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The Journal of Immunology, 2005, 175: 2548-2554.
Copyright © 2005 by The American Association of Immunologists

A Soluble Factor Secreted by an HIV-1-Resistant Cell Line Blocks Transcription through Inactivating the DNA-Binding Capacity of the NF-{kappa}B p65/p50 Dimer1

Adam Lesner, Yuchang Li, Jadwiga Nitkiewicz, Guanhua Li, Alex Kartvelishvili, Magdalena Kartvelishvili and Malgorzata Simm2

Molecular Virology Division, St. Luke’s-Roosevelt Hospital Center, Columbia University Medical Center, New York, NY 10019


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The identity and activity of several anti-HIV soluble factor(s) secreted by CD8 and CD4 T lymphocytes have been determined; however, some of them still await definition. We have established an HIV-1-resistant, transformed CD4 T cell line that secretes HIV-1 resistance protein(s). Our studies indicate that this protein(s), called HIV-1 resistance factor (HRF), inhibits transcription of the virus by interfering with the activity of NF-{kappa}B. In the present report we identified the site at which HRF exerts this inhibition by evaluating a set of discrete events in NF-{kappa}B action. We tested the {kappa}B oligonucleotide binding activity in nuclei of resistant cells, nuclear translocation and binding to the HIV-1 long terminal repeat of p65 and p50 proteins from susceptible cells after exposure to HRF, and the binding of recombinant p50 to the {kappa}B oligonucleotide in vitro as affected by prior or simultaneous exposure to HRF. The results of this experimental schema indicate that HRF interacts with p50 after it enters the nucleus, but before its binding to DNA and that this interaction impedes the formation of an NF-{kappa}B-DNA complex required for the promotion of transcription. These findings suggest that HRF mediates a novel innate immune response to virus infection.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Recent years brought much attention to novel mechanisms of defense against HIV-1 by both CD8 and CD4 T cells (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11). Although much of this interest was dedicated to studying the antiviral responses of CD8 T lymphocytes, it has been well known that some infected individuals mount vigorous HIV-1-specific CD4 T cells responses, resulting in the elaboration of IFN-{gamma} and antiviral {beta}-chemokines (12). Furthermore, a number of studies suggested that CD4 T cells mediated the secretion of unidentified protein factor(s), acting after virus entry to restrict virus replication (10, 13, 14, 15).

In our previous studies we described a cellular model based on a transformed CD4 T cell line induced to secrete soluble antiviral factor(s), called HIV-1 resistance factor (HRF)3 (10, 15). The HRF+ cells permitted virus entry, RT, and translocation of HIV-1 DNA into the nucleus, but restricted transcription of viral DNA (10). Subsequent analysis of gene expression profiles in HIV-1-resistant HRF+ cells showed modulation of genes involved in transcription and indicated global changes in the cellular mechanisms that might be involved in the maintenance of the acquired resistance phenotype (16). Several genes that are differentially expressed in HRF+ cells are implicated in either susceptibility of cells to HIV-1 or promotion of HIV-1 transcription itself. Parallel studies of chromatographic separation of proteins secreted by HRF+ cells identified one of the cofactors of HRF, ubiquitinated histone H1B (H1B) (17). Interference with the expression of H1B reversed the acquired resistance phenotype, suggesting that it contributes to HRF activity and providing verification that the acquisition of the resistant phenotype induced global changes leading to novel mechanisms imposing a block of virus transcription. One such mechanism was described recently for an antiviral factor secreted by CD8 T cells, CD8 antiviral factor (CAF), acting as an inducer of STAT-1, that was postulated to inhibit viral transcription (4).

The HIV-1 long terminal repeat (LTR) serves as a common point for regulation by many cellular and viral proteins (18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29), and these factors are attractive targets for antiviral treatment. We reported previously that HRF+ cells did not permit HIV-1 LTR-promoted transcription (10), and additional studies were directed to define the mechanism of this inhibition. In the present study we investigated transcription factors active in resistant cells as well as the identities of factors affected by HRF treatment that confer HIV-1 resistance upon HIV-1-susceptible cells. We traced the changes caused by HRF in the nuclear compartment, and finally, we used an in vitro assay to resolve the protein/DNA interactions in the presence of HRF. Our studies demonstrate that HRF interacts with p50 NF-{kappa}B after it enters the nucleus, but before its binding to DNA, thus obstructing the formation of the NF-{kappa}B-DNA complex required for initiation of transcription.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Cell cultures, viruses, and transfection procedures

