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Kidney Disease Section, Metabolic Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892; and the Human Retrovirus Section, ABL Basic Research Program, National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, Maryland 21702
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Key Words: nuclear receptors • AIDS • mouse mammary tumor virus • p300/CBP • steroid receptor coactivator 1
Abbreviations used: CAT, chloramphenicol acetyltransferase; CBP, CREB-binding protein; CREB, cAMP-response element binding protein; GFP, green fluorescence protein; GR, glucocorticoid receptor; GRE, glucocorticoid response element; GST, glutathione S-transferase; h, human; MMTV, mouse mammary tumor virus; NF, nuclear factor; PKA, protein kinase A; RSV, Rous sarcoma virus; SRC, steroid receptor coactivator; SV40, simian virus 40.
The HIV-1 protein Vpr, a 96–amino acid virion-associated accessory protein, has multiple functions (for reviews, see references 1–4). Vpr enhances the replication of HIV-1 virus in lymphocyte- and monocyte-derived cell lines (5), is a weak transcriptional activator of several viral promoters (6), causes host cell arrest in the G2/M phase of the cell cycle (7–10), and induces terminal differentiation in some cell lines (11). Vpr has been proposed to increase the translocation of the HIV-1 preintegration complex into the nucleus, and promotes efficient infection of nondividing macrophages (12–15). Vpr was also reported to bind to a host 41-kD cytosolic Vpr interacting protein (Rip-1) and to associate with the activated glucocorticoid receptor (GR)1 (16).
Since Vpr has been shown to circulate at detectable levels in HIV-1–infected individuals, its effects may be extended to cells not infected by HIV-1 (17, 18). To explore the potential involvement of Vpr in the pathogenesis of AIDS, we examined the effect of Vpr on glucocorticoid-responsive promoters and the interactions of Vpr with GR and components of the GR-induced transcription complex in lymphoid and rhabdomyosarcoma cell lines. We show that Vpr is a virus-encoded coactivator of the GR, suggesting that it may contribute to the development of symptoms in patients with AIDS such as muscle wasting in the absence of increased glucocorticoid levels.
Glucocorticoids play major roles in maintaining resting and stress-related homeostasis (19) and also exert antiinflammatory and immunosuppressive effects, which have made them invaluable therapeutic agents in numerous diseases (20). Host tissue sensitivity to glucocorticoids may be altered in several disease states, becoming one of the determinants of disease outcome; both glucocorticoid hypersensitivity and resistance have been reported (21). Glucocorticoid hypersensitivity could be involved in the immunosuppression and myopathy and muscle wasting observed in patients with AIDS, even in the presence of normal plasma cortisol concentrations. Glucocorticoids exert their ubiquitous and pleiotropic effects through the GR, a ligand-dependent transcription factor. Binding of the hormone to the receptor causes it to dissociate from a heterooligomer of heat shock proteins and to translocate into the nucleus, where it binds as a homodimer to specific DNA enhancer elements, the glucocorticoid response elements (GREs), or to other transcription factors, such as AP-1 and nuclear factor (NF)-
Several host coactivators of the GR have been described that directly interact with the GR and components of the transcription initiation complex to enhance the glucocorticoid signal to the transcription machinery (23, 24). Different signal transduction systems share several of these newly described coactivators, which may act not only in a synergistic but also in an inhibitory fashion (25–27). It recently became known that some coactivators possess histone acetyltransferase activity, which helps loosen promoter DNA from the tightly bound histone octamers by acetylating the free NH2 termini of lysine residues, facilitating the access of components of the transcriptional initiation complex to the promoter region (28–30). Here we show that Vpr enhances the activation of a glucocorticoid-inducible gene. We studied the mechanism of this effect and especially the interactions of Vpr with other components of the transcription machinery. It was determined that Vpr interacts directly with components of the RNA polymerase holoenzyme and contributes to the formation of a large complex competent for transcription.
Cell Transfections.
Magnetic Cell Sorting and Immunoblots for the GR and Vpr.
