Tat Trans-Activates the Human lmmunodeficiency Virus ... - Cell Press

Cell, Vol. 59, 273-282, October 20, 1989,Copyright 0 1989 by Cell Press

Tat Trans-Activates the Human lmmunodeficiency Virus through a Nascent RNA Target Ben Berkhout,’ Robert l-l. Silverman,t and Kuan-Teh Jeang’ Laboratory of Molecular Microbiology National Institute of Allergy and Infectious Diseases Bethesda, Maryland 20892 t Department of Pathology Uniformed Services University of the Health Sciences Bethesda, Maryland 20814 l

Summary Expression of the human immunodeficiency virus type 1 (HIV-l) genome is greatly dependent on the viral frans-activator protein Tat. Tat functions through the TAR element, which is represented in both viral DNA and RNA. At present, there is no definitive evidence that determines whether Tat acts through a DNA or RNA form of TAR. We have used an intramolecular mutagenesis approach to change selectively the RNA secondary structure of TAR without affecting its primary sequence. We show that a specific RNA secondary structure for TAR is needed for biological activity. Furthermore, transcripts that only transiently form a native TAR RNA hairpin, which is not maintained in the mature mRNA, are completely frans-activated by Tat, suggesting that TAR is recognized as a nascent RNA. Introduction Human immunodeficiency virus type 1 (HIV-l) is the etiological agent for acquired immunodeficiency syndrome (AIDS) (for reviews see Gallo, 1987; Fauci, 1988). The HIV1 long terminal repeat (LTR) contains promoter elements responsible for the initiation of viral transcription. After synthesis, the full-length viral RNA is either transported directly into the cytoplasm and becomes translated into structural proteins, or it is spliced into subgenomic RNAs that encode the several viral regulatory proteins (Feinberg et al., 1986; Rosen et al., 1986; Malim et al., 1988; Felber et al., 1989; Hammarskjold et al., 1989). One of these regulatory proteins is the Tat protein, which is localized within the nucleus (Hauber et al., 1987, 1988). Tat is essential for viral replication (Dayton et al., 1986; Fisher et al., 1986), and high level expression of HIV requires activation in Pans by Tat (Sodroski et al., 1985; Arya et al., 1985; Rosen et al., 1985; Cullen, 1986; Peterlin et al., 1988; Muesing et al., 1987; Wright et al., 1986). Depending on the strain of HIV-l, this protein varies in length from 86 to 101 amino acids. Only the first 58 amino acids are necessary for its frans-activation function in transfection experiments (Seigel et al., 1986). Within Tat, a cysteine-rich domain modulates the trans-activation function, while a second stretch of basic amino acids dictates nuclear transport (Garcia et al., 1988; Sadaie et al., 1988; Ruben et al., 1989; Hauber et al., 1989). Tat can form a

homodimer in the presence of zinc (Frankel et al., 1988). There is, as yet, no direct evidence that this divalent cation influences the ability of Tat to bind to nucleic acids. Since no specific binding of Tat to nucleic acid has been documented, Tat probably acts in an indirect manner either by interacting with preexisting cellular proteins (Jeang et al., 1988a) or by inducing the new expression of cellular genes. The LTR region needed for Tat-mediated trans-regulation has been termed the TAR @fans-acting responsive) element. Extensive mutagenesis has defined a minimally essential length for TAR of approximately 25 nucleotides (Hauber et al., 1987; Jakobovits et al., 1988). Since TAR is positioned downstream of the mRNA cap site, it is represented in both viral DNA and RNA. Because mutagenesis within the TAR sequence affects both nucleic acid forms simultaneously, our understanding of whether TAR functions as a DNA or RNA molecule is incomplete. In the presence of Tat, the amount of steady-state HIV mRNAs originating from the viral LTR is dramatically increased (Cullen, 1986; Muesing et al., 1987; Hauber et al., 1987; Rice and Mathews, 1988; Jakobovits et al., 1988). Although the TAR DNA sequence is in close proximity to the TATA element and is involved in the binding of cellular transcription factors (Dinter et al., 1987; Garcia et al., 1987; Haubei and Cullen, 1988; Wu et al., 1988; Jones et al., 1988; Garcia et al., 1989; Malim et al., 1989), many studies have shown that these DNA-protein interactions do not correlate with trans-activation (Jones et al., 1988; Malim et al., 1989). Because TAR functions in a position- and orientation-dependent manner (Peterlin et al., 1986; Muesing et al., 1987), this sequence motif likely functions in its RNA form. Recently, it has been proposed that Tat frans-activates the HIV-l LTR, not through increasing transcriptional initiations, but by overcoming a block to transcriptional elongation (Kao et al., 1987; Selby et al., 1989). In this model, TAR is believed to be an RNA molecule. In addition, indirect support for a functional role for TAR as RNA comes from in vitro experiments showing that TAR RNA can fold into an extended hairpin structure (Muesing et al., 1987) and from in vivo assays showing that the TAR hairpin is important for frans-activation (Hauber and Cullen, 1988; Jakobovits et al., 1988; Feng and Holland, 1988; Garcia et al., 1989; Selby et al., 1989). To determine directly whether TAR functions as a DNA or an RNA molecule, we have developed a mutagenesis approach that is selective for RNA secondary structures. Our procedure does not alter the primary nucleotide sequence of TAR either as DNA or RNA. The approach instead introduces antisense sequences in cis in order to engineer a refolding of the TAR RNA structure. Stretches of 10 nucleotides bordering the wild-type TAR hairpin were changed to generate antisense sequences directed against the native TAR secondary RNA structure. When transcribed into RNA, these new sequences are capable of rearranging the base pairing scheme to create alterna-

