Nuclear Ikappa Bbeta Maintains Persistent NF-kappa B Activation in HIV-1-infected Myeloid Cells*

Carmela DeLucaDagger §, Louisa PetropoulosDagger parallel , Dana ZmeureanuDagger , and John HiscottDagger §parallel **Dagger Dagger

From the Dagger  Lady Davis Institute for Medical Research, Sir Mortimer B. Davis Jewish General Hospital and the Departments of parallel  Microbiology & Immunology and § Medicine and the ** McGill AIDS Centre, McGill University, Montreal, Quebec H3T 1E2, Canada

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Monocytic cells exhibit constitutive NF-kappa B activation upon infection with human immunodeficiency virus-1 (HIV-1). Because Ikappa Bbeta has been implicated in maintaining NF-kappa B·DNA binding, we sought to investigate whether Ikappa Bbeta was involved in maintaining persistent NF-kappa B activation in HIV-1-infected monocytic cell lines. Ikappa Bbeta was present in the nucleus of HIV-1-infected cells and participated in the ternary complex formation with NF-kappa B and DNA. In contrast to uninfected cells, the addition of recombinant glutathione S-transferase-Ikappa Balpha protein to preformed NF-kappa B·DNA complexes from HIV-1-infected cell extracts did not completely dissociate the complexes, suggesting that Ikappa Bbeta may protect NF-kappa B complexes from Ikappa Balpha -mediated dissociation. Immunodepletion of Ikappa Bbeta resulted in an NF-kappa B·DNA binding complex that was sensitive to Ikappa Balpha -mediated dissociation, thus demonstrating the protective role of Ikappa Bbeta . In addition, co-transfection studies with an NF-kappa B-dependent reporter construct demonstrated that Ikappa Bbeta co-expression partially alleviated inhibition of NF-kappa B-mediated gene expression by Ikappa Balpha , implying that Ikappa Bbeta can maintain transcriptionally active NF-kappa B·DNA complexes. Furthermore, constitutive phosphorylation of Ikappa Balpha was observed. Immunoprecipitation of the Ikappa B kinase (IKK) complex followed by in vitro analysis of kinase activity demonstrated that IKK was constitutively activated in HIV-1-infected myeloid cells. Thus, virus-induced constitutive IKK activation, coupled with the maintenance of a ternary NF-kappa B·DNA complex by Ikappa Bbeta , maintains persistent NF-kappa B activity in HIV-1-infected myeloid cells.

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HIV-11-infected cells of the myeloid lineage serve as intracellular reservoirs for virus dissemination (1-4). Infection of monocytic cells leads to the deregulation of numerous immunoregulatory functions and aberrant expression of inflammatory cytokines (5, 6), which may further their ability to spread virus and cause disease progression. The NF-kappa B/Rel family of transcription factors participates in the activation of a number of host immunoregulatory cytokine genes (reviewed in Refs. 7 and 8), and its perturbation by HIV-1 infection leads to altered gene expression. In HIV-1-infected myeloid cell lines that express constitutive NF-kappa B·DNA binding activity (9-12), NF-kappa B strongly induces HIV long terminal repeat-driven gene expression (9, 13, 14) and maintains cell viability (15).

NF-kappa B consists of five family members including RelA and c-Rel, which contain transcriptional activation domains p100 and p105 precursor proteins, which are cleaved to the active members, p52 and p50, respectively, and RelB (reviewed in Refs. 7, 8, and 16). In most cells, NF-kappa B is found sequestered in the cytoplasm by inhibitory Ikappa B proteins. Several Ikappa B proteins have been identified including Ikappa Balpha , Ikappa Bbeta , and most recently, Ikappa Bepsilon (17). The precursor proteins p100 and p105 can also serve as functional Ikappa B molecules, retaining NF-kappa B in the cytoplasm although their regulation is less well understood. Serine phosphorylation of Ikappa Balpha at Ser-32 and Ser-36 represents the critical regulatory signal leading to ubiquitin-dependent, proteosome-mediated degradation of Ikappa B, which allows NF-kappa B to translocate to the nucleus and activate NF-kappa B-dependent genes. Recently, several groups have identified the Ikappa B kinase complex (IKK) (18-23), a multisubunit complex that phosphorylates both Ikappa Balpha and Ikappa Bbeta (24, 25). Several pathways of NF-kappa B activation are thought to converge at the level of IKK activation, implicating this complex as a critical regulator of NF-kappa B transcriptional regulation.

The multimeric IKK complex includes two subunits, IKKalpha and IKKbeta , which are responsible for phosphorylating Ikappa B molecules. Several other components of the IKK complex have been identified including the regulatory subunit NEMO (NF-kappa B essential modulator, also called IKKgamma ) (26, 27) and a scaffolding protein, IKAP (IKK-associated protein) (28), which binds the IKK subunits and, together with NF-kappa B-inducing kinase (NIK), assembles them into an active kinase complex. The IKK complex is rapidly stimulated by TNFalpha , IL-1, and PMA, although the mechanism of activation requires further elucidation. Recently, NIK was found to activate IKKalpha directly (29), confirming earlier reports that NIK co-expression leads to IKKalpha phosphorylation (19). MEKK-1 has also been found tightly associated in the IKK complex (21) and has been shown to stimulate IKK activity (30). Further studies are required to determine whether these kinases are essential upstream regulators of IKK activity.

Rapid resynthesis of Ikappa Balpha establishes an autoregulatory loop whereby NF-kappa B activation is self-limited. Unlike Ikappa Balpha , Ikappa Bbeta is not an NF-kappa B-regulated gene and is not rapidly resynthesized after inducer-mediated degradation (31). Several inducers, however, result in persistent NF-kappa B activation, which has been associated with the additional release of NF-kappa B from Ikappa Bbeta complexes and its resynthesis in a hypophosphorylated form that sustains NF-kappa B activation (32, 33). Hypophosphorylated Ikappa Bbeta can bind NF-kappa B without masking its nuclear localization signal (32), thus acting as a chaperone for NF-kappa B nuclear entry and preventing its sequestration by Ikappa Balpha . Recently, two isoforms of Ikappa Bbeta that differ in their C-terminal PEST domain as a consequence of alternative splicing have been identified in human cells (34). The larger protein, approximately 43 kDa, is homologous to the murine Ikappa Bbeta , whereas the 41-kDa form is unique to human cells. The 43-kDa protein degrades upon stimulation and enters the nucleus when hypophosphorylated, whereas the 41-kDa protein is resistant to degradation by several inducers and is found only in the cytoplasm (34).

