NF-κB Activation Stimulates Transcription and Replication of Retrovirus XMRV in Human B-Lineage and Prostate Carcinoma Cells

0022-538X/11/$12.00 doi:10.1128/JVI.02333-10

Copyright © 2011, American Society for Microbiology. All Rights Reserved.

NF-

␬

B Activation Stimulates Transcription and Replication of

Retrovirus XMRV in Human B-Lineage and Prostate

Carcinoma Cells

䌤

Shuhei Sakakibara,

* Kaori Sakakibara,

and Giovanna Tosato

Laboratory of Cellular Oncology1_{and Laboratory of Cancer Biology and Genetics,}2_{Center for Cancer Research,}

National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892

Received 8 November 2010/Accepted 20 January 2011

Xenotropic murine leukemia virus-related virus (XMRV) is a gammaretrovirus linked to prostate carcinoma and chronic fatigue syndrome. Here we report that NF-␬B activation can markedly increase XMRV production. The inflammatory cytokine tumor necrosis factor alpha (TNF-␣), which activates NF-␬B, significantly aug-mented viral Gag protein production in XMRV-infected cells. Reporter assays showed that TNF-␣ and Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1), an intrinsic NF-␬B activator, increased long terminal repeat (LTR)-dependent XMRV transcription. We identified two NF-␬B binding sites (designated

␬B-1 and ␬B-2) in the LTR U3 region of XMRV and demonstrated that both sites bind to the NF-␬B component p65/RelA. Mutation of the␬B-1 site, but not the␬B-2 site, impaired responsiveness to TNF-␣and LMP1 in reporter assays. A mutant XMRV with a mutation at the ␬B-1 site replicated significantly less efficiently than the wild-type XMRV in the prostate carcinoma LNCaP, DU145, and PC-3 cell lines, HEK293 cells, the EBV-immortalized cell line IB4, and the Burkitt’s lymphoma cell line BJAB. These results demon-strate that TNF-␣ and EBV LMP1 enhance XMRV replication in prostate carcinoma and B-lineage cells through the␬B-1 site in the XMRV LTR, suggesting that inflammation, EBV infection, and other conditions leading to NF-␬B activation may promote XMRV spread in humans.

Xenotropic murine leukemia virus-related virus (XMRV) is a gammaretrovirus that was first identified in prostate cancer specimens from patients with a homozygous muta-tion of RNase L, a mediator of innate antiviral responses (36). More recently, two studies have reported that approximately 20% of prostate cancers are infected with XMRV within the malignant epithelial cells, independently of RNase L gene mu-tation (6, 31). Additional studies have detected XMRV (24) and other murine leukemia virus (MLV)-like viruses (23) in blood mononuclear cells from a high proportion of patients with chronic fatigue syndrome, an illness of unknown etiology and defined by clinical criteria (14), but infrequently from volunteer blood donors. However, other studies have failed to detect XMRV in European patients with prostate cancer (3, 12, 18) or in European or American patients with chronic fatigue syndrome (11, 15, 17, 34, 37). Thus, the association of XMRV with human diseases is controversial (21). The human prostate carcinoma cell line 22Rv1, which was passaged in mice, contains XMRV proviral DNA and secretes high-titer XMRV in culture (22), but other prostate carcinoma cell lines spontaneously release little (VCaP [22]) or no (LNCaP and DU145 [9]) virus. However, XMRV could infect and replicate in the prostate carcinoma LNCaP and DU145 cell lines, the ovarian carcinoma Hey1b, and the cervical carcinoma cell line HeLa (9).

As in other retroviruses, transcription of the XMRV genome is controlled by elements in the U3 region of the 5⬘ long terminal repeat (LTR) (36). The U3 region of XMRV contains a glucocorticoid-responsive element (GRE) that is critical to androgen-stimulated XMRV U3 transcriptional activity (10, 30). The human prostate carcinoma cell line LNCaP, which expresses a functional androgen receptor (19) and displays increased LTR-mediated transcriptional activity in response to andro-gen, replicates exogenous XMRV more efficiently than other prostate cell lines that do not express the androgen receptor (30). The cellular receptor for XMRV, XPR1 (xenotropic and polytropic murine leukemia virus receptor 1), is ubiquitously expressed (4, 36). Therefore, many cell types are susceptible to XMRV infectionin vitro(16), but efficient XMRV replication appears to be limited to few cell lines and is linked to steroid-induced viral transcription (30).

