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J. Biol. Chem., Vol. 277, Issue 7, 4918-4924, February 15, 2002

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Ku Represses the HIV-1 Transcription

IDENTIFICATION OF A PUTATIVE Ku BINDING SITE HOMOLOGOUS TO THE MOUSE MAMMARY TUMOR VIRUS NRE1 SEQUENCE IN THE HIV-1 LONG TERMINAL REPEAT^*

Laurence Jeanson and Jean-François Mouscadet

From the CNRS UMR8532, Institut Gustave-Roussy, PR2, 39 rue Camille Desmoulins, 94805 Villejuif, France

Received for publication, November 12, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Ku has been implicated in nuclear processes, including DNA break repair, transcription, V(D)J recombination, and telomere maintenance. Its mode of action involves two distinct mechanisms: one in which a nonspecific binding occurs to DNA ends and a second that involves a specific binding to negative regulatory elements involved in transcription repression. Such elements were identified in mouse mammary tumor virus and human T cell leukemia virus retroviruses. The purpose of this study was to investigate a role for Ku in the regulation of human immunodeficiency virus (HIV)-1 transcription. First, HIV-1 LTR activity was studied in CHO-K1 cells and in CH0-derived xrs-6 cells, which are devoid of Ku80. LTR-driven expression of a reporter gene was significantly increased in xrs-6 cells. This enhancement was suppressed after re-expression of Ku80. Second, transcription of HIV-1 was followed in U1 human cells that were depleted in Ku by using a Ku80 antisense RNA. Ku depletion led to a increase of both HIV-1 mRNA synthesis and viral production compared with the parent cells. These results demonstrate that Ku acts as a transcriptional repressor of HIV-1 expression. Finally, a putative Ku-specific binding site was identified within the negative regulatory region of the HIV-1 long terminal repeat, which may account for this repression of transcription.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The Ku protein, a heterodimer containing both the Ku70 and Ku80 factors, was originally identified as an autoantigen recognized by the sera of patients with autoimmune disorders (1). Ku is the regulatory DNA binding part of the DNA-dependent protein kinase (DNA-PK)¹ that has been implicated in several nuclear processes, including double-stranded DNA break repair (NHEJ) (for a review, see Ref. 2), V(D)J recombination (3), transcription (4), and, more recently, chromosome maintenance (5). DNA-PK_cs, the catalytic kinase subunit of DNA-PK is recruited to DNA by Ku binding (6) and subsequently phosphorylates DNA-bound proteins (7, 8). Although the Ku protein was first shown to possess a strong affinity for DNA ends (9) and peculiar DNA structures such as nicks, gaps, and hairpins (10, 11), it was more recently reported to bind with sequence specificity to DNA (for review, see Ref. 12). Accordingly, two different mechanisms were proposed for Ku/DNA-PK_cs DNA-dependent activity. First, Ku may bind to DNA ends, thereby recruiting and subsequently activating the DNA-PK_cs kinase to this site (13). Ku was shown to adopt different DNA-dependent conformations that control the recruitment and activation of DNA-PK_cs (14). This mechanism accounts for the role of Ku/DNA-PK during the repair process of double strand breaks (for a review, see Ref. 15). Second, Ku may recruit and activate the kinase at specific sites (4). Putative Ku-specific binding sites were proposed in a variety of genes such as c-myc r (16), the transferrin receptor (17), collagen III (18), the U1 snRNA (19), and also in retroviral sequences, notably in the long terminal repeat (LTR) sequences of intracisternal A particle (20), HTLV-1 (21, 22), and mouse mammary tumor virus (MMTV) (4). In the latter two cases, these sequences were suggested to be involved in the negative regulation of transcription. In particular, the direct binding of Ku to the negative regulatory element 1 (NRE1) contained in the MMTV LTR was clearly demonstrated, resulting in the repression of glucocorticoid-induced MMTV transcription (4). DNase I footprinting, as well as electrophoretic mobility shift assays using microcircles as substrates devoid of free DNA extremities, have permitted the identification of a polypurine tract (4) located within NRE1 as a consensus target sequence for Ku. Accordingly, sequences with similarity to this consensus compete efficiently for Ku binding even in the presence of DNA ends, whereas sequences lacking similarity to NRE1 are not recognized by Ku in the absence of DNA ends (22). The glucocorticoid receptor is efficiently phosphorylated by DNA-PK_cs in the presence of MMTV DNA sequences containing specific binding sites for both the receptor and Ku. This sequence-specific phosphorylation leads to the specific regulation of the GR-dependent promoter (14, 22). It has also been shown that the activation of the kinase at the specific site correlates with a Ku-mediated change in the local DNA structure (14).

