High-affinity Rep-binding is not required for the replication of a geminivirus DNA and its satellite.

The 682-nt satellite DNA (sat-DNA) of Tomato leaf curl virus (TLCV) depends on the helper virus for its replication. In contrast to the strict specificity that exists in each geminivirus for its cognate replication associated protein (Rep), TLCV sat-DNA can utilize Rep encoded by distinct geminiviruses. We have used a combination of protein-binding assays and mutagenesis to show that repeat motifs in TLCV and sat-DNA are essential for Rep-binding in vitro. Surprisingly, mutants of TLCV and sat-DNA impaired in their ability to bind TLCV Rep in vitro were infectious in tomato. Thus, in contrast to other geminiviruses reported, TLCV and sat-DNA replication is independent of the high-affinity in vitro Rep binding. These results prompt a reassessment of the current model of geminivirus replication where Rep/DNA interaction is a highly specific step in the initiation of rolling circle replication.

Identification of the replication-associated protein binding domain within the intergenic region of tomato leaf curl geminivirus.

The geminiviral replication-associated protein (Rep) is the only viral protein required for viral DNA replication. Tomato leaf curl virus (TLCV) Rep was expressed in Escherichia coli as a histidine-tagged fusion protein and purified to homogeneity in non-denaturing form. The fusion protein was used in in vitro binding experiments to identify the Rep-binding elements within the origin of replication of TLCV. Electrophoretic mobility shift assays demonstrated that the Rep binds specifically to a 120 bp fragment within the TLCV intergenic region. Fine resolution of the binding regions within the 120 bp fragment, using DNase I footprinting, demonstrated two footprints covering the sequences GCAATTGGTGTCTCTCAA and TGAATCGGTGTCTGGGG containing a direct repeat of the motif GGTGTCT (underlined). Our results suggest that the repeated motif is involved in virus-specific Rep-binding, but may not constitute the entire binding element. This is the first demonstration of geminivirus sequence elements involved in Rep-binding by direct protein-DNA interaction assays.