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Differential Effects of IκB Molecules on Tat-Mediated Transactivation of HIV-1 LTR
VIROLOGY
216, 284–287 (1996) 0062
ARTICLE NO.
SHORT COMMUNICATION Differential Effects of IkB Molecules on Tat-Mediated Transactivation of HIV-1 LTR EDWARD HARHAJ, JOSEPH BLANEY, SCOTT MILLHOUSE, and SHAO-CONG SUN1 Department of Microbiology and Immunology, The Pennsylvania State University College of Medicine, Hershey Medical Center, P.O. Box 850, Hershey, Pennsylvania 17033 Received August 22, 1995; accepted December 8, 1995 The tat gene product of the human immunodeficiency virus type 1 (HIV-1) strongly induces the transcription directed by the viral long terminal repeat (LTR). Tat acts by interacting with a target RNA element located immediately downstream of the initiation site. In addition, the action of Tat appears to be assisted by the upstream DNA enhancer elements, including the binding sites for the NF-kB/Rel family of host transcription factors. In the present study, we demonstrate that Tat transactivation of the HIV-1 LTR is markedly inhibited by several cytoplasmic inhibitors of NF-kB/Rel, suggesting the critical involvement of these host transcription factors in the function of the viral Tat protein. Furthermore, the various NF-kB inhibitors appear to have differential effects on Tat. While IkBa, IkBb, and p100 potently inhibit Tat-mediated transactivation, p105 fails to inhibit, but even moderately synergizes, the action of Tat. We further demonstrate that the action of these NFkB/Rel inhibitors on Tat correlates with their inhibitory activities on the RelA subunit of NF-kB. Finally, we show that a degradation-resistant IkBa mutant is able to potently inhibit Tat-mediated activation of the HIV-1 LTR in both untreated and tumor necrosis factor a-stimulated T cells, thus suggesting that such an IkBa mutant may serve as a constitutive repressor of HIV-1 LTR. q 1996 Academic Press, Inc.
The tat gene product of human immunodeficiency virus type 1 (HIV-1) is a potent transcriptional activator of viral gene expression directed by the long terminal repeat (LTR) of HIV-1 (reviewed in Ref. 1). Tat acts by specific binding to the transactivation response region located immediately downstream of the initiation site (1). Tat appears to increase both the rate of transcriptional initiation and the efficiency of transcriptional elongation ( refs. 2– 4, reviewed in Ref. 5). Tat-mediated stimulation of transcription involves interactions between this viral transactivator and cellular transcription factors such as Sp-1 and the basal transcription factor TFIID (6, 7). In addition, the NF-kB/Rel family of inducible transcription factors has been suggested to contribute to the enhanced expression of HIV-1 LTR in activated T cells (8). The NF-kB/Rel family contains various dimeric complexes composed of a set of structurally related polypeptides, including p50 (NF-kB1), p52 (NF-kB2), RelA (previously named p65), RelB, and the proto-oncoprotein c-Rel (reviewed in Ref. 9). In resting T cells, these NFkB/Rel complexes are sequestered in the cytoplasm as latent precursors by a family of ankyrin motif-rich inhibitory proteins (10) including IkBa (11, 12), IkBb (13), p105 (14–16), and p100 (16–18). Various immunological stimuli, such as the cytokine tumor necrosis factor a (TNFa), induce the degradation of one or more of these cyto-
plasmic inhibitors and the concomitant nuclear expression of select members of NF-kB/Rel (19). The nuclear NF-kB/Rel in turn binds to the kB enhancer elements in HIV-1 LTR and participates in the activation of viral gene expression (8). It has been proposed that NF-kB/Rel plays a role in the initial induction of HIV-1 expression, and the viral Tat regulator serves to amplify the viral gene expression. It is not clear, however, whether the action of Tat strictly requires the participation of the NF-kB/Rel transcription factors. To better understand the function of NF-kB/Rel in the activation of the HIV-1 LTR, we investigated the effects of various NF-kB/Rel inhibitors on Tat-mediated activation of the HIV-1 LTR using transient transfection and reporter gene assays. For these studies, human Jurkat T cells were transfected with a luciferase reporter plasmid driven by the HIV-1 LTR (HIV-1 LTR-luciferase) together with the tat cDNA expression vector either in the absence or in the presence of various IkB expression vectors (Figs. 1A and 1B). As expected, Tat potently activated the LTR, leading to the enhanced expression of the luciferase reporter gene (Fig. 1A, column 2). However, Tat only moderately activated the HIV-1 LTR that lacks the kB enhancer elements (HIV-1 LTR DkB, column 3). To examine whether Tat-mediated activation of the wild-type HIV-1 LTR requires NF-kB/Rel factors present in these cells, the effect of various NF-kB/Rel inhibitory proteins on the transactivation activity of Tat was investigated by cotran-
