Determination of the titer of infectious LNCE in the presence of XMRV.

<p>The supernatant from DU145LNCE cells exposed to the medium from either DU145 (control medium) or DU145XMRV cells was applied in various dilutions to subconfluent cultures of naïve DU145 cells grown in 6-well plates. The treated cells were selected for G418 resistance and the surviving colonies were visualized by methylene blue staining. Neo-transducing particles were readily detectable in the supernatant of XMRV-exposed cells at 1,000-fold (panel A) and 10,000-fold (panel B), but not 100,000-fold (panel C) dilutions. The supernatant from DU145LNCE exposed to control medium was not toxic by itself (panel E), but it failed to transduce the resistance marker even when used without dilution (panel D).</p

XMRV facilitates the transfer of MLV-based LNCE to naïve cells.

<p>A. The scheme of the experimental approach. DU145LNCE and DU145XMRV were generated by infecting DU145 cells with LNCE and XMRV respectively. The supernatant from DU145XMRV or fresh DU145 cells was applied onto DU145LNCE cultures. The treated cells were cultivated for additional week, and the presence of infection LNCE particles in the supernatant was tested by applying the latter to naïve DU145 cells (designated DU145XL or DU145CL respectively). The cells were selected in the presence of G418. No DU145CL cells survived the selection. B. Structure of LNCE<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003144#pone.0003144-Kandel1" target="_blank">[6]</a>. LTR-MLV long terminal repeat; psi-MLV packaging signal; neo-neomycin resistance gene; CMV–cytomegalovirus promoter/enhancer region; GFP–the gene for enhanced green fluorescent protein. Positions of hybridization probes (thick lines) and PCR products (dotted lines) are shown below and above the diagram respectively. The predicted sizes of the PCR fragments and the distance between the two KpnI sites are indicated. C. Amplification of LNCE LTR from DU145XL cells. The expected product was obtained from the DNA of the pooled G418-resistant cells (lane 1) and several individually expanded clones (lanes 2–4), but not the original DU145 (lane 5). D. Amplification of an internal LNCE provirus fragment from DU145XL cells. The product was obtained from the DNA of DU145XL (lane 1), but not the naïve DU145 (lane 2). E. Detection of transmission of LNCE in the presence of XMRV by Southern blotting. Hybridization with the probe derived from the GFP fragment was performed on KpnI-digested DNA from DU145 (lane 1), DU145XMRV (lane 2), DU145LNCE (lane 3) and DU145XL (lane 4).</p

Base composition surrounding XMRV integration sites.

<p>Base compositions of the 4-bp target site duplication (positions D1 to D4; demarcated by the thick vertical lines) and 10 bp upstream (positions −1 to −10) and downstream (positions +1 to +10) of the direct repeat were calculated. The datasets include the 13 integration sites with correct 4-bp direct repeat (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010255#pone-0010255-t001" target="_blank">Table 1</a>), 472 integration sites from acutely infected DU145 cells (GenBank accession numbers EU981292 to EU981799) and 14 integration sites from human prostate cancer tissues (GenBank accession numbers EU981800 to EU981813) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010255#pone.0010255-Kim1" target="_blank">[14]</a>. Integration occurs between positions −1 and D1 on the top strand, and between positions D4 and +1 on the bottom strand (blue arrows). Any base in a position that is significantly overrepresented than the random dataset (<i>P</i><0.0001) is highlighted in green, while any base in a position that is significantly underrepresented than the random dataset (<i>P</i><0.0001) is highlighted in red.</p

Positions of XMRV integration sites and lengths of the target site sequence duplication.

<p>*The nucleotide position corresponds to the position of viral DNA insertion at the top strand of the chromosome indicated. Symbols + and – within the parenthesis indicate the orientation of the viral transcription is the same and opposite, respectively, to the polarity of the top strand. GenBank accession numbers for the integration site sequences are GU816075 to GU816104.</p><p>†The left LTR of the provirus contains a 5-bp deletion that includes the conserved CA dinucleotide at the viral end.</p><p>ψThe target DNA contains a T to A transversion immediately adjacent to the left LTR (position 4).</p

Integration of retroviral DNA and generation of short direct repeats flanking the provirus.

<p>(A) DNA breaking and joining steps during integration. Viral and target DNA strands are represented by thick black and parallel lines, respectively, and the viral long terminal repeats (LTRs) are depicted as grey boxes. Nucleotides at the top and bottom strands are denoted by uppercase and lowercase letters, respectively. During 3′-end processing, IN removes two nucleotides from the 3′ end of each strand of linear viral DNA so that the viral 3′ ends terminate with a conserved CA dinucleotide. Closed arrowheads denote the positions of strand transfer, a concerted cleavage-ligation reaction during which IN makes a staggered break in the target DNA. Host DNA repair enzymes fill in the resulting single-stranded gaps, denoted by D1 to D4 in the upper strand and d1 to d4 in the lower strand of target DNA, and remove the two unpaired nucleotides at the 5′ ends of the viral DNA (open arrowheads), thereby generating the short direct repeats flanking the provirus. (B) A potential pathway for generating a base transversion in the short direct repeat during XMRV integration. A coordinated integration of the two viral ends occurred at the 4-bp staggered positions as depicted by the closed arrowheads. During repair of the single-stranded gap adjacent to the upstream LTR, an adenine nucleotide was introduced at the D4 position either by misincorporation or aberrant processing of the unpaired AA-dinucleotide at the viral 5′ end. Subsequent repair of the mismatch resulted in the observed transversion (denoted by bold types).</p