INTRODUCTION

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NF-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- B activity is regulated in part at the level of subcellular localization. In unstimulated cells, NF-B heterodimers are retained in the cytoplasm by inhibitory IB proteins (4, 5), also a family of proteins that include IB (6), IB (7), IB (8), IB (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, IB is rapidly phosphorylated on Ser-32 and Ser-36; N-terminal phosphorylation of IB 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 IB, and blocked the activation of NF-B (20-22). These mutations also blocked in vitro ubiquitination of the IB protein (19, 23, 24). Although the N terminus of IB is necessary for signal-induced degradation, degradation of IB also requires the C-terminal PEST domain of the protein (25-30). Once released, NF-B is able to activate target genes until new IB is synthesized. Since IB contains NF-B binding sites in its promoter, NF-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-B/Rel pathway plays a central role in HIV-1 LTR-driven transcription. The HIV-1 LTR contains two adjacent high affinity NF-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-B DNA binding activity with stimulators such as TNF and interleukin-1 or upon co-infection with other pathogens (reviewed in Refs. 1 and 2).

The dependence of HIV-1 on its NF-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-B expression (1, 39-41), increased proteasome-mediated turnover of IB, and elevated expression of NF-B1, NF-B2, and c-Rel proteins (42, 43). This modulation of intracellular NF-B levels may contribute to enhanced NF-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-B and in primary T cells by infecting with wild type virus or virus containing NF-B enhancer site mutations. These studies demonstrated that NF-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 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-B sites (37). Interestingly, pathogenic viral isolates with NF-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-B sites in vivo (37).

In the present study, we generated Jurkat T cell lines that inducibly express transdominant repressors of IB (TD-IB), or IB super-repressors, and examined the effect of Tet-inducible expression of (TD-IB) on NF-B DNA binding activity, NF-B-dependent gene expression and de novo HIV-1 multiplication. The time course of de novo HIV-1 infection in TD-IB-expressing Jurkat cells was altered by doxycycline (Dox) induction of TD-IB, 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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Generation of Plasmids-- CMV_t-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 CMV_t from the CMV_tBL 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 CMV_t 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 CMV_t BL (contains the CMV_t promoter) was cloned into the EcoRI (blunt)/NotI sites of neo BL. CMV_t-IB2N and IB2N4 were constructed by cloning an EcoRV (blunt)/BamHI IB-2N mutant cDNA fragment downstream of CMV_t at the EcoRI (blunt, filled with Klenow enzyme)/BamHI site of neo CMV_t BL.

Cell Culture and Generation of IB-expressing Cell Lines-- Jurkat cells were transfected with CMV_t-rtTA plasmid by the DEAE-dextran method. The precipitated CMV_t-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 CMV_t-rtTA plasmid (rtTA-Jurkat cells) were then transfected with CMV_t-Neo, CMV_t-2N, and CMV_t-2N4 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-IB-2N-, and rtTA-IB-2N4-expressing Jurkat cells were analyzed for inducible IB expression; subsequently, 6-10 individual clones from each transformant pool were selected for further analysis. To analyze growth kinetics, rtTA-neo-, rtTA-IB-2N-, and rtTA-IB-2N4-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-IB-2N, and rtTA-IB-2N4 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 IB monoclonal antibody MAD 10B (29), anti-NF-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-2N4 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 -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'.

Analysis of HIV-1 LTR- and NF-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- (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-IB-2N, and rtTA-IB-2N4 Jurkat T cells after induction with TNF- (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- 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-IB-2N-, and rtTA-IB-2N4-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 IB MAD10B monoclonal antibody (29), p24-specific antibody (ID Laboratories), anti-RelA(p65) antibody (46), or actin monoclonal antibody (ICN).

