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The geminivirus AC2 gene product transactivates the expression of the coat and movement protein (CP and BV1) genes, and this effect seems to be mediated by specific although hitherto unknown cis-acting elements. In this work we examined regions from the CP and BV1 gene promoters of pepper huasteco virus (PHV) to define the sequence elements involved in regulation by AC2. Results from transient gene expression and transgenic plant assays suggest that a truncated 115-nt CP promoter is still responsive to the viral transactivator. This promoter contains three elements similar to a sequence motif termed conserved late element (CLE), which is found in the regulatory regions of many geminiviruses and that was previously suggested, on a theoretical basis, to be a potential functional target for AC2 (Argüello-Astorga et al. (1994), Virology 203, 90-100). To confirm these results, an oligonucleotide containing two CLE motifs was synthesized and characterized in gain-of-function experiments. Transient expression assays showed that this 29-nt sequence is able to confer AC2 responsiveness to heterologous promoters. A smaller oligonucleotide (16 nt) containing a single CLE also conferred this activity. In addition, when the CLE motifs were mutated in their original context (truncated 115-nt promoter), this modified promoter lost its ability to be transactivated by AC2. All these results support the involvement, at least in the case of PHV, of CLE sequences in the process of transactivation., (Copyright 1999 Academic Press.)

A phylogenetic and structural analysis of the intergenic region of 22 dicot-infecting and 8 monocot-infecting geminiviruses was carried out. The analysis allowed the identification of iterative sequence motifs 8-12 nucleotides in length, whose organization (number, orientation, and spacing) is highly conserved within each of the three major lineages of dicot-geminiviruses, according to the phylogeny derived from the amino acid sequences of the replication-associated protein (AL1). The iterated elements differ in sequence even between closely related viruses, and are found in the vicinity of the putative TATA box of the AL1 gene in all dicot-infecting geminiviruses. Analogous elements were identified also in monocot-infecting geminiviruses, but the arrangement was different, since one of the iterative sequences is part of the conserved hairpin structure essential for replication of all the members of this viral family. We propose here that the iterated sequences are the specific binding sites of the geminiviral replication-associated proteins and show that the hypothesis is in agreement with the experimental data available to date. Additionally, a model of geminivirus replication that involves the participation of host transcription factors in the process is presented.