A tandem arrangement of the genes encoding the approximately 6-kDa hydrophobic protein (p6) and Hsp70 homolog (Hsp70h) is conserved among the members of the Closterovirus genus. It was not known, however, if these movement proteins are expressed from one or two subgenomic (sg) RNAs. Here we employ RNA ligase-mediated RACE to show that the Beet yellows virus (BYV), a prototype Closterovius, produces separate sgRNAs encoding p6 and Hsp70h. This result is further supported by generation of the recombinant BYV in which the truncated variants of these sgRNAs are resolved by Northern analysis. The 5'-termini of the p6 and Hsp70h sgRNAs are localized to BYV nucleotides G-9402 and A-9467, respectively. Each of the sgRNAs was generated in vitro and found to direct the expected product upon translation in wheat germ extract. Inactivation of the first start codons in these sgRNAs abolished translation of the each product. The polyclonal antibodies raised to synthetic C-terminal peptides of p6 and Hsp70h specifically recognized corresponding translation products, as well as p6 and Hsp70h produced in BYV-infected plants. Taken together with the previous work, our data demonstrate that expression of the BYV genome involves the formation of as many as seven sgRNAs.
(c) 2002 Elsevier Science (USA).