Introduction

The Kaposi's sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus 8, is a gammaherpesvirus originally identified in Kaposi's sarcoma (KS) tissue (Chang et al., 1994). KSHV has been identified in HIV-related and HIV-unrelated cases of KS (Moore and Chang, 1995), primary effusion lymphoma (PEL) (Cesarman et al., 1995), and multicentric Castleman's disease (MCD) (Soulier et al., 1995). In KS, evidence based on serologic and molecular studies suggests that KSHV may play a causative role (Gao et al., 1996). The relative rarity of PEL and MCD render these diseases somewhat more difficult to establish KSHV as an etiologic agent based on seroepidemiologic studies. Nonetheless, the association of KSHV infection with PEL is unique among B-cell lymphomas, and the severity of MCD correlates with KSHV viral load (Grandadam et al., 1997).

A common denominator among the KSHV-associated neoplasms is that they all employ cellular interleukin-6 (cIL-6) as a growth factor. For example, cIL-6 is an autocrine growth factor for KS cells (Miles et al., 1990), and the growth and survival of PEL cells is dependent upon the availability of cIL-6 (Asou et al., 1998). In MCD, cIL-6 levels are elevated in the lymph nodes and serum, and cIL-6 appears to be important for the development of MCD in mice (Leger-Ravet et al., 1991; Screpanti et al., 1996). In addition, cIL-6 levels are elevated in the tissues of patients with KSHV-associated neoplasms. KS cells express cIL-6 RNA transcripts and secrete high levels of cIL-6 protein that binds to IL-6 receptors on KS cells (Ensoli et al., 1989). cIL-6 levels are elevated in the lymph nodes and serum of MCD patients (Leger-Ravet et al., 1991), and PEL cells express high levels of cIL-6 and also use this cytokine as an autocrine growth factor (Foussat et al., 1999). Given the importance of cIL-6 as a growth factor for KSHV-associated diseases and that acute KSHV infection induces cIL-6 expression in primary endothelial cells (Panyutich et al., 1998), we postulated that a KSHV-encoded protein might modulate cIL-6 expression.

The KSHV genome contains over 80 open reading frames (ORFs), many of which may play a role in the pathogenesis of KSHV-related diseases. One of these, ORF K13, encodes the viral homologue of the FADD-like interferon-converting enzyme (FLICE) inhibitory protein (vFLIP). Both vFLIP RNA transcripts as well as vFLIP protein are expressed in KSHV-infected cells (Dittmer et al., 1998; Sarid et al., 1998,1999; Talbot et al., 1999; Grundhoff and Ganem, 2001; Low et al., 2001). vFLIP contains two death effector domains (DEDs) that mediate its physical interaction with other DED-containing proteins (Thome et al., 1997). vFLIP blocks death receptor-induced apoptosis by binding to the DED-containing FLICE (caspase 8) and preventing its activation (Thome et al., 1997). By preventing apoptosis, vFLIP may participate in tumor progression. In addition, vFLIP can activate the nuclear factor B (NF-B) pathway (Chaudhary et al., 1999), which may also play an important antiapoptotic role. For example, inhibition of NF-B induces apoptosis in KSHV-infected PEL cells (Keller et al., 2000).

When members of the tumor necrosis factor receptor (TNFR) family are engaged by ligand, the TNFRs undergo trimerization and recruit death domain-containing proteins, such as TNFR-associated death domain protein (TRADD). TRADD also binds tumor necrosis factor associated factor 2 (TRAF2), which in turn is the branch point for activation of the NF-B and c-jun N-terminal kinase (JNK) pathways in response to binding of TNF family members to TNFRs (Reinhard et al., 1997; Song et al., 1997; Baud et al., 1999). JNK is a MAP kinase that activates members of the activation protein 1 (AP1) transcription family, such as jun and fos.

A previous study demonstrated that vFLIP physically interacts with TRAF2 and activates NF-B. (Chaudhary et al., 1999). Since TRAF2 is the bifurcation point for activation of the NF-B and JNK pathways, we hypothesized that vFLIP also activates the JNK/AP1 pathway in a TRAF-dependent fashion. Given the known role of the NF-B and JNK/AP1 pathways in modulating cIL-6 gene expression (Libermann and Baltimore, 1990; Dendorfer et al., 1994), we further postulated that vFLIP activates the cIL-6 promoter and induces cIL-6 protein expression. We recently demonstrated that another KSHV-encoded protein, latency-associated nuclear antigen (LANA), activates the cIL-6 promoter and induces cIL-6 protein expression (An et al., 2002). Thus, in this report, we also investigated the potential interactions between LANA and vFLIP in the regulation of cIL-6 promoter activity.

Results

vFLIP can activate AP1 response element (RE) through JNK

We performed transient cotransfection experiments to determine if vFLIP could induce reporter gene expression from an AP1 reporter construct that has five tandem copies of a consensus AP1 response element (RE) that regulates expression of firefly luciferase (pAP1-luc). The pAP1-luc plasmid was cotransfected into R1T cells (a bone marrow stromal cell line; An et al., 2002) or human embryonic kidney 293 cells with an enhanced green fluorescent protein (EGFP)-tagged vFLIP fusion construct (pEGFP-vFLIP) or the EGFP blank vector. Bone marrow stromal cells endogenously express cIL-6 and complement the use of 293 cells, which lack cIL-6 expression. In both cell lines, vFLIP markedly induced luciferase reporter gene expression in a dose-dependent fashion (Figure 1a, b), indicating that vFLIP activates transcription factors that transactivate the AP1 RE.

Figure 1.

JNK is required for vFLIP induction of gene expression from the AP1 RE. (a) 293 cells were transiently transfected with pAP1-luc (10 ng) and the EGFP-vFLIP expression vector or the vector control. Total DNA (EGFP-vFLIP+empty vector) was held constant (50 ng). Firefly luciferase was assayed at 48 h. Results are reported as the means of three experiments, and s.d. are shown. (b) Same as (a) but in R1T cells, and 100 ng of pAP1-luc was used. Results were reported in relative luciferase units (RLU), which were normalized to the group with EGFP vector control only. (c) JNK is required for vFLIP activation of the AP1 RE. 293 cells were transiently transfected with pAP1-luc (10 ng). EGFP-vFLIP, or the EGFP control vector (10 ng), and a JNK-DN construct or the control vector. The total amount of JNK-DN or empty vector DNA was held constant (100 ng). Luciferase activity was assayed at 48 h; experiments were performed in triplicate. RLU were normalized to vector control only group. (d) The JNK-DN does not inhibit vFLIP-mediated activation of the NF-B pathway. 293 cells were transiently transfected with either EGFP-vFLIP or the EGFP control vector (10 ng), pB-luc (10 ng), or the JNK-DN or control vector (100 ng). Firefly luciferase was assayed at 48 h and results are means of three experiments s.d. RLU were normalized to vector control only group. (e) Inhibition of p38 MAP kinase by SD203580 does not affect vFLIP activation of the AP1 reporter. 293 cells were transfected with EGFP-vFLIP or the EGFP control vector (10 ng) and the pAP1-luc vector with or without SD203580 (40 M). SD203580 or vehicle control (DMSO, final concentration of 0.1%) was added 24 h after transfection. Luciferase activity was assayed at 48 h; experiments were performed in triplicate. RLU were normalized to vector control only group

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Activation of AP1 transcription factors is accomplished by upstream kinases. In particular, JNK is the only known kinase capable of phosphorylating the transactivation domain of c-jun, which enhances jun-mediated transactivation. Given that vFLIP induces gene expression from an AP1-driven reporter plasmid, we postulated that vFLIP is an upstream activator of JNK. To determine whether vFLIP-induced transactivation of the AP1 RE was mediated by JNK, we transiently cotransfected a JNK dominant-negative construct (JNK-DN) or the empty vector with the pEGFP-vFLIP and pAP1-luc into 293 cells. The JNK-DN contains mutations at the phosphorylation sites (Thr¹⁸³ to Ala and Tyr¹⁸⁵ to Phe; Liu and Shuai, 2001). Whereas reporter gene expression induced by vFLIP was unaffected by the empty vector, it was completely abrogated by the JNK-DN, indicating that vFLIP transactivation of the AP1 RE works through JNK (Figure 1c). Given that vFLIP has been previously shown to activate the NF-B pathway, we performed transient transfections in 293 cells with a B-RE-driven reporter construct (pB-luc) to demonstrate the specificity of the JNK-DN. As shown in Figure 1d, the JNK-DN did not affect vFLIP-mediated activation of the NF-B pathway. The above transient transfection experiments were repeated at least twice, and similar results were obtained, except for variations in the relative luminescence units in the control groups. To confirm that vFLIP activation of the AP1 reporter was specifically mediated by JNK and not other MAP kinases, we attempted to inhibit vFLIP-induced activation of the AP1 reporter with SB203580, a chemical inhibitor of the p38 MAP kinase. SB203580 had no effect on vFLIP activation of the AP1 reporter (Figure 1e), which supports the notion that this effect of vFLIP is mediated through the JNK MAP kinase.

