Bousios et al. BMC Genomics 2010, 11:89 http://www.biomedcentral.com/1471-2164/11/89 a common binding site targeted by a single DBF. It appears that minor variation, notably the presence of one intervening base or a substitution in the symmetrical motif, may confer different binding properties that may enable interactions with different nuclear factors. Future promoter analysis experiments could elucidate the role, if any, of the RMs in the Sirevirus activation network. Clues into the functionality of the envelope gene The function of the ENV-gene in plant retroelements has not been experimentally proven and due to the plant cell wall its role remains highly controversial [42]. So far it has been known that ENV-containing plant ret- roelements express their ENV either through a spliced ENV-like mRNA, for example splice acceptor sites have been predicted for the Ty3-gypsy BAGY2 and Athila ele- ments [35,43], or through stop codon suppression in SIRE1 [44]. However, SIRE1 is an atypical Sirevirus, as pol and ENV are separated only by a stop codon. The sequence of the Sirevirus ENV-gene is highly variable [18]. Peterson-Burch and Voytas (2002) hypothesized that the Sirevirus ENV may be expressed from an inter- nal promoter, although they were not able to identify promoters or transcription factors-binding sites upstream of the ENV. Our work indicates that specific motifs, which are also the epicentre of CpG islands, may have a regulatory role in the 5’ vicinity of ENV, which offers support to the internal promoter hypothesis for ENV expression, although we were not able to conclu- sively identify one. Intriguingly, CpG islands are pre- dicted in the same domain for six ENV-containing Sireviruses that lack the RM boxes, even in the pol/ENV junction of SIRE1-1. Collectively, we believe our obser- vations to be the first set of evidence concerning the Sirevirus ENV expression and their potential capacity to become virulent during their life cycle. Are Sireviruses capable of horizontal transmission? Sireviruses are abundant and widely dispersed in plants [19]. Moreover, it is the only Pseudoviridae genus to contain an ENV gene, which is hypothesized as being funtional. Has horizontal transmission, which is a spora- dic incident, aided their proliferation? Possibly as plant viruses do, Sireviruses accumulate in the cytoplasm within their envelope and wait for a feeding invertebrate to ingest and transfer them to another plant [35]. If hor- izontal transmission has occurred at high rates, then one would expect to frequently encounter elements with high nucleotide similarity in diverse plant phyla, which does not seem to be the case. However, this observation does not rule out the possibility that horizontal transfer can rarely happen, but when it does occur, Sireviruses quickly colonize the new host assisted by their unique genomic organization. Alternatively, the Sirevirus Page 11 of 14 envelope or gag capsid may act as a retrovirus move- ment protein and enable VLP transport outside the cell to infect different tissues of the same host, including meristem or germ cells, thereby enhancing the vertical transmission rate [45,46]. The distinctive relationship of the maize Sireviruses OPIE- 2 and PREM-2 The conservation of the motifs is exemplified in the clo- sely related OPIE-2 and PREM-2 maize elements. The alignment of their LTRs provides striking results, as the microdomains are the only highly similar segments amidst diverse sequences (Figure S5 in Additional file 2), often even higher than regions of pol and particularly gag. These contradict the sequence differentiation that has been reported for the Tnt1 superfamily and to the best of our knowledge this is the first time such a pat- tern of sequence conservation is shown. The Tnt1 superfamily consists of retrotransposons with surpris- ingly homologous genomes that colonize species of the Solanaceae family [47,48]. The only region that exempli- fies high genomic variability is the LTR domain that harbors the cis-acting motifs [49,50]. The sequence divergence and evolution of the motifs has equipped the Tnt1 superfamily with high adaptive regulatory capacity that allowed subgroups to evolve differently and colo- nize new Solanaceae genomes [51,52]. It appears, there- fore, that the OPIE-2/PREM-2 conservation pattern contradicts those of the Tnt1 superfamily and implies that the two elements may respond to the same stimuli and have the same transmission strategy. Conclusions Aided by the novel computational analysis which was orchestrated by the TEIRESIAS pattern discovery algo- rithm, we identified a plethora of highly conserved motifs in specific non-coding regions of the Sirevirus genome (Figure 6) and we hypothesized that these may be among the factors that underlie the extensive propa- gation of the genus in plant species. The motifs may be involved in the activation of the element and the ENV- gene, but also influence the folding of the RNA and DNA strands, suggesting their interaction with gag and the pol enzymes during VLP assembly, reverse transcrip- tion and integration. Based on the extreme sequence divergence outside the coding domain, we proposed that Sireviruses form an ancient lineage, which challenges previous research that argued in favour of the young evolutionary origin of the genus. Amidst their diverse non-coding genome, strict functional pressures appar- ently act on the motifs to maintain their sequence integ- rity. The degree of conservation is astonishing, as the Sirevirus elements of our dataset reside in plant species that diverged 140-150 Mya (monocots/dicots) [53].