time PCR in MA104 cells that were 48 h pre-incubated with filtered stool suspensions of three experimentally infected juvenile macaques. The peak of virus load was detected at PCD 4-5, followed by decreased load at PCD 6-11, and very low levels at PCD 12. Such a pattern corresponded to virus shedding in stools as reported recently based on enzyme-linked immunosorbent assay (ELISA) results. In addition, plasma and cerebrospinal fluids (CSF) from six infected animals were tested for the presence of rotavirus. Rotavirus extraintestinal escape was revealed in three out of six animals by a combination of real-time and nested PCR. However, very low quantities of detected viral RNA (approximately 20 copies/ng of total RNA) were not suggestive of viremia. Thus, the rhesus model of rotavirus infection can be exploited further in studies with vaccine candidates designed to prevent or abrogate rotavirus infection.

Animals↗
PubMed2004

Cloning and sequence analysis of dsRNA segments 5, 6 and 7 of a novel non-group A, B, C adult rotavirus that caused an outbreak of gastroenteritis in China.

A diarrhoeal outbreak among adults in China was caused by a new rotavirus, termed ADRV-N, that does not react with antisera directed against group A, B or C rotaviruses [Zhonghua Liu Xing Bing Xue Za Zhi (Chin. Epidemiol.) 19 (1998) 336]. ADRV-N can be propagated in cell cultures [Zhonghua Yi Xue Za Zhi (Natl. Med. J. China) 82 (2002) 14]. We present the complete sequences for ADRV-N genome segments 5 and 6, and a full ORF sequence of genome segment 7. The deduced amino acid sequences suggest that these segments encode NSP1, VP6 and NSP3, respectively. These three ADRV-N genome segments have a unique -ACCCC-3' terminal sequence. The 5'-GG- terminus of segments 5 and 6 is the same as that of other rotaviruses. The amino acid similarity between VP6 and NSP3 of ADRV-N and the cognate sequences of their closest counterpart, group B IDIR, was 37 and 35%, respectively. The ADRV-N NSP1 has a double-stranded RNA binding motif (DSRM) and a putative autoproteolytic cleavage motif upstream from the DSRM. The putative ADRV-N NSP3 has a truncated C-terminus compared to the cognate protein of group B rotaviruses. All the available data demonstrate that ADRV-N differs significantly from the known rotaviruses and strongly suggest that ADRV-N is the first recognized member of a new group of rotaviruses infecting humans.

Adult↗
PubMed2002

Caspase 9 activation by the dsRNA-dependent protein kinase, PKR: molecular mechanism and relevance.

The double-stranded RNA-dependent protein kinase (PKR) induces apoptosis by activation of the FADD/caspase 8 pathway. Here we show that upon PKR expression, caspase 9 is processed and activated, correlating with the translocation of cytochrome c to the cytoplasm and breakdown of mitochondrial potential upon Bax insertion. However, treatment of cells with an inhibitor of caspase 9 could not prevent PKR-induced apoptosis. During PKR-induced apoptosis, caspase 9 is activated downstream of caspase 8. Our findings revealed that caspase 9, although dispensable, is a mediator of PKR-induced cell death.

Apoptosis↗
PubMed2003

Unique properties of the inner core of bacteriophage phi8, a virus with a segmented dsRNA genome.

The inner core of bacteriophage phi8 is capable of packaging and replicating the plus strands of the RNA genomic segments of the virus in vitro. The particles composed of proteins P1, P2, P4, and P7 can be assembled in cells of E. coli that carry plasmids with cDNA copies of genomic segment L. The gene arrangement on segment L was found to differ from that of other cystoviruses in that the gene for the ortholog of protein P7 is located at the 3' end of the plus strand rather than near the 5' end. In place of the normal location of gene 7 is gene H, whose product is necessary for normal phage development, but not necessary for in vitro genomic packaging and replication. Genomic packaging is dependent upon the activity of an NTPase motor protein, P4. P4 was purified from cell extracts and was found to form hexamers with little NTPase activity until associated with inner core particles. Labeling studies of in vitro packaging of phi8 RNA do not show serial dependence; however, studies involving in vitro packaging for the formation of live virus indicate that packaging is stringent. Studies with the acquisition of chimeric segments in live virus indicate that phi8 does package RNA in the order s/m/l. The inner core of bacteriophage phi8 differs from that of its relatives in the Cystoviridae in that the major structural protein P1 is able to interact with the host cell membrane to effect penetration of the inner core into the cell.

Adenosine Triphosphatases↗