Cell. Vol.

63, 791-602,

November

16, 1990, Copyright

0 1990 by Cell Press

HIV-1 Tat Protein Tram-Activates Transcription In Vitro

Robert A. Marciniak,t Barbara J. Calnan,* Alan D. Frankel,* and Phillip A. Sharp * Center for Cancer Research and Department of Biology Massachusetts Institute of Technology Cambridge, Massachusetts 02139 rHarvard-MIT Division of Health Sciences and Technology Cambridge, Massachusetts 02139 * Whitehead Institute for Biomedical Research Cambridge, Massachusetts 02142 analyzed, it was found to increase only the efficiency of transcription elongation (Kao et al., 1987; Selby et al., 1989). The c&-acting sequences required for Tat tfans-activation (called TAR) are located downstream of the site of transcription initiation (Rosen et al., 1985) between positions +14 and +44 (Jakobovits et al., 1988; Garcia et al., 1989; Selby et al., 1989). TAR sequences are present in the all HIV mRNAs and can assume a stable stem-loop structure (Muesing et al., 1987). The results of mutational analysis of the TAR sequences necessary for Tat transactivation support the idea that the TAR stem-loop structure is recognized in this process (Feng and Holland, 1988; Hauber and Cullen, 1988; Serkhout and Jeang, 1969; Garciaet al., 1989; Selbyet al., 1989), and the ability to form this structure is required for the Tat response in vivo (Berkhout et al., 1989). Although purified Tat protein will bind TAR RNA in vitro (Dingwall et al., 1989; Roy et al., 1990a), the specificity of this binding appears insufficient to explain the observed specificity of the mutational analysis of TAR element function in vivo; alterations restricted to the loop sequences of the TAR structure (Figure 1B) abolish Tat Pans-activation in vivo but have no effect on the binding of purified Tat protein in vitro. A potential solution to this dilemma lies in the identification of a cellular protein that exhibits specific binding to TAR RNA that is sensitive to nucleotide changes restricted to the TAR loop (Marciniak et al., 1990). Several proteins have been identified by means of a specific interaction with TAR DNA (Garcia et al., 1987; Jones et al., 1988; Wu et al., 1988), TAR RNA (Gatignol et al., 1989; Gaynor et al., 1989; Marciniak et al., 1990) or Tat protein (Nelbock et al., 1990). The role of these proteins in the Tat response has not yet been determined. To clarify the roles of Tat protein, TAR element recognition, and cellular factors in the mechanism of Tat transactivation, we have developed a soluble reaction that duplicates this process. Results Tat TransActivation In Vitro Since many studies have shown that Tat exerts its effect, at least in part, at a transcriptional level, we examined whether purified Tat protein could stimulate transcription in an in vitro system (Dignam et al., 1983). When 200 ng of purified Tat protein was added to a transcription reaction containing an HIV-l TAR template (Figure IA), we observed a 14-fold increase in the level of correctly initiated 767 nucleotide (nt) transcripts (Figure 2A, lanes 1 and 2). A derivative HIV template that contained a deletion of nucleotides +35 to +38 of the TAR region (ATAR, shown in Figure lB, and inactive for Tat frans-activation in vivo responded to Tat with a less than P-fold increase (lanes 3 and 4), within the range of recovery error of control transcriptions performed in parallel (data not shown). The stimulation of transcription by Tat was also observed with

Summary Tat protein of human immunodeficiency virus 1 is a potent trans.activator of viral gene expression. We show that purified Tat protein stimulates transcription from viral promoters greater than lo-fold in vitro. A Tat protein mutant that does not trans.activate in vivo did not stimulate transcription in vitro. Tat trans.activation required a functional TAR RNA sequence; frans-activation was competed by the addition of in vitro synthesized wild-type TAR RNA but not by mutant TAR RNAs. That Tat protein directly interacts with the TAR RNA during frans-activation in vitro was suggested by competition with Tat peptides. Preliminary evidence suggests the involvement of a cellular factor in recognition of TAR RNA during Tat trans.activation. Analysis of Tat trans-activation in vitro will provide new mechanistic insights into this process and allow a more detailed study of the relationship between Tat protein structure and function.

Human immunodeficiency virus 1 (HIV-l), the causative agent of acquired immunodeficiency syndrome (AIDS) (Barre-Sinoussi et al., 1983; Gallo et al., 1984; Popovic et al., 1984), encodes several novel regulatory genes (reviewed in Varmus, 1988; Cullen and Greene, 1989). One of these genes encodes a tfans-activator, Tat, that greatly increases expression of viral RNA and is essential for viral replication (Dayton et al., 1986; Fisher et al., 1986). Tat functions at least in part by increasing the level of transcription (Hauber et al., 1987; Kao et al., 1987; Jakobovits et al., 1988; Jeang et al., 1988; Rice and Mathews, 1988; Sadaie et al., 1988; Laspia et al., 1989). The transcriptional effects of Tat protein in vivo have been analyzed in detail. When the activity of Tat protein in HeLa cells was studied by transfection using plasmids containing the HIV long terminal repeat (LTR) (Roy et al., 1990b) or by infection using a recombinant adenovirus containing the HIV LTR (Laspia et al., 1989), it was concluded that Tat protein increases both the rate of transcription initiation and the efficiency of transcription elongation. In contrast, when the effect of Tat on transcription in COS cells was

B. $;A TARjdl,54, ($2, ,% c-c, nTw3iw

Figure 1. Templates Used for In Vitro Transcription, and Structure of TAR and TAR Mutants

(A) Diagram of templates used for in vitro transcription. pHIV-TAR-112 contains HIV (strain HXBS) LTR sequences from an Xhol site at pog.. sition -640 to the Hindlll site at position +64. c-c m A- Sequences downstream of HIV in this conc-c Ac-c c-c struct are derived from the human interA- .A leukin 2 gene. pCMV-TAR contains a hybrid .A c-c a% promoter homologous to human CMV-IE gene C-U-50B * 10 ~.A c.c - EI;:w sequences from position -640 to position -20 ;:: A-u (relative to the CMV-IE transcript start) and HIV cY-A $ .A sequences from -29 to +84 (relative to the c-c c c-c UC -c&y rl HIV transcript start), including the HIV TATA sequence. pT7-TAR contains the DNA sequences encoding TAR adjacent to the bacteriophage T7 RNA polymerase promoter. Restriction enzyme sites utilized and destroyed in cloning are shown in parentheses. (6) Structure of TAR and TAR mutants. The structure shown is that proposed by Muesing et al. (1967). The entire sequence of the T7 transcribed RNAs u&d in the competition experiments of Figure 5 is shown.

