Biomedical subjects

E Truve

Publications and source records attributed to E Truve.

10 recordsLinked to original sources

PubMed2000

RNA-binding activities of cocksfoot mottle sobemovirus proteins.

Cocksfoot mottle virus (CfMV) has a positive-sense ssRNA genome containing four open reading frames (ORFs). ORF1 encoded protein (P1) is the putative movement protein; the product of ORF2a (P2a) contains VPg and the motifs characteristic of serine proteases. P2b, encoded by ORF2b, is the putative RNA-dependent RNA polymerase. P3, the coat protein, is encoded by ORF3. CfMV P1, P2a, P2b, and P3, containing a six histidine tag at the amino terminus, were expressed in Escherichia coli, purified and their RNA-binding activities were analysed. The northwestern blot assay showed that His-tagged P1, P2a, P2b, and P3 were able to interact with ssRNA transcripts in a sequence-nonspecific manner. The filter-binding assay confirmed the ssRNA-binding capacity of recombinant P1, P2a, and P3. The RNA-binding activities of His-tagged P3 and native coat protein were similar. P1 and P2a binding to ssRNA decreased markedly by increasing NaCl concentrations. In contrast, P3 had the RNA-binding optimum at 100-200 mM NaCl. We discuss the possible amino acid motifs involved in the RNA-binding of CfMV proteins.

Binding, Competitive↗

PubMed2000

Sobemoviruses.

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Genome, Viral↗

PubMed1999

Identification of genes encoding for the cocksfoot mottle virus proteins.

Cocksfoot mottle sobemovirus (CfMV) has a monopartite single-stranded positive-sense RNA genome. In wheat germ extract, in vitro translation of CfMV virion RNA resulted in the production of four major proteins of 100, 71, 34 and 12 kDa. In this paper we show the individual genes from which these polypeptides are synthesised. Polyclonal antisera against the proteins encoded by all open reading frames (ORFs) of CfMV were produced. Antibodies were used to immunoprecipitate the in vitro translation products of CfMV genomic RNA as well as the coupled in vitro transcription/translation products of individual viral genes. We demonstrate that the 12, 71, and 100 kDa CfMV proteins are synthesised from the bicistronic genomic RNA of the virus. The CfMV 12 kDa protein is produced from ORF1, the 71 kDa protein from ORF2a (and does not result from the proteolytic cleavage of the polyprotein) and the 100 kDa protein is a polyprotein encoded by ORFs 2a and 2b by a -1 ribosomal frameshift. ORF2b alone does not direct any in vitro protein synthesis. CfMV 34 kDa protein is a coat protein synthesised from the virion-packed subgenomic RNA. The translational strategies of different sobemoviruses are also discussed in this paper.

Animals↗

PubMed1995

The putative replicase of the cocksfoot mottle sobemovirus is translated as a part of the polyprotein by -1 ribosomal frameshift.

The polyprotein of cocksfoot mottle sobemovirus (CfMV) is encoded by two overlapping open reading frames (ORF). The ORF 2a codes for the putative VPg and serine protease and the ORF 2b codes for the putative replicase. The consensus signals for a -1 ribosomal frameshifting event are found at the very beginning of the overlapping region of these ORFs. The shifty heptanucleotide in CfMV is UUUAAAC, and the secondary structure after the shifty sequence is predicted to be a stem-loop. In vitro translation of the CfMV RNA in wheat germ extract produced proteins of several sizes, including one of 100 kDa. According to the nucleotide sequence data, no single ORF is capable of directing the synthesis of a 100-kDa protein. A chimeric beta-glucuronidase-CfMV cDNA containing the entire ORF 2a and 2b overlap region including frameshift signals was constructed. A trans-frame protein of 108 kDa was produced from this construct with an efficiency of 26-29% by in vitro translation in wheat germ extract. CfMV is the first sobemovirus in which the putative replicase is reported to be produced as a part of a polyprotein by a -1 frameshift event. The replicases of the sobemoviruses are related to the luteovirus subgroup II replicases, which are known to be produced by -1 ribosomal frameshift. The reported amino acid sequences of the putative replicases of sobemo- and subgroup II luteoviruses were compared to that of the putative replicase of CfMV. This comparison revealed more extensive homology between these groups than previously reported.

Amino Acid Sequence↗

PubMed1995

Impairment of intracellular antiviral defense with age: age-dependent changes in expression of interferon-induced and double-stranded RNA-activated 2-5A synthetase in rat.

The 2',5'-oligoadenylate (2-5A) system is involved in the defense of mammalian cells against virus infection. In a previous study [25], we demonstrated that the activities of the enzymes which synthesize and degrade 2-5A [2-5A synthetase (2-5OAS) and 2',3'-exoribonuclease] and of the enzyme that is activated by 2-5A (ribonuclease L) change during aging and development in different tissues of rat. The age-dependent decrease in 2-5OAS activity and increase in 2-5A nuclease activity results in a decrease in the cellular 2-5A content, suggesting that the efficiency of the antiviral 2-5A system is impaired in aged rats. Here we determined the age-dependent changes in the level of mRNA coding for the class I isoenzyme of 2-5OAS (M(r) 40-46 kDa) in rat liver and brain using a cDNA which was recently cloned from rat hippocampus. We found that the decrease in 2-5OAS activity is accompanied by a decrease in the level of 2-5OAS mRNA; in old animals (32-33 months old), the amount of 2-5OAS mRNA was reduced to 20-30% compared to young adult (2-3 months old) (100%) and middle-aged adult animals (12 months old) (110-120%). In addition, Western-blotting experiments revealed that the amount of class I 2-5OAS capable of binding to its activator, poly(I).poly(C), is also diminished in the livers and brains of old rats compared to those of young adult and middle-aged adult animals.