Recently, a possible involvement of Ku/DNA-PK_cs at a pre- and/or integrative step during the HIV replication cycle was evoked (23-25). To date, the experimental data supporting such a role of Ku that would involve its nonspecific binding properties remain controversial. However, in the course of their study, Debyser and co-workers (25) noted an enhancement of the expression of an HIV-derived vector in xrs-6-rodent cells. xrs-6 cells are characterized by their lack of a functional Ku80 protein (26-28), thus suggesting that Ku may play a role in HIV transcription repression. To address this possibility, we investigated the capability of the HIV-1 LTR to drive the transcription of a reporter gene in cells either deficient or proficient in the Ku80 factor. We observed that the LTR-driven expression was inversely correlated to the presence of Ku. This result was confirmed in HIV-1 chronically infected U1 human cells by using an antisense strategy to repress Ku expression. A decrease in the amount of Ku80 led to a stimulation of HIV-1 transcription, thus demonstrating that Ku is actually involved in the transcription repression of HIV-1. Finally, we report the identification of a NRE1-related site in the HIV LTR, which may account for the Ku-mediated repression of HIV transcription.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Cell Culture-- All cell culture media contained 10% fetal bovine serum, glutamax, penicillin, and streptomycin. NIH3T3 and GP+envAm 12 cells were grown in Dulbecco's modified Eagle's medium. GP+envAm 12 (gift of Dina Markowitz) is a packaging cell line providing the viral gag, pol, and env functions. U1 cells were grown in RPMI 1640 medium. U1 (NIH 165) is a subclone of U937 chronically infected with HIV-1 and was obtained from Dr. Thomas Folks through AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, National Institutes of Health (29). CHO and xrs cells were cultured in minimal essential medium supplemented with nonessential amino acids.

Massive HIV-1 production from U1 cells was induced by adding 10 ng/ml TNF. Basal or induced viral production was evaluated by measuring the level of p24 antigen in culture supernatant by ELISA (PerkinElmer Life Sciences). Cell viability was evaluated by standard MTT biotransformation assay.

Vectors-- The Moloney murine leukemia virus-based pLNCX retroviral vector contains the retroviral LTRs and PSI sequences, the bacterial neomycin resistance gene, and a multiple cloning site downstream of the human cytomegalovirus immediate early promoter. Ku80 cDNA (gift of Muriel Le Romancer), cloned in reverse orientation in pCDNA3 multicloning site, was excised by XhoI/BamHI and cloned into XhoI/BglII previously digested pLNCX to generate the pLNCX-ASKu vector. Vector DNA was transfected into packaging cell line GP+envAm 12 by using Superfect reagent (Qiagen). After G418 selection, retroviral titers were determined on NIH3T3 cells. Supernatant titer of GP+envAm 12 cells transfected with shuttle vector was 2.10⁵ particles/ml.

Plasmid pBLCAT3-LTR was created by inserting the HIV-1 LTR in the pBLCAT3 multicloning site. HIV-1 LTR was obtained after PCR amplification from the pNL4-3 genomic clone of HIV-1 with primer oligonucleotides LTR-Pst5 (5'-CTG CAG TGG AAG GGC TAA TTT GGT CCC-3') and LTR-Xba3 (5'-TCT AGA TGC TAG AGA TTT TCC ACA CTG-3') followed by TA cloning in pGemT vector (Promega). PstI-XbaI fragment from pGemT-LTR was cloned into pBLCAT3 digested with PstI-XbaI. Plasmid EF1-gal is a gift from Corinne Nicolas-Cabanne. Plasmid pcDNA3-Ku80 contains the human Ku80 cDNA under the control of cytomegalovirus promoter and the neo gene coding for G418 resistance.

Selection of Ku80-depleted U1 Cells-- On the day of infection, 5 × 10⁵ U1 cells were centrifuged and re-suspended in 2 ml of medium containing viral particles (multiplicity of infection = 0.01). At 24 h, cells were transferred into fresh medium containing 800 µg/ml G418 for selection. G418-resistant cells were analyzed 3 weeks later.

Transfections and CAT Assays-- 5 × 10⁴ CHO-K1 or xrs-6 cells were transiently transfected with 1.8 µg of pBLCAT3-LTR and with 0.2 µg of EF1-gal per well in six-well plates using Superfect transfecting reagent (Qiagen). Cells were assayed for CAT and -galactosidase contents 48 h post-transfection by CAT and -galactosidase ELISA kits (Roche Molecular Biochemicals). Stable transfection of xrs-6 cells with pcDNA3-Ku80 was carried out with Superfect. 3 × 10⁵ cells were seeded in 60-mm dish and transfected with 5 µg of DNA. Transfected cells were selected 3 days later with G418 (0.8 mg/ml) and grown as a polyclonal population or as independent clones.