In this report, the mechanism underlying constitutive activation of NF-kappa B in HIV-1-infected myeloid cells has been examined. We demonstrate that the association of nuclear Ikappa Bbeta with NF-kappa B·DNA complexes maintains persistent NF-kappa B·DNA binding activity, and we show that the IKK complex is constitutively activated in HIV-1-infected cells. Constitutive IKK activity and protection of NF-kappa B·DNA activity from Ikappa Balpha -mediated dissociation by nuclear Ikappa Bbeta may explain the persistent NF-kappa B-dependent gene activation observed in HIV-1-infected cells.

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Cell Culture-- Promonocytic U937 and HIV-1-infected U9-IIIB cells, as well as myelomonoblastic PLB-985 and HIV-1-infected PLB-IIIB cells (infected with HIV strain IIIB), were maintained in RPMI 1640 medium (Life Technologies, Inc.) supplemented with 5% Fetal Clone I (Hyclone), 2 mM L-glutamine, and 20 µg/ml gentamicin (Schering Canada). Human embryonic kidney 293 cells were cultured in modified Eagle's medium (alpha -MEM) plus 10% fetal bovine serum (Biomedia Canada), 2 mM L-glutamine, and 20 µg/ml gentamicin.

GST Fusion Proteins-- Ikappa Balpha human cDNA bearing a 22-amino acid C-terminal truncation in the PEST domain (Ikappa B-Delta 4, which inhibits NF-kappa B binding as efficiently as wild type) was subcloned into pGEX 2T as described previously (35). GST·Ikappa Balpha -(1-55) and GST·Ikappa Balpha -(1-55) (S32A/S36A) were a kind gift from Antonis Koromilas. DH5alpha bacteria expressing GST·proteins were grown in Luria broth, washed with phosphate-buffered saline, resuspended in 10% Triton in phosphate-buffered saline, and sonicated. Protein was recovered using Sepharose 4B glutathione beads (Amersham Pharmacia Biotech) and eluted with 20 mM GSH (Calbiochem) in 50 mM Tris, pH 8.0. Isolation purity and quantity were confirmed by SDS-polyacrylamide gel electrophoresis, Coomassie Blue staining, and a visual comparison with bovine serum albumin standards.

Electromobility Shift Assay-- Nuclear extracts were prepared from untreated cells or cells treated for varying times with one of the following inducers: TNFalpha (10 ng/ml, R & D Systems), PMA (50 ng/ml, Sigma), or IL-1beta (5 ng/ml, R & D Systems). An electrophoretic mobility shift assay was carried out using an NF-kappa B 32P-labeled probe corresponding to the PRDII region of the IFN-beta promoter (5'-GGAAATTCCGGGAAATTCC-3') as described (5). In supershift experiments, the antibody (36) and its corresponding peptide where indicated (Santa Cruz Biotechnology), were incubated with 5 µg of nuclear extract for 20 min. Poly(dI·dC) (5 µg) was added for an additional 10 min followed by incubation with labeled probe for 20 min. All steps were carried out at room temperature. In other experiments in which GST fusion proteins were used, nuclear extracts were incubated with poly(dI·dC) for 10 min and then incubated with labeled probe for 20 min. Increasing amounts of GST·Ikappa Balpha Delta 4 (10 ng/µl) were added for an additional 20 min. The resulting protein-DNA complexes were resolved by a 5% Tris/glycine gel and exposed to x-ray film. To demonstrate the specificity of protein-DNA complex formation, 125-fold molar excess of unlabeled oligonucleotide was added to the nuclear extract before adding the labeled probe.

Immunoblot Analysis-- Whole cell extracts were prepared by resuspending the cells in Nonidet P-40 lysis buffer and examined by Western blot analysis as described previously (12). Ikappa Bbeta antibodies C20 (recognizes the 43-kDa isoform) and G20 (recognizes both the 43- and 41-kDa forms) were purchased from Santa Cruz Biotechnology. The phosphoserine 32 Ikappa Balpha antibody was purchased from New England Biolabs, and the monoclonal Ikappa Balpha antisera MAD-10B was a kind gift from Ron Hay (37). The alpha -tubulin antibody was obtained from ICN and the actin antisera from Sigma. The ECL-Western blotting detection system (NEN Life Science Products) was used according to manufacturer's instructions to visualize the specific signals.

Transfections and CAT Assays-- Human embryonic kidney 293 cells were transfected in 100-mm plates by the calcium phosphate DNA precipitate transfection method. Each plate was transfected with 7 µg of NF-kappa B CAT (PRDII element of the IFNbeta gene linked to CAT) and either 4 µg of pSVK3-Ikappa Balpha and 4 µg of pSVK3 empty vector, with 4 µg of pSVK3-Ikappa Balpha and 4 µg of pSVK3-Ikappa Bbeta , or 8 µg of empty vector. Cells were incubated with precipitate for 12 h after which time they were washed with phosphate-buffered saline and fed with fresh medium. Cells were maintained for an additional 36 h and stimulated with PMA (50 ng/ml) for the last 24, 16, or 8 h of the transfection. CAT activity was determined using 100 µg of total cell extract assayed for 2 h at 37 °C. Quantitation of activities was performed using NIH Image 1.60 software. The fold activation reported is the average of a minimum of three experiments with error bars representing the standard deviation.

IKK Assay-- Cells were pelleted by centrifugation and washed with ice-cold phosphate-buffered saline. Pellets were resuspended in TNN buffer (20 mM Tris, pH 7.5, 200 mM NaCl, 0.5% Nonidet P-40, 0.5 mM phenylmethylsulfonyl fluoride, 5 µg/ml each of aprotinin, leupeptin, and pepstatin, 0.5 mM spermine, 0.15 mM spermidine, 1 mM NaF, and 1 mM NaVO4) and incubated on ice for 10 min. Supernatants were removed after centrifugation (14,000 rpm, 10 min, 4 °C) and assayed for protein concentration. 150-300 µg of protein extract was incubated with 5 µl of IKKalpha antibody (Santa Cruz Biotechnology) or 2 µl of normal rabbit serum for 2 h while rotating at 4 °C. 20 µl of protein A-conjugated Sepharose beads, washed three times with TNN buffer, were added to the mixture and incubated with rotation at 4 °C for an additional hour. Beads were pelleted (1000 rpm) and washed once with TNN and twice with kinase buffer (50 mM Tris, pH 8.0, 100 mM NaCl, 2 mM MgCl2, 0.5 mM phenylmethylsulfonyl fluoride, 4 µg/ml chymostatin, 5 µg/ml each of pepstatin, leupeptin, and aprotinin, and 1 mM each dithiothreitol, NaF, and NaVO4). Beads were resuspended in 20 µl of kinase mix containing 2 µg of GST·Ikappa Balpha -(1-55) or Ikappa Balpha -(1-55) (S32A/S36A) and 0.5 µCi of gamma -ATP and incubated for 30 min at 30 °C. 20 µl of 2× SDS loading dye was added to each reaction, boiled for 5 min, and separated on a 10% SDS-polyacrylamide gel. The gel was fixed and Coomassie-stained (5% acetic acid, 10% ethanol, Coomassie Blue dye). It was subsequently destained (5% acetic acid, 10% ethanol), dried, and exposed to film for 1-3 h at -80 °C.

