Human GLI-2 Is a Tat Activation Response Element-Independent Tat Cofactor

Abstract

Zinc finger-containing GLI proteins are involved in the development of Caenorhabditis elegans, Xenopus, Drosophila, zebrafish, mice, and humans. In this study, we show that an isoform of human GLI-2 strongly synergizes with the Tat transactivating proteins of human immunodeficiency virus types 1 and 2 (HIV-1 and -2) and markedly stimulates viral replication. GLI-2 also synergizes with the previously described Tat cofactor cyclin T1 to stimulate Tat function. Surprisingly, GLI-2/Tat synergy is not dependent on either a typical GLI DNA binding site or an intact Tat activation response element but does require an intact TATA box. Thus, GLI-2/Tat synergy results from a mechanism of action which is novel both for a GLI protein and for a Tat cofactor. These findings link the GLI family of transcriptional and developmental regulatory proteins to Tat function and HIV replication.

Infection with human immunodeficiency virus type 1 or 2 (HIV-1 or -2) causes AIDS, one of the leading causes of death in young adults globally. Tat-1 and Tat-2, the transactivator proteins of HIV-1 and HIV-2, respectively, are crucial for effective viral replication. Though their modes of action are still not completely understood, the Tat proteins are unique among eukaryotic viral transactivators in that they bind to the 5′ end of the viral RNA transcript in the Tat activation response (TAR) region and mediate transactivation by affecting multiple levels of transcriptional regulation. While early studies focused on the direct interactions between Tat and TAR in both HIV-1 and HIV-2, it has become increasingly clear that Tat also requires cellular cofactors to allow it to function as an initiator of transcription and elongator of viral transcripts in vivo (16, 34). Though the TAR regions of HIV-1 and HIV-2 differ, both appear to mediate transactivation of the HIV promoters by recruiting Tat and cellular cofactors to the nascent RNA transcript, where they can interact with the RNA polymerase II (Pol II) complex. DNA elements in the promoter are also needed for effective Tat function, further supporting the involvement of cellular accessory proteins in the transactivation of HIVs by Tat. For example, the TATA box, part of the core DNA promoter region of HIV, is necessary for effective Tat function (22, 23), and Tat has been shown to associate with the TATA box binding protein (TBP) (29, 64).

We have been studying the effect on retroviral transcription of a protein first identified as binding to a TG-rich element in the human T-lymphotropic virus type 1 (HTLV-1) promoter, the Tax helper protein (THP) (60). Subsequent analysis has shown that THP, a protein with five zinc fingers, is highly likely to be an isoform of human GLI-2 (59). GLI family members are widely conserved in nature, being found in nematodes (tra-1 [45, 71, 72]), Drosophila (cubitus interruptus [2, 12, 25]), Xenopus (36), mice (23, 39, 65), zebrafish (28), and humans (GLI-1, GLI-2/THP, and GLI-3 [27, 55]). They are involved in sex determination (tra-1), multiple aspects of Drosophila development controlled by Hedgehog signaling (cubitus interruptus), and craniofacial, limb, lung, and/or esophagus development in mice (GLI-2 and GLI-3 [41]), zebrafish (GLI-2 [28]), and humans (GLI-3 and presumably GLI-2). In addition, GLI-1 and GLI-2 show increased expression in certain glioblastoma multiforme tumors, although a causal relationship has not been clearly established (32, 48, 51, 55, 67). More recently, overexpression of GLI-1 has been linked to basal cell carcinomas (10, 25).

GLI-2/THP has been shown to interact with a DNA promoter element in HTLV-1 that is similar to the peri-ets (pets) site of the HIV-2 enhancer, the latter being an enhancer element that is induced following T-cell and monocytic activation (8, 21, 37). As we found that GLI-2/THP could also bind to the HIV-2 pets site, we tested the effects of GLI-2/THP on HIV-2 promoter activity and found that it caused a large increase in HIV-2 gene expression in cells also stimulated with phorbol 12-myristate 13-acetate (PMA). However, surprisingly our studies revealed that pets and other previously delineated enhancer elements of HIV-2 were not needed for the GLI-2/THP activation function, suggesting that the GLI-2/THP effect may be mediated by more central mechanisms (56a). In investigating the mechanism of action by which GLI-2/THP activates the HIV-2 promoter, we found that GLI-2/THP can physically interact with TBP and with Tat, two proteins previously shown to associate with each other (29). Further, GLI-2/THP and Tat strongly synergize to activate both the HIV-1 and HIV-2 promoters. In addition, GLI-2/THP and Tat synergize with the previously described Tat cofactor cyclin T (14, 66). It was also observed that overexpression of GLI-2/THP markedly stimulates viral replication. Interestingly, synergy between GLI-2/THP and Tat is seen even in the absence of the Tat binding element TAR. However, the TATA box, the site of TBP interaction with the promoter, is needed for this synergy to occur, as is the TBP binding site of the Tat protein. These data suggest that GLI-2/THP is a Tat cofactor which markedly activates HIV transcription via a completely unexpected mechanism.

