Octamer Transcription Factors Up-regulate the Expression of CCR5, a Coreceptor for HIV-1 Entry*

T cell activation can induce expression of CCR5, a major coreceptor for macrophage-tropic (R5) human immunodeficiency virus type 1 (HIV-1). Here we report that overexpression of the Oct-2 transcription factor and octamer coactivator BOB.1/OBF/OCA-B, both of which are induced in T cells following T cell receptor signaling, synergistically up-regulates CCR5 promoter activity via interaction with an octamer motif on the promoter. We also show that the octamer transcription factors can increase cell surface expression of CCR5 and fusogenicity of the cells with R5 HIV-1 Env. These results suggest that octamer transcription factors may play a critical role in the induction of CCR5 expression on, and thereby susceptibility to, R5 HIV-1 of T cells following antigenic stimulation.

Oct-2 is a transcription factor that binds specifically to octamer motifs on its target promoter and/or enhancer regions including immunoglobulin and interleukin (IL) 1 -2 genes (1). Oct-2 is expressed predominantly in the B-cell lineage; however, recent studies demonstrated that B cell-restricted gene regulation is dependent on B cell-specific coactivator BOB.1/ OBF/OCA-B (2)(3)(4)(5). Expression of Oct-2 and BOB.1/OBF/OCA-B is also induced by antigenic stimulation of T cells (6,7). Thus, the octamer transcription system may play a role in gene expression during T cell activation.
The CC chemokine receptor CCR5 is preferentially expressed in type 1 T helper cells (8) and serves as a receptor for the CC chemokines regulated upon activation, normal T cell expressed and secreted (RANTES), macrophage inflammatory proteins 1␣ and 1␤ (9 -11). CCR5 is also essential for cellular entry of macrophage-tropic (R5) human immunodeficiency virus type 1 (HIV-1) (12)(13)(14)(15)(16)(17), and levels of its expression appear to be critical for infectability by R5 HIV-1 (18). We have recently cloned and characterized the promoter region of CCR5 and identified an octamer motif on the promoter region (19); however, it remained to be determined whether the octamer motif has a role in the regulation of CCR5 expression on T cells or macrophages, the two major target cells for HIV-1.
In this study, we demonstrate that the octamer motif plays a critical role in the induction of CCR5 expression in activated T cells and that octamer-mediated overexpression of CCR5 increases susceptibility of the cells to R5 HIV-1 infection.

EXPERIMENTAL PROCEDURES
Plasmids-Plasmid pGL-CCR5 WT contains the CCR5 promoter sequence spanning Ϫ770 to ϩ61 relative to the transcription start site (19), followed by the luciferase reporter gene. Plasmid pGL-CCR5 ⌬Oct has mutations (shown in boldface letters below) on the octamer motif (indicated by underline) around Ϫ230 relative to the transcription start site (ATGAATGTAAATGTTC 3 ATGAATGTATACGTTC), which was generated by PCR-oriented site-directed mutagenesis (20). Plasmids pCG-Oct2, pCGN-OCA-B, and their parent plasmid pCGN were generous gifts of W. Herr (Cold Spring Harbor Laboratory) (21,22). Plasmid pMT-GATA1 and its parent plasmid pMT2T were kindly provided by S. H. Orkin (Harvard Medical School) (23); plasmid pMT-p65 (which encodes the NF-B p65 subunit) was a gift of U. Siebenlist (NIAID, National Institutes of Health) (24). Plasmids encoding Env from HIV-1 R5 strain JR-FL or X4 strain HXB2 or Env from amphotropic murine leukemia virus (AMLV) were kindly provided by N. Landau (Aaron Diamond AIDS Research Center) (14).