1 To whom correspondence and reprint requests should be addressed. Fax: (717) 531-6522.
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sfection studies. As shown in Fig. 1B, the transactivation activity of Tat was significantly and dose-dependently inhibited by several NF-kB/Rel inhibitors, including IkBa (columns 1–4), IkBb (columns 5–8), and p100 (columns 9–12). This inhibitory action was not due to the inhibition of Tat expression since Tat-mediated activation of the HIV-1 LTR DkB was not significantly influenced by IkBa (Fig. 1C) as well as the other inhibitors (data not shown). These results indicated that the transactivation function of Tat required the basal activity of NF-kB/Rel that could be detected in these cells (data not shown). Of note, although p105 had been demonstrated to be one of the cytoplasmic inhibitors of NF-kB/Rel (14–16), it failed to inhibit Tat-mediated activation of the LTR (Fig. 1B, columns 13–16). In fact, at low concentration, p105 exhibited a moderate, but significant, synergistic effect on Tat (column 14). We next explored the possible mechanism underlying the differential effects of the various IkBs on the action of Tat. Since RelA has been shown to be a potent transcriptional activator of HIV-1 kB enhancer (20), the inhibitory function of the different IkB molecules on the kB binding activity of the RelA subunit of NF-kB was analyzed. For these studies, we used COS7 cells which had high efficiency of transfection that allowed us to detect the DNA binding activity of transfected RelA using electrophoresis mobility shift assays (EMSA). As shown in Fig. 2, the cells were transfected with either a parental vector (pCMV4) lacking a cDNA insert (lane 1) or the RelA cDNA expression vector in the absence (lane 2) or presence of varied amounts of different IkB expression vectors (lanes 3–14). EMSA analyses of the nuclear extracts revealed specific binding of the transfected RelA to the kB probe, which formed a DNA/protein complex (lane 2). As expected, the RelA DNA binding activity was potently inhibited by IkBa (lanes 3–5), as well as IkBb (lanes 6–8) and p100 (lanes 9–11). In sharp contrast, expression of p105 in the cells did not inhibit but even enhanced the DNA binding activity of RelA (lanes 12– 14). Parallel reporter gene assays revealed that unlike other IkB molecules, pl05 failed to inhibit RelA-mediated transactivation of the HIV-1 kB enhancer (data not shown). While the underlying mechanism remains to be further investigated, these results demonstrate that the effects of the various IkB molecules on Tat correlated with their functional specificity on the RelA transcriptional activator. FIG. 1. Inhibition of Tat-mediated activation of HIV-1 LTR by different NF-kB/Rel inhibitors. (A) Human Jurkat T cells (5 1 106) were transfected using DEAE-Dextran (24) with 0.4 mg of the HIV-1 LTRluciferase reporter plasmid [HIV-1 LTR was transferred from the HIV1 LTR CAT (31) into the pGL2 basic vector (Promega) in front of the luciferase gene] in the absence (column 1) or presence (column 2) of 0.2 mg tat cDNA expression vector, pSV2Tat72 (32). In column 3, a luciferase reporter driven by the HIV-1 LTR lacking the kB elements (HIV-1 LTR DkB-luciferase) was cotransfected with pSV2Tat72. At 40 hr posttransfection, the cells were collected for luciferase assay (Promega). Luciferase activity is presented as fold induction relative to
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the basal level measured in cells transfected with the parental vectorp CMV4. The values shown in this and all the following luciferase assays are representative of three independent experiments. The absolute values for the wild-type HIV-1 LTR luciferase activity in the mock-and Tat-transfected cells are about 1.5 1 104 and 4.5 1 105 cpm, respectively. (B) Luciferase assays of cells transfected with 0.4 mg HIV-1 LTRluciferase reporter and 0.2 mg pSV2Tat72, along with the indicated amounts of the various IkB molecules. (C) Luciferase assays of cells transfected with 0.4 mg HIV-1 LTRDkB-luciferase reporter and 0.2 mg pSV2Tat72, along with the indicated amounts of IkBa.