HRF+ and HRF– cells, distinguished by HIV-1 resistance and HIV-1 susceptibility, have been described previously (10, 16, 17, 30, 31). 1G5 cells stably expressing the luciferase gene driven by the HIV-1 LTR (32) were obtained through National Institutes of Health AIDS Research and Reagent Program Division of AIDS, National Institute of Arthritis and Infectious Diseases. PBL from healthy, HIV-1-negative volunteers obtained by elutriation from whole blood were cultured for 2 days in DMEM supplemented with 10% FBS, PHA (5 µg/ml; Sigma-Aldrich), IL-2 (10 U/ml; R&D Systems), antibiotics, and glutamate. Subsequently, cells were cultured without PHA. Continuous cultures of 1G5, HRF+, HRF–, and 293T cell lines were maintained in RPMI 1640 (Sigma-Aldrich) supplemented with 5% FBS, antibiotics, and glutamate at 37°C in a 5% CO2, 95% air-humidified incubator. To prepare biologically active supernatants, HRF+ and control HRF– cells were cultured overnight in protein-free Hybridoma medium (Sigma-Aldrich) supplemented with glutamate. Subsequently, cells were removed by centrifugation, and supernatants were filtered through 0.45-mm pore size membranes (Millipore) and collected for future applications. Infectious HIV-1/NL4–3env–luc (33) pseudotyped with the vesicular stomatitis virus G (VSV-G) glycoprotein (HIV-1-Luc/VSV-G) was prepared by transfection of 293T cells as described previously (34). Virus stocks were titered by p24 ELISA using the HIV Ag kit (Coulter).

Evaluation of HRF-mediated inhibition of HIV-1 LTR-promoted transcription of luciferase reporter gene in primary blood lymphocytes and a transformed T cell line

PBL were cultured overnight in 50% HRF+ or HRF– conditioned hybridoma medium or in the presence of HRF+ or HRF– cells in a 1:1 ratio in a Transwell apparatus (Costar). Subsequently, PBL were challenged with HIV-Luc/VSV-G pseudotype at a multiplicity of infection of 0.1. After 1-h infection, cells were washed in PBS and cultured in the presence of HRF+ or HRF– cells or 50% HRF+ or HRF– conditioned medium. Untreated infected and uninfected cells served as experimental controls. Virus replication was tested by the expression of luciferase gene 1, 2, and 3 days after infection.

1G5 cells stably transfected with an inducible luciferase gene driven by HIV-1 LTR (32) were washed in PBS and resuspended in hybridoma medium to a concentration of 5 x 106 cells/ml. One-hundred-microliter aliquots of 1G5 cells were supplemented with a 0.01–50% volume of HRF+ or HRF– supernatants or with medium alone, brought to a final volume of 200 µl, and incubated for 3 h at 37°C. Subsequently, all cells were induced with PMA at a concentration of 5 ng/ml. Two control tubes containing 1G5 cells were resuspended in hybridoma medium with or without PMA. Three hours later, cells were collected by centrifugation and lysed in the same tubes using reporter lysis buffer (Promega). The expression of luciferase protein was tested based on the manufacturer’s protocol. Briefly; 1 x 106 PBLs or 0.5 x 106 1G5 cells each were collected by centrifugation and lysed in the same tubes using reporter lysis buffer (Promega). Twenty microliters of lysate was mixed with the beetle luciferin substrate (Promega) and tested in a luminometer (Turner Designs Instruments).

Evaluation of HRF-mediated inhibition of limited NF-{kappa}B promoted transcription of luciferase reporter gene

For the experiments testing HRF-mediated NF-{kappa}B inhibition in HIV-1-susceptible cells, we used the p5xNF-{kappa}B-luc plasmid (Stratagene). HRF– cells were transfected by Lipofectamine using DMRE-C reagent (Invitrogen Life Technologies) at a concentration of 1 µg DNA/1 x 106 cells according to the manufacturer’s instructions. Subsequently, transfected cells were aliquoted into separate wells in six-well plates and exposed for 24 h to soluble products of HRF+ or HRF– cells in a double chamber (Costar) or incubated with 50% conditioned medium containing HRF+ or HRF– supernatants. After 30-min induction with PMA at a concentration of 5 ng/ml, equal number of cells was harvested for luciferase assay and lysed in a reporter lysis buffer (Promega), and the expression of luciferase was tested as described above.