Cell 274

tive RNA stem-loop structures. Thus we can effectively change the secondary structure of the TAR RNA without changing its primary sequence. By placing the antisense stretch either 5’or 3’to the HIV1 TAR, we can prevent the wild-type TAR hairpin from ever forming (the 5’ situation) or allow for the transient formation, but not the stable maintenance, of the natural hairpin structure (the 3’situation). In both instances, the wild-type TAR secondary structure would not be present in the mature transcript. We tested these constructions and found that the 5’ antisense, but not the 3’ antisense, sequences abolished the ffans-activation response to Tat. These findings support the idea that the momentary presence in a nascent transcript of a wild-type TAR structure, which is not maintained in the mature RNA, is sufficient for Tat regulation. Results

The 5% sequences were designed to induce a nor&AR structure by virtue of their complementarity to the remnant A sequences (designated as A’, Figure 1B). This 5%-A’ base pairing is energetically more favorable than the residual TAR-like structure (A-B). Thus it is expected to be preferentially maintained in the mature RNA. For the 3’C mutants, a functional version of the natural TAR structure (A-B’) can initially form in the nascent RNA (Figure 1C). However, after transcription of the downstream 3’C sequences, an RNA structural rearrangement is expected, forming an energetically more stable B’-3’C hairpin (Figure 1C). Hence, the 5’C clones are expected to block the formation of the TAR hairpin from the beginning of transcription, whereas the 3% mutants are delayed in their interfering effects until transcription proceeds beyond the 3% segment. These constructs can thus permit the differentiation of TAR functioning during the initial moments of transcriptional elongation or at a later point.

An Intramolecular Antisense Approach The necessary and sufficient sequences for Tat-mediated trans-activation have been shown to map between nucleotides +19 and +42 in the HIV-1 LTR (Figure 1). Transcripts containing this TAR element can form an RNA structure with an extended stem-loop by the base pairing of flanking sequences (designated as A and B; Figure 1A). This hairpin structure is expected to form during the process of transcription and to be maintained in the mature transcript. We designed two different sets of antisense sequences in order to rearrange the natural RNA structure of TAR. Sequence substitutions, either upstream (5’competitor: 5’C the dotted box in Figure 1B) or downstream (3’C, the hatched box in Figure 1C) of TAR (+19 to +42), were introduced. These alterations, while not affecting the primary nucleotide sequence of TAR DNA or TAR RNA, contribute to the intramolecular refolding of the RNA molecules.

Antisense Sequences 5’ but Not 3’ to TAR Inhibit 7kans-Activation The 5% and 3% antisense constructs that were biologically tested are listed in Figure 2A with their respective base pairing free energies. Four control mutants were made that contained either a deletion in the region used for the antisense substitutions (5’d and 3’d) or a substitution with randomly chosen nucleotides (5’s and 3’s). All the constructs were changed in the lower part of the TAR hairpin. However, unlike the antisense clones, the four control clones do not create alternative base pairing, and are expected to retain a natural, albeit abbreviated, TAR hairpin. These sequence changes were placed into LTR-CAT (Figure 28). The resulting plasmids were transfected into cells in the presence or absence of a plasmid encoding the HIV-1 Tat protein. Production of CAT mRNA was analyzed by Sl nuclease protection (Figure 3A) and by North-

