The human herpes virus 8-encoded viral FLICE inhibitory protein physically associates with and persistently activates the Ikappa B kinase complex.

The human herpesvirus 8 (HHV8, also called Kaposi's sarcoma-associated herpesvirus) has been linked to Kaposi's sarcoma and primary effusion lymphoma (PEL) in immunocompromised individuals. We demonstrate that PEL cell lines have a constitutively active NF-kappaB pathway, which is associated with persistent phosphorylation of IkappaBalpha. To elucidate the mechanism of NF-kappaB activation in PEL cell lines, we have investigated the role of viral FLICE inhibitory protein (vFLIP) in this process. We report that stable expression of HHV8 vFLIP in a variety of cell lines is associated with persistent NF-kappaB activation caused by constitutive phosphorylation of IkappaBalpha. HHV8 vFLIP gets recruited to a approximately 700-kDa IkappaB kinase (IKK) complex and physically associates with IKKalpha, IKKbeta, NEMO/IKKgamma, and RIP. HHV8 vFLIP is incapable of activating NF-kappaB in cells deficient in NEMO/IKKgamma, thereby suggesting an essential role of an intact IKK complex in this process. Our results suggest that HHV8 vFLIP might contribute to the persistent NF-kappaB activation observed in PEL cells by associating with and stimulating the activity of the cellular IKK complex.

The human herpesvirus 8 (HHV8, also called Kaposi's sarcoma-associated herpesvirus) has been linked to Kaposi's sarcoma and primary effusion lymphoma (PEL) in immunocompromised individuals. We demonstrate that PEL cell lines have a constitutively active NF-B pathway, which is associated with persistent phosphorylation of IB␣. To elucidate the mechanism of NF-B activation in PEL cell lines, we have investigated the role of viral FLICE inhibitory protein (vFLIP) in this process. We report that stable expression of HHV8 vFLIP in a variety of cell lines is associated with persistent NF-B activation caused by constitutive phosphorylation of IB␣. HHV8 vFLIP gets recruited to a ϳ700-kDa IB kinase (IKK) complex and physically associates with IKK␣, IKK␤, NEMO/IKK␥, and RIP. HHV8 vFLIP is incapable of activating NF-B in cells deficient in NEMO/ IKK␥, thereby suggesting an essential role of an intact IKK complex in this process. Our results suggest that HHV8 vFLIP might contribute to the persistent NF-B activation observed in PEL cells by associating with and stimulating the activity of the cellular IKK complex.
Nuclear factor B (NF-B) 1 is a heterodimeric transcription factor that is primarily composed of 50-and 65-kDa subunits of the Rel family and that is required for regulated expression of several genes involved in inflammation and immune response (1)(2)(3). NF-B is present in the cytoplasm of cells in association with a family of inhibitory proteins, called IB (1,4). IB proteins retain NF-B in the cytoplasm by masking its nuclear localization signal. Stimulation by a number of cytokines, such as TNF␣ and interleukin-1, results in the activation of a large molecular mass (600 -900 kDa), IB kinase complex that leads to inducible phosphorylation of the IB proteins at two Nterminal serine residues (5,6). This complex consists of two catalytic subunits, IKK␣ (IKK1) and IKK␤ (IKK2), and a regulatory subunit, NEMO/IKK␥ (7)(8)(9)(10)(11)(12)(13). The phosphorylation of IB proteins results in their rapid ubiquitination and proteasome-mediated degradation, which releases NF-B from their inhibitory influence. Once released, NF-B is free to migrate to the nucleus and activate transcription of its target genes.
Some of the noteworthy genes activated by NF-B include those for cytokines and growth factors, chemokines, cell adhesion molecules, acute phase proteins, anti-apoptotic proteins, and transcription factors p53 and c-Myc (2). The NF-B pathway has been also shown to play a key role in the control of cell proliferation and oncogenesis. Several members of the NF-B family have been associated with the development of tumors as a result of overexpression, gene amplification, or gene rearrangement (14). Activation of the NF-B has been shown to be responsible for the transforming ability of human T-cell leukemia virus type I Tax and Epstein-Barr virus latent membrane protein 1 (15). Caspase 8 (FLICE/MACH or Mch5) is one of the apical caspases of the caspase cascade, which is activated by signaling via the death receptors belonging to the TNF receptor family (16 -18). Caspase 8 is recruited to the multimerized deathinducing signaling complex of these receptors via its N-terminal prodomain, which contains two homologous copies of a death effector domain. Death effector domain-containing prodomains are also found in two additional cellular proteins: caspase 10 (Mch4 and FLICE2) (18,19), and MRIT (c-FLIP, Caspar, I-FLICE, FLAME, CASH, and CLARP), a caspase 8 homolog that is devoid of protease activity (20 -26).
