Inducible Expression of Ikappa Balpha Repressor Mutants Interferes with NF-kappa B Activity and HIV-1 Replication in Jurkat T Cells*

Hakju KwonDagger §, Nadine PelletierDagger §, Carmela DeLucaDagger §, Pierre GeninDagger §, Sonia CisternasDagger , Rongtuan LinDagger §, Mark A. WainbergDagger §par , and John HiscottDagger §par **

From the Dagger  Lady Davis Institute for Medical Research and Departments of  Microbiology and § Medicine, par  McGill AIDS Center, McGill University, Montreal, Quebec H3T 1E2, Canada

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Human immunodeficiency virus (HIV-1) utilizes the NF-kappa B/Rel proteins to regulate transcription through NF-kappa B binding sites in the HIV-1 long terminal repeat (LTR). Normally, NF-kappa B is retained in the cytoplasm by inhibitory Ikappa B proteins; after stimulation by multiple activators including viruses, Ikappa Balpha is phosphorylated and degraded, resulting in NF-kappa B release. In the present study, we examined the effect of tetracycline-inducible expression of transdominant repressors of Ikappa Balpha (TD-Ikappa Balpha ) on HIV-1 multiplication using stably selected Jurkat T cells. TD-Ikappa Balpha was inducibly expressed as early as 3 h after doxycycline addition and dramatically reduced both NF-kappa B DNA binding activity and LTR-directed gene activity. Interestingly, induced TD-Ikappa Balpha expression also decreased endogenous Ikappa Balpha expression to undetectable levels by 24 h after induction, demonstrating that TD-Ikappa Balpha repressed endogenous NF-kappa B-dependent gene transcription. TD-Ikappa Balpha expression also sensitized Jurkat cells to tumor necrosis factor-induced apoptosis. De novo HIV-1 infection of Jurkat cells was dramatically altered by TD-Ikappa Balpha induction, resulting in inhibition of HIV-1 multiplication, as measured by p24 antigen, reverse transcriptase, and viral RNA. Given the multiple functions of the NF-kappa B/Ikappa B pathway, TD-Ikappa Balpha expression may interfere with HIV-1 multiplication at several levels: LTR-mediated transcription, Rev-mediated export of viral RNA, inhibition of HIV-1-induced pro-inflammatory cytokines, and increased sensitivity of HIV-1-infected cells to apoptosis.

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

NF-kappa B/Rel transcription factors serve essential roles in the regulation of the immunomodulatory genes and activate genes including cytokines, cell surface receptors, and acute phase proteins, as well as viral genes including the HIV-11 LTR (for review, see Refs. 1-3). NF- kappa B activity is regulated in part at the level of subcellular localization. In unstimulated cells, NF-kappa B heterodimers are retained in the cytoplasm by inhibitory Ikappa B proteins (4, 5), also a family of proteins that include Ikappa Balpha (6), Ikappa Bbeta (7), Ikappa Bepsilon (8), Ikappa Bgamma (9, 10), and Bcl-3 (11, 12), as well as the precursor proteins p105 (13) and p100 (14). Upon stimulation by many activating agents, including cytokines (TNF and interleukin-1), viruses, and double-stranded RNA, Ikappa Balpha is rapidly phosphorylated on Ser-32 and Ser-36; N-terminal phosphorylation of Ikappa Balpha represents a signal for ubiquitination and degradation via the 26 S proteasome pathway (15-19). Substitution of alanine for Ser-32 and Ser-36 completely abolished signal-induced phosphorylation and degradation of Ikappa Balpha , and blocked the activation of NF-kappa B (20-22). These mutations also blocked in vitro ubiquitination of the Ikappa Balpha protein (19, 23, 24). Although the N terminus of Ikappa Balpha is necessary for signal-induced degradation, degradation of Ikappa Balpha also requires the C-terminal PEST domain of the protein (25-30). Once released, NF-kappa B is able to activate target genes until new Ikappa Balpha is synthesized. Since Ikappa Balpha contains NF-kappa B binding sites in its promoter, NF-kappa B autoregulates the transcription of its own inhibitor (31-34).

The intracellular efficiency of HIV-1 gene expression and replication is due in part to the ability of HIV-1 to utilize host signaling pathways to mediate its own transcriptional regulation. In this regard, the NF-kappa B/Rel pathway plays a central role in HIV-1 LTR-driven transcription. The HIV-1 LTR contains two adjacent high affinity NF-kappa B binding sites in the enhancer region of its LTR (-109 to -79) (35). Transient transfection studies using HIV LTR or HIV enhancer reporter constructs demonstrate that HIV gene expression increases upon induction of NF-kappa B DNA binding activity with stimulators such as TNFalpha and interleukin-1 or upon co-infection with other pathogens (reviewed in Refs. 1 and 2).

The dependence of HIV-1 on its NF-kappa B sites in the LTR for virus replication has been examined in several studies (see Refs. 36-38, and references therein). HIV-1 infection of primary macrophages or myeloid cell lines leads to constitutive NF-kappa B expression (1, 39-41), increased proteasome-mediated turnover of Ikappa Balpha , and elevated expression of NF-kappa B1, NF-kappa B2, and c-Rel proteins (42, 43). This modulation of intracellular NF-kappa B levels may contribute to enhanced NF-kappa B-directed gene expression and increased HIV-1 replication. Recently, Chen et al. (37) measured virus multiplication in T cell lines with different basal levels of NF-kappa B and in primary T cells by infecting with wild type virus or virus containing NF-kappa B enhancer site mutations. These studies demonstrated that NF-kappa B sites play a central role in enhancing HIV-1 growth, although the virus was still able to grow more slowly in the absence of kappa B sites. In primary T cells, an intact enhancer element contributed to an earlier and higher peak titer of virus, thus reflecting the selective advantage conferred by the maintenance and utilization of functional NF-kappa B sites (37). Interestingly, pathogenic viral isolates with NF-kappa B site deletions have been described that contain a duplication of a putative TCF-1 site in the U3 region of the LTR (44); compensatory mutations such as the TCF-1 repeat may decrease the dependence of HIV-1 on intact NF-kappa B sites in vivo (37).

