The RNA Element Encoded by the Trans-Activation-Responsive

Proc. Natl. Acad. Sci. USA Vol. 92, pp. 2408-2412, March 1995 Biochemistry

The RNA element encoded by the trans-activation-responsive region of human immunodeficiency virus type 1 is functional when displaced downstream of the start of transcription (RNA-protein interaction/trans-activation/Tat protein/transcription elongation) MARK J. CHURCHER, ANTHONY D. LOWE, MICHAEL J. GAIT, AND JONATHAN KARN* Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 20H, United Kingdom Communicated by Sydney Brenner, University of Cambridge School of Clinical Medicine, Cambridge, United Kingdom, December 7, 1994

ABSTRACT The human immunodeficiency virus type 1 a TAR RNA element placed anywhere downstream of the (HIV-1) trans-activator protein, Tat, specifically stimulates promoter. To examine whether TAR is functional at a dis- transcription from the viral long terminal repeat. Tat binds to tance, we prepared templates carrying duplicated TAR ele- an RNA stem-loop structure encoded by the trans-activation ments. The contributions ofvarious control elements encoded response region (TAR). To test whether TAR is functional by TAR were then measured by introducing mutations into when displaced downstream of the start of transcription, we either the upstream or the downstream TAR elements. The assayed a series of templates carrying duplicated TAR ele- results demonstrate that TAR RNA elements can allow effi- ments in cell-free transcription systems. When the normally cient recruitment of Tat and the activation of transcriptional positioned TAR element (TAR-1) is inactivated by mutations elongation even when they are placed several hundred nucle- in either the Tat binding site or the apical loop sequence, otides downstream of the transcription start site. which acts as the binding site for a cellular factor, trans- activation can be rescued by a wild-type TAR element placed downstream (TAR-2). The TAR-2 element is functional even MATERIALS AND METHODS when placed >200 nt downstream of TAR-1. TAR comple- Template DNAs. Test plasmids were derived from the mentation experiments have also shown that a functional TAR pMAG-10 plasmid (5), which carries a synthetic terminator element requires both an intact Tat binding site and an intact sequence (T) located downstream of the viral LTR (Fig. 1). apical loop sequence. For example, if TAR-1 carries a muta- Duplicated TAR elements were inserted between the HindIII tion in the loop element it cannot be rescued by a TAR-2 and Nar I sites- downstream of the viral LTR. Sequences element carrying a mutation in the Tat binding site. Substi- derived from the bacterial chloramphenicol acetyltransferase tution mutations in TAR-1 show that the 5' half of TAR also (CAT) gene were used to replace sequences in TAR in order encodes an essential DNA element which is required for to map critical DNA elements and to provide "spacers" to efficient transcription initiation. These results strongly sug- move TAR-2 to different positions. gest that Tat and cellular cofactors which bind TAR RNA Cell-Free Transcription. Transcription reactions (50 ,ul) associate with the transcription complex during its transit were performed essentially as described (5). Templates were through TAR. linearized by cleavage with Xba I. After preincubation for 15 or 20 min, the reaction mixtures were incubated for 30 min in The human immunodeficiency virus type 1 (HIV-1) Tat protein the presence of [a-32P]UTP. Transcripts were fractionated by provides the first example of a viral protein that regulates electrophoresis in 6% polyacrylamide gels containing 7 M transcriptional elongation in eukaryotic cells. In the absence of urea, 90 mM Tris base, 89 mM boric acid, and 2 mM EDTA Tat, the majority of the transcriptional complexes formed at the (pH 8.3). The gels were dried and then exposed to x-ray film HIV promoter stall or disengage near the start of transcription, for the times indicated in the figure legends. whereas in the presence of Tat there is a dramatic increase in the production of full-length viral transcripts (1-7). Deletion analysis of the viral