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• W2048656467 abstract "Acquired immunodeficiency syndrome (AIDS) is a result of replication of the human immunodeficiency virus type 1 (HIV-1) predominantly in CD4+ T lymphocytes and macrophages. However, most of these cells in vivo are immunologically quiescent, a condition restricting HIV-1 replication. Vpr is an HIV-1 virion protein suspected to enhance HIV-1 replication in vivo. We demonstrate in this report that Vpr specifically activates HIV-1 long terminal repeat (LTR)-directed transcription. This effect is most pronounced on a minimal promoter from HIV-1 LTR containing the TATA box and binding motifs for the ubiquitous cellular transcription factor Sp1. Evidence is presented that Vpr interacts with Sp1 when Sp1 is bound to the Sp1 motifs within the HIV-1 LTR. Both Vpr-Sp1 interaction and Vpr trans-activation require a central Leu/Ile-rich domain in Vpr. Our findings suggest that Vpr trans-activation through Sp1 is most critical for the immediate early transcription of HIV-1 when other positive regulators, such as NF-κB, are limited or inactive, a condition presumably present in vivo. By interacting with Sp1, Vpr also has the potential to influence cellular gene expression and cellular functions. Thus, therapeutic approaches directed toward blocking the Vpr trans-activation function could prove valuable in treating AIDS. Acquired immunodeficiency syndrome (AIDS) is a result of replication of the human immunodeficiency virus type 1 (HIV-1) predominantly in CD4+ T lymphocytes and macrophages. However, most of these cells in vivo are immunologically quiescent, a condition restricting HIV-1 replication. Vpr is an HIV-1 virion protein suspected to enhance HIV-1 replication in vivo. We demonstrate in this report that Vpr specifically activates HIV-1 long terminal repeat (LTR)-directed transcription. This effect is most pronounced on a minimal promoter from HIV-1 LTR containing the TATA box and binding motifs for the ubiquitous cellular transcription factor Sp1. Evidence is presented that Vpr interacts with Sp1 when Sp1 is bound to the Sp1 motifs within the HIV-1 LTR. Both Vpr-Sp1 interaction and Vpr trans-activation require a central Leu/Ile-rich domain in Vpr. Our findings suggest that Vpr trans-activation through Sp1 is most critical for the immediate early transcription of HIV-1 when other positive regulators, such as NF-κB, are limited or inactive, a condition presumably present in vivo. By interacting with Sp1, Vpr also has the potential to influence cellular gene expression and cellular functions. Thus, therapeutic approaches directed toward blocking the Vpr trans-activation function could prove valuable in treating AIDS. INTRODUCTIONHIV-1 1The abbreviations used are: HIVhuman immunodeficiency virusSIVmacsimian immunodeficiency virus macaqueLR-domainLeu/Ile-rich domainaaamino acidLTRlong terminal repeatCATchloramphenicol acetyltransferaseRSVRous sarcoma virusAdMLadenovirus major late promoter. is the etiological agent of AIDS. The hallmark of AIDS is the slow but progressive depletion of CD4+-T cells, a class of T cells crucial for immune functions. Depletion of CD4+-T cell results in immunodeficiency and AIDS-related disorders, including encephalopathy, dementia, and malignancies(1Levy J.A. Microbiol. Rev. 1993; 57: 189-289Crossref Google Scholar). Despite tremendous efforts in the past, the mechanism of these AIDS-related disorders has remained unclear. However, it is clear that these are a consequence of function of HIV-1 encoded gene products. For example, the HIV-1 envelope glycoprotein was implicated to be involved in toxic effects on neuronal cells(2Giulian D. Wendt E. Vaca K. Noonan C.A. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2769-2773Crossref PubMed Scopus (202) Google Scholar). Recently, the HIV-1 Vpr protein in peripheral blood of HIV-1-infected people was shown to activate HIV-1 replication in latently infected cells(3Levy D.N. Refaeli Y. MacGregor R.R. Weiner D.B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10873-10877Crossref PubMed Scopus (221) Google Scholar, 4Levy D.N. Refaeli Y. Weiner D.B. J. Virol. 1995; 69: 1243-1252Crossref PubMed Google Scholar). This effect of Vpr was suggested to contribute to HIV-1 pathogenesis in vivo.The HIV-1 genome encodes structural as well as regulatory gene products(5Greene W.C. Annu. Rev. Immunol. 1990; 8: 453-475Crossref PubMed Scopus (80) Google Scholar, 6Cullen B. Microbiol. Rev. 1992; 56: 375-394Crossref PubMed Google Scholar). Recently, great efforts have been made toward understanding the function of the so-called accessory regulatory genes, namely vif, vpr, vpu and nef. These genes are generally non-essential for HIV-1 to replicate in activated T cells. Yet, animal model studies with two of these genes, vpr and nef, suggested that they are required for in vivo replication and pathogenesis of the simian immunodeficiency virus(7Lang S.M. Weeger M. Stahl-Hennig C. Coulibaly C. Hunsmann G. Muller J. Muller-Hermelink H. Fuchs D. Wachter H. Daniel M.M. Desrosiers R.C. Fleckenstein B. J. Virol. 1993; 67: 902-912Crossref PubMed Google Scholar, 8Kestler III, H.W. Ringler D.J. Mori K. Panicali D.L. Sehgal P.K. Daniel M.D. Desrosiers R.C. Cell. 1991; 65: 651-662Abstract Full Text PDF PubMed Scopus (1420) Google Scholar). The paradox between HIV-1 replication in vitro and that in vivo suggests that HIV-1 replication may be subjected to different modes of regulation in vivo compared to in vitro. For example, in vitro studies have shown that HIV-1 replication is highly dependent on cellular activation and availability of activated NF-κB transcription factor(5Greene W.C. Annu. Rev. Immunol. 1990; 8: 453-475Crossref PubMed Scopus (80) Google Scholar, 6Cullen B. Microbiol. Rev. 1992; 56: 375-394Crossref PubMed Google Scholar, 9Virelizier J.L. Curr. Opin. Immunol. 1990; 2: 409-413Crossref Scopus (21) Google Scholar). However, in vivo, the majority of the susceptible cells are immunologically quiescent and do not have a high level of NF-κB activity to support a productive HIV-1 replication. Nevertheless, HIV-1 replication in vivo has been demonstrated to be relatively rapid(10Ho D.D. Neumann A.U. Perelson A.S. Chen W. Leonard J.M. Markowitz M. Nature. 1995; 373: 123-126Crossref PubMed Scopus (3779) Google Scholar).To understand the role of HIV-1 accessory regulatory genes during HIV-1 replication and pathogenesis, we focused on the vpr gene product, which is a 96-aa protein produced late in the virus life cycle and assembled into the virion through binding to Gag(11Wong-Staal F. Chanda P.K. Ghrayeb J. AIDS Res. Hum. Retroviruses. 1987; 3: 33-39Crossref PubMed Scopus (104) Google Scholar, 12Yuan X. Matsuda Z. Matsuda M. Essex M. Lee T.H. AIDS Res. Hum. Retroviruses. 1990; 6: 1265-1271Crossref PubMed Scopus (114) Google Scholar, 13Cohen E.A. Dehni G. Sodroski J.G. Haseltine W.A. J. Virol. 1990; 64: 3097-3099Crossref PubMed Google Scholar). Function of Vpr appears to be critical for HIV-1 to replicate in macrophages(14Balotta C. Lusso P. Crowley R. Gallo R.C. Franchini G. J. Virol. 1993; 67: 4409-4414Crossref PubMed Google Scholar). In lymphocytes, the effect of Vpr on HIV-1 replication is difficult to detect(15Dedera D. Hu W. Vander-Heyden N. Ratner L. J. Virol. 1989; 63: 3205-3208Crossref PubMed Google Scholar). Our earlier studies and results from others showed that Vpr has a tendency to localize in the nucleus (16Zhao L.