MultipleCisElements Contribute to Geminivirus Origin Function

Abstract

The genome of the geminivirus tomato golden mosaic virus (TGMV) consists of two circular DNA molecules which are dissimilar in sequence except for a highly conserved 200-bp common region that includes the origin for rolling circle replication. To better characterize the plus-strand origin, we analyzed the capacities of various TGMV common region sequences to support episomal replication in tobacco protoplasts when the viral replication proteins AL1 and AL3 were supplied intrans.These experiments demonstrated that the minimal origin is located in an 89-bp common region fragment that includes the known AL1 binding motif and a hairpin structure containing the DNA cleavage site. Analyses of mutant origin sequences identified two additionalciselements—one that is required for origin activity and a second that greatly enhances replication. In contrast, a conserved partial copy of the AL1 binding site did not contribute to origin function. Mutational analysis of the functional AL1 binding site showed that both spacing and sequence of this motif are important for replicationin vivoand AL1/DNA bindingin vitro.Spacing changes between the AL1 binding site and hairpin also negatively impacted TGMV origin function in a position-dependent manner. Together, these results demonstrated that the organization of TGMV plus-strand origin is complex, involving multipleciselements that are likely to interact with each other during initiation of replication.