To confirm that JNK activation was responsible for the AP1 activation by vFLIP, we employed an in vivo signal transduction trans-reporting system. In this system, a GAL4 responsive element can be transactivated by a GAL4 DNA binding domain/c-jun transactivation domain fusion protein. Activation of the fusion protein occurs when JNK phosphorylates the c-jun transactivation domain. The system consists of several components: (1) a reporter plasmid under the regulation of five tandem repeats of the GAL4 response element, (2) a GAL4 DNA binding domain–c-jun transactivation domain fusion (GAL4DNAbd–jun-TAD) under regulation of the CMV promoter, (3) a negative control, which contains the GAL4 DNA binding domain (GAL4DNAbd), only, and (4) a MEKK construct, an upstream activator of JNK, which serves as a positive control. Transient cotransfections of various combinations of these constructs into 293 cells with either pEGFP-vFLIP or the EGFP blank vector demonstrated that EGFP-vFLIP potently induces luciferase expression compared to EGFP only (Figure 2). Since JNK is the only upstream kinase that can phosphorylate and activate c-jun, these results indicate that vFLIP activates the JNK pathway.

Figure 2.

An in vivo signal transduction reporting system demonstrates that vFLIP activates the c-jun transactivation domain. EGFP-vFLIP or the EGFP control vector was cotransfected with a GAL4DNAbd–jun-TAD and a luciferase reporter driven by five copies of the GAL4 RE. MEKK, an upstream activator of JNK, cotransfected with the GAL4 reporter and GAL4DNAbd–jun-TAD or GAL4DNAbd only (GAL4DNAbd) served as the positive and negative controls, respectively. A measure of 25 ng of each vector was used for transient transfections. Results are means of three experiments s.d., and luciferase was assayed at 48 h. RLU values were normalized to the negative control (MEKK+GAL4DNAbd)

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As another means to verify that vFLIP can activate the JNK pathway, we performed an in vitro kinase assay for JNK as well as Western blotting for immunoprecipitated JNK and phosphorylated JNK. For these studies, we extracted total cellular protein from 293 cells transiently transfected with EGFP-vFLIP or the EGFP control vector. There was a significant increase in phosphorylation of JNK when EGFP-vFLIP was expressed compared to control transfected cells (Figure 3a); total JNK expression was equivalent in the two groups. For the in vitro kinase assay, we immunoprecipitated JNK and then assessed phosphorylation of a GST-jun substrate using a kit. Expression of EGFP-vFLIP led to phosphorylation of the GST-jun substrate compared to the EGFP control (Figure 3b), and thus indicates that vFLIP expression leads to activation of the JNK MAP kinase.

Figure 3.

vFLIP induces phosphorylation of JNK. (a) Immunoblots for phospho-JNK demonstrate that vFLIP leads to increased phosphorylation of JNK without affecting total JNK levels. EGFP-vFLIP or the EGFP control vector was transfected into 293 cells in 10 cm dishes. TPA treatment of 293 cells served as the positive control for JNK phosphorylation. Total protein was extracted and immunoprecipitated with GST-jun using a kit. The immunoprecipitated protein was then subjected to PAGE and immunoblotted with both phospho-JNK (top panel) and total JNK (bottom panel) antibodies. Total JNK was unaffected by any of the indicated treatments, and two different isoforms were detected by the anti-JNK antibody. In contrast, EGFP-vFLIP increased phospho-JNK levels in a dose-dependent fashion (one isoform of phosphorylated JNK was detected). Total protein extract prior to immunoprecipitation was subjected to immunoblotting for actin to document equivalent protein loading (not shown). (b) JNK in vitro kinase assay. EGFP-vFLIP or EGFP control vector was transiently transfected into 293 cells in 10 cm dishes, and total protein was extracted and subjected to immunoprecipitation on agarose beads conjugated to GST-jun. The immunoprecipitates were immunoblotted for total JNK (bottom panel) and demonstrate equivalent pull-down in all groups. The immunoprecipitates were subjected to a kinase reaction and then immunoblotted with an antibody to phospho-jun (top panel), which shows that EGFP-vFLIP induces phosphorylation of the jun transactivation domain compared to the EGFP control

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vFLIP induces binding of jun and fos to the AP1 RE

Many transcription factors, including the AP1 family of transcription factors (e.g. jun, fos, and ATF) and the cyclic-AMP response element binding protein (CREB), are capable of binding to the AP1 RE. We sought to identify specific transcription factors that are activated by vFLIP and subsequently bind to the AP1 RE. We performed electrophoretic mobility shift assays (EMSAs) with nuclear protein extracted from 293-TetOff-EGFP-vFLIP cells in which EGFP-vFLIP expression was either induced or suppressed by the absence or presence of doxycycline, respectively. The induction of an appropriately sized band representing the EGFP-vFLIP fusion protein is shown in Figure 4a. The EMSAs demonstrated that expression of EGFP-vFLIP resulted in the formation of an AP1 protein complex that retarded the electrophoretic mobility of the AP1 probe (Figure 4b). No gel shift was observed when expression of EGFP-vFLIP was suppressed with doxycycline or with nuclear protein from 293 cells transfected with EGFP only (Figure 4b). The specificity of the gel retardation was demonstrated in cold competition experiments. A 100-fold molar excess of the cold wild-type AP1 probe abrogated the band induced by vFLIP, whereas excess of the cold mutant AP1 probe had no effect (Figure 4b). To identify the specific proteins that were present in the shifted band, we preincubated nuclear extracts with anti-jun and -fos antibodies that interfere with the DNA binding of jun and fos, respectively. As shown in Figure 4b, the DNA binding of the vFLIP-induced complex was eliminated with the jun and fos antibodies, but not with an isotype control antibody. Thus, the AP1 protein complex induced by vFLIP is composed of jun and fos heterodimers. When we performed the EMSA in the BCBL-1 PEL cell line, which is endogenously infected with KSHV but not EBV, we determined that an AP1 complex which also consists of jun–fos heterodimers was constitutively present and bound to the AP1 probe (Figure 4c).

Figure 4.

EMSA demonstrates that vFLIP induces binding of jun–fos heterodimers to the AP1 RE. (a) Immunoblot with anti-EGFP antibody on nuclear protein extracts from 293-TetOff-EGFP-vFLIP cells, which display inducible expression of EGFP-vFLIP with doxycycline (1 g/ml). (b) EMSA with AP1 oligonucleotide on nuclear protein from 293-TetOff-EGFP-vFLIP cells in the presence or absence of a suppressive concentration of doxycycline. 293 cells expressing EGFP only do not exhibit gel shift of AP1 oligonucleotide, indicating that the EGFP moiety of the EGFP-vFLIP fusion is not responsible for gel shift. Cold competition experiments with cold wild-type or cold mutant AP1 oligonucleotide demonstrate specificity of binding induced by EGFP-vFLIP. Experiments with anti-jun, anti-fos, and isotype control antibodies show that EGFP-vFLIP induces binding of jun–fos heterodimers to the AP1 oligonucleotide. Note that the jun and fos antibodies bind to the DNA binding regions of jun and fos and consequently result in elimination of DNA binding of the protein complex rather than a supershifted band. (c) EMSA on nuclear extracts from the BCP-1 PEL cell line, which is infected with KSHV, demonstrates presence of jun–fos heterodimer that binds to the AP1 RE

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vFLIP activation of the JNK/AP1 pathway is dependent upon TRAF

As vFLIP physically interacts with TRAF2 (Chaudhary et al., 1999), and TNF activation of the JNK/AP1 pathway is mediated through TRAF2, we sought to establish that activation of the JNK pathway by vFLIP was dependent upon TRAFs. We transfected pAP1-luc, pEGFP-vFLIP, and either a TRAF2-dominant negative (TRAF2-DN) or the empty vector into 293 cells. The TRAF2-DN has a deletion of the N-terminal zinc ring finger and adjacent zinc-finger, which results in the inability of this TRAF2 deletion mutant to activate either the NF-B or JNK/AP1 pathways (Dadgostar and Cheng, 1998). The TRAF2-DN completely abrogated reporter gene expression (Figure 5a), indicating that vFLIP activation of JNK is TRAF-dependent. As the TRAF2-DN can inhibit all TRAF family members and not just TRAF2, we cannot conclude that activation of JNK by vFLIP is specifically dependent upon TRAF2.

