Cell, Vol. 62, 757-767,

August

24, 1990, Copyright

0 1990 by Cell Press

TAR-Independent Activation of the HIV-1 LTR: Evidence That Tat Requires Specific Regions of the Promoter Ben Berkhout, Anne Gatignol, Arnold B. Rabson, and Kuan-Teh Jeang Laboratory of Molecular Microbiology National Institute of Allergy and Infectious Diseases National Institutes of Health Bethesda, Maryland 20892

Replication of HIV-1 requires Tat, which stimulates gene expression through a target sequence, TAR. It is known that TAR is a Tat-responsive target. Since Tat increases transcriptional initiations from the HIV-1 LTR promoter, it is unclear mechanistically how Tat utilizes an RNA target. Here we show that TAR RNA is only one component of the Tat-responsive target. Efficient Tat frans-activation was observed only when TAR was present in conjunction with the HIV-l LTR NFKB/SPI DNA sequences. TAR RNA outside of this context produced a suboptimal Tat response. We propose that TAR RNA serves an attachment function directing Tat to the LTR. A Tat protein engineered to interact with LTR DNA could frans-activate through a TAR-independent mechanism. This suggests that Tat also has a DNA target. Introduction HIV-l Tat is an essential gene product for the life cycle of the virus (Dayton et al., 1988; Fisher et al., 1988). Tat has been shown to increase dramatically the initiation of transcription from the HIV-1 LTR (Muesing et al., 1987; Hauber et al., 1987; Jakobovits et al., 1988; Rice and Matthews, 1988; Jeang et al., 1988a; Laspia et al., 1989). There is additional evidence that Tat can also affect other aspects of mRNA processing and utilization (Rosen et al., 1986; Cullen, 1986; Peterlin et al., 1986; Hauber et al., 1987; Kao et al., 1987; Selby et al., 1989; Braddock et al., 1989). Various direct and indirect studies have delineated a Tat-responsive element (TAR) within the R region of the 5 viral long terminal repeat (LTR). TAR is located between nucleotides +19 and +42 with respect to the initiation site (+l) of viral transcription (Sodroski et al., 1985; Hauber and Cullen, 1988; Jakobovits et al., 1988; Garcia et al., 1989; Selby et al., 1989; Roy et al., 1990). Although TAR is present as both DNA and RNA, experimental results support that it is functional in its RNA form. Specifically, its secondary structure (Muesing et al., 1987; Feng and Holland, 1988; Berkhout et al., 1989) as well as nucleotide sequence within its single-stranded loop and bulge domains (Berkhout and Jeang, 1989; Roy et al., 1990), are important for Tat response. In addition, both Tat (Dingwall et al., 1989) and cellular proteins (Gaynor et al., 1989; Gatignol et al., 1989) are known to associate with TAR RNA in vitro, consistent with the concept that transactivation utilizes an RNA target. Thus, one unprecedented

aspect of Tat-mediated regulation is the use of a downstream RNA to upregulate activity at the corresponding upstream promoter. The proposition that a nascent TAR RNA-Tat complex feeds back to increase transcriptional initiations at the LTR promoter has recently been suggested (Sharp and Marciniak, 1989). In this context, TAR is thought to act possibly as an RNA enhancer. Mechanistically, how an RNA mediates an increase in initiation of transcription is unclear. A plausible scenario is that an initial TAR RNA-Tat complex forms and then contacts promoter elements to influence subsequent rounds of transcription. A previous observation consistent with this model is the finding that TAR rapidly loses functional activity when its position is moved progressively downstream within the transcript (Selby et al., 1989). This suggests a requirement for optimal spacing between TAR and promoter elements and is an expected condition for a protein-mediated TAR RNApromoter DNA interaction. An essential proof for a feedback model would be the demonstration that Tat trans-activates through an LTRspecific DNA promoter element. In this setting, TAR RNA is envisioned to function as a “location indicator,” optimally positioning Tat to influence the promoter. Thus, TAR RNA is physically not the activation site but rather the attachment site for a functional Tat protein. The finding that TAR is only marginally active when coupled to heterologous promoters (Rosen et al., 1985; Peterlin et al., 1988; Muesing et al., 1987; Berkhout et al., 1990) supports the prediction of a HIV-1 promoter-specific Tat-responsive element. We have explored two approaches to examine the interactions between Tat and the HIV-1 LTR promoter. First, we verified that TAR RNA, in certain instances, can be entirely dispensable for Tat frans-activation. Tat transcriptional function was preserved when it was targeted to the HIV-1 LTR independent of TAR. This suggests that TAR, while normally an essential LTR component for Tat function, is not the actual regulatory sequence mediating increased transcriptional initiations. Second, we constructed several U3 deletion/substitution mutants that transcribed intact TAR leader RNAs. We found that a novel HIV-l LTR, deleted for the NF-KB and SPl sequences but preserved in basal promoter activity and ability to synthesize wildtype TAR RNA, was poorly responsive to Tat trans-activation. This result indicates that efficient rrans-activation through the Tat-TAR system can occur only within the context of specific promoter DNA sequences. In the HIV-l LTR, the appropriate DNA sequences are circumscribed within the NF-KBISP-1 region. Results A Hybrid Tat-Jun Protein Accumulating evidence indicates that HIV-1 Tat plays a role in the initiation of transcription from the LTR promoter (Muesing et al., 1987; Hauber et al., 1967; Jakobovits et al., 1988; Rice and Matthews, 1988; Jeang et al., 1988a;