In the present study, we generated Jurkat T cell lines that inducibly express transdominant repressors of Ikappa Balpha (TD-Ikappa Balpha ), or Ikappa Balpha super-repressors, and examined the effect of Tet-inducible expression of (TD-Ikappa Balpha ) on NF-kappa B DNA binding activity, NF-kappa B-dependent gene expression and de novo HIV-1 multiplication. The time course of de novo HIV-1 infection in TD-Ikappa Balpha -expressing Jurkat cells was altered by doxycycline (Dox) induction of TD-Ikappa Balpha , resulting in a dramatic transcriptional inhibition of HIV-1 multiplication, as measured by p24 antigen, reverse transcriptase, and viral RNA analyses.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Generation of Plasmids-- CMVt-rtTA contains the Moloney murine leukemia virus-based pBABE vector backbone, which contains a puromycin resistance gene under the control of the CMV promoter. Plasmid construction consisted of the consecutive insertion of three components into the polylinker site: the doxycycline-responsive promoter CMVt from the CMVtBL vector (a kind gift from A. Cochrane), the rtTA gene from the pUHD172-1neo plasmid (45), and the poly(A) fragment from the pSVK3 vector. neo CMVt BL was constructed in two steps. First, an intermediary plasmid (neo BL) was generated by ligation of a 3-kilobase pair XhoI/EcoRI fragment from the pMV7 vector (contains the neomycin (neo) resistance gene) to a 3.8-kilobase pair XhoI/EcoRI fragment from the CMV BL vector (contains the poly(A) site and the ampicillin (Amp) resistance gene). Second, a 450-bp XhoI (blunt)/NotI fragment of CMVt BL (contains the CMVt promoter) was cloned into the EcoRI (blunt)/NotI sites of neo BL. CMVt-Ikappa B2N and Ikappa B2NDelta 4 were constructed by cloning an EcoRV (blunt)/BamHI Ikappa Balpha -2N mutant cDNA fragment downstream of CMVt at the EcoRI (blunt, filled with Klenow enzyme)/BamHI site of neo CMVt BL.

Cell Culture and Generation of Ikappa Balpha -expressing Cell Lines-- Jurkat cells were transfected with CMVt-rtTA plasmid by the DEAE-dextran method. The precipitated CMVt-rtTA plasmid (15 µg) was resuspended in TS solution (8 mg/ml NaCl, 0.38 mg/ml KCl, 0.1 mg/ml Na2HPO4·7H2O, 3.0 mg/ml Tris, 0.1 mg/ml MgCl2, 0.1 mg/ml CaCl2, pH 7.4) and subsequently DEAE-dextran (Amersham Pharmacia Biotech) was added. For transfection, 1 × 107 cells in exponential phase were washed once in TS, resuspended with the DNA solution and incubated at room temperature for 20 min, and then incubated at 37 °C for 30 min in 10 ml with RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine, 10 µg/ml gentamicin (Schering Canada, Pointe Claire, Quebec, Canada), and 0.1 mM chloroquine (Sigma), after which they were centrifuged and resuspended in fresh medium. Cells were selected beginning at 24 h after transfection in RPMI 1640 medium containing 10% FBS, 2 mM glutamine, 10 µg/ml gentamicin, and 2.5 µg/ml puromycin (Sigma). Resistant clones carrying the CMVt-rtTA plasmid (rtTA-Jurkat cells) were then transfected with CMVt-Neo, CMVt-2N, and CMVt-2NDelta 4 plasmids by DEAE-dextran. Cells were selected beginning at 24 h after transfection for approximately 4 weeks in RPMI containing 10% FBS, 2 mM glutamine, 10 µg/ml gentamicin, 2.5 µg/ml puromycin, and 400 µg/ml G418 (Life Technologies, Inc.). Initially, pools of transformants corresponding to rtTA-, rtTA-neo-, rtTA-Ikappa B-2N-, and rtTA-Ikappa B-2NDelta 4-expressing Jurkat cells were analyzed for inducible Ikappa B expression; subsequently, 6-10 individual clones from each transformant pool were selected for further analysis. To analyze growth kinetics, rtTA-neo-, rtTA-Ikappa B-2N-, and rtTA-Ikappa B-2NDelta 4-expressing Jurkat cells were cultured in the presence of 1 µg/ml Dox for various times at an initial cell density of 1 × 105cells/ml and then counted every other day. All cell lines grew well in the above medium with doubling times of 50 ± 4 h. Values obtained were the average of two experiments.

Analysis of Apoptosis-- To identify apoptotic cells, cells were treated with TNF after 24 h culture in the absence or presence of 1 µg/ml Dox, and stained using the TUNEL assay (Boehringer Mannheim) and Hoechst dye 33258. The mixture was then viewed under UV illumination using a Leica fluorescent microscope. To calculate percent apoptosis, a minimum of 200-400 cells were counted. Apoptosis was also analyzed by DNA fragmentation assay. ~A total of 3 × 106 cells were collected, resuspended in 0.25 ml of TBE containing 0.25% Nonidet P-40 and 0.1 mg/ml RNase A, and incubated for 30 min at 37 °C. Extracts were then treated with 1 mg/ml proteinase K for 30 min at 37 °C. DNA preparations (30 µl) were loaded on 1.8% agarose gel; DNA fragmentation was visualized under UV light.

Western Blot Analysis-- To characterize kinetics of expression, rtTA-neo, rtTA-Ikappa B-2N, and rtTA-Ikappa B-2NDelta 4 Jurkat T cells were cultured in the presence of 1 µg/ml Dox (Sigma) for various times. Cells were then washed with phosphate-buffered saline and lysed in Western Lysis Buffer (WLB) containing 10 mM Tris-Cl, pH 8.0, 60 mM KCl, 1 mM EDTA, 1 mM dithiothreitol (DTT), 0.5% Nonidet P-40, 0.5 mM phenylmethylsulfonyl fluoride (PMSF), 10 µg/ml leupeptin, 10 µg/ml pepstatin, and 10 µg/ml aprotinin. Equivalent amounts of whole cell extract (20 µg) were subject to SDS-polyacrylamide gel electrophoresis in a 10% or 15% polyacrylamide gel. After electrophoresis, the proteins were transferred to Hybond transfer membrane (Amersham Pharmacia Biotech) in a buffer containing 30 mM Tris, 200 mM glycine, and 20% methanol for 1 h. The membrane was blocked by incubation in phosphate-buffered saline (PBS) containing 5% dried milk for 1 h and then incubated overnight with N-terminal Ikappa Balpha monoclonal antibody MAD 10B (29), anti-NF-kappa B antibodies (46), p24 (ID Laboratories), or anti-actin antibody (Sigma) in 5% milk/PBS, at dilutions of 1:500 or 1:1000. These incubations were done at 4 °C overnight. After four 10-min washes with PBS, membranes were reacted with a peroxidase-conjugated secondary goat anti-rabbit or anti-mouse antibody (Amersham Pharmacia Biotech) at a dilution of 1:1000. The reaction was then visualized with the enhanced chemiluminescence detection system (ECL) as recommended by the manufacturer (Amersham Pharmacia Biotech).

