IN PLANTA TRANSCRIPTION OF A 2ND SUBGENOMIC RNA INCREASES THE COMPLEXITY OF THE SUBGROUP 2 LUTEOVIRUS GENOME

The genetic information of potato leafroll virus (PLRV), a typical mem ber 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' cl ustered viral open reading frames ORF3,ORF3/5 and ORF4, Here we demons trate by Northern blot analyses of polysomal RNAs from PLRV-infected S olanum tuberosum and Physalis floridana plants that, as with luteoviru ses belonging to subgroup 1, in planta synthesis of a second 0.8 kb su bgenomic RNA (sgRNA2) increases the complexity of subgroup 2 luteovira i 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 cucu rbit aphid-borne yellows virus (CABYV) a sgRNA2 of similar size and po sition (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 PL RV 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 exp ression of ORF6 and low translational efficiency for synthesis of ORF7 .