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FIG. 2. Inhibition of RelA DNA binding activity by IkBa, IkBb, and p100, but not by p105. Monkey kidney COS7 cells were transfected by DEAE-Dextran (25) with either the parental vector pCMV4 (33) (lane 1) or 0.4 mg pCMV4-RelA cDNA expression vector (25) either alone (lane 2) or together with the indicated amounts of various pCMV4-based IkB expression constructs subcloned from the corresponding cDNAs, including IkBa (lanes 1–4, Ref. 12), IkBb (lanes 5–8, Ref. 13), p100 (lanes 9–12, Ref. 34), and p105 (lanes 13–16, Ref. 35) (lanes 3–14). At 40 hr posttransfection, nuclear extracts were isolated (26) and subjected to electrophoresis mobility shift assays (EMSA) using a 32Pradiolabeled palindromic kB probe, kB-pd (27).
Recent studies have suggested that IkBa is the major inhibitor regulating the immediate early nuclear expression of the RelA-containing NF-kB complex, which occurs in response to various cytokines and mitogens (21). These immunological inducers stimulate the phosphorylation of IkBa at its N-terminal region, which leads to rapid degradation of IkBa (22, 23). Interestingly, an IkBa mutant lacking the N-terminal 71 amino acids (IkBaDN) becomes resistant to cellular stimulation and serves as a constitutive inhibitor of NF-kB (22). To further correlate the action of IkBa on Tat and RelA, we next compared the Tat-inhibitory activities of IkBaDN and its wild type. For these studies, human Jurkat T cells were cotransfected with the HIV-1 LTR-luciferase reporter, Tat, and either the wild-type IkBa (Fig. 3A, columns 2 and 3) or IkBaDN (columns 4 and 5). Remarkably, these studies revealed that the degradation-resistant IkBa mutant (lanes 4 and 5) exhibited a markedly more potent inhibitory effect on Tat compared to that of the wild-type IkBa (lanes 2 and 3). Of note, the two forms of IkBa had similar inhibitory activities on RelA-mediated activation of the kB enhancer (Fig. 3B). These findings raised the possibility that the HIV-1 Tat protein might override the inhibitory action of IkBa. To test this possibility, luciferase reporter gene assays were performed using increasing amounts of Tat expression vector (Fig. 4A). These luciferase assays revealed that expression of increasing amounts of Tat protein in the cells significantly overrode the inhibitory effect of the wild-type IkBa (columns 1–5). In sharp contrast, Tat induced only a slight increase of the luciferase activity in cells expressing the degradation-resistant IkBa mutant (IkBaDN, lanes 6–10). Similar reporter gene studies revealed that IkBaDN potently inhibited Tat-mediated activation of HIV-1 LTR in both untreated (Fig. 4B, columns 1 and 2) and TNF-a-stimulated (columns 3 and 4) cells.
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While it remains to be further investigated how Tat overrides the action of IkBa, our studies strongly suggest that Tat-mediated transactivation of the HIV-1 LTR critically requires the action of NF-kB/Rel, especially the transactivator RelA. Our results are in support of the previous studies showing the involvement of RelA in the activation of HIV-1 LTR (28, 29). Our studies also have confirmed and extended a recent report (30) demonstrating that the function of Tat is tightly dependent on the kB enhancers present in the HIV-1 LTR. Together, these studies strongly suggest that the host transcription factor NF-kB is critically required for HIV1 gene expression.
FIG. 3. Inhibition of Tat-mediated activation of the HIV-1 LTR (A) and RelA-mediated activation of the HIV-1 kB enhancer (B) by the wild-type IkBa (IkBaWT) and its degradation-resistant mutant, IkBaDN. Jurkat cells were transfected with pSV2Tat72 (A) or RelA (B), either alone or together with the indicated amounts of IkBa or its mutant IkBaDN (the N-terminal 36 amino acids were removed from the wild-type IkBa by polymerase chain reaction), along with the HIV-1 LTR-luciferase (A) or the kB-TATA-luciferase reporter [the insert of kB-TATA-CAT (36), containing the HIV-1 kB enhancer and TATA box, was transferred into the pGL2 basic plasmid 5* of the luciferase gene (Promega)]. (B). Luciferase was assayed as described in the legend for Fig. 1.