MATERIALS AND METHODS

Plasmids.

The HIV-1 pHXB2 infectious clone has been described elsewhere (18). The NL4-3-derived HIV-1 infectious clone was provided by Kathleen Collins, University of Michigan. The HIV-1 and HIV-2 long terminal repeat-chloramphenicol acetyltransferase (CAT) reporter constructs (HIV-1/CAT and HIV-2/CAT) have been described previously (13, 57). The HIV-1 mutation ΔTATA plasmid was provided by Gary Nabel (41). The HIV-2 Δstem plasmid and the HIV-1 (TAR del) plasmid have been described elsewhere (5, 52). The GLI-2/THP expression plasmid pCG-THP-2 was constructed and provided by Mitsuaki Yoshida (60). The GLI-2/THP–glutathione S-transferase (GST) bacterial fusion protein construct was made by using PCR to add in-frame BamHI sites to the end of the GLI-2/THP coding sequence and cloning the full-length THP-2 isoform BamHI fragment (63) into pGEX-2TK (Pharmacia). The clone expressing full-length human cyclin T1 under the control of the cytomegalovirus immediate-early promoter is a modification of a similar construct (14). The Tat-1 plasmid, in which Tat expression is driven by the Rous sarcoma virus promoter, has been described elsewhere (11). The Tat-2 plasmid containing the entire Tat gene without introns and under the control of the Rous sarcoma virus promoter was a gift from Sandra Tong-Starksen (63). The Tat exon 1 from the HIV-1 B strain of an infected individual (40) and mutant Tat clones were generated by PCR and ligated into the HindIII-PstI site of the expression vector pcDNA3.1/Neo (Invitrogen). Two mutations in the TBP binding domain were introduced in different constructs. The B.E. mutation changes the conserved lysine at position 41 to a glutamic acid residue, and the B.T. mutation alters it to a threonine. These mutations are analogous to those in the Tat-1 point mutants that were shown by Kashanchi et al. (29) to be defective in TBP binding, and both Tat-B.E. and Tat-B.T. proteins fail to interact with TBP in in vitro binding assays (data not shown). The molecular clones were confirmed by sequencing using a model 373 ABI automated sequencer. The sequences of the DNA oligonucleotides used to create the wild-type and mutant Tat plasmids are as follows: external primer Hind III-Btat+, 5′-CCA AGC TTA CCT GCC ATG GAG CCA GTA GAT CCT AGA CTA GAG CCC-3′; external primer Pst I-Btat-, 5′-A AAC TGC AGT TAC TGC TTT GAT AAA AAA ACT TGA TGA GTC-3′; internal primer K41T+, 5′-T TTC ATA ACA ACA GGC CTA GGC A 3′; internal primer K41T−, 5′-T GCC TAG GCC TGT TGT TAT GAA A-3′; internal primer K41E+, 5′-T TTC ATA ACA GAA GGC CTA GGC A-3′; and internal primer K41E-, 5′-T GCC TAG GCC TTC TGT TAT GAA A-3′.

GST pull-down assays.

