Betasatellite-encodedPC1 protein regulates helper virus accumulation by interfering with the ATP hydrolysis activity of geminivirus-encoded replication initiator protein

Geminivirus-betasatellite disease complexes are an epidemic threat to the majority of economically important crops across the world. Plant virus satellites including betasatellites are maintained by their associated helper virus. Geminivirus-betasat-ellites influence viral pathogenesis by substantially increasing or decreasing their helper virus accumulation. In the present study, we attempted to understand the mechanistic details of the geminivirus-betasatellite interaction. Here, we used tomato leaf curl Gujarat virus (ToLCGV) and tomato leaf curl Patna betasatellite (ToLCPaB) as a model system. This study reveals that ToLCGV can efficiently trans- replicate ToLCPaB in Nicotiana benthamiana plants, but ToLCPaB greatly reduced the accumula-tion of its helper virus DNA. For the first time, we have identified that the ToLCPaB- encoded PC1 protein is able to interact with ToLCGV- encoded replication initiator protein (Rep). In addition, we demonstrate that the C -terminal region of PC1 interacts with the C- terminus of Rep (RepC) protein. Our previous study had established that PC1 proteins encoded by diverse betasatellites possess a novel ATP hydrolysis activity and the conserved lysine/arginine residues at positions 49 and 91 are necessary for this function. Here, we show that mutating lysine at positions 49 to alanine of PC1 (PC1K49A) protein did not affect its ability to interact with RepC protein. Biochemical studies performed with ATP hydrolysis activity- deficient K49A mutated PC1 (PC1K49A) and RepC proteins revealed that Rep-PC1 interaction interferes with the ATP hydrolysis activity of Rep protein. Further, we dem-onstrate thatPC1 protein is able to interact with D227A and D289A mutated RepC proteins but not with D262A, K272A or D286A mutated RepC proteins, suggesting that thePC1- interacting region of Rep protein encompasses its Walker -B and BMODIFIER LETTER PRIME motifs. The results of docking studies supported that the PC1- interacting region of Rep protein encompasses its motifs associated with ATP binding and ATP hydrolysis activities. Docking studies also provided evidence that the Rep-PC1 interaction interferes with the ATP binding activity of Rep protein. Together, our findings suggest thatPC1 protein regulates helper virus accumulation by interfering with the ATP hydrolysis activity of helper virus Rep protein.