Complete replication of a eukaryotic virus RNA in vitro by a purified RNA-dependent RNA polymerase

• Published in: Cell Vol. 63; no. 2; p. 363
• Main Authors: Hayes, R J, Buck, K W
• Format: Journal Article
• Language: English
• Published: United States 19-10-1990
• Subjects: Base Sequence; Genes, Viral; Molecular Sequence Data; Mosaic Viruses - enzymology; Mosaic Viruses - genetics; Mutagenesis, Site-Directed; Nicotiana; Oligonucleotide Probes; Plants, Toxic; RNA, Viral - biosynthesis; RNA, Viral - genetics; RNA, Viral - isolation & purification; RNA-Dependent RNA Polymerase - isolation & purification; RNA-Dependent RNA Polymerase - metabolism
• ISSN: 0092-8674
• DOI: 10.1016/0092-8674(90)90169-F

A soluble RNA-dependent RNA polymerase was isolated from Nicotiana tabacum plants infected with cucumber mosaic virus (CMV), which has a genome of three positive-strand RNA components, 1, 2, and 3. The purified polymerase contained two virus-encoded polypeptides and one host polypeptide. Polymerase activity was completely dependent on addition of CMV RNA as template, and the products of reaction were single-stranded (ss) RNA and double-stranded (ds) RNA, corresponding to RNAs 1, 2, and 3, and a subgenomic RNA (RNA 4) derived from RNA 3. The ratio of ssRNA to dsRNA was about 5:1, and the ssRNA was shown to be predominantly the positive strand. This demonstrates the complete replication of a eukaryotic virus RNA in vitro by a template-dependent RNA polymerase.