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FIG. 4. Potent inhibition of Tat-mediated activation of HIV-1 LTR by IkBaDN in both untreated and TNF-a-stimulated T cells. (A) Tat overrides the inhibitory action of the wild-type IkBa but not that of IkBaDN. Jurkat cells were transfected with 1 mg of either IkBa wild type (lanes 1–5) or IkBaDN (lanes 6–10) together with the indicated amounts of tat cDNA expression vector, along with 0.4 mg HIV-1 LTR-luciferase. Luciferase activity was assayed as described in the legend for Fig. 1. (B) Jurkat cells were transfected with 0.2 mg tat expression vector, in the presence (/) or absence (0) of IkBaDN, along with the HIV-1 LTRluciferase reporter. At 40 hr posttransfection, the cells were either not treated (0) or incubated for 5 hr with 10 ng/ml of TNF-a and then subjected to luciferase assay.
ACKNOWLEDGMENTS We acknowledge Drs. W. C. Greene, A. D. Frankel, and S. Ghosh for providing us the cDNA expression vectors. This work is supported in part by a National Foundation For Infectious Diseases Young Investigator Matching Grant and by Public Health Service Grant 1 R01 CA6847101 to S.-C.S. S.-C.S. is a scholar of the American Foundation for AIDS Research.
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4. Feinberg, M., Baltimore, D., and Frankel, A., Proc. Natl. Acad. Sci. USA 88, 4045–4049 (1991). 5. Cullen, B., Cell 73, 417–420 (1993). 6. Jeang, K.-T., Chun, R., Lin, N. H., Gatignol, A., Glabe, C. G., and Fan, H., J. Virol. 67, 6224–6233 (1993). 7. Kashanchi, F., Piras, G., Radonovich, M. F., Duvall, J. F., Fattaey, A., Chiang, C.-M., Roeder, R. G., and Brandy, J. N., Nature 367, 295–299 (1994). 8. Nabel, G., and Baltimore, D., Nature 326, 711–713 (1987). 9. Gilmore, T. D., Cell 62, 841–843 (1990). 10. Beg, A. A., and Baldwin, A. S., Jr., Genes Dev. 7, 2064–2070 (1993). 11. Baeuerle, P. A., and Baltimore, D., Science 242, 540–546 (1988). 12. Haskill, S., Beg, A. A., Tompkins, S. M., Morris, J. S., Yurochko, A. D., Sampson-Johannes, A., Mondal, K., Ralph, P., and Baldwin, A. S., Jr., Cell 65, 1281–1289 (1991). 13. Thompson, J. E., Phillips, R. J., Erdjument-Bromage, H., Tempst, P., and Ghosh, S., Cell 80, 573–582 (1995). 14. Rice, N. R., MacKichan, M. L., and Israel, A., Cell 71, 243–253 (1992). 15. Naumann, M., Wulczyn, F. G., and Scheidereit, C., EMBO J. 12, 213–222 (1993). 16. Mercurio, F., DiDonato, J. A., Rosette, C., and Karin, M., Genes Dev. 7, 705–718 (1993). 17. Naumann, M., Nieters, A., Hatada, E. N., and Scheidereit, C., Oncogene 8, 2275–2281 (1993). 18. Sun, S.-C., Ganchi, P. A., Beraud, C., Ballard, D. W., and Greene, W. C., Proc. Natl. Acad. Sci. USA 91, 1346–1350 (1994). 19. Siebenlist, U., Franzoso, G., and Brown, K., Annu. Rev. Cell Biol. 10, 405–455 (1994). 20. Perkins, N. D., Schmid, R. M., Duckett, C. S., Leung, K., Rice, N. R., and Nabel, G. J., Proc. Natl. Acad. Sci. USA 89, 1529–1533 (1992). 21. Sun, S.-C., Ganchi, P. A., Ballard, D. W., and Greene, W. C., Science 259, 1912–1915 (1993). 22. Brown, K., Park, S., Kanno, T., Granzoso, G., and Siebenlist, U., Proc. Natl. Acad. Sci. USA 90, 2532–2536 (1993). 23. Brockman, J. A., Scherer, D. C., McKinsey, T. A., Hall, S. M., Qi, X., Lee, W. Y., and Ballard, D. A., Mol. Cell. Biol. 15, 2809–2818 (1995). 24. Holbrook, N., Gulino, A., and Ruscetti, F., Virology 157, 211–219 (1987). 25. Ganchi, P. A., Sun, S.