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

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

Theiler's virus L* protein inhibits the 2–5A/RNase L pathway in infected peritoneal macrophages.

<p>A. Peritoneal macrophages prepared from RNase L ^−/− (central panel) or RNase L^+/+ (left) C57BL/6 mice and, as an additional control, from RNase L^+/+ 129/Sv mice (right), were mock-infected or infected at a MOI of 20 with VV18 (L* WT), TM770 (L* 1–92) or FS58 (L* 1–12). Nine hours post infection, total cell RNA was extracted and analyzed on RNA chips. For control purposes, peritoneal macrophages were also transfected with 2.5 µg/ml poly(I:C) for 7 h before total cell RNA extraction. Prominent rRNA cleavage products are indicated. B. Replication levels of wild-type or L*-mutant viruses in peritoneal macrophages. Peritoneal macrophages isolated from indicated mouse strains were plated for four days and infected as in (A). Viral RNA was quantified by quantitative RT-PCR. Histograms show the mean and SD of viral cDNA copies detected in samples from a representative triplicate infection experiment. Values for mock samples were lower than the detection limit (10 cDNA copies). The experiment was repeated twice, using two independent productions of viruses and macrophages.</p

L* can act on non-macrophage cell lines and in absence of other viral components.

<p>A. L* inhibition of the 2–5A/RNase L pathway is not restricted to macrophages: L929 cells were incubated with 5 U/ml of murine IFN-β for 24 h or left untreated prior to infection with 2 PFU per cell of indicated viruses. Histograms show the mean +/− SD of viral genome copies detected by quantitative RT-PCR in RNA samples isolated 16 h after infection. Values for mock samples were lower than the detection limit (10 cDNA copies). B. L* inhibits the 2–5A/RNase L pathway in the absence of other viral components: L929 cells stably expressing L* (L929-L*) or the empty vector (L929-NEO) were primed with 5 U/ml of murine IFN-β and transfected with poly(I:C) for 7 hours. Alternatively, the cells were directly transfected without priming, with crude 2–5A at a concentration of 5 uM. Total cell RNA was then isolated and analyzed on RNA chips.</p

TMEV L* protein directly interacts with the murine RNase L.

<p>A. Cartoon showing expected inhibition of RNase L activity in the case of direct or indirect L*-RNase L interaction, in murine and human cells or in human cells expressing the murine RNase L. B. HeLa M cells stably expressing either an empty vector (Puro) or L* were transfected with pcDNA3 or with pcDNA3 derivatives expressing indicated RNase L constructs: wild-type (lanes 3–4), flagged (lanes 5–6) and catalytically inactive (R666A)(lanes 7–8) murine RNase L, or wild-type (lanes 9–10) and catalytically inactive (R667A)(lanes 11–12) human RNase L. Selection with 1 mg/ml of G418 was applied for 3 passages. Cells were then incubated with 100 U/ml human recombinant IFN-α-2a for 24 h. Poly(I:C) was then transfected 6 h before total cell RNA was extracted and analyzed on RNA chips. Arrows indicate lack of RNA degradation due to L* expression. C. L* inhibits RNase L <i>in vitro</i>. FRET assay performed with recombinant human (hu) RNase L at 100 nM or murine (mu) GST-RNase L at 180 nM, in the presence or absence of 2–5A at 2 nM and of recombinant His-tagged L* protein at 10 µg/ml. Histograms show mean relative fluorescence units (RFU) resulting from degradation of the FRET RNA probe.</p

Expression plasmids used in this study.

1<p>Neo: G418/Geneticin, puro: puromycin, hygro: hygromycin.</p

L* fails to block apoptosis but prevents RNA degradation in infected macrophages.

<p>A–C. Impaired replication of L*-mutant viruses in J774-1 macrophages: J774-1 cells were infected with VV18 (L* WT)(black columns) or L*-mutant viruses TM770 (L* 1–92)(dark grey columns) and FS58 (L* 1–12)(light grey columns) at a MOI of 20, or left uninfected (white columns). Viral genomes were quantified at indicated times, by quantitative RT-PCR. Values for mock samples were lower than the detection limit (10 cDNA copies) (A). Infectious virus yield in the supernatant was measured by plaque assay. Panel B shows plaques formed by equivalent supernatant dilutions and panel C shows the quantification of the plaques (mean and SD). D. Caspase 3/7 activities in J774-1 infected macrophages were quantified at indicated times on the basis of their ability to cleave a luminogenic substrate. Values represent the mean of relative light unit (RLU) +/− SD calculated from a representative experiment performed in triplicate. E. Cellular RNA degradation: Total cell RNA extracted from J774-1 macrophages that were either mock-infected (lanes 10–12) or infected for 10 h with VV18 (L* WT, lanes 1–3), TM770 (L* 1–92, lanes 4–6) or FS58 (L* 1–12, lanes 7–9) was run on a native 1% agarose gel and stained with ethidium bromide (bands corresponding to 28S and 18S rRNA are indicated).</p

Plasmids carrying TMEV full length cDNA used in this study.

1<p>Viruses derived from these plasmids carry point mutations in the capsid that increase the efficiency of L929 cell infection.</p>2<p>Viruses derived from these plasmids carry point mutations in the capsid that increase the efficiency of macrophage infection.</p

Characteristics of plasma and EPS samples tested for infectious XMRV and related retroviruses.

*<p>Patient identifiers shown in bold indicate patients who had previously tested positive for XMRV. See Discussion for details.</p>†<p>Nucleotides at position 1385 of the RNase L coding regions of both patient alleles are shown. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036073#pone-0036073-t001" target="_blank">Table 1</a> footnotes for additional details.</p

Primers used for real-time PCR.

<p>Primers used for real-time PCR.</p