EMSA

For EMSA studies, nuclear translocation of transcription factors in HRF+, HRF–, or 1G5 cells cultured in the presence of HRF+ or HRF– cells was induced with 5 ng/ml PMA (Sigma-Aldrich) for 30 min at 37°C. Subsequently, induced and uninduced cells were washed in PBS and lysed in lysis buffer (20 mM HEPES (pH 7.9), 10 mM NaCl, 3 mM MgCl2, 1 mM DTT, 0.5 mM EDTA, 10% glycerol, 0.1% Nonidet P-40, 1 mM Na3VO4, and protease inhibitor mixture), and nuclear fractions were collected by sedimentation at 500 x g. Nuclear proteins were extracted with extraction buffer (20 mM HEPES, 400 mM NaCl, 1 mM DTT, 0.5 mM EDTA, 20% glycerol, 1 mM Na3VO4, and protease inhibitors) and tested for their ability to bind labeled nucleotides representing consensus NF-{kappa}B, AP1, and specificity protein 1 (Sp1) binding sites provided by manufacturer (Promega) or representing HIV-1 LTR binding sites (NF-{kappa}B, 5'-CTACAAGGGACTTTCCGCTG-3'; Sp1, 5'-TTCCAGGGAGGCGTGGCCTG-3'). All DNA-protein binding reactions were based on the manufacturer’s protocol (Promega). Briefly, 0.5 ng of labeled probe was incubated for 20 min at 4°C with 5 µg of nuclear extracts in the presence of 1x gel shift buffer (Promega). Subsequently, 1.5 µl of a 10x loading buffer was added to the reaction, followed by separation by electrophoresis on a 5% SDS-polyacrylamide gel until free probe was close to the bottom of the gel.

Recombinant p50 NF-{kappa}B, and AP2 protein extract (Promega) were used for the experiments testing their DNA binding efficiency in the presence of HRF+ supernatants and partially purified HRF. Briefly, 10 ml of each of the clarified supernatants and partially purified HRF (see Partial purification of HRF below) derived from 150 ml of culture supernatant were dialyzed for 16 h against GS buffer (5 µM MgCl2, 2.5 µM EDTA, 2.5 mM NaCl, and 0.5 mM Tris-Cl (pH 7.5)). Subsequently, all samples were subjected to concentration by lyophilization and then resuspended in water as 100x concentrate. Increasing amounts of HRF+ and HRF– supernatants (i.e., 1 µl = 100 µl; 2.5 µl = 250 µl, and 5 µl = 500 µl of starting material) and purified HRF were mixed with 1 gel shift unit of human recombinant p50 NF-{kappa}B or AP2 extract (Promega) in the presence of GS buffer. One gel shift unit is the amount of protein required to gel shift the NF-{kappa}B oligonucleotide under the conditions described above (titrations data not shown). After 10-min incubation at room temperature, 0.5 ng of labeled oligonucleotide probe representing the {kappa}B binding site was added to each tube and incubated for another 20 min. AP2 extract and relevant DNA probe (all from Promega) were used in parallel reactions as a control. Subsequently, all reactions were resolved through 6% polyacrylamide gel as described above.

Investigation of NF-{kappa}B proteins in the nuclear compartment of cells exposed to HRF

HRF– and HRF+ cells were cultured adjacent to each other in a double-chamber apparatus (Costar). Two days later, cells exposed to HRF were induced, or not, with 5 ng/ml PMA for 30 min, followed by separation of the nuclear fraction as described previously (17). Briefly, 5 x 106 of HRF– cells/system were subjected to swelling in a hypotonic buffer (10 mM Tris-Cl (pH 7.9), 1.5 mM MgCl2, 10 mM KCl, and 0.5 mM DTT) supplemented with protease inhibitors (Sigma-Aldrich) and lysed in a glass Dounce homogenizer (type A; Kontes). The efficiency of cell breakage was monitored by dye exclusion using a phase contrast microscope. Lysed cells were centrifuged at 500 x g for 5 min to harvest nuclei. Nuclear pellets were subjected to the second round of centrifugation at 13,000 x g for 5 min to remove any residual cytoplasmic material and lysed in RIPA buffer. Each sample was standardized by total protein concentration in a standard protein assay (Bio-Rad). Nuclear proteins were resolved in SDS-PAGE, immobilized onto nitrocellulose membrane, and probed with Abs against p50, p65 NF-{kappa}B, and nucleophosmin (all from Santa Cruz Biotechnology).

