Identification of replication specificity determinants in two strains of tomato leaf curl virus from New Delhi

We used two strains of tomato leaf curl virus from New Delhi to investigate specificity in replication of their cognate genomes. The strains share 94% sequence identity and are referred to as severe and mild on the basis of s ymptoms on tomato and tobacco. Replication assays in tobacco protoplasts an d plants showed that a single amino acid change, Asn10 to Asp in the N term inus of Rep protein, determines specificity for replication of the two stra ins based upon its interaction with the origin of replication (ori) sequenc es. The change of Asp10 to Asn in Rep protein of the mild strain coupled wi th point mutations at the 3rd and 10th nucleotides of the 13-mer binding si te altered its replication ability, resulting in increased levels of virus accumulation. Similarly, changing Asn10 to Asp in Rep protein of the severe strain impaired replication of the virus and altered its severe phenotype in plants. Site-directed mutations made in ori and Asn10 of Rep protein sug gested that Asn10 recognizes the third base pair of the putative binding si te sequence GGTGTCGGAGTC in the severe strain.