1995
DOI: 10.1099/0022-1317-76-6-1483
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Isolation and characterization of an RNA-dependent RNA polymerase from Nicotiana clevelandii plants infected with red clover necrotic mosaic dianthovirus

Abstract: A template-bound RNA polymerase was isolated from Nicotiana clevelandii plants infected with red clover necrotic mosaic dianthovirus (RCNMV) by differential centrifugation, solubilization with dodecyl //-D-maltopyranoside, and chromatography on columns of Sephacryl S-400 and Q-Sepharose. Analysis of the purified polymerase by SDS--polyacrylamide gel electrophoresis, followed by silver staining or immunoblotting, showed that it contained virus-encoded proteins of molecular masses 27 kDa and 88 kDa together with… Show more

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Cited by 38 publications
(30 citation statements)
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“…The coexistence of p27 and p88 in the purified RdRP fraction from RCNMV-infected tissue (2) suggests that the formation of functional RCNMV RNA replication complexes requires interactions between p27 and p88. To address this issue, we analyzed interactions between RCNMV replicase proteins by coimmunoprecipitation using C-terminally hemagglutinin (HA)-tagged p27 (p27-HA), C-terminally FLAG-tagged p27 (p27-FLAG), and C-terminally T7-tagged p88 (p88-T7).…”
Section: Resultsmentioning
confidence: 99%
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“…The coexistence of p27 and p88 in the purified RdRP fraction from RCNMV-infected tissue (2) suggests that the formation of functional RCNMV RNA replication complexes requires interactions between p27 and p88. To address this issue, we analyzed interactions between RCNMV replicase proteins by coimmunoprecipitation using C-terminally hemagglutinin (HA)-tagged p27 (p27-HA), C-terminally FLAG-tagged p27 (p27-FLAG), and C-terminally T7-tagged p88 (p88-T7).…”
Section: Resultsmentioning
confidence: 99%
“…A strong link between cap-independent translation and replication of RNA2 suggests that only de novo-synthesized RNA2 can function as an mRNA template for translation (26). Both p27 and p88 are to be found in a partially purified RdRP fraction extracted from RCNMV-infected tissue (2), and green fluorescence protein (GFP)-fused p27 and p88 localize to the endoplasmic reticulum (ER) (44). These findings suggest that ER-localized p27 and p88 function coordinately in RCNMV RNA replication.…”
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confidence: 99%
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“…The replicase complex consists of virus-coded proteins, such as the RNA-dependent RNA polymerase (RdRp), auxiliary proteins, and possibly host-derived proteins, and the RNA template (1,4,5,20,27). To study the mechanism of viral RNA replication, functional replicases are purified from virus-infected hosts (3,10,12,16,23,26,38,41,42,53,55) or from heterologous systems, including Escherichia coli (17,19,21,24,44,45), yeast (40), insect (22,24,58), Xenopus (13), and mammalian cells (14,24). The advantage of the heterologous systems is that expression of the replicase proteins can be achieved without dependence on virus replication, thus facilitating mutational analysis of the replicase genes.…”
mentioning
confidence: 99%
“…RNA1 encodes a functionally unknown 27-kDa protein (p27) and an 88-kDa protein (p88) with an RNA-dependent RNA polymerase (RdRp) motif (23) translated by the extension of p27 because of a ribosome frameshift (22,52). Proteins p27 and p88 are components of the partially purified template-dependent RNA polymerase extracted from RCNMV-infected tissue (1). RNA1 also encodes a 37-kDa coat protein (CP) expressed from subgenomic RNA (53).…”
mentioning
confidence: 99%