Efficient transcription obtained by a new procedure of the tRNA-like structure of turnip yellow mosaic virus by a template-dependent and specific viral RNA polymerase

• Published in: Journal of virological methods Vol. 64; no. 2; pp. 181 - 195
• Main Authors: Deiman, B.A.L.M., Séron, K., Jaspars, E.M.J., Pleij, C.W.A.
• Format: Journal Article
• Language: English
• Published: London Elsevier B.V 01.03.1997; Amsterdam Elsevier; New York, NY
• Subjects: ARN; Biological and medical sciences; Fundamental and applied biological sciences. Psychology; Generalities. Techniques. Transmission, epidemiology, ecology. Antiviral substances, control; Microbiology; Phytopathology. Animal pests. Plant and forest protection; PLANT VIRUSES; Plant viruses and viroids; Replication; RNA; RNA-dependent RNA polymerase; Techniques used in virology; tRNA-like structure; Turnip yellow mosaic virus; Virology; VIRUS DE LAS PLANTAS; VIRUS DES VEGETAUX; Replication; Turnip yellow mosaic virus; RNA-dependent RNA polymerase; tRNA-like structure; Virus; In vitro transcription; Nucleotidyltransferases; Tymovirus; Plant pathogen; RNA; Enzyme; Transferases; Primer; Genome; RNA-directed RNA polymerase
• ISSN: 0166-0934; 1879-0984
• DOI: 10.1016/S0166-0934(96)02166-0

The RNA-dependent RNA polymerase (RdRp) of turnip yellow mosaic virus (TYMV) was isolated by a simple, new method. An active, template-dependent and specific enzyme was obtained. Although the genomic RNA of TYMV could not be transcribed completely during an in vitro RdRp assay, a complete double-stranded product was obtained when a 3′ terminal RNA fragment of 83 nucleotides was used as a template. The reaction product was identified as being of negative polarity by complete digestion with ribonuclease T l. Antibodies directed to part of the N-terminal (Ab140) or C-terminal (Ab66) in vitro autocleavage products of the large non-structural polyprotein of TYMV, could both partially inhibit RdRp activity. Further purification of the RdRp preparation by ion-exchange chromatography resulted in two activity peaks with different protein compositions. Both peak fractions retained high specificity for transcription of TYMV RNA. A protein of approximately 115 kDa was detected by both Ab140 and Ab66.