long terminal repeat (LTR) RESULTS showed that Tat activity requires the trans-activation-respon- Phenotypes ofTAR Mutants. Detailed analysis of the role of sive region (TAR) (8-12). TAR encodes an RNA stem-loop TAR in the trans-activation mechanism is now possible be- structure which acts as a binding site for the Tat protein (13). cause of the development of efficient cell-free transcription Extensive mutagenesis studies have now defined the key systems that respond to Tat (4-7, 20). In an earlier paper (5) elements required for TAR recognition and demonstrated we used a cell-free system derived from HeLa cells to dem- that there is a direct correlation between Tat binding to TAR onstrate that the Tat-stimulated RNA polymerase can read RNA and trans-activation (reviewed in ref. 14). In addition to through a synthetic terminator sequence (T) placed down- acting as a binding site for Tat protein, the apical portion of stream of the start of transcription. The presence of T caused TAR RNA also acts as a binding site for cellular RNA-binding "50% of the transcribing polymerases to disengage prema- proteins that participate in trans-activation (15, 16). DNA turely from the template. Addition of recombinant Tat protein control elements essential for transcription initiation may also to the reaction stimulated the production of run-off product overlap TAR (3, 17, 18). (p) by >30-fold (Fig. 1). One likely mechanism for trans-activation is that Tat forms Tat-dependent trans-activation in vitro has the same se- a modified transcription complex together with RNA poly- quence requirements seen in vivo (5). In the experiments that merase, TAR RNA, and cellular factors (1, 5, 19-21). If this follow, we have inactivated TAR by either the G26-C39 -> C-G model is correct, then it should be possible to introduce Tat via Abbreviations: CAT, chloramphenicol acetyltransferase; HIV, human The publication costs of this article were defrayed in part by page charge immunodeficiency virus; LTR, long terminal repeat; TAR, trans- payment. This article must therefore be hereby marked "advertisement" in activation-responsive region. accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 2408 Downloaded by guest on September 24, 2021 Biochemistry: Churcher et al Proc. Natl. Acad. Sci. USA 92 (1995) 2409 LTR TA TAR-1 TAR-2 p LTR \ gag

-453 +1 224 666 ----.-w_ -453 + 1 102 222 664 uG G1 u Template Template [ Template C-GA (mLG) MTX MTX MTX I~~~~~~~~~~~~~~~~~ C-G 1 63 64 MTX MTX MTX MTX MTX MTX MTX MTX G-C ITAR WT mGCmL 53 60 61 62 1 55 54 56 A-U TAR-1 WT WT mGC mGC WT WT mLG mLG (mGC) TAR-22 WT mmGC WT mGC [ - mLG WT mLG A-U -622 Tat G-C -1+1-1+1-1+1-1+.:++ .... A-U 527 -404 TAR RNA .- AU -309 A U G-C G-C G-C U-A ..w.=a C-G --217 C-G C- C-G _201 A-U -190 G-C -180 G-C G-C C-GUUUUUUUUU -160 Terminator (X) I FIG. 2. Rescue of mutant TAR elements by a wild-type TAR element downstream of the promoter. (Left) Assay of templates FIG. 1. Cell-free transcription of templates carrying the HIV LTR. placed carrying the mGC mutation in either TAR-1 or TAR-2. Note that (Upper) Structure of template DNAs. Each plasmid contains a syn- MTX-61, which carries the mGC mutation in TAR-1 and a wild-type of the start of thetic terminator (T) inserted downstream transcription. TAR-2, responds strongly to Tat, whereas the control template Templates carried either the mGC (G26.C39 -> C.G) mutation, which MTX-62, in which both TAR-1 and TAR-2 are inactivated by the mGC reduces Tat >14-fold, or the mLG -- UUU at binding by (GGG mutation, is unable to respond to Tat. MTX-53 is a control template 32-34) mutation, which does not affect Tat binding. Transcription carrying wild-type TAR-1 and TAR-2 elements. MTX-60 carries a reactions were performed in the presence of 0 (-) or 200 ng (+) wild-type TAR-1 element and a TAR-2 element with the mGC recombinant Tat protein. p, Runoff transcript; T, transcripts ending at mutation. (Right) Assay of templates carrying the mLG mutation in terminator. Gel was exposed to x-ray film at -70°C for 15 hr. either TAR-1 or TAR-2. The MTX-54 template, which carries the mLG mutation in TAR-1 and a wild-type TAR-2, responds