GEMINIVIRUS REPLICATION ORIGINS HAVE A MODULAR ORGANIZATION

Tomato golden mosaic virus (TGMV) and bean golden mosaic virus (BGMV) are closely related geminiviruses with bipartite genomes. The A and B DNA components of each virus have cis-acting sequences necessary for r eplication, and their A components encode trans-acting factors require d for this process. We showed that virus-specific interactions between the cis- and trans-acting functions are required for TGMV and BGMV re plication in tobacco protoplasts. We also demonstrated that, similar t o the essential TGMV ALI replication protein, BGMV ALI binds specifica lly to its origin in vitro and that neither TGMV nor BGMV AL1 proteins bind to the heterologous origin. The in vitro AL1 binding specificiti es of the B components were exchanged by site-directed mutagenesis, bu t the resulting mutants were not replicated by either A component. The se results showed that the high-affinity AL1 binding site is necessary but not sufficient for virus-specific origin activity in vivo. Gemini virus genomes also contain a stem-loop sequence that is required for o rigin function. A BGMV B mutant with the TGMV stem-loop sequence was r eplicated by BGMV A, indicating that BGMV AL1 does not discriminate be tween the two sequences. A BGMV B double mutant, with the TGMV AL1 bin ding site and stem-loop sequences, was not replicated by either A comp onent, indicating that an additional element in the TGMV origin is req uired for productive interaction with TGMV AL1. These results suggeste d that geminivirus replication origins are composed of at least three functional modules: (1) a putative stem-loop structure that is require d for replication but does not contribute to virus-specific recognitio n of the origin, (2) a specific high-affinity binding site for the AL1 protein, and (3) at least one additional element that contributes to specific origin recognition by viral trans-acting factors.