Escherichia coli JM109 cells were transformed with a pGEX-2TK vector containing the sequence for GLI-2/THP or an empty pGEX-2TK vector. Recombinant GLI-2/THP-GST protein or GST alone was induced by treatment of the culture with 1 mM isopropyl-β-d-thiogalactopyranoside for 3 h, and recombinant protein was extracted as suggested by the manufacturer (Amersham Pharmacia Biotech). Extracts were analyzed by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) on a 10% gel and Coomassie blue staining to confirm expression of recombinant protein. Crude GLI-2/THP-GST extract was incubated with a 10-μl bed volume of glutathione-Sepharose 4B resin (Pharmacia) for 1 h at 4°C and washed three times with 200 μl of incubation buffer A (20 mM HEPES [pH 7.9], 75 mM KCl, 2.5 mM MgCl₂, 1 mM dithiotheitol, 0.1% NP-40). An equivalent amount of GST-only extract was similarly incubated with glutathione-Sepharose 4B resin and washed for use as a negative control. A 10% slurry of GST or GLI-2/THP-GST bound to glutathione-Sepharose 4B was incubated at 4°C for 2 h with 8 μl of [³⁵S]methionine-labeled, in vitro-transcribed/translated protein (TBP, Tat-1, Sp1, or the TFIIE α or TFIIE β subunit) which had been made using a TNT kit (Promega). The mixture was washed three times with 0.5 ml of incubation buffer B (identical to incubation buffer A but containing 150 mM KCl) to remove any protein not attached to GST. The resin and attached proteins were suspended in SDS loading buffer and boiled for 1 min, and the released protein was resolved by SDS-PAGE (10% gel). The gel was enhanced by treatment with Amplify (Amersham) and subjected to autoradiography.

Antibodies.

The anti-GLI-2/THP polyclonal serum was generated in a rabbit using the purified GST fusion protein of the THP-2 isoform.

Cell culture and transfections.

The 293 and NIH 3T3 cell lines were transfected by the calcium phosphate method (38). The Jurkat T-cell line and the U937 monocytic cell line were grown in RPMI 1640 supplemented with 10% fetal bovine serum, 2 μM l-glutamine, and penicillin-streptomycin. For the reporter gene (CAT) assays, 5 × 10⁶ cells were transfected by the DEAE-dextran method (47), stimulated where indicated with 16 nM PMA after 20 h, and harvested after an additional 24 h of incubation. Cell lysates were prepared by multiple freeze-thaw cycles in 0.25 M Tris-Cl (pH 7.5), and CAT activity was assayed by standard methods (17). Transfection efficiencies were normalized for protein concentration using the Bio-Rad reagent. CAT activity was quantitated on a Betagen beta scanner.

Viral replication.

HIV-1 replication was assessed using the reverse transcriptase (RT) assay as described elsewhere (1).

RESULTS

GLI-2/THP interacts with Tat.

Previous experiments suggested that GLI-2/THP modulated HIV transcription not through the predicted TG-rich GLI binding sites but rather through interaction with basal transcription factors (56a). In the process of investigating the interactions between GLI-2/THP and the basal transcription factors, we analyzed a HeLa nuclear extract that had been passed over a Tat-1 affinity column. A Western blot showed that GLI-2/THP or a related protein was in the fraction which bound to Tat (not shown), suggesting that Tat might associate biochemically with GLI-2/THP. The ability of Tat and GLI-2/THP to physically interact was confirmed by GST pull-down experiments (Fig. 1A and B). This biochemical interaction suggested that GLI-2/THP might potentiate transactivation by Tat.

FIG. 1.

GLI-2/THP interacts biochemically with TBP and Tat-1. Apparent mass in kilodaltons is indicated at the left in each panel. (A) ³⁵S-labeled reticulocyte lysate-translated input proteins used in GST pull-down assays. Approximately 25% of the quantity of Tat-1, TBP, Sp1, TFIIE α, or TFIIE β used in the binding reactions in panels B and C is shown in lanes 1 through 5. Proteins in lanes 1 to 4 were resolved on an SDS–12% polyacrylamide gel; in vitro transcribed-translated HIV-1 Tat (lane 5) was run on an SDS–15% polyacrylamide gel. (B) GLI-2/THP interacts specifically with Tat-1. ³⁵S-labeled reticulocyte lysate-translated Tat-1 was incubated with resin-bound GLI-2/THP-GST (lane 1) or GST alone (lane 2), the reaction mixture was washed, and residual bound proteins were resolved on an SDS–15% polyacrylamide gel. Tat-1 bound to GLI-2/THP-GST but not to GST alone. (C) GLI-2/THP binds specifically to TBP. Resin-bound GLI-2/THP-GST was incubated with ³⁵S-labeled reticulocyte lysate-translated Sp1 (lane 1), TBP (lane 2), TFIIE α (lane 3), or TFIIE β (lane 4). The reaction mixtures were washed, and residual bound protein was resolved on an SDS–10% polyacrylamide gel. Only TBP bound GLI-2/THP-GST.