RT-PCR Analysis of HIV RNA-- Total RNA was isolated from rtTA-Neo and rtTA-2NDelta 4 Jurkat cells infected with HIV in the presence or absence of Dox using the RNeasy Mini Kit (QIAGEN) and treated with 1 unit of RNase-free DNase (RQ1 DNase; Promega Biotech, Madison, WI) for 30 min at 37 °C, phenol:chloroform:isoamyl alcohol-extracted, and ethanol-precipitated. RT was performed on 2 µg of RNA and 0.2 pmol of random hexamers using 200 units of Moloney murine leukemia virus reverse transcriptase (Life Technologies, Inc., Burlington, Ontario, Canada) in buffer containing 50 mM Tris-HCl, pH 8.3, 75 mM KCl, 10 mM dithiothreitol, 3 mM MgCl2, 500 nM dNTP, 0.1 mg/ml bovine serum albumin, 272.5 units/ml RNase inhibitor (Amersham Pharmacia Biotech). PCR assays were performed using 7 µl of RT product, in PCR buffer containing 100 mM Tris-HCl, pH 8.4, 500 mM KCl, 15 mM MgCl2, 200 mM dNTP, 15 pmol of gamma -32P-labeled primers, and 1.25 units of Taq DNA polymerase (Amersham Pharmacia Biotech). Nucleotide sequences of primers used are as follows: M667, 5'-GGCTAACTAGGGAACCCACTG-3'; M668, 5'-CAGGTCCCTGTTCGGGCGCC-3'; LA45, 5'-GCCTTAGGCATCTCCTATGGC-3'; LA41, 5'-TGTCGGGTCCCCTCGTTGCTGG-3'; M669, 5'-GTGTGCCCGTCTGTTGTGTGACTCTGGTAAC-3'; LA23, 5'-GCCTATTCTGCTATGTCGACACCC-3'.

The PCR reaction mixture was subjected to 24 cycles of denaturation for 1 min at 95 °C, annealing for 2 min at 61 °C, and polymerization for 2 min at 72 °C. PCR products were then ethanol-precipitated and analyzed on a 6% denaturing polyacrylamide gel. Primers for glyceraldehyde-3-phosphate dehydrogenase were used as described previously (47) to normalize all reactions.

Analysis of HIV-1 LTR- and NF-kappa B-dependent Gene Expression-- Cells were transiently transfected with HIV LTR CAT or HIV enhancer containing CAT reporter plasmids (15 µg) by the DEAE-dextran method. In some experiments, pSVexTat plasmid (wtTat) (48) was also cotransfected together with the reporter plasmids. At 24 h after transfection, cells were incubated with or without Dox in the presence of 10 ng/ml TNF-alpha (Boehringer Mannheim) or PMA (100 ng/ml, ICN) plus PHA (1 µg/ml, ICN). At 24 h after induction, cells were harvested and lysed. Extracts (200-400 µg) were assayed for CAT activity for 4-8 h, depending on the experiment. The percent acetylation was determined by ascending thin layer chromatography as described previously (49) and quantified using the Bio-Rad Gelscan phosphor imager and the Molecular Analyst (Bio-Rad) software program.

Electrophoretic Mobility Shift Assay-- Following the addition of 1 µg/ml Dox to the culture medium for times ranging from 1 to 48 h, nuclear extracts were prepared from rtTA-neo, rtTA-Ikappa B-2N, and rtTA-Ikappa B-2NDelta 4 Jurkat T cells after induction with TNF-alpha (10 ng/ml) or PMA (100 ng/ml)/PHA (1 µg/ml) for 4 h. Cells were washed in Buffer A (10 mM HEPES, pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM dithiothreitol (DTT), and 0.5 mM phenylmethylsulfonyl fluoride (PMSF)) and were resuspended in Buffer A containing 0.1% Nonidet P-40. Cells were then chilled on ice for 10 min before centrifugation at 10,000 × g. Pellets were then resuspended in Buffer B (20 mM HEPES, pH 7.9, 25% glycerol, 0.42 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM DTT, 0.5 mM PMSF, 5 µg/ml leupeptin, 5 µg/ml pepstatin, 0.5 mM spermidine, 0.15 mM spermine, and 5 µg/ml aprotinin). Samples were incubated on ice for 15 min before being centrifuged at 10,000 × g. Nuclear extract supernatants were diluted with Buffer C (20 mM HEPES, pH 7.9, 20% glycerol, 0.2 mM EDTA, 50 mM KCl, 0.5 mM DTT, and 0.5 mM PMSF). Nuclear extracts were subjected to EMSA by using a 32P-labeled probe corresponding to two copies of the PRDII region of the IFN-beta promoter (5'-GGGAAATTCCGGGAAATTCC-3'). The resulting protein-DNA complexes were resolved by 5% Tris-glycine gel and exposed to x-ray film. To demonstrate the specificity of protein-DNA complex formation, a 125-fold molar excess of unlabeled oligonucleotide was added to the nuclear extract before adding labeled probe.

Analysis of HIV-1 Multiplication-- rtTA-neo-, rtTA-Ikappa B-2N-, and rtTA-Ikappa B-2NDelta 4-expressing Jurkat cells after preincubation with or without Dox for 24 h were infected with HIV-IIIB, derived from the HXB2D molecular clone of HIV-1 (50) in serum-free medium for 2 h at a m.o.i. of 0.01 pfu/ml and then grown in complete medium for 36 days. Cell supernatants (precleared by centrifugation at 3000 rpm for 30 min at 4 °C) were collected every 4 days and analyzed for virus RT activity as described previously (51). The relative amount of virion protein p24 present in the medium was determined by ELISA (52). Proteins were extracted from a portion of the collected cells by resuspending them in WLB. Proteins were analyzed by immunoblotting as described above using Ikappa Balpha MAD10B monoclonal antibody (29), p24-specific antibody (ID Laboratories), anti-RelA(p65) antibody (46), or actin monoclonal antibody (ICN).

