Search results for "RNA-Dependent RNA Polymerase"
showing 10 items of 40 documents
Nucleotide sequence of a ssRNA phage from Acinetobacter: kinship to coliphages.
2002
The complete nucleotide sequence of ssRNA phage AP205 propagating in Acinetobacter species is reported. The RNA has three large ORFs, which code for the following homologues of the RNA coliphage proteins: the maturation, coat and replicase proteins. Their gene order is the same as that in coliphages. RNA coliphages or Leviviridae fall into two genera: the alloleviviruses, like Qβ, which have a coat read-through protein, and the leviviruses, like MS2, which do not have this coat protein extension. AP205 has no read-through protein and may therefore be classified as a levivirus. A major digression from the known leviviruses is the apparent absence of a lysis gene in AP205 at the usual positio…
RNA dependent DNA polymerase in cells of xeroderma pigmentosum
1971
Abstract Cells from X.P. ∗ skin contain an RNA dependent DNA polymerase, while in cells from normal skin this enzyme is lacking. This finding stimulates the thought that carcinogenesis in X.P. cells is due to an infection with an oncogenic RNA virus.
Biochemical and Kinetic Analyses of NS5B RNA-Dependent RNA Polymerase of the Hepatitis C Virus
1998
The biochemical properties of the RNA-dependent RNA polymerase (RdRp) of the hepatitis C virus were analyzed. A hexahistidine affinity-tagged NS5B fusion protein was expressed with recombinant baculoviruses in insect cells and purified to near homogeneity. Enzymatic activity of the purified protein was inhibited by KCl or high concentrations of NaCl and was absolutely dependent on Mg2+, which could be replaced by Mn2+. NS5B was found to be processive and able to copy long heteropolymeric templates with an elongation rate of 150-200 nucleotides/min at 22 degreesC. Kinetic constants were determined for all four nucleoside triphosphates and different templates. In case of a heteropolymeric RNA…
RNA In Vitro Synthesis by Phage T7 DNA-Dependent RNA Polymerase
1998
The purpose of this chapter is to introduce beginners in molecular biology to RNA transcription by phage T7 DNA-dependent RNA polymerase. The work outlined here includes the transcription procedure of plasmid vectors or PCR-amplified DNA templates, the purification and identification of RNA products by sequencing with reverse transcriptase.
Bleomycin, a selective inhibitor of DNA-dependent DNA polymerase from oncogenic RNA viruses.
1972
Abstract Bleomycin, an antibiotic, inhibits the DNA-dependent DNA polymerase from Rauscher murine leukemia virus. Higher concentrations of BLM ∗ are required to inhibit it's RNA-dependent DNA polymerase. These inhibition effects of the non-competitive type are not altered by preincubation of the DNA with BLM. Under comparable conditions neither the DNA-dependent DNA polymerase activity from E. coli and mouse liver nor the DNA-dependent RNA polymerase activity from mouse lymphoma cells are affected by BLM.
Single-chain antibodies against a plant viral RNA-dependent RNA polymerase confer virus resistance.
2004
Crop loss due to viral diseases is still a major problem for agriculture today. We present a strategy to achieve virus resistance based on the expression of single-chain Fv fragments (scFvs) against a conserved domain in a plant viral RNA-dependent RNA polymerase (RdRp), a key enzyme in virus replication. The selected scFvs inhibited complementary RNA synthesis of different plant virus RdRps in vitro and virus replication in planta. Moreover, the scFvs also bound to the RdRp of the distantly related hepatitis C virus. T(1) and T(2) progeny of transgenic lines of Nicotiana benthamiana expressing different scFvs either in the cytosol or in the endoplasmic reticulum showed varying degrees of r…
Biochemical and structural analysis of the NS5B RNA-dependent RNA polymerase of the hepatitis C virus.
2000
Hepatitis C virus (HCV), the major causative agent of chronic and sporadic non-A, non-B hepatitis worldwide, is a distinct member of the Flaviviridae virus family. These viruses have in common a plus-strand RNA genome that is replicated in the cytoplasm of the infected cell via minus-strand RNA intermediates. Owing to the lack of reliable cell culture systems and convenient animal models for HCV, the mechanisms governing RNA replication are not known. As a first step towards the development of appropriate in vitro systems, we expressed the NS5B RNA-dependent RNA polymerase (RdRp) in insect cells, purified the protein to near homogeneity and studied its biochemical properties. It is a primer…
Enhancement of hepatitis C virus RNA replication by cell culture-adaptive mutations.
2001
ABSTRACT Studies of the Hepatitis C virus (HCV) replication cycle have been made possible with the development of subgenomic selectable RNAs that replicate autonomously in cultured cells. In these replicons the region encoding the HCV structural proteins was replaced by the neomycin phosphotransferase gene, allowing the selection of transfected cells that support high-level replication of these RNAs. Subsequent analyses revealed that, within selected cells, HCV RNAs had acquired adaptive mutations that increased the efficiency of colony formation by an unknown mechanism. Using a panel of replicons that differed in their degrees of cell culture adaptation, in this study we show that adaptive…
Viral and cellular determinants of hepatitis C virus RNA replication in cell culture.
2003
Studies on the replication of hepatitis C virus (HCV) have been facilitated by the development of selectable subgenomic replicons replicating in the human hepatoma cell line Huh-7 at a surprisingly high level. Analysis of the replicon population in selected cells revealed the occurrence of cell culture-adaptive mutations that enhance RNA replication substantially. To gain a better understanding of HCV cell culture adaptation, we characterized conserved mutations identified by sequence analysis of 26 independent replicon cell clones for their effect on RNA replication. Mutations enhancing replication were found in nearly every nonstructural (NS) protein, and they could be subdivided into at …
Biochemical properties of hepatitis C virus NS5B RNA-dependent RNA polymerase and identification of amino acid sequence motifs essential for enzymati…
1997
The NS5B protein of the hepatitis C virus (HCV) is an RNA-dependent RNA polymerase (RdRp) (S.-E. Behrens, L. Tomei, and R. De Francesco, EMBO J. 15:12-22, 1996) that is assumed to be required for replication of the viral genome. To further study the biochemical and structural properties of this enzyme, an NS5B-hexahistidine fusion protein was expressed with recombinant baculoviruses in insect cells and purified to near homogeneity. The enzyme was found to have a primer-dependent RdRp activity that was able to copy a complete in vitro-transcribed HCV genome in the absence of additional viral or cellular factors. Filter binding assays and competition experiments showed that the purified enzym…