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(A) Schematic representation of the fusion of the v-Jun DNA binding domain to Tat. A fragment containing the Jun DNA binding domain was ligated in frame (SVTatj) or out of frame (SVTatjf) to a DNA fragment containing the first 66 amino acids of Tat. ORF, open reading frame. (6) Expression of the SVTatj and SVTatjf proteins. pSVTatj (5 ug; lanes 2, 3, 5, and 6) or p.SVTatjf (5 ug; lanes 8 and 9) was individually transfected into HeLa monolayer cells. Cellular proteins were radiolabeled and precipitated with preimmune serum (lanes 2, 5, and 8). antiserum against Tat (lanes 1, 3, 7, and 9) or antiserum directed against the Jun DNA binding domain (lanes 4 and 6). Arrows point to the Tatj and Tatjf proteins. (C) Preservation of bans-activation function in Tatj and Tatjf. A HIV-1 LTR-CAT target (1 frg) was cotransfected with pBR322 (1 rrg; +mock, lane l), with pSVTatj (1 ug; +SVTatj, lane 2) or with pSVTatjf (1 pg; +SVTatjf, lane 3). CAT enzyme assays were performed 48 hr after transfection. A more extensive titration series of CAT assays showed an eauivalence in frans-activation ability between SVTatj and SVTatjf (data not shown). AcCm. acetylated chloramphenicol; Cm, chloramphenicol

Laspia et al., 1989). At the same time, numerous studies have also documented that Tat requires a correctly structured TAR RNA for its trans-activating function (Feng and Holland, 1988; Garcia et al., 1989; Berkhout et al., 1989). The ability of Tat, interacting through a downstream RNA target, to increase mRNA initiations at the upstream promoter presents a novel and seemingly confusing paradigm. A clarifying explanation considers both a downstream RNA and an upstream DNA element as the complete HIV-l Tat-responsive ensemble. In that context, TAR RNA simply serves as an attachment site for positioning Tat. To verify this proposition we designed an approach that allowed Tat to attach to the HIV-l LTR in a TAR-independent manner. We built a hybrid gene, driven by the SV40 early promoter, that encoded the first 88 amino acids of Tat followed by the DNA binding domain of v-Jun (Maki et al., 1987; SVTatj, Figure 1A). A similarly organized control gene (SVTatjf, Figure lA), containing a 1 nucleotide insertion resulting in a frameshift in the Jun domain, was also constructed. When transfected into HeLa cells, SVTatj directed the synthesis of a 32 kd protein that was equally immunoprecipitated by a polyclonal antiserum to Tat (Figure lB, lane 3) or by a peptide antiserum directed to the Jun DNA binding epitope (Figure lB, lane 6). In the same experiment, SVTatjf produced a shorter Tat protein (Figure lB, lane 9) that, when normalized for incorporation of radiolabeled amino acids, was quantitatively equivalent to the amount of Tatj. Both Tatj and Tatjf efficiently activated a HIV-1 LTR-CAT target (Figure lC), consistent with the fact that only the first 58 amino acids of Tat are needed for