GLI-2/THP can strongly synergize with Tat-1 or Tat-2 to activate HIV gene expression.

Using cotransfection assays, we next examined whether GLI-2/THP might function as a Tat cofactor for HIV-1 or HIV-2. For these studies, we used the Tat expression vectors and the HIV promoter-driven CAT plasmids at limiting concentrations, in order to detect potentiation of the Tat effect. When these limiting quantities are used, there was only a weak Tat-induced boost in HIV-1 promoter activity, even in cells treated with PMA (Fig. 2A and B). However, transactivation was markedly increased by the expression of GLI-2/THP, demonstrating synergy between Tat and GLI-2/THP in the HIV-1 system (Fig. 2A and B). Stimulation of the cells with PMA did not increase the expression of GLI-2/THP (56a) but was necessary to potentiate the activity of GLI-2/THP, consistent with our prior observations (56a) and the previously described regulation of GLI protein function by phosphorylation events (3, 6, 50). Indeed, cubitus interruptus is closely associated with a serine threonine kinase, Fused, which is a vital part of the functional Hedgehog-responsive complex (reviewed in reference 53). The boost in HIV-1 promoter-driven gene expression by GLI-2/THP occurred over a range of Tat expression vector concentrations in a dose-dependent manner (Fig. 2B). This synergistic effect was similarly seen with the HIV-2 promoter and HIV-2 Tat (Fig. 2C). These studies demonstrated that the Tat proteins of HIV-1 and HIV-2 can act synergistically with GLI-2/THP to stimulate HIV-1 (Fig. 2A and B) or HIV-2 (Fig. 2C) gene expression in T cells. Similar results were seen in monocytic cells (see below). Further, GLI-2/THP expression caused a similar boost in transactivation when a Tat protein cloned from an HIV-1 subtype B primary isolate (40) was used (see Fig. 6), demonstrating that this marked effect occurs with Tat from more than one HIV-1 strain, as well as with HIV-2 Tat.

FIG. 2.

GLI-2/THP synergizes with Tat. (A) An HIV-1/CAT construct was transfected into Jurkat T cells with (striped bars) or without (solid and empty bars) a limiting concentration (50 ng) of a Tat-1 expression vector and the indicated amount of the GLI-2/THP expression vector or control vector. The indicated samples were treated with 16 nM PMA 20 h after transfection. The cells were harvested 24 h later. Protein was standardized, and CAT assays were performed. The lack of activation seen with PMA alone is due to the low amount of reporter plasmid (1 μg). (B) HIV-1 Tat dose response. Jurkat cells were transfected with 1 μg of HIV-1/CAT and either 0, 1, 10, or 25 ng of the HIV-1 Tat expression vector and 100 ng of GLI-2/THP expression vector where indicated. In panels B and C, open bars indicate basal activity of the HIV-1 promoter, and solid bars show the PMA response, widely striped bars represent activity when 100 ng of the GLI-2/THP expression vector was cotransfected, and dark striped bars show activity of the GLI-2/THP cotransfectants after stimulation with PMA. (C) HIV-2 Tat dose response. Jurkat cells were transfected with 1 μg of HIV-2/CAT and either 0, 25, 50, or 100 ng of the HIV-2 Tat expression vector, plus 100 ng of GLI-2/THP expression vector where indicated.

FIG. 6.

GLI-2/THP-Tat synergy requires the TATA box and functional Tat-TBP interaction. (A) HIV-1/CAT (1 μg) or HIV-1/CAT with the TATA box mutated (1 μg) was transfected into U937 cells with either empty vector or 50 ng of Tat-1 expression vector and 100 ng of GLI-2/THP expression vector. The cells were treated and harvested as for Fig. 5. Open bars represent unstimulated samples; solid bars represent PMA-treated samples. The data are representative of four separate experiments. (B) Transfections were performed as for panel A except that 25 ng of expression vectors for wild-type HIV-1 B strain Tat and the point mutants Tat-B.E. and Tat-B.T., which have been shown not to physically interact with TBP, were used instead of the Tat-1 expression vector. Fifty-nanograms of the GLI-2/THP expression vector was cotransfected into samples as indicated. Open bars represent unstimulated samples; solid bars represent PMA-treated samples. The data are representative of four separate experiments.

GLI-2/THP stimulates HIV-1 replication.