Ku80 Protein Analysis-- Cells were collected, washed in phosphate-buffered saline, and 5 × 10⁶ cells/ml were lysed in lysis buffer (50 mM NaF, 20 mM HEPES, pH 8, 450 mM NaCl, 25% glycerol, 0.5 mM dithiothreitol, 0.2 mM EDTA, pH 8) in the presence of a protease inhibitor mix (Roche Molecular Biochemicals). After three freeze/thaw cycles, samples were centrifuged for 30 min at 12,000 rpm at 4 °C. Protein concentration in supernatants was determined using a standard Bradford assay. Total proteins were electrophoresed on 8% polyacrylamide gel and electrotransferred onto nylon membranes. Membranes were washed in TBS, blocked in 5% dry nonfat milk/TBS, and then incubated with 0.2 µg of anti-Ku80 antibody (Serotec) in TBS, 0.05% Tween. Blots were washed in TBS/Tween and incubated with alkaline phosphatase-conjugated secondary antibody at room temperature. Visualization was achieved using a chemiluminescence assay (Bio-Rad).

Nuclear Extracts Preparation-- All buffers contained a complete protease inhibitor mix (Roche Molecular Biochemicals). Cells were washed with phosphate-buffered saline and re-suspended in 3 ml of STM buffer (20 mM Tris, pH 7.85, 250 mM sucrose, 1.1 mM MgCl₂) containing 0.2% Triton X-100 and incubated at 4 °C for 4 min. The cell lysate was then centrifuged at 2000 rpm for 3 min at 4 °C. The pellet, mostly containing the unbroken nuclei, was re-suspended in 1 ml of STM buffer and centrifuged at 2000 rpm for 2 min at 4 °C. The nuclei were then re-suspended in STM buffer containing 0.4 M KCl and incubated 10 min at 4 °C. The extract was centrifuged at 14,000 rpm for 10 min at 4 °C, and the supernatant fraction was collected and used as nuclear extract.

Band Shift Assay-- Nuclear extract (10 µg) was incubated with a ³²P-labeled double-stranded oligonucleotide, 5'-GGG CCA AGA ATC TTA GCA GTT TCG GG-3' in binding buffer (20 mM Tris, pH 8, 2 mM EDTA, 20% glycerol, 0.4 M NaCl, 200 µg/ml plasmid DNA) for 5 min at 4 °C. For supershift, anti-Ku80 antibody was added, and the binding reaction was allowed to proceed 10 min longer. Ku·DNA complexes were separated by electrophoresis on a 5% polyacrylamide gel and visualized on a phosphorimager screen.