Chromatin immunoprecipitation (ChIP) assay

We used ChIP assay, focusing on the availability of HIV-1 LTR for p65 and p50 components of NF-{kappa}B dimmer and control Sp1 in the presence of HRF. Briefly, 1G5 cells were cultured for 24 h in the presence of 50% HRF+ or HRF– conditioned medium. Subsequently, cells were treated with 5 ng/ml PMA to induce nuclear translocation, and 30 or 60 min later, all proteins were cross-linked with 1% HCHO for 10 min at 25°C, followed by 5-min incubation in 0.125 M glycine. Cells were washed in PBS and resuspended in ice-cold lysis buffer (50 mM Tris (pH 8.0), 0.2 mM EDTA, 0.1% Nonidet P-40, 10% glycerol, and a standard mixture of protease inhibitors). Ten minutes later, nuclei were collected by centrifugation at 500 x g and incubated for 10 min in 100 µl of ice-cold 50 mM Tris (pH 8.0) supplemented with 0.1% SDS, 5 mM EDTA, and protease inhibitors. Chromatin was sheared by sonication to the point that 800- to 1000-bp fragments were produced (titration data not shown). All samples were subjected to centrifugation for 10 min at 6700 x g, and supernatants containing DNA-protein fragments were transferred into new tubes and mixed with 400 µl of dilution buffer (50 mM Tris (pH 8.0), 0.5% Nonidet P-40, 0.2 M NaCl, 0.5 mM EDTA, and protein inhibitors). To reduce nonspecific background, all samples were precleared for 30 min with 80 µl of salmon sperm DNA-protein-A agarose slurry. Protein A-agarose beads (Sigma-Aldrich) were collected by brief centrifugation, and 80 µl of each sample (input control) was set aside. The rest of the material was divided into three portions and incubated overnight with 4 µg each of anti-p65, anti-p50 NF-{kappa}B, or anti-Sp1 Abs (Santa Cruz Biotechnology). Immune complexes were collected by salmon sperm DNA-protein A-Sepharose beads after 1 h of incubation, followed by three washes in high salt buffer (20 mM Tris (pH 8.0), 0.1% SDS, 1% Nonidet P-40, 2 mM EDTA, and 0.5 M NaCl) and elution with 1x Tris-EDTA supplemented with 2% SDS. Eluted samples were heated at 65°C for 10 min, and formaldehyde cross-links were reversed by adding NaCl to a final concentration of 0.3 M and incubating at 65°C for 5 h. DNA was purified by PCR purification columns (Qiagen). The DNA concentration was calculated based on OD reading measured by spectrophotometer at a wavelength of 260 nm, and all samples standardized by input control were subjected to 35 cycles of PCR amplification using primers flanking NF-{kappa}B or Sp1 binding sites on the HIV-1 LTR promoter. The following primers were used: NF-{kappa}B primers: forward LTR 331(M), 5'-GCTGACATCGAGCTTGCTACAAGGG-3'; reverse M701, 5'-AGGGCTCGCCACTCCCCAGTC-3'; and probe, 365 5'-CACGCCTCCCTGGAAAGTCC-3'; Sp1 primers: forward LTR331, 5'-GACATCGAGCTTGCTACAAGG-3'; reverse LTR 409, 5'-TGCAGGATCTGAGGGCTC-3'; and probe M701. The HIV-1 LTR titration curve was prepared from serial dilutions of pNL4–3 amplified by the LTR 331(M) and M701 pair of primers under the same conditions as those used for experimental systems.

Partial purification of HRF

Partial purification of HRF was performed essentially as we previously described (17). Briefly; HRF containing supernatants were collected, centrifuged at 1000 x g to remove particulate materials, filtered through a 0.45-µm pore size Millipore filter, and concentrated by lyophilization from 300-ml aliquots using Labconco lyophilizer. Protein powder was resuspended in 10 ml of distilled water and subjected to dialysis against 10 mM Tris-HCl (pH 8.0) using benzoylated cellulose tubing with a Mr cut-off of 1.2 kDa (Sigma-Aldrich). Dialyzed material was concentrated again by lyophilization and stored at 4°C. Subsequently, desalted lyophilisate was dissolved in 10 mM Tris-HCl (pH 8.0) and separated during 25 min with a 0–100% linear gradient of B (Tris-Cl (pH 8.1) and 1 M NaCl) at a flow rate of 3.0 ml/min through a 5-ml ion exchange High Trap Q XL column using the Acta Prime chromatography system (all from Pharmacia). HRF fractions were collected after 20–24 min of elution, pooled, dialyzed against GS buffer (5 mM MgCl2, 2.5 µM EDTA, 2.5 mM NaCl, and 0.5 mM Tris-Cl, pH 7.5), and concentrated by lyophilization.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
HIV-1 LTR-mediated transcription is restricted in primary and transformed CD4 T cells cultured in the presence of HRF

Our previous study showed that the synthesis of HIV-1 RNA was impeded in HRF+ virus-resistant cells, and the inhibition of LTR-driven transcription of chloramphenicol acetyltransferase reporter gene suggested that this block was imposed on the level of transcription (10). We explored this observation, and in direct analogy to our previous study we compared the promoting efficiency of LTR-mediated expression of another reporter gene after HRF treatment in two systems: 1) in primary blood lymphocytes and 2) in transformed CD4+ T cells. The main objective of this analysis was to choose a cell-type model for future experiments to study the mechanism of HRF-mediated inhibition of transcription. Both primary and transformed CD4 T cells were cultured in the presence of HRF-producing cells or their cell culture supernatants. To secure the most efficient delivery of LTR-driven reporter gene, PBL were challenged with HIV-1-Luc pseudotyped with VSV-G glycoprotein, thus allowing observation of the HRF-mediated inhibition of HIV-1 in the single round of infection, and 1G5 CD4 T cells stably transformed with an inducible luciferase gene upon the control of HIV-1 promoter were exposed to serial dilutions of HRF+ or HRF– culture supernatants expanding from 50 to 0.01%/vol of tested sample (Fig. 1). During the course of infection, PBL cultured in the presence of control HRF– cells or their culture medium expressed luciferase at levels comparable to those in control infected, but untreated, cells (Fig. 1A). In stark contrast, the expression of luciferase gene in PBL cultured in the presence of HRF+ cells or their 50% conditioned medium was inhibited by 7- and 17-fold, respectively, at the peak of virus infection. Similar to this, treatment of 1G5 cells with 50 and 10% volumes of HRF+, but not HRF–, supernatant completely blocked the expression of luciferase gene, and addition of 1 or 0.1% vol of HRF+ sample to the reaction mixture still inhibited the expression of the reporter gene by 63 and 45%, respectively. These data suggested that HRF treatment interferes with HIV-1 LTR-mediated transcription efficiently in primary and transformed cells and allowed us to continue our investigation in selected CD4 T cell lines.



