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PubMedDate not supplied

Nucleic acid and structural proteins of infectious bursal disease virus isolates belonging to serotypes I and II.

The nucleic acid and structural proteins of infectious bursal disease virus serotype I (six isolates) were compared with those of serotype II (two isolates). Five of the serotype I isolates originated from chickens, whereas both serotype II isolates were from turkeys. The growth curves of representative isolates of both serotypes were similar, but the latent period and virus yield were different. The seven isolates tested had two segments of double-stranded genomic RNA. The RNA migration patterns of viruses belonging to each serotype were similar, but differences were noticed between the two serotypes. There were differences in the molecular weights of viral proteins (VP) 3 and 4 from the two serotypes, and serotype II isolates lacked VP-2.

Animals↗
PubMed1991

The autocatalytic protease p29 encoded by a hypovirulence-associated virus of the chestnut blight fungus resembles the potyvirus-encoded protease HC-Pro.

Gene expression by a viral-like double-stranded RNA genetic element associated with reduced virulence (hypovirulence) of the chestnut blight fungus was recently shown to involve an autoproteolytic event which resulted in the release of an encoded protease, designated p29, from a polyprotein during translation. Mutational analysis of p29, described in this report, revealed that residues Cys-162 and His-215 are essential for autocatalytic cleavage. The results were also consistent with previous predictions that cleavage occurs between Gly-248 and Gly-249. Interestingly, p29 bears a striking resemblance to the potyvirus-encoded protease HC-Pro. Both proteases autocatalytically cleave at glycine dipeptides. In addition, there is a significant degree of similarity in the amino acid sequences flanking the essential Cys and His residues of the two proteases and in the spacing of these residues from their respective cleavage sites.

Amino Acid Sequence↗
PubMed1996

Possible involvement of double-stranded RNA-activated protein kinase in cell death by influenza virus infection.

We reported previously that influenza virus infection induces the apoptotic death of HeLa cells associated with activation of the Fas gene. In this report, we show that transfection with a PKR having a point mutation in the catalytic domain of K at 296 to R suppressed both the augmented expression of Fas and cell death by influenza virus infection. These results suggested the involvement of PKR in influenza virus-induced cell death.

Apoptosis↗
PubMed1983

A survey of electropherotype relationships of bluetongue virus isolates from the western United States.

The genome profiles of 200 field isolates of bluetongue virus were obtained by extracting the double-stranded RNA genome from each isolate and separating the ten genome segments by polyacrylamide gel electrophoresis. These virus isolates, consisting of representatives of the four bluetongue virus serotypes existing in the U.S.A., were obtained during an epidemiological study commencing in 1979 in four western states of the United States. Analysis of the migration patterns of the ten double-stranded RNA genome segments revealed distinct variations of electrophoretic profiles of many isolates. These variations are discussed in relation to the year and geographical region in which the isolate was made, the species of animal from which the virus was obtained and the electropherotype relationships of the four bluetongue virus serotypes existing in the U.S.A.

Animals↗
PubMed1980

A comparative study on the u.v. resistance of double-stranded and single-stranded encephalomyocarditis virus RNAs: evaluation of the possible contribution of host-mediated repair.

To reveal previously suggested host-mediated repair of u.v.-induced lesions in dsRNA of encephalomyocarditis (EMC) virus, two sets of experiments have been carried out: (i) samples of dsRNA of EMC virus were irradiated with different doses of u.v. light and their infectivity was assayed in krebs II cells, before and after conversion of dsRNA into a ss form: (ii) samples of ssRNA of EMS virus were similarly irradiated and their infectivity was assayed before and after conversion of ssRNA into a ds form. No evidence for a significant host-mediated repair of dsRNA in this virus-cell system has been obtained.

Animals↗
PubMed1986

Enhanced level of double-stranded RNA-dependent protein kinase in peripheral blood mononuclear cells of patients with viral infections.