RT-PCR Analysis-- Total RNA was extracted (Rneasy, Qiagen) from 1 million of nontreated or TNF-induced (2 days after induction) U1 or ASKu cells. 1 µg of total RNA was reverse-transcribed by Moloney murine leukemia virus reverse transcriptase using oligo(dT) primers (M-MLV RT, Promega). 1 µl of the resulting cDNA was used as template for the following PCR. The PCR was performed in PerkinElmer Taq buffer II, supplemented with 2.5 mM MgCl₂, 1 µM concentration of each primer, 200 µM dNTP, and 1.5 units of Gold AmpliTaq polymerase (PerkinElmer Life Sciences). The primers used to amplify HIV-1 multispliced mRNAs were primer GH4 (5'-GAC TGG TGA GTA CGC CAA A-3'), localized immediately downstream of the 5' LTR sequence and primer primRT5 (5'-GCT AAG GAT CCG TTC ACT AA-3'), at a 3' position with respect to the 3'-most splice acceptor site of the full-length mRNA. The PCR conditions used to amplify multispliced HIV-1 mRNAs were: preincubation 10 min at 94 °C, 30 cycles of 30 s at 95 °C, 30 s at 53.4 °C, 1 min 30 s at 72 °C, followed by a final step of 10 min at 72 °C. Parallel PCR reactions were performed to amplify the mRNA of the housekeeping gene G3PDH to normalize the results obtained for HIV-1 messengers. The primers used were: forward 5'-TGA AGG TCG GAG TCA ACG GAT-3', reverse 5'-CAT GTG GGC CAT GAG GTC C-3'. The same conditions as above were used to amplify the G3PDH except that a temperature of 60 °C was used for the annealing step and the number of cycles was comprised between 22 and 27. The G3PDH PCR products were detected with ethidium bromide staining. HIV-1 PCR products were detected with Southern hybridization using a radiolabeled probe made of the entire HIV-1 NL43 genome. Both PCR products were quantified by densitometric analysis.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Ku Represses the Transcription Controlled by the HIV-1 LTR in CHO Cells-- The possible effect of Ku80 on the HIV-1 LTR-driven transcription was investigated by transient transfection assays in CHO-K1 and xrs-6 cells. xrs-6 cells are CHO-K1-derived cells lacking Ku80 expression (26, 27). A plasmid construct containing the CAT gene under the control of the complete HIV-1 LTR was transfected in both cell lines, and expression of the reporter gene was quantified in cells 48 h following the transfection. As presented in Fig. 1, the activity of the HIV-1 LTR promoter underwent a 5-fold increase in xrs-6 Ku-negative cells compared with the expression in parent CHO-K1 cells, thus suggesting a possible role for Ku80 in the repression of the LTR promoter. To confirm that the lack of Ku80 was responsible for expression enhancement, the mutant xrs-6 cells were stably transfected with a pCDNA3 expression vector coding for the full-length human Ku80 sequence. Several G418-resistant clones were isolated and monitored for Ku80 expression by either immunoblot and electrophoretic mobility shift assays (EMSA). Results are shown in Fig. 2. The immunoblot was performed using an antibody directed against the human Ku80 protein. Since Ku is a highly conserved protein, the detection of constitutive hamster Ku80 in CHO extracts was possible (see Fig. 2, panel A). An extract from human CEM cells was used as a control for Ku80 identification on the gel. As expected, no Ku80 was present in xrs-6 cells, whereas the protein was readily detected in CHO-K1 extracts. As seen in Fig. 2, panel A, cells from the CHO-K1 parent cell line, the polyclonal population (xk-pc), as well as from the two independent clones xk4 and xk5 expressed the human Ku80 factor. Interestingly, we also obtained the xk2 clone, which possessed a significant G418 resistance without significant expression of Ku80. This clone was used in further experiments as an experimental control.

View larger version (9K): [in this window] [in a new window]   Fig. 1.   Transcription of HIV-1 LTR-driven CAT gene in CHO-K1 and in xrs-6 cells. CHO-K1 and Ku80-deficient xrs-6 cells were transfected with a LTR-CAT construct. CAT activity was evaluated after transient transfection in three independent experiments. An EF1--galactosidase reporter construct was co-transfected, and -galactosidase activity was used to normalize the values obtained from CAT expression.

View larger version (39K): [in this window] [in a new window]   Fig. 2.   Determination of Ku content and activity in xrs-6 cells stably transfected with Ku80 full-length cDNA. A, immunoblot analysis of 30 µg of total proteins from CHO-K1, xrs-6, and xrs-6 cells stably transfected with human Ku80 cDNA (clones xk-2, xk-4, xk-5, and polyclonal cells xk-p). 5 µg of human cells (CEM) proteins were used as a control for human Ku80 detection. Ku was revealed using an antibody raised against human Ku80. Homogenous protein transfer was assessed by Ponceau red staining. B, DNA end binding activity in CHO, xrs-6, xk-2, xk-4, xk-5 clones, and xk-p polyclonal cells. 10 or 20 µg of nuclear extract were incubated with [-32P]dATP-labeled M1/M2 oligonucleotide probe and separated on 5% polyacrylamide gel. 10 µg of CHO-K1 nuclear extracts were further incubated with anti-Ku80 antibody to confirm the presence of Ku80 in the complex by inducing a supershift of this one.

To demonstrate that the human Ku80 was functional in xrs-6 cells expressing the Ku80 full-length cDNA, EMSA was performed to investigate DNA end binding activity in selected clones. Previous studies indicated that Ku is the major DNA end binding activity in cell extracts (30). As shown in Fig. 2, panel B, a shifted complex was observed with CHO-K1 extracts. The amount of complex was related to the quantity of cell extract and the involvement of Ku80 in this dependent DNA end binding shift was unequivocally confirmed, since it was possible to supershift the band using an anti-Ku80 antibody. In contrast, the shifted complex was not present in the Ku80-deficient cell line xrs-6. Furthermore, the DNA end binding activity was restored in the polyclonal cell population xk-pc and in clonal xk4 and xk5 cells that expressed Ku80 but not Ku80-deficient clone xk2, which showed no detectable Ku80 expression.

