Bousios et al. BMC Genomics 2010, 11:89 http://www.biomedcentral.com/1471-2164/11/89 plus and minus strand cDNA synthesis, respectively. Both steps are followed by template switching events (or jumps) that transfer the partial DNA fragments to the other side of the template, so as to resume and complete cDNA synthesis [16]. During the jumps the intervening nucleo- tides inbetween the 5’LTR-PBS and PPT-3’LTR junctions are ordinarily added to the outer sides of the linear cDNA intermediate, hence generating the attachment sites. In the first step of integration in the new genomic location, namely the 3’ end processing, INT recognizes and cleaves the intervening nucleotides from the 3’ ends of the linear cDNA, thereby preparing the new copy for nucleophilic attack [26]. According to the TEIRESIAS algorithm the two most conserved motifs of the Sirevirus genome are the PPT/ 3’LTR and 5’LTR/PBS junctions. The extreme sequence conservation of the two domains, which appear to be very important for reverse transcription and integration [2], reflects the strict sequence requirements of the Sire- virus replication strategy. Also, the presence of the inte- grase signal is unique among LTR retrotransposons and suggests the tight cooperation of INT with the Sirevirus LTRs. In contrast, the respective regions of classic ele- ments show extensive sequence variation, although a large proportion maintains a common PPT structure that relates to the Sirevirus PPT octamer. We propose that the primer for plus strand cDNA synthesis in the majority of Pseudoviridae consists of eight bases, which may tolerate some variability in classic elements, but is strictly conserved in Sireviruses. Which is the role of the multiple PPTs? The presence of multiple PPTs within retroelements is not unprecedented, however their role in plus strand cDNA synthesis is unclear. One extra PPT is present in the linkers of the ENV-containing Ty3-gypsy elements Athila4 and Calypso [35], whilst one functional PPT has been reported in the coding domains of Ty1 and HIV1 [36]. PPT-primed cDNA synthesis is a rapid event and when extra PPTs occur in the coding domain, synthesis initiates simultaneously with the natural PPT [37]. Yet, the upstream-primed cDNA must synthesize several kilobases of sequence and as a result it is outcompeted by the natural PPT-primed cDNA due to kinetic compe- tition [36]. In Sireviruses kinetic competition is unlikely to prevent strand transfer, since the distance between the PPTs is small. The critical parameter might be the availability of the tRNA primer at the 3’ end of the minus DNA strand that is utilized during the second strand transfer to provide complementarity to the PBS sequence at the 5’ end of the minus DNA strand. Rapid RH degradation of the tRNA after it is copied in the natural PPT-primed cDNA may prohibit strand transfer from internally initiated cDNAs [36]. Page 10 of 14 The upstream PPTs also lack the correct attachment site and the IR motif, which further obscures their functional- ity. The transposition ability of the retroelement is depen- dant on the attachment site [26]. If the PPT is not followed by the correct signal for INT processing, the cDNA intermediate will be unable to insert in the new genomic location. On the other hand, the strict nucleotide integrity of the upstream PPTs, their high copy number and preferential clustering next to the 3’LTR point towards selection. They may facilitate genetic recombina- tion between the two RNA genomes in the VLP [38], or offer a relative replication advantage to the genus [39]. In any way, based on the fact that retrotransposons with ENV genes contain near their 3’LTR repeats that show similarity to PPTs, Havecker et al. (2004) proposed their involvement in reverse transcription. The IR can act as facilitator for the correct PPT primer formation Research in Ty1 has shown that the PPT sequence by itself is not sufficient to promote correct RH processing. Additional recognition signals in the form of secondary structures near the PPT domain may be needed to affect the overall conformation of the region and attract the RH for PPT formation and subsequent DNA elongation [37]. A highly conserved IR that forms a stem loop sec- ondary structure, of which the 8 bp PPT retains its out- most left side, seems as the most suitable candidate for such a recognition signal. Presumably, this configuration preferentially ‘exposes’ the terminal PPT and renders it accessible to RH for precise cleavage, which will auto- matically generate the highly conserved attachment site. Many classic Ty1/copia elements also contain the IR, which suggests that this motif may have a universal role in retrotransposon life cycle. Are the RM boxes involved in the regulation of Sirevirus activity? Sireviruses share highly similar versions of a novel RM, which in conjunction to the exceptional high copy number and flanking sequence divergence, make the conservation remarkable. Symmetry is a frequent feature of regulatory sequences, including box I of Tnt1 [14], an activator in the 5’UTR of Copia element [40] and the Pal elements in the b-glucokinase promoter of neuroendocrine pancreatic, gut and brain cells in humans and mice [41]. The position of the boxes in the LTRs and upstream of the ENV-gene does not seem random and they also form CpG islands that are by default found at the 5’ side of genes regulating their expression [29]. Interestingly, Sireviruses lacking the boxes may have similar repeated structures, as large sec- tions of their LTRs and upstream of the ENV are particu- larly CG-rich and generate CpG islands (data not shown). Despite these unique characteristics, we could not identify