Cells-Peripheral blood mononuclear cells were obtained from healthy volunteers (Department of Transfusion Medicine, Warren Grant Magnuson Clinical Center, National Institutes of Health and Red Cross Blood Center in Nagasaki Prefecture, Japan), and CD4 ϩ T cells were isolated as described previously (25). Where indicated, CD4 ϩ T cells were stimulated with phorbol 12-myristate 13-acetate (PMA) (1 M) plus ionomycin (1 M) or anti-CD3 plus anti-CD28 antibodies immobilized by immunomagnetic beads for 8 h. CCR5-expressing PM1 lymphoid cells were propagated as described previously (19).
RNA-PCR-Total cellular RNA was extracted from cell pellets according to the manufacturer's instructions (RNeasy Mini; Qiagen, Santa Clarita, CA). RNA was reverse transcribed in a 50-l reaction containing 50 mM Tris-HCl (pH 8.4), 75 mM KCl, 3 mM MgCl 2 , 10 mM dithiothreitol, 0.8 mM each dNTP, 1 unit of RNasin (Promega, Madison, WI), 100 pmol of random hexamer oligonucleotides, 2 g of RNA sample, and 400 units of Moloney murine leukemia virus reverse transcriptase (Promega). The reaction was incubated for 60 min at 37°C, heated at 95°C for 5 min, and quick-chilled on ice. The cDNA corresponding to whole coding sequence of Oct-2 or BOB.1/OBF/OCA-B was amplified by PCR for 30 cycles in a 50-l reaction containing 1 l of cDNA product and 1.25 units of Taq Bead TM Hot Start Polymerase (Promega). MgCl 2 concentration in the reaction was 3.0 mM for Oct-2 amplification and 2.0 mM for OCA-B amplification. Primers used for PCR were 5Ј-ATGTCTAAGCCCCTGGAG-3Ј (sense) and 5Ј-CATCAAG-GCAGGTAAGGG-3Ј (antisense) for Oct-2 and 5Ј-CACAGCTCCGGAG-CAAGC-3Ј (sense) and 5Ј-CTAAAAGCCTTCCACAGAG-3Ј (antisense) for OCA-B. A thermal cycle profile was denaturation for 45 s at 94°C, annealing for 1 min at 55°C, and elongation for 1 min 30 s at 72°C, followed by a final elongation at 72°C for 7 min. Primers used for amplification of CCR5 cDNA were 5Ј-TACCAGCCTCCGTATTTC-3Ј (sense) and 5Ј-TTACTATTCCCTCACCTTACC-3Ј (antisense). Control amplification was performed for ␤-actin cDNA. Amplification was performed at denaturation for 45 s at 94°C, annealing for 1 min at 50°C, and elongation for 1 min at 72°C for 20 cycles, followed by a final elongation at 72°C for 7 min. The PCR products (10 l for Oct-2, BOB.1/OBF/OCA-B, or CCR5 and 2 l for ␤-actin) were resolved on a 1% agarose gel and visualized by ethidium bromide staining.
Transfection and Transient Expression Assays-Transfections and luciferase assays for transient expression assays were performed as described previously (26).
Flow Cytometric Analysis-Cell surface expression of CCR5 or CXCR4 was determined by staining cells with monoclonal anti-CCR5 antibody 2D7 phycoerythrin conjugate or anti-CXCR4 antibody 12G5 phycoerythrin conjugate (PharMingen, San Diego, CA), respectively, and by analyzing in a FACScan (Becton Dickinson Immunocytometry, San Jose, CA), as described previously (27).
Viruses and Fusion Assays-Recombinant vaccinia viruses (rVV) were propagated as described previously (27). rVV expressing Oct-2 (vOct2) was a generous gift of Dr. P. C. van der Vliet (28). Fusion assays were performed as described previously (28). In brief, 293T cells were seeded onto 60-mm diameter tissue culture plates, transfected with 10 g of an Env expression vector plasmid using calcium phosphate method, infected with vCB21R (a rVV encoding the lacZ gene under the control of T7 promoter) at a multiplicity of infection of 10 on the following day, and served as fusion effectors. Primary CD4 ϩ T cells obtained from healthy volunteers were infected with vTF7-3 (a rVV expressing T7 RNA polymerase) along with vWT or vOct2 at a multiplicity of infection of 10 each and served as fusion targets. After a 10-h culture at 37°C, both fusion effectors (293T cells) and fusion targets (CD4 ϩ T cells) were mixed and incubated at 37°C for 6 h in the presence of cytosine arabinoside, and the mixed cell culture lysates were subjected to ␤-galactosidase assays.