a shorter template. The level of a 506 nt TAR runoff transcript was stimulated 4-fold upon Tat addition (Figure 2A, lanes 5 and 6), while the level of the corresponding ATAR transcript did not increase upon Tat addition (lanes 7 and 6). Tat tfans-activation was specific for the TAR template when both the TAR and the ATAR templates were included in a single transcription reaction. Stimulation of the TAR transcript relative to the ATAR transcript was 7-fold (long runoff) and 4-fold (short runoff) in reactions analyzed in Figure 2A, lanes 9 and 10 and lanes 11 and 12, respectively. The absolute levels of both the TAR and ATAR transcripts decreased P-fold in the combined reaction, consistent with the P-fold reduction in the amount of each template added to these reaction mixtures. Thus, the stimulatory activity of Tat protein in vitro requires a functional TAR element. The ability of Tat to fmns-activate was also tested using a hybrid promoter derived in part from the human cytomegalovirus immediate early gene (CMV-IE) promoter and in part from the HIV promoter (Figure 28). This hybrid promoter, pCMV-TAR, contains a region of HIV from position -29 to position +64 (see Figure 1A) and includes the HIV TATA box and TAR sequences necessary for Tat transactivation (Cullen, 1966). The sequence downstream of

+64 is derived from the cloning vector pBluescript SK(-). Three TAR sequence variants of this construct were analyzed: wild-type TAR, ATAR, and TAR(31/34) (Figure 1B). The mutant TAR(31/34), in which the loop sequence (+31 to +34) U G G G was replaced by CAAA, is negative for Tat trans-activation in vivo (Garcia et al., 1969; Roy et al., 199Oa). This mutation is particularly interesting since it does not affect binding in vitro of purified Tat protein (Roy et al., 199Oa) but does diminish binding in vitro of the 68 kd TAR binding protein from HeLa cells (Marciniak et al., 1990). The ATAR variant does not bind either protein (Marciniak et al., 1990; Roy et al., 1990a). Tat increased production of the 851 nt (long) runoff transcript of the CMV-TAR template by 6-fold (Figure 28, lanes 1 and 2) while it had no effect on the same runoff of the mutant template, CMVTAR(31/34) (lanes 3 and 4). Furthermore, the CMVTAR(31/34) template did not respond to Tat when included in the same reaction with the wild type TAR template (lanes 9 and 10). The CMV-ATAR template also showed no response to Tat, either when tested in isolation (data not shown) or in combination with a wild-type TAR template (lanes 5 and 6, lanes 7 and 8). Pans-activation of the hybrid CMWHIV promoter was less than that observed with the intact HIV promoter: 6-fold (Figure 28, lanes 1 and 2) or a-fold (lanes 6 and 7) when tested as the 851 nt runoff, or 3-fold when tested as the 344 nt runoff (lanes 7 and 8, lanes 9 and 10). In vivo, this hybrid promoter also shows decreased Pans-activation by Tat (Cullen, 1986). Tat-responsive transcripts (90-100 nt) apparently derived from the added templates were also observed in these reactions (Figure 2). A prominent band of approximately 95 nt was observed in the Tat minus control reaction that contained the HIV-TAR template (Figure 2A, lane 1). This band decreased in intensity by 75% when Tat protein was added to the reaction mixture (lanes 1 and 2, lanes 9 and 10). Reaction mixtures containing the ATAR template yielded a band of similar intensity, but of slightly greater mobility (lanes 3,4,8-12). The difference in mobilities of these transcripts is consistent with the 4 nucleotide deletion in the ATAR template, and thus the RNAs probably possess identical 5 and 3 termini. The ATAR 95 nt transcript did not change in intensity upon addition of Tat protein. The 3 terminus of these 90-100 nt transcripts extended to the human IL-2 sequence present in the template, since the HIV sequences end at position +84. Similar 90-100 nt transcripts that also decreased upon Tat addition were observed with the CMWHIV constructs (labeled W O O nt transcripts in Figure 2B). These 100 nt transcripts differed from those derived from the HIV prdmoter templates in that they were uniformly lower in intensity and were of slightly slower mobility. The 3 terminus of these transcripts appears to be determined by the sequences 3 of the HIV sequences in the template. The amount of transcripts generated in the 90-100 nt range varied with the particular preparation of nuclear extract used; some extracts that showed high levels of Tat Pansactivation generated no transcripts in this size range (data not shown).

Tat Trams-Activation 793

In Vitro

A
Long template Short template Tat
TAR&T

--I

TAR ATAR TAR -I+

622 527 Short runoff-

1 - 1 TAR ATARATAR -L+l-[~+1-1+ - + -I+

-* m mB

Long runotiShort runoff-

- 95 nt transcripts

Figure 2. Tat 7iansActivation

In Vitro

(A) HIV promoter. DNA templates were added to transcription reactions as indicated. The total DNA template concentration used in these reactions was 20 Kg/ml. Two hundred nanograms of Tat protein was added as indicated. The TAR template was the plasmid pHIV-TAR-112. The ATAR template was the plasmid pHIV-ATAR-ILP, which is identical to pHIWARIL2 except for a 4 base deletion of the TAR sequence at positions +35 to +38. The Long template refers to these plasmids cut with BarnHI, which produces a 767 nt runoff RNA. The Short template refers to these plasmids cut with Xbal, which produces a 505 nt runoff RNA. Molecular size markers were obtained by Mspl digestion of pSR322. (E) HlVlCMV promoter. The TAR template was the plasmid pCMV-TAR. The ATAR template was the plasmid pCMV-ATAR, which is identical to pCMV-TAR except for a 4 base deletion of the TAR sequence at positions +35 to +38. The 31134 template was the plasmid pCMV-TAR(31/34) which is identical to the pCMV-TAR except for a 4 base substitution of the TAR sequence at positions +31 to +34. The Long template refers to these plasmids cut with Neil, which produces an 851 nt runoff RNA. The Short template refers to these plasmids cut with Haell, which produces a 344 nt runoff RNA.