function (Seigel et al., 1986). In addition, we noted that the presence of a fragment of the Jun protein attached at the C-terminus of Tat did not affect trans-activation function. TAR-Independent Activation of a HIV-1 LTR Containing AP-1 Binding Sites Tatj represents a novel form of functional Tat protein that may be capable of direct binding to DNA. To test for its ability to activate the HIV-l LTR in a TAR-independent fashion, we inserted four copies of an oligonucleotide containing the AP-1 binding motif (Lee et al., 1987; Angel et al., 1987) into TAR (pLTRAPCAT Figure 2A, left). Based upon previous mutagenesis studies (Hauber and Cullen, 1988; Garcia et al., 1989) this insertion in pLTRAPCAT would be expected to inactivate TAR. Indeed, the putative secondary structure of the pLTRAPCAT leader RNA does not resemble TAR at all (Figure 28, compare TAR with APTAR). Computer folding of this RNA revealed an exceedingly stable structure (-102 kcallmol) that does not preserve the overt configuration of the wild-type TAR structure. Experimentally, we found that pLTRAPCAT was poorly trans-activated by Tat (Figure 2A, lane 3). In contrast, it responded well to Tatj (Figure 2A, lane 2). The ability of Tatj, compared with Tat, to bind to LTR DNA may account for the observed difference in functional activity. Two experiments confirmed this hypothesis. First, we found that pSVTatjf, which differed from pSVTatj by only a single frameshifting nucleotide inserted upstream of the Jun DNA binding domain, was ineffective in transactivating pLTRAPCAT (Figure 3A, compare lane 3 with lane 2; Figure 38, compare lanes 4 and 7 with lanes 3 and

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(A) (Left) Four copies of an AP-1 binding motif (GTGACTCAGCGCC; Lee et al., 1997) were inserted into HIV-1 LTR-CAT in the indicated orientations (arrows) to generate pLTRAPCAT (Right) pLTRAPCAT was activated in a TAR-independent manner. pLTRAPCAT (0.5 ug) was transfected into HeLa cells with pBR322 (lane l), pSVTatj (1 trg, lane 2) or pSVTat (1 ug, lane 3). CAT activities were assayed 43 hr after transfection. (6) Schematic secondary structures of the RNA leader sequences from pLTRCAT (TAR, left) and pLTRAPCAT (APTAR, right). Note that in AP-TAR the exposed nucleotides previously found in the TAR loop are now basepaired. Calculated free energies (Zuker and Stiegler, 1991) for the RNA structures are -35 kcal/mol (left) and -102 kcallmol (right). Because of its tight secondary structure, AP-TAR-CAT mRNAs may be inefficiently translated, which may result in an underestimation of overall expression.

6; Figure 3C). Second, a mutation known to interfere with DNA-protein binding, when introduced into the AP-1 binding sites in pLTRAPCAT, abolished responsiveness to Tatj (Figure 3A, lane 5). These results establish that a DNA binding function coupled with the appropriate presence of a binding site are necessary for Tatj activity. Further experiments controlled for the fact that the Jun DNA binding domain is not in itself sufficient for activity. In parallel cotransfections, the Jun DNA binding domain under the control of the SV40 early promoter (pSVj) or the Rous sarcoma virus (RSV) LTR (pRSVj) was found to be incapable of frans-activating pLTRAPCAT (Figure 36, lanes 8 and 9; Figure 3C, right panel) or pLTRCAT (Figure 3C, middle panel). Similarly, SVTatj did not activate a Jun-responsive reporter construction (5xTRETKCAT Figure 3C, left panel). An LTR U3 DNA Sequence Is a Necessary Target for Tat Tram-Activation One interpretation of the above results is that TAR serves as the physical attachment, but not activation, point within the LTR for Tat. In this model, TAR can be dispensible for function so long as Tat is directed to the LTR via a different

mechanism (i.e., binding to DNA through a Jun domain). That HIV-1 LTR was activated by Tat in a TAR-independent fashion (Figures 2 and 3) implied the existence of a promoter element(s) that specifies Tat responsiveness. To define the HIV-1 DNA sequence(s) that responds to Tat trans-activation, we designed a series of changes within U3 (Figure 4). A major feature of these LTR mutations is that none altered the wild-type mRNA start site. Thus, the nonresponsiveness of a given construction to Tat cannot be explained by a perturbation in TAR RNA. Our mutations included the deletion of all U3 sequences upstream of -159 (Figure 4A, AAat-Ava), all U3 sequences upstream of -105 (Figure 4A, NWSp), both NF-KB motifs (Figure 4A, NF-A), and the three SPl binding sites (Figure 4A, Sp-A). We also substituted the nucleotides between the TATA box and +l with a random sequence (Figure 4A, S-10). When these plasmids were individually cotransfected into cells with a Tat-producing vector, we found that all maintained essentially wild-type levels of activation (see Fold Induction, Figure 4A). Graphic representation of the observed induction over a range of cotransfected Tat-producing plasmid showed a sharp increase of activity at 0.5 pg of Tat plasmid (Figure 48). In