HIV-1

LTR DNA

nascent

rran*Crlptlon

RNA

Full

Length RNA

Figure 1. Schematic Outline of Intramolecular Antisense Base Pairings The wild type (A), Santisense mutants 5% (B), and the 3’antisense mutants 3% (C) are shown as DNAs. nascent RNAs, or full-length RNAs. The predicted RNA secondary structures for both nascent and full-length transcripts are shown. The stability of a particular structure is approximated by the size of the stem and the number of base pairs drawn. The wild-type TAR hairpin (A-B, Figure 1A) is formed by pairing of A sequences (open box) with B sequences (black box). The 5% sequences (dotted box in IB) form an alternative hairpin (5’C-A’; A’is the remnant of the original A sequence). The TARlike hairpin (AI-B) is unlikely to form because it is thermodynamically less stable. Transcription of TAR nucleotide sequences precedes transcription of the 3C sequences (hatched box in panel C), thus accounting for a delayed effect on the configuration of the TAR hairpin (A-E’) in the nascent RNA. Once fully transcribed, the transcript is expected to rearrange into the energetically more stable structure (B’-3%).

HIV-1 Tat Trans-Activation 275

Requires Nascent TAR RNA

TAR -__-__--____-_---_______ 20 30 40

10

wild type

cap ~C"C"C"cc""AGA~"c"~cucoc*cc"c"c"c~c"~c"~ccc~ccc*cuoc""~ccc"c~"~cc~ A B

5'd

..

5'5

..RGCCGA""C"CGRG............

AG............

5'Cl

..C"CAGA"

5'CZ

.. C"CAGR"C"G

5'C3

..C" _

C cc...,

50

60

70

80

+

......................................................

-19.x*

+

......................................................

-19.3+

+

-20.1

-

................................................................

......................................................................

c GR"C"GG ..................................................................... S'C ~ A'..-

3'd

...................... ........................

.......................

3'Cl

.................................................

.."ACCCAGAGAGC"C

3'C2

.................................................

..CRG"CAC"AG""AGCCAGAGCUCC

3'C3

.................................................

..oAGCCAGAGAGC"CCCAC"cc~~""

3'C‘l

.................................................

.."AGCCAGAG

3'C5

.................................................. A

LTR-CAT

C G C U C C .......

.......................

. CUUCGAAC"CGAGCC""AGc""c

t

HIV-1

rTAR -1

.......

C AC"CGAGLKl......

A C"CCCAGGC"CGAc_.

.u~occ~o~~~~cuccc~occ"cc~ 3'C ~

B'

W

-23.6

-

-24.1

-

-*5.3*

+

-*5.3*

+

-32.0

+

-35.8

+

-38.9

+

-50.7

+

-57.8

+

B

3's

.

AC t/a -35.3

....... ...... .... .....

CAT t80

SPG-IFN +I Figure 2. Nucleotide

Sequences

t236

of the Wild-Type and the Mutant TAR Elements

(A) The complete nucleotide sequences of the HIV-1 leader RNAs from position +l (cwG) to position +82 are shown. A dashed line above the wildtype sequence indicates the minimal TAR region (+19 to +42). Underlining indicates the nucleotides involved in the most stable base pairing; gaps indicate non-base-paired nucleotides. The two regions that form the stem of the TAR hairpin (see Figure 1) are labeled A and 8. The calculated free energies (Zuker and Stiegler, 1981) of the stem-loop structures (AG in kcallmol) with their respective responsiveness to Tat-mediated tfansactivation (t/a) are shown in the right-hand columns. Mutations 5’0r3’to the minimally essential TAR region are shown with base substitutions specified by letter, and deletions indicated by gaps. Nucleotides unchanged from the wild-type sequence are represented by dots. All the antisense mutants (SC and 3C) are designed to adopt non-TAR hairpins (5%-A’ and El-3%, respectively). The most stable configuration for the four control mutations (5’d. 5’s, 3’d, and 3’s), which do not contain antisense sequences, is predicted to be an abbreviated TAR-like structure (A’-B and A-B’, respectively, with their AG values marked by asterisks). (6) Schematic representation of the expression vectors LTR-CAT and SPG-IFN. The LTR-CAT plasmid contains the HIV-1 LTR driving a CAT gene (Gendelman et al., 1988). CAT mRNA from this plasmid is a fused transcript containing the +l to +82 of TAR. The SP6-IFN vector was made to produce a transcript that starts at position +l of the TAR region (Muesing et al., 1987). The r-interferon gene was inserted at nucleotide +238 of the HIV-l LTR.