Since XMRV and MLV-like virus sequences have been reported to be amplified from peripheral blood mononu-clear cells of a high proportion of patients with chronic fatigue syndrome and 3.7 to 7% of healthy volunteer blood donors (23, 24) and infectious virus has been recovered from blood cells after cytokine activationin vitro(24), the host range of XMRV appears to be broader than previously suspected. Thus, fac-tors other than steroid hormones may regulate the spread of XMRV and MLV-like virus in humans. Since some of the symptoms of chronic fatigue syndrome are consistent with cy-tokine abnormalities, as has been documented in some studies (13, 27), and prostate inflammation may contribute to the development of prostate cancer (8), we hypothesized that in-flammation may enhance XMRV replication.

In this study, we evaluated the importance of the NF-␬B pathway, an essential mediator of inflammation, in the regu-* Corresponding author. Mailing address: Laboratory of Cellular

Oncology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Building 37, Room 4134, Bethesda, MD 20892-1907. Phone: (301) 594-9597. Fax: (301) 594-9585. E-mail: sakakibs@mail.nih.gov.

䌤_{Published ahead of print on 26 January 2011.}

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lation of XMRV replication. We found that the XMRV LTR (XLTR) contains two binding sites for activated NF-␬B, one of which critically contributes to XMRV replication in cells of prostate carcinoma and B-cell lineage.

MATERIALS AND METHODS

Cells. Human embryonic kidney-derived HEK293 and HEK293T/17 cells (American Type Culture Collection [ATCC]) were propagated in Dulbecco’s modified Eagle’s medium (DMEM)-Glutamax medium (Invitrogen) with 10% fetal bovine serum (FBS) (Atlanta Biologicals). The human prostate carcinoma cell lines, LNCaP, DU145, and PC-3 were purchased from ATCC and propa-gated in RPMI 1640 with 10% FBS. R187, a rat hybridoma producing a mono-clonal antibody (MAb) against the Gag protein of MLV (ATCC) was maintained in RPMI 1640 supplemented with 0.05 mM 2-mercaptoethanol and 10% FBS or in protein-free and chemically defined CD hybridoma medium (Invitrogen). The Epstein-Barr virus (EBV)-immortalized lymphoblastoid cell line IB4 (a kind gift of Elliot Kieff, Brigham and Women’s Hospital, Harvard Medical School) was maintained RPMI 1640 with 10% FBS. All cell culture was carried out at 37°C with 5% CO2.

Reagents.Recombinant human tumor necrosis factor alpha (TNF-␣) at 10 ␮g/ml (R&D Systems) was dissolved in phosphate-buffered saline (PBS) and stored at⫺80°C. Dexamethasone (Dex) (soluble form; Sigma) was dissolved in sterilized water at 1 mM. Anti-Gag antibody was produced from R187 hy-bridoma-conditioned medium prepared in CD hybridoma medium (Invitro-gen). Cell debris was removed by centrifugation and filtration through 0.45-␮m filter discs (Millipore). The rabbit antibody to p65/RelA (C20) was purchased from Santa Cruz Biotechnology.

Plasmids.XMRV VP62 clone pcDNA3.1(⫺)VP62 and LTR reporter con-struct pGL4-XMRV LTR (9, 31) were gifts of Robert Silverman, Cleveland Clinic Foundation. EBV latent membrane protein 1 (LMP1) expression vector pCMVHA-LMP1 (20) was a gift of Bill Sugden, McArdle Laboratory, University of Wisconsin—Madison. To generate the␬B mutant reporter constructs, PCR-based site-directed mutagenesis was performed. pGL4-XLTR mut1 and -XLTR mut2 contain the substitutions G7959C/C7967G (mut1) and G7998C/C8006G (mut2) (nucleotide positions are based on GenBank accession number DQ399707.1; see Fig. 2A and C). For construction of pcDNA3.1(⫺)VP62m␬B-1, the PmlI and HindIII region of pcDNA3.1(⫺)VP62 was replaced with the PCR product containing the G7959C/C7967G mutation. pIL-6-Luc was a kind gift from Arnold Rabson, University of Medicine and Dentistry of New Jersey (UMDNJ)-Robert Wood Johnson Medical School (33).