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3. Activation

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of Wild-Type

or Mutant

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by Tatj or Tatjf

A pLTRAPCAT construction (pLTRAPmCAT) was designed that contained reiterated copies of a mutated AP-1 binding site (GTCCCTGGGCGCC). (A) Cotransfection of pLTRAPCAT (lanes 2 and 3) or pLTRAPmCAT (lanes 5 and 6) with pSVTatj (lanes 2 and 5) or pSVTatjf (lanes 3 and 6). Lanes 1 and 4 are pLTRAPCAT and pLTRAPmCAT, respectively, cotransfected with pBR322. (B) Sl nuclease quantitation of RNA synthesized from pLTRAPCAT. Total cellular RNA was isolated from HeLa cells transfected with 5 ug of LTRAPCAT (lanes 2 and 5) 5 bg of pLTRAPCAT + 2 pg of pSVTatj (lanes 3 and 6) 5 ng of pLTRAPCAT + 2 ng of pSVTatjf (lanes 4 and 7) 5 pg of pLTRAPCAT + 2 pg of pSVj (lane 9) or 5 pg of pLTRAPCAT + 2 ng of pRSVj (lane 9). Synthesis of the Jun DNA binding domain was checked by immunofluoresence of transfected cells. Expression of CAT mRNA was measured by the protection from nuclease of a 256 bp fragment using an antisense probe. Lane 1 contains the input 522 bp probe alone; lane M contains molecular size markers. (C) Quantitation based upon CAT enzyme assays of the relative extent of ffans-activation of 5xTRETKCAT, pLTRCAT, or pLTRAPCAT by the indicated Tat and Jun constructions. Tins-activation of 5xTRETKCAT by pSVTatj, pSVTatjf, or pRSVvjun (left); trans-activation of pLTRCAT by pSVTatj, pSVj, or pRSVj (middle); trans-activation of pLTRAPCAT by pSVTatj, pSVj, or pRSVj (right). Because of its extensively structured leader RNA (-102 kcallmol, Figure 28) expression of pLTRAPCAT may be underestimated by protein assays.

contrast, a deletion within TAR (Figure 4A, ATAR) potently diminished Tat responsiveness even at higher levels of cotransfected Tat plasmid (Figure 48). The above analysis failed to reveal a single DNA element that is absolutely required for Tat response. We next

considered the effect of the simultaneous removal of both NF-KB and Spl binding sites. Deletion of both elements results in a nonfunctional promoter (Rosen et al., 1985; Muesing et al., 1987; Leonard et al., 1989; our unpublished data). Thus, to maintain promoter function, we sub-

5;;

Is a DNA

Trans.Activator

stituted the SV40 72 bp enhancer for HIV-1 sequences upstream of the TATA box in a spatially conservative manner (Figure 4A, pSV-LTR-CAT). pSV-LTR-CAT replaced the exact nucleotides between -199 and -36 with 162 bases containing the SV40 enhancer. It otherwise maintained completely the relative spacing of HIV-1 LTR sequences. Primer extension analysis of cat mRNA transcribed from pSV-LTR-CAT confirmed correct initiation at +l (Figure 4C, lane 5). A second plasmid (pSV-TAR-CAT) had the complete SV40 enhancer-promoter linked to the HIV-1 TAR element, in a spatially nonconservative manner, and was used as a control. This construction, however, had an aberrant mRNA start site (Figure 4C, lane 7). We found that both pSV-LTR-CAT and pSV-TAR-CAT responded poorly to Tat (Figure 4A, Fold Induction). Each showed marginal incremental Tat-induced activity over the negative control ATAR plasmid (Figure 4A, compare 6- and 3-fold with 3-fold). In the case of pSV-TAR-CAT the incorrect length of the TAR leader RNA (Selby et al., 1969; Figure 4C, lane 7) might be one explanation for the decreased activity. Surprisingly, pSV-LTR-CAT, which synthesized a wild-type TAR RNA leader and conserved virtually all U3 DNA sequences except for NF-KB/SP~, was similarly nonresponsive to Tat (Figures 4A and 48). This finding demonstrated that an intact TAR RNA, without the correct context of HIV-1 promoter-enhancer elements, cannot effect optimal Tat activation. Targeting of Tat to a Promoter Is Itself Insufficient for Activity To substantiate that Tat-mediated LTR regulation necessarily involves both correct attachment and promoterspecific activation, we explored the ability of Tat to modulate different promoter elements. First, we asked whether Tatj could activate a HTLV-I promoter with (pUSRAPCAT, Figure 5A) exogenously added AP-1 binding sites. We note that the three copies of Tax-responsive 21 bp sequences normally found in the HTLV-I LTR (Seiki et al., 1962, 1963; Figure 5A, left) could function as AP-1 sites (K.-T. J., unpublished data). pU3RAPCATcontained an additional four copies of AP-1 sequences (Figure 5A, left), inserted in the previously described orientations, and should allow Tatj to bind efficiently to DNA. We found, by Sl nuclease protection analysis, that it was not transactivated by cotransfection with pSVTatj (Figure 5A, lane 4) or pSVTatjf (Figure 5A, lane 5). As expected, it remained Tax responsive (Figure 5A, lane 6). In a second set of experiments we directed wild-type Tat to a HIV-1 LTR promoter unit that was deleted in its TAR element (Figure 56, plasmid A22; see Experimental Procedures for details in construction). We attached a CMV IE promoter-driven transcription unit (Figure 58, plasmid CMVTARA22) to this construction, such that a copy of TAR RNA, albeit synthesized from the opposite DNA strand, would be transcribed in proximity to the HIV-l LTR promoter. In pCMV-TARA22 it is conceivable that Tat binding to the CMV-driven TAR RNA could “swivel” in a manner to still activate HIV-1 U3 elements. We, however, observed no such activity (Figure 58, compare lane 4 with