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Ikappa Bbeta Is Part of the NF-kappa B·DNA Binding Complex in HIV-1-infected Cells-- Previous studies demonstrated that myeloid cell lines PLB-985 and U937 acquire constitutive NF-kappa B·DNA binding activity upon HIV-1 infection (9-11, 15). Analysis of this complex revealed that the DNA binding activity was composed predominantly of RelA and p50 heterodimers with a minor contribution by c-Rel and p50 heterodimers (10-12). To investigate the possibility that Ikappa Bbeta may be involved in maintaining this persistent activation, nuclear extracts prepared from HIV-1-infected PLB-IIIB and U9-IIIB cells were analyzed for DNA binding levels by electrophoretic mobility shift assay. Analysis of NF-kappa B·DNA binding activity, using an Ikappa Bbeta -specific antibody that recognized the 43-kDa isoform of Ikappa Bbeta , demonstrated that Ikappa Bbeta protein was a part of the DNA binding complex (Fig. 1A, lanes 2 and 10) and could be detected in cells stimulated with TNFalpha or IL-1beta for 6 h (Fig. 1A, lanes 5 and 7). Preincubation with the cognate peptide recognized by the Ikappa Bbeta antibody demonstrated the specificity of antibody recognition (Fig. 1A, lanes 3 and 11), whereas incubation with excess unlabeled NF-kappa B probe competed the NF-kappa B-specific complexes (Fig. 1A, lane 8). Similar experiments using Ikappa Balpha did not produce a shifted complex (data not shown), suggesting that Ikappa Bbeta was uniquely present in HIV-1-infected cells. Uninfected PLB-985 and U937 cells stimulated with TNFalpha or PMA for 18 h (Fig. 1B, lanes 9, 14, and 16), but not cells treated for shorter times (Fig. 1B, lanes 3 and 5), likewise exhibited an NF-kappa B·DNA binding complex that could be supershifted with Ikappa Bbeta antibody. Induction of PLB-IIIB cells with TNFalpha or PMA for 0, 2, 4, 8, 12, or 18 h revealed that Ikappa Bbeta remained part of the DNA binding complex over the course of induction (Fig. 1C and data not shown).


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Fig. 1.   Electrophoretic mobility shift assay of HIV-1-infected myeloid cells for Ikappa Bbeta -containing NF-kappa B·DNA binding complexes. A, nuclear extracts from PLB-IIIB cells were subjected to supershift analysis using an Ikappa Bbeta -specific antibody (Ab). Extracts were either unstimulated (control (CON), lanes 1-3) or stimulated for 6 h with IL-1beta (lanes 4 and 5) or TNFalpha (lanes 6 and 7). Ikappa Bbeta supershifted bands are identified by the upper arrow. Antibody specificity was confirmed by preincubating the antibody with cognate peptide (lane 3). Competition of TNFalpha -stimulated extract with excess unlabeled probe demonstrated specificity of binding (lane 8). Unstimulated U9-IIIB nuclear extract (lanes 9-11) was incubated with Ikappa Bbeta antibody (lane 10) or antibody preincubated with peptide (lane 11) and analyzed as above. B, uninfected cells exhibit Ikappa Bbeta NF-kappa B·DNA binding complexes upon stimulation. Nuclear extracts from PLB-985 cells or U937 cells were unstimulated (lanes 1 and 12), stimulated with TNFalpha for 2 h (T2, lanes 2-3) or 18 h (T18, lanes 6, 7, 13, 14, 17, and 18), or stimulated with PMA for 2 h (P2, lanes 4 and 5) or 18 h (P18, lanes 8-11, 15, and 16). Extracts were incubated alone (lanes 1, 2, 4, 6, 8, 11-13, 15, and 18), with Ikappa Bbeta antibody (lanes 3, 5, 7, 9, 14, and 16), or with Ikappa Bbeta antibody and peptide (lanes 10 and 17). PMA-stimulated extract (lane 11) or TNFalpha -induced extract (lane 18) were competed with cold probe. C, Ikappa Bbeta -NF-kappa B·DNA binding complex is not disrupted by stimulation with TNFalpha or PMA. PLB-IIIB cells were stimulated with TNFalpha (lanes 1-6) or PMA (lanes 7-12) for 0 or 18 h. Extracts were incubated alone (lanes 1, 3, 7, and 9) or with Ikappa Bbeta antibody (lanes 2, 4, 8, and 10) or Ikappa Bbeta antibody and peptide (*, lanes 5 and 11). Ikappa Bbeta -containing complexes are identified with an arrow. Specificity was confirmed by incubating with excess unlabeled probe (*, lanes 6 and 12).

The presence of Ikappa Bbeta in the nuclear compartment of HIV-1-infected myeloid cells was confirmed by biochemical fractionation. Cytoplasmic and nuclear extracts from PLB-IIIB and PLB-985 cells stimulated with TNFalpha or PMA were separated by SDS-polyacrylamide gel electrophoresis and immunoblotted for Ikappa Bbeta . Whereas Ikappa Bbeta was present in the cytoplasm and nucleus of HIV-1-infected cells (Fig. 2A, lanes 1 and 6), Ikappa Bbeta was predominantly cytoplasmic in noninfected PLB-985 cells (Fig. 2B, lanes 1 and 6). Ikappa Bbeta was present in the nucleus of HIV-1-infected cells after stimulation with TNFalpha or PMA (Fig. 2A, lanes 7-10), and low levels were also detected in stimulated PLB-985 cells (Fig. 2B, lanes 7-10). Nuclear extracts were shown to be free of cytoplasmic contamination by reprobing with alpha -tubulin antibody (Fig. 2, A and B, lower panels). Similar results were obtained with U937 and U9-IIIB cells (data not shown).


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Fig. 2.   Ikappa Bbeta is found in the nucleus of HIV-1-infected cells. PLB-IIIB (A) or PLB-985 cells (B) were treated with TNFalpha (lanes 2, 3, 7, and 8) or PMA (lanes 4, 5, 9, and 10) for 0, 2, or 18 h. Cytoplasmic extract (40 µg) (lanes 1-5) or nuclear extract (40 µg) (lanes 6-10) was separated by SDS-polyacrylamide gel electrophoresis and immunoblotted for Ikappa Bbeta (upper panels). The blots were stripped and reprobed for alpha -tubulin (lower panels).