Biotinylated DNA binding affinity assays.Double-stranded DNA (dsDNA) for the␬B site from the Ig(␬) chain enhancer (1) and a mutated␬B site from the Ig(␬) chain enhancer were custom synthesized and annealed in TE buffer (10 mM Tris-HCl, 0.1 mM EDTA, pH 8.0) by heating at 95°C for 2 min and then placement at room temperature. The␬B probe sequence, from the Ig(␬) light chain enhancer, was 5⬘-TCTCGGAAAGTCCCCTCTGG-3⬘(core sequence is underlined) (1), and the m␬B probe sequence was 5⬘-TCTCGCAAAGTCGCC TCTGG-3⬘(substituted nucleotides are underlined). XMRV LTR probes con-taining the␬B-1 and␬B-2 sites (for␬B-1, 5⬘ -TTCTGGAAAGTCCCACCTCA-3⬘, and for␬B-2, 5⬘-GACCGGGAAATACCCCAAGC-3⬘[putative␬B sites are underlined]) were prepared. For each probe, one of the strands was chemically conjugated with biotin at the 5⬘end (Integral DNA Technology). Nuclear ex-tracts from the IB4 cell line, prepared as described previously (38), were diluted in NE C buffer [10 mM HEPES (pH 8.0), 0.1% Igepal CA630 (Sigma-Aldrich), 0.05 mg/ml poly(dI-dC) (Sigma-Aldrich), 0.2 mg/ml sheared salmon sperm DNA (Invitrogen), 0.1 mg/ml bovine serum albumin (Sigma-Aldrich)] and adjusted to an NaCl concentration of 100 mM. Five picomoles of dsDNA probe, streptavidin Sepharose (10␮l; GE Amersham), and nuclear extracts (total reaction mixture, 0.5 ml) were incubated at 4°C for 3 h. After five washes with NE C buffer containing 100 mM NaCl, the precipitated protein was analyzed by Western blotting.

Reporter assays. HEK293 cells were seeded onto 12-well plates (200,000 cells/well); after 24 h, the cells were transiently transfected with 100 ng of XMRV LTR (i.e., with XLTR), XMRV LTR mut1, or pGL4-XMRV LTR mut2, together with 20 ng of pRL-TK luc. After 24 h, the cells were harvested, lysed, and assayed with the Dual-Luciferase assay system (Promega). Firefly and renilla luciferase activities (in 10␮l from 100␮l of total lysate from each culture) were read with a FLUOstar Omega microplate reader (BMG Labtech). Firefly luciferase activity was measured in triplicate and normalized to renilla luciferase activity.

Viral infection.Virus was prepared by transfection of HEK293T/17 cells with pcDNA3.1(⫺)VP62 or pcDNA3.1(⫺)VP62m␬B-1 by using Fugene 6 transfec-tion reagent (Roche). After a 5-day incubatransfec-tion, culture supernatants were har-vested. Cell debris was removed by centrifugation and filtration through 0.45-␮m filter discs (Millipore). Clarified/filtered supernatant containing VP62 or VP62m␬B-1 virus was added in conjunction with 4␮g/ml of Polybrene (Sigma) to adherent cells (1⫻105_{cells) preseeded onto 35-mm plates. For infection of}

cells in suspension, the cells (1⫻105

cells) were washed with culture medium and placed onto 35-mm plates. Spin inoculation was carried out by centrifugation at 2,500 rpm at 30°C for 1 h. After being washed, the cells were cultured in fresh culture medium (1.5 ml/plate). To maintain infected suspension cells, fresh medium was added to achieve a cell density of approximately 0.5⫻106_{to 1⫻}

106

cells/ml. To monitor the release of XMRV particles, culture supernatant (0.5 ml at each time point) was cleared by centrifugation (1,000⫻gfor 5 min) and further spun by ultracentrifugation in polyallomer centrifugation tubes (13 by 51 mm; Beckman) by using a SW55 Ti rotor (Beckman) at 100,000⫻gfor 1 h. Pellets were suspended in SDS-PAGE loading buffer (LDS sample buffer [In-vitrogen]) and subjected to Western blot analysis for Gag capsid protein (p30CA

) (31). For reverse transcriptase measurement, pellets from ultracentrifugation were suspended with buffer provided in the kit (Roche) and stored at⫺80°C until assayed.