Figure 5.

vFLIP activation of the JNK/AP1 pathway is dependent upon TRAF, and TRAF represents the bifurcation point of vFLIP activation of the NF-B and JNK/AP1 pathways. (a) 293 cells were transfected with pAP1-luc (10 ng), EGFP-vFLIP, or the EGFP control vector (10 ng), and a TRAF2-DN construct that inhibits NF-B and JNK/AP1 signaling or the empty vector control (100 ng). Experiments were performed in triplicate. Luciferase activity was assayed at 48 h and RLU were normalized to the vector control group. (b) Same as (a), but with TRAF2-B-DN (100 ng), which selectively inhibits the NF-B but not the JNK/AP1 pathway. Using the identical transient transfection conditions, the TRAF2-B-DN did block vFLIP-mediated activation of the B-RE (data not shown)

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Since there may be crosstalk between the NF-B and JNK pathways, it is conceivable that the inhibition of the JNK/AP1 pathway by the TRAF2-DN was a result of inhibition of the NF-B pathway and not a direct inhibition of upstream signaling proteins in the JNK pathway. To evaluate this possibility, we employed a second dominant-negative TRAF2 construct, (termed TRAF2-B-DN), which has a deletion of the N-terminal zinc ring finger only and is incapable of activating the NF-B pathway, but does activate the JNK/AP1 pathway (Dadgostar and Cheng, 1998). The TRAF2-B-DN did not inhibit vFLIP activation of AP1 in 293 cells (Figure 5b). The TRAF2-B-DN did block vFLIP-mediated activation of the B-RE (data not shown). Thus, inhibition of the NF-B pathway does not affect JNK/AP1 activation by vFLIP, and bifurcation of JNK and NF-B activation by vFLIP is at the level of TRAF. The experiments shown in Figure 6 were performed several times, and the results were similar each time except for variations in the relative luminescence in the control group.

Figure 6.

vFLIP activates the cIL-6 promoter. (a) 293 cells were transiently transfected with the IL-6 promoter reporter vector (pIL6-1200/SEAP, 25 ng) and the EGFP-vFLIP expression vector or the vector control (10 ng). Luciferase was assayed at 48 h. Results are reported as means of three experiments s.d., and RLU were normalized to the negative control group (EGFP vector). (b) Same as (a) but in R1T cells and dose-dependent effect of vFLIP on activation of the IL-6 promoter was assessed by increasing the amount of EGFP-vFLIP, while maintaining the total amount of DNA (EGFP-vFLIP+EGFP) constant. (c) The 293-TetOff-EGFP-vFLIP stable line was transiently transfected with pIL6-1200/SEAP (25 ng) in the presence or absence of a suppressive concentration of doxycycline (1 g/ml), and luciferase activity was assayed at 48 h. Results are means of three experiments s.d. and were normalized to the group with suppressed expression of EGFP-vFLIP (dox +)

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vFLIP activates the cIL-6 promoter

Activation of the NF-B and AP1 pathways can induce gene expression from the cIL-6 promoter (Ray et al., 1988; Libermann and Baltimore, 1990; Dendorfer et al., 1994). Given the established role of vFLIP in activating NF-B (Chaudhary et al., 1999) and our evidence that vFLIP activates the JNK/AP1 pathway, we investigated the ability of vFLIP to activate the cIL-6 promoter. We performed transient transfection studies in R1T and 293 cells. Transient cotransfection of our cIL-6 promoter construct (pIL6-1200/SEAP) with either pEGFP-vFLIP or the EGFP empty vector demonstrated that vFLIP induces reporter gene expression (Figure 6a, b). As further evidence that vFLIP activates the cIL-6 promoter, we transiently cotransfected pIL6-1200/SEAP into the 293-TetOff-EGFP-vFLIP cell line in the presence or absence of suppressive concentrations of doxycycline. Expression of EGFP-vFLIP resulted in augmentation of reporter gene expression from the cIL-6 promoter (Figure 6c). Thus, in both R1T and 293 cells, vFLIP functions to activate the cIL-6 promoter.

vFLIP activates the cIL-6 promoter in an NF-B- and AP1-dependent fashion

We next sought to investigate the roles of the NF-B and JNK/AP1 pathways in vFLIP induction of the cIL-6 promoter. We first confirmed that vFLIP activates the NF-B. vFLIP potently activated the B reporter construct (Figures 1d, 7b). As further confirmation of the ability of vFLIP to activate NF-B, we performed EMSAs on nuclear protein extracts from 293-TetOff-EGFP-vFLIP cells. These cells were grown in the presence or absence of suppressive concentrations of doxycycline, and nuclear protein was extracted. NF-B was clearly activated when EGFP-vFLIP was expressed, whereas no detectable NF-B activation was observed when EGFP-vFLIP expression was suppressed (Figure 7a). Cold competition experiments and nuclear protein from EGFP-expressing 293 cells confirmed the specificity of the reaction.

Figure 7.

vFLIP activation of the cIL-6 promoter is dependent upon both NF-B and AP1 activation. (a) EMSA demonstrates that vFLIP induces binding of a complex to the B RE. EMSA with B oligonucleotide on nuclear protein from 293-TetOff-EGFP-vFLIP cells in the presence or absence of a suppressive concentration of doxycycline. 293 cells expressing EGFP only do not exhibit gel shift of the B oligonucleotide, indicating that the EGFP moiety of the EGFP-vFLIP fusion is not responsible for gel shift. Cold competition experiments with cold wild-type or cold mutant NF-B oligonucleotide demonstrate specificity of binding induced by EGFP-vFLIP. (b) The IB-DA effectively blocks vFLIP-mediated NF-B activation, but has no effect on JNK/AP1 activation. Top panel: 293 cells were transiently transfected with EGFP-vFLIP or the EGFP control (10 ng), a B RE-driven reporter plasmid (10 ng), and the IB-DA expression plasmid or the corresponding control vector (100 ng). Luciferase was assayed at 48 h, experiments were performed in triplicate, and RLU were normalized to the value of the group containing control vectors only. Bottom panel: Same as top panel, but B RE driven reporter plasmid was replaced with the AP1 RE plasmid. (c) The jun-DN effectively blocks vFLIP-mediated JNK/AP1 activation, but has no effect on NF-B activation. Top panel: 293 cells were transiently transfected with EGFP-vFLIP or the EGFP control vector (10 ng), pAP1-luc (10 ng), and the jun-DN or the control (100 ng). Luciferase was assayed at 48 h, experiments were performed in triplicate, and RLU were normalized to the value of the group containing control vectors only. Bottom panel: Same as top panel, but pB-luc replaced the pAP1-luc reporter construct. (d) Inhibition of the NF-B pathway partially abrogates vFLIP-mediated activation of the cIL-6 promoter. 293 cells were transfected with pIL6-1200/SEAP (10 ng), EGFP-vFLIP, or the EGFP control (10 ng), and the IB-DA expression vector or the control vector (100 ng). SEAP activity was measured in culture supernatants at 48 h. Experiments were performed in triplicate and results normalized to the group containing the control vectors. (e) Same as (d), but jun-DN expression vector was used in place of the IB-DA vector. (f) 293-TetOff-EGFP-vFLIP cells were transfected with either the wild-type cIL-6 promoter or one in which point mutations in the AP1 RE were created (10 ng) in the presence or absence of suppressive concentrations of doxycycline (1 g/ml). SEAP activity was measured as in (d). (g) 293 cells were transfected with EGFP-vFLIP or EGFP control vectors (10 ng), pIL6-1200/SEAP (10 ng), and the IB-DA plus the jun-DN (100 ng each) or the control vector (200 ng total). SEAP measured and reported as in (d). (h) A TRAF2-DN blocks activation of the cIL-6 promoter by vFLIP. 293 cells were transfected as in (d), but the TRAF2-DN was employed in place of the IB-DA