Ikappa Bbeta Protects NF-kappa B·DNA Complexes from Ikappa Balpha -mediated Dissociation-- Because previous in vitro studies have demonstrated that NF-kappa B·DNA complexes are sensitive to dissociation by Ikappa Balpha (38, 39), the possibility that Ikappa Bbeta protects NF-kappa B·DNA complexes from Ikappa Balpha dissociation was evaluated. The NF-kappa B·DNA binding complex from HIV-1-infected U9-IIIB cells was resistant to GST·Ikappa Balpha -mediated dissociation (Fig. 3A, lanes 1-3). Furthermore, GST·Ikappa Balpha did not reduce NF-kappa B binding to levels lower than those observed in unstimulated HIV-1-infected cells (Fig. 3A, lanes 4-15). Similar results were obtained with PLB-IIIB cells (Fig. 3B), suggesting this phenomenon was a property of HIV-1-infected myeloid cells. In contrast, NF-kappa B·DNA complexes from TNFalpha - or PMA-stimulated PLB-985 and U937 cells were completely dissociated by GST·Ikappa Balpha (Fig. 3, C and D, lanes 2, 3, 5, and 6).


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Fig. 3.   Recombinant Ikappa Balpha does not dissociate preformed NF-kappa B·DNA complexes in HIV-1-infected cells. U9-IIIB (A) and PLB-IIIB (B) cells were untreated (control (CON), lanes 1-3), treated with TNFalpha for 2 h (T2, lanes 4-6) or 18 h (T18, lanes 7-9) or with PMA for 2 h (P2, lanes 10-12) or 18 h (P18, lanes 13-15). Nuclear extracts were incubated with labeled PRDII probe followed by incubation with increasing amounts of GST·Ikappa Balpha . PLB-985 (C) and U937 (D) cells were stimulated with TNFalpha for 2 h (T2, lanes 1-3) or 18 h (T18, lanes 4-6) and treated as described in A.

Next, nuclear extracts were immunodepleted of Ikappa Bbeta using an Ikappa Bbeta -specific antibody and analyzed for NF-kappa B·DNA binding. Incubation of PMA-stimulated U9-IIIB nuclear extracts with GST·Ikappa Balpha reduced the amount of NF-kappa B·DNA binding complexes but did not completely dissociate NF-kappa B binding activity (Fig. 4A, lanes 1-6). In contrast, extracts immunodepleted for Ikappa Bbeta were sensitive to GST·Ikappa Balpha -mediated dissociation (Fig. 4A, lanes 9-14). In this case, levels of NF-kappa B binding were reduced to the levels observed in uninfected U937 cells (Fig. 3D), indicating that Ikappa Bbeta played a role in maintaining NF-kappa B·DNA binding activity in infected cells. Furthermore, the use of control serum for the immunodepletion step did not increase the sensitivity of the protein-DNA complex to Ikappa Balpha -mediated dissociation (Fig. 4A, lanes 7 and 8), indicating that the effect was specific. Similar results were obtained with untreated and PMA-induced PLB-IIIB cells (Fig. 4B). Like U9-IIIB cells, Ikappa Bbeta -depleted PLB-IIIB extracts were sensitive to GST·Ikappa Balpha -mediated dissociation of NF-kappa B·DNA binding activity (Fig. 4B, compare lanes 2 and 4 with lanes 6 and 8), indicating that nuclear Ikappa Bbeta maintained Ikappa Balpha -insensitive NF-kappa B·DNA binding activity in HIV-1-infected cells. Supershift analysis further revealed that similar NF-kappa B·DNA binding complexes were present before and after Ikappa Bbeta immunodepletion in PMA-stimulated (Fig. 4C, lanes 1-4 and 9-12) and TNFalpha -stimulated (Fig. 4C, lanes 5-8 and 13-16) U9-IIIB (Fig. 4C) and PLB-IIIB (data not shown) cells, arguing against the specific immunodepletion of an Ikappa Balpha -insensitive NF-kappa B complex.


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Fig. 4.   Depletion of Ikappa Bbeta -containing complexes sensitizes HIV-1 nuclear extracts to dissociation by Ikappa Balpha . A, DNA binding activity in U9-IIIB cells stimulated with PMA for 0, 2, or 18 h and depleted for Ikappa Bbeta by immunoprecipitation (Ikappa Bbeta IP, lanes 9-15) were compared with undepleted nuclear extracts (NE, lanes 1-6). Extracts were incubated with labeled probe only (lanes 1, 3, 5, 7 9, 11, and 13) or with labeled probe and GST·Ikappa Balpha (lanes 2, 4, 6, 8, 10, 12, and 14). Immunoprecipitation with normal rabbit serum was used as a control for specificity (lanes 7 and 8). B, PLB-IIIB NF-kappa B·DNA binding activity in cells stimulated with PMA for 0 or 2 h and depleted for Ikappa Bbeta (Ikappa Bbeta IP, lanes 1-4) was compared with the activity in undepleted, similarly treated nuclear extracts (NE, lanes 5-8). Extracts were incubated with only labeled probe (lanes 1, 3, 5, and 7) or with labeled probe and GST·Ikappa Balpha (lanes 2, 4, 6, and 8). C, the NF-kappa B subunit composition of U9-IIIB nuclear extracts stimulated with PMA or TNFalpha for 18 h and immunodepleted of Ikappa Bbeta (lanes 9-16) was compared with that of undepleted control extracts (lanes 1-8) by supershift analysis. RelA (lanes 2, 6, 10, and 14), p50 (lanes 3, 7, 11, and 15), or c-Rel (lanes 4, 8, 12, and 16) antibodies were utilized to identify the NF-kappa B·DNA binding subunits present.

Ikappa Bbeta Co-expression Increases NF-kappa B Transcriptional Activation-- Next, a series of co-transfection experiments were performed to determine whether Ikappa Bbeta co-expression affected NF-kappa B-mediated transcription and to examine whether Ikappa Bbeta blocked the inhibitory effect of Ikappa Balpha . Human embryonic kidney 293 cells were transfected with an NF-kappa B-driven CAT reporter plasmid and empty vector or reporter plasmid and pSVK3-Ikappa Balpha and/or pSVK3-Ikappa Bbeta expression plasmids. PMA (50 ng/ml) was added for 0, 8, 16, or 24 h, and transactivation was assessed by comparing CAT activity levels. PMA-stimulated cells transfected with both Ikappa Balpha and Ikappa Bbeta partially alleviated the Ikappa Balpha -mediated repression of transcription (Fig. 5, compare 24-h Ikappa Balpha , Ikappa Balpha and Ikappa Bbeta , and pSVK3 levels) and exhibited a 50% increase in NF-kappa B-dependent expression compared with cells transfected with Ikappa Balpha alone (Fig. 5). This result indicated that Ikappa Bbeta expression partially reversed the inhibitory effects of Ikappa Balpha and increased NF-kappa B-mediated transcription.