efficiently (mGC) mutation in the Tat binding site or by the GGG to Tat, whereas MTX-56, which carries the mLG mutation in both UUU (mLG) mutation at 32-34 in the apical loop sequence TAR-1 and TAR-2, does not respond significantly to Tat. MTX-1 is (Fig. 1). The mGC mutation reduces Tat binding to TAR RNA a control template carrying a single wild-type TAR element, whereas by >14-fold and inactivates the viral LTR in reporter systems, MTX-55 is a control template carrying a wild-type TAR-1 element and whereas the mLG mutation does not affect Tat binding but an inactive TAR-2 element. -, No Tat protein; +, 200 ng of Tat inactivates trans-activation in vivo (22). protein; p, runoff transcripts; T, transcripts ending at the synthetic terminator. Transcripts were fractionated in a 6% polyacrylamide gel Tat Can Be Introduced via a Displaced TAR RNA Element. and exposed to x-ray film at -70°C for 15 hr. If Tat binds to the TAR RNA which is produced by an elongating RNA polymerase, then it should also be possible to contrast, MTX-62, which carries the mGC mutation in both introduce Tat when TAR is placed considerably downstream TAR-1 and TAR-2, has negligible activity. of the promoter. We were concerned that displacing the TAR TAR RNA Elements Require Intact Tat Binding-Site and RNA element by introducing nucleotide spacers near the start Loop Sequences. Active TAR elements also require an intact of transcription (12) could simultaneously disrupt DNA ele- loop sequence (Fig. 2 Right). As expected, the MTX-55 ments critical for transcription initiation. Therefore, to exam- template, which carries a wild-type TAR-1 element and a ine whether Tat could be introduced at a distance under TAR-2 element inactivated by the mLG mutation, responds conditions where the sequences near the start of HIV tran- normally to Tat. In the MTX-54 template, TAR-1 is inacti- scription were unaltered, we prepared templates carrying vated by the mLG mutation, but a Tat response can still be duplicated TAR elements (Fig. 2). demonstrated by introducing a wild-type TAR-2 element. By contrast, the MTX-56 template, which carries the mLG mu- TAR RNA elements carrying inactivating mutations can be tation in both TAR-1 and TAR-2, is unable to respond to Tat. rescued by a wild-type element placed downstream (Fig. 2). To test whether the Tat binding-site and loop sequences The control MTX-53 template carries two wild-type TAR provide separate and complementary functions, a series of elements. As expected, readthrough transcription is stimulated templates carrying the mGC mutation in TAR-1 and various >30-fold following addition of recombinant Tat to the cell- mutations in TAR-2 was constructed (Fig. 3 Left). The control free system. Similarly, the MTX-60 template, which carries a template was MTX-61, which carries a wild-type TAR-2 wild-type TAR-1 element and the mGC mutation in TAR-2, sequence and is transcriptionally active. Introduction of the also shows a normal response to Tat. Thus, the TAR-2 element mLG mutation into the TAR-2 element (MTX-70) blocked the does not inhibit transcriptional elongation. Tat response, even though Tat could, in principle, bind to In the MTX-61 and MTX-62 templates, the TAR-1 element TAR-2 and a cellular factor could bind to TAR-1. The small has been inactivated by the mGC mutation. As shown in Fig. increase in readthrough transcription seen in this experiment 2 (Lower Left), in MTX-61, which carries a wild-type TAR-2 does not appear to be TAR-mediated, since the control element, Tat is able to efficiently stimulate transcription. By template MTX-67, which carries a TAR-2 element inactivated Downloaded by guest on September 24, 2021 2410 Biochemistry: Churcher et al Proc. NatL Acad ScL USA 92 (1995)

TAR-1 TAR-2 p LTR \1 102 222 gag

-453 +1 102 222 664 Template Template I I I ______I _____1r_|__|____|___ MTX MTX MTX MTX MTX MTX MTX MTX Template 61 62 70 67 54 56 87 88 MTX MTX MTX MTX TAR-1 mGC mGC mGC mGC mLG mLGT mLG mLG 119 61 120 121 TAR-2 WT mGC mLG mGC WT mLG mGC mGC Spacer (nt) 36 43 91 165 l I---T- Tat j4 - 1111 Tat -+ I-I+ -+-II + ... . .:::: ::.: ::::.: :: :." .. :.:: ;.: -p p _ s w ...