-J. Mukherjee S. Narayan O. J. Biol. Chem. 1994; 269: 15577-15582Abstract Full Text PDF PubMed Google Scholar, 17Lu Y.-L. Spearman P. Ratner L. J. Virol. 1993; 67: 6542-6550Crossref PubMed Google Scholar) without utilizing a classical nuclear localization signal(16Zhao L.-J. Mukherjee S. Narayan O. J. Biol. Chem. 1994; 269: 15577-15582Abstract Full Text PDF PubMed Google Scholar). These results are consistent with the notion that Vpr may play a role during the nuclear migration of the pre-integration complex (18Heinzinger N.K. Bukrinsky M.I. Haggerty S.A. Ragland A.M. Kewalramani V. Lee M.-A. Gendelman H.E. Ratner L. Stevenson M. Emerman M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7311-7315Crossref PubMed Scopus (750) Google Scholar). However, they were also consistent with the hypothesis that Vpr can function in the nucleus to trans-activate HIV-1(19Cohen E.A. Terwilliger E.F. Jalinoos Y. Proulx J. Sodroski J.G. Haseltine W.A. J. Acquir. Immune Defic. Syndr. 1990; 3: 11-18PubMed Google Scholar). We report here that Vpr trans-activates HIV-1 LTR through interaction with the cellular transcription factor Sp1. Sp1 is an O-glycosylated transcription factor which binds to decanucleotide Sp1 motifs (consensus core sequence: GGGCGG) through three zinc finger domains (20Jackson S.P. Tjian R. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 1781-1785Crossref PubMed Scopus (141) Google Scholar, 21Kadonaga J.T. Courey A.J. Ladika J. Tjian R. Science. 1988; 242: 1566-1569Crossref PubMed Scopus (279) Google Scholar, 22Jackson S.P. Tjian R. Cell. 1988; 55: 125-133Abstract Full Text PDF PubMed Scopus (648) Google Scholar). It is ubiquitously expressed and is involved in transcription of a variety of cellular genes including the proto-oncogenes Ha-ras-1 (23Ishii S. Kadonaga J.T. Tjian R. Brady J.N. Merlino G.T. Pastan I. Science. 1986; 232: 1410-1413Crossref PubMed Scopus (106) Google Scholar) and pim-1(24Meeker T.C. Loeb J. Ayres M. Sellers W. Mol. Cell. Biol. 1990; 10: 1680-1688Crossref PubMed Scopus (51) Google Scholar). Purified Sp1 protein was shown to bind to all three Sp1 motifs within the HIV-1 LTR(25Jones K.A. Kadonaga J.T. Luciw P.A. Tjian R. Science. 1986; 232: 755-758Crossref PubMed Scopus (444) Google Scholar). We found that Vpr trans-activation was more dramatic when the transcriptional activity of HIV-1 LTR was lower. Our results are consistent with the notion that Vpr function is most critical for the immediate early transcription of HIV-1 when other positive regulators, such as NF-κB, are limited or inactive, a condition presumably present in vivo.MATERIALS AND METHODSPlasmid Constructs and Transcription TemplatesThe EcoRI/HindIII fragment of the plasmid pU3RCATIII, 2C.-Z. Giam, unpublished data. containing the HIV-1 LTR plus 500-base pair cellular sequence, was cloned into pUC18 vector to generate pUC-HIV-1-LTR, which was digested with EcoRI/NdeI and used as the LTR transcription template (Fig. 1A). To obtain the NF-κB+Sp1 template (Fig. 1A), the pUC-HIV-1-LTR was digested with ScaI (immediately upstream of the NF-κB motifs) and PvuI (in the pUC18 vector). The three Sp1 motifs together with the first 82-base pair transcribed region of the HIV-1 LTR was amplified by polymerase chain reaction and cloned at the EcoRI/HindIII sites of pUC18 to generate plasmid pUC-Sp1-LTR, which was digested with EcoRI/PvuI to generate the Sp1 template. Sequence from the TATA box to position +82 was amplified by polymerase chain reaction and cloned the same way as pUC-Sp1-LTR to generate pUC-TATA-LTR, which was digested with EcoRI/PvuI to serve as the TATA template. Plasmid pDN-AdML (26Conaway J.W. Travis E. Conaway R.C. J. Biol. Chem. 1990; 265: 7564-7569Abstract Full Text PDF PubMed Google Scholar) containing the TATA box basal promoter region of the adenovirus major late promoter was digested with EcoRI and NdeI to generate a 270-base transcript.The p3×Sp1-CAT plasmid was obtained by insertion of the EcoRI-HindIII region of pUC-Sp1-LTR plasmid into a vector containing the CAT reporter gene. The vpr(wt) gene in pET-vpr(wt) was transferred to the RSV-HnefPFH plasmid (16Zhao L.