lane 2). Thus, the inappropriate association of Tat to any given promoter does not suffice for function, supporting the concept of promoter-specific and spatially constrained organization of the HIV-1 LTR.

An essential role for the TAR element in HIV-1 Iransactivation has been documented in many studies (Hauber and Cullen, 1966; Jakobovits et al., 1966; Garcia et al., 1969; Selby et al., 1969; Roy et al., 1990; Berkhout and Jeang, 1969). Although TAR is functionally recognized as an RNA (Feng and Holland, 1966; Berkhout et al., 1969), a result of Tat trans-activation is increased transcriptional initiations (Rice and Matthews, 1966; Jakobovits et al., 1966; Jeang et al., 1966a; Laspia et al., 1969). It is unclear how recognition through an RNA target could increase transcription from a promoter. In this study we present evidence implicating an attachment function for TAR RNA that serves to bring Tat into a promoter proximal position in the LTR transcriptional unit. Once anchored to the LTR, through TAR RNA, Tat functionally interacts with factors bound to the NF-KB/S~~ region to influence promoter activity. We propose that Tat resembles generic transcriptional activators, in that it has a domain responsible for targeting to nucleic acids and a second domain for activation (for a review see Ptashne, 1966). During the life cycle of HIV, the initial round of transcription is Tat independent (i.e., one copy of mRNA encoding Tat needs to be transcribed first). Upon synthesis, Tat, in association with a cellular protein(s), utilizes TAR RNA as a “scaffolding” from which it influences the activity of the upstream promoter (see Figure 6A). The unique difference between general transcriptional activators and Tat is that while the former recognize DNA attachment sites (for a review see Dynan, 1969), Tat recognizes an RNA attachment site. The small size of Tat makes a demonstration of discrete activation and targeting domains difficult. A recent report, however, suggests that the activation function of Tat might be entirely dictated by its first 13 amino acids (Rappaport et al., 1969). Our present approach predicts and provides evidence that if Tat is able to directly bind HIV-1 DNA in an appropriate context, then TAR RNA could become a dispensible component of the trans-activation responsive target (Figure 66). In contrast, we found that the NF-~6ISpl segment of the HIV-1 promoter (Figure 4) is an indispensible HIV-1 LTR DNA element for Tat responsiveness. We were initially surprised that the isolated removal of large segments of HIV-1 U3 sequences had little (SPA, see also Harrich et al., 1969) or no (AAat-Ava, NWSp, NF-A, S-10) effect on Tat responsiveness (Figure 4A). Given that sequences downstream from -105 (see NF/Sp, which preserves only the NF-KB/S~~ and TATA elements from the HIV-1 U3, Figure 4A) maintained wild-type levels of Tat response, we were impeded in further analysis since more extensive deletions in the -105/-l region resulted in nonfunctional promoters. This difficulty was circumvented by preserving