Reverse transcriptase assay. Reverse transcriptase was measured with the Roche colorimetric reverse transcriptase assay kit by following the manufac-turer’s protocol, with the addition of 10 mM MnCl2to the reaction mixture.

Pellets from each conditioned medium (0.5 ml) were assayed in triplicate. HIV-1 reverse transcriptase provided in the kit was used for generation of a standard curve.

Indirect immunofluorescence staining.Cells were washed with PBS, fixed with 4% paraformaldehyde, and permeabilized with 1% Triton X-100–PBS. The cells were incubated for 1 h at 37°C with rat R187 hybridoma culture supernatant diluted (1:5) in PBS containing 0.1% bovine serum albumin (BSA) and 0.2% Tween 20. After being washed, the cells were stained with anti-rat Alexa 567-conjugated antibody (Invitrogen; 1:200 dilution); DAPI (4⬘ ,6-diamidino-2-phe-nylindole) was used for nuclear staining. After being washed, the slides were mounted with fluorescent mounting medium (Dako) and observed through a confocal laser scanning microscope (LSM510) equipped with the objective lens Plan Neofluar 10⫻/0.3 (Carl Zeiss MicroImaging, Thornwood, NY).

RESULTS

TNF-␣increases XMRV production in HEK293 cells.To ex-amine whether proinflammatory signals can influence XMRV production, we infected the human embryonic kidney HEK293 cell line with an aliquot (100␮l/35-mm plate) of recombinant XMRV VP62 (produced as described in Materials and Meth-ods). After cells were cultured for 10 days, the XMRV-infected cells were washed, seeded on new culture plates, and incubated with TNF-␣(1.3 to 20 ng/ml) for 24 h. The Gag protein content in the culture supernatants was measured by immunoblotting with the Gag-specific monoclonal antibody R187. As shown in Fig. 1A, 24-h exposure to 20 ng/ml TNF-␣increased XMRV release in the culture supernatant approximately 10-fold. We used a XMRV LTR (XLTR) luciferase reporter construct to test whether TNF-␣increases XMRV transcription in HEK293. As shown in Fig. 1B, we found that TNF-␣(5 ng/ml) augments the reporter’s activity by approximately 8-fold, providing evi-dence that TNF-␣can enhance XMRV replication by enhanc-ing the XMRV LTR transcriptional activity.

The XMRV LTR contains a binding site for NF-␬B critical for responsiveness to TNF-␣.TNF-␣can activate several sig-naling pathways emanating from the TNF receptor, including the NF-␬B pathway, which mediates various cytokine-induced inflammatory responses (35). To test for the possibility that NF-␬B activation leads to increased XMRV LTR transcrip-tional activity, we examined the effects of Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1), which activates

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NF-␬B by engaging signaling proteins for the TNF receptor (26). We cotransfected HEK293 cells with various amounts of an LMP-1 construct and the luciferase reporter XMRV LTR construct. The results of reporter assays showed that EBV LMP1 activates XMRV LTR transcription by approximately 9-fold (Fig. 1C).

Based on these results, we looked for the presence of NF-␬B binding sites on the XMRV LTR. Sequence analysis showed that there are two potential NF-␬B binding sites, which we named ␬B-1 (5⬘-GGGACTTTCCA-3⬘) at nucleotides (nt) 7968 to 7958 in the VP62 strain of XMRV (9) and ␬B-2 (5⬘-GGGTATTTCCC-3⬘) at nt 8007 to 7997 (Fig. 2A). We tested for NF-␬B binding to XMRV␬B-1 and␬B-2 sequences by incubating biotinylated double-stranded DNA (containing the␬B-1 or␬B-2 site of XMRV) with nuclear extracts from the EBV-infected IB4 lymphoblastoid cell line, in which NF-␬B is constitutively active (26). For a positive control, we used the ␬B site from the immunoglobulin␬light chain enhancer (5⬘ -GGGACTTTCCG-3⬘) (1), and for a negative control, we used

its minimally mutated sequence (5⬘-GCGACTTTGCG-3⬘ [mu-tations are underlined]). We looked for NF-␬B p65/RelA bind-ing to biotinylated DNA after precipitation with streptavidin Sepharose and immunoblotting the precipitate with antibodies to NF-␬B p65/RelA. Under conditions where the␬chain en-hancer, but not the mutated␬chain (mut␬B) oligonucleotide, precipitated NF-␬B p65/RelA, both the␬B-1 and␬B-2 XMRV oligonucleotides similarly bound NF-␬B p65/RelA (Fig. 2B).