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  		FIGURE 1. Exposure to HRF+ cells or their culture supernatant inhibits HIV-1 LTR-mediated expression of reporter gene in primary blood lymphocytes and the transformed 1G5 cell line. A, After 24-h exposure to HRF+ or HRF– cells in a double-chamber apparatus or in 50% HRF+ or HRF– conditioned hybridoma medium, PBL were infected with HIV-1 Lucenv– pseudotyped with VSV-G envelope at a multiplicity of infection of 0.1 and cultured under the same conditions for another 3 days. Untreated infected and uninfected PBL served as experimental controls. B, 1G5 cells were cultured in the presence of serial dilutions of HRF+ or HRF– supernatants. The expression of luciferase reporter gene was induced by PMA treatment as described in the text. Untreated induced and uninduced cells served as experimental controls.

 
Formation of the NF-{kappa}B-DNA complex is restricted in HRF+ and HIV-1-susceptible cells treated with HRF

The HRF-mediated inhibition of LTR-driven transcription of the reporter gene indicated that the one of the complex host factors or transcription regulatory mechanisms could be affected by the acquired resistance phenotype and that the continuous exposure to HRF in resistant cells themselves might interfere with one of the steps involved in protein-LTR binding. Although multiple transactivators bind directly to HIV-1 promoter, early studies suggested that three Sp1, two NF-{kappa}B, and AP1 motifs were indispensable for HIV Tat responsiveness (20, 35), so we tested the DNA binding efficiency of these factors isolated from nuclear fractions of HRF+ cells and HIV-1-susceptible cells exposed to HRF under induced and constitutive culture conditions (Fig. 2). Both HRF+ and HRF– cell types showed similar levels of constitutive AP1, Sp1, and NF-{kappa}B-DNA binding for all tested transactivators (Fig. 2A). Also, PMA induction produced comparable AP1 and Sp1 gel shifts in HRF+ and HRF– cells, suggesting that HRF did not affect the nuclear translocation or DNA binding activity of these factors. In contrast, the PMA-induced binding of nuclear NF-{kappa}B to its target DNA was strikingly inhibited in HRF+, but not HRF–, cells indicating that the acquired resistance to HIV-1 was mediated through the interference with NF-{kappa}B function, which suggested the need for additional experiments to elucidate this mechanism, but left open the question of whether the exposure of HIV-1-susceptible cells to HRF has a similar outcome on the formation of the NF-{kappa}B-DNA complex and consequently whether HRF treatment prevents binding of NF-{kappa}B to cognate sequences present on HIV-1 LTR.



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  		FIGURE 2. The formation of NF-{kappa}B/DNA complex is blocked in HIV-1-resistant HRF+ cells and HIV-1-susceptible cells exposed to HRF. HRF+ and control HRF– cells (annotated as (–) and (+); A) and 1G5 cells cultured for 3 days in the presence of HRF+ or HRF– cells (B) were induced, or not, for 30 min with 5 ng/ml PMA, and nuclear fractions were isolated. Five micrograms of nuclear proteins each were incubated with labeled DNA probes representing consensus AP1, NF-{kappa}B, and Sp1 DNA binding sequences (A) and HIV-1 LTR NF-{kappa}B and Sp1 DNA binding sequences (B).

 
We tested these possibilities using probes representing HIV-1 LTR NF-{kappa}B and control Sp1 binding sites in 1G5 cells cultured in the presence or the absence of HRF (Fig. 2B). Similar to before, we observed that NF-{kappa}B-DNA, but not Sp1-DNA, binding was inhibited in nuclear extracts isolated from cells exposed to HRF. Taken together, these data suggested that HRF is an efficient inhibitor of NF-{kappa}B-DNA binding in both HIV-1-resistant and HRF-treated, HIV-1-susceptible cells and that HRF blocks formation of the NF-{kappa}B-DNA complex with cognate sequence on HIV-1 LTR. Based on this observation we chose to investigate further the mechanism through which HRF interferes with NF-{kappa}B activity using HIV-1-susceptible cells exposed to soluble HRF because of the convenience of experimentation at the point of inducing viral resistance.