We and others have previously demonstrated that overexpression of HHV8 vFLIP can protect against death receptorinduced apoptosis in vitro and to promote tumor growth in vivo (33)(34)(35). Furthermore, unlike MC159L and E8, HHV8 vFLIP was found to activate the NF-B pathway when overexpressed in 293T and NIH3T3 cells by transient transfection (33). The present study was undertaken to better understand the mechanism of NF-B activation by HHV8 vFLIP.
Cell Culture and Protein Extraction-Human non-small cell lung cancer cell line H460 (a kind gift of Dr. John Minna) was cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum and penicillin/streptomycin. Murine pre-B-cell line 70Z/3 and its NEMOdeficient mutant 1.3E2 (a kind gift of Dr. Carol Sibley) were cultured in RPMI 1640 medium supplemented with 7% fetal bovine serum and 50 M ␤-mercaptoethanol. Retrovirus-infected H460 cells were maintained in medium with 1000 g/ml of G418, whereas clones of 70Z/3 and 1.3E2 cells were selected in 300 g/ml of G418 (Invitrogen).
To obtain cytoplasmic proteins, the cells were washed with cold phosphate-buffered saline (pH 7.2), resuspended in buffer C (10 mM HEPES, pH 7.6, 0.1 mM EDTA, 10 mM KCl, 1 mM DTT, 50 mM NaF, 50 mM ␤-glycophosphate, 5% glycerol, 1ϫ protease inhibitor mixture (Roche Molecular Biochemicals)), and incubated on ice for 15 min. At the end of incubation, 1:20 volume of 10% Nonidet P-40 was added. The cells were vortexed for 30 s and then subjected to centrifugation for 30 s. The supernatants were collected as cytoplasmic extracts. The protein concentrations of the cytoplasmic extracts were determined by using Bio-Rad protein assay reagent.
To prepare whole cell extracts, the cells were washed with cold phosphate-buffered saline twice and lysed in lysis buffer containing 20 mM Tris-HCl, pH 8.0, 500 mM NaCl, 0.25% Triton X-100, 1 mM EDTA, 10 mM ␤-glycophosphate, 10 mM NaF, 1 mM DTT, 1ϫ protease inhibitor mixture (Roche Molecular Biochemicals) at 4°C for 30 min. After incubation, the mixture was pippetted five or six times to disperse the cells followed by centrifugation at 14,000 rpm at 4°C for 10 min. The supernatants were collected as whole cell extracts, and protein concentration was determined as described above.
Electrophoretic Mobility Shift Assay-10 g of each nuclear extract sample was incubated with 0.1 pmol of 32 P-labeled double-stranded B binding oligonucleotide (5Ј-GCTGGGGACTTTC-3Ј) or SP1 binding oligonucleotide (5Ј-ATTCGATCGGGGCG GGGCGAGC-3Ј) in buffer containing 1 g of poly(dI-dC), 1 g of bovine serum albumin, 10 mM HEPES, pH 7.6, 0.5 mM DTT, 0.1 mM EDTA, 60 mM KCl, 0.2 mM PMSF, 5 mM MgCl 2 , and 12% glycerol at room temperature for 30 min. The samples were analyzed by 5% native PAGE followed by autoradiography. For competition and antibody-mediated supershift experiments, B-specific or -nonspecific oligonucleotides or specified antibodies were added to reaction for 15 min at room temperature before the addition of radio isotope-labeled B probe.
Immunoprecipitation of vFLIP or MRIT/cFLIP-Immobilized monoclonal antibody against FLAG (M2, Sigma) or mouse IgG-Sepharose beads as control was added to cellular extracts (4 mg) prepared from virus-infected H460 cells and incubated at 4°C for 1 h. In the case of immunoprecipitation with ␣-NEMO antibody, 10 l of soluble antibody were added to cellular extracts for 30 min on ice. Protein G-Sepharose Cl-4B were then added to the mix, and incubation was performed at 4°C for 1 h. The beads were washed three times with buffer containing 40 mM HEPES, pH 7.9, 500 mM NaCl, 0.2 mM EDTA, 1 mM DTT, 10 mM NaF, 10 mM ␤-glycophosphate, and 0.1% Nonidet P-40. In vitro kinase assay or Western blotting experiments were then performed.
