Partial Purification and Characterization of Foxtail Mosaic Potexvirus RNA-Dependent RNA Polymerase

• Published in: Virology (New York, N.Y.) Vol. 197; no. 2; pp. 695 - 703
• Main Authors: Rouleau, Michèle, Bancroft, J.B., Mackie, George A.
• Format: Journal Article
• Language: English
• Published: San Diego, CA Elsevier Inc 01.12.1993; Elsevier
• Subjects: ACTIVIDAD ENZIMATICA; ACTIVITE ENZYMATIQUE; Agronomy. Soil science and plant productions; ARN; Base Sequence; Biological and medical sciences; CHENOPODIUM QUINOA; Fabaceae - microbiology; foxtail mosaic virus; Fundamental and applied biological sciences. Psychology; Genome, Viral; Hordeum - microbiology; Microbiology; Micrococcal Nuclease - metabolism; Molecular Sequence Data; Morphology, structure, chemical composition, physicochemical properties; Plants - microbiology; Plants, Medicinal; Plasmids; POTEXVIRUS; Potexvirus - enzymology; PURIFICACION; PURIFICATION; Ribonuclease, Pancreatic - metabolism; RNA Replicase - isolation & purification; RNA Replicase - metabolism; RNA, Messenger - biosynthesis; RNA, Viral - biosynthesis; Single-Strand Specific DNA and RNA Endonucleases - metabolism; TRANSFERASAS; TRANSFERASE; Virology; VIRUS DE LAS PLANTAS; VIRUS DES VEGETAUX; Virus; Nucleotidyltransferases; Purification; Plant pathogen; Enzymatic activity; Enzyme; Potexvirus; Transferases; Substrate specificity; In vitro; RNA-directed RNA polymerase
• ISSN: 0042-6822; 1096-0341
• DOI: 10.1006/viro.1993.1645

The RNA-dependent RNA polymerase (RdRp) of foxtail mosaic virus (FMV) was partially purified from infected leaves of Chenopodium quinoa. The membrane fraction of crude plant extracts contained most of the FMV RdRp activity. Additional purification was obtained by solubilization of the RdRp using KCl and dodecyl-sucrose and by centrifugation through a glycerol gradient. The RNA template endogenous to RdRp preparations could be removed using micrococcal nuclease but the resulting fraction was unable to copy added template purified from FMV virions. However, supplementation of fractions containing RdRp activity with FMV RNA resulted in a significant decrease in the level of RNA synthesis. This effect was specific to potexviral RNAs since a similar interference was also observed with clover yellow mosaic virus RNA but not with brome mosaic virus RNA or yeast RNA. RNA transcripts corresponding to various regions of the FMV genome were tested for their ability to inhibit RNA synthesis on endogenous template. The simultaneous presence of both 5′ and 3′ terminal regions of the viral genome was necessary to interfere with RNA synthesis suggesting that this inhibition resulted from competition for the binding of component(s) of the RdRp complex.