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We studied the contribution of NF-B and AP1 activation to vFLIP-mediated cIL-6 induction. Our strategy was to transfect vFLIP and selectively inhibit NF-B activation with a dominant active I kappa B (IB-DA) construct and/or AP1 activation with a jun-dominant negative (jun-DN). The IB-DA contains mutations at the phosphorylation sites (Ser³² to Ala and Ser³⁶ to Ala), which prevent its phosphorylation, dissociation from NF-B, and subsequent degradation by the ubiquitin–proteasome pathway (Lee et al., 1999). The jun-DN construct contains an insertion at nucleotide 282 in the dimerization domain of c-jun, which results in a frame-shift downstream of the insertion (Raitano et al., 1995). The specificities of the IB-DA and jun-DN constructs were demonstrated as follows: the IB-DA completely inhibited vFLIP induction of the pB-luc plasmid (Figure 7b, top panel), but had no effect on activation of the JNK/AP1 pathway (Figure 7b, bottom panel), and the jun-DN inhibited vFLIP activation of the JNK/AP1 pathway (Figure 7c, top panel) but not the NF-B pathway (Figure 7c, bottom panel). We then studied the contribution of the NF-B and AP1 pathways to activation of the cIL-6 promoter, which was cloned as a 1200 bp region upstream of the transcription start site, as previously described (An et al., 2002). The IB-DA only partially inhibited vFLIP transactivation of cIL-6 promoter (pIL6-1200/SEAP) (Figure 7d), and similar results were obtained with the jun-DN (Figure 7e), suggesting that both the NF-B and AP1 pathways contribute to vFLIP-induced activation of the cIL-6 promoter. As further support for the role of the AP1 RE in vFLIP-induced activation of the cIL-6 promoter, we transfected the inducible 293-TetOff-EGFP-vFLIP cells in the presence or absence of suppressive concentrations of doxycycline with either the wild-type IL-6 promoter or one that contains point mutations of the AP1 RE (Figure 7f; An et al., 2002). The presence of the mutated AP1 RE led to partial abrogation of vFLIP-induced activation of the cIL-6 promoter (Figure 7f). When we blocked both the JNK/AP1 and NF-B pathways simultaneously with the JUN-DN and IkB-DA constructs, respectively, vFLIP activation of the wild-type cIL-6 promoter was completely inhibited, indicating that activation of both pathways is required for vFLIP induction of gene expression from the cIL-6 promoter (Figure 7g). As further evidence of the importance of the NF-B and JNK/AP1 pathways, we next examined the ability of vFLIP to activate the cIL-6 promoter in the presence or absence of the TRAF2-DN. Coexpression of EGFP-vFLIP with the TRAF2-DN, which blocks both the NF-B and JNK/AP1 activation, effectively blocked induction of the cIL-6 promoter (Figure 7h).

Figure 7.

(continued)

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vFLIP induces cellular IL-6 protein expression

We next sought to evaluate the potential for vFLIP to induce cIL-6 protein expression from the endogenous cIL-6 promoter. For these experiments, we used R1T cells, because they manifest endogenous cIL-6 expression, and the endogenous cIL-6 promoter is thus amenable to heightened stimulation. R1T cells were transiently transfected with the EGFP-vFLIP expression vector or the vector control, and supernatants were harvested at 48 h. cIL-6 expression in culture supernatants was significantly induced by EGFP-vFLIP, whereas the EGFP control had no effect on cIL-6 production (Figure 8). IL-2 and TNF- concentrations were unaffected by EGFP-vFLIP expression (data not shown), a finding that demonstrates that vFLIP-mediated IL-6 upregulation is not because of a generalized increase in protein expression.

Figure 8.

IL-6 ELISA showing that vFLIP induces cellular IL-6 expression in R1T cells. R1T cells were transiently transfected with EGFP-vFLIP or the EGFP control vector (0.5 g in six-well plate). Culture supernatants were harvested at 48 h; results represent means of two experiments

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Reciprocal potentiation of cIL-6 promoter activation by vFLIP and LANA

We have previously demonstrated that another KSHV-encoded latent protein, LANA, activates the cIL-6 promoter in an AP1-dependent fashion (An et al., 2002). Thus, given that vFLIP also activates the cIL-6 promoter through, at least in part, activation of the JNK/AP1 pathway, we examined the interactions between LANA and vFLIP on cIL-6 promoter activation. For these experiments, vFLIP was expressed as a red fluorescent protein tag (RFP-vFLIP) and LANA as an EGFP tag (EGFP-LANA). Using two different fluorescent tags allowed us to confirm transfection efficiency using appropriate UV filters. In cotransfection studies with pIL6-1200/SEAP in 293 cells, we kept the quantity of the EGFP-LANA construct constant and varied the amount of RFP-vFLIP. The total amount of DNA was held constant with the RFP empty vector. As shown in Figure 9a, increasing the amount of RFP-vFLIP resulted in reporter gene expression that was greater than the additive effect of the individual proteins. Moreover, reporter gene expression increased with amounts of RFP-vFLIP (0.5–10 ng) that did not transactivate the cIL-6 promoter by itself. Thus, vFLIP potentiates LANA-induced transactivation of the cIL-6 promoter. We performed reciprocal studies by keeping the amount of RFP-vFLIP constant and varying EGFP-LANA and obtained similar results (Figure 9b).

Figure 9.

Reciprocal potentiation of cIL-6 promoter activation by vFLIP and LANA. (a) The pIL6-1200/SEAP (25 ng) vector was cotransfected with a constant amount of EGFP-LANA or EGFP control vector (50 ng) and increasing amounts of RFP-vFLIP as indicated. Total DNA was held constant with the RFP empty vector. Luciferase was assayed at 48 h, and experiments were performed in triplicate. (b) Same as in (a), but the amount of RFP-vFLIP or RFP empty vector was held constant, and increasing amounts of EGFP-LANA were cotransfected

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Effects of vFLIP in endogenously infected PEL cells

To extend the biological context of the effects of vFLIP on the JNK/AP1 pathway and cIL-6 expression to a more relevant system, we investigated the biochemical effects of vFLIP in PEL cells. For this purpose, we employed RNA interference of vFLIP to post-transcriptionally silence vFLIP gene expression. We generated a small interfering RNA (siRNA) duplex that targets a specific region of the vFLIP mRNA and compared the effects of vFLIP siRNA to that of a scrambled control siRNA. We transfected siRNA duplexes into the BCBL-1 PEL cell line, which is endogenously infected with KSHV, but not EBV or other human herpesviruses. A specific gene silencing effect of vFLIP siRNA was assayed by Northern blotting for vFLIP, which demonstrated a decrease in the predominant 1.7 kb vFLIP transcript compared to the control siRNA, but no significant difference in the larger 5.8 kb message that also contains the LANA open reading frame (Figure 10a, left); Northern blot for GAPDH, a housekeeping gene, gave the same result for vFLIP and control siRNA (Figure 10a, left). Since LANA can also induce AP1 reporter activity (An et al., 2002) and is transcribed as a polycistronic message with vFLIP in the larger 5.8 kb transcript (Dittmer et al., 1998; Sarid et al., 1999), we determined whether vFLIP silencing could also affect expression of LANA. vFLIP siRNA had no effect on LANA expression as determined by Western blotting with a monoclonal antibody to LANA (Figure 10a, right panel). Next, we cotransfected BCBL-1 PEL cells with the AP1 or B reporter with the vFLIP or control siRNA and assessed reporter gene expression. As shown in Figure 10b, the vFLIP siRNA completely abrogated the reporter gene expression from the AP1 construct compared to the control siRNA.

Figure 10.