Dependence of 7hnsActivation In Vitro on Tat Protein The dependence of Pans-activation on the concentration of wild-type Tat protein was determined (Figure 3A). The level of Vans-activation by Tat protein began to decrease when the Tat concentration was lower than 63 ng per reaction (300 nM monomeric concentration; Figure 3A, lane 3). Since the fraction of Tat protein added that is biologically active is difficult to determine, the significance of the minimal active concentration of Tat in this assay cannot be readily interpreted. The level of Tat rrans-activation observed remained essentially constant over an approximate lo-fold range in Tat concentration (Figure 3A, lanes 3-6). When 1000 ng of Tat was added to a reaction, the absolute amount of transcription was reduced (lane 7) although the ratio of the RNAs from the TAR and ATAR tem-

plates was still elevated over the reaction without Tat (lane 1). At Tat concentrations equal to or greater than 500 ng per reaction, a general inhibition of transcription was also observed in control reactions containing unrelated templates such as the adenovirus major late promoter (data not shown). To confirm that the observed trans-activation in vitro resulted from Tat activity, a Tat mutant containing a single amino acid substitution at position 41, lysine-alanine, was tested. This mutant protein, Tat (ala 41), was expressed and purified under the same conditions a8 wildtype Tat. The ala 41 mutation eliminates Tat activity in vivo (Rice and Carlotti, l990). In contrast to wild-type Tat, Tat (ala 41) caused no increase in TAR-dependent transcription when titrated over the same concentration range (Figure 36). As with wild-type Tat, a general inhibition of tran-

Cell 794

A. Tat-s F w ,$ 8

B.

c.

TAR-

P,,

234567

1234563

123456<

Figure 3. Tat Dependence

of in Vitro Pans-Activation

The amount (nanograms) of Tat or mutant Tat protein was added to transcription reactions as indicated. The TAR runoff was the 851 nt transcript produced by pCMV-TAR cut with Neil. The ATAR runoff was the 340 nt transcript produced by pCMV-ATAR cut with Haell. (A) Tat concentration dependence of @ens-activation. (8) Tat (ala 41) concentration dependence. (C)Tat (ala 41) competition. Fifty nanograms of wild-type Tat protein and the indicated amount of Tat (ala 41) were premixed and added to transcription reactions in vitro. (D) In vivo Pans-activation of Tat and Tat (ala 41) proteins. The indicated amounts of Tat protein were scrape-loaded into HeLa cells and assayed as described (Frankel et al., 1989).

scription was observed at Tat (ala 41) concentrations greater than 500 ng per reaction. The ability of the Tat (ala 41) protein to function as an inhibitor of Tat frans-activation in vitro was tested by titrating the concentration of this protein in the presence of a fixed (50 ng) amount of wild-type Tat (Figure 3C). A decrease in the level of specific Tat tfans-activation was observed, albeit only at a high Tat (ala 41) to wild-type Tat molar ratio of 10 (Figure 3C, lane 5). The decrease in the ratio of TAR vs. ATAR RNAs stimulated by addition of Tat in this reaction was 30%. At concentrations of Tat (ala 41) above 500 ng per reaction, it was difficult to ascertain the specificity of inhibition, as transcription was generally inhibited (see Figure 38, lanes 6 and 7). Thus, this protein appears to bind TAR RNA without activation of transcription. The frans-activation activity of the wild-type Tat and Tat (ala 41) proteins used for transcription stimulation in vitro was also tested in vivo using a scrape-loading assay (Frankel et al., 1989). Wild-type Tat protein was highly active in vivo (Figure 3D, lanes 2-4), while no tfans-activation was detected with Tat (ala 41) (lane 5). Time Course of Tat 7MwActlvationlRNA Stability The optimal signal for Tat trans-activation was observed when RNA synthesized in the last 30 min of a 1 hr reaction was specifically assayed (Figure 4A). These conditions are those used in the above studies. To separately analyze transcripts synthesized early in the reaction from those synthesized later, transcription was allowed to initiate and proceed in the absence of any radidlabeled nucleotide. At a subsequent time, UTP labeled to high specific activity was added to the reactions containing a total concentration of 40 pfvl UTI? The [a-32P]UTP constitutes only 0.3% of the total UTP in these reactions. Specific Tat &ens-activation was P-fold when all RNAs synthesized during a 1 hr incubation were analyzed (Figure 4A, lanes 1 and 2). The specific Tat Vans-activation in

the last 45 min of a 1 hr incubation was 5-fold (lanes 3 and 4), and further increased to O-fold when RNAs synthesized during the last 30 min of a 1 hr incubation were analyzed (lanes 5 and 6). Multiple possible explanations for this increase in response to Tat during the incubation exist. Among the most plausible are the following: First, Tat may function by binding RNA to influence a step at initiation, and generation of a nascent transcript provides a substrate for its action. Second, a labile factor may be present in the nuclear extracts that prevents Tat action early in the reaction. Third, there may be a rate-limiting association of Tat with cellular factors. Preliminary experiments suggest that the second possibility may be the primary cause of the effects of preincubation (data not shown). However, this does not exclude the possibility that Tat may require a nascent transcript for activity. Tat does not function in vitro by selectively stabilizing RNAs containing a functional TAR element. Figure 48 shows the result of a pulse-chase experiment in which [aJ2P]UTP was added at time zero and excess cold UTP was either not added (lanes 1 and 2) or added after 15 min of incubation (lanes 3 and 4). The TAR-containing RNA synthesized during the first 15 min of the 1 hr reaction was specifically increased only 1.4-fold by addition of Tat protein (Figure 4B, lanes 3 and 4). In contrast, the TAR RNA synthesized during the last 45 min of the 1 hr reaction was stimulated B-fold (Figure 4A, lanes 3 and 4). Although some degradation of the RNAs, especially of the ATAR runoff RNA, occurred during the 45 min chase (Figure 48, lanes 3 and 4), no Tat-dependent difference in the degradation patterns was observed. This indicates that Tat does not function by selectively stabilizing TAR-containing transcripts. The typical rate of degradation, which is made more obvious by the cold chase, is dependent on the nature of the terminus of the transcript and the particular preparation of nuclear extract used, but not on the presence of the TAR sequence (data not shown).

Tat Trans-Activation 795

In

Vitro

TARTAR-

622

&TAR-

0 4 rNTPs

15 I 32

,30 / 4 P-UTP

template
NE

Figure 4. Time Course of Tat Tlans-Activation The templates used for nuclear extract transcription were as described in Figure 3. (A) Effect of presynthesis. [aJ2P]UTP (10 pCi; 3000 Cilmmol) was added at the time (minutes) indicated and incubation continued for a total of 1 hr. Two hundred nanograms of Tat protein was added at the start of the reaction as indicated. (6) Pulse-chase analysis. Transcription reactions were performed as described in Experimental Procedures, except [a-3*P]UTP was added at the start of the incubation, and 25 nmol of cold UTP was added at the time (minutes) indicated.

*
Competitors ( 90

3456 8

Figure 5. RNA Inhibition of Tat 7ian.s.Activation Sixty nanograms of competitor RNAs and 200 ng of purified Tat protein were added to the in vitro transcription reactions as indicated. The templates used were as described in Figure 3. The competitor RNAs were T7 RNA polymerase transcripts produced from the plasmids pT7-TAR (82 nt), pT7-ATAR (78 nt), and pT7-TAR(31134) (82 nt). The competitor RNAs were synthesized at low specific activity, and are therefore visible and labeled Competitors.