To test for functional correlates of XMRV ␬B-1 or ␬B-2 binding to␬B p65/RelA, we compared the transcriptional ac-tivity of the wild-type (WT) XMRV LTR (XLTR) with the transcriptional activity of a mutant XMRV LTR with a muta-tion at the␬B-1 (XLTR mut1) or␬B-2 (XLTR mut2) site (Fig. 2A). With luciferase reporter assays, we found that XLTR mut1 lost responsiveness to TNF-␣and LMP1 transcriptional activation, while XLTR mut2 retained partial activity in this assay (Fig. 2C). These results provide evidence that the␬B-1 site is a critical mediator of XMRV LTR transcriptional re-sponse to TNF-␣and EBV LMP-1.

Mutation of the␬B-1 site severely impairs XMRV replica-tion.To examine the role of the␬B-1 site in XMRV replication, we constructed a recombinant XMRV with a mutated␬B-1 se-quence in the cytomegalovirus (CMV) vector pcDNA3.1 (Fig. 3A). The HEK293T/17 cell line transfected with the parental VP62 XMRV and that transfected with the ␬B mutant VP62m␬B-1 construct produced similar amounts of virus, as judged by immunoblotting with the Gag-specific antibody R187 (Fig. 3B). Using VP62 and VP62m␬B-1 virus (500 ␮l) released from the transfected HEK293T/17 cells, we infected the human prostate LNCaP cell line, in which NF-␬B is con-stitutively active, at least in part (33). As shown in Fig. 3C, we detected similar levels of Gag protein in culture supernatants of LNCaP cells infected with the WT and VP62m␬B-1 viruses (Fig. 3C, upper panel). Since Gag protein levels in the condi-tioned medium peaked 3 days postinfection, we considered the possibility that viral infection was maximal and that this may have hidden quantitative differences. When we reduced the viral inoculum by 10-fold (50␮l), Gag protein was not clearly detectable in 3-day culture supernatants of LNCaP cells in-fected with either WT or VP62m␬B-1 mutant virus (Fig. 3C, middle panel). However, 6 days after infection, culture super-natants from LNCaP cells infected with the WT virus con-tained much greater amounts of Gag protein than LNCaP cells infected with the m␬B-1 mutant virus; the amount of Gag protein produced by the mutant-infected cells was 83% lower than the value for the WT as judged by a comparison of band intensities (Fig. 3C, middle panel). Consistently, we detected higher levels of reverse transcriptase activity in the culture supernatants of LNCaP cells infected with the WT than in those of cells infected with the m␬B-1 mutant virus (Fig. 3D). XMRV replicates in the androgen receptor-independent prostate DU145 cell line (30), in which NF-␬B is constitutively active (33). Four days after infection with WT VP62 XMRV (500␮l culture supernatant of the HEK293T/17 cell line; Fig. 3B), culture supernatants of DU145 cells contained clearly detectable Gag protein (Fig. 3C, lower panel), whereas 4-day culture supernatants of DU145 infected with the␬B mutant VP62m␬B-1 virus under the same conditions contained signif-icantly less Gag protein. A similar difference in levels of Gag protein production was observed in the androgen receptor-FIG. 1. TNF-␣and LMP1 enhance LTR-mediated viral

transcrip-tion. (A) XMRV-infected HEK293 cells were incubated with TNF-␣at the indicated concentrations. After 24 h, the conditioned media were harvested and ultracentrifuged (100,000⫻gfor 1 h), and each pellet was subjected to Western blot analysis for Gag protein (p30CA_{) 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-␣(5 ng/ml) was added to HEK293 cells transiently cotrans-fected 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 nor-malized to RL activity. The results are presented as fold increases (⫾ standard deviations [SD] for triplicate cultures) in normalized FL in cultures with TNF-␣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.

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independent PC-3 prostate carcinoma cell line infected with WT or VP62m␬B-1 virus (data not shown). PC-3 also displays constitutively active NF-␬B (33). Unlike the situation with XMRV infection in LNCaP (Fig. 3D), reverse transcriptase activity was undetectable in the culture supernatant of DU145 and PC-3 cells infected with XMRV (data not shown), indi-cating that LNCaP cells are higher-level producers of XMRV than the other cell lines. Together, these results show that introduction of a mutation at the␬B-1 site in the viral LTR markedly reduces XMRV replication in prostate cell lines, providing evidence that NF-␬B activity contributes to XMRV replication in these cell lines.

