XMRV replicates efficiently in Raji B cells and causes a cytopathic effect.

<p>A) Raji cells were infected with the indicated dilutions of XMRV-GFP and expression of the fluorescence protein was analyzed by flow cytometry at indicated time points. B, C) Formation of multinucleated syncytia in XMRV-GFP infected Raji cells as shown by light microscopy (B) and confocal microscopy (C). Scale bar 5 µm.</p

Utilization of Replication-Competent XMRV Reporter-Viruses Reveals Severe Viral Restriction in Primary Human Cells

<div><p>The gammaretrovirus termed xenotropic murine leukemia virus-related virus (XMRV) was described to be isolated from prostate cancer tissue biopsies and from blood of patients suffering from chronic fatigue syndrome. However, many studies failed to detect XMRV and to verify these disease associations. Data suggesting the contamination of specimens in particular by PCR-based methods and recent reports demonstrating XMRV generation via recombination of two murine leukemia virus precursors raised serious doubts about XMRV being a genuine human pathogen. To elucidate cell tropism of XMRV, we generated replication competent XMRV reporter viruses encoding a green fluorescent protein or a secretable luciferase as tools to analyze virus infection of human cell lines or primary human cells. Transfection of proviral DNAs into LNCaP prostate cancer cells resulted in readily detectably reporter gene expression and production of progeny virus. Inoculation of known XMRV susceptible target cells revealed that these virions were infectious and expressed the reporter gene, allowing for a fast and highly sensitive quantification of XMRV infection. Both reporter viruses were capable of establishing a spreading infection in LNCaP and Raji B cells and could be easily passaged. However, after inoculation of primary human blood cells such as CD4 T cells, macrophages or dendritic cells, infection rates were very low, and a spreading infection was never established. In line with these results we found that supernatants derived from these XMRV infected primary cell types did not contain infectious virus. Thus, although XMRV efficiently replicated in some human cell lines, all tested primary cells were largely refractory to XMRV infection and did not support viral spread. Our results provide further evidence that XMRV is not a human pathogen.</p> </div

Overview of proviral XMRV reporter viruses.

<p>Shown are the genomic organizations of the parenteral XMRV provirus (XMRV-∆5U3), the provirus with the reconstituted 5`LTR (XMRV), and the reporter viruses coexpressing env and <i>GFP</i> or <i>GLUC</i> via an IRES element. The env* construct contains a frameshift in <i>env</i>. Restriction sites used for cloning are shown in italics.</p

XMRV reporter gene expression and virion production.

<p>A) Flow cytometry analysis of LNCaP cells transfected with <i>env</i> intact or <i>env</i> defect XMRV-GFP proviral DNA. B) GLUC activities in the supernatants of LNCaP cells transfected with <i>env</i> intact or <i>env</i> defect XMRV-GLUC constructs. C) Reverse transcriptase activities and D) Genomic RNA copy numbers (D) in supernatants of LNCaP cells transfected with XMRV wt, XMRV-GFP and XMRV-GLUC proviral DNA. E) qPCR and F) <i>Gaussia</i> luciferase activities of serial dilutions of an XMRV-GLUC virus stock. Values shown are average values derived from triplicate measurements ± standard deviation. Abbreviations and symbols: RLU/s (relative light units per second), n.s. (not significant), E (medium only), * (p<0.001), ** (p<0.00001).</p

XMRV reporter viruses are infectious.

<p>A, B) Serial dilutions of XMRV-GFP (A) or XMRV-GLUC (B) were used to inoculate Raji cells. Infection rates were determined by measuring GFP expression 3 days later (A) or by quantifying GLUC activities in cellular supernatants obtained at indicated time points (B). RLU/s (relative light units per second).</p

Use of different activity-based probes to visualize CatG activity.

<p>A) The structure of MARS116, MARS123, and MARS125. B) 800 ng/ml of CatG, NE, or PR3 with or without 250 μg/ml (3.27 μM) LF were quantified by using MARS116, MARS123, or MARS125. One representative out of three independent experiments is shown (left panel). The CatG band intensity was quantified and summarized in the bar diagram (right panel).</p

Analysis of pH dependent enhancement of CatG activity.

<p>CatG (800 ng/ml) or both LF (250 μg/ml, 3.27 μM) and CatG were incubated with MARS116 for the indicated time points under A) pH 5 or B) pH 7.4 conditions at 37°C. Samples were resolved by SDS-PAGE, transferred to PVDF membrane, and visualized by streptavidin-HRP. C) CatG and MARS116 were independently preincubated for 0, 30, or 60 min. After 60 min, in one set of samples LF was added and further incubated. The band intensity was analyzed by ImageJ and summarized (right panel). Three independent experiments were performed.</p

Detection of CatG activity under the control of LF or heparin.

<p>800 ng/ml CatG was incubated with or without LF or heparin (250 μg/ml) and MARS116 for 0 h or 2 h. CatG activity was detected via SDS-PAGE and streptavidin-HRP blot (upper panel). One representative out of two independent experiments is shown. The intensity of the respective bands were determined by ImageJ (lower panel).</p

Functional assay to determine physiological relevance of LF-mediated enhancement of CatG activity.

<p>A) Granulocytes were treated with PMA, Zymosan, or kept in medium (control). The granulocyte-derived supernatant was incubated with MARS116 and CatG activity was detected after SDS-PAGE and streptavidin-HRP blot. LF was visualized by LF-specific immunoblot (left panel). The CatG band intensity was quantified by ImageJ and summarized (right panel). One representative out of two independent experiments is shown. B) Human blood from buffy coats were incubated with CatG (8, 4, 2, or 1 μg/ml), with or without LF (250 μg/ml, 3.27 μM), CatG with CatG inhibitor (10 μM) and LF, or CatG and LF with DMSO for 30 min at RT. Platelet activation was determined by the increase of cell surface levels of CD62P analyzed by flow cytometry (n = 5 different donors; for CatG+LF+CatGinh. and CatG+LF+DMSO, n = 3).</p

Degradation of LF.

<p>Recombinant human LF (50 μg/ml) was incubated with CatG (50 μg/ml) or CatS (5 μg/ml) at pH 7.4 and CatL (5 μg/ml) at pH 5.0 for 6 h at 37°C. The degradation products were visualized by SDS-PAGE and Coomassie staining. A representative result of three independent experiments is shown.</p