As GLI-2/THP is able to strongly synergize with Tat to activate the HIV-1 and HIV-2 promoters, we next assessed whether GLI-2/THP also stimulates HIV-1 replication. This was first examined by cotransfecting the HIV-1 infectious clone pHXB2 along with GLI-2/THP into the 293 cell line. These experiments clearly demonstrate that GLI-2/THP can stimulate replication of the HXB2 isolate (Fig. 3). GLI-2/THP was also able to stimulate replication in 293 cells of another isolate of HIV-1, an NL4-3-based infectious clone (data not shown).

FIG. 3.

GLI-2/THP stimulates single-round HIV-1 replication in 293 cells. 293 cells were transfected with 2 μg of pHXB2 and 5 μg of GLI-2/THP or control plasmid. Supernatants were collected 1, 2, 3, and 5 days after transfection and assayed in triplicate for RT activity. This experiment is representative of three independent experiments.

We next tested whether GLI-2/THP can activate HIV-1 replication in monocytic cells, which are more biologically relevant than 293 cells. In these experiments, the pHXB2 infectious clone was again cotransfected with GLI-2/THP, this time using the U937 monocytic cell line. As shown in Fig. 4, HXB2 replication was again strongly stimulated by GLI-2/THP. While PMA was not strictly necessary to demonstrate this stimulation, it did lead to much more rapid induction of viral replication in the presence of GLI-2/THP. GLI-2/THP was also able to markedly stimulate replication of the NL4-3-based clone, and with this isolate the presence of PMA was necessary for stimulation (data not shown). Therefore, in these cotransfection experiments involving infectious HIV-1 clones, similar to the experiments using reporter gene constructs, the presence of PMA greatly augmented the GLI-2/THP effect in monocytic cells. It must also be pointed out that similar to what is seen in reporter gene assays, the GLI-2/THP effect on infectious virus shows a dose-response relationship (data not shown). In both the reporter gene and infectious clone experiments, once GLI-2/THP is present beyond a certain concentration, the synergistic effect with Tat, and the ability to activate viral replication, is lost (data not shown).

FIG. 4.

GLI-2/THP stimulates HIV-1 replication in U937 monocytic cells. U937 cells were transfected with 2 μg of pHXB2 and 5 μg of either a control vector or a vector which expresses GLI-2/THP. The indicated cells were stimulated with PMA at a concentration of 16 nM 20 h after transfection. RT activity was measured in supernatants collected each day after transfection. Data represent mean RT activity in triplicate wells. This experiment is representative of three independent experiments.

GLI-2/THP-Tat synergy is independent of TAR.

Our data demonstrated that GLI-2/THP can markedly stimulate HIV-1 replication and powerfully synergize with Tat, and our biochemical studies suggested that GLI-2/THP might directly interact with Tat in the transcription complex. We next tested whether TAR is necessary for the GLI-2/THP synergy, as it is for synergy with other reported Tat cofactors (9, 15, 43, 46, 66, 69, 74), using an HIV-1 promoter construct containing a deletion in the TAR region. The results of these cotransfection experiments, here shown in U937 monocytic cells, demonstrated that GLI-2/THP-Tat synergy is, surprisingly, independent of TAR-1 (Fig. 5A). An HIV-2 TAR mutant, Δstem-CAT (5), was also responsive to the combination of GLI-2/THP plus HIV-2 Tat (data not shown), despite its weak transactivation by even large amounts of HIV-2 Tat alone (5). Consistent with these findings is the observation that HIV-2 Tat, which does not interact well with TAR-1 and does not transactivate the HIV-1 promoter (49), markedly boosts HIV-1 promoter activity in the presence of GLI-2/THP (Fig. 5B). Thus, GLI-2/THP synergizes with Tat in T cells and monocytic cells and operates through a TAR-independent mechanism. TAR-independent transactivation of HIV promoters has been seen in experiments using artificial systems such as GAL4 to tether Tat to the HIV promoter (58) but has not been observed previously with the wild-type promoter and any cloned Tat cofactor.

FIG. 5.

