Bousios et al. BMC Genomics 2010, 11:89 http://www.biomedcentral.com/1471-2164/11/89 transcription. pol encodes the enzymatic proteins required for the production of the DNA copy from the RNA template and the insertion of the new copy in the host genome: an aspartic protease (AP), integrase (INT), reverse transcriptase (RT) and RNaseH (RH) [13]. LTRs flank the retrotransposon genome and contain the cis- acting transcriptional regulators, the promoter and ter- mination transcription points. The cis-acting boxes are often recognition sites of stress-related DNA binding factors (DBFs) and may be organized as arrays of two or three repeated motifs (RM) in tandem [14,15]. A 5’ untranslated region (5’UTR) serves as the tether domain between the 5’LTR and gag, while the linker domain connects pol and the 3’LTR. At the junctions of the 5’ and 3’LTR with the internal retrotransposon genome reside the primer binding site (PBS) and the polypurine tract (PPT), respectively, that prime cDNA synthesis during reverse transcription [16]. The International Committee on the Taxonomy of Viruses (ICTV) has classified Sireviruses into the Pseu- doviridae family [17] together with the Pseudovirus and Hemivirus genera. It is the most recently described genus named after the SIRE1 element from soybean, and as they have colonized only plant species, they were originally named Agroviruses [13]. Sireviruses have putative retroviral properties, since many ele- ments contain an ENV-like gene in their linker domain [18], which differentiates them from the other Ty1/ copia genera (herein referred as ‘classic’ Ty1/copia ele- ments, following the analysis of Havecker et al. 2005). Sireviruses have successfully proliferated within plant genomes, comprising a large proportion of the avail- able Ty1/copia populations [19]. Examples of such ele- ments are OPIE-2 and PREM-2 with more than 127,000 (fragmented) copies each in the maize gen- ome, of which 3,530 and 4,093 are full length elements respectively [9], and Osr8 that is the second most abundant element in rice [8]. Phylogenetic analysis based on the RT domain revealed that Sireviruses are less diverse than classic Ty1/copia retrotransposons, indicating a possible recent evolutionary origin and colonization of their host genomes [20]. Sireviruses differ in the sequence and genomic organi- zation compared to other elements. Whereas the vast majority of plant retrotransposons encode gag and pol as a single open reading frame (ORF), Sireviruses exhibit variation in respect to their organization. Besides the consensus, many elements have pol in two unusual +1 frameshifts relative to gag, of which the one suggests novel ways of translational recoding for pol expression, involving internal ribosomal entry or a bypass strategy [20]. For the bypass mechanism a highly conserved inverted repeat (IR) at the gag/pol boundary may pro- vide a favorable secondary structure at the RNA level. Page 2 of 14 Sireviruses also have a much larger gag gene compared to classic retrotransposons, which is characterized by a central RNA-binding CCHC zinc knuckle and a down- stream predicted coiled-coil domain [13,18]. The larger capsid protein interacts with the plant Light Chain 8 protein family, which can bind to cargo like cellular pro- teins and aid their transportation, suggesting it may facilitate VLP assembly or movement towards the nucleus. So far, the evolutionary history of Sireviruses, their life cycle and why they are so successful in invad- ing plant genomes remains unclear. In this study, the in depth comparative analysis of Pseudoviridae genomes enabled the identification of sev- eral short but highly conserved sequence motifs in the otherwise diverse Sirevirus genome. The motifs are found in key regions of the non-coding genome, regions which are necessary for the activation, reverse transcrip- tion and integration of the element, or even its virulence capacity through the expression of the ENV gene. Inter- estingly, one of the motifs is semi-conserved in classic retrotransposons, implying a global fundamental role of its function. Our results suggest that Sireviruses are an ancient retrotransposon lineage that maintains core domains with absolute or high similarity. These domains appear to attribute novel features to the Sirevirus life cycle and may partially explain their distribution within and across plants. Results Sireviruses represent an ancient Ty1/copia genus In order to establish a representative dataset for the comparative sequence analysis of Pseudoviridae, 21 Sir- eviruses were collected from various monocot and eudicot species including (Table S1 in Additional file 1): the five elements in the ICTV Sirevirus database (OPIE-2, PREM-2, SIRE1-1, ToRTL1, Endovir1-1); ret- rotransposons previously identified as Sireviruses, such as Osr7, Osr8, Osr9, Osr10 from rice [8], HOPIE from maize [21], Lotus2 from lotus, and one element each from sorghum, medicago and citrus [18,22]; plus Tnd- 1 from tobacco [23], one Vitis vinifera retrotransposon and the Maximus lineage recently reported by Wicker and Keller (2007) that are re-classified as Sireviruses during this work. The classic Ty1/copia retrotranspo- son population consisted of 30 elements (25 from plants and five from other organisms), representing the other two genera that make up the Pseudoviridae family: 26 Pseudoviruses and four Hemiviruses (Table S1 in Additional file 1). Phylogenetic analysis based on the RT segment, which includes the 5th to 7th RT con- served domains [24] and continues through RH to the end of pol, separates the Sireviruses in a distinct clade with 100% bootstrap support (Figure S1 in Additional file 2). The relative shorter branch lengths of the