ern blotting (Figure 36). Cell lysates were also tested for CAT enzyme activity (Figure 3D). The wild-type TAR construct showed a dramatic increase in CAT mRNA and CAT protein in response to Tat (Figure 3A, compare lanes 2 and 3, lanes 10 and 11; Figure 38, compare lanes 1 and 2, lanes 7 and 8; Figure 3D, compare lanes 1 and 2, lanes 6 and 7). We quantitated trans-activation levels of approximately 200-fold. The 5’C mutations dramatically reduced the level of Tat-induced trans-activation (Figure 3A, lanes 4, 5, and 6; Figure 36,

lanes 3,4, and 5; Figure 3D, lanes 3,4, and 5). This reduction in Pans-activation was specific for the 5% antisense sequences since the randomly substituted control mutant (5’s, Figure 3A, lane 7 and Figure 38, lane 6) and the deletion mutant (5’d, not shown) both produced wild-type amounts of CAT RNA in response to Tat. The inhibitory effects of the 5% antisense sequences also correlated with the stability of the alternative hairpin. For example, a quantitation of the results in Figure 3D showed that the 5’Cl mutant had a 21% trans-activation efficiency compared

Cell 276

D)

Figure 3. Tat-Mediated

Trens-Activation

of Wild-Type and Antisense

TAR Constructs

COS cells were transfected with equal amounts (0.5 ug) of LTR-CAT DNA (indicated on top of the panels) in the absence (-) or presence (+) of pSVtat. Total cellular RNAs were isolated and analyzed by Sl nuclease protection (A), by Northern blotting (B), and by transcriptional run-ons (C), 46 hr after transfection. In addition, cell lysates were tested for CAT enzyme activity (D). The basal level of transcription from each plasmid was found to be equivalent to that of the wild-type construct, and primer extension experiments indicated that all mutant constructs use the wild-type transcription initiation site (data not shown). (A) The Sl nuclease protection analysis used a uniformly labeled 456 nucleotide single-stranded DNA probe (lanes 1 and 9) that contained a part of the CAT mRNA. A 256 nucleotide fragment (indicated by an arrow) is the expected protected product from Sl nuclease digestion in the presence of CAT mFtNA. An end-labeled pBR322-Hpall digest was used as molecular weight marker (lanes 6 and 17). (6) The Northern blot analysis used a asP-nick-translated CAT-specific probe. The two CAT-specific RNA species indicated by arrows are the spliced and unspliced forms of the CAT mRNA (Okayama and Berg, 1963). An actin probe was used as an internal control to normalize for the amount of RNA. (C) Transcriptional run-on experiments using isolated nuclei. In vitro elongated radiolabeled CAT transcripts were hybridized to 20 ug of CAT DNA immobilized on a nitrocellulose filter. Phage A DNA was used as a negative hybridization control; actin DNA was used to normalize the CAT signals. (D) CAT enzyme activity was monitored using a standard CAT assay analyzed by TLC. The positions of nonacetylated [r4C]chloramphenicol (Cm) and acetylated chloramphenicol (AC-3-Cm) are indicated.