A soluble factor secreted by HRF+ cells inhibits NF-{kappa}B activity in HIV-1-susceptible target cells

To explore the possibility that HRF exposure interferes with NF-{kappa}B activity, we used a reporter system that allowed us to study selectively its transactivation efficiency from the limited promoter composed only of five tandem NF-{kappa}B binding sites. To this end, HRF– cells were transfected with p5xNF-{kappa}B-luc construct and cultured in 50% conditioned medium from HRF+ or control HRF– cells or next to HRF+ or HRF– cells in a double-chamber apparatus. After exposure to HRF, the expression of luciferase gene was induced with PMA, and the NF-{kappa}B-mediated transactivation of the reporter gene was tested by measurement of luciferase activity in cell lysates (Fig. 3). PMA treatment induced the expression of luciferase gene in the systems where HRF was absent; however, similar to previous observations, there was a significant reduction in the expression of the reporter gene in cells exposed to HRF. We observed a total shut down of the expression of luciferase in cells treated with HRF+ supernatant, and its expression was 27-fold lower in cells exposed to HRF+ cells through the porous membrane, suggesting that HRF treatment interferes with NF-{kappa}B-mediated transactivation also in HIV-1-susceptible cells. Although NF-{kappa}B might be inactivated on multiple levels, we focused our investigation of HRF-mediated interference with two of the most obvious and critical steps: nuclear translocation of NF-{kappa}B and efficiency of forming a DNA-protein complex. It has been shown previously that the NF-{kappa}B moiety involved in transactivation of HIV-1 LTR promoter is composed of p65/p50 NF-{kappa}B dimer (36); therefore, we tested these possibilities, focusing on p65 and p50 NF-{kappa}B components.



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  		FIGURE 3. Exposure to HRF+ cells or their culture supernatant blocks the expression of luciferase gene driven by limited NF-{kappa}B promoter. HRF– cells were transfected with p5xNF-{kappa}B-luc plasmid and exposed to HRF treatment by cell coculture in a double-chamber apparatus ({blacksquare}) or 50% conditioned medium ({square}) or were cultured in the absence of HRF ({cjs2108}). The inhibition of NF-{kappa}B-mediated transactivation was measured by the expression levels of luciferase reporter gene.

 
HRF exposure does not affect the nuclear translocation of NF-{kappa}B, but inhibits recruitment of NF-{kappa}B complex to HIV-1 LTR promoter

To test the first possibility, that exposure to HRF interferes with the nuclear translocation of NF-{kappa}B, we evaluated the presence of p50/p65 NF-{kappa}B subunits in the nuclear compartment of HRF– cells cultured for 2 days in the presence of HRF. HRF– cells were exposed to PMA to induce nuclear translocation of NF-{kappa}B in the presence or the absence of HRF, and their nuclear extracts were subjected to Western blot analysis (Fig. 4A). PMA treatment induced nuclear translocation of p50 and p65 NF-{kappa}B in all tested systems, which suggested that HRF does not interfere with nuclear import of NF-{kappa}B and excluded the cytosolic compartment as a site of HRF activity.



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  		FIGURE 4. Exposure to HRF does not inhibit the nuclear translocation of NF-{kappa}B, but disorders the recruitment of p50/p65 NF-{kappa}B to HIV-1 LTR promoter. A, HRF– cells were cultured in the presence of HRF+ cells in a double-chamber apparatus for 2 days. After PMA induction, nuclear lysates were subjected to Western blot analysis with Abs raised to p50 and p65 NF-{kappa}B and nucleophosmin. B, Recruitment of Sp1 or p50/p65 NF-{kappa}B dimer to HIV-1 LTR promoter was tested in 1G5 cells cultured for 24 h in 50% HRF+ or HRF– conditioned medium. NF-{kappa}B occupancy on HIV-1 LTR was tested 30 and 60 min after PMA induction. 0, uninduced samples. HIV-1 LTR titration curve was prepared from pNL4–3 amplified with a pair of primers used for detection of NF-{kappa}B binding.