Effects of gene silencing of vFLIP in BCBL-1 PEL cells by RNA interference. (a) BCBL-1 cells were transfected with vFLIP or scramble (control) siRNA, and RNA was extracted at 48 h. Northern blot demonstrated reduction in the predominant 1.7 kb vFLIP message by vFLIP siRNA, but not scramble siRNA (control) or untreated cells (top, left panel). vFLIP siRNA did not affect expression of the GAPDH housekeeping gene (bottom, left panel) In addition. vFLIP siRNA did not affect expression of LANA as demonstrated by Western blot (top, right panel; 40 g of total protein loaded per lane); equal loading of protein was demonstrated by a Western blot for actin (bottom, right panel). (b) Gene silencing of vFLIP markedly reduces AP1 and B reporter gene expression. BCBL-1 cells were plated in 24-well plates and transfected with 60 pmol of vFLIP or control siRNA and the indicated reporter plasmids (1 g) with Lipofectamine 2000 (2 l) and the pRL-SV40 plasmid (1 ng) for normalization of transfection efficiency. A dual luciferase assay was performed on protein extracted at 48 h. Relative reduction in firefly luciferase expression because of vFLIP siRNA compared to scramble siRNA is reported. Results are means of three experiments reported as s.d. (c) JNK in vitro kinase assay showing decreased JNK activity with vFLIP siRNA. BCBL-1 cells (6 10⁷ cells) were plated in 10 cm dishes and transfected with vFLIP or scramble siRNA (1800 pmol per dish) with Oligofectamine (15 l). Protein was extracted at 48 h for JNK in vitro kinase assay. Experiments with siRNA were performed in triplicate; also shown are results from untreated cells and cells treated with Oligofectamine only. (d) cIL-6 expression is decreased upon vFLIP gene silencing with siRNA. BCBL-1 cells (2 10⁵) were plated in 24-well plates and transfected with siRNA (150 pmol) with Oligofectamine (1.5 l). Supernatants were harvested at 48 h for ELISA assays. Results are reported as the means of three experiments s.d. There were no significant differences in cIL-6 levels between scramble siRNA-transfected cells and untreated cells or cells treated with transfection reagent only (not shown)

Full figure and legend (89K)

In addition, the vFLIP siRNA also reduced B-regulated gene expression by approximately two-thirds compared to the control siRNA. As a negative control, we transfected a glucocorticoid responsive element (GRE) plasmid, which resulted in equivalent reporter gene expression in the presence of vFLIP or control siRNA. As the reduction in AP1 reporter activity could be attributed to effects on kinases other than JNK, we specifically assessed the effect of vFLIP siRNA on JNK activity. As measured by in vitro kinase activity, vFLIP siRNA potently inhibited JNK activity in BCBL-1 cells compared to the control siRNA and untreated cells (Figure 10c). In vitro kinase experiments in BCP-1 cells, another KSHV-infected PEL cell line, also demonstrated constitutive activation of JNK (data not shown). Given that vFLIP activates both the NF-B and JNK/AP1 pathways, we examined the effects of vFLIP gene silencing on cellular IL-6 expression in BCBL-1 supernatants. vFLIP siRNA significantly downregulated expression of cellular IL-6 expression, but not TNF-, compared to control siRNA and untreated cells (Figure 10d and data not shown).

Discussion

In this report, we have used both exogenous expression of vFLIP and inhibition of endogenous vFLIP in KHSV-infected PEL cells to show that the KSHV-encoded vFLIP activates the JNK/AP1 pathway, activates the cIL-6 promoter, and induces cellular IL-6 expression. In vFLIP transfection experiments, we demonstrated that maximal activation of the cIL-6 promoter by vFLIP is dependent upon both the NF-B and JNK/AP1 pathways, and vFLIP and another KSHV-encoded latent protein, LANA, potentiate each other's ability to activate the cIL-6 promoter. The ability of vFLIP to activate the JNK/AP1 pathway has not been previously described. In fact, one study reported that vFLIP did not induce gene expression from an AP1 responsive plasmid (Chaudhary et al., 1999). We believe that the discrepancy in these results may be attributable to the conditions of transient transfection used by the other investigators, who transfected 293 cells with up to 500 ng of the vFLIP expression plasmid and 75 ng of the AP1 responsive plasmid. When we utilized the same high concentrations of DNA as previously published, we also did not observe AP1-driven reporter gene expression induced by vFLIP. We optimized our transfection conditions and determined that optimal reporter gene expression in 293 cells requires far less DNA (10–20 ng of vFLIP and 10–25 ng of the AP1 responsive plasmid). In addition, the previously published study demonstrated activation of the NF-B pathway in reporter experiments; in those studies, vFLIP induced only approximately sevenfold activation of the B-driven reporter in 293 cells transfected with the larger quantities of DNA as described (Chaudhary et al., 1999). We found similar relative induction with vFLIP at those conditions (data not shown), yet, when we utilized smaller amounts of DNA (i.e. quantities that demonstrated vFLIP-induced activation of the AP1-driven reporter), we observed an approximately 100-fold activation of the B-driven reporter. Moreover, our transient transfection results that vFLIP can induce expression from an AP1-driven reporter construct were corroborated by other assays and in a bone marrow stromal cell line, as well as by RNA interference studies. Regardless of the quantity of DNA used in transient transfection studies, there appears to be greater sensitivity of the B reporter compared to the AP1 reporter in 293 cells. The explanation for this difference remains uncertain, although it is possible that 293 cells manifest constitutive activation of pathways that by themselves do not activate NF-B but function synergistically with vFLIP in this regard.

We also explored the mechanism of JNK/AP1 activation by vFLIP. TRAF2 represents the bifurcation point of TNFR activation of both the NF-B and AP1 pathways (Reinhard et al., 1997; Song et al., 1997). A TRAF2-DN construct that selectively inhibits the NF-B pathway did not reduce vFLIP activation of an AP1 responsive plasmid, indicating that components of the NF-B pathway are not involved in crosssignaling to the JNK/AP1 pathway. In contrast, a TRAF2-DN that is known to inhibit upstream signaling of both the NF-B and AP1 pathways effectively blocked vFLIP activation of an AP1-driven reporter construct. Thus, vFLIP-mediated activation of the NF-B and JNK pathways diverges at the level of TRAF, which is analogous to the role of TRAF2 in death receptor-induced signaling to these pathways.

We considered that our initial findings that vFLIP can activate the JNK/AP1 pathway and induce cIL-6 expression and the previous report that vFLIP activates the NF-B pathway (Chaudhary et al., 1999) in cell lines that are transfected with the vFLIP open reading frame may represent results emanating from artificial systems in which vFLIP is exogenously overexpressed. To address this possibility, we used RNA interference to inhibit endogenous vFLIP expression in BCBL-1 PEL cells, which are infected with KSHV and manifest basal vFLIP expression. Using RNA interference to post-transcriptionally silence vFLIP gene expression, we first demonstrated that vFLIP siRNA significantly reduced the expression of the predominant 1.7 kb vFLIP transcript, which contains the v-cyclin message, but not the larger 5.8 kb transcript, which also includes the LANA open reading frame (Figure 10a; Dittmer et al., 1998; Sarid et al., 1999). We used Western blotting to confirm that the vFLIP siRNA does not interfere with LANA expression (Figure 10a). This latter finding is particularly significant in the light of the fact that LANA functions to activate an AP1-driven reporter (An et al., 2002). A potential explanation for the lack of interference with the expression of the 5.8 kb message is that the smaller 1.7 kb message is considerably more abundant and may sequester the transfected vFLIP siRNA. Although we did not specifically address the effects of vFLIP siRNA on v-cyclin, there has been no empiric data or theoretical rationale to invoke v-cyclin as a modulator of JNK/AP1 or NF-B activity.

Our RNA interference studies in BCBL-1 cells also demonstrated that knockdown of vFLIP expression results in virtually complete abrogation of AP1-driven reporter gene expression and significant reduction in B-driven reporter gene activity (Figure 10b). The specificity of these findings was confirmed by the inability of vFLIP RNA interference to inhibit a GRE-driven reporter (Figure 10b) or reporter gene expression mediated by a basal promoter (data not shown). In addition, JNK in vitro kinase assays demonstrated that vFLIP gene silencing results in significantly reduced JNK activity. Finally, vFLIP RNA interference resulted in reduced cIL-6 protein expression, an expected finding given the known role of the JNK/AP1 and NF-B pathways in activating the cIL-6 promoter (Libermann and Baltimore, 1990; Dendorfer et al., 1994).