Tat TransActivation Requires TAR RNA To address directly the question of whether the TAR RNA or DNA is recognized during Tat tmns-activation, wild-type and mutant TAR FiNAs, synthesized by T7 RNA polymerase, were tested for competition for Tat frans-activation in vitro (Figure 5). The T7 promoter templates produced competitor RNAs with the same S termini as those generated by the HIV promoter (Marciniak et al., 1990) (see Figure 1A) and are therefore identical (from nucleotides 1 to 82) to the RNAs transcribed from the CMV/HIV templates in these reactions (Figure 1B). The competitor RNAs were synthesized to a low specific activity by incorporation of a small amount of [u-~*P]ATP, to permit accurate determination of their concentrations and to assess their stabilities during incubation. They are visible in the autoradiogram in Figure 5, labeled Competitors. When 60 ng of wild-type TAR l-82 RNA (approximately 100 nM final concentration) was included in the transcription reaction in vitro, Tat stimulation of the TAR template was markedly inhibited (Figure 5, lanes 3 and 4). The inhibition was specific for Tat trans.activation, since no decrease in transcription of the TAR template was observed in the absence of Tat (lanes 1 and 3). Neither ATAR nor TAR(31/34) RNAs functioned efficiently as transcompetitors (lanes 5-8). This difference in the ability to compete for Tat trans.activation did not result from a difference in the stability of the competitors in the nuclear extract. Some decrease in Tat trans.activation is evident in the ATAR and TAR(31/34) competition; however the concentration of RNAs added to these reactions was in excess of that needed for quantitative competition of Tat trans.activation

by wild-type TAR RNA (data not shown). The specificity of this RNA competition directly demonstrates that TAR RNA is recognized during Tat trans.activation. It is important to recognize that although TAR(31/34) RNA binds purified Tat protein with the same affinity as wild-type TAR RNA (Roy et al., 199Oa), it did not function efficiently in trans.competition. This suggests that the added RNAs are competing by sequestering a factor other than Tat. Furthermore, the amount of Tat protein, 200 ng, added to the reactions analyzed in Figure 5 was in excess of that required for maximal trans.activation (see Figure 3A). When suboptimal concentrations of Tat protein were added to the reaction, TAR(31/34) RNA still did not function efficiently as a competitor (data not shown). Binding of Tat Protein to TAR RNA Is Necessary for 7?ans-Activation Recent results have shown that surprisingly small peptides containing the basic region of Tat can bind specifically to TAR RNA (Weeks et ai., 1990; B. J. C., S. Biancalana, D. Hudson, and A. D. F., submitted). Such peptides should compete for Tat trans.activation if recognition of TAR RNA is essential in this reaction. A 12 amino acid peptide, Tat 47-58, binds TAR with high affinity and retains the full binding specificity of intact Tat protein (6. J. C., S. Bian-

Cell 796

A a, 9 E 47-58
12 3 510

B 47-58 (ala52ala53)
12 3 510100

Figure 6. Peptide Activation

Inhibition

of

Tat

pans-

g-w
?

Peptide (ng)

Tat W 101
47 YGRKKRRQRRRP 58 TARATAR$2 123456

86

(A) Binding of arginine-rich Tat peptides to TAR RNA. Complexes were formed at the indicated peptide to TAR ratios and analyzed by gel shift. A schematic representation of Tat and the sequence of Tat 47-56 are shown. Tat 47-56 (ala 52 ala 53) contains a double mutation in which alanine has been substituted for arginine at positions 52 and 53. The binding of Tat 47-56 is specific for TAR RNA (B. J. C., S. Biancalana, D. Hudson, and A. D. E, submitted). (B) Fifty nanograms of Tat protein was mixed with the indicated amount of the wild-type Tat 47-56 peptide or the mutant Tat 47-56 (ala 52 ala 53) peptide, and then added to in vitro transcription reactions. The TAR runoff refers to the 767 nt transcript produced by the SamHI digest of pHIWAR-ILP. The ATAR runoff refers to the 502 nt transcript produced by the Xbal digest of pHIV-ATAR-ILP.

calana, D. Hudson, and A. D. F., submitted). As shown in Figure 6A, the Tat 47-58 peptide specifically bound to wildtype TAR RNA as assayed by gel shift analysis. A control peptide in which arginine 52 and arginine 53 were substituted with alanine did not bind TAR RNA (Figure 6A). Tat 47-58 and Tat 47-58 (ala 52 ala 53) peptides were analyzed for specific inhibition of Tat rrans-activation of the HIV promoter (Figure 86). As the amount Tat 47-58 peptide was increased to 500 ng per reaction (approximately 80-fold molar excess of competitor peptide over wild-type Tat protein), Tat-dependent traps-activation of the TAR template decreased by over 80% (Figure 8B, lane 4). This inhibition was specific as there was no decrease in the level of transcription of the ATAR template. In contrast, no effect on transcription or Tat trans-activation was seen with Tat 47-48 (ala 52 ala 53) (lanes 5 and 8), even when the amount of peptide was increased to 1 pg per reaction (data not shown). Increasing the concentration of Tat 47-58 peptide to 750 ng per reaction or above reproducibly abolished all transcription under these reaction conditions (data not shown). These transcription reactions contain 250 ng of poly(l) poly(C) double-stranded RNA. The narrow concentration range over which specific peptide inhibition was observed may reflect the titration of this RNA binding peptide against this large pool of nonspecific RNA. If this is the case, then the binding of Tat protein during the rrans-activation of the TAR template is significantly more specific than the binding of competitor peptide Tat 47-58. Tat protein specifically trans-activated in vitro transcription over a broad range of poly(l).poly(C) concentrations (data not shown). Transcript Length Dependence of in Vitro clansActivation Since background labeling of small nucleic acids present in the nuclear extract obscured detection of short (55-80 nt) template-derived RNAs (see Figure 2) the products of a Tat trans-activation reaction were analyzed by hybridization (Figure 78). The RNA probe was synthesized from the

