Organization of the 3'-terminal half of beet yellow stunt virus genome and implications for the evolution of closteroviruses.

The 3'-terminal half of the beet yellow stunt virus (BYSV) genome 10,545 nt, has been cloned and sequenced. The sequenced portion of the BYSV genome encompasses 10 open reading frames (ORFs) and 241 nt of the 3' untranslated region. The sequence spans, in the 5' to 3' direction, the C-terminal region of the replication-associated polyprotein gene (ORF 1a) which includes the set of motifs typical of helicases (HEL), the entire 53-kDa polymerase (RdRp) gene (ORF 1b), and genes encoding 30-kDa (ORF 2), 6-kDa (ORF 3), 66-kDa (ORF 4), 61-kDa (ORF 5), 25-kDa (ORF 6), 23.7-kDa (coat protein, CP) (ORF 7), 18-kDa (ORF 8), and 22-kDa (ORF 9) proteins. The double-stranded RNA "replicative form" of the BYSV was demonstrated to have a nontemplate G residue at the 3' terminus of the (+) strand. The RdRp of BYSV is presumably expressed via a +1 ribosomal frameshift. The five-gene module conserved among closteroviruses was identified in BYSV; it includes a gene array coding for a 6-kDa small hydrophobic protein, a 66-kDa homolog of the cellular HSP70 heat shock proteins, a 61-kDa protein, and a 25-kDa diverged copy of the CP followed by the CP gene itself. Phylogenetic analysis of the replication-associated HEL and RdRp domains as well as proteins from the five-gene module demonstrated the closest relationship between BYSV and two other closteroviruses, beet yellows (BYV) and citrus tristeza (CTV) viruses. Like CTV, the BYSV genome contains a 30-kDa protein gene between the RdRp and the 6-kDa protein genes, and like BYV it has only two genes downstream of the CP gene. The organization of the BYSV genome appears to be intermediate between BYV and CTV, which suggests that these three viruses might represent three distinct but probably close stages in the closterovirus evolution.