Independent expression of Rep and RepA and their roles in regulating bean yellow dwarf virus replication.

Bean yellow dwarf virus (BeYDV) is a mastrevirus specific for dicotyledenous hosts. It contains four ORFs encoding a movement protein, a coat protein, and two Rep gene products, Rep and RepA, which are encoded by two overlapping ORFs. In this study, the roles of Rep and RepA in regulating replication of the BeYDV-based replicon were investigated by uncoupling them and placing Rep and RepA each under constitutive promoter control. Constitutive expression of both Rep and RepA supported replication and enhanced gene expression. When a reporter plasmid containing the Rep gene in the context of its native promoter was supplemented with additional Rep protein, replication was enhanced but the increase in gene expression was found to be more modest. Furthermore, expression of constitutively expressed RepA alone was found to reduce replication of this reporter construct as well as delay BeYDV replication in general. The effect of a RepA mutant with an altered retinoblastoma-related-protein binding motif on the efficiency of BeYDV replication was also examined. This mutant was found to severely diminish replication efficiency. Finally, the relationship of BeYDV coat protein to virus replication and reporter gene expression was investigated. Addition of coat protein increased accumulation of single-stranded DNA and had a detrimental effect on reporter gene expression.