Synthesis of full-length transcripts of beet western yellows virus RNA: messenger properties and biological activity in protoplasts

Abstract

Full-length cDNA of beet western yellows virus genomic RNA has been cloned behind the bacteriophage T7 RNA polymerase promoter of the transcription vector BS(-). The in vitro run-off transcription product obtained in the presence of T7 RNA polymerase and m7GpppG cap has the same messenger properties as natural viral RNA in in vitro translation systems. The full-length transcript was also able to infect Chenopodium quinoa protoplasts inoculated by electroporation. Infection could be followed by the appearance of viral coat protein in the inoculated protoplasts and the de novo synthesis of viral RNA. Site-directed mutagenesis experiments revealed that expression of beet western yellows virus open reading frame 1 and the C-terminal portion of open reading frame 6 were not required for infection of protoplasts. Additional experiments with these mutants and mutants in the other viral open reading frames should provide information concerning the requirements for beet western yellows virus replication and, ultimately, the role of virus genes in other important steps in the virus infection cycle, such as aphid transmission.