Location of the 5ʹ Termini of Tobacco Necrosis Virus Strain D Subgenomic mRNAs

Northern blot analysis of double-stranded (ds) RNA from bean-leaf tissue infected with tobacco necrosis virus strain D (TNV-D) detected the 4 kb genomic RNA and two subgenomic RNAs of about 1.5 kb and 1.2 kh; RNA extracted from virus particles only contained the genomic species. Blotting and probing with a range of probes indicated the approximate locations of the 5ʹ ends of subgenomic RNA so that primers to fine-map the ends could be designed. When both singlestranded and ds RNA, extracted from TNV-D infected and healthy bean leaves were used as templates for primer extension using primers complementary to sequences at, or upstream of, the initiation codons of, respectively, the coat protein and the p7a genes, major infectionspecific products were detected. Both subgenomic RNAs start at G residues. The larger subgenomic RNA is 1547 nucleotides in length with a leader sequences of 36 nucleotides upstream ofthe p7a gene, and the smaller subgenomic RNA has a 90 nucleotide leader upstream of the coat protein AUG and is 1202 nucleotides long. An analysis ofthe 5ʹ terminal locations of both subgenomic RNAs and the previously mapped analogous subgenomic RNAs associated with infection with the related TNV-A isolate, revealed a marked degree of homology downstream of the initiation sites for each RNA, This homology was maintained at the 5ʹ termini of both virion RNAs and could be extended to another isolate of TNV for which partial sequence data, but not subgenomic mapping RNA data are available.