To assess further the contribution of NF-␬B activity to XMRV replication, we examined the relationship between levels of XMRV replication and levels of NF-␬B activity, as assessed by measuring NF-␬B-directed transcription of the reporter pIL-6-Luc plasmid, which carries three copies of the NF-␬B-responsive

element, 5⬘-GGGATTTTCCC-3⬘, before the interleukin-6 (IL-6) promoter basal element (33). We found that DU145 cells show a much higher level of constitutive luciferase activity than HEK293, and LNCaP cells (Fig. 3E). TNF-␣enhanced luciferase activity in DU145 and HEK293 cells but only minimally in LNCaP (Fig. 3E). These results provide evidence that levels of NF-␬B ac-tivity are not the sole determinant of XMRV replication in prostate cancer cell lines.

NF-␬B is constitutively active in EBV-immortalized cell lines due to the expression of EBV LMP1, which activates NF-␬B (26). We selected the IB4 cell line as a representative EBV-immortalized cell line to test whether it is permissive for XMRV replication. The IB4 cell line was infected with WT VP62 or mutant VP62m␬B-1 XMRV (500␮l culture superna-tant of the producer HEK293T/17 cell line) (Fig. 3B) and incubated for 13 days. At the 3- and 6-day time points, Gag protein was minimally detectable in the conditioned medium, FIG. 2. Identification of NF-␬B binding sites␬B-1 and␬B-2 in the XMRV LTR and functional analysis of NF-␬B binding to the XMRV LTR. (A) DNA sequences of consensus and NF-␬B binding sites in the immunoglobulin (Ig)␬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␬B-1 and␬B-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-␣. The cells were also cotransfected with pCMV-LMP1 (0.1

␮g/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.

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but at the 13-day time point, IB4 cells infected with WT VP62 virus produced much greater amounts of Gag protein than IB4 cells infected with the VP62m␬B-1 mutant virus (Fig. 4A). These results provide evidence that the EBV-immortalized IB4 cell line, in which NF-␬B is constitutively active, can replicate XMRV and that the presence of the␬B-1 site within the viral LTR contributes to viral replication. The IB4 cells infected with the mutant VP62m␬B-1 virus did produce Gag protein in the culture supernatant (albeit reduced compared to the amount produced by WT virus-infected cells), suggesting that the␬B-2 site or other elements within the XMRV LTR may contribute to XMRV replication in IB4 cells.

A similar analysis was carried out with the EBV-negative Burkitt lymphoma BJAB cell line, which displays constitutive, but weak, NF-␬B activity that derives from p50 and c-Rel

rather than from p50 and p65, as observed in many other cell types (2). Gag protein was not detectable in the conditioned medium of cells infected 6 and 8 days earlier with either WT VP62 or mutant VP62m␬B-1 XMRV (500␮l culture superna-tant of the producer HEK293T/17 cell line) (Fig. 3B). How-ever, at the 13-day time point, WT VP62 virus produced de-tectable Gag protein, but the mutant VP62m␬B-1 virus did not (Fig. 4B), providing evidence that the␬B-1 site is critical for XMRV replication in BJAB cells. Immunofluorescence stain-ing showed that the WT VP62 virus induced expression of Gag protein in about 90% of BJAB cells after 2 weeks (Fig. 4C), but the mutant VP62m␬B-1 virus induced Gag expression in a lower percentage (⬃3%) of cells after 2 weeks, and the inten-sity of Gag protein staining was reduced (Fig. 4C). Taken together, these results demonstrate that the␬B-1 site is impor-FIG. 3. Mutation of the ␬B-1 site reduces XMRV replication. (A) Schematic representation of the XMRV provirus constructs [pcDNA3.1(⫺)VP62 and pcDNA3.1(⫺)VP62m␬B-1]; the WT␬B-1 site and the mutant m␬B-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;⌿, packaging signal for XMRV. Lowercase letters represent mutations. (B) Conditioned medium from 293T/17 cells transfected with pcDNA3.1(⫺)VP62 or pcDNA3.1(⫺)VP62m␬B-1 was spun at 100,000⫻gfor 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␮l [top and bottom panels] and50

␮l [middle panel] viral preparation shown in panel B) of WT XMRV (VP62) or mutant virus (VP62m␬B-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 (p30CA_{) 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

␮l) with VP62 or VP62m␬B-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-␣(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.