with wild type, while the 5%2 and 5C3 mutants were even more affected, with 7% and 2% efficiencies, respectively. These results suggest that a perturbation of the specific TAR RNA configuration affects Tat responsiveness. To test whether the inhibitory effect, as seen for the 5% mutants, is due to a decrease in transcription or to RNA stability, we performed a nuclear run-on experiment (Figure 3C). Nuclei were isolated from transfected COS cells, and preinitiated transcriptional complexes were extended in vitro in the presence of (3zPJUTP These transcripts were hybridized against nitrocellulose filters containing phage X DNA, CAT DNA, or actin DNA. The 5%3 mutant construct produced far fewer CAT transcripts than did the wild-type plasmid (Figure 3C). These results suggest that the differences in the steady-state RNAs (Figures 3A and 38) reflect increased transcription and not RNA destabilization. In contrast to the 5% mutants, none of the 3% antisense mutations affected Tat-dependent LTR activation. The induction of RNA synthesis from the 3% LTR-CAT mutants was comparable to that observed for the wild-type HIV-1 LTR-CAT (Figures 3A and 38). Increasing the stability of the alternative hairpin, and thereby increasing the probability that it will be successfully maintained at the expense of the native TAR structure, did not affect the overall level of transcription. For instance, 3%5, predicted to adopt an extremely stable hairpin of AG=-57.8 kcallmol, did not influence trans-activation as measured by RNA synthesis (Figure 3A, lane 14; Figure 38, lane 11). Evidence for Alternative RNA Structures In Mature lkanscripts of the 3% Mutants While all 3% mutants produced wild-type levels of CAT mRNA in the presence of Tat (Figures 3A and 38) a noticeable decrease was measured in some of the CAT enzyme activities (Figure 3D). For instance, we measured 25% for 3%3 (lane 10) and only 2% for 3%5 (lane 12) compared with 100% activity for wild type (Figure 3D, lane 7). These results are consistent with previous studies on the translational inhibitory effect of hairpin structures in the 5’ leader of mRNAs (Pelletier and Sonenberg, 1985; Kozak, 1988). The correlation between the reduction in translation and the stability of the mutant B-3% hairpin was a first indication that these predicted alternative structures indeed formed. To verify this directly, we cloned 3’C TAR mutations into an SP8-IFN vector (Figure 2B), and translated in vitro synthesized RNAs using a rabbit reticulocyte lysate (Figure 4). We found a substantial decrease in the 18 kd interferon-specific translation product for the 3% mutant mRNAs compared with the wild-type mRNA (Figure 4; compare lanes 3-5 with lane 2). This decrease was more pronounced for the mutants expected to carry a more stable RNA hairpin. We further noticed that translation of the mutant 3%3-derived mRNAs could be stimulated at a higher temperature (37oC instead of 3oOC, results not shown). These results agreed with our in vivo transfection experiments and suggested that decreased translational efficiency, due to mRNA secondary structure, accounted for the observed drop in CAT activity (Figure 3D).

-18 -14

1

2

3

4

5

6

Figure 4. In Vitro Translation of Wild-Type and 3% Mutant RNAs Capped transcripts were synthesized in vitro using SP6 RNA polymerase and linearized DNA templates. Equal amounts (15 ug) of wild-type or mutant RNAs (indicated on top of the panel) were added to a reticulocyte lysate in the presence of [%]methionine, incubated at 3ooC for 45 min, and analyzed by SDS-PAGE. Endogenous protein synthesis by the lysate in the absence of added RNA is shown in lane 1. The 18 kd interferon-specific protein is indicated by an arrowhead. Size standards (in kd) are presented in lane 6.

We also probed the RNA structure of one 3% mutant in detail. In vitro generated transcripts of 3%3 and wild type were compared. The secondary structure of each was analyzed using several single-strand-specific reagents (see Experimental Procedures). These single-stranded RNA cleavage sites were resolved in a denaturing gel (Figure 5A; wild type, lanes 5-8; mutant 3%3, lanes 9-12). For reference, dideoxy sequencing reactions were electrophoresed with the treated RNA samples (Figure 5A; wild type, lanes 1-4; mutant 3%3, lanes 13-18 and 17-20). We found that the digestion profile of wild-type TARcontaining RNA was indeed consistent with the A-B TAR hairpin shown in Figure 58. Most of the residues susceptible to diethylpyrocarbonate (DEP; Figure 5A, lane 5) ribonuclease Tl (lane 7) and Sl nuclease (lane 8, compare with the untreated sample in lane 8) were found in the hairpin-loop sequences around position +31. The data are summarized in Figure 58. In the analysis of the 3%3 mutant, one should note that its sequence from position +l to position +51 is identical to its wild-type counterpart. Yet a very different sensitivity pattern toward singlestrand-specific reagents was found in this region (Figure 5A, compare lanes 5-8 with 9-12), suggesting a dissimilar RNA configuration. The nucleotides corresponding to the loop of the wild-type TAR hairpin were seen to be completely protected in the mutant RNA (Figure 5A, compare lanes 9, 11, and 12 with lane lo), indicating that these sequences were now base paired. Instead, we saw novel highly reactive nucleotides around position +49 on the

Cell 276

A)

Figure 5. Secondary Structure Analysis Wild-Type and 3’C3 Mutant RNAs

I

-

B)