T3 polymerase promoter that was present in antisense orientation in the plasmid pCMV-TAR (see Figure 1A). This probe, which is 220 nt long, protects a full-length fragment of 91 nt for RNAs initiated correctly at the CMVlHlV promoter. Analyzing the ATAR runoff transcript with this unlabeled antisense riboprobe protects a fragment 4 nt shorter than the fragment protected when the same hybridization analysis was performed on the wild-type TAR runoff transcript. Since the assay for maximal Tat trans-activation includes a 30 min pretranscription step, the RNAs analyzed by hybridization were radiolabeled by inclusion of [a-ssP]UTP for the last 30 min in the nuclear extract transcriptions. Thus, RNAs produced in a single transcription reaction were analyzed both by runoff analysis (Figure 7A) and by hybridization (Figure 78). One-eighth of a 100 f.~lTat trans-activation reaction was analyzed for runoff transcription on a 8% denaturing polyacrylamide gel (Figure 7A). The results were essentially identical to those shown in Figure 28. Tians-activation of the 851 nt TAR transcripts was 4-fold (Figure 7A, lanes 2 and 3, lanes 8 and 7) while no increase in the level of the 344 nt ATAR transcripts was observed (lanes 4-7). The remainder of each Tat trans-activation reaction was hybridized to the T3-produced antisense riboprobe and analyzed on a 10% denaturing polyacrylamide gel (Figure 78). The most striking result of the hybridization analysis of the short transcripts in these reactions is that no Tat stimulation was evident. In the internally controlled reactions, containing both TAR and ATAR templates, 4-fold specific frans-activation of the 851 nt TAR-containing transcript was seen when the RNAs were analyzed by runoff analysis (Figure 7A, lanes 6 and 7). When an aliquot of the same RNAs was subjected to hybridization analysis, protecting 87 and 91 nt RNAs, no Tat frans-activation was detected from the two templates (Figure 78, lanes 8 and 7). The level of the protected 91 and 87 nt RNAs was in molar excess (minimally 4-fold) of the detected levels of the 851 nt or 344 nt runoff transcripts. Similar results were obtained when this hybridization probe was used to assay

Tat Trans-Activation 797

In Vitro

Tat

-j_

+I--+!

TAR-

ShoR

rund-

ATAREl.
110

12

Figure 8. Effect of Addition of ~68 TAR Binding Protein

.\TAR fragment r
90

76

67

Transcription reactions were supplemented with 8 ul of buffer D containing 190 m M KCI (lanes 1 and 2). or 8 ul of HeLa ~68 TAR RNA binding protein fraction (lanes 3 and 4) in this buffer. The final KCI concentration was 97 mM, as compared with 68 m M under standard conditions. [r@P]UTP was present from the start of the incubation. Templates used and transcripts produced were as described in Fig ure 3.

34

12

34

Figure 7. Length Dependence

of Tat Tins-Activation

Transcription reactions were performed as described in Experimental Procedures, except a 100 ul final reaction volume was used. (A) Autoradiogram of transcripts produced during fmns-activation in vitro after electrophoresis in a 6% denaturing polyacrylamide gel. One-eighth of the products of a 100 ul transcription reaction was analyzed. The templates used and transcripts produced are as described in Figure 3. A 72 hr exposure is shown, (6) Autoradiogram of the hybridization analysis of transcripts produced during Warts-activation in vitro after electrophoresis in a 10% denaturing polyacrylamide gel. The remainder of each transcription reaction analyzed in Figure 7A was hybridized to a 220 nt antisense TAR riboprobe. This probe protects both a 91 nt readthrough transcript containing the wild-type TAR sequence (TAR fragment) and a 87 nt readthrough transcript containing the ATAR sequence (ATAR fragment). A 24 hr exposure is shown.

concentration of this protein was increased approximately 3-fold (estimated by quantitative UV cross-linking) over the amount present in the standard nuclear extract. Addition of the p6Bcontaining fraction had no effect on the level of transcription in the absence of Tat (compare the TAR transcript in Figure 8, lane 1, to that in lane 3). Specific Tat rrans-activation increased from P-fold in the absence of ~68 (lanes 1 and 2) to approximately 5fold when partially purified ~68 was added (lanes 3 and 4). The level of Pans-activation in the absence of p68 was only P-fold in this experiment because no pretranscription step was included (as in Figure 4A, lanes 1 and 2). These results indicate that a component in a column fraction containing p68 specifically stimulates Tat Pans-activation in vitro; the demonstration that this component is indeed the HeLa p68 TAR RNA binding protein awaits the further purification and characterization of this protein. Discussion We have described rrans-activation of transcription in vitro mediated by bacterially expressed and purified Tat protein. A previous study has suggested that a component(s) found in extracts of HIV-infected cells could stimulate HIV LTR-directed transcription in vitro (Okamoto and WongStaal, 1986) but when the &-acting sequence requirements of this stimulation were later determined, no clear TAR dependence was observed (Okamoto et al., 1990). In our analysis, pure Tat protein Pans-activated transcription in vitro of the HIV regulatory sequences greater than lofold. This Pans-activation required functional Tat protein; a single amino acid substitution mutant of Tat in which lysine 41 was converted to alanine showed no trans-activation activity in vitro. The Tat (ala 41) protein, which has no trans-activation activity in vivo, was expressed and purified in a procedure identical to that used for the active wild-type Tat protein. This trens-activation also required a

transcripts derived from the HIV promoter, although the pattern of protected fragments was complicated by the fact that the HIV promoter template used contained a TAR sequence that had been mutated at positions +lO and +ll to create a Xhol restriction site (data not shown). Effect of Addition of TAR Binding Protein We have previously identified a 68 kd protein (~68) in nuclear extracts of HeLa cells that binds TAR RNA in a sequence- and structure-specific fashion (Marciniak et al., 1990). The effect of addition of partially purified fractions of p68 TAR binding protein on Tat Pens-activation in vitro was tested (Figure 6). In reactions receiving partially purified ~68 TAR binding protein (Figure 8, lanes 3 and 4), the