The cell biologic effects of vFLIP have been well studied, and vFLIP is an antiapoptotic protein. vFLIP prevents death receptor-induced FLICE (caspase 8) activation by blocking the interaction and autoactivation of FLICE with a death receptor–adaptor complex (Hu et al., 1997; Thome et al., 1997). The ability of vFLIP to inhibit FLICE activation results in inhibition of apoptosis in vitro (Hu et al., 1997; Thome et al., 1997). In immunocompetent mice, vFLIP prevents death receptor-induced apoptosis triggered by T cells (Djerbi et al., 1999). In addition, vFLIP activates the NF-B pathway, and inhibition of NF-B results in apoptosis of PEL cells, suggesting that vFLIP's antiapoptotic function may be partially attributable to NF-B activation (Chaudhary et al., 1999; Keller et al., 2000). Thus, vFLIP may function as a tumor progression factor by inhibiting apoptosis. Although the role of vFLIP as an antiapoptotic protein is well established, it has been reported that the vFLIP of the closely related herpesvirus saimiri is dispensable for transformation and pathogenicity of T cells in a New World primate model (Glykofrydes et al., 2000). However, this latter finding may not be applicable to the KSHV-encoded vFLIP, given that infected cell types in KSHV-associated diseases are not T cells, and the milieu of both cellular and viral proteins in KSHV-infected cells is apt to differ from that of T cells infected with herpesvirus saimiri.

In addition to its antiapoptotic effects, vFLIP manifests the ability to activate the cIL-6 promoter in an NF-B- and JNK/AP1-dependent fashion and induce cIL-6 protein expression. This biological effect of vFLIP may be crucial in tumor oncogenesis, given that cIL-6 represents an important growth factor for KSHV-associated neoplasms (Miles et al., 1990; Screpanti et al., 1996). Thus, vFLIP may be a tumor growth factor in part because of its ability to upregulate cIL-6. When we investigated the mechanisms responsible for vFLIP activation of the cIL-6 promoter, we found that both the NF-B and JNK pathways are involved in this activity. In addition, activation of both pathways is required for maximal activation of the cIL-6 promoter by vFLIP.

The significance of JNK/AP1 activation by vFLIP may extend beyond that of activation of the cIL-6 promoter. JNK/AP1 activation results in cellular proliferation in many cell systems. In contrast, JNK/AP1 activation may represent a proapoptotic stimulus in some cell systems (Butterfield et al., 1997), although the overriding antiapoptotic effects of vFLIP (via its inhibition of FLICE activation and possibly its ability to activate NF-B) abrogate any proapoptotic effects of JNK/AP1 activation. Since vFLIP activates the NF-B and AP1 family of transcription factors, vFLIP may induce expression of many genes through transactivation of the respective response elements, which are present in the promoters of many cytokines (e.g. the vascular endothelial growth factor promoter) and growth factors (Diaz et al., 2000).

As demonstrated by gel shift assay, vFLIP induces jun–fos heterodimers to bind to the AP1 RE in 293 cells, and jun–fos heterodimers are constitutively present in BCBL-1 cells. Activated JNK phosphorylates serine residues in the transactivation domain of c-jun and thereby enhances the transcriptional activity of c-jun (Smeal et al., 1991,1992). JNK, however, does not directly affect dimerization or DNA binding of c-jun. Thus, the formation of a jun–fos–AP1 RE complex must involve alternative biochemical pathways. Given that c-jun autoregulates its own expression via transactivation of the c-jun promoter, one possibility is that vFLIP, by enhancing the transcriptional activity of c-jun, upregulates transcription of the c-jun gene (Karin, 1994). Alternatively, vFLIP expression may result in enhanced DNA binding, which results from dephosphorylation of carboxy-terminal serine and threonine residues in the jun DNA binding domain. For example, vFLIP may directly or indirectly modulate the activity of protein phosphatase 2A, which is known to dephosphorylate the relevant carboxy-terminal serine and threonine residues involved in DNA binding (Black et al., 1991). Yet another explanation for the vFLIP-induced jun–fos–AP1 complex relates to the recent discovery that the short form of cellular FLIP induces c-fos expression independent of NF-B and JNK activation (Siegmund et al., 2001).

In our cotransfection experiments with vFLIP and LANA, we found that these proteins potentiate each other to activate the cIL-6 promoter. This may have particular significance, given that both proteins are expressed in latently infected cells and may, therefore, function together to induce expression of cIL-6 protein in vivo (Rainbow et al., 1997; Grundhoff and Ganem, 2001; Low et al., 2001). The mechanisms that underlie this potentiation of cIL-6 promoter activity are unknown, but may involve the ability of LANA to function as a transcriptional coactivator, as has been described for LANA and the retinoblastoma-E2F pathway (Radkov et al., 2000).

Materials and methods

Cells and reagents

Human embryonal kidney cells 293 cells were obtained from the American Type Culture Collection (Manassus, VA, USA) and maintained in modified Eagle's medium (MEM, Omega Scientific, Thousand Oaks, CA, USA) supplemented with 10% fetal bovine serum (FBS, Omega Scientific). 293 cells were chosen because they are semipermissive for KSHV infection (Foreman et al., 1997). For our studies, we also used an immortalized bone marrow stromal cell line, termed R1T, which was generated as previously described and maintained in IMDM plus 10% FBS (An et al., 2002). Bone marrow stromal cells endogenously express cIL-6 and complement the use of 293 cells, which lack cIL-6 expression. The BCBL-1 primary effusion lymphoma cell line was maintained in RPMI plus 10% FBS and antibiotics.

The anti-jun, -fos, and -actin antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti-EGFP antibody was from Clontech (Palo Alto, CA, USA). The anti-JNK and antiphospho-JNK antibodies were from New England Biolabs. The anti-LANA monoclonal antibody was from Novacastra Laboratories (Newcastle upon Tyne, UK). The JNK-DN, the TRAF2-DN constructs, and the IB-DA were kind gifts of Dr Genhong Cheng. The pAP1-luc, pB-luc, and pGRE-luc reporter plasmids were purchased from Clontech. The JNK-DN contains mutations at the phosphorylation sites (Thr¹⁸³ to Ala and Tyr¹⁸⁵ to Phe; Liu and Shuai, 2001). Two TRAF2-DN constructs were used for our studies: the first construct (termed TRAF2-DN) has a deletion of the N-terminal zinc ring finger and adjacent zinc-finger, which results in the inability of this TRAF2 deletion mutant to activate either the NF-B or JNK/AP1 pathways; the second dominant negative of TRAF2 (termed TRAF2-B-DN) has a deletion of the N-terminal zinc ring finger only, and is incapable of activating the NF-B pathway, but does activate the JNK/AP1 pathway (Dadgostar and Cheng, 1998). The IB-DA contains mutations at the phosphorylation sites (Ser³² to Ala and Ser³⁶ to Ala), which prevent its phosphorylation, dissociation from NF-B, and subsequent degradation by the ubiquitin–proteasome pathway (Lee et al., 1999). A jun-DN construct, a gift of Dr Charles Sawyers, contains an insertion at nucleotide 282 in the dimerization domain of c-jun, which results in a frame-shift downstream of the insertion (Raitano et al., 1995). The jun-DN open reading frame was subcloned into the pcDNA3.1 mammalian expression vector (Invitrogen, Carlsbad, CA, USA). The plasmids for the JNK in vivo signal transduction trans-reporting system were purchased from Stratagene (see Results). We generated an expression construct for EGFP-LANA as previously described (An et al., 2002). The p38 MAK kinase inhibitor SD203580 was obtained from BIOMOL Research Laboratories (Plymouth Meeting, PA, USA).

Cloning of vFLIP

Genomic DNA from the KSHV-positive PEL cell line, KS-1, was used as a template to PCR amplify vFLIP using the expand high fidelity DNA polymerase (Roche Molecular Biochemicals, Indianapolis, IN, USA). The following primers were used: forward 5'-TCTGGAATTCACCATGGCCACTTACGAGGT-3' and reverse 5'-GATGGGATCCCTATGGTGTATGGCGATAGTGT-3'; EcoRI and BamHI sites are underlined, and nonspecific nucleotides were positioned 5' of restriction sites to facilitate enzyme digestion. The amplification product was digested with EcoRI and BamHI and cloned into the pEGFP-C2 plasmid vector (Clontech). The construct. pEGFP-vFLIP, results in EGFP fused in-frame to the 5' end of vFLIP. We confirmed the orientation of selected clones by restriction analysis and the reading frame by partial sequencing. A similar strategy was used to clone the vFLIP open reading frame into pDs-Red-C1 (Clontech), which results in expression of a red fluorescent protein tag fused in-frame to the 5' end of vFLIP (RFP-vFLIP).