The protein kinase activity dependent on double-stranded RNA was assayed in extracts of peripheral blood mononuclear (PBM) cells from healthy volunteers and in patients with different types of virus infections. The protein kinase was assayed after one-step purification on an immunoaffinity column containing monoclonal antibody against the 68,000 Mr protein, a subunit of the protein kinase. In healthy individuals, the activity of the protein kinase remains constant. In contrast, the activity of the protein kinase is enhanced significantly in patients with viral infections and is decreased during the course of the disease in parallel with clinical ameliorations and reversal of clinical symptoms. There is a strong correlation between the enhanced levels of the protein kinase activity and another interferon-mediated enzyme, 2-5A synthetase. Both of these enzymes, therefore, could be used as markers to evaluate the state of the disease and recovery. In the different populations of lymphocytes, most of the protein kinase activity was found to be present in T and B lymphocytes, T lymphocytes showing a higher activity than B lymphocytes.

2',5'-Oligoadenylate Synthetase↗
PubMed2001

Partial characterisation of citrus leaf blotch virus, a new virus from Nagami kumquat.

Citrus leaf blotch virus (CLBV) was purified from leaves of Nagami kumquat SRA-153 that showed bud union crease when propagated on Troyer citrange. Virions were filamentous particles (960 x 14 nm) containing a 42 kDa protein and a single-stranded RNA (ssRNA) of about 9,000 nt (Mr 3 x 10(6)). Infected tissue contained three species of double-stranded RNA (dsRNA) of Mr 6, 4.5 and 3.4 x 10(6). The nucleotide sequence of several complementary DNA (cDNA) clones showed significant similarities with replication-related proteins from plant filamentous viruses in several genera. A digoxigenin-labelled probe from one of these cDNA clones hybridised in Northern blots with ssRNA from virions and with the three dsRNA species, suggesting that the ssRNA is the genomic RNA of the virus, the largest dsRNA is its replicative form, and the two smaller dsRNAs probably replicative forms of 5' co-terminal subgenomic RNAs. CLBV was also detected in several citrus cultivars from Spain and Japan including Navelina sweet orange field trees propagated on Troyer citrange showing bud union crease; however, no virus could be detected in other citrus trees with similar symptoms. This indicates that CLBV is not restricted to kumquat SRA-153, but its involvement in causing the bud union disorder remains unclear.

Citrus↗
PubMed1989

Extrahepatic replication of woodchuck hepatitis virus in chronic infection.

We describe studies of woodchuck hepatitis virus nucleic acids in liver and other tissues of chronically infected woodchucks, using Southern and Northern blot hybridization techniques. Single-stranded and covalently closed circular replicative DNA molecules were distinguished from partly double-stranded virus genomes. In most animals the liver contained more virus than any other organ, but all extrahepatic organs studied (spleen, thymus, pancreas, and kidney) contained viral DNA and significant amounts of viral RNA. In the spleen, partly double-stranded virus genomes were present at higher levels than in any other organ but liver, but nonetheless replicative intermediates were not detected. The observation of RNA transcripts in the absence of detectable covalently closed circular DNA template suggests that a small proportion of cells in extrahepatic tissues are infected with WHV.

Animals↗
PubMed1991

Interferon action: binding of viral RNA to the 40-kilodalton 2'-5'-oligoadenylate synthetase in interferon-treated HeLa cells infected with encephalomyocarditis virus.

The 40-kDa 2'-5'-oligoadenylate [(2'-5') (A)n] synthetase isoenzyme was proven to be a mediator of the inhibition of encephalomyocarditis virus (EMCV) replication by interferon (IFN). When activated by double-stranded RNA, this enzyme converts ATP into 2'-5'-oligoadenylate [(2'-5') (A)n], and (2'-5') (A)n was found to accumulate in IFN-treated, EMCV-infected cells. The only known function of (2'-5') (A)n is the activation of RNase L, a latent RNase, and this was also implicated in the inhibition of EMCV replication. Intermediates or side products in EMCV RNA replication, presumed to be partially double stranded, were shown to activate (2'-5') (A)n synthetase in vitro. These findings served as the basis of the long-standing hypothesis that the activator of (2'-5') (A)n synthetase in IFN-treated, EMCV-infected cells is the viral RNA. To test this hypothesis, we have generated a polyclonal rabbit antiserum to the human 40-kDa (2'-5') (A)n synthetase. The antiserum immunoprecipitated, from IFN-treated HeLa cells that had been infected with EMCV, the 40-kDa (2'-5') (A)n synthetase protein in complex with both strands of EMCV RNA. The immunoprecipitate was active in (2'-5') (A)n synthesis even without addition of double-stranded RNA, whereas the immunoprecipitate from IFN-treated, uninfected cells was not. These and other results demonstrate that in IFN-treated, EMCV-infected cells, viral RNA is bound to the (2'-5') (A)n synthetase and suggest that the agent activating the (2'-5') (A)n synthetase is the bound viral RNA.