Cell 798

functional TAR element; only DNA templates that contained the wild-type TAR element efficiently responded to Tat rrans-activation. Two subtle mutations of the TAR element sequence that are negative for Tat trans-activation in vivo (Hauber and Cullen, 1988; Garcia et al., 1989) were tested and found to be negative for Tat frans-activation in vitro. The TAR specificity of the Tat response in vitro was most clearly demonstrated when templates containing the wild-type and mutant TAR sequences were mixed in a single transcription reaction. Thus, analysis of Tat frans-activation in vitro fully supports the mutational analysis of Tat activity in vivo. Tat frans-activation is mediated through recognition of the TAR RNA structure. This has been directly demonstrated by competition for Tat trans-activation in vitro by addition of short RNAs containing an intact TAR element. Comparable short RNAs containing mutant TAR elements did not effectively compete for Tat trans-activation. The basis of the RNA specificity of Tat Wins-activation is indicated by failure of these two RNAs to compete. More specifically, changes in the sequence of the TAR loop (mutant 31134) inactivate Tat trans-activation in vivo (Garcia et al., 1989; Roy et al., 1990a). A similar loss of activity in vivo was observed when base pairs in the hairpin stem were disrupted (mutant ATAR) (Hauber and Cullen, 1988). These two mutant TAR RNAs are recognized differently by Tat protein. Tat protein binding is not affected by changes in the loop sequence as in TAR(31/34) but is abolished by deletions in the stem as in mutant ATAR (Dingwall et al., 1989; Roy et al., 1990a). The fact that a short RNA containing a loop mutation, TAR(31/34), failed to effectively compete for Tat trans-activation suggests that a cellular protein, perhaps in conjunction with Tat, is titrated by the addition of wild-type TAR RNA. Several cellular proteins have been reported to bind to TAR RNA (Gatignol et al., 1989; Gaynor et al., 1989; Marciniak et al., 1990), but, with one exception, this binding has not been shown to be specific for the loop sequence of TAR (Marciniak et al., 1990). Binding of a 68 kd protein (~68) defined by UV crosslinking in the presence of the polyanion heparin is specific for the loop sequence. It is possible that the added TAR RNA is competing for the binding of this protein in conjunction with Tat protein. An important aspect of the availability of a soluble system for study of a process is the opportunity to test compounds for antagonistic activities. Short peptides containing Tatrelated sequences that bind TAR RNA in a structure-and bulge loop sequence-specific fashion inhibit Tat ffansactivation in vitro. In particular, addition of a peptide consisting of Tat amino acids 47-58 suppressed Tat tfansactivation in vitro without altering the level of Tat-independent transcription. Addition of comparable concentrations of a related mutant peptide containing two arginine-alanine substitutions did not result in inhibition. This mutant peptide also did not specifically bind TAR RNA. Thus, Tat rrans-activation requires protein binding to the TAR RNA at sites overlapping that of Tat binding. Furthermore, Tat possesses at least two functional activities: an RNA binding activity and a trans-activation activity (Selby and Peterlin, 1990; Southgate et al., 1990).

The mechanism of Tat trans-activation in vivo has been intensively studied (Cullen, 1988; Kao et al., 1987; Muesing et al., 1987; Rice and Mathews, 1988; Braddock et al., 1989; Laspia et al., 1989; Miiller et al., 1990; Selby et al., 1989). A model of an RNA enhancer has been proposed that, in the barest of details, pictured the Tat-mediated process as recognizing the TAR RNA and stimulating initiation of transcription (Sharp and Marciniak, 1989). More recently, several experiments designed on the premise of an RNA enhancer have reinforced this model. For example, fusing Tat protein sequences to another protein that specifically binds an RNA element and substitution of the sequences for this RNA in the position of TAR results in a system capable of trans-activation of the modified HIV promoter (Selby and Peterlin, 1990; Southgate et al., 1990). Furthermore, fusion of Tat protein to sequences encoding a DNA binding protein and insertion of sequences for the binding site in the position of TAR results in an HIV promoter trans-activated by recognition of DNA (Berkhout et al., 1990). The preliminary characterization of Tat trans-activation in vitro suggests that the primary effect is an increase in the efficiency of elongation of transcription. In the same reaction, where Tat frans-activation of product RNAs approximately 800 nt in length was 4-fold, there was no detectable increase in the level of transcription of the first 91 nt. This suggests that the total level of initiation of transcription of TAR RNA was not stimulated by Tat addition but that the fraction of TAR RNAs elongating beyond 91 nt was stimulated. Detection in vivo of changes in the levels of promoterproximal short RNAs upon trans-activation of the HIV LTR by Tat suggested that Tat might function by suppressing TAR-dependent termination. However, this was unlikely since deletions of TAR sequences did not result in an increase in transcription (Rosen et al., 1985; Muesing et al., 1987; Hauber and Cullen, 1988; Jakobovits et al., 1988). Recent studies of the density of nascent RNA polymerase II molecules across a TAR-containing transcription unit revealed that only one-tenth of the newly initiated polymerases elongated beyond 1200 nt (Laspia et al., unpublished data). In the presence of Tat, the efficiency of elongation across the unit increased lo-fold. This increase in efficiency of elongation was apparently not due to suppression of termination at a specific site but rather a uniform increase in the efficiency of elongation across the unit. This suggests that transcription complexes formed in the presence of Tat are less prone to terminate at uniformly distributed sites than complexes formed in its absence. Another study in vivo also suggests that elongating complexes formed in the presence of Tat and TAR are different (Ratnasabapathy et al., 1990). In this case, Tat stimulated complexes elongated through a specific termination site while complexes directed by the same promoter in the absence of Tat did not. The suggestion that Tat activity increases the efficiency of elongation in vivo is consistent with the Tat activity observed in vitro. Changes in the efficiency of elongation in vivo and in vitro have been characterized (reviewed in Spencer and Groudine, 1990). Perhaps interesting in this regard is the

Tat TransActNation 799

In Vitro

of the drug 5,6-dichloro-1-6-b-ribofuranosylbenzimidazole (DRB). Addition of low levels of DRB to transcription reactions in vitro uniformly reduces the efficiency of elongation of RNA polymerase II across templates unrelated to HIV (Chodosh et al., 1989). This again suggests that elongating complexes frequently terminate and this termination event can be stimulated by inhibition of a common cellular process. Since Tat activity increases the efficiency of elongation, it is possible that it stimulates the same process inhibited by DRB. How can processes affecting both elongation and initiation by RNA polymerase II be jointly regulated? Tat Pans-activation apparently involves regulation of both of these temporally distinct processes. The unitary hypothesis that both levels of regulation are the consequence of a single event at initiation is most attractive and puzzling. Further studies of the mechanism of Tat trans-activation in vitro may prove enlightening in this regard. The addition of a fraction containing a partially purified cellular protein, ~68, increased the level of trans-activation by Tat protein. As mentioned previously, the binding of p68 to TAR depends on the integrity of both the loop sequence and the hairpin stem structure (Marciniak et al., 1990). The significance of the specificity of p68 recognition of TAR is brought into question by the results mentioned above that show that Tat protein contains a Pans-activation domain and thus promoter activation in vivo requires only the physical proximity of Tat. Thus Tat-specific binding to the TAR element should be adequate for trans-activation. Since mutations in the TAR loop sequence do not affect Tat binding in vitro (Dingwall et al., 1989; Royet al., 199Oa) but do block Tat Pans-activation in vivo (Muesing et al., 1987; Garcia et al., 1989; Roy et al., 1990a) and in vitro, cellular factors with specificity for TAR must be important in this process. A possible role for such a cellular factor would be to promote the specific binding of Tat to TAR
action Experimental Procedures

