The HIV-1 Virion-associated Protein Vpr Is a Coactivator of the Human Glucocorticoid Receptor

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)GRα protein fused to B42-activation domain expression vector was constructed by in-frame insertion of human GRα cDNA into pJG4-5 (pJG45-GRα). The other plasmids used were as follows: pRc/RSV, pCR3.1, and pHook™-1 (Invitrogen); pSV40-β-Gal (Promega Corp.); β-actin-luc, containing −472 to +49 of human β-actin promoter, pRShGRα and pGR107, containing the full-length coding region of hGRα, and pRS-erbA⁻¹, containing a thyroid receptor cDNA in inverse orientation but otherwise similar to pRShGRα (all gifts from Dr. R. Evans, Salk Institute, La Jolla, CA); pF25-GFP-hGRα, expressing green fluorescent protein (GFP) tagged at the NH₂ terminus of hGRα; 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 α (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 × 10⁷ cells) were enriched by the pHook™-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, Complete™ 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-GRα antibody (36), HIV-1_NL4-3 Vpr antiserum (National Institutes of Health AIDS Research and Reference Reagent Program), or an anti-FLAG (M2) antibody (Eastman Kodak Co.).

³⁵S-labeled human GRα 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-GRα 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 GRα 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, Complete™ 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-GRα 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.

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).

To examine the dependence of the coactivator effect of Vpr on the glucocorticoid ligand–receptor interaction, we conducted several experiments in A204 cells using the GR antagonist RU 486. The effect of Vpr on the MMTV promoter was antagonized by RU 486 in a dose-dependent fashion (Fig. 1 F). Vpr-enhanced luciferase activity was completely abolished by 10⁻⁵ M RU 486, and returned to the levels seen in the absence of dexamethasone.

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 GRα expression vector pRShGRα (Fig. 2 D).

To rule out the possibility that Vpr might cause activation of the MMTV promoter by changing the levels of the GR or the ratio of the GR α 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-Tec™ 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 hGRα (GFP-GRα [39, 40]). Vpr did not change the translocation rate or efficiency of dexamethasone-activated GFP-GRα (data not shown). In addition, dexamethasone did not affect localization of GFP-tagged Vpr at concentrations sufficient to translocate the GFP-GRα from the cytosol into the nucleus (data not shown).

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 α-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.

To determine the specificity of the Vpr coactivator effect on the glucocorticoid and other nuclear receptor signal transduction pathways, we examined the effect of this protein on the progesterone, estrogen, and cAMP signal transduction pathways in A204 cells. In the first two systems, using the MMTV and the vitellogenin ERE-containing promoters, respectively, we detected a small but significant potentiation effect of Vpr by two- and fivefold, respectively. In the third system, using a cAMP-responsive promoter, Vpr showed no coactivator effect, even though the GR and cAMP signal transduction pathways share p300/ CBP as a coactivator (25; Fig. 3 B). These findings suggest that the coactivator effect of Vpr is exerted on the glucocorticoid as well as other nuclear receptor signal transduction pathways, and are in agreement with the effects reported for other coactivators of steroid nuclear receptors (25, 26, 41). We also tested the coactivator activity of HIV-2 and SIVmac239 Vpr and Vpx, since these proteins are evolutionary related to HIV-1 Vpr (43), but do not have any LXXLL coactivator signature motifs. None of these proteins showed GR coactivator activity (data not shown).

To test the direct interaction of Vpr and GR, we used in vitro–translated hGRα 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.

It has been shown that Vpr interacts with the RNA polymerase II ancillary factor TFIIB (44). The sequence necessary for this interaction was mapped between amino acids 15 and 77. We examined the coactivator function of an NH₂-terminal deletion mutant of Vpr using FLAG-Vpr(36–96), which contains a deletion of the first 35 amino acids of Vpr and does not bind to TFIIB. FLAG-Vpr(36–96) did not show any coactivator effect on the MMTV promoter in A204 cells (Fig. 4 C). In contrast, Vpr and FLAG-Vpr showed similar coactivator activities on this promoter. Vpr, VprL64A, FLAG-Vpr, and FLAG-Vpr(36–96) were expressed at similar levels and could be detected in Western blots of extracts from A204 cells transfected with the corresponding plasmids (Fig. 4, D and E). These results are consistent with the hypothesis that the effect of Vpr as a coactivator depends also on the presence of an intact TFIIB-binding domain.

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.

Vpr stimulates HIV replication in lymphocytes and is important for the efficient viral replication in macrophages due to both transcriptional activation and its role in nuclear targeting of the preintegration complex (5, 12–15). The present work suggests that, in addition, Vpr may render cells more sensitive to glucocorticoids. Though it is not immediately clear what advantage such an activity may give to the virus, this new effect may be of importance for AIDS pathogenesis.

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).

The relation of nuclear receptor coactivator activity of Vpr described in this work to previously described transcriptional effects of this HIV protein is not clear. The stimulatory effect of Vpr on several promoters, including HIV-1 LTR, correlates with Vpr's ability to block cell cycle. It was proposed that G2 arrest in T cells was sufficient to enhance LTR transcription (5). The ability of Vpr mutants to arrest dividing cells correlated also with transcription stimulation in nondividing macrophages, suggesting that cell cycle arrest is not a prerequisite for transcriptional activation by Vpr (15). In either case, it appears that the Vpr effect on nuclear receptors is distinct from HIV-1 LTR activation and cell cycle arrest. Our mutational analysis showed that there is no correlation between the ability of Vpr to stimulate glucocorticoid response and its cell cycle–blocking activity. VprL64A, a mutant unable to activate GR, was proficient in the cell cycle arrest, whereas VprR80A, known to be inactive in the arrest (49), was fully able to activate glucocorticoid response (33).

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 IκB 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.

We thank Drs. R.H. Goodman, B.W. O'Malley, D. Livingston, J.A. Cidlowski, J. Segars, A. Vottero, and S.S. Simons for plasmids and antibodies, Dr. Z.J. Chen for advice and FACS^® analysis, and G. Gragerova, K. Zachman, K. Alexander, M. Margetis, and A. Verbalis for technical assistance.

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