-J. Mukherjee S. Narayan O. J. Biol. Chem. 1994; 269: 15577-15582Abstract Full Text PDF PubMed Google Scholar) to generate RSV-vpr(wt) plasmid. The construction of RSV-vpr-LR-mu and RSV-S-vpr-T has been described(16Zhao L.-J. Mukherjee S. Narayan O. J. Biol. Chem. 1994; 269: 15577-15582Abstract Full Text PDF PubMed Google Scholar).Cell Culture, Preparation of Nuclear Extracts, and TransfectionHeLa cells were grown in suspension medium (Joklik's modified minimal essential medium, Life Technologies Inc.) supplemented with 5% horse serum. CEM×174 and U937 cells were cultured in RPMI medium supplemented with 10 and 20% fetal bovine serum, respectively. Nuclear extracts were prepared from cell lines according to established procedures (32Dignam J.D. Lebovitz R.M. Roeder R.G. Nucleic Acids Res. 1983; 11: 1475-1489Crossref PubMed Scopus (9142) Google Scholar) except KCl was used to replace NaCl. CEM×174 cells were transfected with 3 μg of the p3×Sp1-CAT plasmid in the presence or absence of activator plasmids by using the DEAE-dextran method, and CAT assay was performed at 48 h post-transfection with a reaction time of 8 h following established protocols(29Zhao L.-J. Giam C.-Z. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7070-7074Crossref PubMed Scopus (296) Google Scholar).In Vitro Transcription ReactionIn vitro transcription reaction was carried out as described (22Jackson S.P. Tjian R. Cell. 1988; 55: 125-133Abstract Full Text PDF PubMed Scopus (648) Google Scholar) using 1 μg of template DNA and radiolabeled UTP. All Vpr related proteins were purified as described before(16Zhao L.-J. Mukherjee S. Narayan O. J. Biol. Chem. 1994; 269: 15577-15582Abstract Full Text PDF PubMed Google Scholar). Transcription reaction was carried out in a 50-μl volume with 20 μl of the crude nuclear extract (approximately 200 μg of total protein) at 30°C for 45 min. The reaction was stopped with 50 μl of a buffer containing 0.6 M sodium acetate, 2 mg/ml yeast tRNA, and 20 mM EDTA, and then extracted with phenol twice and precipitated with ethanol. The precipitated RNA was dissolved in a formamide loading buffer, electrophoresed on a sequencing gel, and transcription activity was quantitated with the help of a PhosphoImager (Molecular Dynamics).Gel Electrophoretic Mobility Shift Assay and Co-precipitation AssayConditions for gel electrophoretic mobility shift assay was essentially as described(29Zhao L.-J. Giam C.-Z. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7070-7074Crossref PubMed Scopus (296) Google Scholar). Briefly, proteins and radiolabeled probes were incubated in a 10-μl volume for 30 min at room temperature in a buffer containing 25 mM Hepes (pH 7.9), 50 mM KCl, 5% glycerol, 0.5 mg/ml bovine serum albumin, 5 μM zinc sulfate, and 0.5 mM dithiothreitol, and then loaded to a 6% polyacrylamide gel prepared with a Tris glycine buffer(29Zhao L.-J. Giam C.-Z. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7070-7074Crossref PubMed Scopus (296) Google Scholar). PolydI•polydC was used at 500 ng/ml as a nonspecific competitor. The Sp1 protein was purified by Promega following established protocols (20Jackson S.P. Tjian R. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 1781-1785Crossref PubMed Scopus (141) Google Scholar), involving wheat germ agglutinin affinity chromatography followed by a Sp1-specific DNA column chromatography. For co-precipitation assay, Sp1-DNA complexes were assembled as per gel shift assay. Then the volume was diluted to 100 μl with the incubation buffer containing 20 μl of the Flag IgG-Affi-Gel (16Zhao L.-J. Mukherjee S. Narayan O. J. Biol. Chem. 1994; 269: 15577-15582Abstract Full Text PDF PubMed Google Scholar) which recognizes the Flag tag at the C terminus of Vpr-T. After shaking at 4°C for 30 min, the Affi-Gel beads were washed and bound probe eluted with a TE buffer containing 1 M NaCl. The probe was analyzed on a 10% polyacrylamide gel. Alternatively, Sp1-DNA complex was assembled with 100 ng of unlabeled 3×Sp1 oligo (Fig. 1B), and the co-precipitated proteins examined by Western blot with the Flag antibody (16Zhao L.-J. Mukherjee S. Narayan O. J. Biol. Chem. 1994; 269: 15577-15582Abstract Full Text PDF PubMed Google Scholar) and the Sp1 monoclonal antibody (Santa Cruz, CA) following conditions as described(29Zhao L.-J. Giam C.-Z. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7070-7074Crossref PubMed Scopus (296) Google Scholar).RESULTSIn Vitro Transcriptional Activation of HIV-1 LTR by Vpr through Sp1 Binding MotifsThe nuclear localization of Vpr and its presence in the virion is consistent with the notion that Vpr functions in the nucleus to trans-activate immediate early transcription of HIV-1. To examine this possibility, we performed in vitro transcription experiments with the HIV-1 LTR template and purified Vpr-T which carries a C-terminal tag (14Balotta C. Lusso P. Crowley R. Gallo R.C. Franchini G. J. Virol. 1993; 67: 4409-4414Crossref PubMed Google Scholar) (Fig. 1A). The LTR template was prepared by digestion of the plasmid pUC-HIV-1-LTR with restriction enzymes such that a 293-base run-off transcript could be generated. With nuclear extracts prepared from HeLa, CEM×174, and U937 cell lines, Vpr-T increased HIV-1 LTR-directed transcription by more than 100% (Fig. 2, A and B). The transcript was RNA-polymerase II-derived since it was specifically inhibited by α-amanitin (data not shown). In addition, as described next, progressive deletion of HIV-1 LTR also progressively reduced this transcriptional activity.Figure 2In vitro trans-activation of the HIV-1 LTR by Vpr. A, in vitro transcription with nuclear extracts prepared from different cell lines. Where indicated, HIV-1 Vpr-T protein (16Zhao L.-J. Mukherjee S. Narayan O. J. Biol. Chem. 1994; 269: 15577-15582Abstract Full Text PDF PubMed Google Scholar) (0.5 μg) was included in the transcription reaction. M, 1-kilobase DNA ladder (Life Technologies, Inc.) end-labeled with 32P. B quantitation of transcripts by PhosphoImager (Molecular Dynamics, Inc.). Values were graphed as when the activity in lane 1 of A was normalized to 100. C, in vitro transcription with templates (Fig. 1A) indicated on the right-hand side. Upper panel, transcription with 0.5 μg each of the LTR template and the NF-κB+Sp1 template. Lower panel, transcription with 0.5 μg each of the LTR template and the Sp1 template. Both the Sp1+NF-κB template and the Sp1 template yielded a 202-base transcript. Lanes 2-5 contained 0.05, 0.25, 0.5, and 1 μg of Vpr-T, respectively. D quantitation of transcripts in C the same way as in B. Activation index was expressed as the gain in transcription in the presence of Vpr-T divided by the control transcription without Vpr-T.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The HIV-1 LTR contains as positive regulatory elements two binding motifs for the transcription factor NF-κB followed by three binding motifs for the Sp1 transcription factor(5Greene W.C. Annu. Rev. Immunol. 