2',5'-Oligoadenylate Synthetase↗

PubMed1995

Characterization of cocksfoot mottle sobemovirus genomic RNA and sequence comparison with related viruses.

The genome of cocksfoot mottle virus (CfMV) is a positive-sense ssRNA molecule of 4082 nucleotides as revealed by sequencing the entire genome. The 5'-untranslated region of the genome is 69 nucleotides and the 3'-untranslated region is 225 nucleotides in length. The coding region contains four open reading frames (ORFs). The organization of CfMV ORFs differs significantly from that of the previously sequenced sobemoviruses southern bean mosaic virus and rice yellow mottle virus. ORF1 encodes a protein having a calculated molecular mass of 12.3 kDa. The function of this protein is unknown. The next ORF codes for the putative VPg and serine protease. The ORF2a product consists of 568 amino acids, with a calculated molecular mass of 60.9 kDa. The replicase of CfMV is translated as part of a polyprotein by--1 ribosomal frameshifting in ORF2a. The calculated molecular mass of the transframe protein is 103.4 kDa. ORF3 encodes the 27.6 kDa coat protein. This has been verified by amino acid sequencing of the CfMV coat protein N terminus. Northern blots of total RNA from CfMV-infected barley leaves reveal the 4.1 kb genomic RNA band and one virus-specific band of 1.2 kb, which may represent a subgenomic RNA for coat protein synthesis.

Amino Acid Sequence↗

PubMed1994

Homologies between different forms of 2-5A synthetases.

Sequence analyses of 2-5A synthetases of class I (M(r) 40,000-46,000) revealed high homology among them. The cDNA coding for the M(r) 69,000 2-5A synthetase of class II displayed in the second half a likewise high homology to the complete sequences of class I enzymes. This high degree of conservation of the 2-5A synthetases supports the assumption that these enzymes play important roles during virus infection (Williams et al. 1979; Coccia et al. 1990) and in the control of growth and differentiation of mammalian cells (Williams and Silverman 1985).

2',5'-Oligoadenylate Synthetase↗

PubMed1994

Principles and background for the construction of transgenic plants displaying multiple virus resistance.

We investigated the possibility of reconstructing the 2'-5' oligoadenylate (2-5A) pathway into the plant kingdom to achieve multiple virus resistance. Differently phosphorylated 2-5A trimers and tetramers inhibited TMV RNA translation in cell-free systems. In wheat germ extracts the most potent inhibitors were nonphosphorylated forms of 2-5A. Triphosphorylated forms of 2-5A were deposphorylated and hydrolysed in plant extracts. Since we could not detect homologous DNA to mammalian 2-5A synthetase cDNA in tobacco or potato, we cloned rat 2-5A synthetase cDNA and transformed it by the Agrobacterium-mediated mechanism into tobacco and potato. Transformed tobacco plants were resistant to PVS infection and propagation of PVX was reduced. In transgenic potatoes tolerance to PVX and, in one transgenic clone, also to PVY was observed.

2',5'-Oligoadenylate Synthetase↗

PubMed1994

Rapid reduction of mRNA coding for 2'-5'-oligoadenylate synthetase in rat pheochromocytoma PC12 cells during apoptosis.

Apoptosis is a form of physiological cell death, characterized by DNA fragmentation, which often depends on RNA and protein synthesis. Because cellular RNA is also degraded during apoptosis we studied the role of the 2'-5'-oligoadenylate (2-5A) synthetase in this process. The product of the synthetase, 2-5A, stimulates endoribonuclease-L-mediated controlled RNA degradation. Here we show that apoptosis is induced in rat phenochromocytoma PC12 cells by tributyltin (TBT) at low concentrations (1 nM); already 5-10 min. after addition of this compound DNA fragmentation resulting in a stepladder-like gel pattern was observed. The level of mRNA coding for 2-5A synthetase was determined using a cloned cDNA from rats. Sequence analyses of the rat 2-5A synthetase (M(r) 40-46,000) revealed high homology to other members of class I synthetase cloned from mouse and human. Applying the rat cDNA as a probe we found that parallel with degradation of DNA the level of mRNA coding for 2-5A synthetase decreased already 7.5 min. after induction of apoptosis by TBT the amount of 2-5A synthetase mRNA was reduced by 60%. This finding indicates that this enzyme is among those mRNAs which are degraded during apoptosis and it suggests that 2-5A synthetase, which is involved in the antiviral response of cells and most likely in the control of cell growth and differentiation, does not play an active role during this process.

2',5'-Oligoadenylate Synthetase↗

PubMed1993

Transgenic potato plants expressing mammalian 2'-5' oligoadenylate synthetase are protected from potato virus X infection under field conditions.

We cloned and sequenced a rat cDNA encoding the 2'-5' oligoadenylate synthetase, a component of the mammalian interferon-induced antiviral response, and used Agrobacterium-mediated transformation to generate transgenic potato clones expressing this mammalian enzyme. In transgenic plants infected with potato virus X and followed under field conditions, virus concentrations in leaves and in tubers were significantly lower than in nontransgenic controls. Additionally, virus concentration in the leaves of five transgenic clones and in tubers of one clone was also lower than in transgenic potatoes expressing potato virus X coat protein.

2',5'-Oligoadenylate Synthetase↗