-C., Greene, W. C., and Ballard, D. W., Mol. Biol. Cell 3, 1339–1352 (1992). 26. Schreiber, E., Matthias, P., Muller, M. M., and Schaffner, W., Nucleic Acids Res. 17, 6419 (1989). 27. Ballard, D. W., Bohnlein, E., Hoffman, J., Bogerd, H., Dixon, E. P., Franza, B. R., and Greene, W. C., New Biol. 1, 83–92 (1989). 28. Liu, J., Perkins, N. D., Schmid, R. M., and Nabel, G. J., J. Virol. 66, 3883–3887 (1992). 29. Duckett, C. S., Perkins, N. D., Kowalik, T. F., Schmid, R. M., Huang, E.-S., Baldwin, A. S., Nabel, G. J., Mol. Cell. Biol. 13, 1315–1322 (1993). 30. Alcami, J., Lain de Lera, T., Folgueira, L., Pedraza, M.-A., Jacque´, J.-M., Bachelerie, F., Noriega, A. R., Hay, R. T., Harrich, D., Gaynor, R. B., Virelizier, J.-L., Arenzana-Seisdedos, F., EMBO J. 14, 1552–1560 (1995). 31. Doerre, S., Sista, P., Sun, S.-C., Ballard, D. W., and Greene, W. C., Proc. Natl. Acad. Sci. USA 90, 1023–1027 (1993). 32. Frankel, A. D., and Pabo, C. O., Cell 55, 1189–1193 (1988). 33. Andersson, S., Davis, D., Dahlback, H., Jornvall, H., and Russell, K. W., J. Biol. Chem. 264, 8222–8229 (1989). 34. Schmid, R. M., Perkins, N. D., Duckett, C. S., Andrews, P. C., and Nabel, G. J., Nature 352, 733–736 (1991). 35. Kieran, M., Blank, V., Logeat, F., Vandekerckhove, J., Lottspeich, F., Le Bail, O., Urban, M. B., Kourilsky, P., Baeuerle, P. A., and Israel, A., Cell 62, 1007–1018 (1990). 36. Stein, B., Rahmsdorf, H., Steffen, A., Litfin, M., and Herrlich, P., Mol. Cell. Biol. 9, 5169–5181 (1989).
AP-Virology
216, 284–287 (1996) 0062
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SHORT COMMUNICATION Differential Effects of IkB Molecules on Tat-Mediated Transactivation of HIV-1 LTR EDWARD HARHAJ, JOSEPH BLANEY, SCOTT MILLHOUSE, and SHAO-CONG SUN1 Department of Microbiology and Immunology, The Pennsylvania State University College of Medicine, Hershey Medical Center, P.O. Box 850, Hershey, Pennsylvania 17033 Received August 22, 1995; accepted December 8, 1995 The tat gene product of the human immunodeficiency virus type 1 (HIV-1) strongly induces the transcription directed by the viral long terminal repeat (LTR). Tat acts by interacting with a target RNA element located immediately downstream of the initiation site. In addition, the action of Tat appears to be assisted by the upstream DNA enhancer elements, including the binding sites for the NF-kB/Rel family of host transcription factors. In the present study, we demonstrate that Tat transactivation of the HIV-1 LTR is markedly inhibited by several cytoplasmic inhibitors of NF-kB/Rel, suggesting the critical involvement of these host transcription factors in the function of the viral Tat protein. Furthermore, the various NF-kB inhibitors appear to have differential effects on Tat. While IkBa, IkBb, and p100 potently inhibit Tat-mediated transactivation, p105 fails to inhibit, but even moderately synergizes, the action of Tat. We further demonstrate that the action of these NFkB/Rel inhibitors on Tat correlates with their inhibitory activities on the RelA subunit of NF-kB. Finally, we show that a degradation-resistant IkBa mutant is able to potently inhibit Tat-mediated activation of the HIV-1 LTR in both untreated and tumor necrosis factor a-stimulated T cells, thus suggesting that such an IkBa mutant may serve as a constitutive repressor of HIV-1 LTR. q 1996 Academic Press, Inc.