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basal promoter activity of the HIV-1 LTR through a spatially conserved substitution using a heterologous enhancer element (Figure 4A). In this manner, we could analyze the response to Tat of the entire HIV-1 LTR when deleted for both the NF-KB and SP-1 sequences. Although pSV-LTR-CAT maintained virtually all HIV-1 LTR U3 sequences (except the NF-KB and Sp-1 motifs) and used the wild-type +l transcriptional initiation site (Figure 4A), it was poorly responsive to Tat. Simply interpreted, this result suggested that, in the absence of NF-KB and Sp-1 sites, TAR RNA is insufficient for Tat response. We note that pSV-LTR-CAT has a 4-fold higher basal activity (1,210) than pLTR-CAT (371; Figure 4A). However, we saw little evidence (see Figure 48) that this increase in basal activity saturated the cellular transcriptional machinery in a manner making measurements of induced expression unreliable. Our pSV-LTR-CAT results contrast somewhat with the findings of Muesing et al. (1987). Those investigators found that HIV-1 LTR sequences between -29 and +232 (containing essentially TATA and TAR elements) conferred a >139-fold activation to Tat. However, in the same study “wild-type” levels of activation were measured to be 2,879-fold (Muesing et al., 1987). A similar construction in our assays produced no measurable basal activity, rendering no reliable assessment of fold induction. Our results substantially agree with those of Rosen et al. (1985) in which LTR sequences between -104 and -45, shown in one assay, seem to be important for &arts-activation. We cannot easily explain why in the same report an RSV LTR fused to HIV-1 sequences from -44 to +80 was activated by virus infection in HS cells. We interpret our results to indicate that frans-activation requires a specific DNA sequence in U3 for interaction with TAR RNA and Tat. A HIV-l LTR deleted for NF-KB/SP~, but synthesizing unaltered TAR RNA, is poorly trans-activated by Tat. What are the ramifications for using RNA as an attachment point? Proteins that bind to a continuously elongating TAR RNA have far greater constraints on their potential interactions with upstream promoter elements than proteins that bind to a static DNA site. An RNA binding site will increasingly distance itself from the LTR promoter during transcription. This suggests that there exists only a narrow time window for nascent TAR RNA and Tat to feed back to the promoter. This is consistent with previous

Figure

4. T-arks-Activation

results demonstrating that Tat action requires only a transitory nascent TAR RNA (Berkhout et al., 1989). The actual time a nascent TAR RNA is in close proximity to the promoter can be increased when transcriptional pausing occurs. We could imagine that a transcriptional pause would “freeze” TAR RNA in the optimal spatial position and would lend time for proper RNA folding and assembly of the RNA-protein-DNA complex. Indeed, transcriptional elongation over the HIV-1 genome has been reported to stall at a position just downstream of TAR (Kao et al., 1987; Selby et al., 1989). We cannot presently define how a TAR RNATat-U3 DNA complex physically enhances transcriptional initiation. Three possibilities come to mind. First, this Tat complex may functionally influence the promoter such as to allow increased loading of RNA polymerase II molecules in a manner similar to DNA enhancers. Second, Tat in concert with cellular proteins may act to stabilize initiated polymeras8 complexes, preventing a premature termination event (Kao et al., 1987; Selby et al., 1989). Finally, Tat could functionally displace a constitutive transcriptional repressor of the LTR promoter. This would explain the extremely low basal activity of the HIV-1 LTR and could provide an explanation for how a single feedback event activates the LTR promoter for multiple subsequent cycles. What would be the advantage for HIV to use such a complex Tat-TAR regulatory system? One observation is that Tat fmns-activation of the HIV LTR is highly specific. Unlike other transcriptional activators, Tat, to our knowledge, does not appreciably activate eukaryotic promoters other than the HIV LTR. We consider that the use of TAR as an RNA target adds a further level of specificity to the regulated expression of the LTR. TAR RNA, in addition, needs to be positioned in a manner more than simply to direct Tat to a U3 proximal location (Figure 58). This may, in part, explain our qualitative but less than quantitative reproduction of Tat frans-activation using a DNA site for targeting. It also suggests that suboptimal targeting could possibly be compensated for by extremely high concentrations of Tat protein. In the absence of Tat, transcription from the viral LTR is low and is dependent on the available pool of cellular transcription factors. General activation of the host cell results in increased synthesis of TARcontaining RNA, including that coding for Tat protein. Once a threshold amount of Tat has accumulated, frans-

