Nuclear IκBβ Maintains Persistent NF-κB Activation in HIV-1-infected Myeloid Cells*

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

HIV-1 1 -infected cells of the myeloid lineage serve as intracellular reservoirs for virus dissemination (1)(2)(3)(4). Infection of monocytic cells leads to the deregulation of numerous immunoregulatory functions and aberrant expression of inflamma-tory cytokines (5,6), which may further their ability to spread virus and cause disease progression. The NF-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-B⅐DNA binding activity (9 -12), NF-B strongly induces HIV long terminal repeat-driven gene expression (9,13,14) and maintains cell viability (15).
NF-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-B is found sequestered in the cytoplasm by inhibitory IB proteins. Several IB proteins have been identified including IB␣, IB␤, and most recently, IB⑀ (17). The precursor proteins p100 and p105 can also serve as functional IB molecules, retaining NF-B in the cytoplasm although their regulation is less well understood. Serine phosphorylation of IB␣ at Ser-32 and Ser-36 represents the critical regulatory signal leading to ubiquitin-dependent, proteosomemediated degradation of IB, which allows NF-B to translocate to the nucleus and activate NF-B-dependent genes. Recently, several groups have identified the IB kinase complex (IKK) (18 -23), a multisubunit complex that phosphorylates both IB␣ and IB␤ (24,25). Several pathways of NF-B activation are thought to converge at the level of IKK activation, implicating this complex as a critical regulator of NF-B transcriptional regulation.
The multimeric IKK complex includes two subunits, IKK␣ and IKK␤, which are responsible for phosphorylating IB molecules. Several other components of the IKK complex have been identified including the regulatory subunit NEMO (NF-B essential modulator, also called IKK␥) (26,27) and a scaffolding protein, IKAP (IKK-associated protein) (28), which binds the IKK subunits and, together with NF-B-inducing kinase (NIK), assembles them into an active kinase complex. The IKK complex is rapidly stimulated by TNF␣, IL-1, and PMA, although the mechanism of activation requires further elucidation. Recently, NIK was found to activate IKK␣ directly (29), confirming earlier reports that NIK co-expression leads to IKK␣ 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 IB␣ establishes an autoregulatory loop whereby NF-B activation is self-limited. Unlike IB␣, IB␤ is not an NF-B-regulated gene and is not rapidly resynthesized after inducer-mediated degradation (31). Several inducers, however, result in persistent NF-B activation, which has been associated with the additional release of NF-B from IB␤ complexes and its resynthesis in a hypophosphorylated form that sustains NF-B activation (32,33). Hypophosphorylated IB␤ can bind NF-B without masking its nuclear localization signal (32), thus acting as a chaperone for NF-B nuclear entry and preventing its sequestration by IB␣. Recently, two isoforms of IB␤ 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 IB␤, 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-B in HIV-1-infected myeloid cells has been examined. We demonstrate that the association of nuclear IB␤ with NF-B⅐DNA complexes maintains persistent NF-B⅐DNA binding activity, and we show that the IKK complex is constitutively activated in HIV-1-infected cells. Constitutive IKK activity and protection of NF-B⅐DNA activity from IB␣-mediated dissociation by nuclear IB␤ may explain the persistent NF-B-dependent gene activation observed in HIV-1-infected cells.
