Generation of subgenomic RNA directed by a satellite RNA associated with bamboo mosaic potexvirus: Analyses of potexvirus subgenomic RNA promoter

Satellite RNA of bamboo mosaic potexvirus (satBaMV), a single-stranded posi tive-sense RNA encoding a nonstructural protein of 20 kDa (P20), depends on bamboo mosaic potexvirus (BaMV) for replication and encapsidation. A full- length cDNA clone of satBaMV was used to examine the sequences required for the synthesis of potexvirus subgenomic RNAs (sgRNAs). Subgenomic promoter- like sequences (SGPs), 107 nucleotides (nt) upstream from the capsid protei n (CP) gene of BaMV-V, were inserted upstream of the start codon of the P20 gene of satBaMV. Insertion of SGPs gave rise to the synthesis of sgRNA of satBaMV in protoplasts of Nicotiana benthamiana and leaves of Chenopodium q uinoa when coinoculated with BaMV-V genomic RNA. Moreover, both the satBaMV cassette and its sgRNA were encapsidated. From analysis of the SGPs by del etion mutation, we concluded that an SGP contains one core promoterlike seq uence (nt -30 through +16), two upstream enhancers (nt -59 through -31 and -91 through -60), and one downstream enhancer (nt +17 through +52), when th e transcription initiation site is taken as +1. Site-directed mutagenesis a nd compensatory mutation to disrupt and restore potential base pairing in t he core promoter-like sequence suggest that the stem-loop structure is impo rtant for the function of SGP in vivo. Likewise, the insertion of a putativ e SGP of the BaMV open reading frame 2 gene or a heterologous SGP of potato virus X resulted in generation of an sgRNA. The satBaMV cassette should be a useful tool to gain insight into sequences required for the synthesis of potexvirus sgRNAs.