For tetracycline-regulated expression, the EGFP-vFLIP fusion gene was subcloned into the pTRE vector (Clontech). pEGFP-vFLIP was digested with NheI, treated with Klenow fragment to create blunt ends and subsequently digested with BamHI. The pTRE vector was prepared by EcoRI digestion, followed by blunt ending and then BamHI digestion. ORF73 was cloned as previously described (An et al., 2002).

Cloning of the cIL-6 promoter

The cloning of LANA and the cIL-6 promoter, including constructs that contain mutations of the AP1 RE, was previously described (An et al., 2002).

Transient transfections

R1T and 293 cells were plated at 10⁵ cells/well in 24-well plates the day prior to transfection. All plasmids were transfected with Lipofectamine Plus (Life Technologies) in serum-free medium according to the manufacturer's instructions. Transfection efficiency was determined by the percentage of EGFP-expressing cells as determined by cell counting with an inverted phase-contrast UV microscope (TS100-F, Nikon, Melville, NY, USA). Supernatants or cell lysates were harvested at 48 h for reporter gene expression.

Reporter gene assays

Luciferase assays were performed on cell lysates 48 h after transfection with a luciferase assay kit (Promega Corporation) according to the manufacturer's instructions. SEAP assays were performed on supernatants 48 h post-transfection with a SEAP assay kit (Clontech). For both luciferase and SEAP assays, luminescence was measured on the TD20/20 tube luminometer (Turner Designs, Sunnyvale, CA, USA). Results of reporter assays were normalized to the transfection efficiency results as described above for 293 and R1T cells. Firefly luciferase expression in BCBL-1 cells was normalized to that of Renilla luciferase using the Dual Luciferase Assay System (Clontech).

Stable cell lines

293 cells were transfected with pEGFP-C2 in 10 cm dishes with Lipofectamine Plus reagent. At 48 h after transfection, 800 g/ml of G418 (Gibco) was added for selection of stable transformants. Medium was changed every 4 days until stable transformants were observed. Colonies demonstrating green fluorescence were isolated with cloning cylinders and subsequently combined. Cells were maintained in 400 g/ml of G418.

The 293 Tet-Off cell line was purchased from Clontech. The 'Tet-Off' inducible mammalian expression system allows for regulated expression of a gene of interest by altering the concentration of tetracycline (or doxycycline), such that increasing antibiotic concentrations result in repression of transcription of the gene of interest. The EGFP-vFLIP fusion gene was cloned into the tetracycline-responsive vector pTRE, as described above. pTRE-EGFP-vFLIP was cotransfected with pTK-Hyg (to allow for antibiotic selection with hygromycin) into 293 Tet-Off cells in 10 cm dishes. At 48 h after transfection, cells were selected in 50 g/ml of hygromycin. Green fluorescing colonies were identified after 4 weeks, isolated and expanded. Colonies were tested for basal and induced expression of EGFP-vFLIP by immunoblotting with an anti-GFP antibody (Clontech). One colony with absent background and high induced expression of EGFP-vFLIP was chosen for future studies and was termed 293-TetOff-EGFP-vFLIP. These cells were maintained in 25 g/ml of hygromycin in 10% FBS (Tet-approved serum, Clontech).

Immunoprecipitation and JNK in vitro kinase assay

Immunoprecipitation of JNK and the JNK in vitro kinase assay were performed with a kit according to the manufacturer's instructions (Cell Signaling Technology, Beverly, MA, USA). Briefly, 200 g of total cellular protein was immunoprecipitated on GST-jun-agarose beads. The immunoprecipitated protein was then either subjected to immunoblotting with anti-JNK or antiphosphorylated-JNK antibodies or a kinase assay followed by immunoblotting with an antiphosphorylated-jun antibody.

Western blot

Protein was electrophoresed on a polyacrylamide gel. Protein was transferred to a nitrocellulose membrane with the Trans Blot system (Bio-Rad). The membrane was blocked with TBS with 1% bovine serum albumin and 1% nonfat powdered milk. Membranes were immunoblotted with relevant primary antibodies. Horseradish peroxidase-conjugated secondary antibodies (Santa Cruz) were used at 1 : 2000 concentration. Bands were identified by chemiluminescence (ECL Western blotting detection reagents, Amersham Biosciences, Piscataway, NJ, USA).

Cytokine assays

IL-6, TNF-, and interleukin-2 (IL-2) were measured on ELISA plates (R&D Systems, Minneapolis, MN, USA) for IL-6 and TNF- and Biotech Diagnostic, Laguna Niguel, CA, USA for IL-2) according to the manufacturer's instructions.

EMSA

Wild-type and mutant AP1 and B oligonucleotide probes were purchased from Santa Cruz Biotechnology. A 15 g weight of nuclear protein was combined with end-labeled, double-stranded oligonucleotide probe, 1 g of poly-dIdC (Amersham Pharmacia Biotech, Piscataway, NJ, USA), 1 g of BSA, and 5 mM spermidine in a final reaction volume of 20 l for 20 min at room temperature. The DNA–protein complex was run on a 4% nondenaturing polyacrylamide gel with 0.4 TBE running buffer prior to subsequent autoradiography. Cold-competition experiments were performed with a 100-fold molar excess of cold wild-type or cold mutant B or AP1 oligonucleotides. For supershift assays, nuclear protein was preincubated with 2 l of antibody for 20 min at RT.

RNA interference (RNAi)

siRNA (Dharmacon Research, Lafayette, CO, USA) for vFLIP with the following target sequence was used: 5'-AACGUGUUCAUACCUCAACCC-3'. A control siRNA termed Scramble II (target sequence: 5'-AAGCGCGCUUUGUAGGAUUCG-3') was also purchased from Dharmacon and has no significant homology to any known human or viral sequence. For reporter gene assays, 6 10⁵ BCBL-1 cells were plated in 24-well plates in 500 l of Opti-MEM (Invitrogen) without serum and antibiotics. Cells were cotransfected with reporter plasmids (1 g), including the pRLSV40 plasmid (1 ng) for normalization for transfection, and siRNA (60 pmol) with 2 l of Lipofectamine 2000 according to the manufacturer's instructions. At 5 h after transfection, medium was removed and replaced with Opti-MEM plus 10% FBS. At 48 h after transfection, protein was extracted for reporter gene assays. For ELISAs, siRNA (150 pmol) was transfected into BCBL-1 cells (2.5 10⁵ cells/well) with Oligofectamine (1.5 l; Invitrogen) according to the manufacturer's instructions, and supernatant was harvested at 48 h.

For the JNK in vitro kinase assays and Northern blots. 5 10⁷ BCBL-1 cells were plated in 4 ml of Opti-MEM without antibiotics or serum in a 10 cm dish. siRNA (1800 pmol) was transfected with 15 l of Oligofectamine. At 6 h after transfection, the medium was changed to Opti-MEM plus 10% FBS. Protein or RNA was extracted 48 h after transfection.

Northern blots

Total RNA (15 g) was electrophoresed on a 1% agarose gel and transferred to a nitrocellulose membrane, which was hybridized to a vFLIP probe encoding the entire open reading frame of ORFK13, the gene that encodes vFLIP, which was labeled by random priming. The membrane was subjected to autoradiography overnight.