In Vitro Binding Assay.
Yeast Two Hybrid Assay.
Coimmunoprecipitation of Vpr.
Statistical Analyses.
B (22).
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Plasmids.
The pCDNA3-VPR and pGEX-4T3-VPR vectors for expression of Vpr and GST-Vpr, respectively, were constructed using PCR-amplified Vpr sequence in pNLA1 (a gift from Dr. K. Strebel, National Institutes of Health, Bethesda, MD), which contains the full coding sequence of the wild-type Vpr (31). The plasmids used were pCDNA3 (Invitrogen) and PGEX-4T3 (Amersham Pharmacia Biotech), respectively. The pCMV-FLAG-VPR and pCMV-FLAG-VPR(36–96) vectors were generated by in-frame insertion of PCR-amplified Vpr cDNA corresponding to amino acids 1–96 or 36–96 between the HindIII and XbaI sites of pFLAG-CMV-2 (Eastman Kodak Co.). These plasmids express Vpr fragments tagged at the NH2 terminus by the FLAG epitope under the control of the CMV promoter. The 64 leucine to alanine–substituted mutant Vpr cDNAs were prepared by PCR-assisted in vitro mutagenesis of the Vpr expression vectors and named pCDNA3-VPRL64A, PGEX-4T3-VPRL64A, and pCMV-FLAG-VPRL64A, respectively. Similarly, a vector expressing Vpr with an arginine to alanine substitution at position 80 was named pCDNA3-VPRR80A. For the yeast two hybrid assay, DNA-binding LexA fusion vector pEG202, B42-activation domain fusion vector pJG4-5, and lacZ reporter plasmid pSH18-34 were used (32). Plasmids LexA-Vpr and LexA-Tat express Vpr or Tat fused to LexA DNA-binding domain, respectively (33). LexA-VprL64A and LexA-VprR80A expression vectors were constructed by PCR-assisted in vitro mutagenesis. The full-length human (h)GR
protein fused to B42-activation domain expression vector was constructed by in-frame insertion of human GR cDNA into pJG4-5 (pJG45-GR). The other plasmids used were as follows: pRc/RSV, pCR3.1, and pHookTM-1 (Invitrogen); pSV40-β-Gal (Promega Corp.); β-actin-luc, containing –472 to +49 of human β-actin promoter, pRShGR and pGR107, containing the full-length coding region of hGR, and pRS-erbA–1, containing a thyroid receptor cDNA in inverse orientation but otherwise similar to pRShGR (all gifts from Dr. R. Evans, Salk Institute, La Jolla, CA); pF25-GFP-hGR, expressing green fluorescent protein (GFP) tagged at the NH2 terminus of hGR; pSVLPRA, containing the full-length coding region of human progesterone receptor A (a gift from S.S. Simons, Jr., National Institutes of Health); pRc/RSV-CREB341, containing the full-length coding region of cAMP-responsive element binding protein (CREB); RSV-PKA, which expresses the constitutive active form of protein kinase A (PKA); p(–71)SRIF-CAT, containing nucleotides –71 to +53 of rat somatostatin 5' flanking region (all gifts from Dr. R.H. Goodman, Vollum Institute, Portland, OR); CMVβ-p300-CHA expression vector (a gift from Dr. D. Livingston, Dana-Farber Cancer Institute, Boston, MA); pCR-SRC-1a (a gift from Dr. B.W. O'Malley, Baylor College of Medicine, Houston, TX); HE0, containing the full-length coding region of human estrogen receptor (a gift from Dr. P. Chambon, University of Strasbourg, Strasbourg, France); pRSV-β-Gal and ERE-tk-luc, containing synthetic vitellogenin A2 ERE sequence from –336 to –310 (gifts from Dr. J. Segars, National Institutes of Health), pMMTV-luc (a gift from Dr. G. Hager, National Institutes of Health), pHH-luc and pM-luc (American Type Cell Collection), containing either full-length MMTV-LTR, –223 to +105, or –109 to +105 of the MMTV-LTR, respectively; and the synthetic GRE-containing plasmids, pGRE2-NF1-E1B-CAT, pNF1-E1B-CAT, pGRE2-E1B-CAT, and pE1B-CAT (gifts from Dr. J.A. Cidlowski, National Institutes of Health, Research Triangle Park, NC).