GLI-2/THP-Tat synergy is independent of TAR. (A) The TAR region is not required for synergy. The human monocytic cell line U937 was transfected with 1 μg of either HIV-1/CAT or HIV-1 (TAR del)/CAT and 100 ng of either the GLI-2/THP expression vector or empty vector; 50 ng of an HIV-1 Tat expression vector was also cotransfected where indicated. Twenty hours after transfection, the indicated samples (striped bars) were treated with 16 nM PMA. The cells were harvested 24 h later; samples were normalized for protein concentration and used in the CAT assay. (B) Tat-2 is able to synergize with GLI-2/THP to activate the HIV-1 promoter. HIV-1/CAT (1 μg) was transfected into U937 cells with either empty vector, 50 ng of Tat-2 expression vector, 100 ng GLI-2/THP expression vector, or a combination of 50 ng of Tat-2 expression vector and 100 ng of GLI-2/THP expression vector. The transfected cells were treated and harvested as for panel A. These data are representative of three separate experiments.

GLI-2/THP-Tat synergy is dependent on the TATA box and Tat-TBP interactions.

The above findings suggested that as neither TAR (Fig. 5) nor the TG-rich element to which GLI-2/THP binds (not shown) was necessary for GLI-2/THP-Tat synergy, neither Tat nor GLI-2/THP was directly responsible for recruiting the complex to the HIV promoter. Therefore, we investigated other proteins that might serve this function. One such attractive candidate protein was TFIIE, which has been demonstrated to interact with Krüppel, a Drosophila protein related to the GLI proteins (56), and the transcription factors Sp1 and TBP, which have been shown previously to interact physically and functionally with Tat (24, 26, 29). Using GST pull-down experiments (Fig. 1A and C), we found that TBP interacts with GLI-2/THP-GST (Fig. 1C, lane 2) but not with GST alone (not shown), whereas Sp1 and the TFIIE α and β subunits do not interact with GLI-2/THP-GST (Fig. 1C, lanes 1, 3, and 4). As GLI-2/THP can interact with Tat (Fig. 1B) and TBP (Fig. 1C), and TBP can also interact with Tat (29), we hypothesized that a Tat-TBP-GLI-2/THP association might be involved in the activation of HIV transcription by the crucial Tat transactivator. This would imply that the TATA box, the binding site for TBP, was necessary for the GLI-2/THP-Tat synergy to occur. Indeed, when ΔTATA, an HIV-1 promoter construct with a site-directed mutation in the TATA box (42), was transfected into U937 cells, only weak activation was detected in the presence of PMA, GLI-2/THP, and Tat-1 (Fig. 6A). In a parallel transfection using larger amounts of ΔTATA and GLI-2/THP, we demonstrated response to activation by PMA (56a), consistent with previous observations (4) that ΔTATA is still capable of responding to cellular stimulation. Thus, mutation of the TATA box specifically inhibits the response to the GLI-2/THP-Tat synergistic effect. Further, synergy was not seen with two Tat mutants (Tat-B.E. and Tat-B.T.) that contain point mutations in the TBP interaction domain (Fig. 6B), suggesting that direct Tat-TBP interactions must occur for synergy to be seen. The lack of GLI-2/THP-Tat synergy seen with the ΔTATA promoter or with the Tat point mutants suggests that the functional interaction between Tat, GLI-2/THP, and TBP requires tethering of these factors to the promoter via the TBP-TATA interaction. Such tethering through the TATA box would be consistent with TAR-independent activation, as Tat-TAR interactions have been shown to be unnecessary for effective Tat function if the activation domain of Tat can be recruited to the transcription complex by other, artificial means (49, 58). It must also be noted that the requirement for the TATA box is not seen in other circumstances in which GLI-2/THP modulates retroviral transcription (56a), and thus is specific for the Tat synergy. In addition, while the HIV promoters are stimulated by GLI-2/THP, HTLV-1 promoter-driven transcription is suppressed and HTLV-2-driven transcription is unaffected by GLI-2/THP (56a), further demonstrating the specificity of the GLI-2/THP-Tat-TBP interaction.

GLI-2/THP synergizes with cyclin T1 to augment Tat function.