2',5'-Oligoadenylate Synthetase↗
PubMed1989

Coordinate viral induction of tumor necrosis factor alpha and interferon beta in human B cells and monocytes.

Human tumor necrosis factor alpha (TNF-alpha) gene expression can be induced primarily in cells of the monocyte/macrophage lineage by a variety of inducers, including lipopolysaccharide, phorbol esters such as phorbol 12-myristate 13-acetate, and virus or synthetic double-stranded RNA [poly(I).poly(C)]. In this paper we show that the TNF-alpha gene also responds to virus and phorbol 12-myristate 13-acetate in B lymphocytes and that virus is the most potent inducer of TNF-alpha mRNA in both monocyte and B-cell lines. In addition, we show that viral infection coinduces the expression of TNF-alpha and interferon beta mRNA and that viral induction of both genes is blocked by the kinase inhibitor 2-aminopurine. Inhibition of protein synthesis with cycloheximide had no effect on mRNA expression of the genes in one of three cell lines tested (U937) but blocked the viral induction of both genes in another (Namalwa). Thus, the regulatory factors required for mRNA induction of both genes are present prior to the addition of virus in U937 but not in Namalwa cells. However, in a third cell line (JY), cycloheximide blocked viral induction of the interferon beta gene but not the TNF-alpha gene. Taken together, these observations suggest that viral induction of TNF-alpha and interferon beta gene expression may involve overlapping pathways with both common and distinct regulatory factors.

2-Aminopurine↗
PubMed1989

A double-stranded RNA-inducible fish gene homologous to the murine influenza virus resistance gene Mx.

We cloned and sequenced a 2.35-kilobase EcoRI fragment of genomic DNA from a local freshwater fish (Perca fluviatilis) that strongly hybridized to probes derived from the murine influenza virus resistance gene Mx. The cloned fish DNA contained blocks of sequences related to Mx gene exons 3 to 8, which appeared to represent exons of a bona fide fish gene because they were separated by intron sequences flanked by consensus splice acceptor and donor sites. Injection of double-stranded RNA into the peritoneal cavity of trouts resulted in 5- to 10-fold elevated levels of two liver mRNAs of about 2.0 to 2.5 kilobases in length that hybridized to the cloned genomic DNA. High sequence similarity between this fish gene and the murine Mx gene, identical exon lengths, and similar inducibilities in vivo by double-stranded RNA indicate that we isolated a fragment of a fish Mx gene.

Amino Acid Sequence↗
PubMed2004

Mapping the site of guanylylation on VP1, the protein primer for infectious pancreatic necrosis virus RNA synthesis.

VP1, the putative virion-associated RNA-dependent RNA polymerase (RdRp) of infectious pancreatic necrosis virus (IPNV) can be guanylylated in vitro whereupon it becomes a primer for in vitro RNA synthesis [Virology 208 (1995) 19]. The role of a template or other virion polypeptides in the reaction is unknown. To shed light on this question, his-tagged recombinant VP(1) (rVP1) was expressed both in Escherichia coli and insect cells and used in the guanylylation reaction. Unlike other viral VPg polypeptides, the purified rVP1 alone could guanylylate itself in vitro in a template-independent manner. Chemical and enzymatic cleavage in combination with site-directed mutagenesis mapped the site of guanylylation to serine 163. The purified rVP1 functioned as a primer as well as an RdRp in vitro, producing labeled dsRNA in the presence of [alpha(32)P] NTP and synthetically produced viral ss + RNA as a template. Only a single cycle of replication was observed and labeled VPg could be recovered from the dsRNA by RNase V(1) digestion. Denaturation of the dsRNA yielded genome-length labeled ssRNA, indicating that RNA synthesis was not initiated by 3'-end snap-back self-priming. Mutating serine 163 to alanine of rVP1 abolished both its self-guanylylating and polymerizing activity.