of HIV-1 U3 Mutants

(A) Schematic representation of the LTR is shown at the top. HIV-1 U3 contains two NF-KS sites (position -105/-94 and -91/-60) three Sp-1 sites (-77/-67, -65/-56, and -M-44), and the TATA element (-26/-24). The transcriptional initation site is marked by an arrow and by +I. The TAR element, indicated by a solid box, is located within the transcribed sequences (+19/+42). Nucleotide deletions and substitutions such as in S-10 are indicated. pSV-LTR-CAT has part of the U3 region (-199/-36, including all NF-KS and SP-1 sites) substituted by the SW0 enhancer (SV-E box). pSWAR-CAT contains the SV40 enhancer-promoter (SV-EP box) coupled to the wild-type TAR element. CAT enzyme levels were measured in the absence (-) or presence (+) of Tat, and the ratio between the -Tat and +Tat values reflect bans-activation efficiency (Fold Induction). (6) Graphic representations of CAT enzyme levels synthesized by pLTR-CAT pNF/Sp, pNF-A, pSpA, ATAR, and pSV-LTR-CAT in response to 0, 0.5, 1.0, and 1.5 ug of Tat plasmid. (C) Primer extension analysis of cat mRNA synthesized by the indicated plasmids. (-) and (+) indicate the absence or presence of Tat. Cells were transfected with pLTR-CAT alone (lane 2) pLTR-CAT with pSVTat (lane 3) pSV-LTR-CAT (lane 4) or pSVLTR-CAT with pSVTat (lane 5) pSMAR-CAT (lane 6) or pSVTAR-CAT with pSVFat (lane 7). A 31nucleotide primer, complementary to cat mRNA, was used (primer alone is shown in lane 8). The expected primer extension products are indicated by arrowheads and are 163 (pLTR-CAT and pSV-LTR-CAT) and 273 nucleotides (pSVTAR-CAT). End-labeled pBR322-Hpall fragments were used as molecular size markers (lane 1). A longer exposure of lanes 6 and 7 is shown (right).

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5. Inappropriate

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5

in pans-Activation

(A) Targeting of Tat to the HTLV-I LTR via AP-1 sites does not cause activation. (Left) Schematic representation of an HTLV-I LTR-CAT construction (not drawn to scale) containing four inserted AP-1 sites (pU3RAPCAT) in the indicated orientation (arrows). The HTLV-I LTR naturally contains three copies of the 21 bp Tax-responsive element that function as AP-1 sites (K.-T J., unpublished data). (Right) Sl nuclease protection analysis of RNAs from cotransfections of pU3RAPCAT with pSVTatj (lane 4) pSVTatjf (lane 5) or an HTLV-I Tax-producing plasmid (lane 6). Lane 3 represents cells transfected with pU3RAPCAT and pBR322; lane 2 contains input Sl probe alone; lanes 1 and 7 contain molecular size markers. CAT mRNA expression is measured by the appearance of a protected 256 bp band (arrow). (8) cis positioning of Tat using TAR RNA does not activate an adjacent HIV-l LTR transcription unit. (Left) Schematic representations of proposed interactions between an intragenic TAR RNA and its upstream LTR promoter elements (pLTR-CAT) or a TAR RNA (driven by a CMV promoter) with an adjacent LTR promoter deleted in its TAR sequence (CMWARA22). A22 is pLTR-CAT with 22 bases spanning TAR (+16 to +32) deleted; CMVTARA is A22 with a CMV IE promoter-driven TAR transcription unit attached in the opposite direction. (Right) Primer extension analysis of Tat rrans-activation of pLTRCAT (lane 2) A22 (lane 3) or CMWARA22 (lane 4). (+) or (-) indicates presence or absence of Tat. Lane 5 contains molecular size markers. An expected product of 163 bp is seen in lane 2. Lane 1 represents analysis of basal expression from pLTRCAT,

Tat Is a DNA Trams-Activator 765

A)

similarly deleted for 22 nucleotides between +16 to +32. Both constructions remove sequences important for trans-activation. CMVTARA has a Simian CMV IE promoter driving the synthesis of a functional TAR RNA attached to A22 in an opposite orientation to the HIV-1 LTR promoter. pSV-LTR-CAT was derived from pLTR-CAT and contains a spatially conserved replacement of HIV-1 LTR sequences (position -199/-36) with SV40 sequences containing the two 72 bp repeats. This construct was made by the polymerase chain reaction, using the method of gene splicing by overlap extension (Horton et al., 1989). pSV-LTR-CAT contains the exact 72 bp repeat with 18 flanking SV40 nucleotides substituting for HIV-l sequences between -199 and -38. pSV-TAR-CAT contains the complete SV40 enhancer-promoter region coupled to the HIV-l TAR sequences. Compared with HIV-l transcripts, RNA made from this construct has the TAR element at a position 111 nucleotides further downstream. pSVTat contains a Tat cDNA (strain SF2) under the transcriptional control of the SV40 early promoter (Kao et al., 1987). pSVTatj was made by ligating, using an in-frame linker, the Ncol to EcoRl fragment from RSVjun (Angel et al., 1967) into the Hindlll site of pSVTat. pSVTatjf was similarly constructed, except an out-of-frame linker was used. pSVj and pRSVj (RSV LTR promoter) were made from pSVTatj, in which the Tat sequences have been deleted and an in-frame methionine is the first amino acid for the Jun DNA binding domain.