A204, HS729, and CV-1 cells were transfected using lipofectin (Life Technologies); CEM and Jurkat cells were transfected using electroporation (960 µF, 250 mV [34]). The cells were treated with dexamethasone 24 h after transfection, and cell lysates were collected after a 24-h incubation with the steroid. Luciferase activity, chloramphenicol acetyltransferase (CAT) activity, and protein concentrations were determined as described (35–37). All measurements of the reporter gene activity were conducted in triplicate transfections and averaged. Cells from the same transfection were used with and without dexamethasone whenever the effects of the steroid were studied.
The transfection-positive cells (0.5–1 x 107 cells) were enriched by the pHookTM-1 plasmid method following the company's recommendations, and homogenized and centrifuged at 300 g for 5 min in Taps buffer (25 mM Taps [pH 8.0], 2 mM dithiothreitol, CompleteTM tablets 1 Tab/50 ml [Boehringer Mannheim], 10% glycerol). The supernatants were used in Western blot analyses, with affinity-purified polyclonal rabbit anti–human GR antibodies (Affinity Bioreagents), anti-GR antibody (36), HIV-1NL4-3 Vpr antiserum (National Institutes of Health AIDS Research and Reference Reagent Program), or an anti-FLAG (M2) antibody (Eastman Kodak Co.).
35S-labeled human GR were generated by in vitro translation and tested for interaction with GST-Vpr, GST-VprL64A, or glutathione S-transferase (GST) protein, immobilized on glutathione-sepharose beads in the presence or absence of 10–6 M dexamethasone and/or 10–5 M RU 486 in buffer containing 50 mM Tris-HCl (pH 8.0), 50 mM NaCl, 1 mM EDTA, 0.1% NP-40, 10% glycerol, and 0.1 mg/ml BSA at 4°C for 1 h. After vigorous washing with the buffer, proteins were eluted and separated on a 4–20% SDS-PAGE gel. Gels were fixed and exposed on film.
Yeast strain EGY48 (Clontech) was transformed with the lacZ reporter plasmid pSH18-34, pJG45-GR coding sequence fused to B42-activation domain under the control of the galactose-inducible promoter, and pLexA-Vpr coding for wild-type or mutant Vpr-LexA DNA-binding domain fusion in pEG202 vector (32). The cells were grown to the early stationary phase in a selective medium with galactose to induce GR fusion expression, and permeabilized with CHCl3-SDS treatment, and the β-galactosidase activity was measured in the cell suspension using a colorimetric assay with ONPG as a substrate.
3 h before cell lysis, cells were exposed to 10–6 M dexamethasone and/or 10–5 M RU 486. Cell lysis and coimmunoprecipitation were carried out using lysis buffer (50 mM Tris-HCl [pH 7.4], 400 mM NaCl, 0.2% NP-40, CompleteTM tablets 1 Tab/50 ml). Proteins were precipitated by anti–human TFIID (TBP) antibody, anti–human TFIIB antibody (Santa Cruz Biotechnology, Inc.), or by anti-GR antibody bound to protein A Trisacryl (Pierce Chemical Co.). After blotting on nitrocellulose membrane, FLAG-tagged Vprs were detected by anti-FLAG (M2) antibody.
Statistical analysis was carried out by analysis of variance, followed by Student's t test with Bonferroni correction for multiple comparisons.
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Vpr Enhances Glucocorticoid Response.