Animals↗
PubMedDate not supplied

Detection of virus-associated dsRNA from leafroll infected grapevines.

A simple procedure is described for reproducible detection of double stranded (ds) RNAs in leafroll infected grapevines. The procedure involves the extraction of tissues by a medium which preferentially yields dsRNA. The RNA is purified by CF11 cellulose chromatography and gel electrophoresis. The dsRNAs varied in size in different vines. In the cases tested they did not cross hybridize and occurred at higher concentrations in stem cortex tissues than in leaves. They were not detectable in healthy vines, could be passaged with the disease to healthy plants by graft inoculation and removed by virus elimination procedures. These observations indicated that the dsRNAs are of viral origin and that a number of viruses are associated with the grapevine leafroll disease.

Chromatography↗
PubMed1987

Double-stranded cucumovirus associated RNA 5: which sequence variations may be detected by optical melting and temperature-gradient gel electrophoresis?

Sequence variants of the double-stranded form of satellite RNAs of cucumber mosaic virus (dsCARNA 5) were analyzed for the possibility to experimentally detect minor nucleotide sequence changes. Denaturation maps (helix-probability versus position of the nucleotide in the sequence versus temperature) were calculated applying the Poland algorithm. Optical denaturation curves and temperature-gradient gel mobility curves were simulated using the denaturation maps and were compared with experimental results from optical melting and temperature-gradient gel electrophoresis (Tien Po et al., accompanying paper). Melting of the dsRNAs starts from both ends of the molecule in two transitions of low co-operativity, continues in the right part in a highly co-operative transition, and is finished in another highly co-operative transition including strand-separation. Whereas all parts of the molecule contribute uniformly to the optical melting curve, opening of the ends predominates in the retardation transition in gel electrophoresis. Detailed discussion of the influence of base pair changes in the sequence shows that a single base pair change may be detected by temperature-gradient gel electrophoresis, if it is located in certain favorable locations, whereas its detection in optical melting curves is possible only in very special cases. The systematic differences found in the accompanying paper between necrogenic and non-necrogenic dsCARNA 5 could be interpreted on the basis of such nucleotide sequence differences.

Base Composition↗
PubMed1986

Association of tobacco etch virus related RNA with chloroplasts in extracts of infected plants.

The RNA in various subcellular fractions of tobacco etch virus (TEV) infected tissue was analyzed for the presence of complementary viral RNA, and double-stranded viral RNA by hybridization with 32P-labeled viral RNA or cDNA probes. Although viral RNA was detected in several cellular fractions, the complementary RNA of full-length size was found exclusively associated with fractions containing chloroplasts. Treatment of RNAs with RNase before hybridization suggested that the virus-related complementary RNA was present in double-stranded form.

Chloroplasts↗
PubMed1982

Evidence that infectious pancreatic necrosis virus has a genome-linked protein.

The double-stranded RNA segments of infectious pancreatic necrosis virus were extracted from virions by a method which avoids proteinase. In contrast to proteinase-treated RNA, such segments (i) exhibited a lower electrophoretic mobility in sodium dodecyl sulfate-polyacrylamide gels and agarose gels, (ii) had a slightly lower buoyant density, and (iii) demonstrated a marked tendency toward aggregation as observed by electron microscopy. A small amount of protein tightly bound to the RNA could account for the above properties, and a 110,000-dalton protein was liberated from purified virion RNA by sequential digestion with RNase III and RNase A. The amount of radioactivity associated with RNA from virions labeled in vivo with [35S]methionine suggested that an average of 1.4 molecules was bound per RNA segment. Interactions between RNA segments seen in electron micrographs appeared to occur only among the ends of the segments, suggesting these were the exclusive sites of protein attachment.

Centrifugation, Density Gradient↗