In Vitro Kinase Assay-Beads from the immunoprecipitation experiments were used to incubate with soluble GST-IB␣ proteins (10 g/ reaction) in buffer containing 20 mM HEPES, pH 7.6, 100 mM KCl, 10% glycerol, 0.2 mM EDTA, 0.5 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 8 M MgCl 2 , 3.3 g/l bovine serum albumin, 4 M ATP, 10 Ci of [␥-32 P]ATP, 5 mM NaF, 5 mM ␤-glycophosphate, and protease inhibitor mixture (Roche Molecular Biochemicals) at 37°C for 30 min. 30 l of reaction solution was added to each sample. 10 l of 4ϫ SDS-PAGE sample buffer was added at the end of reaction, and the mixture was heated at 95°C for 5 min and pelleted by centrifugation. The supernatants were then resolved on SDS-12% polyacrylamide gels followed by autoradiography. For in-solution kinase assay, protein samples (10 l each of fractions from Superdex 200 column) were incubated with soluble GST-IB␣ protein in the same reaction conditions mentioned above.

Constitutive Activation of NF-B in HHV8-infected PEL Cell
Lines Is Due to Persistent Activation of the IKK Complex-NF-B is normally sequestered in the cytoplasm of cells because of its association with a family of inhibitory proteins, called IB (1, 4). However, NF-B is persistently present in the nuclei of human T-cell leukemia virus type I-and Epstein-Barr virus-transformed cells and has been shown to contribute to the transforming ability of these viruses (15). We were interested in checking whether infection by HHV8 virus might also lead to persistent NF-B activation. To test this hypothesis we used electrophoretic mobility shift assay (EMSA) to examine the DNA binding activity of nuclear NF-B in three HHV8infected PEL cell lines, BC-1, BC-3, and BCBL, respectively. We also used two non-HHV8-infected lymphoid cell lines, CEM and Jurkat, respectively, as controls for the above experiment. Consistent with a recent report (38), persistent NF-B activation was seen in all three HHV8-infected PEL cell lines, with the BC-1 cell line showing the highest and BCBL cell line showing the least NF-B activation. In contrast, NF-B binding activity was absent in the nuclear extracts of CEM and Jurkat cell lines (Fig. 1A).
We next sought to analyze the nature and composition of the observed complex in the BC-1 cell line. As shown in Fig. 1B, the observed complex in the BC-1 cell line could be effectively competed with an excess cold probe containing B binding sites but was unaffected by competition with a nonspecific DNA duplex. Finally, a supershift assay utilizing subunit-specific antibodies demonstrated that the observed complex contained the p65 and p50 subunits of NF-B (Fig. 1B).
To determine the mechanism of persistent NF-B activation in PEL cell lines, we examined the phosphorylation status of IB␣ protein by Western blot analysis. Consistent with the EMSA results, phosphorylation of IB␣ was totally absent in CEM and Jurkat cells (Fig. 1C). In contrast, phosphorylation of IB␣ was readily detected in all three PEL cell lines and was present in the following order of magnitude: BC-1 Ͼ BC-3 Ͼ BCBL (Fig. 1C). Of interest, persistent phosphorylation of IB␣ observed in BC-1 and BC-3 cell lines was significantly stronger than the TNF-induced IB␣-phosphorylation observed in Jurkat cells. Reprobing of the above blot with an IB␣ antibody revealed a decrease in the level of total IB␣ protein in the PEL cell lines (Fig. 1C). The above results suggest that the constitutive NF-B activation in the PEL cell lines is probably due to persistent phosphorylation and subsequent degradation of the IB␣ protein.
Retroviral-mediated Expression of HHV8 vFLIP Leads to Persistent NF-B Activation-We have previously demonstrated that transient transfection-based overexpression of HHV8 vFLIP can lead to NF-B activation (33). We were interested in determining whether the constitutive NF-B activation observed in PEL cell lines might be mediated by HHV8 vFLIP. To test this hypothesis, we began by checking whether stable expression of HHV8 vFLIP can lead to constitutive NF-B activation. For this purpose, we used retroviral-mediated gene transfer to generate mass culture of H460 cells with stable expression of FLAG epitope-tagged HHV8 vFLIP or MRIT/cFLIP. As shown in Fig. 2A, stable expression of HHV8 vFLIP in H460 cells lead to strong NF-B binding activity as measured by gel shift assay. In contrast, cells expressing an empty vector or MRIT/cFLIP demonstrated low level basal NF-B binding activity. The specificity of the NF-B complex seen in the vFLIP-expressing cells was confirmed by competition with a cold NF-B probe or a nonspecific probe ( Fig. 2A). A supershift assay utilizing subunit specific antibodies demonstrated that the complex was a heterodimer of p65 and p50 subunits of NF-B ( Fig. 2A). Essentially similar results were obtained upon stable expression of HHV8 vFLIP in 293T and TF-1 cells (Fig. 2B and data not shown). Finally, expression of vFLIP in 293T and H460 cells was associated with an increase in NF-B transcriptional activity as measured by a luciferasebased reporter assay ( Fig. 2C and data not shown).