..-. .: .;...... w.M,.*,.,::...... FxUt :.....: ...... :..

t * . .. ; ... ,.: ...... i : - T FIG. 3. TAR activity requires...... both a functional Tat binding site and a loop element. (Left) Templates carried the mGC mutation in TAR-1 and the indicated mutations in TAR-2. There is no significant complementation between a TAR-1 element carrying the mGC mutation and a TAR-2 element carrying the mLG mutation (MTX-70). FIG. 4. TAR-2 is functional when placed at a variety of distances (Right) Templates carried the mLG mutation in TAR-1 and the from the start of transcription. Template DNAs carried the mGC indicated mutations in TAR-2. This reciprocal experiment shows no mutation in TAR-1, a duplicated wild-type TAR-2 element, and a significant complementation between a TAR-1 element carrying the variable spacer element derived from the coding sequence of the CAT mLG mutation and a TAR-2 element carrying the mGC mutation gene. The length of the spacer, from the end of TAR-1 (nt 59) to the (MTX-87). -, No Tat protein; +, 200 ng Tat protein; p, runoff start of TAR-2 (nt 1), is indicated. The lengths of transcripts ending transcripts; T, transcripts ending at the synthetic terminator. Gel was at the terminator (T) or the runoff products (p) correspond to those exposed to x-ray film at -70°C for 15 hr. expected for the different spacer lengths. Reactions were supplemented with 1 jig of poly(dIdC). -, No Tat protein; +, 200 ng of Tat by both the mGC and mLG mutations, also shows a weak protein. Gel was exposed to x-ray film at -70°C for 5 hr. response after addition of recombinant Tat protein. A reciprocal experiment is shown in Fig. 3 Right. In this series, the sence of Tat and a dramatic loss of Tat-responsiveness. The starting template was MTX-54, in which TAR-1 is inactivated +11 and +59 replacement mutation (MTX-99) is somewhat by the mLG mutation. As expected, introduction of the mGC more active but still shows impaired transcription in both the mutation into TAR-2 (MTX-87 and MTX-88) blocks the Tat presence and the absence of Tat. By contrast, a strong Tat responses. response is obtained with MTX-115, which carries TAR-1 TAR Is Functional at a Distance. The first nucleotide of sequences up to position +35. The level of transcription from TAR-2 is located 102 nt downstream of the start of transcrip- MTX-115 nearly matches that obtained from MTX-61, which tion in the templates described above. Fig. 4 demonstrates that carries a TAR-1 element that is inactivated by the mGC TAR is also functional as an RNA element when it is placed mutation and a wild-type TAR-2 element. in a variety of different positions. A series of templates was Since MTX-115 carries TAR sequence only up to +35, and constructed in which TAR-1 was inactivated by the mGC the TAR RNA stem-loop structure cannot be formed by the mutation and the spacing between the last nucleotide of replacement sequence, we conclude that an essential DNA TAR-1 and the first nucleotide of TAR-2 was either 36 nt element overlaps the 5' half of TAR. Because the deletions in (MTX-119), 43 nt (MTX-61), 91 nt (MTX-120), or 165 nt the 5' half of TAR reduce transcription in both the presence (MTX-121). In each case, TAR-2 is active and permits effi- and the absence of Tat, it seems likely that the encoded DNA cient Tat responses. element is required for efficient initiation. In experiments to An Essential DNA Element Overlaps TAR. We next tested be described elsewhere, we have shown by RNase protection whether TAR-1 contains DNA elements which are essential assays that deletions and point mutations in the 5' half of TAR for trans-activation. A series of templates was constructed in can specifically reduce transcription initiation in the presence which sequences from TAR-1 were replaced by sequences and absence of Tat. taken from the coding sequence of the CAT gene (Fig. 5). Because the templates contain substitutions rather than deletions, the active TAR-2 element remains in a constant position DISCUSSION with respect to the start of transcription. The sequences chosen TAR Is Functional at a Distance. In an influential paper, do not affect the folding of TAR-2 RNA. When these same Selby et al. (12) reported that maximal promoter activity sequences are inserted between TAR-1 and TAR-2 (Fig. 4), required TAR to be located immediately downstream of the TAR-2 remains active. start