Transcription from HIV-1 LTR was evaluated in xk-pc polyclonal cells and in the three independent clones xk2, xk4, and xk5 cells and compared with both parental Ku80-deficient xrs-6 and Ku80-proficient CHO-K1 cells. All cell lines were transiently transfected with an LTR-CAT construct, and CAT expression was measured 48 h post-transfection. Results from this experiment are shown on Fig. 3. The CAT activity was decreased by a 2-fold factor in cells expressing human Ku80 compared with xrs-6 cells. Again, the Ku80-deficient xk-2 clone showed a different behavior with no inhibition of HIV-1 expression. Altogether these data demonstrate that the HIV-1 LTR-driven CAT expression was inversely correlated to the amount of Ku80 present in the transfected cells, allowing us to conclude that the HIV-1 LTR promoter activity was negatively regulated by the Ku factor.

View larger version (10K): [in this window] [in a new window]   Fig. 3.   Comparison of HIV-1 LTR-driven transcription of CAT in CHO cells either proficient or deficient in Ku. Transcription from HIV-1 LTR was evaluated after transient transfection with an LTR-CAT construct in CHO, xrs-6, xk-2, xk-4, xk-5, and xk-p cells transfected. CAT activity was normalized by using the -galactosidase activity from a EF1--galactosidase reporter construct which was co-transfected. Values are mean of three independent experiments.

Depletion of Ku80 in Human U1 Cells Leads to an Increase of HIV-1 Expression-- Since our data demonstrated a regulation of the HIV-1 LTR promoter in rodent cells, we were interested in assessing next whether this conclusion could be extended to human cells that are a current target for HIV-1. To address that issue, we used an antisense strategy to decrease the expression of Ku80 in human U1 cells, which are chronically infected with HIV-1. This strategy was previously reported to be efficient for studying the effects of a Ku depletion in several human cell lines (30-32). A retroviral expression vector coding for an antisense RNA containing the complete coding sequence of Ku80 was constructed. An amphotropic virus-packaging murine cell line, GPAm112, was transfected with this construct or with the empty vector pLNCX, and cells resistant to geneticin were selected and used as a source of recombinant virus. HIV-1 chronically infected U1 cells were transduced with the retroviral vector and selected for G418 resistance as a polyclonal cell population (U1-AsKu). The expression of Ku80 was measured by Western blotting. As shown on Fig. 4, panel A, an immunoblotting assay with anti-Ku80 antibody indicated a 2-fold depletion in Ku80 content in cells expressing the Ku80 antisense compared with the parent U1 cell line, indicating that Ku80 production was efficiently impaired in the antisense expressing cells. It was not possible to obtain a stronger depletion due to loss of cell viability in deeply depleted cell population. EMSA was then performed to evaluate the DNA end binding activity in the Ku80-depleted U1-AsKu cells. As shown on Fig. 4, panel B, U1-ASKu cells showed a lower DNA end binding activity compared with parent U1 cells. Hence, we conclude that the transfection of the Ku80 antisense was efficient in lowering Ku80 activity in human U1 cells.

View larger version (33K): [in this window] [in a new window]   Fig. 4.   Characterization of Ku80 content and activity in Ku-depleted U1 cells. A, immunoblot analysis of 10 µg of total proteins from either U1 or U1-ASKu80 cellular extracts. Homogenous protein transfer was monitored by Ponceau red staining. B, DNA end binding activity in U1-ASKu cells. 10 (1×) or 20 µg (2×) of nuclear extract of U1 or U1-ASKu cells were incubated with [-32P]dATP labeled M1/M2 oligonucleotide probe and separated by 5% polyacrylamide gel electrophoresis. Ku·DNA complex was supershifted with anti-Ku80 antibody to assess the presence of Ku80 in the complex.