	RESULTS

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Dox-inducible Expression of TD-IB Down-regulates Endogenous IB Expression-- To examine the consequences of overexpression of transdominant repressors of IB (TD-IB) on NF-B-dependent gene expression and virus multiplication, Jurkat T cell lines were generated that inducibly expressed two forms of mutant IB (TD-IB). One form was a full-length IB mutated at Ser-32 and Ser-36 (termed IB-2N), two sites present in the signal response domain of IB required for inducible phosphorylation and degradation of IB (20-22). The other form of IB (IB-2N4) also contained the S32A/S36A mutations, as well as a 22-amino acid deletion of the C-terminal portion of IB (25, 27), a region of the PEST domain that is dispensable with regard to binding of NF-B subunits but is important in IB 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 CMV_t autoregulatory promoter (Fig. 1A), we isolated pools and clones of rtTA-Jurkat cells expressing IB-2N and IB-2N4. The first pool of IB-2N4 cells was disappointing because of the high level of leakiness of the transgene (Fig. 1B, lane 6), compared with endogenous IB (Fig. 1B, lanes 1-4). However, analysis of IB expression revealed an interesting modulation of endogenous IB protein level when IB-2N4 was Dox-induced for 24 h. The level of endogenous IB was decreased about 4-fold in Jurkat cells expressing IB-2N4 compared with the IB levels in either Jurkat or rtTA-Jurkat cells (Fig. 1B, lanes 1-4 and 6). Dox induction resulted in a 5-fold increase in IB-2N4 expression after 24 h and an almost complete inhibition of endogenous IB expression (Fig. 1B, lane 5). To characterize this inhibition further, a representative clone of IB-2N4 Jurkat cells was Dox-induced for 3-48 h and then treated with TNF (10 ng/ml) for 5 min as an inducer of IB phosphorylation, following a 30-min pretreatment with calpain inhibitor I (100 µM) to block inducer-mediated degradation of IB (27, 54). The different forms of IB were resolved by 15% SDS-polyacrylamide gel electrophoresis; both endogenous IB and the slower migrating phosphorylated form of IB (P-IB) were detected by immunoblotting (Fig. 1C, lanes 1 and 2). In the IB-2N4-Jurkat clone without Dox addition, the three forms of IB were detected (Fig. 1C, lane 3); Dox addition for 3-6 h resulted in a 3-5-fold increase in IB-2N4 (Fig. 1C, lanes 4-6), and a progressive decrease in the endogenous IB forms such that, by 24 and 48 h after Dox addition, endogenous IB was undetectable (Fig. 1C, lanes 4-8). Because of the S32A/S36A mutation in IB-2N4, this form of IB was not phosphorylated as a consequence of TNF addition (20-22). Additionally, a clone of IB-2N Jurkat cells was analyzed that demonstrated properties similar to the IB-2N4 Jurkat cells, although it was not possible to distinguish the endogenous from the transfected IB forms (Fig. 1D, lanes 1 and 2). With this clone, the level of leakiness of the TD-IB appears to be significantly lower than that observed with IB-2N4, since the amounts of IB in the control rtTA-Jurkat and IB-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 IB in these cells (Fig. 1D, lanes 2-7). Based on these results, we have isolated clones of Jurkat cells inducibly expressing TD-IB under the control of the Tet-responsive promoter. Furthermore, Dox induction of TD-IB resulted in the inhibition of endogenous IB, consistent with the fact that the IB gene is tightly regulated by an NF-B-dependent mechanism (32).

View larger version (22K): [in this window] [in a new window]   Fig. 1.   Schematic summary of the TD-IB-expressing CMVt-IB vectors and inducibility of TD-IB-expressing Jurkat cells. A, the five ankyrin repeats of IB are indicated by the open boxes. Two phosphorylation sites at Ser-32 and Ser-36 (indicated by triangles) in the N-terminal region of IB required for inducer-mediated degradation of IB. 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. IB-2N4 contains a 22-amino acid C-terminal deletion of IB-2N in which Ser-32 and Ser-36 are mutated to Ala. B, Jurkat, rtTA-Jurkat, and rtTA-IB-2N4 Jurkat cells (pool of 50 clones) were collected at 24 h after Dox addition, and the effect of Dox on IB expression was examined by immunoblotting using the IB MAD10B antibody. C, rtTA-IB-2N4 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 IB was analyzed by immunoblotting using the IB MAD10B antibody. D, rtTA-IB-2N Jurkat cells from a representative clone were harvested after 0-48 h of Dox addition and similarly analyzed by immunoblotting for IB expression. Phosphorylated IB (P-IB), endogenous IB (IB), and IB-2N4 (2N4) are indicated by arrows.

Growth of TD-IB-expressing Cells and Induction of Apoptosis-- All stably transfected TD-IB-expressing clones grew well in 10% serum, and growth was not dramatically retarded as a consequence of Dox addition or, in the case of IB-2N or IB-2N4-Jurkat cells, by up-regulation of TD-IB expression (doubling time of 50 ± 4 h). Based on recent observations that NF-B may play a protective role in apoptotic cell death (55-57), the response of control and TD-IB-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-IB-expressing cells were undergoing apoptotic cell death, whereas in Dox-induced TD-IB-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-IB-expressing cells and 6% apoptotic cells in rtTA-Neo Jurkat cells (Table I). Dox induction of TD-IB 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-IB-expressing cells but not in the control rtTA-Neo cells (data not shown). It appears that Dox induction of TD-IB 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-IB (see below).