The C-terminal domain of RNA Pol II is needed for effective transactivation by Tat (7, 44, 46, 69). Recently, the Pol II-associated cyclin-dependent kinase-activating kinase (CAK) (9, 15, 43, 46) and the Tat-associated kinase (TAK/P-TEFb) (35, 69, 74) have been implicated in the elongation effects of Tat. Both CAK and TAK appear to function by phosphorylating the C-terminal domain of Pol II, an event that allows Pol II to effectively elongate transcripts (19, 20, 68). An 87-kDa cyclin C-related protein, cyclin T1, identified in the TAK/P-TEFb complex, plays a particularly crucial role in Tat-mediated transactivation (14, 66). Unlike GLI-2/THP, cyclin T1 function requires the presence of TAR. As cyclin T1 appears to be the most powerful and biologically relevant Tat cofactor previously described, we tested whether GLI-2/THP could further augment the combined effect of Tat and cyclin T1 on HIV-1 promoter-driven expression in cotransfection studies in NIH 3T3 cells (Fig. 7). As expected, in these cells Tat-1 had a relatively small effect (14-fold activation) on HIV-1 promoter function, and cyclin T1 showed marked synergy (240-fold activation) with Tat-1. A very similar degree of synergy was seen with GLI-2/THP and Tat-1 (280-fold activation). The addition of GLI-2/THP to Tat-1 and cyclin T1 gave a further, marked synergistic effect (2,000-fold activation above baseline). Thus, a TAR-independent Tat cofactor, GLI-2/THP, and cyclin T1, a TAR-dependent cofactor, together greatly augment HIV-1 promoter-driven expression in conjunction with Tat-1.

FIG. 7.

GLI-2/THP synergizes with cyclin T1 to augment Tat function. NIH 3T3 cells were transfected with 5 μg of HIV-1/luciferase, 0.33 μg of a vector expressing Tat-1, 0.33 μg of a vector expressing human cyclin T1 (hCycT1), and/or 0.33 μg of a vector expressing GLI-2/THP. Transfections were normalized with 0.5 μg of Renilla luciferase standard. When necessary, pcDNA3 plasmid was added to keep the total amount of DNA constant at 6.5 μg. Results shown are from two separate transfections (stippled and solid bars) performed at the same time and are representative of two independent transfection experiments.

DISCUSSION

Here we have shown that GLI-2/THP markedly stimulates HIV replication and synergizes with Tat-1, Tat-2, and cyclin T1, potent activators of HIV transcription. As it has become clear that Tat requires cellular cofactors to function in vivo (34), intensive efforts have been made to identify functionally important Tat cofactors which might interact with Tat, the TAR RNA element, DNA promoter elements, the basal transcriptional machinery, or some combination of these elements. Despite the requirement for cellular cofactors for Tat-mediated transactivation, few potential cofactors, other than GLI-2/THP, cyclin T1, and cyclin-dependent kinases, have demonstrated convincing functional effects in promoter and viral replication studies. We have now shown that GLI-2/THP clearly can work with Tat-1 or Tat-2 to stimulate HIV-1 or HIV-2 expression well above the absolute level seen with Tat alone. The Tat-GLI-2/THP synergy is dependent on an intact TATA box in the promoter and does not take place with Tat mutants incapable of interacting with TBP.

An individual GLI protein is found in multiple different forms in the cell, and variants which are less abundant are often biologically very significant (3, 54). GLI-2/THP is an isoform found in low abundance in cells, which we have detected only in cellular extracts that have been enriched through Tat affinity chromatography (M. Smith and D. Markovitz, unpublished data). Several new isoforms of GLI-2/THP have recently been described (56a, 59), and their ability to synergize with Tat is under investigation. However, our findings demonstrate that the GLI-2/THP isoform is able to activate transcription through an unexpected mechanism that has not previously been reported for any other GLI protein family member. These proteins bind TG-rich enhancer elements in vitro, and all previously published studies have implicated these upstream enhancer sites as the mediators of GLI function. Here, we demonstrate that GLI-2/THP can activate transcription through a central, TBP/TATA box-dependent mechanism. Therefore, GLI-2/THP-Tat synergy occurs not only through a mechanism which is unique among Tat cofactors but also through a mechanism not previously reported for GLI family members. As GLI proteins play an important role in the development of multiple species, are involved in the Hedgehog signaling pathway, which is crucial for correct patterning of the embryo, and have been implicated in the genesis of cancer in humans, it will be important to further examine the exact mechanisms by which GLI-2 and other GLI proteins interact with cellular and viral proteins to regulate gene expression.