The tat gene product of human immunodeficiency virus type 1 (HIV-1) is a potent transcriptional activator of viral gene expression directed by the long terminal repeat (LTR) of HIV-1 (reviewed in Ref. 1). Tat acts by specific binding to the transactivation response region located immediately downstream of the initiation site (1). Tat appears to increase both the rate of transcriptional initiation and the efficiency of transcriptional elongation ( refs. 2– 4, reviewed in Ref. 5). Tat-mediated stimulation of transcription involves interactions between this viral transactivator and cellular transcription factors such as Sp-1 and the basal transcription factor TFIID (6, 7). In addition, the NF-kB/Rel family of inducible transcription factors has been suggested to contribute to the enhanced expression of HIV-1 LTR in activated T cells (8). The NF-kB/Rel family contains various dimeric complexes composed of a set of structurally related polypeptides, including p50 (NF-kB1), p52 (NF-kB2), RelA (previously named p65), RelB, and the proto-oncoprotein c-Rel (reviewed in Ref. 9). In resting T cells, these NFkB/Rel complexes are sequestered in the cytoplasm as latent precursors by a family of ankyrin motif-rich inhibitory proteins (10) including IkBa (11, 12), IkBb (13), p105 (14–16), and p100 (16–18). Various immunological stimuli, such as the cytokine tumor necrosis factor a (TNFa), induce the degradation of one or more of these cyto-
plasmic inhibitors and the concomitant nuclear expression of select members of NF-kB/Rel (19). The nuclear NF-kB/Rel in turn binds to the kB enhancer elements in HIV-1 LTR and participates in the activation of viral gene expression (8). It has been proposed that NF-kB/Rel plays a role in the initial induction of HIV-1 expression, and the viral Tat regulator serves to amplify the viral gene expression. It is not clear, however, whether the action of Tat strictly requires the participation of the NF-kB/Rel transcription factors. To better understand the function of NF-kB/Rel in the activation of the HIV-1 LTR, we investigated the effects of various NF-kB/Rel inhibitors on Tat-mediated activation of the HIV-1 LTR using transient transfection and reporter gene assays. For these studies, human Jurkat T cells were transfected with a luciferase reporter plasmid driven by the HIV-1 LTR (HIV-1 LTR-luciferase) together with the tat cDNA expression vector either in the absence or in the presence of various IkB expression vectors (Figs. 1A and 1B). As expected, Tat potently activated the LTR, leading to the enhanced expression of the luciferase reporter gene (Fig. 1A, column 2). However, Tat only moderately activated the HIV-1 LTR that lacks the kB enhancer elements (HIV-1 LTR DkB, column 3). To examine whether Tat-mediated activation of the wild-type HIV-1 LTR requires NF-kB/Rel factors present in these cells, the effect of various NF-kB/Rel inhibitory proteins on the transactivation activity of Tat was investigated by cotran-
