Functional implications of residues of the B' motif of geminivirus replication initiator protein in its helicase activity.

Geminivirus replication initiator protein (Rep) is a multifunctional viral protein required for replication. During the process of viral replication, Rep acts as a site- and strand-specific endonuclease, ligase, ATPase, and helicase. B' motif and ß-hairpin loop of the geminivirus Rep are conserved and important for Rep-mediated helicase activity required for viral replication. To dissect the roles of various amino acid residues of the B' motif and ß-hairpin loop of the geminivirus Rep helicase in its process of unwinding DNA, we investigated eight conserved residues near the ATP active site or the ssDNA contact channel. Our strategy was to mutate these residues to alanines and investigate the effects of these mutations on various biochemical activities associated with DNA unwinding. We looked into the ATP binding, ATP hydrolysis, DNA binding, and DNA unwinding activities of the wild-type and mutant Rep proteins. These investigations showed four residues (Arg279, Asp280, Tyr287, and Pro290) affecting the DNA unwinding activity. A structural model analysis confirmed the B' loop and ssDNA binding loop to be connected through a ß-hairpin structure, suggesting that changes on one loop might affect the other and that these residues function by acting in concert. Viral genomes containing Rep proteins having these mutations in the B' motif did not replicate in planta. Taken together, these results indicated all four residues to be implicated in helicase activity mediated by Rep and demonstrated the significance, for viral replication, of the B' motif and ß-hairpin loop of the C-terminal region of the Rep protein.

Multifunctional roles of geminivirus encoded replication initiator protein.

Geminivirus infection has been a threat to cultivation worldwide by causing huge losses to the crop. The single-stranded DNA genome of a geminivirus possesses a limited coding potential and many of the open reading frames (ORFs) are overlapping. Out of 5-7 ORFs that a geminivirus genome codes for, the AC1 ORF encodes for the replication initiator protein (Rep) which is involved in the replication of virus within the infected plant cell. Rep is the only viral protein absolutely required for the in planta viral replication. Across different genera of the Geminiviridae family, the AC1 ORF exhibits a high degree of sequence conservation thus it has been used as an effective target for developing broad spectrum resistance against the invading geminiviruses. This multifunctional protein is required for initiation, elongation as well as termination of the viral replication process. Rep is also involved in stimulation of viral transcription. In addition, it also functions as suppressor of gene silencing and is involved in the process of transcription by regulating the expression of certain viral genes. Rep protein also interacts with few viral proteins such as coat protein, replication enhancer protein and with several host factors involved in different pathways and processes for its replication and efficient infection. This review will summarise our current understanding about the role of this early viral protein in viral propagation as well as in establishment of pathogenesis in a permissive host.

Mutational analysis of the helicase domain of a replication initiator protein reveals critical roles of Lys 272 of the B' motif and Lys 289 of the ß-hairpin loop in geminivirus replication.

Replication initiator protein (Rep) is indispensable for rolling-circle replication of geminiviruses, a group of plant-infecting circular ssDNA viruses. However, the mechanism of DNA unwinding by circular ssDNA virus-encoded helicases is unknown. To understand geminivirus Rep function, we compared the sequence and secondary structure of Rep with those of bovine papillomavirus E1 and employed charged residue-to-alanine scanning mutagenesis to generate a set of single-substitution mutants in Walker A (K227), in Walker B (D261, 262), and within or adjacent to the B' motif (K272, K286 and K289). All mutants were asymptomatic and viral accumulation could not be detected by Southern blotting in both tomato and N. benthamiana plants. Furthermore, the K272 and K289 mutants were deficient in DNA binding and unwinding. Biochemical studies and modelling data based on comparisons with the known structures of SF3 helicases suggest that the conserved lysine (K289) located in a predicted ß-hairpin loop may interact with ssDNA, while lysine 272 in the B' motif (K272) located on the outer surface of the protein is presumably involved in coupling ATP-induced conformational changes to DNA binding. To the best of our knowledge, this is the first time that the roles of the B' motif and the adjacent ß-hairpin loop in geminivirus replication have been elucidated.