References

1. An J, Lichtenstein AK, Brent G and Rettig MB. (2002). Blood, 99, 649–654. | Article | PubMed | ISI | ChemPort |
2. Asou H, Said JW, Yang R, Munker R, Park DJ, Kamada N and Koeffler HP. (1998). Blood, 91, 2475–2481. | PubMed | ISI | ChemPort |
3. Baud V, Liu ZG, Bennett B, Suzuki N, Xia Y and Karin M. (1999). Genes Dev., 13, 1297–1308. | PubMed | ISI | ChemPort |
4. Black EJ, Street AJ and Gillespie DA. (1991). Oncogene, 6, 1949–1958. | PubMed |
5. Butterfield L, Storey B, Maas L and Heasley LE. (1997). J. Biol. Chem., 272, 10110–10116. | Article | PubMed | ISI | ChemPort |
6. Cesarman E, Chang Y, Moore PS, Said JW and Knowles DM. (1995). N. Engl. J. Med., 332, 1186–1191. | Article | PubMed | ISI | ChemPort |
7. Chang Y, Cesarman E, Pessin MS, Lee F, Culpepper J, Knowles DM and Moore PS. (1994). Science, 266, 1865–1869. | Article | PubMed | ISI | ChemPort |
8. Chaudhary PM, Jasmin A, Eby MT and Hood L. (1999). Oncogene, 18, 5738–5746. | Article | PubMed | ISI | ChemPort |
9. Dadgostar H and Cheng G. (1998). J. Biol. Chem., 273, 24775–24780. | Article | PubMed | ISI | ChemPort |
10. Dendorfer U, Oettgen P and Libermann TA. (1994). Mol. Cell. Biol., 14, 4443–4454. | PubMed | ISI | ChemPort |
11. Diaz BV, Lenoir MC, Ladoux A, Frelin C, Demarchez M and Michel S. (2000). J. Biol. Chem., 275, 642–650. | Article | PubMed | ISI | ChemPort |
12. Dittmer D, Lagunoff M, Renne R, Staskus K, Haase A and Ganem D. (1998). J. Virol., 72, 8309–8315. | PubMed | ISI | ChemPort |
13. Djerbi M. Screpanti V, Catrina AI, Bogen B, Biberfeld P and Grandien A. (1999). J. Exp. Med., 190, 1025–1032. | Article | PubMed | ISI | ChemPort |
14. Ensoli B, Nakamura S, Salahuddin SZ, Biberfeld P, Larsson L, Beaver B, Wong-Staal F and Gallo RC. (1989). Science, 243, 223–226. | Article | PubMed | ISI | ChemPort |
15. Foreman KE, Friborg J, Kong WP, Woffendin C, Polverini PJ. Nickoloff BJ and Nabel GJ. (1997). N. Engl. J. Med., 336, 163–171.
16. Foussat A, Wijdenes J, Bouchet L, Gaidano G, Neipel F, Balabanian K, Galanaud P, Couderc J and Emilie D. (1999). Eur. Cytokine Netw., 10, 501–508. | PubMed | ISI | ChemPort |
17. Gao SJ, Kingsley L, Hoover DR, Spira TJ, Rinaldo CR, Saah A, Phair J, Detels R, Parry P, Chang Y and Moore PS. (1996). N. Engl. J. Med., 335, 233–241. | Article | PubMed | ISI | ChemPort |
18. Glykofrydes D, Niphuis H, Kuhn EM, Rosenwirth B, Heeney JL, Bruder J, Niedobitek G, Muller-Fleckenstein I, Fleckenstein B and Ensser A. (2000). J. Virol., 74, 11919–11927.
19. Grandadam M, Dupin N, Calvez V, Gorin I, Blum L, Kernbaum S, Sicard D, Buisson Y, Agut H, Escande JP and Huraux JM. (1997). J. Infect. Dis., 175, 1198–1201.
20. Grundhoff A and Ganem D. (2001). J. Virol., 75, 1857–1863.
21. Hu S, Vincenz C, Buller M and Dixit VM. (1997). J. Biol. Chem., 272, 9621–9624. | Article | PubMed | ISI | ChemPort |
22. Karin M. (1994). Curr. Opin. Cell Biol., 6, 415–424. | Article | PubMed | ISI | ChemPort |
23. Keller SA, Schattner EJ and Cesarman E. (2000). Blood, 96, 2537–2542. | PubMed | ISI | ChemPort |
24. Lee HH, Dadgostar H, Cheng Q, Shu J and Cheng G. (1999). Proc. Natl. Acad. Sci. USA, 96, 9136–9141. | Article | PubMed | ChemPort |
25. Leger-Ravet MB, Peuchmaur M, Devergne O, Audouin J. Raphael M, Van Damme J, Galanaud P, Diebold J and Emilie D. (1991). Blood, 78, 2923–2930. | PubMed | ChemPort |
26. Libermann TA and Baltimore D. (1990). Mol. Cell. Biol., 10, 2327–2334. | PubMed | ISI | ChemPort |
27. Liu B and Shuai K. (2001). J. Biol. Chem., 276, 36624–36631. | Article | PubMed | ISI | ChemPort |
28. Low W, Harries M, Ye H, Du MQ, Boshoff C and Collins M. (2001). J. Virol., 75, 2938–2945. | Article | PubMed | ChemPort |
29. Miles SA, Rezai AR, Salazar-Gonzalez JF, Vander MM. Stevens RH, Logan DM, Mitsuyasu RT, Taga T, Hirano T, Kishimoto T and Martinez-Maza O. (1990). Proc. Natl. Acad. Sci. USA, 87, 4068–4072. | Article | PubMed | ChemPort |
30. Moore PS and Chang Y. (1995). N. Engl. J. Med., 332, 1181–1185. | Article | PubMed | ISI | ChemPort |
31. Panyutich EA, Said JW and Miles SA. (1998). AIDS, 12, 467–472. | Article | PubMed | ISI | ChemPort |
32. Radkov SA, Kellam P and Boshoff C. (2000). Nat. Med., 6, 1121–1127. | Article | PubMed | ISI | ChemPort |
33. Rainbow L, Platt GM, Simpson GR, Sarid R, Gao SJ, Stoiber H, Herrington CS, Moore PS and Schulz TF. (1997). J. Virol., 71, 5915–5921. | PubMed | ISI | ChemPort |
34. Raitano AB, Halpern JR, Hambuch TM, and Sawyers CL. (1995). Proc. Natl. Acad. Sci. USA, 92, 11746–11750. | Article | PubMed | ChemPort |
35. Ray A, Ratter SB, May LT and Sehgal PB. (1988). Proc. Natl. Acad. Sci. USA, 85, 6701–6705. | Article | PubMed | ChemPort |
36. Reinhard C, Shamoon B, Shyamala V and Williams LT. (1997). EMBO J., 16, 1080–1092. | Article | PubMed | ISI | ChemPort |
37. Sarid R, Flore O, Bohenzky RA, Chang Y and Moore PS. (1998). J. Virol., 72, 1005–1012. | PubMed | ISI | ChemPort |
38. Sarid R, Wiezorek JS, Moore PS and Chang Y. (1999). J. Virol., 73, 1438–1446. | PubMed |
39. Screpanti I, Musiani P, Bellavia D, Cappelletti M, Aiello FB. Maroder M, Frati L, Modesti A, Gulino A and Poli V. (1996). J. Exp. Med., 184, 1561–1566. | Article | PubMed | ISI | ChemPort |
40. Siegmund D, Mauri D, Peters N, Juo P, Thome M, Reichwein M, Blenis J, Scheurich P, Tschopp J and Wajant H. (2001). J. Biol. Chem., 276, 32585–32590. | Article | PubMed | ISI | ChemPort |
41. Smeal T, Binetruy B, Mercola D, Grover-Bardwick A, Heidecker G, Rapp UR and Karin M. (1992). Mol. Cell. Biol., 12, 3507–3513. | PubMed | ISI | ChemPort |
42. Smeal T, Binetruy B, Mercola DA, Birrer M and Karin M. (1991). Nature, 354, 494–496. | Article | PubMed | ISI | ChemPort |
43. Song HY, Regnier CH, Kirschning CJ, Goeddel DV and Rothe M. (1997). Proc. Natl. Acad. Sci. USA, 94, 9792–9796. | Article | PubMed | ChemPort |
44. Soulier J, Grollet L, Oksenhendler E, Cacoub P, Cazals-Hatem D, Babinet P, d'Agay MF, Clauvel JP, Raphael M and Degos L. (1995). Blood, 86, 1276–1280. | PubMed | ISI | ChemPort |
45. Talbot SJ, Weiss RA, Kellam P and Boshoff C. (1999). Virology, 257, 84–94.
46. Thome M, Schneider P, Hofmann K, Fickenscher H, Meinl E, Neipel F, Mattmann C, Burns K, Bodmer JL, Schroter M, Scaffidi C, Krammer PH, Peter ME and Tschopp J. (1997). Nature, 386, 517–521. | Article | PubMed | ISI | ChemPort |

Acknowledgements

We thank Drs Alan K Lichtenstein and Gregory Brent for their critical review of the manuscript and helpful discussions. Supported by research funds of the Veterans Administration, including a Career Development Award and the VA Research Enhancement Award Program (REAP) (both to MBR), the Jonsson Comprehensive Cancer Center at UCLA (to MBR and RS), the American Cancer Society (Grant # RPG-00-305-01-MBC to MBR), the Public Health Service (Grant # 1R01CA80004-01A1 to MBR), and the American Society of Hematology (Junior Faculty Scholar Award to MBR).