GST Fusion Proteins-IB␣ human cDNA bearing a 22-amino acid C-terminal truncation in the PEST domain (IB-⌬4, which inhibits NF-B binding as efficiently as wild type) was subcloned into pGEX 2T as described previously (35). GST⅐IB␣-(1-55) and GST⅐IB␣-(1-55) (S32A/S36A) were a kind gift from Antonis Koromilas. DH5␣ bacteria expressing GST⅐proteins 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: TNF␣ (10 ng/ml, R & D Systems), PMA (50 ng/ml, Sigma), or IL-1␤ (5 ng/ml, R & D Systems). An electrophoretic mobility shift assay was carried out using an NF-B 32 P-labeled probe corresponding to the PRDII region of the IFN-␤ promoter (5Ј-GGAAATTCCGG-GAAATTCC-3Ј) as described (5). In supershift experiments, the antibody (36) and its corresponding peptide where indicated (Santa Cruz Biotechnology), were incubated with 5 g of nuclear extract for 20 min. Poly(dI⅐dC) (5 g) 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(dI⅐dC) for 10 min and then incubated with labeled probe for 20 min. Increasing amounts of GST⅐IB␣ ⌬4 (10 ng/l) 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). IB␤ 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 IB␣ antibody was purchased from New England Biolabs, and the monoclonal IB␣ antisera MAD-10B was a kind gift from Ron Hay (37). The ␣-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 g of NF-B CAT (PRDII element of the IFN␤ gene linked to CAT) and either 4 g of pSVK3-IB␣ and 4 g of pSVK3 empty vector, with 4 g of pSVK3-IB␣ and 4 g of pSVK3-IB␤, or 8 g 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 g 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.

IB␤ Is Part of the NF-B⅐DNA Binding Complex in HIV-1infected Cells-Previous
studies demonstrated that myeloid cell lines PLB-985 and U937 acquire constitutive NF-B⅐DNA 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 IB␤ 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-B⅐DNA binding activity, using an IB␤-specific antibody that recognized the 43-kDa isoform of IB␤, demonstrated that IB␤ protein was a part of the DNA binding complex (Fig. 1A, lanes 2 and 10) and could be detected in cells stimulated with TNF␣ or IL-1␤ for 6 h (Fig. 1A, lanes 5 and 7). Preincubation with the cognate peptide recognized by the IB␤ antibody demonstrated the specificity of antibody recognition (Fig. 1A, lanes 3 and 11), whereas incubation with excess unlabeled NF-B probe competed the NF-B-specific complexes (Fig. 1A, lane 8). Similar experiments using IB␣ did not produce a shifted complex (data not shown), suggesting that IB␤ was uniquely present in HIV-1-infected cells. Uninfected PLB-985 and U937 cells stimulated with TNF␣ 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-B⅐DNA binding complex that could be supershifted with IB␤ antibody. Induction of PLB-IIIB cells with TNF␣ or PMA for 0, 2, 4, 8, 12, or 18 h revealed that IB␤ remained part of the DNA binding complex over the course of induction ( Fig. 1C and data not shown).
The presence of IB␤ in the nuclear compartment of HIV-1infected myeloid cells was confirmed by biochemical fractionation. Cytoplasmic and nuclear extracts from PLB-IIIB and PLB-985 cells stimulated with TNF␣ or PMA were separated by SDS-polyacrylamide gel electrophoresis and immunoblotted for IB␤. Whereas IB␤ was present in the cytoplasm and nucleus of HIV-1-infected cells ( Fig. 2A, lanes 1 and 6), IB␤ was predominantly cytoplasmic in noninfected PLB-985 cells (Fig. 2B, lanes 1 and 6). IB␤ was present in the nucleus of HIV-1-infected cells after stimulation with TNF␣ 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 ␣-tubulin antibody (Fig. 2, A and B, lower panels). Similar results were obtained with U937 and U9-IIIB cells (data not shown).
IB␤ Protects NF-B⅐DNA Complexes from IB␣-mediated Dissociation-Because previous in vitro studies have demonstrated that NF-B⅐DNA complexes are sensitive to dissociation by IB␣ (38,39), the possibility that IB␤ protects NF-B⅐DNA complexes from IB␣ dissociation was evaluated. The NF-B⅐DNA binding complex from HIV-1-infected U9-IIIB cells was resistant to GST⅐IB␣-mediated dissociation (Fig. 3A,  lanes 1-3). Furthermore, GST⅐IB␣ did not reduce NF-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-B⅐DNA complexes from TNF␣-or PMA-stimulated PLB-985 and U937 cells were completely dissociated by GST⅐IB␣ (Fig. 3, C and D, lanes 2, 3, 5, and 6).