 
Knowing now that NF-{kappa}B is translocated to the nucleus in the presence of HRF, we investigated the efficiency of NF-{kappa}B binding to its cognate DNA sequence upon HRF exposure. We used the ChIP assay, focusing on the availability of HIV-1 LTR for p65 and p50 components of NF-{kappa}B dimer and control Sp1 protein in the 1G5 cell line, which is stably transfected with plasmid encoding luciferase promoted by the HIV-1 LTR (32), thus providing easy access to assay proteins bound to HIV-1 promoter upon cell induction. As expected, the DNA binding of Sp1 protein was unaffected in both systems (Fig. 4B). We observed a small fraction of Sp1 protein on transcriptionally inactive LTR, which corresponded to one copy per 2 x 105 cells, and PMA induced the Sp1 nuclear translocation with similar efficiency in the presence or the absence of HRF (Fig. 4B, 30 and 60 min). The recruitment of NF-{kappa}B to the LTR was initiated by PMA induction, and the p65 and p50 NF-{kappa}B dimer was maintained up to 1 h later in 1G5 cells cultured in the presence of HRF– conditioned medium. In contrast, in cells exposed to HRF, the recruitment of both p50 and p65 NF-{kappa}B proteins was blocked and, based on the DNA titration curve, was estimated to be 100-fold lower for p65 (Fig. 4B, 30 and 60 min points). Although both p65 and p50 bound to the LTR less efficiently in the presence of HRF, the binding of p50 was completely blocked, which suggests that HRF also blocked p50 homodimers present in transcriptionally inactive promoters (37). Overall, these results showed that HRF interferes with the efficient formation of NF-{kappa}B-DNA complex, but leave open the question of whether HRF interacts with NF-{kappa}B or with the {kappa}B site on DNA.

To explore these possibilities, we established an in vitro assay using human recombinant p50 and control AP2 proteins. First, we used this system to confirm the previous results and to establish the dose at which HRF+ supernatant induces the complete inhibition of p50-DNA binding. The results of this experiment are shown in Fig. 5A. Consistent with previous observations, supernatant from HRF– cells did not inhibit p50-DNA binding, and the concentration of DNA-protein complex was comparable to that in the untreated control. Conversely, HRF+ supernatant reduced the formation of p50-DNA, but not AP2-DNA, protein complex in a dose-dependent manner, and based on band densitometry analysis, treatment with 5 µl of 100-fold concentrated HRF+ supernatant inhibited >99% of p50 protein from DNA binding. Even the lowest dose of the ion exchange-purified HRF blocked p50-DNA binding, showing enrichment of activity in the isolated material.



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  		FIGURE 5. The formation of human recombinant p50/DNA complex is blocked by HRF. A, Dose-response analysis of the formation of DNA/protein complexes with NF-{kappa}B or AP2 probes, HRFIXC, partially purified HRF, C-competing cold NF-{kappa}B nucleotide. B, HRF/p50 affinity analysis. The sunburst with HRF inside shows the HRF molecule; the gray oval indicates p50 NF-{kappa}B; the chained lines show the DNA probe.

 
Because the observed inhibition of p50 binding was tested in the system in which HRF was introduced before DNA probe was added, we explored the HRF-p50 interactions (Fig. 5B). We used three systems in which we assayed direct p50-DNA binding and HRF/p50 interactions before and after DNA binding. As seen previously, the formation of p50-DNA complex was efficient in the absence of HRF, and incubation of p50 with 2.5 µl of HRF+ concentrate before the probe was added blocked its binding to DNA by 95%, thus confirming the previous results (Fig. 5, lanes 1 and 2). In contrast, HRF+ interference with p50 was reduced after p50 was permitted to bind to DNA and, based on gel densitometric analysis, impeded gel shift by 40% (Fig. 5B, lane 3), suggesting that HRF has a higher affinity to the unbound p50 protein, and this protein-protein interaction might induce changes in the NF-{kappa}B conformation, thus preventing the formation of transcriptionally active complex with DNA.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The presence of soluble noncytolytic antiviral factors secreted by both CD8 and CD4 T lymphocytes has been observed for some 20 years, and during this time, some of these factors were identified (9, 11, 38, 39), but the others still await classification, including CAF and HRF (10, 14, 40). Although their identities are not yet known, the mechanism of their antiviral activities points to the inhibition of transcription of viral genes (4, 10). The findings presented in this study indicate that HRF blocks HIV-1 transcription through direct action on the nuclear NF-{kappa}B p50/p65 dimer, preventing it from forming transcriptionally active DNA/protein complex.

Of 15 known NF-{kappa}B homo- and heterodimers formed in cells, p65/p50 NF-{kappa}B is the most abundant (41) and the most rapidly activated (42); most importantly, it is used by HIV-1 during LTR transactivation (36). Our data showed that the formation of NF-{kappa}B-DNA complex was interrupted in HRF+ cells, and the exposure to HRF reduced recruitment of p65 and p50 NF-{kappa}B proteins to {kappa}B binding sites on synthetic NF-{kappa}B and HIV-1 LTR promoters. These results suggested two possible scenarios for HRF activity: 1) HRF may compete with NF-{kappa}B for binding to DNA motif; or 2) it may inhibit NF-{kappa}B binding by forming a protein-protein complex. The first possibility appears unlikely, because we did not observe that HRF formed a gel shift with {kappa}B DNA probe in the system where NF-{kappa}B was absent. Therefore, the second option for HRF-NF-{kappa}B binding seemed more feasible, and we considered two possible scenarios for its inactivating activity: that HRF may bind to nuclear NF-{kappa}B before or after it reaches its DNA, as was found for I{kappa}B{alpha} (43). This hypothesis was supported by the observation of stronger inhibition of LTR-mediated transcription in PBL or transformed CD4 T cells cultured in the presence of HRF conditioned supernatants representing ready-state HRF as opposed to cell-to-cell culture, where therapeutic concentrations of HRF had to be produced over time by HRF+ cells.

