Organization of thecis-Acting Element Required for Wheat Dwarf Geminivirus DNA Replication and Visualization of a Rep Protein–DNA Complex

Abstract

Initiation of geminivirus DNA replication depends on the activity of the initiator protein (Rep) upon interaction with DNA sequences present in the intergenic region of the viral DNA. In this study, we have analyzed the DNA sequences present in the large intergenic region (LIR) of wheat dwarf virus (WDV), a subgroup I member of the geminivirus family, which are required for viral DNA replication. We have (i) defined the boundaries of the viral cis-acting DNA replication element, (ii) determined the contribution of different domains of the LIR to DNA replication efficiency, and (iii) visualized WDV Rep-DNA complexes. Analysis of unidirectional deletions from both sides of the LIR leads us to establish that a approximately 200-bp cis-acting element (core) is essential for viral DNA replication. It spans approximately 170 and 28 bp upstream and downstream, respectively, from the initiation site (+1), located in the invariant loop. This core element is flanked, at each side, by auxiliary regions (5'-aux and 3'-aux, approximately 70 and approximately 25 bp long, respectively), which contain DNA sequences that stimulate DNA replication. Competition experiments using viral replicating vectors bearing wild-type or mutant WDV LIRs suggest that the auxiliary regions may contribute to the stabilization and/or activity of the initiation complex formed by WDV Rep at the origin. We have visualized DNA-protein complexes by electron microscopy and a high-affinity binding site of WDV Rep protein within the core element has been mapped to approximately 144 +/- 18 bp upstream from the initiation site, between the start site for complementary-sense transcription and the TATA box. Our studies (i) establish the modular structure of the WDV DNA replication cis-acting element and (ii) provide direct evidence for the formation in vitro of a large nucleoprotein complex within the essential cis-acting element.

Geminivirus DNA replication

“…Further deletion analyses have shown that mastreviruses require a large ( 300 bp) cis-acting element for efficient viral DNA replication in cultured cells ( fig. 5; [86]). This element is built up by at least three major components: a central core, 200 bp in size (in WDV between positions − 178/−169 and + 28, taking the A residue at the initiation site as position +1), which is essential for viral DNA replication; the core is flanked by two regions which stimulate replication, the auxiliary regions 5%-aux and 3%-aux, 70 and 25 bp in size, respectively [86].…”

“…5; [86]). This element is built up by at least three major components: a central core, 200 bp in size (in WDV between positions − 178/−169 and + 28, taking the A residue at the initiation site as position +1), which is essential for viral DNA replication; the core is flanked by two regions which stimulate replication, the auxiliary regions 5%-aux and 3%-aux, 70 and 25 bp in size, respectively [86]. The 3%-aux region (spanning from + 28 to + 50/+63 in WDV) contains two phased A/T tracts, part of the region conferring a static DNA curvature to the WDV LIR [118].…”

“…The oligomerization domain has been mapped between residues 134 and 181 in TGMV Rep [85] and between residues 175 and 187 in MSV Rep [62]. Recent electron microscopic studies have served to visualize directly WDV Rep oligomers bound to DNA and suggest that a large protein core, most likely of six to eight monomers, is present in the nucleoprotein complex [86]. Rep-Rep interaction has been proposed to be a prerequisite for DNA binding but not for nicking, since Rep monomers can support the in vitro cleavage and joining reactions [85].…”

Adaptation of the geminivirus bean yellow dwarf virus to dicotyledonous hosts involves both virion-sense and complementary-sense genes.

“…However, two members have been described, namely tobacco yellow dwarf virus (Morris et al, 1992) and bean yellow dwarf virus (BeYDV ; , that are adapted to dicotyledonous species. Mutational analysis of open reading frames (ORFs) predicted from the nucleotide sequence, and the identification of a complementary-sense intron has indicated that BeYDV encodes four genes that have functionally conserved homologues in other mastreviruses (Liu et al, a, 1998 Gene expression occurs bidirectionally from the large intergenic region (LIR), which contains cis-acting promoter elements that bind host factors and control virion-sense gene expression (Fenoll et al, 1988(Fenoll et al, , 1990, as well as the origin of virion-sense DNA replication (ori) (Heyraud et al, 1993 ;Heyraud-Nitschke et al, 1995 ;Sanz-Burgos & Gutie! rrez, 1998).…”

T‐DNA recombination and replication in maize cells

“…It is a single-stranded DNA virus replicating through a rolling circle mechanism in infected monocot plants (Hanley-Bowdoin et al, 2000;Stenger et al, 1991). The essential components of WDV replication are the initiator protein, the (-)strand origin of DNA replication, and the (þ)strand origin of replication (ori) (Sanz-Burgos and Gutierrez, 1998). The Rep protein initiates replication by cutting one strand of the double-stranded viral DNA at the (þ)strand origin of replication, thus generating a free 3 0 -end hydroxyl group that serves as an initiation site for DNA synthesis to be catalyzed by a cellular DNA polymerase and other accessory proteins (Gutierrez, 1999).…”