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The HIV-1 virion-associated protein vpr is a coactivator of the human glucocorticoid receptor.

Kino T, Gragerov A, Kopp JB, Stauber RH, Pavlakis GN, Chrousos GP - J. Exp. Med. (1999)

Bottom Line: We report that Vpr enhances the activity of glucocorticoids in lymphoid and muscle-derived cell lines by interacting directly with the glucocorticoid receptor and general transcription factors, acting as a coactivator.A mutant Vpr molecule with disruption of this coactivator signature motif lost its ability to influence transcription of glucocorticoid-responsive genes and became a dominant-negative inhibitor of Vpr, possibly by retaining its general transcription factor-binding activities.The glucocorticoid coactivator activity of Vpr may contribute to increased tissue glucocorticoid sensitivity in the absence of hypercortisolism and to the pathogenesis of AIDS.

View Article: PubMed Central - PubMed

Affiliation: Section on Pediatric Endocrinology, Developmental Endocrinology Branch, National Institute of Child Health and Human Development, Bethesda, MD 20892, USA. kinot@cc1.nichd.nih.gov

ABSTRACT
The HIV-1 virion-associated accessory protein Vpr affects both viral replication and cellular transcription, proliferation, and differentiation. We report that Vpr enhances the activity of glucocorticoids in lymphoid and muscle-derived cell lines by interacting directly with the glucocorticoid receptor and general transcription factors, acting as a coactivator. Vpr contains the signature motif LXXLL also present in cellular nuclear receptor coactivators, such as steroid receptor coactivator 1 and p300/CREB-binding protein, which mediates their interaction with the glucocorticoid and other nuclear hormone receptors. A mutant Vpr molecule with disruption of this coactivator signature motif lost its ability to influence transcription of glucocorticoid-responsive genes and became a dominant-negative inhibitor of Vpr, possibly by retaining its general transcription factor-binding activities. The glucocorticoid coactivator activity of Vpr may contribute to increased tissue glucocorticoid sensitivity in the absence of hypercortisolism and to the pathogenesis of AIDS.

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(A) Vpr can be directly associated with hGRα. In vitro– translated hGRα and glutathione beads–immobilized GST-Vpr, GST-VprL64A, or GST were incubated in the presence of 10−6 M dexamethasone and/or 10−5 M RU 486. Lane 1, 1/10 vol of 35S-labeled hGRα was  applied. (B) Interaction of Vpr with the GR in the yeast two hybrid assay.  Yeast cells were transformed with lacZ reporter plasmid pSH18-34,  pLexA-Vpr (wild-type or mutants L64A, R80A), and pJG45-GRα coding for the galactose-inducible GRα-activation domain fusion. LexA-Tat  was used as a negative control. β-galactosidase activity was stimulated by  Vpr but not VprL64A. β-galactosidase activity of yeast cells grown with  glucose rather than galactose was for all strains much less than the activity  produced by GR-LexA-Tat interaction viewed by us as a background value. (C) Coactivator function of Vpr, FLAG-Vpr, and FLAG-Vpr(36–96). A204  cells were transfected with different amounts of pCDNA3-VPR, pCMV-FLAG-VPR, or pCMV-FLAG-VPR(36–96), and pMMTV-luc. Each point  shows the mean ± SEM values obtained in the presence of 10−7 M dexamethasone. *P < 0.001. (D and E) Vpr, VprL64A, FLAG-Vpr, and FLAG-Vpr(36–96) expressed and detected by Western blot in A204 cells using anti-Vpr or anti-FLAG (M2) antibody. A204 cells were transfected with  pCDNA3 (control), pCDNA3-VPR, or pCDNA3-VPRL64A, and Vpr was detected after immunoblotting by using HIV-1NL4-3 Vpr antiserum (D).  FLAG-Vpr or FLAG-Vpr(36–96) was detected by using anti-FLAG antibody (E).
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Figure 4: (A) Vpr can be directly associated with hGRα. In vitro– translated hGRα and glutathione beads–immobilized GST-Vpr, GST-VprL64A, or GST were incubated in the presence of 10−6 M dexamethasone and/or 10−5 M RU 486. Lane 1, 1/10 vol of 35S-labeled hGRα was applied. (B) Interaction of Vpr with the GR in the yeast two hybrid assay. Yeast cells were transformed with lacZ reporter plasmid pSH18-34, pLexA-Vpr (wild-type or mutants L64A, R80A), and pJG45-GRα coding for the galactose-inducible GRα-activation domain fusion. LexA-Tat was used as a negative control. β-galactosidase activity was stimulated by Vpr but not VprL64A. β-galactosidase activity of yeast cells grown with glucose rather than galactose was for all strains much less than the activity produced by GR-LexA-Tat interaction viewed by us as a background value. (C) Coactivator function of Vpr, FLAG-Vpr, and FLAG-Vpr(36–96). A204 cells were transfected with different amounts of pCDNA3-VPR, pCMV-FLAG-VPR, or pCMV-FLAG-VPR(36–96), and pMMTV-luc. Each point shows the mean ± SEM values obtained in the presence of 10−7 M dexamethasone. *P < 0.001. (D and E) Vpr, VprL64A, FLAG-Vpr, and FLAG-Vpr(36–96) expressed and detected by Western blot in A204 cells using anti-Vpr or anti-FLAG (M2) antibody. A204 cells were transfected with pCDNA3 (control), pCDNA3-VPR, or pCDNA3-VPRL64A, and Vpr was detected after immunoblotting by using HIV-1NL4-3 Vpr antiserum (D). FLAG-Vpr or FLAG-Vpr(36–96) was detected by using anti-FLAG antibody (E).

Mentions: 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.


