Sequence parameters that determine specificity of binding of the replication-associated protein to its cognate site in two strains of tomato leaf curl virus-New Delhi

The DNA binding sites for the replication-associated protein (Rep) of two strains of tomato leaf curl virus from New Delhi (ToLCV-Nde) were identified using electrophoretic mobility shift assays (EMSAs). The Rep proteins of the two strains were found to exhibit sequence specificity in recognition of their cognate repeat motifs (iterons) in the origin, despite the fact that they share 91% sequence identity. Using a series of synthetic oligonucleotides as probes in EMSAs, the interaction of Rep protein with its binding site was found to be dependent on number, size, and sequence of the two iterons. Mutations in the sequence of the repeat motifs or alteration in the arrangement of the motifs compromised the ability of Rep protein to bind the DNA sequence and reduced accumulation of viral DNA in protoplasts, suggesting that binding of Rep protein to its cognate iterons is an essential step in virus replication. In addition, a difference in sequence of two base pairs in the binding site of two ToLCV-Nde strains was found to affect DNA binding by the corresponding Rep protein and replication of the virus DNA in protoplasts. (C) 2000 Academic Press.