1990; 8: 453-475Crossref PubMed Scopus (80) Google Scholar, 6Cullen B. Microbiol. Rev. 1992; 56: 375-394Crossref PubMed Google Scholar). To decipher which region of the HIV-1 LTR mediated response to Vpr-T, three templates containing: (a) NF-κB and Sp1 motifs (NF-κB+Sp1 template), (b) Sp1 motifs (Sp1 template), or (c) neither NF-κB nor Sp1 motifs (TATA template), were prepared (Fig. 1A). These templates produced a shorter mRNA transcript than the full-length LTR template, and were individually mixed with the LTR template in equal molar amounts for in vitro transcription with different amounts of Vpr-T. It was clear that the NF-κB+Sp1 template and the Sp1 template were both responsive to Vpr-T (Fig. 2, C and D) while the TATA template had no detectable basal activity and no Vpr response (data not shown). Among the three templates shown (Fig. 1A), the Sp1 template had the lowest level of basal activity (Fig. 2C, lane 1). However, it was activated by Vpr the most: the maximum gain in transcription reached 400% (Fig. 2D). Thus, it seemed that the Sp1 motifs in HIV-1 LTR were sufficient to confer Vpr response. Comparison between the results from the NF-κB+Sp1 template and the Sp1 template suggested that presence of NF-κB in the promoter reduced Vpr trans-activation. Thus, the NF-κB activation pathway appears not to be targeted by Vpr. Consistent with this analysis, when the two NF-κB motifs in HIV-1 LTR were cloned immediately upstream of the TATA box, at position −40 of the HIV-1 LTR (refer to Fig. 1), the resulting template did not respond to Vpr (data not shown).Control experiments were carried out with two other Vpr-related proteins: the authentic Vpr (Vpr(wt)), and the Vpr-T mutant: Vpr-LR-mu which contains a mutation of 8 aa residues in the Leu/Ile-rich domain (LR-domain, aa numbers 60-81) of Vpr(16Zhao L.-J. Mukherjee S. Narayan O. J. Biol. Chem. 1994; 269: 15577-15582Abstract Full Text PDF PubMed Google Scholar). For this experiment, the Sp1 template was used alone. It was clear that the authentic Vpr(wt) activated transcription of the Sp1 template to the same extent as Vpr-T while the ability of the Vpr-LR-mu protein to trans-activate was impaired (Fig. 3, A and B). We noticed that under this condition the maximum Vpr trans-activation was lower than when the Sp1 template and the LTR template were used together (Fig. 2C, lower panel). Thus, it seemed that the LTR template competed for the basal transcription machinery much more efficiently than the Sp1 template when Vpr was absent. However, when Vpr was present, the Sp1 template gained a competitive advantage.Figure 3Specificity of Vpr trans-activation. A transcription reaction was carried out with 0.25, 0.5, and 1 μg of Vpr-T (lanes 2-4), Vpr(wt) (lanes 5-7), and Vpr-LR-mu (lanes 8-10) using 1 μg of the Sp1 template. B the highest activation indices observed for the three Vpr proteins were plotted for comparison. C transcription reaction with 0.5 μg each of the Sp1 template and the AdML template. D activation indices for both templates at different Vpr-T concentrations were plotted for comparison.View Large Image Figure ViewerDownload Hi-res image Download (PPT)A template containing the TATA box basal promoter of adenovirus major late promoter (AdML) was also mixed with the Sp1 template in equal molar amounts and tested for potential activation by Vpr-T. As Fig. 3C shows, the Sp1 template gave a much higher level response to Vpr-T than the AdML template, although the latter template was also significantly activated by Vpr-T. Inspection of the AdML sequence revealed two blocks of GC-rich sequences surrounding the TATA box(26Conaway J.W. Travis E. Conaway