1 To whom correspondence and reprint requests should be addressed. Fax: (717) 531-6522.
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sfection studies. As shown in Fig. 1B, the transactivation activity of Tat was significantly and dose-dependently inhibited by several NF-kB/Rel inhibitors, including IkBa (columns 1–4), IkBb (columns 5–8), and p100 (columns 9–12). This inhibitory action was not due to the inhibition of Tat expression since Tat-mediated activation of the HIV-1 LTR DkB was not significantly influenced by IkBa (Fig. 1C) as well as the other inhibitors (data not shown). These results indicated that the transactivation function of Tat required the basal activity of NF-kB/Rel that could be detected in these cells (data not shown). Of note, although p105 had been demonstrated to be one of the cytoplasmic inhibitors of NF-kB/Rel (14–16), it failed to inhibit Tat-mediated activation of the LTR (Fig. 1B, columns 13–16). In fact, at low concentration, p105 exhibited a moderate, but significant, synergistic effect on Tat (column 14). We next explored the possible mechanism underlying the differential effects of the various IkBs on the action of Tat. Since RelA has been shown to be a potent transcriptional activator of HIV-1 kB enhancer (20), the inhibitory function of the different IkB molecules on the kB binding activity of the RelA subunit of NF-kB was analyzed. For these studies, we used COS7 cells which had high efficiency of transfection that allowed us to detect the DNA binding activity of transfected RelA using electrophoresis mobility shift assays (EMSA). As shown in Fig. 2, the cells were transfected with either a parental vector (pCMV4) lacking a cDNA insert (lane 1) or the RelA cDNA expression vector in the absence (lane 2) or presence of varied amounts of different IkB expression vectors (lanes 3–14). EMSA analyses of the nuclear extracts revealed specific binding of the transfected RelA to the kB probe, which formed a DNA/protein complex (lane 2). As expected, the RelA DNA binding activity was potently inhibited by IkBa (lanes 3–5), as well as IkBb (lanes 6–8) and p100 (lanes 9–11). In sharp contrast, expression of p105 in the cells did not inhibit but even enhanced the DNA binding activity of RelA (lanes 12– 14). Parallel reporter gene assays revealed that unlike other IkB molecules, pl05 failed to inhibit RelA-mediated transactivation of the HIV-1 kB enhancer (data not shown). While the underlying mechanism remains to be further investigated, these results demonstrate that the effects of the various IkB molecules on Tat correlated with their functional specificity on the RelA transcriptional activator. FIG. 1. Inhibition of Tat-mediated activation of HIV-1 LTR by different NF-kB/Rel inhibitors. (A) Human Jurkat T cells (5 1 106) were transfected using DEAE-Dextran (24) with 0.4 mg of the HIV-1 LTRluciferase reporter plasmid [HIV-1 LTR was transferred from the HIV1 LTR CAT (31) into the pGL2 basic vector (Promega) in front of the luciferase gene] in the absence (column 1) or presence (column 2) of 0.2 mg tat cDNA expression vector, pSV2Tat72 (32). In column 3, a luciferase reporter driven by the HIV-1 LTR lacking the kB elements (HIV-1 LTR DkB-luciferase) was cotransfected with pSV2Tat72. At 40 hr posttransfection, the cells were collected for luciferase assay (Promega). Luciferase activity is presented as fold induction relative to
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the basal level measured in cells transfected with the parental vectorp CMV4. The values shown in this and all the following luciferase assays are representative of three independent experiments. The absolute values for the wild-type HIV-1 LTR luciferase activity in the mock-and Tat-transfected cells are about 1.5 1 104 and 4.5 1 105 cpm, respectively. (B) Luciferase assays of cells transfected with 0.4 mg HIV-1 LTRluciferase reporter and 0.2 mg pSV2Tat72, along with the indicated amounts of the various IkB molecules. (C) Luciferase assays of cells transfected with 0.4 mg HIV-1 LTRDkB-luciferase reporter and 0.2 mg pSV2Tat72, along with the indicated amounts of IkBa.
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FIG. 2. Inhibition of RelA DNA binding activity by IkBa, IkBb, and p100, but not by p105. Monkey kidney COS7 cells were transfected by DEAE-Dextran (25) with either the parental vector pCMV4 (33) (lane 1) or 0.4 mg pCMV4-RelA cDNA expression vector (25) either alone (lane 2) or together with the indicated amounts of various pCMV4-based IkB expression constructs subcloned from the corresponding cDNAs, including IkBa (lanes 1–4, Ref. 12), IkBb (lanes 5–8, Ref. 13), p100 (lanes 9–12, Ref. 34), and p105 (lanes 13–16, Ref. 35) (lanes 3–14). At 40 hr posttransfection, nuclear extracts were isolated (26) and subjected to electrophoresis mobility shift assays (EMSA) using a 32Pradiolabeled palindromic kB probe, kB-pd (27).