View this table: [in this window] [in a new window]   Table I TNF-induced apoptosis in TD-IB-expressing Jurkat cells rtTA-Neo-, IB-2N-, and IB-2N4-expressing Jurkat cells were treated with Dox (1 µg/ml) for 24 h and then induced with TNF (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-B DNA Binding Activity and Gene Expression by TD-IB-- The induction of NF-B DNA binding activity in control and TD-IB-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 TNF or PMA/PHA for 4 h resulted in a strong induction of NF-B DNA binding activity, irrespective of Dox treatment for 6-48 h (Fig. 2A, lanes 5-14). In contrast, TNF induction of NF-B activity in IB-2N4 Jurkat cells (Fig. 2B, lanes 5 and 6) was completely blocked by the induction of IB-2N4 for 6 h or longer (Fig. 2B, lanes 6-9); the strong induction of NF-B binding activity by PMA/PHA was also more than 95% inhibited by Dox activation of IB-2N4 (Fig. 2B, lanes 10-14). Similarly, Dox induction of IB-2N also inhibited completely the induction of NF-B binding activity by TNF (Fig. 2C, lanes 2-6) and PMA/PHA (Fig. 2C, lanes 7-12). In a subsequent experiment, the kinetics of activation of IB-2N4 and the inhibition of NF-B binding activity were examined (Fig. 2, D and E). Surprisingly, as early as 1 h after Dox addition, expression of IB-2N4 was up-regulated (Fig. 2E, lanes 1 and 2) and NF-B DNA binding activity was inhibited (Fig. 2D, lanes 1 and 2). Interestingly, comparison of the levels of IB-2N4 and endogenous IB revealed that constitutive expression of IB-2N4 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 IB-2N was also observed in IB-2N-Jurkat cells, based on the resistance of the induced IB to TNF-induced degradation (data not shown). TD-IB-expressing Jurkat cells were also resistant to induction of NF-B binding activity by double-stranded RNA (poly(I·C)) treatment (data not shown). Thus, overexpression of TD-IB blocks the induction of NF-B DNA binding activity by multiple inducers. Additionally, continued growth in culture selects for Jurkat cells that display low constitutive expression of TD-IB, possibly due to an increased sensitivity of TD-IB-expressing cells to apoptosis.

View larger version (41K): [in this window] [in a new window]   Fig. 2.   NF-B DNA binding activity in control and TD-IB-expressing Jurkat T cells treated with TNF or PMA/PHA. rtTA-Neo (A), IB-2N4 (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. IB immunoblot analysis (E) was performed on whole cell extracts (20 µg) prepared from IB-2N4 cells induced by Dox for different times and subsequently treated with PMA/PHA for 4 h. Endogenous IB and transfected IB-2N4 are indicated by arrows. Control lanes (C) in panels A, B, C, and D represent the competition of NF-B-DNA complex formation using a 125-fold excess of unlabeled PRDII probe.

View larger version (18K): [in this window] [in a new window]   Fig. 3.   Inhibition of Tat-TNF, Tat, or PMA/PHA-induced HIV-1 LTR and HIV-enhancer activation by IB mutants. A, rtTA-IB-2N4 Jurkat cells were transfected with HIV-enh () or HIV-enh mut CAT () 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-IB-2N4 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 TNF (10 ng/ml) alone, with TNF + 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-IB-expressing Jurkat Cells-- Given the involvement of NF-B in the early transcriptional control of HIV-1 LTR gene expression (reviewed in Ref. 1), the impact of TD-IB expression on the course of de novo HIV-1 protein synthesis and virus production was next examined. Control and TD-IB-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 IB-2N4 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 IB-2N4 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).

View larger version (19K): [in this window] [in a new window]   Fig. 4.   Suppression of HIV-1 infection in IB-2N4. rtTA-Neo and rtTA-IB-2N4 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). , rtTA-Neo + Dox; , IB-2N4  Dox; , IB-2N4  Dox (+Dox12, Dox addition 12 days after infection); , rtTA-2N4 + Dox (Dox addition 24 h before infection); , rtTA-2N4 + Dox (continuous addition of Dox at 1, 8, 18, and 24 days after infection).