IB␤ Co-expression Increases NF-B Transcriptional Activation-Next, a series of co-transfection experiments were performed to determine whether IB␤ co-expression affected NF-B-mediated transcription and to examine whether IB␤ blocked the inhibitory effect of IB␣. Human embryonic kidney 293 cells were transfected with an NF-B-driven CAT reporter plasmid and empty vector or reporter plasmid and pSVK3-IB␣ and/or pSVK3-IB␤ 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 IB␣ and IB␤ partially alleviated the IB␣mediated repression of transcription (Fig. 5, compare 24-h IB␣, IB␣ and IB␤, and pSVK3 levels) and exhibited a 50% increase in NF-B-dependent expression compared with cells transfected with IB␣ alone (Fig. 5). This result indicated that IB␤ expression partially reversed the inhibitory effects of IB␣ and increased NF-B-mediated transcription.
Turnover of IB␤ Isoforms-The regulation of IB␤ 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 IB␤ 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 IB␤ 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-1␤ (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. TNF␣ stimulation led to rapid degradation of 43-kDa IB␤ 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 IB␤ may result in the continuous production of hypophosphorylated IB␤, the form previously shown to shield DNA-bound NF-B from the effects of IB␣ (32,40). (15), we sought to determine whether the IB kinase was constitutively active in HIV-1infected cells. When an antibody that recognizes the phosphoserine 32 of IB␣ was used, HIV-1-infected U9-IIIB and PLB-IIIB cells, but not their uninfected counterparts, contained high levels of phosphorylated IB␣ in the presence or absence of inducer (Fig. 7A, top panel, lanes 4 -6 and 10 -12). Stimulation of uninfected cells with TNF␣ or PMA for 10 min resulted in the appearance of phosphorylated IB␣ 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 IB␣ antibody (middle panel) to confirm IB␣ turnover. As expected, TNF␣ stimulation led to degradation of IB␣ in all cell lines (Fig. 7A, middle panel, lanes 2, 5, 8, and 11). IB␣ levels in PMA-stimulated cells were not reduced (Fig. 7A, middle panel, lanes 3, 6, 9, and 12), although phosphorylated IB␣ was detected, reflecting the longer kinetics of PMA induced IB␣ degradation. As described previously, an IB␣ immunoreactive band of approximately 30 kDa in size was also detected in PLB-985 cells (12). The 30-kDa IB␣ was also recognized by the phosphoserine-specific IB␣ antibody (Fig. 7A, top panel, lower arrow) and degraded similarly to IB␣ (Fig. 7A, middle panel), suggesting that the regulation of the 30-kDa form may be similar to that of full-length IB␣.

Constitutive Activation of the IKK Complex in HIV-1-infected Cells-Because IB␣ (12) and IB␤ turnover are increased in HIV-1-infected cells and the constitutive NF-B⅐DNA binding in HIV-1-infected myeloid cells disappears when oxidant signaling pathways are interrupted
Given the crucial role of the IKK in the activation cascade of NF-B, the possibility of constitutive IKK activity in HIV-1infected cells was also examined. PLB-985 and PLB-IIIB cells were stimulated with TNF␣ for 15 min, extracts were immunoprecipitated with anti-IKK antibody, and immunoprecipitates were analyzed for the ability to phosphorylate N-terminal IB␣-(1-55) in vitro. PLB-985 cells exhibited little or no IKK activity (Fig. 7B, lane 1) unless stimulated with TNF␣ (Fig. 7B,  lane 2), whereas HIV-1-infected cells displayed IKK activity with or without TNF␣ stimulation (Fig. 7B, lanes 3-4). This activity was specific because immunoprecipitation with normal rabbit serum did not result in detectable kinase activity (TNF␣-stimulated PLB-985, Fig. 7B, lane 5) and mutated IB␣-(1-55) (S32A/S36A) substrate