Next, we examined the status of total and phosphorylated IB␣ in H460 cells expressing empty vector, HHV8 vFLIP, or MRIT/cFLIP. Consistent with previous results with the PEL cell lines, expression of HHV8 vFLIP was associated with a decrease in the steady-state level of total IB␣ and an increase in its phosphorylated form (Fig. 2D). Taken together, the above results suggest that HHV8 vFLIP leads to persistent NF-B activation by constitutive phosphorylation of IB␣.
HHV8 vFLIP Complex Possesses IKK Activity-To test the possibility that HHV8 vFLIP leads to persistent IB␣ phosphorylation by interacting with and activating the IKK complex, FLAG-tagged HHV8 vFLIP was immunoprecipitated from the cytosolic extracts of H460-vFLIP cells using FLAG antibody beads and assayed for the IB␣ kinase activity in an in vitro kinase reaction using GST-IB␣ and [␥-32 P]ATP. Parallel experiments utilizing a nonspecific antibody or an antibody against NEMO/IKK␥ served as negative and positive controls. As shown in Fig. 3A, immunoprecipitate of vFLIP with the FLAG antibody was able to phosphorylate GST-IB␣, whereas an immunoprecipitate using a control antibody failed to do so. Similarly, immunoprecipitates of MRIT/cFLIP-or empty vector-expressing cells failed to phosphorylate GST-IB␣ (Fig. 3A,  top and bottom panels). The vFLIP-associated IKK activity was specific for Ser-32 and Ser-36 of IB␣, because it failed to phosphorylate a GST-IB␣ mutant substrate in which both the above residues were mutated to alanine (Fig. 3A, middle panel). Collectively, these results suggest that HHV8 vFLIP associates with a cytosolic complex that possesses IKK activity.
Components of HHV8 vFLIP-associated IKK Complex-To determine the components of the HHV8 vFLIP-associated IKK activity, coimmunoprecipitation experiments were carried out. FLAG-tagged vFLIP and MRIT/cFLIP were immunoprecipitated using a FLAG monoclonal or a control mouse antibody, and the nature of the coimmunoprecipitated proteins was determined by Western analysis. As shown in Fig. 4A, IKK␣, IKK␤, and NEMO/IKK␥ readily coimmunoprecipitated with vFLIP but were not detected in the immunoprecipitate of MRIT/cFLIP. In contrast, RIP was detected in the immunoprecipitates of both vFLIP and MRIT/cFLIP.
HHV8 vFLIP Physically Interacts with an ϳ700-kDa IKK Signalsome Complex-Previous studies have demonstrated that cytokine-induced IKK activity is present in a multiprotein signalsome complex of ϳ700 kDa (5, 6). To determine whether HHV8 vFLIP stimulates IKK activation by interacting with this large molecular mass complex, we compared the chromatographic distribution of vFLIP in extracts prepared from H460-vFLIP cells. A parallel experiment with cellular extracts prepared from H460-MRIT/cFLIP cells served as a negative control. Following Superdex-200 fractionation of the above extracts, the column fractions were immunoprecipitated with FLAG (M2) monoclonal antibody. The immunoprecipitate was subsequently used for Western analysis with a rabbit polyclonal antibody against the FLAG tag to detect the presence of FLAG-tagged vFLIP or MRIT/cFLIP as well as in an in vitro kinase assay using GST-IB␣ as a substrate. As shown in Fig.  5, A and B, the majority of vFLIP was found migrating between 600 and 700 kDa, which also correlated with the fraction containing the IKK activity. In contrast, MRIT/cFLIP was found migrating between 443 and 200 kDa (Fig. 5A).