Activation of CD4 ϩ T Cells Induces Expression of Oct-2 and
BOB.1/OBF/OCA-B-Since we have recently identified an octamer motif on the CCR5 promoter, we wanted to investigate whether this motif plays a role in CCR5 expression on CD4 ϩ T cells, a target for HIV-1. First, we examined whether T cell activation can induce expression of Oct-2 (a B cell-specific octamer factor) and BOB.1/OBF/OCA-B (a B cell-specific octamer coactivator). As shown in Fig. 1, mRNA specific for Oct-2 and BOB.1/OBF/OCA-B was amplified by reverse transcriptase-PCR in CD4 ϩ T cells treated with PMA plus ionomycin or anti-CD3 plus anti-CD28, stimuli mimicking T cell receptor signaling or antigenic stimulation. As previously shown, CCR5 mRNA in activated CD4 ϩ T cells but not that in unstimulated cells was easily detectable under the PCR conditions used (Fig. 1). Thus, these octamer factors may be able to influence expression from octamer motif-bearing promoter (e.g. that for CCR5) in activated CD4 ϩ T cells. Overexpression of Oct-2 Up-regulates the CCR5 Promoter-To investigate whether octamer transcription factors can regulate CCR5 promoter activity, PM1 cells were transfected with pGL-CCR5 WT along with various amounts of an expression vector for Oct-2. As shown in Fig. 2A, overexpression of Oct-2 up-regulated CCR5 promoter activity in a dose-dependent manner. Furthermore, stimulation with PMA plus ionomycin synergistically increased Oct-2-mediated transactivation of CCR5 promoter (data not shown).
To determine whether an octamer motif on the CCR5 promoter is critical for Oct-2-mediated activation of the promoter, the octamer motif was mutated in plasmid pGL-CCR5 WT, and the construct was tested for its reporter activity. Mutation of the octamer motif had minimal effect on the CCR5 promoter activities induced by either GATA1 (Fig. 2C) or NF-B p65 (data not shown); however, it almost abolished the promoter activity induced by Oct-2 (Fig. 2B), indicating that the octamer motif plays a crucial role in the Oct-2-induced activity of the CCR5 promoter.
Oct-2 and the Octamer Coactivator BOB.1/OBF.1/OCA-B Synergistically Up-regulate the CCR5 Promoter-The octamer coactivator BOB.1/OBF/OCA-B plays a critical role in octamerdependent transcriptional activation in B cells and activated T cells (2)(3)(4)(5)7). To determine whether BOB.1/OBF/OCA-B plays a crucial role in the induction of CCR5 promoter activity, Oct-2 and BOB.1/OBF/OCA-B were expressed in PM1 cells individually or in combination. Either Oct-2 or BOB.1/OBF/OCA-B alone had a minimal effect on the CCR5 promoter activity at the concentration used; however, expression of both factors markedly enhanced the promoter activity (Fig. 3).
Overexpression of Oct-2 Up-regulates Cell Surface Expression of CCR5-As shown above, octamer transcription factors can up-regulate the CCR5 promoter. To demonstrate whether cell surface expression of CCR5 is also up-regulated by octamer transcription factors, we overexpressed Oct-2 by infecting CD4 ϩ T cells with vOct2 (a rVV expressing Oct-2), and determined cell surface CCR5 expression by flow cytometry. CCR5 expression in CD4 ϩ T cells that were infected with vWT (a wild type rVV) was modest (3%) but was significantly induced (30%) by infection of CD4 ϩ T cells with vOct2 (Fig. 4A). Thus, overexpression of Oct-2 appears to induce cell surface expression of CCR5. In contrast, overexpression of Oct-2 did not induce cell surface CXCR4 expression (Fig. 4B).