We first examined the effect of Vpr on the LTR promoter of mouse mammary tumor virus (MMTV)-driven luciferase activity in human T lymphoblastoma–derived CEM and Jurkat cells, and human rhabdomyosarcoma–derived A204 and HS729 cells. We cotransfected different amounts of a Vpr expression vector, pCDNA3-VPR, with the dexamethasone-responsive plasmid pMMTV-luc (Fig. 1, A–D). Vpr induced a 3.4-, 4.4-, 20-, and 4.5-fold increase of luciferase activity in the dexamethasone-stimulated cell lines, respectively, whereas it had minimal effects in the absence of dexamethasone. As A204 cells showed the highest increase of luciferase activity, we conducted a dexamethasone titration experiment in the presence or absence of pCDNA3-VPR in this cell line (Fig. 1 E). The dose–response curve of the MMTV promoter to dexamethasone was potently shifted in the presence of Vpr, indicating that this protein potentiates the glucocorticoid signal transduction pathway in a fashion reminiscent of a classic coactivator (23).
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To show the dependency of the Vpr effect on the presence of GREs, we used three MMTV deletion mutants, four GRE-containing promoter constructs, the simian virus 40 (SV40) and Rous sarcoma virus (RSV) promoters, and the human β-actin promoter, which contain no recognizable GREs. Because the NF1 site is important for the full activation of the MMTV promoter, we used promoter constructs containing a synthetic NF1 site (Fig. 2 A). As shown in Fig. 2, B and C, the coactivator effect of Vpr depended on the presence of GREs. Decreasing the numbers of GREs in the MMTV or in synthetic GRE promoters was associated with diminishing Vpr coactivator activity. Vpr had no or minimal effect on the synthetic promoters not containing GRE sites and on the SV40, RSV, and β-actin promoters (data not shown). We also used CV-1 cells, which contain no functional GR, to show the requirement of the GR for the coactivator effect of Vpr on the MMTV promoter. Vpr-dependent activation could be observed only when CV-1 cells were cotransfected with the GR expression vector pRShGR (Fig. 2 D).
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and β isoforms, we examined the protein levels of the two isoforms of the GR in A204 cells (38). We enriched the transfected cell population up to 90% by using Capture-TecTM beads and examined the effect of Vpr on the levels of the GR isoforms by Western blot. As shown in Fig. 2 E, Vpr affected neither the levels of these isoforms nor the isoform ratio in A204 cells.
We also examined the possibility that Vpr influenced the translocation of the GR induced by dexamethasone in A204 cells by using GFP-tagged hGR (GFP-GR [39, 40]). Vpr did not change the translocation rate or efficiency of dexamethasone-activated GFP-GR (data not shown). In addition, dexamethasone did not affect localization of GFP-tagged Vpr at concentrations sufficient to translocate the GFP-GR from the cytosol into the nucleus (data not shown).
Vpr Interacts Directly with GR through the Coactivator Motif LXXLL.
The recent discovery that cellular nuclear receptor coregulators contain one or more signature motifs (LXXLL) through which they interact with nuclear hormone receptors and exert their coregulator effects (41) prompted examination of the Vpr sequence for such motifs. Since Vpr contains the sequence LQQLL at amino acids 64–68, a region of -helical secondary structure (42), we examined the functional importance of this motif by generating mutant Vpr proteins with disrupted sequences. A mutant Vpr containing a leucine to alanine substitution at amino acid 64 (VprL64A) failed to exert any coactivator effect in our assays, and showed a concentration-dependent dominant-negative effect on the wild-type Vpr (Fig. 3 A). In contrast, VprL64A was fully functional in arresting cells in G2/M phase of the cell cycle. A second point mutant, VprR80A, showed the opposite phenotype; it had coactivation function similar to Vpr, but did not arrest cells in G2/M (33). These results show that the glucocorticoid coactivator activity of Vpr is distinct from its cell cycle arrest function and requires an intact LXXLL domain.