View larger version (66K): [in this window] [in a new window]   Fig. 5.   Inhibition of viral p24 protein expression by IB-2N4 mutant. rtTA-Neo and rtTA-IB-2N4 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-IB-2N4Jurkat cells (panels D and E) were treated with Dox for 24 h prior to infection or not treated (panel B). rtTA-IB-2N4Jurkat 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 IB (wtIB), IB-2N4 (2N4), 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-IB-expressing Jurkat Cells-- To link the inhibition of HIV-1 multiplication with NF-B inhibitory effects of TD-IB, the effects of TD-IB induction on HIV-1-induced NF-B binding activity were evaluated by mobility shift analysis. Virus-induced activation of NF-B was blocked by TD-IB expression (data not shown), thus complementing the results described in Fig. 2, and confirming that TD-IB interfered with HIV-induced NF-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-2N4-Jurkat cells were detected at different times after de novo HIV infection in the presence or absence of Dox-induced TD-IB (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-2N4 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-IB, 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-2N4 Jurkat cells treated with Dox (Fig. 6B, lanes 18-20, lower panel). These results demonstrate a transcriptional inhibitory effect of TD-IB expression on HIV mRNA accumulation in Jurkat cells engineered to express TD-IB.

View larger version (50K): [in this window] [in a new window]   Fig. 6.   Suppression of HIV-1 transcription in IB-2N4-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-IB-2N4 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 IB under the control of a Tet-responsive promoter and the first characterization of the impact of TD-IB repressors on the course of de novo HIV-1 infection. We demonstrate that: 1) doxycycline-inducible expression of IB-2N and IB-2N4 was detected as early as 1 h after Dox addition and blocked the induction of NF-B DNA binding by multiple inducers including TNF, PMA/PHA, and double-stranded RNA; 2) expression of TD-IB also repressed the expression of the endogenous IB gene, consistent with the observation that the IB gene contains multiple NF-B binding sites in its promoter and is tightly regulated by NF-B (32); 3) NF-B-dependent reporter gene activity was also inhibited following the induction of TD-IB; and 4) induced expression of the TD-IB 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-IB 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-IB 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-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-B sites. Similarly, conditional inhibition of NF-B in Jurkat T cells by an IB 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- 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-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 IB on the synergistic activation of the HIV-1 LTR by TNF and the HIV-1 transactivator, Tat, was examined in transiently transfected Jurkat T cells (38). Co-expression of IB inhibited Tat-TNF activation of HIV LTR in a dose-dependent manner, and transdominant repressor forms of IB, mutated in serine or threonine residues required for inducer-mediated (S32A/S36A-2N) or constitutive phosphorylation (S283A/T291A/T299A-3C) of IB, possessed different inhibitory potentials for the HIV-1 LTR. Surprisingly, IB-2N (but not IB-2N+3C) was more effective in blocking HIV-1 protein and RNA synthesis in a single cycle infection than wtIB. The observation that mutations in the C-terminal PEST domain of IB decreased the inhibitory potential of IB-2N suggested that an intact C terminus was required for maximal inhibition of HIV-1 multiplication by IB-2N and may reflect a distinct functional activity for the IB C terminus (38). Although the above studies suggest a transcriptional role for IB in the inhibition of HIV-1 LTR gene expression, IB 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). IB also contains a consensus NES element at amino acids 264-281, matching the NES consensus within Rev (65). Furthermore, newly synthesized IB can localize to the nucleus, dissociate NF-B-DNA complexes, and translocate back to the cytoplasm (66). Thus, an additional function of IB is to serve as a shuttle protein to export NF-B from the nucleus. One possible explanation for the inhibitory activity of TD-IB in HIV-1 infection is that the stable form of IB competes effectively for the nuclear export pathway utilized by Rev/Rab.

HIV-1 infection also causes constitutive activation of NF-B DNA-binding activity in infected cells (1). A direct temporal correlation exists between HIV infection and the appearance of NF-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-IB also interferes with HIV-1 infection at the level of expression of HIV-1-induced inflammatory cytokines.³

Recently, NF-B activation was shown to play a protective function in the response to TNF-, 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 TNF plasma levels correlate with disease progression (1), the role of NF-B in HIV-1-induced apoptosis was examined (72). These studies demonstrated that constitutive NF-B activation is required to counteract a persistent apoptotic signal resulting from HIV-1 infection; thus, a previously unrecognized role for constitutive NF-B activation in HIV-1-infected cells is to protect from virus-mediated apoptotic cell death. Similarly, activation of TD-IB sensitized Jurkat cells to TNF-induced cell death (Table I). Thus, given the multiple functions of the NF-B/IB transcription factors, Dox-induced TD-IB 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-IB molecules to interfere with HIV-1 multiplication.