(data not shown). Yeast tRNA functioned efficiently as a carriednonspecific competitor for Tat trens-activation, but had more inhibitory effect on basal transcription than poly(l).poly(C) (data not shown). The transcription reactions contained 800 pM final concentrations of ATP, GTR and CTP and 40 pM LJTP Tat hens-activation was substantially decreased when 50 pM final concentrations of ATP, GTR and CTP and 8 nM UTP were used (data not shown). The reactions contained a final concentration of 15 m M NaCI. The source of this constituent was the poly(l)poly(C) stock solution; it was present to maintain the polymer in a double-stranded structure during storage. The addition of 15 m M NaCl did not otherwise increase obSSNSd Tat tfans-activation (data not shown). Tat protein was stored lyophilized in an anaerobic chamber after purification (Frankel et al., 1988). For use, Tat was dissolved at a concentration of 1 mglml in buffer D (20 m M HEPES-KOH [pH 7.91, 20% [v/v] glycerol, 100 m M KCI, 0.2 m M EDTA, 1 m M OTT) and stored at -70C. No decrease in trens-activation activity was detected when Tat protein was stored for 4 months. The addition of Tat increased the total protein in the transcription reaction by less than 0.2%. To quantify specific Tat tmns-activation, autoradiograms produced from appropriate exposures were analyzed by densitometry using an Ultrascan XL laser densitometer (LKB). In reactions that contained both a wild-type TAR template and a mutant TAR template, the specific Tat trans-activation was calculated as the ratio of the quantity of TAR transcript in the presence of Tat to the quantity of TAR transcript in the absence of Tat, after first normalizing these values by the quantity of the mutant TAR transcripts in those reactions: Specific tfans-activation =

[$j

[g&j

The specific Tat bans-activation calculated in this fashion is most probably an underestimate. In reactions containing a single transcript, trans-activation was calculated as the relative increase in the quantity of the transcript that occurred upon Tat addition. Plasmlds pHIV-TAR-IL2 is the plasmid pBC12IHIVIXhol and is derived from plasmid pBCWHIV/IL-2 (Cullen, 1988). pBCWHIV/Xhol is identical to pBCWHIV/IL-2 except for the introduction of a contiguous 2 base mutation of the TAR element at positions +lO and +ll, TG+GA, to introduce a synthetic Xhol cloning site. This alteration has no affect on measured in vivo Tat trans-activation (Hauber and Cullen, 1988). Downstream of HIV sequences in this construct are human IL-2 sequences. pHIV-ATAR-IL2 is the plasmid pD+35/+38 (Hauber and Cullen, 1988). The plasmids pnTAR, pT7-ATAR, and pT7-TAR(31/34) contain the T7 polymerase promoter adjacent to the TAR DNA sequences +1 to +84 (Marciniak et al., 1990). All cloning procedures followed standard protocols (Maniatis et al., 1982). pCMV-TAR was subcloned from the plasmid pBCWC-H/IL-2 (Cullen, 1988) which contains a chimeric CMV/HIV promoter homologous to CMV-IE sequences (-87l to -20, numbering relative to the CMV transcript start) and to HIV (HXB3) sequences (-29 to +84, numbering relative to the HIV transcript start). This plasmid utilizes the HIV transcript start site in vivo (Cullen, 1988). pCMV-TAR was cloned by ligating a 828 bp Hincll-Bglll fragment of pBCWC-H/IL-2 that contained the CMVIHIV promoter sequences and the TAR element to the 2147 bp Bglll-Sspl fragment of pmAR. In the resulting plasmid, the T7 polymerase promoter of pT7JAR has been replaced with the hybrid CMV/HIV promoter (see Figure IA). pCMVATAR and pCMWAR(31/34) were cloned by ligating the 1052 bp Pvul-Bglll fragment of pCMV-TAR to the 17ll bp (1713 bp) Bglll-Pvul fragment of pT7-ATAR or pTnAR(31/34). The resulting plasmids, pCMV-TAR, pCMV-ATAR, and pCMV-TAR(31/34), are identical in sequence except in the composition of the TAR element. Templates for transcription were prepared by treating 50 pg of plasmid with a 10. to 40-fold unit excess of restriction enzyme for 3 hr in the buffer supplied by the manufacturer (New England BioLabs). The concentration of NaCl in the reactions was adjusted to 208 mM, and the reactions were extracted sequentially with equal volumes of phe nol-chloroform-isoamyl alcohol (20:20:1) and chloroform. The DNA was then precipitated by the addition of 2 volumes of ethanol.

Conditlons for Tat Trens-Activation In Vitro Nuclear extracts were prepared as described (Dignam et al., 1983). Transcription reactions (25 pl) contained 20 pglml template DNA (500 ng per reaction), 14 m M HEPES-KOH (pH 7.9) 14% (v/v) glycerol, 88 m M KCI, 15 m M NaCI, 7 m M MgCls, 250 ng of poly(l).poly(C) (Pharmacia), 300 ng of poly(dl).poly(dC) (Pharmacia), 1 m M DTT, 0.1 m M EDTA, 800 pM ATP, 800 pM GTR 800 pM CTR 40 pM UTR 10 m M creatine phosphate, approximately 120-180 pg of nuclear extract proteins, and 200 ng of purified Tat protein where indicated. Reactions were incubated for 30 min at 3ooc, followed by the addition of 10 t&i of [a3*P]UTP (3000 Ci/mmol;l pl). After a further incubation of 30 min. the reactions were terminated by the addition of 100 pl of stop solution (100 m M Tris-HCI [pH 7.51, 10 m M EDTA, 0.75% SDS, 300 m M sodium acetate, 200 m M NaCI, 25 pg/ml yeast tRNA). Nucleic acids were purified from these reactions as described (Manley et al., 1983). RNA transcripts, typically one-third of the products of the transcription reactions, were resolved by electrophoresis on 0.4 m m thick 8% acrylamide-7 M urea sequencing gels, using a running buffer of Tris-borate-EDTA (Maxam and Gilbert, 1980). Gels were exposed to preflashed Kodak X-Omat XR-5 film at -7OOC using an intensifying screen. The major deviation from standard in vitro transcription conditions was the inclusion of 250 ng of synthetic double-stranded RNA, poly(l)poly(C), as acarriednonspecific competitor in the reaction. This inclusion increased Tat treps-activation from approximately P-fold under otherwise optimized conditions in the absence of carrier to greater than W-fold. The level of Tat-independent transcription was unaltered