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Dox-inducible Expression of TD-Ikappa Balpha Down-regulates Endogenous Ikappa Balpha Expression-- To examine the consequences of overexpression of transdominant repressors of Ikappa Balpha (TD-Ikappa Balpha ) on NF-kappa B-dependent gene expression and virus multiplication, Jurkat T cell lines were generated that inducibly expressed two forms of mutant Ikappa Balpha (TD-Ikappa Balpha ). One form was a full-length Ikappa Balpha mutated at Ser-32 and Ser-36 (termed Ikappa Balpha -2N), two sites present in the signal response domain of Ikappa Balpha required for inducible phosphorylation and degradation of Ikappa Balpha (20-22). The other form of Ikappa Balpha (Ikappa Balpha -2NDelta 4) also contained the S32A/S36A mutations, as well as a 22-amino acid deletion of the C-terminal portion of Ikappa Balpha (25, 27), a region of the PEST domain that is dispensable with regard to binding of NF-kappa B subunits but is important in Ikappa Balpha degradation (Fig. 1A). Starting with Jurkat T cells selected for expression of the reverse tetracycline transactivator protein rtTA (53), expressed under the control of the CMVt autoregulatory promoter (Fig. 1A), we isolated pools and clones of rtTA-Jurkat cells expressing Ikappa Balpha -2N and Ikappa Balpha -2NDelta 4. The first pool of Ikappa Balpha -2NDelta 4 cells was disappointing because of the high level of leakiness of the transgene (Fig. 1B, lane 6), compared with endogenous Ikappa Balpha (Fig. 1B, lanes 1-4). However, analysis of Ikappa Balpha expression revealed an interesting modulation of endogenous Ikappa Balpha protein level when Ikappa Balpha -2NDelta 4 was Dox-induced for 24 h. The level of endogenous Ikappa Balpha was decreased about 4-fold in Jurkat cells expressing Ikappa Balpha -2NDelta 4 compared with the Ikappa Balpha levels in either Jurkat or rtTA-Jurkat cells (Fig. 1B, lanes 1-4 and 6). Dox induction resulted in a 5-fold increase in Ikappa Balpha -2NDelta 4 expression after 24 h and an almost complete inhibition of endogenous Ikappa Balpha expression (Fig. 1B, lane 5). To characterize this inhibition further, a representative clone of Ikappa Balpha -2NDelta 4 Jurkat cells was Dox-induced for 3-48 h and then treated with TNFalpha (10 ng/ml) for 5 min as an inducer of Ikappa Balpha phosphorylation, following a 30-min pretreatment with calpain inhibitor I (100 µM) to block inducer-mediated degradation of Ikappa Balpha (27, 54). The different forms of Ikappa Balpha were resolved by 15% SDS-polyacrylamide gel electrophoresis; both endogenous Ikappa Balpha and the slower migrating phosphorylated form of Ikappa Balpha (P-Ikappa Balpha ) were detected by immunoblotting (Fig. 1C, lanes 1 and 2). In the Ikappa Balpha -2NDelta 4-Jurkat clone without Dox addition, the three forms of Ikappa Balpha were detected (Fig. 1C, lane 3); Dox addition for 3-6 h resulted in a 3-5-fold increase in Ikappa Balpha -2NDelta 4 (Fig. 1C, lanes 4-6), and a progressive decrease in the endogenous Ikappa Balpha forms such that, by 24 and 48 h after Dox addition, endogenous Ikappa Balpha was undetectable (Fig. 1C, lanes 4-8). Because of the S32A/S36A mutation in Ikappa Balpha -2NDelta 4, this form of Ikappa Balpha was not phosphorylated as a consequence of TNF addition (20-22). Additionally, a clone of Ikappa Balpha -2N Jurkat cells was analyzed that demonstrated properties similar to the Ikappa Balpha -2NDelta 4 Jurkat cells, although it was not possible to distinguish the endogenous from the transfected Ikappa Balpha forms (Fig. 1D, lanes 1 and 2). With this clone, the level of leakiness of the TD-Ikappa Balpha appears to be significantly lower than that observed with Ikappa Balpha -2NDelta 4, since the amounts of Ikappa Balpha in the control rtTA-Jurkat and Ikappa Balpha -2N-Jurkat cells in the absence of Dox were similar (Fig. 1D, lanes 1 and 2). Nevertheless, Dox induction resulted in a 20-25-fold increase in the overall level of Ikappa Balpha in these cells (Fig. 1D, lanes 2-7). Based on these results, we have isolated clones of Jurkat cells inducibly expressing TD-Ikappa Balpha under the control of the Tet-responsive promoter. Furthermore, Dox induction of TD-Ikappa Balpha resulted in the inhibition of endogenous Ikappa Balpha , consistent with the fact that the Ikappa Balpha gene is tightly regulated by an NF-kappa B-dependent mechanism (32).


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Fig. 1.   Schematic summary of the TD-Ikappa Balpha -expressing CMVt-Ikappa Balpha vectors and inducibility of TD-Ikappa Balpha -expressing Jurkat cells. A, the five ankyrin repeats of Ikappa Balpha are indicated by the open boxes. Two phosphorylation sites at Ser-32 and Ser-36 (indicated by triangles) in the N-terminal region of Ikappa Balpha required for inducer-mediated degradation of Ikappa Balpha . Within the highly acidic C-terminal region, several potential casein kinase II (CKII) phosphorylation sites are clustered around Ser-283, Thr-291, and Thr-299. Ikappa Balpha -2NDelta 4 contains a 22-amino acid C-terminal deletion of Ikappa Balpha -2N in which Ser-32 and Ser-36 are mutated to Ala. B, Jurkat, rtTA-Jurkat, and rtTA-Ikappa Balpha -2NDelta 4 Jurkat cells (pool of 50 clones) were collected at 24 h after Dox addition, and the effect of Dox on Ikappa Balpha expression was examined by immunoblotting using the Ikappa Balpha MAD10B antibody. C, rtTA-Ikappa Balpha -2NDelta 4 cells from a representative clone were incubated with Dox for different intervals (0-48 h) and then treated with calpain inhibitor (100 µM) for 30 min and subsequently induced with TNF (10 ng/ml) for 5 min; expression of Ikappa Balpha was analyzed by immunoblotting using the Ikappa Balpha MAD10B antibody. D, rtTA-Ikappa Balpha -2N Jurkat cells from a representative clone were harvested after 0-48 h of Dox addition and similarly analyzed by immunoblotting for Ikappa Balpha expression. Phosphorylated Ikappa Balpha (P-Ikappa Balpha ), endogenous Ikappa Balpha (Ikappa Balpha ), and Ikappa Balpha -2NDelta 4 (2NDelta 4) are indicated by arrows.

Growth of TD-Ikappa Balpha -expressing Cells and Induction of Apoptosis-- All stably transfected TD-Ikappa Balpha -expressing clones grew well in 10% serum, and growth was not dramatically retarded as a consequence of Dox addition or, in the case of Ikappa Balpha -2N or Ikappa Balpha -2NDelta 4-Jurkat cells, by up-regulation of TD-Ikappa Balpha expression (doubling time of 50 ± 4 h). Based on recent observations that NF-kappa B may play a protective role in apoptotic cell death (55-57), the response of control and TD-Ikappa Balpha -expressing Jurkat cells to TNF-induced apoptosis was examined by TUNEL assay. At 16 h after TNF (100 ng/ml) treatment in Dox-induced cells, 65-80% of the TD-Ikappa Balpha -expressing cells were undergoing apoptotic cell death, whereas in Dox-induced TD-Ikappa Balpha -expressing cells without TNF treatment, less than 2% of the cells were apoptotic (Table I). Similarly, TNF treatment alone resulted in 2% apoptosis in TD-Ikappa Balpha -expressing cells and 6% apoptotic cells in rtTA-Neo Jurkat cells (Table I). Dox induction of TD-Ikappa Balpha expression resulted in an increased sensitivity to TNF-induced apoptosis, as detected by DNA fragmentation analysis at 8 or 16 h after TNF addition in the TD-Ikappa Balpha -expressing cells but not in the control rtTA-Neo cells (data not shown). It appears that Dox induction of TD-Ikappa Balpha expression does not lead to apoptosis per se but, instead, dramatically sensitizes Jurkat cells to TNF-induced apoptosis. This observation may explain the observed selection against cells that constitutively express TD-Ikappa Balpha (see below).