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tant for XMRV replication in B-lineage and prostate carci-noma cell lines, suggesting that NF-␬B activation in host cells can contribute to XMRV replication.

Dex enhances replication of an NF-␬B mutant XMRV in HEK293 cells.Previous studies have shown the presence of a functional glucocorticoid-responsive element (GRE) in the U3 region of the XMRV LTR, which contributes to viral transcrip-tion (30) and replicatranscrip-tion (10) in LNCaP. Dong and Silverman first reported that the LTR-driven XMRV transcription in LNCaP and DU145 cells is enhanced by dexamethasone (Dex) (10). Since the results presented here indicate that the␬B-1 site in the XMRV LTR can also contribute to viral replication in prostate carcinoma and B-cell lines, we examined the rela-tionship between the Dex-glucocorticoid receptor-GRE and NF-␬B pathways in XMRV replication. We selected the hu-man embryonic kidney HEK293 cell line for these experiments because it expresses glucocorticoid receptors␣and ␤(5, 28) and displays increased XMRV replication in the presence of TNF-␣(Fig. 1A) attributable to NF-␬B activation (Fig. 2C). As shown in Fig. 5A, 1.3␮M Dex enhanced the replication of WT XMRV in HEK293 cells by approximately 10-fold as judged by Gag protein levels in the culture supernatants, likely attribut-able to the previously identified GRE within the XMRV LTR (10, 30).

We compared the effects of Dex on WT VP62 and mutant VP62m␬B-1 virus replication in HEK293 cells. As expected, WT VP62 virus replicated in HEK293 cells and replication was enhanced in the presence of 1.5␮M Dex (Fig. 5B). The mutant VP62m␬B-1 virus did not replicate or replicated minimally in 293T cells cultured in medium only, but the addition of Dex (1.5␮M) clearly enhanced the replication of the VP62m␬B-1 mutant virus (Fig. 5B) so that levels of Gag protein were now

similar to those found in the culture supernatants of HEK293 cells infected with WT virus cultured in medium only. These results indicate that the Dex-glucocorticoid receptor-GRE and NF-␬B pathways (Fig. 5C) can independently contribute to XMRV replication in these cells.

DISCUSSION

We report that the proinflammatory cytokine TNF-␣ mark-edly increases XMRV replication in host cells, and by a variety of approaches, we demonstrated that activation of the NF-␬B pathway by TNF-␣, EBV LMP1, and other mechanisms con-tributes to XMRV replication in prostate carcinoma and B-lineage cell lines. We found that the XMRV LTR U3 region contains two ␬B sites, which we designated ␬B-1 and ␬B-2, which bind the NF-␬B component p65/RelAin vitro, and we showed that the XMRV ␬B-1 site is critical for increased XMRV transcription and replication induced by NF-␬B ac-tivation. Thus, the results presented here extend the previ-ously described spectrum of target cells capable of replicat-ing XMRV to include B-lineage cells. In addition, we identify the NF-␬B pathway as an inducer of XMRV replication in

vitro.

Two recent studies have identified XMRV and MLV-like virus sequences in the blood mononuclear cells of a high pro-portion of patients with chronic fatigue syndrome and 4 to 7% of healthy blood donors (23, 24). One of these studies addi-tionally reported the identification of XMRV antigens in acti-vated peripheral blood B and T cells from two patients with chronic fatigue syndrome (24). We now show that it is possible to successfully propagate XMRV in B-lineage cells, provided that NF-␬B is activated, raising the possibility that B-cell in-FIG. 4. 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-␮l XMRV-containing supernatants (VP62 or VP62m␬B-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 (p30CA_{) (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 p30CA_{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 VP62m␬B-1.

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fection with EBV or activation by inflammatory mediators may play a role in the spread of XMRV in humans. It was previ-ously reported that the human EBV-negative lymphoblastoid DG-75 cell line is infected with the DG-75 MLV, which is closely related to XMRV. We found that the DG-75 MLV contains a potential ␬B-2 site in the LTR (29), raising the additional possibility that NF-␬B activation may promote the replication of other MLV-like viruses. Also, since we found that NF-␬B activation contributes to XMRV replication in prostate carcinoma and HEK293 cells, the current results pro-vide epro-vidence for an androgen receptor-independent pathway of XMRV spread in prostate-derived cells and other cells that do not express steroid receptors.