U1 cells are chronically infected cells that display a low basal viral production. Massive HIV-1 production can be induced by adding TNF. In a first experiment, shown in Fig. 5, panel A, the basal production in U1 and U1-ASKu cells was followed over 168 h after medium change. U1 cells infected with an empty pLNCX vector were also selected and characterized for HIV-1 production as a control of an eventual effect on G418 resistance. In a second experiment shown in Fig. 5, panel B, the induction of HIV-1 production was followed over a 48-h period following TNF addition. Results shown in Fig. 5 demonstrate that, in either experiment, HIV-1 production was ~2-fold higher in U1 cells presenting a Ku80 depletion than for parental cells. The level of viral production of pLNCX control cells (U1-pLN) was also significantly lower than the one obtained for U1 depleted cells under all conditions. In parallel, the cell growth was monitored to eliminate a bias that could arise from a modification in the multiplication rate. As shown in Fig. 5, panel C, the three cell populations behaved similarly with indistinguishable growth rates in all experiments. Furthermore, the Ku80 depletion already gave rise to an increase in viral production in noninduced cells (see Fig. 5, panel A), thus indicating that the increase in viral production levels in Ku80-depleted cells was not due to a modulation of the TNF induction pathway. Finally, to determine whether the enhancement of HIV-1 expression in Ku80-depleted U1 cells was due to a stimulation of transcription, we performed a RT-PCR analysis to follow the variation of the HIV-1 mRNA amounts. Results of the experiment are shown in Fig. 6. mRNAs of the homing gene G3PDH were PCR-amplified to normalize the signal obtained with HIV-1 RNAs (Fig. 6, panel A). A significant increase in the overall level of HIV-1 messengers was obtained in noninduced or TNF-induced U1-ASKu cells compared with parent U1 cells in both conditions (Fig. 6, panel B). Ku80 depletion resulted in a 9-fold increase in HIV-1 mRNA for noninduced cells and in a 2-fold increase for TNF-induced cells over nondepleted cells (see Fig. 6, panel C). In conclusion, a depletion in Ku80 production actually led to an increase in HIV-1 expression at the level of mRNA in chronically infected U1 cells, thus confirming that Ku was involved in the negative regulation of the HIV-1 LTR-driven transcription in human cells.

View larger version (24K): [in this window] [in a new window]   Fig. 5.   HIV-1 production in U1 cells underexpressing Ku80. A, HIV-1 production in U1, U1-pLN, and U1-ASKu cells. Basal production was evaluated 72, 120, and 168 h after seeding (left panel). B, TNF-induced production was measured 24 and 48 h post-induction (right panel). Viral production was evaluated by measuring the level of p24 antigen in culture supernatant by ELISA. C, cell number was evaluated by MTT assay either in noninduced or in induced cell cultures. Black squares, U1-AsKu; gray squares, U1 transfected with empty pLN vector; white squares, parent U1 cells.

View larger version (37K): [in this window] [in a new window]   Fig. 6.   RT-PCR analysis of HIV-1 mRNAs in either noninduced or TNF-induced U1 and U1-ASKu cells. 1 µg of total RNA was reverse-transcribed, and the resulting cDNAs were PCR-amplified for 22, 24, and 27 cycles (G3PDH) or 30 cycles (HIV-1). G3DPH expression was used for normalization of the HIV-1 RNA content. A, amplification of G3PDH mRNA. Products were loaded on an agarose gel and stained with ethidium bromide. B, amplification of HIV-1 multispliced RNAs. RNAs were detected after agarose gel electrophoresis of RT-PCR products and Southern blotting using a specific HIV-1 probe. C, quantitative analysis HIV-1 mRNA content after normalization using values of G3PDH amplification obtained for 24 PCR cycles.

A Potential Ku Binding Site Is Present in HIV-1 LTR-- Although numerous sequence-specific binding sites have been proposed for Ku, to date, only the NRE1 sequence of the MMTV GR strain has been unambiguously shown to be recognized by Ku in the absence of DNA ends (12). Among proposed Ku binding sites, DNA elements identified upstream of the c-myc gene and in the HTLV LTR are implicated in transcription repression and show strong homologies with MMTV NRE1 sequence. After analysis of the HIV-1 LTR sequence, we found a potential Ku binding site (217, 197) showing strong homologies with both GR-MMTV NRE1 and the Ku binding site in Col3A1 promoter. The proposed alignment between the HIV-1 (217, 197) sequence, the GR-MMTV NRE1 sequence, and four NRE1-related sequences is given in Fig. 7. More than 50% of the whole HIV sequence is homologous to the MMTV NRE1 with a strict conservation of the purine tract located in the central part of the sequence. Since this purine stretch is the main determinant of Ku binding to MMTV NRE1, it is very likely that Ku will bind to the HIV LTR as well. Furthermore this sequence is part of the modulator region of the HIV-1 LTR, which has been proposed to contain a NRE between 340 and 184. Indeed, deletions that affected this region stimulated HIV-1 LTR-directed transcription and viral replication (33, 34). To our knowledge this putative specific binding site for Ku within this HIV-1 negative regulatory element has not been reported to date.