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Fig. 5.   Ikappa Bbeta protects NF-kappa B transcriptional activity from inhibition by Ikappa Balpha . Human embryonic kidney 293 cells were transfected with an NF-kappa B CAT (7 µg) reporter construct and pSVK3 control vector (8 µg) or pSVK3-Ikappa Balpha (4 µg) and/or pSVK3-Ikappa Bbeta (4 µg) and stimulated with PMA (50 ng/ml) for 0, 8, 16, or 24 h. Equal amounts of protein were assayed for CAT activity. Results are the average of a minimum of three experiments ± S.E.

Turnover of Ikappa Bbeta Isoforms-- The regulation of Ikappa Bbeta protein turnover was investigated next. Antibodies recognizing the 43-kDa isoform or both the 43- and 41-kDa isoforms were used to analyze the rate of Ikappa Bbeta basal turnover and stimulus-induced degradation. U937 and U9-IIIB cells were treated with cycloheximide alone or with different inducers for 0, 2, 6, or 8 h. The 43-kDa isoform of Ikappa Bbeta exhibited faster constitutive turnover in HIV-1-infected U9-IIIB cells (compare Fig. 6, A and C to B and D, lanes 1-4) and also degraded more quickly following IL-1beta (compare Fig. 6, A and C to B and D, lanes 9-12) or PMA (compare Fig. 6, A and D, lanes 5-8) stimulation. TNFalpha stimulation led to rapid degradation of 43-kDa Ikappa Bbeta in both infected and uninfected cells (Fig. 6, B and C, lanes 5-8). The faster migrating 41-kDa form was not degraded in U9-IIIB or U937 cells (Fig. 6, C and D, respectively, lower bands). Thus, the dynamic state of degradation and resynthesis of the 43-kDa form of Ikappa Bbeta may result in the continuous production of hypophosphorylated Ikappa Bbeta , the form previously shown to shield DNA-bound NF-kappa B from the effects of Ikappa Balpha (32, 40).


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Fig. 6.   Ikappa Bbeta turnover is increased in HIV-1-infected cells. A, U9-IIIB cells were incubated with cycloheximide (CHX) alone, cycloheximide and PMA (lanes 5-8), or cycloheximide and IL-1beta (lanes 9-12) for 0, 2, 6, or 8 h. Whole cell extracts were separated by SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and probed with an Ikappa Bbeta antibody that recognizes only the 43-kDa isoform. B, U937 cells were incubated with cycloheximide alone, cycloheximide and TNFalpha , or cycloheximide and IL-1beta for 0, 2, 6, or 8 h. Whole cell extracts were immunoblotted with anti-Ikappa Bbeta (43-kDa isoform). C, U9-IIIB cells were treated as described in B, immunoblotted, and probed with an antibody that recognizes both the 43- and 41-kDa Ikappa Bbeta isoforms. D, U937 cells were treated as described in A, immunoblotted, and probed with an antibody that recognizes both isoforms of Ikappa Bbeta .

Constitutive Activation of the IKK Complex in HIV-1-infected Cells-- Because Ikappa Balpha (12) and Ikappa Bbeta turnover are increased in HIV-1-infected cells and the constitutive NF-kappa B·DNA binding in HIV-1-infected myeloid cells disappears when oxidant signaling pathways are interrupted (15), we sought to determine whether the Ikappa B kinase was constitutively active in HIV-1-infected cells. When an antibody that recognizes the phosphoserine 32 of Ikappa Balpha was used, HIV-1-infected U9-IIIB and PLB-IIIB cells, but not their uninfected counterparts, contained high levels of phosphorylated Ikappa Balpha in the presence or absence of inducer (Fig. 7A, top panel, lanes 4-6 and 10-12). Stimulation of uninfected cells with TNFalpha or PMA for 10 min resulted in the appearance of phosphorylated Ikappa Balpha in U937 (Fig. 7A, top panel, lanes 2 and 3) and PLB-985 (Fig. 7A, lanes 8 and 9) cells. This blot was reprobed with monoclonal Ikappa Balpha antibody (middle panel) to confirm Ikappa Balpha turnover. As expected, TNFalpha stimulation led to degradation of Ikappa Balpha in all cell lines (Fig. 7A, middle panel, lanes 2, 5, 8, and 11). Ikappa Balpha levels in PMA-stimulated cells were not reduced (Fig. 7A, middle panel, lanes 3, 6, 9, and 12), although phosphorylated Ikappa Balpha was detected, reflecting the longer kinetics of PMA induced Ikappa Balpha degradation. As described previously, an Ikappa Balpha immunoreactive band of approximately 30 kDa in size was also detected in PLB-985 cells (12). The 30-kDa Ikappa Balpha was also recognized by the phosphoserine-specific Ikappa Balpha antibody (Fig. 7A, top panel, lower arrow) and degraded similarly to Ikappa Balpha (Fig. 7A, middle panel), suggesting that the regulation of the 30-kDa form may be similar to that of full-length Ikappa Balpha .


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Fig. 7.   The Ikappa B kinase complex is constitutively active in HIV-1-infected cells. A, cells were untreated or treated with TNFalpha (T) or PMA (P) for 10 min. Whole cell extracts were immunoblotted using an Ikappa Balpha antibody that recognizes phosphoserine 32 of Ikappa Balpha (upper panel). The blot was first reprobed for Ikappa Balpha using a monoclonal Ikappa Balpha antibody (middle panel) and then reprobed with actin antibody as a control for equal loading (lower panel). B, HIV-1-infected myeloid cells are constitutively activated for IKK. PLB-985 (PLB, lanes 1, 2, and 5) and PLB-IIIB cells (P-IIIB, lanes 3, 4, and 6) were treated with TNFalpha for 0 or 15 min and assayed for IKK using GST·Ikappa Balpha -(1-55) as a substrate. Specificity was confirmed using normal rabbit serum as the immunoprecipitating antibody (lane 5) or by using GST·Ikappa B-(1-55) (S32A/S36A) as substrate (lane 6). Coomassie Blue staining of the gel (bottom panel) reveals that equal amounts of recombinant protein were used in each reaction. C, U937 (lanes 1-3 and 7) and U9-IIIB (lanes 4-6 and 8) cells were stimulated with TNFalpha for 1 or 12 h and analyzed for IKK activity using GST·Ikappa Balpha -(1-55) as a substrate. Specificity was confirmed using normal rabbit serum as the immunoprecipitating antibody (lane 7) and GST·Ikappa Balpha -(1-55) (S32A/S36A) as substrate (lane 8).