We tested these hypotheses in the in vitro assay and found that after exposure to HRF, p50 failed to bind its DNA motif and was less active in the system where the protein-DNA complex was allowed to form. The stronger interaction of HRF with unbound p50 suggests that HRF may compete with DNA for binding to p50 or that once p50 has bound DNA, it assumes a new conformation that prevents HRF binding. This observation is supported by the work of Chen-Park et al. (44), who showed variable conformations of NF-{kappa}B p50/p65 dimer on different {kappa}B binding sites. The high specificity of HRF to form complex with the free NF-{kappa}B also suggests that its activity is directed to rapidly induced and translocated p50/p65 dimers, which might be caused by HIV-1 and does not affect the expression of genes that are already in the process of transcription, thus ensuring the survival of cells. A similar observation was made for another antiviral factor, CAF, secreted by CD8+ T cells (4). Although CAF-mediated activity is regulated through different molecules, its antiviral protection was most effective in cell cultures where nuclear translocation was induced in its presence, indicating that the virus inhibitory activity of CAF was not mediated by proteins already present in the nucleus.

The results of our previous studies indicated that the acquisition of HIV-1 resistance triggered a panel of changes in gene expression (16), and one of these changes included the expression of HRF. Concurrent with proposition that CAF might be a collection of known antiviral cytokines with redundant functions (45), the increasing body of evidence suggests that HRF is not a single molecule. Our recent studies uncovered the presence of ubiquitinated H1B copurifying in a biologically active fraction, and the exposure to HRF induced the ubiquitination and expression of H1B from HIV-susceptible cells, suggesting that the acquisition of the resistance phenotype is intimately linked to histone activity (17). Furthermore, the biological activity of histones in the transcription regulation is well acknowledged (46), and histone modifications were implicated in a stepwise recruitment of transcription regulators, including NF-{kappa}B (47).

The direct interaction of HRF with NF-{kappa}B suggests that this protein is present in the nuclear compartment, and the antiviral activity of HRF+ cell culture supernatants indicates that HRF is also secreted into the extracellular space, which makes it a versatile protein and allows us to hypothesize that HRF treatment of HIV-1-susceptible cells induces its own expression through a mechanism involving the ubiquitination of H1B. It is possible that once expressed, a portion of HRF is transported to the nuclear compartment, where it inhibits NF-{kappa}B-DNA binding, and another portion of HRF together with ubiquitinated H1B is exported from the cells, where it has a protective function on HIV-1-susceptible cells.

Considerable research has been devoted to identification of Ag-specific immune responses that might control or prevent HIV-1 infection. More recently, it has become clear that the human genome encodes several innate immune effectors that possess potent antiviral activity, including APOBEC3G (48) and Ref1 (49, 50). This work exposes yet another piece of the puzzle of the innate cellular responses to HIV-1, an inducible product of a human gene that is secreted from HIV-1 resistant cells and confers the antiviral protection upon other cells in culture. We showed in this study that the end-point target of HRF is NF-{kappa}B, and final identification of this molecule will promote understanding of the entire mechanism of this acquired antiviral activity involving H1B, its ubiquitination, NF-{kappa}B, and HRF.


   Acknowledgments
 
1G5 cells were obtained through the National Institutes of Health AIDS Research and Reagent Program, Division of AIDS, National Institute of Arthritis and Infectious Diseases, National Institutes of Health. We thank Dr. Mary Jane Potash for critical reading of this manuscript.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by Grants RO1AI48388, MH070282, AI061286, and AI43913 from the National Institutes of Health. Back

2 Address correspondence and reprint requests to Dr. Malgorzata Simm, Molecular Virology Division, St. Luke’s-Roosevelt Hospital Center, 432West 58th Street, Room 709, New York, NY 10019. E-mail address: ms130{at}columbia.edu Back

3 Abbreviations used in this paper: HRF, HIV-1 resistance factor; CAFc CD8 cell antiviral factor ChIP, chromatin immunoprecipitation; H1B, histone H1B; LTR, long terminal repeat; Sp1, specificity protein 1; VSV-G, vesicular stomatitis virus G. Back

Received for publication November 22, 2004. Accepted for publication June 3, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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