In planta transcription of a second subgenomic RNA increases the complexity of the subgroup 2 luteovirus genome

The genetic information of potato leafroll virus (PLRV), a typical member of the subgroup 2 luteoviruses, is contained in a single-stranded (+/-) sense RNA of similar to 5.9 kb, A single subgenomic RNA (sgRNA1) of similar to 2.3 kb has been characterized as the mRNA for the 3' clustered viral open reading frames ORF3,ORF3/5 and ORF4, Here we demonstrate by Northern blot analyses of polysomal RNAs from PLRV-infected Solanum tuberosum and Physalis floridana plants that, as with luteoviruses belonging to subgroup 1, in planta synthesis of a second 0.8 kb subgenomic RNA (sgRNA2) increases the complexity of subgroup 2 luteovirai genomes significantly, PLRV-specific hybridization probes as well as primer extension experiments map sgRNA2 to the 3'-end of the PLRV RNA genome (positions 5190-5987), Similarly, for the closely related cucurbit aphid-borne yellows virus (CABYV) a sgRNA2 of similar size and position (positions 4888-5669) was identified, PLRV sgRNA2 may code for two viral proteins of 7.1 (ORF6) and 14 kDa (ORF7) respectively, while the CABYV proteins are 8.7 (ORF6) and 8.3 kDa (ORF7) In size, with PLRV ORF7 displaying nucleic acid binding activity, fn vivo experiments by transient expression of chimeric GUS fusions in potato protoplasts demonstrated that sgRNA2 functions as a bicistronic mRNA with high expression of ORF6 and low translational efficiency for synthesis of ORF7.