Overexpression of Oct-2 Increases Fusogenicity of Peripheral Blood Lymphocytes with R5 HIV-1 Env-Levels of CCR5 expression appear to correlate well with infectability of CD4 ϩ T cells by R5 HIV-1 (18). We therefore investigated whether up-regulation of CCR5 expression by Oct-2 can increase efficiency of fusion/entry of R5 HIV-1. Primary CD4 ϩ T cells (fusion targets) were infected with vWT or vOct2 as well as vTF7-3 (expressing T7 RNA polymerase). 293T cells (fusion effectors) were transfected with a plasmid expressing HIV-1 JR-FL (R5) Env or HIV-1 HXB2 (X4) Env or a plasmid expressing AMLV Env and infected with vCB21R (encoding the lacZ gene under the control of the T7 promoter). Fusion efficiency between the two cells was assayed by ␤-galactosidase activity in the mixed cell culture lysates. Overexpression of Oct-2 markedly increased fusogenicity of CD4 ϩ T cells with R5 HIV-1 Env ( Fig. 5), and pretreatment of the cells with AOP-RANTES (a CCR5 ligand) abolished R5 HIV-1 Env-mediated cell fusion (data not shown), indicating that CCR5 is required for the Oct-2-mediated effect. Fusogenicity with X4 HIV-1 Env was modestly increased upon overexpression of Oct-2 at a level comparable with that with unrelated AMLV Env (Fig. 5), suggesting that Oct-2 has no effect on CXCR4-mediated cell fusion per se, although it may somehow increase cellular fusogenicity in a nonspecific manner. Thus, the octamer transcription system may play a critical role in R5 HIV-1 infection by upregulating expression of CCR5. DISCUSSION In this study, we have demonstrated that octamer transcription system can up-regulate expression of CCR5 at the promoter level, leading to increased efficiency of R5 HIV-1 entry into CD4 ϩ T cells. Levels of CCR5 expression appear to correlate well with infectability of CD4 ϩ T cells by R5 HIV-1 (18), and expression of CCR5 appears to be highly regulated (9, 30 -36). Activation with phytohemagglutinin and IL-2 increases expression of CCR5 on lymphocytes (30). Treatment of macrophages with IL-10, granulocyte-macrophage colony-stimulating factor, or macrophage colony-stimulating factor upregulates CCR5 expression (31,32), while CCR5 expression on macrophages is down-regulated by treatment with IL-4, IL-13, or prostaglandin E 2 (32,33). Furthermore, CCR5 expression in cells of monocyte/macrophage lineage appears to increase upon differentiation into a more mature phenotype (34). At the promoter level, several transcription factors have been demonstrated to regulate CCR5 expression, including p65 (RelA) (35) and GATA-1 (36). The present study has extended our understanding of molecular mechanism of regulation of CCR5 promoter activity by demonstrating that Oct-2 and BOB.1/OBF/ OCA-B, both of which are induced by antigenic stimulation of T cells (6,7), synergistically transactivate the CCR5 promoter.
Antigenic stimulation has been associated with increased viral load in HIV-infected individuals (37,38). A recent study has demonstrated that most HIV-1 strains isolated from patients who were stimulated with tetanus toxoid, a common recall antigen, were R5 (39), suggesting that antigenic stimu-lation may provide a favorable environment for replication of R5 HIV-1. Therefore, one mechanism that renders antigenically stimulated CD4 ϩ T cells highly susceptible to HIV-1 infection is probably up-regulation of CCR5 expression by the octamer transcription system. Further investigation to delineate the molecular and cellular mechanisms that regulate expression of HIV coreceptors is warranted.