was not phosphorylated (TNF␣-stimulated PLB-IIIB, Fig. 7B, lane 6). Coomassie Blue staining revealed that equal amounts of IB␣ substrate were  (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 GST⅐IB␣. PLB-985 (C) and U937 (D) cells were stimulated with TNF␣ for 2 h (T2, lanes 1-3) or 18 h (T18, lanes 4 -6) and treated as described in A. used in each reaction (Fig. 7B, bottom panel). Similarly, IKK activity was inducible by TNF␣ in U937 cells (Fig. 7C, lanes  1-3) but was constitutively activated in U9-IIIB cells (Fig. 7C,  lanes 4 -6). DISCUSSION Previously, we have shown that HIV-1-infected primary monocytes and myeloid cell lines PLB-985 and U937 exhibit constitutive NF-B activation as a consequence of virus infection (10, 11, 15). In addition, levels of NF-B subunits p105, p100, and c-Rel are elevated compared with uninfected cells, and constitutive turnover of IB␣ is increased (12). These cells also express a low level of cytokines such as TNF␣ and IL-1␤, which are able to activate NF-B (Refs. 5, 6, and 41-44 and references therein). Together these results suggest that the constitutive activation of NF-B in HIV-1-infected myeloid cells is caused by the continuous signal-induced degradation of IB.

FIG. 5. IB␤ protects NF-B transcriptional activity from inhibition by IB␣.
Human embryonic kidney 293 cells were transfected with an NF-B CAT (7 g) reporter construct and pSVK3 control vector (8 g) or pSVK3-IB␣ (4 g) and/or pSVK3-IB␤ (4 g) 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 Ϯ S.E.

FIG. 6. IB␤ 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-1␤ (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 IB␤ antibody that recognizes only the 43-kDa isoform. B, U937 cells were incubated with cycloheximide alone, cycloheximide and TNF␣, or cycloheximide and IL-1␤ for 0, 2, 6, or 8 h. Whole cell extracts were immunoblotted with anti-IB␤ (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 IB␤ isoforms. D, U937 cells were treated as described in A, immunoblotted, and probed with an antibody that recognizes both isoforms of IB␤. cells and in uninfected cells stimulated with various inducers. NF-B⅐DNA complexes could not be completely dissociated by GST⅐IB␣ in HIV-1-infected cells, whereas complexes induced by TNF␣ or PMA were readily dissociated in uninfected cells. Depletion of IB␤ from HIV-1-infected nuclear extracts resulted in NF-B⅐DNA binding complexes that were completely sensitive to inhibition by GST⅐IB␣-mediated dissociation, indicating that IB␤ protects DNA-bound NF-B from dissociation by IB␣. Furthermore, co-expression experiments demonstrated that IB␤ increased NF-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 IB␣-resistant IB␤⅐NF-B⅐DNA 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 IB␣ or supershifted with NF-B antisera (11). Although these proteins could be specifically competed with unlabeled probe, it was considered that these NF-B-like proteins might not specifically belong to the NF-B family. The present findings suggest that the NF-B-like binding activity may in fact be IB␤bound NF-B complexes that are protected from IB␣mediated dissociation.
IB␤ was detected in NF-B⅐DNA binding complexes of uninfected U937 and PLB-985 cells after long periods of TNF␣ or PMA stimulation. In contrast to HIV-1-infected cells, NF-B complexes from uninfected cells were sensitive to IB␣-mediated dissociation. The reason for this discrepancy may lie in the additional pathways that are activated or inhibited in HIV-1infected cells. IB␤ protection of NF-B may require additional factors (such as high mobility group proteins) that are activated in HIV-1-infected myeloid cells. Alternatively, IB␤-mediated protection in uninfected cells may require longer induction periods than those used in this study.