                              
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Table I
TNFalpha -induced apoptosis in TD-Ikappa Balpha -expressing Jurkat cells
rtTA-Neo-, Ikappa Balpha -2N-, and Ikappa Balpha -2NDelta 4-expressing Jurkat cells were treated with Dox (1 µg/ml) for 24 h and then induced with TNFalpha (10 ng/ml) for 16 h. Cells were fixed and stained for TUNEL (Boehringer Mannheim) and with Hoechst 33258. The percentage of total cells staining positive by TUNEL assay was determined by counting 200-400 cells/sample. The values are presented as a percentage of positive cells by TUNEL divided by the total number of Hoechst 33258-stained nuclei.

Inhibition of NF-kappa B DNA Binding Activity and Gene Expression by TD-Ikappa Balpha -- The induction of NF-kappa B DNA binding activity in control and TD-Ikappa Balpha -expressing cells was examined following treatment of cells with several different inducers (Fig. 2). Treatment of rtTA-neo-Jurkat (Fig. 2A) or rtTA-Jurkat (data not shown) with TNFalpha or PMA/PHA for 4 h resulted in a strong induction of NF-kappa B DNA binding activity, irrespective of Dox treatment for 6-48 h (Fig. 2A, lanes 5-14). In contrast, TNFalpha induction of NF-kappa B activity in Ikappa Balpha -2NDelta 4 Jurkat cells (Fig. 2B, lanes 5 and 6) was completely blocked by the induction of Ikappa Balpha -2NDelta 4 for 6 h or longer (Fig. 2B, lanes 6-9); the strong induction of NF-kappa B binding activity by PMA/PHA was also more than 95% inhibited by Dox activation of Ikappa Balpha -2NDelta 4 (Fig. 2B, lanes 10-14). Similarly, Dox induction of Ikappa Balpha -2N also inhibited completely the induction of NF-kappa B binding activity by TNFalpha (Fig. 2C, lanes 2-6) and PMA/PHA (Fig. 2C, lanes 7-12). In a subsequent experiment, the kinetics of activation of Ikappa Balpha -2NDelta 4 and the inhibition of NF-kappa B binding activity were examined (Fig. 2, D and E). Surprisingly, as early as 1 h after Dox addition, expression of Ikappa Balpha -2NDelta 4 was up-regulated (Fig. 2E, lanes 1 and 2) and NF-kappa B DNA binding activity was inhibited (Fig. 2D, lanes 1 and 2). Interestingly, comparison of the levels of Ikappa Balpha -2NDelta 4 and endogenous Ikappa Balpha revealed that constitutive expression of Ikappa Balpha -2NDelta 4 was dramatically decreased with time in culture (compare lane 6 in Fig. 1B and lane 1 in Fig. 2E), whereas the cells remained highly inducible in response to Dox addition (Fig. 2E, lanes 2-9). Low background, high inducibility of Ikappa Balpha -2N was also observed in Ikappa Balpha -2N-Jurkat cells, based on the resistance of the induced Ikappa Balpha to TNFalpha -induced degradation (data not shown). TD-Ikappa Balpha -expressing Jurkat cells were also resistant to induction of NF-kappa B binding activity by double-stranded RNA (poly(I·C)) treatment (data not shown). Thus, overexpression of TD-Ikappa Balpha blocks the induction of NF-kappa B DNA binding activity by multiple inducers. Additionally, continued growth in culture selects for Jurkat cells that display low constitutive expression of TD-Ikappa Balpha , possibly due to an increased sensitivity of TD-Ikappa Balpha -expressing cells to apoptosis.


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Fig. 2.   NF-kappa B DNA binding activity in control and TD-Ikappa Balpha -expressing Jurkat T cells treated with TNF or PMA/PHA. rtTA-Neo (A), Ikappa Balpha -2NDelta 4 (B, D, and E), and rtTA-2N (C) Jurkat cells were incubated with Dox for different times as indicated in the figure and subsequently induced by TNF (10 ng/ml) or PMA/PHA (100 ng/ml and 1 µg/ml, respectively) for 4 h. EMSA was performed on nuclear extracts (5 µg) prepared from cells. Ikappa Balpha immunoblot analysis (E) was performed on whole cell extracts (20 µg) prepared from Ikappa Balpha -2NDelta 4 cells induced by Dox for different times and subsequently treated with PMA/PHA for 4 h. Endogenous Ikappa Balpha and transfected Ikappa Balpha -2NDelta 4 are indicated by arrows. Control lanes (C) in panels A, B, C, and D represent the competition of NF-kappa B-DNA complex formation using a 125-fold excess of unlabeled PRDII probe.

To complement the observation that overexpression of TD-Ikappa Balpha inhibited NF-kappa B binding activity, the effect of TD-Ikappa Balpha on NF-kappa B-dependent reporter gene expression was also examined. A CAT reporter gene driven by the SV40 minimal promoter, linked to the HIV-1 -109 to -79 region of the LTR, was transfected into Ikappa Balpha -2NDelta 4-expressing Jurkat cells together with a control plasmid mutated in the two NF-kappa B binding sites of the HIV-1 LTR (41). As shown in Fig. 3A, treatment of these cells at 24 h after transfection with PMA/PHA (24 h) resulted in a 4-fold stimulation of reporter gene activity above background level with the wild type but not mutated promoter; Dox addition simultaneously with PMA/PHA inhibited NF-kappa B-dependent induction of reporter gene activity. The HIV-1 LTR-CAT reporter plasmid was also transfected into Ikappa Balpha -2NDelta 4-expressing Jurkat cells, and the effect of TD-Ikappa Balpha induction on the Tat-TNF synergistic activation of the HIV-1 LTR was examined (38) (Fig. 3B). In this experiment, Tat-TNF activation of the LTR represented an 80-fold activation of gene activity that was inhibited more than 5-fold with Dox-induced Ikappa B expression; the residual LTR reporter gene activity probably represents NF-kappa B independent activation of the HIV-1 LTR. Co-expression of Tat protein also stimulated the HIV-1 LTR about 10-fold; somewhat surprisingly, expression of the TD-Ikappa Balpha also inhibited induction of the HIV-1 LTR mediated by Tat alone (Fig. 3B).