R.C. J. Biol. Chem. 1990; 265: 7564-7569Abstract Full Text PDF PubMed Google Scholar). Preliminary studies suggested that Sp1 could bind to these sequences (data not shown). Thus, it may be possible that the observed activation of AdML promoter by Vpr also involved Sp1 binding to AdML. This may be in contrast to an earlier report which showed that Sp1 significantly activated a promoter in the absence of an identifiable Sp1 motif (27Courey A.J. Holtzman D.A. Jackson S.P. Tjian R. Cell. 1989; 59: 827-836Abstract Full Text PDF PubMed Scopus (389) Google Scholar).Protein-Protein Interaction between Vpr and the Ubiquitous Transcription Factor Sp1Many transcription activators, such as the herpes simplex virus Vp16 (28Triezenberg S.J. LaMarco K.L. McKnight S.L. Genes & Dev. 1988; 2: 730-742Crossref PubMed Scopus (182) Google Scholar) and the HTLV-I Tax(29Zhao L.-J. Giam C.-Z. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7070-7074Crossref PubMed Scopus (296) Google Scholar), function by targeting specific transcription factors bound to upstream promoter elements. Since the Sp1 motifs were sufficient to confer Vpr response, we examined if Vpr can directly target the cellular transcription factor Sp1. Sp1 is an O-glycosylated transcription factor which binds to decanucleotide Sp1 motifs (consensus core sequence: GGGCGG) through three zinc finger domains(20Jackson S.P. Tjian R. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 1781-1785Crossref PubMed Scopus (141) Google Scholar, 21Kadonaga J.T. Courey A.J. Ladika J. Tjian R. Science. 1988; 242: 1566-1569Crossref PubMed Scopus (279) Google Scholar, 22Jackson S.P. Tjian R. Cell. 1988; 55: 125-133Abstract Full Text PDF PubMed Scopus (648) Google Scholar). It is ubiquitously expressed and is involved in transcription of a variety of cellular genes including the proto-oncogenes Ha-ras-1 (23Ishii S. Kadonaga J.T. Tjian R. Brady J.N. Merlino G.T. Pastan I. Science. 1986; 232: 1410-1413Crossref PubMed Scopus (106) Google Scholar) and pim-1(24Meeker T.C. Loeb J. Ayres M. Sellers W. Mol. Cell. Biol. 1990; 10: 1680-1688Crossref PubMed Scopus (51) Google Scholar). Purified Sp1 protein was shown to bind to all three Sp1 motifs within the HIV-1 LTR(25Jones K.A. Kadonaga J.T. Luciw P.A. Tjian R. Science. 1986; 232: 755-758Crossref PubMed Scopus (444) Google Scholar). These Sp1 motifs were synthesized as a double-stranded oligonucleotide (3×Sp1, Fig. 1B), radiolabeled, and used in a gel electrophoretic mobility shift assay with purified Sp1 protein and Vpr-T (Fig. 4A). Under the limiting concentration of purified Sp1 protein, Sp1 bound to the probe and generated a predominant Sp1-probe complex which was most likely due to Sp1 binding to one Sp1 motif in the probe (lane 1). In addition, a weak band corresponding to Sp1 binding to two motifs was also visible. The Sp1-probe complex was recognized by a monoclonal Sp1 antibody (lane 7). Interestingly, inclusion of Vpr-T also caused a mobility shift of the Sp1-probe complex (lane 2). Since Vpr-T itself did not bind to DNA (lane 8), it seemed possible that Vpr-T physically incorporated into the Sp1-probe complex. Alternatively, Vpr-T induced Sp1 binding to more Sp1 motifs in the probe. Incubation of the Vpr-T-induced complex with the Flag IgG-Affi-Gel for the tag in Vpr-T (16Zhao L.-J. Mukherjee S. Narayan O. J. Biol. Chem. 1994; 269: 15577-15582Abstract Full Text PDF PubMed Google Scholar) depleted this complex (data not shown), suggesting that the first possibility was correct. This interpretation was also supported by the co-precipitation results (see next).Figure 4Vpr-Sp1 interaction. A gel mobility shift assay with Sp1 protein (25 ng, Promega) for lanes 1-7 and 9-14, and ATF-1 protein19 (25 ng) for lanes 15 and 16. Sp1 Ab is a mouse monoclonal for Sp1 (1 μg, Santa Cruz Biotechnology). The assay (10 μl) contained 0.1 ng of labeled probe and 5 ng of polydI•polydC under a condition as described earlier(29Zhao L.