The TAR independence of the GLI-2/THP-Tat synergy might suggest that GLI-2/THP stimulates initiation of transcription, rather than elongation. It is possible that a GLI-2/THP-mediated increase in initiated RNA Pol II complexes could potentiate a cryptic TAR-independent recruitment mechanism for Tat. However, under the conditions tested here, which employ limiting concentrations of promoter and Tat, it does not appear that GLI-2/THP simply initiates HIV transcription independently of Tat, as this explanation would not be compatible with the fact that the TAR-independent Tat synergy seen with GLI-2/THP is such a high proportion of the wild-type activity (Fig. 5). Use of limiting concentrations of Tat, as seen in the present studies, would seem to mirror the in vivo situation, in which low to limiting levels of Tat are seen in HIV-1-infected cells (reviewed in reference 31). It must also be noted that for optimal effect of GLI-2/THP, PMA appears to be necessary. This is not surprising, in view of the dependence of most GLI proteins on phosphorylation changes to function. As noted above, the Drosophila protein cubitus interruptus is actually accompanied by a kinase in a complex in the cell. Clearly, PMA simply mimics a natural kinase or other unidentified signal transduction mechanism. However, it should be noted that GLI-2/THP stimulation of HIV-1 replication in 293 cells is not at all dependent on PMA (Fig. 3). In addition, while PMA potentiates the effect of GLI-2/THP in U937 monocytic cells, GLI-2/THP alone can also augment viral replication to a lesser degree (Fig. 4). In addition, PMA is not necessary to demonstrate the GLI-2/THP effect, or its marked synergy with cyclin T1, in NIH 3T3 cells (Fig. 7). Thus, while it makes sense that activation of monocytic or T cells will augment viral transcription and replication, GLI-2/THP can also function without cellular stimulation. Thus, GLI-2/THP is a strong modulator of Tat function and HIV-1 replication, which is under the control of signal transduction pathways.

The Tat-GLI-2/THP synergy is dependent on an intact TATA box in the promoter and does not take place with Tat mutants incapable of interacting with TBP. In addition, GLI-2/THP can interact physically with both Tat and TBP, and Tat and TBP also interact. Thus, a Tat-TBP-GLI-2/THP complex might be an intermediary in the process of Tat transactivation, with GLI-2/THP augmenting the Tat-TBP functional interaction and TBP recruiting the complex to the promoter, though other mechanisms of GLI-2/THP/Tat corecruitment are certainly possible. As the mere presence of a TATA box does not indicate that a promoter will be stimulated by GLI-2/THP (56a), other factors must further confer specificity. How GLI-2/THP works with cyclin T1 to give such marked stimulation of HIV promoter-driven transcription is currently being studied. The use of GLI-2/THP, in conjunction with cyclin T1, to overcome the Tat block in murine cells and thus facilitate the development of a mouse model of HIV infection is also under investigation.

One of the most intriguing aspects of our findings is the observation that GLI-2/THP can potentiate Tat transactivation of HIV gene expression in the absence the TAR element. To our knowledge, no other TAR-independent Tat cofactor has been clearly characterized at the molecular level in lymphocytic or monocytic cell types, although TAR-independent Tat activation of the HIV-1 promoter has been observed in neuronal cells and linked to variations in the NF-κB complex in this cell type (61, 62). Previous studies have clearly shown that when Tat is brought to the HIV-1 promoter by use of heterologous constructs, it can activate gene expression in the absence of TAR (26, 58). Thus, if GLI-2/THP does indeed bring Tat to the HIV promoter via TBP or another corecruited factor, it would be expected to readily transactivate in the absence of TAR. In this light, it is of interest to note that Kashanchi et al. recently showed that Tat-dependent, TAR-independent transactivation of the HIV-1 promoter can be seen at specific times in the cell cycle (30). Our studies demonstrate that the Tat cofactor GLI-2/THP can markedly augment HIV-1 replication in a TAR-independent manner (Fig. 3 and 4). Whether or not GLI-2/THP contributes to cell cycle stage-specific TAR-independent activation of HIV-1 transcription is now under study.

Our findings clearly demonstrate that HIV transactivation by the viral Tat protein can be potentiated by the THP isoform of the human GLI-2 protein. This synergistic activation is dependent on other cellular signaling processes, which can occur following stimulation of the cells by either mitogens or cyclin T1 overexpression. Further, GLI-2/THP is able to enhance Tat transactivation of HIV even in the absence of the TAR element, suggesting that GLI-2/THP can provide an alternate recruitment method for Tat to the core HIV promoter. Clearly, HIV gene expression can be triggered by a variety of stimuli, and further studies on how cellular signaling pathways interconnect and interface with viral regulatory elements are needed to effectively model this complex process.