Recent studies have suggested that IkBa is the major inhibitor regulating the immediate early nuclear expression of the RelA-containing NF-kB complex, which occurs in response to various cytokines and mitogens (21). These immunological inducers stimulate the phosphorylation of IkBa at its N-terminal region, which leads to rapid degradation of IkBa (22, 23). Interestingly, an IkBa mutant lacking the N-terminal 71 amino acids (IkBaDN) becomes resistant to cellular stimulation and serves as a constitutive inhibitor of NF-kB (22). To further correlate the action of IkBa on Tat and RelA, we next compared the Tat-inhibitory activities of IkBaDN and its wild type. For these studies, human Jurkat T cells were cotransfected with the HIV-1 LTR-luciferase reporter, Tat, and either the wild-type IkBa (Fig. 3A, columns 2 and 3) or IkBaDN (columns 4 and 5). Remarkably, these studies revealed that the degradation-resistant IkBa mutant (lanes 4 and 5) exhibited a markedly more potent inhibitory effect on Tat compared to that of the wild-type IkBa (lanes 2 and 3). Of note, the two forms of IkBa had similar inhibitory activities on RelA-mediated activation of the kB enhancer (Fig. 3B). These findings raised the possibility that the HIV-1 Tat protein might override the inhibitory action of IkBa. To test this possibility, luciferase reporter gene assays were performed using increasing amounts of Tat expression vector (Fig. 4A). These luciferase assays revealed that expression of increasing amounts of Tat protein in the cells significantly overrode the inhibitory effect of the wild-type IkBa (columns 1–5). In sharp contrast, Tat induced only a slight increase of the luciferase activity in cells expressing the degradation-resistant IkBa mutant (IkBaDN, lanes 6–10). Similar reporter gene studies revealed that IkBaDN potently inhibited Tat-mediated activation of HIV-1 LTR in both untreated (Fig. 4B, columns 1 and 2) and TNF-a-stimulated (columns 3 and 4) cells.
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While it remains to be further investigated how Tat overrides the action of IkBa, our studies strongly suggest that Tat-mediated transactivation of the HIV-1 LTR critically requires the action of NF-kB/Rel, especially the transactivator RelA. Our results are in support of the previous studies showing the involvement of RelA in the activation of HIV-1 LTR (28, 29). Our studies also have confirmed and extended a recent report (30) demonstrating that the function of Tat is tightly dependent on the kB enhancers present in the HIV-1 LTR. Together, these studies strongly suggest that the host transcription factor NF-kB is critically required for HIV1 gene expression.
FIG. 3. Inhibition of Tat-mediated activation of the HIV-1 LTR (A) and RelA-mediated activation of the HIV-1 kB enhancer (B) by the wild-type IkBa (IkBaWT) and its degradation-resistant mutant, IkBaDN. Jurkat cells were transfected with pSV2Tat72 (A) or RelA (B), either alone or together with the indicated amounts of IkBa or its mutant IkBaDN (the N-terminal 36 amino acids were removed from the wild-type IkBa by polymerase chain reaction), along with the HIV-1 LTR-luciferase (A) or the kB-TATA-luciferase reporter [the insert of kB-TATA-CAT (36), containing the HIV-1 kB enhancer and TATA box, was transferred into the pGL2 basic plasmid 5* of the luciferase gene (Promega)]. (B). Luciferase was assayed as described in the legend for Fig. 1.
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FIG. 4. Potent inhibition of Tat-mediated activation of HIV-1 LTR by IkBaDN in both untreated and TNF-a-stimulated T cells. (A) Tat overrides the inhibitory action of the wild-type IkBa but not that of IkBaDN. Jurkat cells were transfected with 1 mg of either IkBa wild type (lanes 1–5) or IkBaDN (lanes 6–10) together with the indicated amounts of tat cDNA expression vector, along with 0.4 mg HIV-1 LTR-luciferase. Luciferase activity was assayed as described in the legend for Fig. 1. (B) Jurkat cells were transfected with 0.2 mg tat expression vector, in the presence (/) or absence (0) of IkBaDN, along with the HIV-1 LTRluciferase reporter. At 40 hr posttransfection, the cells were either not treated (0) or incubated for 5 hr with 10 ng/ml of TNF-a and then subjected to luciferase assay.
ACKNOWLEDGMENTS We acknowledge Drs. W. C. Greene, A. D. Frankel, and S. Ghosh for providing us the cDNA expression vectors. This work is supported in part by a National Foundation For Infectious Diseases Young Investigator Matching Grant and by Public Health Service Grant 1 R01 CA6847101 to S.-C.S. S.-C.S. is a scholar of the American Foundation for AIDS Research.
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