-J. Giam C.-Z. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7070-7074Crossref PubMed Scopus (296) Google Scholar). Human Sp1 protein was purified from HeLa cells by Promega following an established protocol(20Jackson S.P. Tjian R. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 1781-1785Crossref PubMed Scopus (141) Google Scholar). The probe sequences are listed in Fig. 1B. B co-precipitation of the 3×Sp1 probe, but not the Sp1(I) probe, with Sp1 and Vpr-T by the Flag antibody Affi-Gel (IBI) that recognizes the C-terminal tag in Vpr-T. Lane 1 shows 20% of the input probe mixture without precipitation. Radioactive bands in between the two probes and below the Sp1(I) probe are single-stranded oligos. C co-precipitation as in B except unlabeled 3×Sp1 oligo (100 ng) was used and the precipitated proteins examined by Western blot with Sp1 antibody and the Flag antibody. Lane 1 was 100% of the input proteins directly examined.View Large Image Figure ViewerDownload Hi-res image Download (PPT)In the same assay, the authentic Vpr(wt) protein also induced a mobility shift of the Sp1-probe complex (Fig. 4A, lane 5). The apparently higher efficiency of Vpr(wt) was most likely due to the higher amount of Vpr(wt) used as compared to Vpr-T, since doubling the amount of Vpr-T also generated the same pattern. The Vpr-LR-mu protein, which failed to trans-activate the Sp1 template (Fig. 3A), had a severely reduced activity (lane 3) and VprΔ43-96 had no significant activity (lane 4). Interestingly, the SIVmac Vpr protein (S-Vpr-T) expressed and purified the same way as HIV-1 Vpr-T(16Zhao L.-J. Mukherjee S. Narayan O. J. Biol. Chem. 1994; 269: 15577-15582Abstract Full Text PDF PubMed Google Scholar), was not active (lane 6), suggesting that there is a host specificity in the Sp1 protein or a viral specificity in the Vpr protein. The potential existence of host specificity in Sp1 was consistent with the observation that with a HeLa nuclear extract S-Vpr-T did not trans-activate either HIV-1 LTR or SIVmac LTR, while HIV-1 Vpr trans-activated both templates (data not shown). With a control system, the ATF-1 (29Zhao L.-J. Giam C.-Z. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7070-7074Crossref PubMed Scopus (296) Google Scholar) bound to the cyclic AMP-responsive element (Fig. 4A, lane 15), but was not affected by Vpr-T (lane 16).The Vpr-Sp1 interaction was further examined by co-immunoprecipitation assays. First, the DNA-protein complex formed as per the gel shift assay was co-precipitated by the Flag IgG-Affi-Gel which recognizes the C-terminal tag in Vpr-T(16Zhao L.-J. Mukherjee S. Narayan O. J. Biol. Chem. 1994; 269: 15577-15582Abstract Full Text PDF PubMed Google Scholar). The precipitated DNA probe was examined by gel analysis and autoradiography. It was clear that the probe was co-precipitated only with Vpr-T (Fig. 4B, lane 3), but not with Vpr-LR-mu (lane 4) or S-Vpr-T (lane 5). Second, the Sp1 protein and Vpr-T protein were directly mixed and then co-precipitated with the Flag IgG-Affi-Gel. The precipitated proteins were examined for the presence of Vpr-T and Sp1 proteins by Western blot. Under this condition, Sp1 and Vpr-T did not co-precipitate (data not shown). However, when during the co-precipitation an excess of unlabeled 3×Sp1 oligo was included, Sp1-Vpr-T co-precipitation was observed (Fig. 4C, lane 3). Under this condition, Sp1 did not co-preci" @default.
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