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Fig. 3.   Inhibition of Tat-TNF, Tat, or PMA/PHA-induced HIV-1 LTR and HIV-enhancer activation by Ikappa Balpha mutants. A, rtTA-Ikappa Balpha -2NDelta 4 Jurkat cells were transfected with HIV-enh () or HIV-enh mut CAT (square ) reporter plasmid (10 µg); at 24 h after transfection, cells were treated with PMA/PHA or PMA/PHA + Dox for an additional 24 h. B, rtTA-Ikappa Balpha -2NDelta 4 Jurkat cells were transfected with pTZIIICAT (10 µg) and in some cases with pSVexTat (10 µg); at 24 h after transfection, some cells were treated with TNFalpha (10 ng/ml) alone, with TNFalpha  + Dox (), or with Dox alone (). The level of HIV-1 LTR-driven reporter gene expression was determined by CAT assay on the whole cell extracts (200 µg), assayed for 2 h. The negative control (lane -) was obtained by transfection with the HIV-1 LTR reporter construct only without TNF induction.

Inhibition of de Novo HIV-1 Multiplication in TD-Ikappa Balpha -expressing Jurkat Cells-- Given the involvement of NF-kappa B in the early transcriptional control of HIV-1 LTR gene expression (reviewed in Ref. 1), the impact of TD-Ikappa Balpha expression on the course of de novo HIV-1 protein synthesis and virus production was next examined. Control and TD-Ikappa Balpha -expressing Jurkat cells after preincubation with or without Dox for 24 h were infected with the HIV-1 strain IIIB (derived from the molecular clone HXB2D) at an m.o.i. of 0.01 pfu/ml and HIV-1 infection was monitored by RT assay, p24 ELISA, and p24 antigen accumulation over periods varying from 16 days to 36 days depending on the infection. Both RT (Fig. 4A) and p24 ELISA analyses (Fig. 4B) demonstrated that continuous Dox-induced expression of Ikappa Balpha -2NDelta 4 resulted in a dramatic delay in the onset of HIV-1 multiplication. In control rtTA-Neo-Jurkat cells, RT and p24 expression were detected as early as 8 days post-infection (p.i.), whereas in Ikappa Balpha -2NDelta 4 cells infection was first detected at day 12; addition of Dox at different times during the infection delayed the onset of detectable HIV-1 multiplication until 16-20 days (Fig. 4, A and B). The most effective inhibitory regimen was Dox addition 24 h before infection (day -1) and subsequent Dox addition to the medium at days 8, 18, and 24. One addition of Dox at day -1 or addition of Dox at day 12 also effectively blocked the onset of HIV-1 replication. However, with these treatments, breakthrough of HIV-1 multiplication was observed at day 36, as detected by RT and p24 ELISA. On the other hand, intermittent replenishment of Dox in the medium dramatically repressed HIV-1 RT and p24 expression throughout the course of infection (Fig. 4, A and B).


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Fig. 4.   Suppression of HIV-1 infection in Ikappa Balpha -2NDelta 4. rtTA-Neo and rtTA-Ikappa Balpha -2NDelta 4 Jurkat cells were infected with HIV-IIIB for 2 h at an m.o.i. of 0.01 pfu/ml. Cells were then grown for 36 days and HIV-1 infection was monitored by RT assay (A, 200 µl) and p24 ELISA (B, 50 µl). bullet , rtTA-Neo + Dox; open circle , Ikappa Balpha -2NDelta - Dox; triangle , Ikappa Balpha -2NDelta - Dox (+Dox12, Dox addition 12 days after infection); square , rtTA-2NDelta 4 + Dox (Dox addition 24 h before infection); diamond , rtTA-2NDelta 4 + Dox (continuous addition of Dox at -1, 8, 18, and 24 days after infection).

The intracellular accumulation of p24 antigen was also monitored during the course of de novo HIV-1 infection, together with the expression of Ikappa Balpha and beta -actin (Fig. 5). p24 accumulation was weakly detected as early as 4 day p.i. (Fig. 5A, lane 2); high level expression was detected thereafter throughout the course of infection (Fig. 5A, lanes 3-9) in HIV-1-infected rtTA-Neo-Jurkat cells. Also with the analysis of HIV-1 replication in Jurkat cells, a decrease in the level of Ikappa Balpha was detected during the exponential phase of virus multiplication at days 12-20 (Fig. 5A, lanes 4-6), reflecting the HIV-1-mediated degradation of Ikappa Balpha , an effect that contributes to constitutive NF-kappa B DNA binding activity in HIV-1-infected cells (39-43). In the Ikappa Balpha -2NDelta 4-expressing cells, the appearance of p24 antigen was delayed until day 12 after infection and peaked at day 20-24 p.i. (Fig. 5B, lanes 4-7). In the TD-Ikappa Balpha -expressing cells, the decrease in Ikappa Balpha was also delayed until later times after infection. Again, the basal level of Ikappa Balpha transgene was significantly reduced relative to endogenous Ikappa Balpha , reflecting the ongoing selection of low background expressing cells (Fig. 2E). However, Dox addition to the infected cell culture at day 12 p.i. resulted in a dramatic increase in Ikappa Balpha -2NDelta 4 expression (Fig. 5C, lanes 4 and 5); this time of addition was not sufficient to inhibit the appearance of p24 antigen at day 12 (Fig. 5C, lane 4). However, induction of TD-Ikappa Balpha completely inhibited endogenous Ikappa Balpha expression by day 16 (Fig. 5C, lane 5) and partially blocked the subsequent intracellular accumulation of p24 antigen (Fig. 5, compare B and C, lanes 5-9). Addition of Dox to TD-Ikappa Balpha -expressing Jurkat cells 24 h before infection (Fig. 5D) or intermittent Dox addition at days -1, 8, 18, and 24 (Fig. 5E) delayed the onset of detectable p24 antigen until day 20 p.i. (Fig. 5, D and E, lane 6). In contrast to the results obtained by RT and p24 ELISA assays, both one-time addition and intermittent addition of Dox delayed the accumulation of intracellular p24 antigen to an equivalent extent. Overall, Dox induction of TD-Ikappa Balpha expression resulted in a 10-20-fold decrease in the production of p24 antigen (Fig. 5), as well as inhibition of HIV-1 virion release (Fig. 4). Similar results were obtained using the Ikappa Balpha -2N-expressing cells (data not shown), thus demonstrating a dramatic inhibitory effect of TD-Ikappa Balpha on the course of de novo HIV-1 infection in Jurkat T cells.