of transcription. For example, they reported that tran- Replacement of TAR-1 sequences between +4 and +59 scriptional activity was reduced by >80% when TAR was (MTX-111) results in a reduction in transcription in the ab- displaced by only 88 nt. This experiment has often been Downloaded by guest on September 24, 2021 Biochemistry: Churcher et aL Proc. NatL Acad Sci USA 92 (1995) 2411 TAR-1 TAR-2 p region of the first TAR element could be partially rescued by LTR an intact downstream TAR element (25, 26). T gag Cellular Cofactors for Tat. The TAR RNA element is functional only when both an intact Tat binding site and a -453 85 205 647 functional apical loop sequence are present on the same TAR RNA element. By contrast to previous experiments performed SubstitutionI with single TAR elements (27), the experiments reported here +1 exclude the possibility that the loop sequence acts as a DNA element. For example, if the loop sequence were acting as a Template DNA element alone, then it should be possible to rescue a MTX MTX MTX MTX MTX TAR-1 element carrying the mGC mutation in the Tat binding 53 61 111 99 115 site by a TAR-2 element carrying the mLG mutation. TAR-1 |WT |GC +3 +10 .+35 A number ofcellular proteins that interact specificallywith the TAR-2 WT WT WT WT WT TAR loop sequence have been identified (15, 16). TRP-185 (or Tat TRP-1) is a protein of 185 kDa which can be UV crosslinked to -I+I- i+ TAR RNA. Complex formation involves sequences of the TAR loop and lower molecular weight cellular cofactors. One puzzling feature of TRP-185 activity is that Tat appears to be able to compete with TRP-185 for TAR binding. Although this obser- vation does not by itself rule out a role for TRP-185 in the trans-activation mechanism, it is inconsistent with the prediction that a cellular cofactor for Tat should be able to form a stable ternary complex together with Tat and TAR and suggests that additional factors may be required. A demonstration of whether TRP-185, or any other candidate cofactor, is required for trans- activation will have to await reconstitution of the system in vitro with purified components. T Overlapping DNA and RNA Elements in TAR. In addition to these RNA elements, TAR appears to encode an essential DNA element. Since the TAR-2 element is functional when it is placed downstream ofthe start oftranscription, we were able FIG. 5. The 5' half of TAR contains an essential DNA element. to replace sequences in TAR-1 without disrupting Tat re- Template DNAs carried substitutions in TAR-1 and a wild-type sponses. These experiments demonstrate that a DNA element TAR-2 element. In MTX-111, nt +4 to +59 were replaced by sequence which is essential for a trans-activation the derived from the coding region of the CAT gene. In MTX-99, nt + 11 response overlaps to +59 were replaced, and in MTX-115 nt +36 to +59 were replaced. 5' half of TAR and extends no further than +36. These MTX-53 and MTX-61 are control templates carrying intact TAR-1 observations are consistent with previous studies that have and TAR-2 elements. In MTX-61, TAR-1 is inactivated by the mGC suggested that the 5' half of TAR encodes a sequence very mutation. Reactions were supplemented with 1 ,ug of poly(dIFdC). -, similar to the initiator (INR) sequence found in the terminal No Tat protein; +, 200 ng of Tat protein; p, runoff transcripts; T, deoxynucleotidyltransferase gene and adenovirus major late transcripts ending at the synthetic terminator. Gel was exposed to x-ray promoter (Ad-MLP) (18, 28). Mutations in the HIV INR can film at -70°C for 5 hr. dramatically reduce initiation rates in the absence ofTat (3, 18, 28, 29). Our results also demonstrate that the HIV TAR DNA interpreted to mean that TAR needs to be adjacent to the start element acts independently of the TAR RNA element and is of transcription in order to allow Tat to interact with the essential for both Tat-independent and Tat-activated tran- upstream elements of the promoter (23). scription. In agreement with Selby et al. (12), we have also observed Transcription from the HIV LTR in vivo characteristically that transcription is reduced when the TAR element is dis- produces a population of short transcripts ending near TAR. placed downstream. The amount of runoff transcripts pro- Hernandez and colleagues (3, 29) have shown that short duced from the MTX-62 template in the presence