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To test the direct interaction of Vpr and GR, we used in vitro–translated hGR and bacterially expressed GST-tagged Vpr. Some binding of GR to GST-Vpr was detected in the absence of dexamethasone, whereas binding was increased in the presence of the steroid. In contrast, GR did not bind to the mutant GST-VprL64A (Fig. 4 A). RU 486 antagonized the dexamethasone-induced interaction of GR to GST-Vpr. We also detected interaction of GR with Vpr in the yeast two hybrid system (Fig. 4 B). Wild-type Vpr and VprR80A, which retains coactivator activity, interacted with the GR and induced β-galactosidase activity, whereas the dominant-negative mutant VprL64A produced no such effect, further supporting a direct interaction between the two molecules in the cell.
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We also studied the interactions of Vpr with GR and components of the transcription complex in dexamethasone- and mock-treated pCMV-FLAG-VPR–transfected cells by coimmunoprecipitations of cell extracts. As shown in Fig. 5, FLAG-Vpr was coimmunoprecipitated by anti-TFIID (TBP), anti-TFIIB, or anti-GR antibodies in dexamethasone-treated cells, suggesting that Vpr binds to components of the transcription machinery and to the GR, as part of the glucocorticoid-activated transcription initiation complex (23). FLAG-VprL64A was coimmunoprecipitated by anti-TFIIB antibody, suggesting that the TFIIB-binding site of this mutant remains functional, whereas FLAG-Vpr(36–96) was not precipitated by either GR, TFIID, or TFIIB antibodies. Coprecipitation of FLAG-Vpr was not efficient by TFIIB antibodies in the absence of dexamethasone, whereas it increased in its presence. FLAG-VprL64A was similarly precipitated by TFIIB antibodies, but this did not increase in the presence of dexamethasone. These results suggest that Vpr binds directly to TFIIB through the NH2-terminal part of the molecule and to the GR through the LXXLL coactivator domain. Binding to both factors leads to enhanced incorporation of Vpr into a large transcription complex also including other transcription factors such as TFIID. The binding of the VprL64A mutant to TFIIB but not to the GR may explain its transdominant negative phenotype as competition of the mutant with wild-type Vpr for the TFIIB-binding site. If Vpr becomes part of a bigger transcription complex in the presence of dexamethasone, then it may be coprecipitated with antibodies for other proteins known to be in the GR transcription complex, such as the coregulators p300. In coimmunoprecipitation experiments using lysate of FLAG-Vpr–transfected A204 cells, Vpr and p300 were either weakly or strongly coprecipitated by anti-FLAG antibody in the absence or presence of dexamethasone, respectively (data not shown). This may reflect the presence of Vpr and p300 in the same complex. Alternatively, it may indicate additional contacts of Vpr with p300. It was recently suggested that Vpr transactivation on the HIV promoter is mediated through p300 (45). To study any potential interactions of Vpr and p300, we compared the effects of transfected Vpr and p300/CBP coregulators on the MMTV promoter (Fig. 6 A). We also studied the interaction of Vpr with another GR coactivator, steroid receptor coactivator (SRC)-1, by cotransfecting pCDNA3-VPR with an SRC-1 expression vector. As shown in Fig. 6, A and B, Vpr potentiated dexamethasone activity >20-fold. Vpr and p300 or SRC-1a synergistically enhanced ligand-activated GR activity on the MMTV promoter. No enhancement was observed in the absence of glucocorticoid. Therefore, Vpr appears to synergize with other coactivators in the activation of the MMTV promoter.
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We found that in several lymphoid (CEM, Jurkat), rhabdomyosarcoma (A204, HS729), and kidney (CV1) cell lines Vpr dramatically increases the effect of glucocorticoids on the MMTV promoter (Fig. 1). This effect of Vpr is mediated by binding to the GR through a helical part of the molecule containing the LQQLL sequence; disruption of the motif is detrimental for both GR binding and the transcriptional effect of Vpr (Figs. 3 and 4). This region has been shown to interact with transcription factor Sp1 (46). Mutations in this region also decrease the nuclear localization of Vpr (47). Similar motifs, LXXLL, were shown to be involved in binding of several coactivator molecules to nuclear receptors (41). These findings suggest that Vpr is a virally encoded coactivator of the GR. Vpr also works as a coactivator for other nuclear receptors, such as the progesterone and estrogen receptors, as would be expected from the presence of a functional LXXLL motif. A previous report has associated Vpr to the activated GR complex, presumably through interaction with a cytosolic protein, named Rip-1 (16). Our results suggest that the observed association to the activated GR complex is through a direct interaction to GR.