We also examined the distribution of NEMO/IKK␥ in the cell extracts prepared from vFLIP-and MRIT/cFLIP-expressing cells. Although the majority of NEMO/IKK␥ was found migrating between 600 and 700 kDa, a smaller peak migrating near 450 kDa was detected in both cell lines (Fig. 6, A and B). As compared with MRIT-expressing cells, a relatively larger amount of NEMO/IKK␥ in vFLIP-expressing cells was found in the ϳ700-kDa fraction. Because this fraction has been previously shown to contain the IKK activity (5, 6), these results suggest that expression of vFLIP leads to incorporation of NEMO/IKK␥ into a constitutively active high molecular mass IKK complex. Finally, we examined the elution profile of IKK␣ and IKK␤ in the cell extracts prepared from vFLIP-expressing cells using Western blot analysis. As shown in Fig. 6C, both of the above kinases were found to coelute with vFLIP in the column fractions 8 -10, thereby demonstrating that vFLIP coelutes with both the catalytic and regulatory subunits of the IKK complex.
Because the majority of vFLIP and the IKKs were detected in the same elution fractions, we next examined whether they are physically associated with each other. For this purpose, FLAGtagged vFLIP or MRIT/cFLIP present in the various column fractions obtained following Superdex-200 fractionation were immunoprecipitated using the M2 FLAG antibody, and the presence of any associated NEMO/IKK␥ was detected using Western blot analysis. As shown in Fig. 7A, NEMO/IKK␥ was found to coimmunoprecipitate with vFLIP in the column fractions 8 -10, suggesting that the two proteins not only comigrate but are the components of the same IKK signalsome complex. Similarly, both IKK␣ and IKK␤ were found to coimmunoprecipitate with vFLIP (see below). Consistent with previous results, no NEMO/IKK␥ was found to coimmunoprecipitate with MRIT/cFLIP (Fig. 7B).
We next examined the proportion of the IKKs that associate with vFLIP in various column fractions. For this purpose, various column fractions were immunoprecipitated with M2 FLAG antibody beads, and the amount of IKKs found to coimmunoprecipitate with the beads and the unbound fraction remaining in the supernatant was examined by Western blot analysis. As shown in Fig. 7C, significant proportions of IKK␣, IKK␤, and NEMO/IKK␥ were found to coimmunoprecipitate with vFLIP in the column fractions 8 -10. A densitometry analysis revealed that 27 and 38% of IKK␣, 36 and 42% of IKK␤, and 34 and 51% of NEMO/IKK␥ were associated with vFLIP in the column fractions 8 and 9, respectively. NEMO/IKK␥ Is Essential for HHV8 vFLIP-induced NF-B-We were next interested in demonstrating that the IKK complex not only associates with HHV8 vFLIP but is also essential for its ability to activate NF-B. For this purpose we took advantage of the murine pre-B-cell lines 70Z/3 and 1.3E2, respectively. The 1.3E2 cell line is a NEMO-deficient mutant of 70Z/3 cells and has been previously shown to be incapable of activating NF-B in response to multiple stimuli (39). We used retroviral-mediated gene transfer to generate stable clones of the above cell lines expressing empty vector  (Fig. 8D) and the lack of NEMO expression in 1.3E2 cells (Fig. 8C). DISCUSSION KS is the most common malignancy found in the patients with HIV infection. The isolation of a novel gamma herpesvirus, designated HHV8, as a potential etiological agent for KS was a major step in understanding the pathogenesis of KS (40). HHV8 genomes have also been consistently found in patients with PEL, also known as body cavity-associated lymphoma, a rare form of B-cell lymphoma characterized by malignant pleural, pericardial, or peritoneal effusion in the absence of a tumor mass (41). In addition to KS and PEL, HHV8 genome has been detected in multicentric Castleman's disease, angioimmunoblastic lymphadenopathy, and some cases of reactive lymphadenopathies (42)(43)(44).
Despite the increasing evidence linking the presence of KSassociated herpesvirus/HHV8 with KS and lymphoproliferative disorders, the mechanism by which this virus leads to a transformed phenotype is still unknown. In the present study, we have demonstrated that IB␣ is persistently phosphorylated in the PEL cell lines and is associated with constitutive NF-B activation in these cells. Because constitutive NF-B activation has been previously implicated in cellular transformation seen in association with infection by Epstein-Barr virus and human T-cell leukemia virus type I (15), it may play a causative role in the pathogenesis of KS and HHV8-associated lymphoproliferative disorders as well.