Given the crucial role of the IKK in the activation cascade of NF-kappa B, the possibility of constitutive IKK activity in HIV-1-infected cells was also examined. PLB-985 and PLB-IIIB cells were stimulated with TNFalpha for 15 min, extracts were immunoprecipitated with anti-IKK antibody, and immunoprecipitates were analyzed for the ability to phosphorylate N-terminal Ikappa Balpha -(1-55) in vitro. PLB-985 cells exhibited little or no IKK activity (Fig. 7B, lane 1) unless stimulated with TNFalpha (Fig. 7B, lane 2), whereas HIV-1-infected cells displayed IKK activity with or without TNFalpha stimulation (Fig. 7B, lanes 3-4). This activity was specific because immunoprecipitation with normal rabbit serum did not result in detectable kinase activity (TNFalpha -stimulated PLB-985, Fig. 7B, lane 5) and mutated Ikappa Balpha -(1-55) (S32A/S36A) substrate was not phosphorylated (TNFalpha -stimulated PLB-IIIB, Fig. 7B, lane 6). Coomassie Blue staining revealed that equal amounts of Ikappa Balpha substrate were used in each reaction (Fig. 7B, bottom panel). Similarly, IKK activity was inducible by TNFalpha in U937 cells (Fig. 7C, lanes 1-3) but was constitutively activated in U9-IIIB cells (Fig. 7C, lanes 4-6).

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Previously, we have shown that HIV-1-infected primary monocytes and myeloid cell lines PLB-985 and U937 exhibit constitutive NF-kappa B activation as a consequence of virus infection (10, 11, 15). In addition, levels of NF-kappa B subunits p105, p100, and c-Rel are elevated compared with uninfected cells, and constitutive turnover of Ikappa Balpha is increased (12). These cells also express a low level of cytokines such as TNFalpha and IL-1beta , which are able to activate NF-kappa B (Refs. 5, 6, and 41-44 and references therein). Together these results suggest that the constitutive activation of NF-kappa B in HIV-1-infected myeloid cells is caused by the continuous signal-induced degradation of Ikappa B.

Because Ikappa Bbeta has been implicated in persistent NF-kappa B activation (31-33, 45-47), we sought to characterize its role in maintaining constitutive NF-kappa B activation. Ikappa Bbeta was found complexed to NF-kappa B in nuclear extracts from HIV-1-infected cells and in uninfected cells stimulated with various inducers. NF-kappa B·DNA complexes could not be completely dissociated by GST·Ikappa Balpha in HIV-1-infected cells, whereas complexes induced by TNFalpha or PMA were readily dissociated in uninfected cells. Depletion of Ikappa Bbeta from HIV-1-infected nuclear extracts resulted in NF-kappa B·DNA binding complexes that were completely sensitive to inhibition by GST·Ikappa Balpha -mediated dissociation, indicating that Ikappa Bbeta protects DNA-bound NF-kappa B from dissociation by Ikappa Balpha . Furthermore, co-expression experiments demonstrated that Ikappa Bbeta increased NF-kappa B-dependent gene activity. Finally, IKK was constitutively activated in HIV-1-infected myeloid cells. Interestingly, Sendai virus infection of U937 also leads to prolonged activation of IKK.2 It is possible that activation of IKK and formation of Ikappa Balpha -resistant Ikappa Bbeta ·NF-kappa B·DNA ternary complexes is a common mechanism exploited by several viruses to regulate host and viral gene expression.

In earlier studies, chronic HIV-1 infection or Sendai virus infection of PLB-985 cells resulted in the induction of protein DNA complexes that could not be dissociated with recombinant Ikappa Balpha or supershifted with NF-kappa B antisera (11). Although these proteins could be specifically competed with unlabeled probe, it was considered that these NF-kappa B-like proteins might not specifically belong to the NF-kappa B family. The present findings suggest that the NF-kappa B-like binding activity may in fact be Ikappa Bbeta -bound NF-kappa B complexes that are protected from Ikappa Balpha -mediated dissociation.

Ikappa Bbeta was detected in NF-kappa B·DNA binding complexes of uninfected U937 and PLB-985 cells after long periods of TNFalpha or PMA stimulation. In contrast to HIV-1-infected cells, NF-kappa B complexes from uninfected cells were sensitive to Ikappa Balpha -mediated dissociation. The reason for this discrepancy may lie in the additional pathways that are activated or inhibited in HIV-1-infected cells. Ikappa Bbeta protection of NF-kappa B may require additional factors (such as high mobility group proteins) that are activated in HIV-1-infected myeloid cells. Alternatively, Ikappa Bbeta -mediated protection in uninfected cells may require longer induction periods than those used in this study.

Several studies have implicated Ikappa Bbeta in maintaining persistent NF-kappa B activation (31-33, 45-47). B cells stimulated with lipopolysaccharide or IL-1 experienced a persistent degradation of Ikappa Bbeta that correlated with sustained NF-kappa B activation, whereas inducers that did not degrade Ikappa Bbeta produced only a transient activation of NF-kappa B (31). A similar correlation between Ikappa Bbeta degradation and persistent NF-kappa B activation was also reported in human vascular endothelial cells (47). Ikappa Bbeta degradation has been also implicated in the synergistic activation of NF-kappa B observed in TNFalpha - and IFN-gamma -stimulated cells (48). Although others have observed Ikappa Bbeta degradation during transient activation of NF-kappa B (49), persistent activation of NF-kappa B is generally associated with Ikappa Bbeta degradation. The increased rate of Ikappa Bbeta turnover seen in our HIV-1-infected cells supports a role for Ikappa Bbeta in maintaining persistent NF-kappa B activation.

Several groups (32, 40, 50, 51) have demonstrated that hypophosphorylated Ikappa Bbeta can bind NF-kappa B·DNA complexes without inhibiting DNA binding. Hypophosphorylated Ikappa Bbeta did not mask the nuclear localization signal of RelA, permitting NF-kappa B·Ikappa Bbeta complexes to enter the nucleus and bind DNA (32). Sites important in regulating the ability of Ikappa Bbeta to chaperone NF-kappa B into the nucleus were identified in the C-terminal PEST domain. Phosphorylation of Ser-313 and Ser-315 by casein kinase II prevented Ikappa Bbeta from associating with NF-kappa B·DNA complexes (40), and conversely mutation of these sites to alanine permitted Ikappa Bbeta to form ternary complexes with NF-kappa B and DNA. Other serines in the PEST domain also appear to be important, because replacing Ser-313 and Ser-315 with a phosphomimetic amino acid (Glu) was not sufficient to block the ternary complex formation (40). The Ikappa Bbeta CKII mutant (S313A/S315A) also blocked the capacity of Ikappa Balpha to dissociate NF-kappa B from DNA (40). Based on these results, it seems likely that the Ikappa Bbeta complexed with nuclear NF-kappa B in HIV-1-infected cells is hypophosphorylated.

