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

The Journal of general virology

PubMedID: 24728710

George B, Ruhel R, Mazumdar M, Sharma VK, Jain SK, Gourinath S, Chakraborty S. Mutational analysis of the helicase domain of a replication initiator protein reveals critical roles of Lys 272 of B' motif and Lys 289 of ß-hairpin loop in geminivirus replication. J Gen Virol. 2014;.
Replication initiator protein (Rep) is indispensible for rolling-circle replication of geminiviruses, a group of plant infecting circular single stranded DNA (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 blot from 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 comparison 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.