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Fig. 5.   Inhibition of viral p24 protein expression by Ikappa Balpha -2NDelta 4 mutant. rtTA-Neo and rtTA-Ikappa Balpha -2NDelta 4 Jurkat cells were incubated in the presence or absence of Dox and then infected with HIV-IIIB for 2 h at an m.o.i. of 0.01 pfu/ml. Cells were collected every 4 days for 36 days, and whole cell extracts (20 µg) were analyzed by immunoblotting. rtTA-Neo Jurkat cells (panel A) and rtTA-Ikappa Balpha -2NDelta 4Jurkat cells (panels D and E) were treated with Dox for 24 h prior to infection or not treated (panel B). rtTA-Ikappa Balpha -2NDelta 4Jurkat cells (panels B and C) were infected without Dox pretreatment, but Dox was added 12 days after the infection (panel C). Dox was also intermittently added at 8, 18, and 24 days after infection in panel E. Endogenous Ikappa Balpha (wtIkappa Balpha ), Ikappa Balpha -2NDelta 4 (2NDelta 4), RelA(p65), viral p24, and actin proteins were detected by immunoblotting as described under "Materials and Methods" and are indicated by arrows.

Decreased Levels of HIV-1 mRNA in TD-Ikappa B-expressing Jurkat Cells-- To link the inhibition of HIV-1 multiplication with NF-kappa B inhibitory effects of TD-Ikappa B, the effects of TD-Ikappa B induction on HIV-1-induced NF-kappa B binding activity were evaluated by mobility shift analysis. Virus-induced activation of NF-kappa B was blocked by TD-Ikappa B expression (data not shown), thus complementing the results described in Fig. 2, and confirming that TD-Ikappa B interfered with HIV-induced NF-kappa B binding. To further characterize the block in HIV-1 multiplication, accumulation of HIV-1 viral RNA species was evaluated by semi-quantitative RT-PCR, using the primers and procedures described previously (58). The primer pair M667/M668 detected total viral RNA with a fragment length of 161 bp; M669/LA23 identified a fragment of 214 bp, which represented singly spliced (env) and doubly spliced tat/rev RNA; and LA41/LA45 amplified a fragment of 123 bp, corresponding to doubly spliced tat/rev RNA (Fig. 6A). Viral RNA species in rtTA-Neo-Jurkat and rtTA-2NDelta 4-Jurkat cells were detected at different times after de novo HIV infection in the presence or absence of Dox-induced TD-Ikappa B (Fig. 6B). In rtTA-Neo Jurkat cells, HIV mRNA transcripts were detected as early as 2-4 days after infection (Fig. 6B, lanes 2, 3, 7, and 8) and their levels increased at days 6 and 10 post-infection (Fig. 6B, lanes 4, 5, 9, and 10), regardless of Dox addition. In rtTA-2NDelta 4 Jurkat cells, without Dox addition, a low level of full-length viral RNA was detected at day 2 (Fig. 6B, lane 12, upper panel) and by day 4 unspliced and spliced viral RNA was detected (Fig. 6B, lane 13) and accumulated thereafter at days 6 and 10 (Fig. 6B, lanes 14 and 15). However, with Dox induction of TD-Ikappa B, viral RNA levels were reduced (Fig. 6B, lanes 17-20). Full-length RNA was detected at day 2 (Fig. 6B, lane 17, upper panel), but the overall levels of unspliced and spliced RNA were decreased at days 6 and 10, relative to the levels detected in the absence of Dox or in rtTA-Neo cells. Strikingly, doubly spliced tat/rev mRNA was not detected in rtTA-2NDelta 4 Jurkat cells treated with Dox (Fig. 6B, lanes 18-20, lower panel). These results demonstrate a transcriptional inhibitory effect of TD-Ikappa B expression on HIV mRNA accumulation in Jurkat cells engineered to express TD-Ikappa B.


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Fig. 6.   Suppression of HIV-1 transcription in Ikappa Balpha -2NDelta 4-expressing Jurkat cells. A, schematic representation of full-length and spliced mRNAs (env, tat/rev) of HIV-1 with specific primers used for RT-PCR analysis (58). M667/M668 detects both full-length and spliced mRNAs and generates a 161-bp fragment. The primer pair M669/LA23 detects only spliced mRNA (env, tat/rev) and produces a 214-bp fragment. tat/rev mRNA is detected by LA41/LA45 primer pair, and a 123-bp fragment is generated following PCR. B, RT-PCR analysis of HIV-1 RNA. rtTA-Neo and rtTA-Ikappa Balpha -2NDelta 4 Jurkat cells were incubated in the presence or absence of Dox and then infected with HIV-IIIB for 2 h at an m.o.i. of 0.01 pfu/ml. Cells were collected 2, 4, 6, and 10 days after infection, and total RNA (2 µg) was analyzed by RT-PCR, as described under "Materials and Methods." All results were normalized to the levels of glyceraldehyde-3-phosphate dehydrogenase using a Bio-Rad GS250 phosphor imager and the NIH Image densitometric analysis program version 1.6.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

The present studies describe, for the first time, the selection of Jurkat T clones engineered to inducibly express two transdominant repressor forms of Ikappa Balpha under the control of a Tet-responsive promoter and the first characterization of the impact of TD-Ikappa B repressors on the course of de novo HIV-1 infection. We demonstrate that: 1) doxycycline-inducible expression of Ikappa Balpha -2N and Ikappa Balpha -2NDelta 4 was detected as early as 1 h after Dox addition and blocked the induction of NF-kappa B DNA binding by multiple inducers including TNFalpha , PMA/PHA, and double-stranded RNA; 2) expression of TD-Ikappa Balpha also repressed the expression of the endogenous Ikappa Balpha gene, consistent with the observation that the Ikappa Balpha gene contains multiple NF-kappa B binding sites in its promoter and is tightly regulated by NF-kappa B (32); 3) NF-kappa B-dependent reporter gene activity was also inhibited following the induction of TD-Ikappa Balpha ; and 4) induced expression of the TD-Ikappa Balpha successfully inhibited de novo HIV-1 infection in Jurkat cells as measured by RT assay, p24 antigen accumulation, and viral RNA analysis by RT-PCR. During these studies, the levels of CD4 and fusin were also measured by flow cytometry to determine if TD-Ikappa Balpha expression repressed cell surface expression of the HIV-1 co-receptors (59); no modulation of either CD4 or fusin levels was observed after 48-96 h of Dox-induced TD-Ikappa Balpha expression.2 Thus, HIV-1 infection was not inhibited at the level of virus attachment and entry.

