Tagging of the BYV ORFs by insertion of the self-processing reporter combined with traditional hybridization analysis allowed us to reveal a complex pattern of transcriptional regulation in a prototype closterovirus. Comparison of the GUS expression under the control of three subgenomic promoters demonstrated earliest activation of the HSP70h and CP promoters and a delayed activation of the p20 promoter. Since the direct quantitation of sgRNAs expressing HSP70h, CP, and p20 was problematic, the utilization of reporter activity offered a more reliable approach for the analysis of these promoters’ regulation. The potential drawback of this system, however, is the high stability of GUS that may result in masking the fine details of regulation.
Hybridization analysis of the remaining three sgRNAs revealed an early and relatively fast accumulation of the p21 and CPm sgRNAs in contrast to a much slower accumulation of the p64 sgRNA. Indeed, the latter RNA species reached 50% of its maximal level after 3 d.p.t., whereas p21 and CPm sgRNAs reached that level before 1.5 d.p.t. A decline in the levels of these two sgRNAs seen at 2.5 d.p.t. adds further complexity to corresponding patterns of temporal regulation. Although direct superposition of the results obtained with tagged and nontagged viruses would be incorrect, the same timing of product appearance driven by the CP promoter in the wild-type virus and in the CPGUS variant allows one to relate these two experimental systems. Hence, we can classify the HSP70h, CPm, CP, and p21 promoters as early and the p64 and p20 promoters as late. It is obvious that the order of promoter activation does not correspond to the order of the ORFs in the BYV genome (Fig. A).
The pattern of temporal regulation of multiple transcriptional units demonstrated here for a closterovirus is unique among RNA viruses. Animal coronaviruses that also possess large RNA genomes and produce multiple sgRNAs exhibit no temporal regulation of the transcription (5
). It should be remembered, however, that the time scale of coronavirus reproduction is within hours, compared to days for closteroviruses. Among plant viruses producing more than one sgRNA, no temporal regulation was found for two sgRNAs of turnip crinkle carmovirus (36
). Strikingly, the plant tobamovirus that also expresses two sgRNAs does regulate the timing of their accumulation (9
What is common among diverse groups of positive-strand RNA viruses is tight quantitative regulation of subgenomic promoter activities. Several elegant studies have previously demonstrated the roles of the core promoter and additional elements located in adjacent regions of the template (e.g., see references 15
, and 36
) or even on a distinct RNA molecule (34
) in regulating transcription levels. Promoter strength was also shown to depend on the location of the promoter and the presence of additional subgenomic promoters (9
). It seems that all these factors modulate closterovirus transcription as well. First, the CP promoter remains the strongest among BYV promoters even though its position varies among four analyzed variants. Specifically, the distance of this promoter from the 3′ end of genomic RNA is 1.8 kb in the wild-type and HSP70hGUS variants, 4.1 kb in the CPGUS and p20GUS variants, and 2.8 kb in the GUS-p21 variant. Second, the activity of the CP promoter decreases in CPGUS and p20GUS but not in HSP70hGUS, likely due to the increased distance from the 3′ end in the first two variants but not in the last variant. Third, the deletion of the adjacent promoters in the truncated GUS-p21 variant results in a dramatic activation of the CP promoter even though it is located farther from the 3′ end than in the wild-type virus.
The ability of BYV to accommodate a 2.3-kb reporter ORF at different positions of the genome demonstrates its utility as a high-capacity gene expression vector. A potential advantage provided by a self-processing reporter is the production of a reporter and a viral product from the same expression unit that minimizes modification of a virus genetic makeup. Preservation of all genes and control regions in a tagged virus appears to be advantageous for studies of viral replication, assembly, and translocation (11