View larger version (16K): [in this window] [in a new window]   Fig. 7.   Alignment between (217, 197) HIV-1 LTR sequence, MMTV NRE1 (nuclear regulatory element of the MMTV LTR), and four NRE1-like sequences. Dark gray shaded bases are perfect matches with the consensus sequence. Light gray shaded bases are purine residues matching the consensus sequence.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Ku has been shown to be involved in the transcriptional regulation of different genes such as the late UL38 promoter of HSV-1 (35), the promoter of human COL3A1 gene (18), and the human U1 snRNA gene. Furthermore, it has been suggested that Ku may act as a transcriptional repressor in the context of the expression of retroviral elements either endogenous such as the intracisternal A particle (20) or exogenous as the GR strain of MMTV (36) and the HTLV (21). Although, to our knowledge, a putative role for Ku in lentiviral, and in particular for HIV-1 transcription, has not been considered to date, we noticed that the expression of a reporter gene was increased in Ku80-deficient cells that had been transduced with an HIV-derived vector in the course of a study addressing a possible role of Ku during the early steps of HIV-1 replication (25). Thus, we decided to investigate the potential role of Ku in the regulation of HIV-1 LTR transcription. Ku is a highly conserved factor in eukaryotic cells, and its functions can be conveniently studied in rodent cell lines, since several hamster and mice Ku null mutant cell lines are available. We first evaluated the HIV-1 LTR activity in a Ku-negative context by transfecting an LTR-CAT construct either in the CHO-K1 or in the Ku80(/) xrs-6 hamster cell lines. The LTR-driven expression level of the CAT reporter gene was significantly enhanced in xrs-6 cells. This increase could be leveled off after restoring the expression of Ku80 by stable transfection of human Ku80 cDNA in xrs-6 cells. We concluded that Ku was capable of negatively modulating the transcription of the reporter gene. However, although rodent cell lines defective in Ku are widely used in such studies, no Ku null mutant cell line of human origin was isolated to date, emphasizing the fact that rodent and human cells differ in their regulation of the mechanisms known to require the Ku factor such as DNA repair and chromosome maintenance. This difference is reinforced by the fact that a second protein coded by Ku80 gene, KARP, which is also implicated in the DNA-PK activity regulation (37, 38), is present solely in primates. Thus, to address that issue in human cells, chronically HIV-1-infected U1 cells bearing integrated proviruses were transduced with a retroviral vector carrying a Ku80 full-length antisense RNA. Indeed, the antisense strategy can be used to study the effect of Ku depletion in human cells, as it was demonstrated that antisense sequences directed against Ku80 were capable of impairing the expression of the protein in human cell lines, notably human colon carcinoma cells HCT116 (31) and human fibroblast cells MRC5V1 (30). Antisense directed against Ku70 were also used to deplete human T cell lymphoma MT2 cells (32). In every tested cell line, the expression of the antisense gave rise to a partial inhibition of Ku expression, 50% of wild-type level being the minimum amount of Ku required to assure the cell viability. In the present study, we obtained chronically HIV-1-infected U1 cells with a 2-fold decrease in Ku80 expression using the full-length genomic antisense RNA. The functional effect of the Ku80 depletion in U1-AsKu cells was confirmed by a specific mobility shift assay. This result is in agreement with previous studies in human cells, demonstrating that this 50% depletion was sufficient to affect both Ku and DNA-PK function significantly (32, 39). The quantification of the viral RNA in both U1 and U1-ASKu cells with or without expression stimulation by TNF revealed that Ku depletion stimulated the viral production at a transcriptional level. Altogether our data allowed us to conclude that Ku is involved in the repression of HIV-1 LTR transcription.

Many sequences have been proposed as being specific binding sites for Ku (19, 21, 40-46). The most extensively studied sequence, NRE1, is located in the MMTV LTR region and is capable of specific binding to the Ku heterodimer complex (18, 22). The GR-MMTV NRE1 element was shown to be, in vitro, the highest affinity Ku DNA binding site. Ku binding to NRE1-like sequences is markedly preferred over DNA end binding, whereas other proposed binding sites lacking homology to NRE1 did not function as direct, internal, high affinity Ku DNA binding sites. NRE1-like sequences comprise DNA elements in the LTR of HTLV, C3H strain of MMTV, a sequence flanking the murine c-myc gene, and a partially homologous sequence to GR-MMTV NRE1 in the human collagen III promoter. The best characterized activity of Ku in transcription is the regulation of MMTV LTR promoter through NRE1 sequence, which is dependent upon the phosphorylation of target proteins by DNA-PK_cs. Thus, Ku binding to DNA activates DNA-PK_cs, which is in turn autophosphorylated. DNA-PK_cs becomes subsequently capable of phosphorylating different target transcription factors. Activation of DNA-PK could then repress the positive effects of transcriptional co-activators recruited to HIV-1 LTR.