Several studies have implicated IB␤ in maintaining persistent NF-B activation (31)(32)(33)(45)(46)(47). B cells stimulated with lipopolysaccharide or IL-1 experienced a persistent degradation of IB␤ that correlated with sustained NF-B activation, whereas inducers that did not degrade IB␤ produced only a transient activation of NF-B (31). A similar correlation between IB␤ degradation and persistent NF-B activation was also reported in human vascular endothelial cells (47). IB␤ degradation has been also implicated in the synergistic activation of NF-B observed in TNF␣-and IFN-␥-stimulated cells (48). Although others have observed IB␤ degradation during transient activation of NF-B (49), persistent activation of NF-B is generally associated with IB␤ degradation. The increased rate of IB␤ turnover seen in our HIV-1-infected cells supports a role for IB␤ in maintaining persistent NF-B activation.
Several groups (32,40,50,51) have demonstrated that hypophosphorylated IB␤ can bind NF-B⅐DNA complexes without inhibiting DNA binding. Hypophosphorylated IB␤ did not mask the nuclear localization signal of RelA, permitting NF-B⅐IB␤ complexes to enter the nucleus and bind DNA (32). Sites important in regulating the ability of IB␤ to chaperone NF-B into the nucleus were identified in the C-terminal PEST domain. Phosphorylation of Ser-313 and Ser-315 by casein kinase II prevented IB␤ from associating with NF-B⅐DNA complexes (40), and conversely mutation of these sites to alanine permitted IB␤ to form ternary complexes with NF-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 IB␤ CKII mutant (S313A/S315A) also blocked the capacity of IB␣ to dissociate NF-B from DNA (40). Based on these results, it seems likely that the IB␤ complexed with nuclear NF-B in HIV-1-infected cells is hypophosphorylated.
In accord with studies conducted by Hirano and colleagues (34), two isoforms of IB␤ of 43 and 41 kDa were also detected. The 41-kDa isoform of IB␤ 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 IB␤. Similarly, lipopolysaccharide induced a prolonged NF-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 2 C. Heylbroeck and J. Hiscott, unpublished results.

FIG. 7. The IB kinase complex is constitutively active in HIV-1-infected cells.
A, cells were untreated or treated with TNF␣ (T) or PMA (P) for 10 min. Whole cell extracts were immunoblotted using an IB␣ antibody that recognizes phosphoserine 32 of IB␣ (upper panel). The blot was first reprobed for IB␣ using a monoclonal IB␣ 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 TNF␣ for 0 or 15 min and assayed for IKK using GST⅐IB␣-(1-55) as a substrate. Specificity was confirmed using normal rabbit serum as the immunoprecipitating antibody (lane 5) or by using GST⅐IB-(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 TNF␣ for 1 or 12 h and analyzed for IKK activity using GST⅐IB␣-(1-55) as a substrate. Specificity was confirmed using normal rabbit serum as the immunoprecipitating antibody (lane 7) and GST⅐IB␣-(1-55) (S32A/S36A) as substrate (lane 8).
of NF-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 IB␤ isoform (compared with the 41-kDa isoform) in HIV-1-infected cells. Other mechanisms likely exist to maintain IB␤ in a hypophosphorylated form, because antioxidant treatment or proteosome inhibition did not affect the nuclear localization of hypophosphorylated IB␤ in WEHI 231 cells (33). Maintenance of hypophosphorylated IB␤ in these cells may result from the activation of a phosphatase, because treatment with the phosphatase inhibitor okadaic acid led to IB␤ hyperphosphorylation. This is the first report demonstrating that IB␤ is present in nuclear NF-B⅐DNA complexes in HIV-1-infected cells and contributes to constitutive NF-B activation. We suggest that the induction of IKK, arising as a consequence of the low level TNF␣ production or the increased pro-oxidant state of HIV-1infected cells (53), leads to increased phosphorylation and degradation of IB␣ and IB␤. Both proteins release active NF-B, which translocates to the nucleus and transcriptionally activates responsive genes. In addition, newly synthesized IB␤ enters the nucleus and prevents IB␣-mediated termination of the NF-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-B (15, 54 -56). Blocking this pathway may be an important strategy in targeting long-lived HIV-1-infected myeloid cells.