Complementary DNA cloning and characterization of cymbidium ringspot virus RNA

DNA complementary to the 4.7 kb cymbidium ringspot virus (CyRSV) genome was cloned in Escherichia coli. Recombinant plasmids were screened with a cDNA probe synthesized from randomly primed CyRSV genomic RNA, and two clones were chosen for further analysis. Two additional subclones were prepared after deletion of two sequences in the 3 ' region and one clone containing 5 ' virus genome sequences was constructed by primer extension. Three different RNA species associated with CyRSV were detected in purified virions and virus-infected tissue. The largest RNA was genomic RNA and the other two subgenomic. Northern blot hybridization indicated that the two subgenomic RNAs had in common a region corresponding tothe 3 ' region of genomic RNA. All three RNAs were found in tissue in both single- and double-stranded forms. In vitro translation experiments with fractionated virus RNA indicated that the coat protein gene is internally located and that it is followed by a T-terminal gene coding for a 22K protein