The HIV-1 virion-associated protein vpr is a coactivator of the human glucocorticoid receptor.

Kino T, Gragerov A, Kopp JB, Stauber RH, Pavlakis GN, Chrousos GP - J. Exp. Med. (1999)

(A) Vpr can be directly associated with hGRα. In vitro– translated hGRα and glutathione beads–immobilized GST-Vpr, GST-VprL64A, or GST were incubated in the presence of 10−6 M dexamethasone and/or 10−5 M RU 486. Lane 1, 1/10 vol of 35S-labeled hGRα was  applied. (B) Interaction of Vpr with the GR in the yeast two hybrid assay.  Yeast cells were transformed with lacZ reporter plasmid pSH18-34,  pLexA-Vpr (wild-type or mutants L64A, R80A), and pJG45-GRα coding for the galactose-inducible GRα-activation domain fusion. LexA-Tat  was used as a negative control. β-galactosidase activity was stimulated by  Vpr but not VprL64A. β-galactosidase activity of yeast cells grown with  glucose rather than galactose was for all strains much less than the activity  produced by GR-LexA-Tat interaction viewed by us as a background value. (C) Coactivator function of Vpr, FLAG-Vpr, and FLAG-Vpr(36–96). A204  cells were transfected with different amounts of pCDNA3-VPR, pCMV-FLAG-VPR, or pCMV-FLAG-VPR(36–96), and pMMTV-luc. Each point  shows the mean ± SEM values obtained in the presence of 10−7 M dexamethasone. *P < 0.001. (D and E) Vpr, VprL64A, FLAG-Vpr, and FLAG-Vpr(36–96) expressed and detected by Western blot in A204 cells using anti-Vpr or anti-FLAG (M2) antibody. A204 cells were transfected with  pCDNA3 (control), pCDNA3-VPR, or pCDNA3-VPRL64A, and Vpr was detected after immunoblotting by using HIV-1NL4-3 Vpr antiserum (D).  FLAG-Vpr or FLAG-Vpr(36–96) was detected by using anti-FLAG antibody (E).
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Figure 4: (A) Vpr can be directly associated with hGRα. In vitro– translated hGRα and glutathione beads–immobilized GST-Vpr, GST-VprL64A, or GST were incubated in the presence of 10−6 M dexamethasone and/or 10−5 M RU 486. Lane 1, 1/10 vol of 35S-labeled hGRα was applied. (B) Interaction of Vpr with the GR in the yeast two hybrid assay. Yeast cells were transformed with lacZ reporter plasmid pSH18-34, pLexA-Vpr (wild-type or mutants L64A, R80A), and pJG45-GRα coding for the galactose-inducible GRα-activation domain fusion. LexA-Tat was used as a negative control. β-galactosidase activity was stimulated by Vpr but not VprL64A. β-galactosidase activity of yeast cells grown with glucose rather than galactose was for all strains much less than the activity produced by GR-LexA-Tat interaction viewed by us as a background value. (C) Coactivator function of Vpr, FLAG-Vpr, and FLAG-Vpr(36–96). A204 cells were transfected with different amounts of pCDNA3-VPR, pCMV-FLAG-VPR, or pCMV-FLAG-VPR(36–96), and pMMTV-luc. Each point shows the mean ± SEM values obtained in the presence of 10−7 M dexamethasone. *P < 0.001. (D and E) Vpr, VprL64A, FLAG-Vpr, and FLAG-Vpr(36–96) expressed and detected by Western blot in A204 cells using anti-Vpr or anti-FLAG (M2) antibody. A204 cells were transfected with pCDNA3 (control), pCDNA3-VPR, or pCDNA3-VPRL64A, and Vpr was detected after immunoblotting by using HIV-1NL4-3 Vpr antiserum (D). FLAG-Vpr or FLAG-Vpr(36–96) was detected by using anti-FLAG antibody (E).
Mentions: 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.

Bottom Line: We report that Vpr enhances the activity of glucocorticoids in lymphoid and muscle-derived cell lines by interacting directly with the glucocorticoid receptor and general transcription factors, acting as a coactivator.A mutant Vpr molecule with disruption of this coactivator signature motif lost its ability to influence transcription of glucocorticoid-responsive genes and became a dominant-negative inhibitor of Vpr, possibly by retaining its general transcription factor-binding activities.The glucocorticoid coactivator activity of Vpr may contribute to increased tissue glucocorticoid sensitivity in the absence of hypercortisolism and to the pathogenesis of AIDS.

View Article: PubMed Central - PubMed

Affiliation: Section on Pediatric Endocrinology, Developmental Endocrinology Branch, National Institute of Child Health and Human Development, Bethesda, MD 20892, USA. kinot@cc1.nichd.nih.gov

ABSTRACT
The HIV-1 virion-associated accessory protein Vpr affects both viral replication and cellular transcription, proliferation, and differentiation. We report that Vpr enhances the activity of glucocorticoids in lymphoid and muscle-derived cell lines by interacting directly with the glucocorticoid receptor and general transcription factors, acting as a coactivator. Vpr contains the signature motif LXXLL also present in cellular nuclear receptor coactivators, such as steroid receptor coactivator 1 and p300/CREB-binding protein, which mediates their interaction with the glucocorticoid and other nuclear hormone receptors. A mutant Vpr molecule with disruption of this coactivator signature motif lost its ability to influence transcription of glucocorticoid-responsive genes and became a dominant-negative inhibitor of Vpr, possibly by retaining its general transcription factor-binding activities. The glucocorticoid coactivator activity of Vpr may contribute to increased tissue glucocorticoid sensitivity in the absence of hypercortisolism and to the pathogenesis of AIDS.

Show MeSH
Related in: MedlinePlus