Tat

NFkB/Sp

1

Site

r NFkB/Sp

1

Tat

AP-1

sites

L Activation Tat Binding

Figure

6. Hypothetical

Modeling

of Tat Interaction

with DNA and RNA

(A) Tat attaches to a nascently transcribed TAR RNA and feeds back to influence transcription factors that bind to the NF-rcB/SPl sequences. Tat, in this model, is shown with a cellular cofactor(s) in its association to TAR. (6) The ability of Tat to target directly to the LTR transcriptional unit bypasses the use of TAR RNA. In this instance, appropriately positioned AP-1 sites allow a Tat-Jun fusion protein to bind to DNA. This type of interaction substitutes for the role of TAR RNA and can reconstitute tfans-activation function.

activation through TAR and the promoter alemen& rapidly occurs. Transcription using a feedback mechanism is expected to result dramatically in higher amounts of Tat. This positive cycling integrates the functions of viral Tat and cellular proteins and can likely be a trigger for progression from a quiescent to a fulminant viral infection. Experimental

Procedures

Plasmid Constructions All plasmids were constructed by standard techniques. Nucleotide numbers refer to the position on the HIV-l transcript, with +I being the capped G residue. LTR-CAT isas previously described (Gendelman et al., 1966, pBennCAT) with the exception of a deletion of pBR322 sequences between the Accl sites (nucleotides 651 and 2246; pBR322 coordinates). LTRCAT contains HIV-1 LTR sequences (BRU strain) up to the Hindlll site at position +77 fused to the CAT reading frame. TAR mutants were generated by cloning synthetic double-stranded DNA oligomers into the LTR-CATvector between the unique sites Sacl(+33)-Hindlll(+77) or between an introduced Xhol site (-9) and Bglll(+19). AAat-Ava was generated by ligating the unique Aatll site present in pBR322 sequences (pBR322 coordinate 4286) to the unique LTR Aval site (position -159). Both sites were first blunt ended using Klenow enzyme. NF/Sp plasmid contains HIV-1 U3 sequences up to -105. The NF-A (position -105/-77) deletion was previously described (Leonard et al., 1989). The SpA (position -77/-41) deletion was made in a similar manner. S-10 is asubstitution mutant (Berkhout et al.. 1990). ATAR carries a 12 nucleotide deletion (+23/+34) in the TAR element; A22 is

lVansfectlon, RNA, and Pmtein Analysis COS or HeLa cells were transfected using 400 uglml DEAE-dextran in the presence of 100 &ml chloroquine and Nu serum (Seed and Aruffo, 1987). HeLa cell transfections were performed using 100 uglml DEAE-dextran. Radioimmunoprecipitation and protein labeling have been previously described (Jeang et al., 1988b). Antibody generated against a peptide containing the DNA binding domain of Jun was purchased from Oncogene Science (Manhasset, NY). For RNA analysis, we used 5 wg of pLTR-CAT plasmid in the absence or presence of 2 pg of pSVTat per 100 mm dish of cultured cells. Total cellular RNA was isolated by the hot phenol method. A single-stranded DNA probe was generated from an M13CAT construct (Jeang et al., 1988a) for use in Sl nuclease protection assays. Primer extension analysis was performed using a 31-base oligonucleotide complementary to cat mRNA (Berkhout et al., 1990). For CAT enzyme assays (Gorman et al., 1982) 0.5 pg of both pLTR-CAT and pSVTat plasmids was transfected per 80 mm dish. Cell lysates were made by freeze thawing, and CAT enzyme was quantitated using the phase-extraction protocol (Seed and Sheen, 1988) or by scintillation counting of the different forms of ‘4C-chloramphenicol separated by thin layer chromatography. CAT activities were determined within the linear range of the assay, and transfections were repeated three times. Cell Culture COS and HeLa cells were maintained medium containing 10% fetal bovine

in Dulbeccos serum.

modified

Eagle’s

Acknowledgments We thank Alicia Buckler-White for oligonucleotide synthesis, John Leonard and Carmen Parrot for plasmids NF-A and SpA, and Peter Angel and Michael Karin for plasmids RSVvjun and 5xTRETKCAT. We are grateful to Malcolm A. Martin, Keith Peden, and Warren Leonard for critical reviews of the manuscript. This work was supported in part by the Intramural AIDS targeted anti-viral program from the Office of the Director of the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received

April

19, 1990; revised

June

15, 1990.

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TAR-independent activation of the HIV-1 LTR: evidence that tat requires specific regions of the promoter.

Replication of HIV-1 requires Tat, which stimulates gene expression through a target sequence, TAR. It is known that TAR is a Tat-responsive target. S...
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