We have discovered that stable expression HHV8 vFLIP in both hematopoietic and nonhematopoietic cell lines can lead to constitutive NF-B activation. We would like to point out that although we have used a retroviral vector to express vFLIP, it did not result in the expression of abnormally high levels of this protein. On the contrary, vFLIP protein was undetectable in the cellular lysates using a highly sensitive Western blot analysis and could be detected only after severalfold concentration of the protein by immunoprecipitation. Furthermore, PEL cell lines are known to harbor multiple copies (50 -100) of HHV8 genome (41,45). Therefore, taken together, it is highly unlikely that the NF-B activation observed in the present study is due to expression of abnormally high and supraphysiological levels of vFLIP protein. In addition to vFLIP, two other HHV8-encoded proteins have been shown to lead to NF-B activation, i.e. K1 and viral G-protein-coupled receptor (vGPCR), respectively (46 -50). However, among these proteins, only vFLIP is expressed in latently infected KS spindle and PEL cells (51)(52)(53)(54), making it a prime candidate for the constitutive NF-B activation observed in the PEL cell lines.
Our study suggests that persistent NF-B activation seen in the PEL cell lines is due to constitutive phosphorylation of IB␣, a feature also seen in vFLIP-expressing cells. Inducible phosphorylation of IB␣ at Ser-32 and Ser-37 followed by its destruction by the ubiquitin-proteasome-dependent pathway is a known mechanism for NF-B activation by cytokines, such as TNF and interleukin-1. This signal-dependent phosphorylation of IB␣ has been shown to be mediated by the activation of a ϳ700-kDa signalsome complex comprising IKK␣, IKK␤, and IKK␥/NEMO (7)(8)(9)(10)(11)(12)(13). Our results suggest that HHV8 vFLIP leads to constitutive NF-B activation by associating with this high molecular mass complex. In this regard, HHV8 vFLIP may resemble human T-cell leukemia virus type I Tax protein, which has been also shown to lead to NF-B activation by associating with and persistently activating the IKK complex (55)(56)(57)(58)(59)(60).
The mechanism by which interaction of HHV8 vFLIP with the IKK complex results in persistent increase in IKK activity remains to be determined. It is conceivable that HHV8 vFLIP recruits an upstream kinase to the IKK complex. For example, FIG. 7. HHV8 vFLIP physically interacts with components of the ϳ700-kDa IKK signalsome complex. A and B, column fractions containing extracts prepared from vFLIP-and MRIT/cFLIP-expressing H460 cells were immunoprecipitated using FLAG antibody and resolved on a SDS-polyacrylamide gel, and the presence of NEMO/IKK␥ in the immunoprecipitates was detected by Western blot analysis. Lane C, control antibody; lane F, FLAG antibody. C, column fractions containing extracts prepared from vFLIP-expressing H460 cells were immunoprecipitated using FLAG antibody beads. The supernatant (S) and the pellet (P) fractions were resolved by a SDS-polyacrylamide gel followed by Western blot analysis using antibodies directed against NEMO/IKK␥, IKK␣, and IKK␤, respectively. IN, input.
FIG. 8. NEMO/IKK␥ is essential for vFLIP-induced NF-B activation. A, EMSA demonstrating increased NF-B DNA binding activity in nuclear extracts prepared from vFLIP-expressing 70Z/3 cells but not in the corresponding 1.3E2 cells or those expressing empty vector or MRIT. The specificity of the NF-B complex present in the vFLIPexpressing 70Z/3 cells is demonstrated by competition with excess cold B or nonspecific (NS) probes. B, control EMSA demonstrating equivalent SP1 DNA binding activity in the nuclear extracts prepared from 70Z/3 and 1.3E2 cells. C, Western blot analysis confirming the lack of NEMO expression in 1.3E2 cells. D, control experiment demonstrating equivalent expression of vFLIP in 70Z/3 and 1.3E2 clones. Cellular lysates (L) from the indicated cells were immunoprecipitated using control mouse IgG beads (C) or FLAG beads (F), and the presence of vFLIP was detected using a rabbit polyclonal antibody against the FLAG tag.
we have demonstrated that in addition to the various IKKs, HHV8 vFLIP also interacts with RIP, a protein kinase known to be crucial for TNF␣-mediated NF-B activation. RIP in turn may recruit and activate NF-B-inducing kinase or mitogeneactivated protein kinase kinase kinase, which are known to activate the IKK complex (61)(62)(63)(64). Studies to address the role of RIP, NF-B-inducing kinase, and mitogene-activated protein kinase kinase kinase in vFLIP-induced NF-B activation are currently in progress.