Our results are complementary to the recent studies of Chen et al. (37), demonstrating the importance of NF-kappa B sites in enhancing the growth of primary HIV-1 isolates; nonetheless, the virus was still able to grow slowly in the absence of NF-kappa B sites. Similarly, conditional inhibition of NF-kappa B in Jurkat T cells by an Ikappa Balpha repressor significantly interfered with virus multiplication, but could not suppress HIV-1 growth indefinitely. Many earlier studies in lymphoid cells have also shown that mutation of NF- kappa B motifs reduced gene expression in the presence or absence of HIV-1 Tat (see Refs. 36-38, and references therein). Strikingly different requirements for maximal LTR activation were observed in primary CD4+ T cells and the J-Jhan T lymphoid cells line (36). In unstimulated CD4+ T lymphocytes, a low basal level of LTR activity was detected, whereas, in the lymphoblastoid cell line, a high spontaneous level of LTR activity was found that was essentially independent of the NF-kappa B responsive elements. In lymphoblastoid cell lines, HIV infection resulted in active replication in the absence of other stimuli, whereas, in primary T cells, replication was dependent upon T cell activation for triggering of viral replication (36).

In previous studies, the effect of transdominant repressors of Ikappa Balpha on the synergistic activation of the HIV-1 LTR by TNFalpha and the HIV-1 transactivator, Tat, was examined in transiently transfected Jurkat T cells (38). Co-expression of Ikappa Balpha inhibited Tat-TNF activation of HIV LTR in a dose-dependent manner, and transdominant repressor forms of Ikappa Balpha , mutated in serine or threonine residues required for inducer-mediated (S32A/S36A-2N) or constitutive phosphorylation (S283A/T291A/T299A-3C) of Ikappa Balpha , possessed different inhibitory potentials for the HIV-1 LTR. Surprisingly, Ikappa Balpha -2N (but not Ikappa Balpha -2N+3C) was more effective in blocking HIV-1 protein and RNA synthesis in a single cycle infection than wtIkappa Balpha . The observation that mutations in the C-terminal PEST domain of Ikappa Balpha decreased the inhibitory potential of Ikappa Balpha -2N suggested that an intact C terminus was required for maximal inhibition of HIV-1 multiplication by Ikappa Balpha -2N and may reflect a distinct functional activity for the Ikappa Balpha C terminus (38). Although the above studies suggest a transcriptional role for Ikappa Balpha in the inhibition of HIV-1 LTR gene expression, Ikappa Balpha may act at a distinct level in the HIV-1 life cycle, at the post-transcriptional level of Rev function (60, 61). HIV Rev contains an RNA binding domain, required for interaction with HIV-1 RNA, and an effector domain, required for RNA-bound Rev to function in export. The Rev effector domain contains a nuclear export signal (NES) and interacts with the nucleoporin Rab/Rip, a protein that mediate nucleocytoplasmic transport (62-64). Ikappa Balpha also contains a consensus NES element at amino acids 264-281, matching the NES consensus within Rev (65). Furthermore, newly synthesized Ikappa B can localize to the nucleus, dissociate NF-kappa B-DNA complexes, and translocate back to the cytoplasm (66). Thus, an additional function of Ikappa B is to serve as a shuttle protein to export NF-kappa B from the nucleus. One possible explanation for the inhibitory activity of TD-Ikappa B in HIV-1 infection is that the stable form of Ikappa B competes effectively for the nuclear export pathway utilized by Rev/Rab.

HIV-1 infection also causes constitutive activation of NF-kappa B DNA-binding activity in infected cells (1). A direct temporal correlation exists between HIV infection and the appearance of NF-kappa B DNA-binding activity in myeloid cells (39-43, 67), which may in turn prime or stimulate cytokine release (59). HIV-1-induced cytokine release may account for the elevated levels of multiple cytokines and chemokines present in the sera of AIDS patients in late stage disease and may exacerbate symptoms (reviewed in Ref. 1). Preliminary data suggest that TD-Ikappa Balpha also interferes with HIV-1 infection at the level of expression of HIV-1-induced inflammatory cytokines.3

Recently, NF-kappa B activation was shown to play a protective function in the response to TNFalpha -, ionizing irradiation- and daunorubicin-induced apoptosis (55-57). Since apoptosis has been suggested to be one of the major mechanisms of CD4+ T cell depletion in HIV-1-infected patients (68-71) and because TNFalpha plasma levels correlate with disease progression (1), the role of NF-kappa B in HIV-1-induced apoptosis was examined (72). These studies demonstrated that constitutive NF-kappa B activation is required to counteract a persistent apoptotic signal resulting from HIV-1 infection; thus, a previously unrecognized role for constitutive NF-kappa B activation in HIV-1-infected cells is to protect from virus-mediated apoptotic cell death. Similarly, activation of TD-Ikappa Balpha sensitized Jurkat cells to TNF-induced cell death (Table I). Thus, given the multiple functions of the NF-kappa B/Ikappa B transcription factors, Dox-induced TD-Ikappa Balpha expression may interfere with HIV-1 multiplication at several levels: LTR-mediated transcription, Rev-mediated export of viral RNA, inhibition of HIV-1-induced pro-inflammatory cytokines, and increased sensitivity of HIV-1-infected cells to apoptosis. Experiments are in progress to assess the relative contribution of these processes to the capacity of TD-Ikappa Balpha molecules to interfere with HIV-1 multiplication.

    ACKNOWLEDGEMENTS

We thank Dr. Ron Hay for the MAD10B antibody and members of the McGill AIDS Center for helpful discussions.

    FOOTNOTES

* This work was supported by a grant from the Medical Research Council of Canada (to J. H. and M. A. W.), a grant from the National Cancer Institute (to J. H.), a grant from the Canadian Foundation for AIDS Research (to J. H. and M. A. W.), a FCAR studentship (to H. K.), a NHRDP studentship (to C. D. L.), a Fraser Monat McPherson fellowship from McGill University (to R. L.), and a Medical Research Council Scientist award (to J. H.).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.

** 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.

1 The abbreviations used are: HIV-1, human immunodeficiency virus type 1; LTR, long terminal repeat; TNF, tumor necrosis factor; RT, reverse transcription; PCR, polymerase chain reaction; ELISA, enzyme-linked immunosorbent assay; pfu, plaque-forming unit(s); m.o.i., multiplicity of infection; PMA, phorbol 12-myristate 13-acetate; PHA, phytohemagglutinin; Dox, doxycycline; CMV, cytomegalovirus; FBS, fetal bovine serum; DTT, dithiothreitol; PMSF, phenylmethylsulfonyl fluoride; PBS, phosphate-buffered saline; CAT, chloramphenicol acetyltransferase; bp, base pair(s); p.i., post-infection; NES, nuclear export signal; TD-Ikappa Balpha , transdominant repressor of Ikappa Balpha .

2 H. Kwon, data not shown.

3 H. Kwon and P. Genin, unpublished data.

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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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