In accord with studies conducted by Hirano and colleagues (34), two isoforms of Ikappa Bbeta of 43 and 41 kDa were also detected. The 41-kDa isoform of Ikappa Bbeta resisted degradation by several inducers in both infected and uninfected cells, whereas the constitutive protein turnover of the 43-kDa form was increased in HIV-1-infected cells. One plausible explanation is that virus infection represents the persistent activation signal required for the continuous degradation of Ikappa Bbeta . Similarly, lipopolysaccharide induced a prolonged NF-kappa B activation in 7OZ/3 pre-B cells as a result of a persistent activating signal that could be blocked by employing antioxidants (31). Constitutive activation of NF-kappa B may also result from decreased cellular antioxidant levels (52). HIV-1 infection can lead to decreases in antioxidant levels (53). Increased turnover because of constitutive stimulation could also explain the decreased steady state level of the inducer-sensitive 43-kDa Ikappa Bbeta isoform (compared with the 41-kDa isoform) in HIV-1-infected cells. Other mechanisms likely exist to maintain Ikappa Bbeta in a hypophosphorylated form, because antioxidant treatment or proteosome inhibition did not affect the nuclear localization of hypophosphorylated Ikappa Bbeta in WEHI 231 cells (33). Maintenance of hypophosphorylated Ikappa Bbeta in these cells may result from the activation of a phosphatase, because treatment with the phosphatase inhibitor okadaic acid led to Ikappa Bbeta hyperphosphorylation.

This is the first report demonstrating that Ikappa Bbeta is present in nuclear NF-kappa B·DNA complexes in HIV-1-infected cells and contributes to constitutive NF-kappa B activation. We suggest that the induction of IKK, arising as a consequence of the low level TNFalpha production or the increased pro-oxidant state of HIV-1-infected cells (53), leads to increased phosphorylation and degradation of Ikappa Balpha and Ikappa Bbeta . Both proteins release active NF-kappa B, which translocates to the nucleus and transcriptionally activates responsive genes. In addition, newly synthesized Ikappa Bbeta enters the nucleus and prevents Ikappa Balpha -mediated termination of the NF-kappa B response. This occurrence would create an environment conducive to viral replication, promoting HIV-1 long terminal repeat-driven gene transcription and maintaining cell survival resulting from the antiapoptotic effects of NF-kappa B (15, 54-56). Blocking this pathway may be an important strategy in targeting long-lived HIV-1-infected myeloid cells.

    ACKNOWLEDGEMENTS

We thank Ron Hay for the kind gift of the Ikappa Balpha monoclonal antibody and Dmitrios Thanos for supplying Ikappa Bbeta cDNA. We thank members of the McGill Aids Center for helpful comments and numerous reagents.

    FOOTNOTES

* This work was supported by the Medical Research Council (MRC) of Canada and by CANFAR.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Recipient of a National Health Research Development Program Fellowship.

Dagger Dagger Recipient of an MRC Senior Scientist award. To whom correspondence should be addressed: Lady Davis Institute for Medical Research, 3755 Cote Ste. Catherine, Montreal, Quebec H3T 1E2, Canada. Tel.: 514-340-8222, Ext. 5265; Fax: 514-340-7576; E-mail: mijh{at}musica.mcgill.ca.