Classical nuclear receptor coactivators, like SRC-1 and CBP/p300, were shown to interact with the receptors in the presence of the appropriate ligand as well as with general transcription factors, components of the RNA polymerase II complex (29, 48). They also possess histone acetyltransferase activity that may overcome the inhibitory effect of chromatin on gene expression, leading to efficient transcription (28, 30).
Coprecipitation experiments (Fig. 5) showed that in the presence of glucocorticoid Vpr became associated not only with the GR, but also with TFIIB and TFIID, consistent with its incorporation into a stable transcription initiation complex, reminiscent of other nuclear receptor coactivators. Notably, mutant VprL64A, which is unable to enhance the glucocorticoid response, was excluded from such a complex, but was still able to bind to TFIIB. This observation is consistent with the reported ability of the NH2-terminal part of Vpr to interact with TFIIB (44) and may explain the dominant-negative phenotype of the L64A mutation that disrupts GR binding but retains the ability to bind, and thus block, a general transcription factor.
These results suggest that Vpr functions by bridging the ligand-bound nuclear receptor and general transcription factors, resulting in the stabilization of the transcription preinitiation complex. It is also highly probable that Vpr cooperates with and enhances the activity of other coactivators, the same way coactivators cooperate with each other. We found synergistic effects of Vpr with both p300 and SRC-1 on the MMTV promoter (Fig. 6). The simplest interpretation of these results is that each coactivator, including Vpr, contributes to the efficiency and stability of the transcription initiation complex (Fig. 7).
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The contribution of the glucocorticoid coactivator effect of Vpr on the replication of HIV-1 remains to be defined. The reported effects of glucocorticoids on HIV-1 expression have been controversial and both mildly stimulatory and inhibitory (5, 16, 50–53). However, there is a role for the Vpr coactivator activity in the pathophysiology of HIV-1 infection. Patients with AIDS have clinical manifestations compatible with glucocorticoid hypersensitivity, reflected in severe immune suppression and profound myopathy and muscle wasting, all recognized effects of chronically elevated levels of glucocorticoids. Thus, the glucocorticoid coactivator actions of Vpr may contribute, along with actions of other viral proteins, to the development of HIV-1–associated pathologies. Furthermore, Vpr may mimic glucocorticoid effects on apoptosis and on immune system suppression through induction of IB transcription (54). The role of HIV-1 on muscle wasting cannot be explained by direct effects of the virus. However, Vpr can be detected outside of infected cells and in the plasma of HIV-1–infected patients (17, 18). Like Tat, Vpr appears also to affect uninfected cells in a paracrine or endocrine fashion (17, 18, 55, 56). If Vpr contributes to increased tissue sensitivity to glucocorticoids, our data suggest a role for steroid hormone receptor antagonists, such as RU 486, or Vpr antagonists in the treatment of HIV-1 disease, even in the absence of hypercortisolism.
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Submitted: 14 August 1998
Revised: 21 October 1998
Address correspondence Tomoshige Kino, Section on Pediatric Endocrinology, Developmental Endocrinology Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bldg. 10, Rm. 10N262, 10 Center Drive MSC 1862, Bethesda, MD 20892-1862. Phone: 301-496-6909; Fax: 301-402-0574; E-mail: kinot{at}cc1.nichd.nih.gov; or Dr. George N. Pavlakis, ABL Basic Research Program, National Cancer Institute-Frederick Cancer Research and Development Center, Bldg. 535, Rm. 210, Frederick, MD 21702. Phone: 301-846-1474; Fax: 301-846-6368; E-mail: pavlakis{at}ncifcrf.gov
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