Identification of NF-κB binding sites κB-1 and κB-2 in the XMRV LTR and functional analysis of NF-κB binding to the XMRV LTR. (A) DNA sequences of consensus and NF-κB binding sites in the immunoglobulin (Ig) κ chain enhancer and in the XMRV LTR. Mutated (mut) sequences are listed below the WT sequences; complementary sequences (c) are noted. Lowercase letters represent mutations. (B) Representative immunoblotting results from DNA affinity binding assays. Individual biotinylated double-stranded DNA probes (sequences shown in panel A) were incubated with nuclear extracts from the EBV-infected lymphoblastoid IB4 cell line as a source of p65/RelA. After precipitation with streptavidin Sepharose, the binding of p65/RelA to biotinylated DNA was revealed by Western blotting (WB) with a specific antibody to p65/RelA. NE, nuclear extracts only. (C) Effects of TNF and LMP1 on WT and mutant XMRV LTR transcriptions as measured in luciferase reporter assays. Mutations in the κB-1 and κB-2 sites (shown in panel A) were introduced in the XMRV LTR reporter construct. Each firefly luciferase (FL) LTR reporter construct (the WT is XLTR, and mutants 1 and 2 are XLTR mut1 and mut2, respectively) was transiently transfected into HEK293 cells; the cells were cultured in medium alone or with 5 ng/ml of TNF-α. The cells were also cotransfected with pCMV-LMP1 (0.1 μg/well). After 16 h of incubation, FL activity was measured and normalized for renilla luciferase (from cotransfection with pTK-RL). The results are presented as relative luciferase activities (± SD for triplicate wells) in comparison to HEK293 cells transfected with XLTR and cultured in medium alone. R, repeat sequence.

XMRV replicates in human B-cell lines. The EBV-immortalized lymphoblastoid cell line IB4 (A) and the Burkitt's lymphoma BJAB cell line (B and C) were infected with 500-μl XMRV-containing supernatants (VP62 or VP62mκB-1) (analyzed in Fig. 3B). At the indicated time points, culture supernatants (0.5 ml) were ultracentrifuged and analyzed by Western blotting for Gag protein (p30^CA) (A and B). The bar graphs reflect quantitative analysis of duplicate band intensities from the nitrocellulose membranes displayed on the left. The results are expressed as relative band intensities. The results are presented as fold increases in p30^CA expression relative to the value for WT VP62 virus at day 6. (C) The images reflect representative BJAB cells immunostained for Gag protein (rat MAb R187) 12 days after mock infection (Mock), infection with XMRV VP62, or infection with VP62mκB-1.

TNF-α and LMP1 enhance LTR-mediated viral transcription. (A) XMRV-infected HEK293 cells were incubated with TNF-α at the indicated concentrations. After 24 h, the conditioned media were harvested and ultracentrifuged (100,000 × g for 1 h), and each pellet was subjected to Western blot analysis for Gag protein (p30^CA) by using the rat MAb R187. The bar graph reflects quantitative analysis of duplicate band intensities; the results are expressed as relative band intensities, and the error bars reflect the ranges of measurements. (B) TNF-α (5 ng/ml) was added to HEK293 cells transiently cotransfected with the XMRV LTR construct fused to firefly luciferase (FL) and with a renilla luciferase (RL) plasmid (pTK-RL). After a 16-h incubation, FL and RL activities were measured. FL activity was normalized to RL activity. The results are presented as fold increases (± standard deviations [SD] for triplicate cultures) in normalized FL in cultures with TNF-α relative to that in medium only. (C) HEK293 cells were cotransfected with the pCMV-LMP1 plasmid at the indicated concentrations and the XMRV LTR FL reporter. For normalization, we also transfected with pTK-RL. FL and RL activities were measured from cells harvested 16 h after transfection. The results are presented as fold increases (± SD for triplicate wells) of normalized FL induced by LMP1 transfection relative to that without LMP1 transfection.

Mutation of the κB-1 site reduces XMRV replication. (A) Schematic representation of the XMRV provirus constructs [pcDNA3.1(−)VP62 and pcDNA3.1(−)VP62mκB-1]; the WT κB-1 site and the mutant mκB-1 site are expanded. The mutated bases (G to C at position 7959 and C to G at position 7967) are shown. CMV, human cytomegalovirus immediate early promoter; Ψ, packaging signal for XMRV. Lowercase letters represent mutations. (B) Conditioned medium from 293T/17 cells transfected with pcDNA3.1(−)VP62 or pcDNA3.1(−)VP62mκB-1 was spun at 100,000 × g for 1 h, and virus-like particles were subjected to Western blotting for Gag protein by using the rat MAb R187. (C) Duplicate semiconfluent cultures of the prostate carcinoma cell lines LNCaP and DU145 were infected with equal amounts (500 μl [top and bottom panels] and50 μl [middle panel] viral preparation shown in panel B) of WT XMRV (VP62) or mutant virus (VP62mκB-1). After culture for the indicated periods (2 to 6 days), individual conditioned media were ultracentrifuged and the pellets analyzed by Western blotting for Gag protein (p30^CA) by using the rat MAb R187. The bar graphs reflect quantitative analysis of duplicate band intensities from the membranes shown above; the results are expressed as relative band intensities, and error bars reflect the ranges of measurements. U, sample from uninfected cells. Each experiment was repeated at least three times, and representative results are shown. (D) Reverse transcriptase (RT) activity in the culture supernatants of LNCaP cells left uninfected or infected (50 μl) with VP62 or VP62mκB-1. The results reflect RT activity (ng/ml; ± SD for triplicate cultures) in the precipitates from conditioned media (0.5 ml) tested in duplicate. Exp., experiment. (E) Relative luciferase activity after transfection of the pIL-6-Luc plasmid into the cell lines HEK293, LNCaP, and DU145, with or without the addition of TNF-α (20 ng/ml) to the cultures. For normalization, each cell line was cotransfected with pTK-RL. FL and RL activities were measured from cells harvested 18 h after transfection.

Relative effects of dexamethasone and NF-κB on XMRV production. (A) HEK293 cells were infected with WT XMRV (pcDNA-VP62). Ten days after infection (∼75% of cells expressed Gag protein, per immunostaining), cells were harvested, washed, and seeded onto 6-well plates in fresh medium supplemented with dexamethasone (Dex) at the indicated concentrations. After 24 h, the conditioned media (0.5 ml) were harvested and ultracentrifuged (100,000 × g for 1 h), and each pellet was analyzed by Western blotting for Gag protein (p30^CA) by using the rat MAb R187. (B) HEK293 cells were infected with WT or κB-1 mutant XMRV (VP62 or VP62mκB-1) with or without Dex (1.5 μM). After 6 days of incubation, the conditioned media (0.5 ml) were harvested and ultracentrifuged, and each pellet was analyzed by Western blotting for Gag protein (p30^CA). The bar graphs below the nitrocellurose membrane images in panels A and B reflect quantitative analysis of duplicate band intensities; the results are expressed as relative band intensities, and the error bars reflect the ranges of measurements. The dotted line in panel B demarks the mean relative band intensity from culture supernatants of XMRV VP62-infected HEK293 cells cultured in medium. Each experiment was repeated three times, and representative results are shown. (C) Schematic representation of XMRV LTR-mediated replication regulated by the Dex-glucocorticoid receptor (GR)-GRE and TNF-α/LMP1-NF-κB-κB-1 pathways. TNFR, tumor necrosis factor receptor; IKK, IκB kinase.