High-frequency reversion of geminivirus replication protein mutants during infection

The geminivirus replication protein AL1 interacts with retinoblastoma-related protein (RBR), a key regulator of the plant division cell cycle, to induce conditions permissive for viral DNA replication. Previous studies of tomato golden mosaic virus (TGMV) AL1 showed that amino acid L148 in the conserved helix 4 motif is critical for RBR binding. In this work, we examined the effect of an L148V mutation on TGW replication in tobacco cells and during infection of Nicotiana benthamiana plants. The L148V mutant replicated 100 times less efficiently than wild-type TGW in protoplasts but produced severe symptoms that were delayed compared to those of wild-type infection in plants. Analysis of progeny viruses revealed that the L148V mutation reverted at 100% frequency in planta to methionine, leucine, isoleucine, or a second-site mutation depending on the valine codon in the initial DNA sequence. Similar results were seen with another geminivirus, cabbage leaf curl virus (CaLCuV), carrying an L145A mutation in the equivalent residue. Valine was the predominant amino acid recovered from N. benthamiana plants inoculated with the CaLCuV L145A mutant, while threonine was the major residue in Arabidopsis thaliana plants. Together, these data demonstrated that there is strong selection for reversion of the TGW L148V and CaLCuV L145A mutations but that the nature of the selected revertants is influenced by both the viral background and host components. These data also suggested that high mutation rates contribute to the rapid evolution of geminivirus genomes in plants.