2 C. Heylbroeck and J. Hiscott, unpublished results.

    ABBREVIATIONS

The abbreviations used are: HIV-1, human immunodeficiency virus-1; IKK, Ikappa B kinase; IL-1, interleukin-1; PMA, phorbol 12-myristate 13-acetate; PEST, proline-, glutamic acid-, serine-, and threonine-rich domain; TNFalpha , tumor necrosis factor alpha ; GST, glutathione S-transferase; CAT, chloramphenicol acetyltransferase; NIK, NF-kappa B-inducing kinase; PRD, positive regulatory domain.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
  1. Crowe, S. M., and Kornbluth, R. S. (1994) J. Leukocyte Biol. 56, 215-422[Medline] [Order article via Infotrieve]
  2. Kornbluth, R. S. (1995) J. Leukocyte Biol. 56, 247-256[Abstract]
  3. Perno, C. F., Crowe, S. M., and Kornbluth, R. S. (1997) J. Leukocyte Biol. 62, 1-143[Medline] [Order article via Infotrieve]
  4. Orenstein, J. M., Fox, C., and Wahl, S. M. (1997) Science 276, 1857-1860[Abstract/Free Full Text]
  5. D'Addario, M., Wainberg, M. A., and Hiscott, J. (1992) J. Immunol. 148, 1222-1229[Abstract/Free Full Text]
  6. Roulston, A., Lin, R., Beauparlant, P., Wainberg, M. A., and Hiscott, J. (1995) Microbiol. Rev. 59, 481-505[Abstract]
  7. Baeuerle, P. A., and Henkel, T. (1994) Annu. Rev. Immunol. 12, 141-179[CrossRef][Medline] [Order article via Infotrieve]
  8. Baldwin, A. S., Jr. (1996) Annu. Rev. Immunol. 14, 649-681[CrossRef][Medline] [Order article via Infotrieve]
  9. Bachelerie, F., Alcami, J., Arenzana-Seisdedos, F., and Virelizier, J.-L. (1991) Nature 350, 709-712[CrossRef][Medline] [Order article via Infotrieve]
  10. Roulston, A., D'Addario, M., Boulerice, F., Caplan, S., Wainberg, M., and Hiscott, J. (1992) J. Exp. Med. 175, 751-763[Abstract]
  11. Roulston, A., Beauparlant, P., Rice, N. R., and Hiscott, J. (1993) J. Virol. 67, 5235-5246[Abstract]
  12. DeLuca, C., Roulston, A., Koromilas, A., Wainberg, M. A., and Hiscott, J. (1996) J. Virol. 70, 5183-5193[Abstract]
  13. Griffin, G. E., Leung, K., Folks, T. M., Kunkel, S., and Nabel, G. (1989) Nature 339, 70-73[CrossRef][Medline] [Order article via Infotrieve]
  14. Osborn, L., Kunkel, S., and Nabel, G. J. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 2336-2340[Abstract]
  15. DeLuca, C., Kwon, H. J., Pelletier, N., Wainberg, M. A., and Hiscott, J. (1998) Virology 244, 27-38[CrossRef][Medline] [Order article via Infotrieve]
  16. Baeuerle, P. A., and Baltimore, D. (1996) Cell 87, 13-20[Medline] [Order article via Infotrieve]
  17. Whiteside, S. T., Epinat, J., Rice, N. R., and Israel, A. (1997) EMBO J. 16, 1413-1426[Abstract/Free Full Text]
  18. Chen, Z. J., Parent, L., and Maniatis, T. (1996) Cell 84, 853-862[Medline] [Order article via Infotrieve]
  19. Régnier, C., Song, H. Y., Gao, X., Goeddel, D. V., Cao, Z., and Rothe, M. (1997) Cell 90, 373-383[Medline] [Order article via Infotrieve]
  20. DiDonato, J. A., Hayakawa, M., Rothwarf, D. M., Zandi, E., and Karin, M. (1997) Nature 388, 548-554[CrossRef][Medline] [Order article via Infotrieve]
  21. Mercurio, F., Zhu, H., Murray, B. W., Shevchenko, A., Bennett, B. L., Li, J. W., Young, D. B., Barbosa, M., and Mann, M. (1997) Science 278, 860-866[Abstract/Free Full Text]
  22. Zandi, E., Rothwarf, D. M., Delhase, M., Hayakawa, M., and Karin, M. (1997) Cell 91, 243-252[Medline] [Order article via Infotrieve]
  23. Woronicz, J. D., Gao, X., Cao, Z., Rothe, M., and Goeddel, D. V. (1997) Science 278, 866-869[Abstract/Free Full Text]
  24. Maniatis, T. (1997) Science 278, 818-819[Free Full Text]
  25. Stancovski, I., and Baltimore, D. (1997) Cell 91, 299-302[Medline] [Order article via Infotrieve]
  26. Rothwarf, D. M., Zandi, E., Natoli, G., and Karin, M. (1998) Nature 395, 297-300[CrossRef][Medline] [Order article via Infotrieve]
  27. Yamaoka, S., Courtois, G., Bessia, C., Whiteside, S. T., Weil, R., Agou, F., Kirk, H. E., Kay, R. J., and Israel, A. (1998) Cell 93, 1231-1240[Medline] [Order article via Infotrieve]
  28. Cohen, L., Henzel, W. J., and Baeuerle, P. A. (1998) Nature 395, 292-296[CrossRef][Medline] [Order article via Infotrieve]
  29. Ling, L., Cao, Z., and Goeddel, D. V. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 3792-3797[Abstract/Free Full Text]
  30. Lee, F. S., Hagler, J., Chen, Z. J., and Maniatis, T. (1997) Cell 88, 213-222[Medline] [Order article via Infotrieve]
  31. Thompson, J. E., Phillips, R. J., Erdjument-Bromage, H., Tempst, P., and Ghosh, S. (1995) Cell 80, 573-582[Medline] [Order article via Infotrieve]
  32. Suyang, H., Phillips, R., Douglas, I., and Ghosh, S. (1996) Mol. Cell. Biol. 16, 5444-5449[Abstract]
  33. Phillips, R. J., and Ghosh, S. (1997) Mol. Cell. Biol. 17, 4390-4396[Abstract]
  34. Hirano, F., Chung, M., Tanaka, H., Maruyama, N., Maruyana, N., Makino, I., Moore, D., and Scheidereit, C. (1998) Mol. Cell. Biol. 18, 2596-2607[Abstract/Free Full Text]
  35. Lin, R., Beauparlant, P., Makris, C., Meloche, S., and Hiscott, J. (1996) Mol. Cell. Biol. 16, 1401-1409[Abstract]
  36. Garoufalis, E., Kwan, I., Lin, R., Mustafa, A., Pepin, N., Roulston, A., Lacoste, J., and Hiscott, J. (1994) J. Virol. 68, 4707-4715[Abstract]
  37. Jaffray, E., Wood, K. M., and Hay, R. T. (1995) Mol. Cell. Biol. 15, 2166-2172[Abstract]
  38. Zabel, U., and Baeuerle, P. A. (1990) Cell 61, 255-265[Medline] [Order article via Infotrieve]
  39. Arenzana-Seisdedos, F., Thompson, J., Rodriguez, M. S., Bachelerie, F., Thomas, D., and Hay, R. T. (1995) Mol. Cell. Biol. 15, 2689-2696[Abstract]
  40. McKinsey, T. A., Chu, Z. L., and Ballard, D. W. (1997) J. Biol. Chem. 272, 22377-22380[Abstract/Free Full Text]
  41. D'Addario, M., Roulston, A., Wainberg, M. A., and Hiscott, J. (1990) J. Virol. 64, 6080-6089[Medline] [Order article via Infotrieve]
  42. Lenardo, M. J., and Baltimore, D. (1989) Cell 58, 227-229[Medline] [Order article via Infotrieve]
  43. Molina, J.-M., Schindler, R., Ferriani, R., Sakaguchi, M., Vannier, E., Dinarello, C. A., and Groopman, J. E. (1990) J. Invest. Dermatol. 161, 888-893
  44. Peters, A., Jager, F.-S., Warneke, A., Muller, K., Brunkhorst, U., Schedel, I., and Gahr, M. (1991) Clin. Immunol. Immunopathol. 61, 343-352[Medline] [Order article via Infotrieve]
  45. Good, L., and Sun, S-C. (1996) J. Virol. 70, 2730-2735[Abstract]
  46. Attar, R. M., MacDonald-Bravo, H., Raventos-Suarez, C., Durham, S. K., and Bravo, R. (1998) Mol. Cell. Biol. 18, 477-487[Abstract/Free Full Text]
  47. Johnson, D. R., Douglas, I., Jahnke, A., Ghosh, S., and Pober, J. S. (1996) J. Biol. Chem. 271, 16317-16322[Abstract/Free Full Text]
  48. Cheschire, J. L., and Baldwin, A. S. (1997) Mol. Cell. Biol. 17, 6746-6754[Abstract]
  49. Kalli, K., Huntoon, C., Bell, M., and McKean, D. J. (1998) Mol. Cell. Biol. 18, 3140-3148[Abstract/Free Full Text]
  50. Chu, Z.-L., McKinsey, T. A., Liu, L., Qi, X., and Ballard, D. (1996) Mol. Cell. Biol. 16, 5974-5984[Abstract]
  51. Tran, K., Merika, M., and Thanos, D. (1997) Mol. Cell. Biol. 17, 5386-5399[Abstract]
  52. Jin, D. Y., Chae, H. Z., Rhee, S. G., and Jeang, K. T. (1997) J. Biol. Chem. 272, 30952-30961[Abstract/Free Full Text]
  53. Staal, F. J., Ela, S. W., Roederer, M., Anderson, M. T., and Herzenberg, L. A. (1992) Lancet 339, 909-912[CrossRef][Medline] [Order article via Infotrieve]
  54. Beg, A. A., and Baltimore, D. (1996) Science 274, 782-784[Abstract/Free Full Text]
  55. Van Antwerp, D. J., Martin, S. J., Kafri, T., Green, D. R., and Verma, I. M. (1996) Science 274, 787-789[Abstract/Free Full Text]
  56. Wang, C. Y., Mayo, M. W., and Baldwin, A. S. (1996) Science 274, 784-787[Abstract/Free Full Text]


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