- Volume 91, Issue 12, 2010
Volume 91, Issue 12, 2010
- Plant
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Identification and characterization of RNA-binding activity in the ORF1-encoded replicase protein of Pelargonium flower break virus
More LessPelargonium flower break virus (PFBV) belongs to the genus Carmovirus (family Tombusviridae) and, as with the remaining members of the group, possesses a monopartite genome of single-stranded, positive-sense RNA that contains five ORFs. The two 5′-proximal ORFs (ORFs 1 and 2) encode two polypeptides of 27 and 86 kDa (p27 and p86), respectively, that show homology with replication proteins. The p27 does not present any motif to explain its presumed involvement in replication, while p86 has the motifs conserved in RNA-dependent RNA polymerases. In this work, we have confirmed the necessity of p27 and p86 for PFBV replication. To gain insights into the function(s) of p27, we have expressed and purified the protein from Escherichia coli and tested its ability to bind RNA in vitro. The results have shown that p27 is able to bind ssRNA with high affinity and in a cooperative fashion and that it is also capable of binding other types of nucleic acids, though to a lesser extent. Additionally, competition experiments suggest that p27 has a preference for PFBV-derived ssRNAs. Using truncated forms of p27, it can be concluded that several regions of the protein contribute to its RNA-binding properties and that this contribution is additive. This study is the first to show nucleic acid-binding ability of the ORF1 product of a carmovirus and the data obtained suggest that this product plays an essential role in selection and recruitment of viral RNA replication templates.
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- Fungal
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Mycoviruses related to chrysovirus affect vegetative growth in the rice blast fungus Magnaporthe oryzae
Mycoviruses causing impaired growth and abnormal pigmentation of the host were found in the rice blast fungus, Magnaporthe oryzae. Four dsRNAs, dsRNA 1 (3554 bp), dsRNA 2 (3250 bp), dsRNA 3 (3074 bp) and dsRNA 4 (3043 bp), were detected in isolate S-0412-II 1a of M. oryzae. By picking up single conidia of S-0412-II 1a, cured strains of the fungus were isolated that had completely lost the mycovirus. The cured strains had normal mycelial growth and pigmentation, suggesting that this mycovirus modulates host traits. The buoyant densities of isometric virus particles (∼35 nm diameter) containing these dsRNAs in CsCl ranged from 1.37 to 1.40 g cm−3. The single ORF (3384 nt) of dsRNA 1 encoded a gene product highly homologous to the viral RNA-dependent RNA polymerase of members of the family Chrysoviridae. It is noteworthy that mycovirus S-0412-II 1a was detected not only in host cells but also in culture supernatant. Furthermore, abnormal aggregation of mycelia was observed after adding the mycovirus-containing culture supernatant to an uninfected strain of M. oryzae and mycoviral dsRNAs were detectable from the aggregated mycelia. This novel dsRNA mycovirus was named Magnaporthe oryzae chrysovirus 1.
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- Other Agents
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Epitope-specific anti-prion antibodies upregulate apolipoprotein E and disrupt membrane cholesterol homeostasis
The mechanisms of neuronal degeneration induced by the transformation of normal cellular prion protein (PrPC) into disease-associated PrPSc are not fully understood. Previous reports have demonstrated that cross-linking cellular prion protein by anti-PrPC antibodies can promote neuronal apoptosis. In this report, we now show that treatment of neuronal cells with anti-prion antibodies leads to sequestration of free cholesterol in cell membranes, significant overexpression of apolipoprotein E, and to cytoplasmic phospholipase A2 activation as well as to production of prostaglandin. These results confirm the in vivo toxic effects and indicate that anti-prion antibody treatment of neurons lead to deleterious effects. Finally, great caution should be exerted when adopting antibody-based therapy for prion diseases.
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Digestion and transportation of bovine spongiform encephalopathy-derived prion protein in the sheep intestine
Bovine spongiform encephalopathy (BSE) is acquired orally and the mechanisms involved in the absorption and transportation of infectivity across the gut wall are therefore critical. Isolated gut loops were created in lambs, massaged to remove intestinal contents (flushed) or left non-flushed, inoculated with cattle BSE homogenate and excised at different time-points. Gut loops were examined by immunohistochemistry (IHC) for disease-associated prion protein (PrPd), and the contents were analysed by Western blotting (WB) to determine the degradation rate of protease-resistant PrP (PrPres). The contents of scrapie-inoculated gut loops from a previous experiment were analysed by WB, and these in vivo digestion results were compared with those of an in vitro experiment on the same transmissible spongiform encephalopathy homogenates. BSE-inoculum-derived PrPd was detected by IHC in the gut lumen between 15 min and 3.5 h. It was found in the intestinal lymphatic system from 30 min onwards and was present at the highest frequency at 120 min post-inoculation. In vivo degradation of PrPres in the BSE inoculum had a significantly (P=0.006) different pattern compared with scrapie-derived PrPres, with the BSE PrPres degrading more rapidly. However, the overall amount of degradation became similar by 120 min post-challenge. The results of the in vitro digestion experiments showed a similar pattern, although the magnitude of PrPres degradation was less than in the in vivo environment where absorption could also take place. BSE-derived PrPres is less protease resistant than scrapie PrP over a short time-course and the disappearance of detectable PrPres from the gut lumen results from both absorption and digestion by intestinal contents.
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- Phage
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Structural analysis of a Synechococcus myovirus S-CAM4 and infected cells by atomic force microscopy
More LessA tailed cyanophage, S-CAM4 (family Myoviridae) from California coastal waters that infects Synechococcus, was characterized by atomic force microscopy. Capsomeric clusters of protein composing the 85 nm diameter icosahedral head were resolved and indicated a triangulation number of T=16. The 140 nm tail assembly, exhibiting a helical appearance with a 13 nm pitch, was seen in both extended and contracted states, the latter exposing the injection tube within. Attached below the base plate were six 50 nm long fibres, and six fibres 275–300 nm in length protruded from the periphery of the base plate. Protein-free DNA was abundant from ruptured heads. Virus attached en masse, in clusters and individually to cells, and cell fragments were recorded, as were perforated cells lysed by the phages. The capsid structure appears most closely related to that of the cyanophage Syn9 and the Bacillus subtilis phage SPO1, which may, in turn, be evolutionarily related to herpesvirus.
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Volumes and issues
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Volume 105 (2024)
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Volume 2 (1968)
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Volume 1 (1967)