The NF-␬B pathway regulates gene transcription in re-sponse to a variety of signals generated in the course of in-flammation, such as cytokines, free radicals, and bacterial or viral antigens. Epidemiological and histopathological evidence indicates that chronic inflammation may contribute to prostate carcinogenesis (7). Cytokine imbalance in cohorts of patients with chronic fatigue syndrome has been documented (13, 27), and EBV infection has been suspected to be an initiating or contributing event in chronic fatigue syndrome (32). Thus, prostate tissue inflammation and systemic cytokine imbalances

may result in activation of NF-␬B and contribute to XMRV replication in prostate and blood.

Sequence alignment and phylogenetic analyses have con-cluded that the genome of XMRV is more similar to the genomes of xenotropic and polytropic viruses than to those of ecotropic MLVs (23, 36). The LTR of XMRV has the highest degree of nucleotide identity to the LTRs from xenotropic MLVs NSF-Th-1 and NZB-9-1 while containing structural and regulatory elements shared by other MLVs, including the up-stream conserved region (UCR), leukemia factor b (LVb), the GRE, and the CCAAT box (36). The previously identified GRE site in XMRV is found in the genomes of other MLVs, where it similarly drives LTR-directed transcription in re-sponse to steroids (25, 36). The␬B-1 site of XMRV, identified here, is conserved in several MLVs, including MCF13, where it is responsible for viral tumorigenesis in vivo(39), but is not found in the LTR of the MCF1233 murine leukemia virus, the AKV murine leukemia virus, and neuro-2a-associated retrovi-rus (NeRV) (GenBank accession numbers U13766, J01998, and DQ366149, respectively). It will be interesting to deter-mine whether a␬B-1 site is present in the recently described MLV-like viruses identified in patients with chronic fatigue FIG. 5. Relative effects of dexamethasone and NF-␬B 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⫻gfor 1 h), and each pellet was analyzed by Western blotting for Gag protein (p30CA_{) by using the rat} MAb R187. (B) HEK293 cells were infected with WT or␬B-1 mutant XMRV (VP62 or VP62m␬B-1) with or without Dex (1.5␮M). 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 (p30CA_{). 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-␣/LMP1–NF-␬B–␬B-1 pathways. TNFR, tumor necrosis factor receptor; IKK, I␬B kinase.

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syndrome (23) and to what extent it contributes to xenotropic MLV spread into humans.

The association of XMRV with prostate carcinoma, chronic fatigue syndrome, and subsets of healthy blood donors is con-troversial, underlying the need for additional studies. We show here that XMRV can replicate in human B cellsin vitroand that XMRV replication is enhanced by inflammation and other activators of NF-␬B. This observation may explain some of the differences in detection of XMRV in different studies and may prove useful in the design of future studies aimed at clarifying the potential of XMRV to spread in humans.

ACKNOWLEDGMENTS

This study was supported by the intramural research program of the Center for Cancer Research (CCR), National Cancer Institute (NCI), National Institutes of Health, Bethesda, MD.

We thank R. Silverman (Cleveland Clinic Foundation) for the XMRV VP62 clone and reporter construct, B. Sugden (University of Wisconsin) for the EBV LMP1 plasmid, A. B. Rabson (UMDNJ-Robert Wood Johnson Medical School) for providing the pIL-6-Luc plasmid, E. Kieff (Brigham and Women’s Hospital, Harvard Medical School) for the IB4 cell line, R. Yarchoan for scientific discussions and critical review of the manuscript, the DNA Minicore Facility (Labo-ratory of Experimental Carcinogenesis [LEC], CCR, NCI) for DNA sequencing, the Confocal Microscopy Core Facility (LEC, CCR, NCI) for laser confocal microscopy, and K. Nagashima (NCI—Frederick, SAIC), L. Sierra (National Center for Complementary and Alternative Medicine [NCCAM]), P. Gasperini, P. McCormick, M. Segarra, K. Jiang, G. Espígol-Frigole´, O. Salvucci, and other members in the Lab-oratory of Cellular Oncology (LCO) branch for their help on various aspects in this work.

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