of Tat is transcripts are produced whenever the HIV TAR (-6 to +59) -50% that obtained from templates where TAR-1 is active is placed downstream of a heterologous promoter. These (MTX-53 and MTX-60). However, we do not interpret this observations have also suggested that this region of the result to mean that TAR needs to be positioned precisely. promoter includes a DNA element which acts as an inducer of Since elongation is very inefficient in the absence of Tat (1, 2, short transcripts, an IST element (3, 29). It seems likely that 21, 24), we believe this decrease in TAR activity indicates that the TAR DNA element we have mapped overlaps the IST a substantial fraction of the transcribing RNA polymerases element as well as the INR element. We suggest that the TAR never reach the displaced TAR and are therefore unable to DNA element is a key feature of the promoter which allows recruit Tat. initiation by a Tat-responsive polymerase. The IST activity is Our demonstration that the displaced TAR remains capable partly a consequence of this, since polymerases that initiate on the HIV LTR tend to disengage from the template and give of recruiting Tat is based on comparisons with mutant TAR rise to short transcripts after encountering TAR RNA or any elements placed in the downstream position. Controls of this other sequence with a high secondary structure (1, 3, 5, 29-32). type were not available at the time of the studies by Selby et al. Trans-Activation Mechanism. Currently there are two alter- (12). Using an identical experimental design, we have also native models for the mechanism of action of Tat. According to demonstrated that displaced TAR elements are functional in the anti-termination model, Tat and cellular cofactors associate vivo (data not shown). Similarly, HIV-2 carries a TAR element directly with TAR RNA and the transcribing RNA polymerase which resembles a tandem duplication of the stem-loop struc- to produce a modified transcription complex (reviewed in ref. ture of TAR RNA from HIV-1 (25, 26). Consistent with the 33). An analogous mechanism is used by the bacteriophage A N results reported here, in vivo experiments have shown that the anti-terminator protein (1, 5, 19). Alternatively, it has been addition of the second TAR hairpin increased the responsive- suggested that Tat selectively stimulates initiation by a poly- ness of the HIV-2 TAR to Tat 3-fold. Mutations in the loop merase with altered elongation properties (23, 34-36). One Downloaded by guest on September 24, 2021 2412 Biochemistry: Churcher et alP Proc. Natl Acad ScL USA 92 (1995) implication of this initiation model is that because Tat is intro- 13. Dingwall, C., Ernberg, I., Gait, M. J., Green, S. M., Heaphy, S., duced to the HIV promoter via its interactions with TAR RNA, Karn, J., Lowe, A. D., Singh, M., Skinner, M. A. & Valerio, R. itwould have to be free to "loop back" and make contact with the (1989) Proc. Natl. Acad. Sci. USA 86, 6925-6929. cellular transcription factors bound near the promoter. 14. Gait, M. J. & Karn, J. (1993) Trends Biochem. Sci. 18, 255-259. At present it is difficult to distinguish between these two 15. Sheline, C. T., Milocco, L. H. & Jones, K. A. (1991) Genes Dev. models experimentally. We favor the anti-termination model, 5, 2508-2520. since it is known that Tat can efficiently stimulate elongation 16. Wu, F., Garcia, J., Sigman, D. & Gaynor, R. (1991) Genes Dev. 5, 2128-2140. in cell-free systems under conditions where reinitiation of 17. Garcia, J. A., Wu, F. K., Mitsuyasu, R. & Gaynor, R. B. (1987) transcription is limited (4-6). The results reported in this EMBO J. 6, 3761-3770. paper argue against one of the central premises of the initia- 18. Zenzie-Gregory, B., Sheridan, P., Jones, K. A. & Smale, S. T. tion model-namely, that TAR is positionally constrained. We (1993) J. Biol. Chem. 268, 15823-15832. have shown that the TAR RNA element remains active even 19. Dingwall, C., Ernberg, I., Gait, M. J., Green, S. M., Heaphy, S., when it is displaced by >200 nt from the start site of tran- Karn, J., Lowe, A. D., Singh, M. & Skinner, M. A. (1990) EMBO scription. The simplest interpretation of the data is that Tat is J. 9, 4145-4153. introduced into the transcription complex during its transit 20. Laspia, M. F., Wendel, P. & Mathews, M. B. (1993) J. Mol. 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