Cell 800

Purification and Actlvlty of 81 and Tal (ala 41) Plotsins

The ala 41 mutation was introduced into the synthetic fat gene of ptacl2 (Frankel and Pabo, 1968) by cassette mutagenesis. BL21(DE3) ply& cells (Studier et al., 1990) containing the wild-type or mutant plasmids were induced for protein expression, and the Tat proteins were purified as described (Frankel et al., 1988; Frankel and Pabo, 1988). The Tat and Tat (ala 41) proteins behaved identically during purification. liinsactivation activity was determined by scrape-loading several concentrations of protein into HeLa cells that contain an integrated HIV LTR CAT reporter (HL3Tl cells; Felber and Pavlakis, 1988), in the presence of 100 uM chloroquine and assaying for CAT activity as previously described (Frankel et al., 1989).

produced in the nuclear extract transcriptions, we initially used a hybridization temperature of 55OC. This temperature had been determined earlier to be optimum for the hybridization analysis of TAR-length transcripts (Roy et al., 1990b). In other reports, a loss of detection of <82 nt fragments occurred when the hybridization temperature (at a constant 400 mM NaCl concentration) was increased above 3pC (Selby et al., 1989). To determine if the lack of detection of 55-80 nt RNAs was due to the elevated hybridization temperature, the hybridization was repeated using a temperature of 3PC. This set of experiments gave results identical to those in Figure 7 (data not shown).

p68 Addition
The preparation of HeLa p88 TAR binding protein used was the pooled, peak heparin column fraction 26-28 (Marciniak et al., 1990). Eight microliters of buffer D, or 8 ~1 of p68 fraction, was mixed with 8 ~1 of HeLa nuclear extract before addition of the remaining components. Total HeLa protein in reactions supplemented with p88 was increased approximately 16%. The reactions were processed as described above, except [a-zP]UTP was added at the start of the incubation.

Blndlng of Tat Peptldea to TAR RNA

Radiolabeled TAR RNA was transcribed in vitro from a synthetic oligonucleotide template containing a T7 promoter and sequences +I8 to +44 of the HIV LTR TAR site, using T7 RNA polymerase (Milligan and Uhlenbeck, 1989). The RNA was purified on a 10% polyacrylamide-8 M ureagel, visualized byautoradiography, eluted in buffer containing 0.5 M ammonium acetate, 10 mM magnesium acetate, 1 mM EDTA, 0.1% SDS, and extracted twice with phenol. Purified TAR RNA concentrations were calculated from the specific activity. Peptides were synthesized and purified as described (6. J. C., S. Biancalana, D. Hudson, and A. D. F., submitted). Peptide-TAR binding reactions were in 10 PI of 10 mM Tris-HCI (pH 7.5), 70 mM NaCI, 0.2 mM EDTA, 5% (v/v) glycerol. Reactions containing 2 nM RNA and various peptide concentrations were incubated on ice for 10 min and electrophoresed on 10% polyacrylamide-0.5x TBE native gels (Konarska and Sharp, 1988). Gels were run at 200 V for 3 hr at 4OC.

Acknowledgments
Our special thanks to M. Garcia-Blanc0 for much encouragement and helpful discussions. We thank Bryan Cullen for providing plasmids containing HIV and CMV sequences used in these analyses, Sara Biancalana and Derek Hudson for peptide synthesis, and David Mann for help with CAT assays. We are grateful to N. Hernandez, M. Mathews, and N. Sonenberg for communicating results prior to publication, to G. Kaufmann, M. Timmers, and J. Kjems for critical readings of the manuscript, and to M. Siafaca for her help in its preparation. R. A. M. is a predoctoral fellow of the Whitaker Health Sciences Fund and is partially supported by a Johnson and Johnson Research Fund Fellowship. This work was supported by United States Public Health Service grants from the National Institutes of Health and the National Cancer Institute, by Ajinomoto General Support for the Center for Cancer Research, Ajinomoto Co., Inc., Japan (P A. S.), and by the Lucille P. Markey Charitable Trust and National Institute of Allergy and Infectious Diseases grant Al29135 (A. D. F.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby with 18 USC Section 1734 marked advertisement in accordance solely to indicate this fact. Received August 16, 1990; revised September 19, 1990.

Competltlons
The TAR and TAR mutant RNAs were synthesized and prepared as described (Marciniak et al., 1990). For the TAR RNA competitions, the final component added to start transcription was nuclear extract or nuclear extract that had been supplemented with Tat protein. For peptide and Tat (ala 41) competitions, Tat protein was diluted to 50 ng/2 ul into buffer D that contained 100 pglml acetylated BSA (New England BioLabs). Serial dilutions of the peptide, or Tat (ala 41), were made into this same buffer, and the peptide and Tat solutions mixed before addition of nuclear extract. The remaining reaction components were added as a mixture to the Tat-peptide-nuclear extract to initiate transcription.

Hybridizations
Transcription reactions (100 ul) were performed by uniformly increasing the volume of all added components 4-fold. The precipitated transcription products were washed once with 70% ethanol, dried, and dissolved in 100 ul of a solution containing 10 mM Tris, 5 mM Mg&, and 1 mM DTT. Twenty-five units of DNAase I (Pharmacia) was then added and incubated for 15 min at 3oOC. The reactions were adjusted to a concentration of 10 mM EDTA and 200 mM NaCI, and extracted sequentially with phenol-chloroform-isoamyl alcohol and chloroform. Two volumes of ethanol were added, and a one-eighth volume of the precipitation mix was removed and processed for runoff analysis as described above. The antisense TAR riboprobe was transcribed from the Aatll digest of pCMV-TAR using T3 polymerase, following the protocol supplied by the manufacturer (Stratagene). The antisense TAR RNA was purified as described (Marciniak et al., 1990). The precipitated pellet after DNAase I digestion was washed with 70% ethanol and dissolved in 30 ~1 of a solution of 45 mM PIPES (pH 8.7), 400 mM NaCI, 1 mM EDTA, 80% formamide that contained 500 ng of antisense riboprobe. After hybridizing overnight at 55OC, 300 ~1 of an ice-cold solution containing 10 mM Tris-HCI (pH 7.5), 5 mM EDTA, 300 mM NaCI, 40 uglml RNAase A (Boehringer), and 10 U/ml RNAase Tl (Pharmacia) was added and incubated 80 min at 30C. Twenty microliters of 10% SDS and 50 ug of proteinase K (Boehringer) were added, and incubation continued for 15 min at rPC. After sequential extractions with phenol-chloroform-isoamyl alcohol and chloroform, 20 wg of yeast tRNA was added and the nucleic acids were precipitated by the addition of 1 volume of ethanol. The protected products were analyzed on a 10% denaturing polyacrylamide gel. To minimize background due to the large amount of labeled tRNA

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The work referred to in the Discussion as Laspia et al. (unpublished data) is now in press: Laspia, M. F., Rice, A. P., and Mathews, M. B. (1990). Synergy between HIV-l Tat and adenovirus ElA is principally due to stabilization of transcriptional elongation. Genes Dev., in press.