After performing an homology search, we have identified an LTR HIV-1 sequence located at position (217, 197) in the viral promoter, which displays a strong homology to MMTV NRE1-like sequences. Taking into account the available data on the NRE1 sequences in LTRs from both MMTV and HLTV, it is highly likely that the HIV-1 sequence functions in a similar manner. Reinforcing this hypothesis is the fact that a specific interaction was recently observed between Ku and the octamer transcription factors 1 and 2 (Oct1 and Oct2) (47). Strikingly, Oct1 and Oct2 were shown to repress both the basal activity of the HIV-1 LTR and its transactivation by Tat (48). We thus favor a model in which Ku binding that allows the recruitment of these proteins to DNA and enhances their phosphorylation by DNA-PK could be involved in Ku-mediated repression of HIV-1 transcription. This activity would be similar to the NRE1-dependent regulation of MMTV LTR repression.

On the other hand, the negative effect of Ku on HIV-1 transcription may be related to the ability of this factor to regulate the chromatin structure and function. The HIV-1 LTR encompasses a NRE that down-regulates the LTR-dependent HIV gene expression (49). Strikingly, this NRE contains a nuclear matrix attachment region (MAR) from position 220 to 160 (49), spanning the Ku consensus sequence derived from MMTV NRE1. Raziuddin and co-workers (49) proposed that the NRE role in influencing HIV-1 expression may be through a MAR-nuclear matrix interaction exerting a negative effect on NF-B activity and hence down-regulating HIV gene expression. This group isolated a nuclear matrix-specific factor that binds the NRE and consequently inhibits HIV expression. And indeed, Ku is well known to bind the primary base unpairing regions contained in MARs (50). It is thus tantalizing to identify Ku to this factor. Furthermore, Ku binding to MAR involves a functional interaction with another cellular factor, PARP (50, 51), the inhibition of which leads to the activation of HIV-1 expression most probably by increasing the binding activity of NF-B to the HIV-1 LTR (52, 53), thus giving strength to this model.

Another mechanism would involve the Ku-dependent methylation of regulatory sequences. Such examples exist in the literature. For instance, it was shown that the heavy metal-induced metallothionein-I gene expression was specifically repressed in a rat fibroblast cell line (Ku-80) overexpressing the 80-kDa subunit of Ku autoantigen due to the hypermethylation of the promoter in response to overexpression of Ku80 (54). Falzon et al. (20) also reported the relationship between the methylation state of an intracisternal A particle LTR promoter and the capability of Ku80 of binding and subsequently activate the promoter. These results lead to the possibility that the gene silencing effect of Ku80 overexpression may be related to the hypermethylation of regulatory sequences.

Finally, it is worth noting that Ku is also known to be associated with an RNA polymerase II complex (55). The helicase activity of Ku could play a role in transcription initiation with localized DNA unwinding (56).

More work is obviously needed to settle the question of the Ku-driven repression of HIV-1 transcription. From this viewpoint, we are currently investigating the role of the identified potential Ku binding site (217, 197) in the transcriptional repression observed in cells as well as other possibly important sequences like MAR contained in HIV-1 NRE.

	ACKNOWLEDGEMENTS

We thank Dr. Hervé LEH for fruitful discussions and Dr. Malcolm Buckle for manuscript reading.

	FOOTNOTES

^* This work was supported by grants from Ensemble contre le Sida and the Agence Nationale de Recherche sur le SIDA.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: CNRS UMR8532, Institut Gustave Roussy, PR2, 39 rue Camille Desmoulins, 94805 Villejuif, France. Tel.: 33-1-42-11-50-43; Fax: 33-1-42-11-52-76; E-mail: jfm@igr.fr.

Published, JBC Papers in Press, November 30, 2001, DOI 10.1074/jbc.M110830200

	ABBREVIATIONS

The abbreviations used are: DNA-PK, DNA-dependent protein kinase; DNA-PK_cs, the catalytic kinase subunit of DNA-PK; CAT, chloramphenicol acetyltransferase; LTR, long terminal repeat; HTLV, human T cell leukemia virus; MMTV, mouse mammary tumor virus; NRE, negative regulatory element; GR, glucocorticoid receptor; HIV, human immunodeficiency virus; ELISA, enzyme-linked immunosorbent assay; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; TBS, Tris-buffered saline; RT, reverse transcriptase; TNF, tumor necrosis factor; G3PDH, glyceraldehyde-3-phosphate dehydrogenase; CHO, Chinese hamster ovary; EMSA, electrophoretic mobility shift assay; MAR, matrix attachment region.

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