-60

3’C3

of

(A) Run-off transcripts generated by SP6 RNA polymerase were gel purified. The RNAs were treated under native conditions with limiting amounts of single-strand-specific reagents (DEP = diethylpyrocarbonate. Tl = ribonuclease Tl, Sl = nuclease Sl, - = untreated sample) and the cleavage sites were visualized using reverse transcriptase in the presence of a s2P-labeled oligonucleotide primer (Hu Qu et al., 1963). Reaction products were resolved in an 6% acrylamide-6 M urea gel (lanes 5-6, wild type; lanes 9-12, mutant 3%3). Cleavages are indicated with small arrowheads on the autoradiogram, and the interpretation is shown in Figure 58. Dideoxy sequencing of the corresponding DNA constructs using the same primer was aligned with the digestion data (lanes l-4, wild type; lanes 13-20,3%3). Numbering along the sequence lanes corresponds to original TAR coordinates. The position of the stem-loop regions is schematically indicated alongside the sequence lanes (nomenclature is as in Figure 1; A-B for wild type and B’-3’C3 for the 3%3 hairpin). Strong DNA band compression, which typically occurs on the side of a hairpin distal to the priming site (Ahlquist et al.. 1961) was seen for the sequence analysis of the 3%3 mutant in a region that corresponded precisely to the B’ stem (lanes 17-20). The identical sequences in the context of the wildtype DNA do not show such compression (lanes i-4). The DNA compression was relieved by substituting dlTP for dGTP in the sequencing reactions (compare lanes 13-16 [ITP] with lanes 17-20 IGTP]). (B) Summary of secondary structures derived from the data in (A). The results obtained for wild-type TAR RNA are in agreement with previous work (Muesing et al., 1967). Strong and weak cleavages are indicated with symbols as follows: DEP filled and open circles; ribonuclease Tl, large and small arrowheads: nuclease Sl, filled and open diamonds, respectively. The mutant sequences from 3%3, which show base pairing with B’sequences. are marked by hatching.

WILD TYPE

mutant RNA, which coincide with the predicted loop domain of the 8’3%3 hairpin (Figure 56). These in vitro results showed that the downstream antisense sequences do indeed force the TAR RNA into an alternative hairpin. Discussion The HIV-1 TAR element contains a unique array of inverted repeat sequences. This set of sequences is potentially capable of organizing into a cruciform structure at the

DNA level or a hairpin configuration at the RNA level. It has been assumed that TAR is functional as an RNA element, although the evidence in support of this has so far been indirect. Our observation of a differential effect on Pans-activation based on antisense mutations positioned either on the 5’side or the 3’side of TAR argues directly against TAR functioning as a DNA element. We base this conclusion on the fact that a DNA target should be equally affected by antisense sequences either 5’ or 3’ to the target element. Instead, we found that 5’ antisense se-



quences had a very different biological effect than 3’ antisense sequences. We can explain this positional effect by an RNA model. The different phenotype observed for the 5’ and 3’ antisense mutants can be understood when interpreted in a kinetic transcription model. In such a case, the effect of the 5’ interfering sequences is manifest at the onset of transcription, while the 3’ antisense sequence is only effective after transcription has progressed through the TAR sequence. In the former situation, no TAR hairpin structure can ever form (Figure 16). In the latter condition, the sequential events of transcription stipulate that the 3’ antisense stretch can interfere only after a TAR hairpin has had a chance to form, however transiently (Figure 1C). We can thus understand the fully trans-activated phenotype of the 3% mutants, because these transcripts can form, albeit transiently, a wild-type TAR hairpin (see Figure lC, hairpin A-B’). In these RNAs the 3’ antisense sequences, once transcribed, can rapidly alter the native TAR hairpin through an RNA structural rearrangement (forming the B’-3% hairpin). This non-TAR structure is indeed what we measured to be present in mature, in vitro generated transcripts. The apparent ability of these constructs to be trans-activated indicates that trans-activation must occur concurrently with transcriptional elongation, using a nascent TAR RNA as target. Our experiments argue against a posttranscriptional mechanism for tramactivation. Assuming that a native TAR configuration is the target for Tat, the rapidity of TAR RNA refolding, as it occurs for the 3% transcripts, would predict a brief time span for trans-activation. The fact that a trans-activation-competent phenotype is seen for these transcripts suggests that Tat needs to interact only transiently with TAR for functional activity. We note that the time available for Tat-TAR interaction may be much longer if pausing of transcription occurs. It has been reported that transcription of the natural HIV-1 LTR is a discontinuous process with pausing at approximately nucleotide +59 (the antitermination model; Kao et al., 1987; Selby et al., 1989). Should this occur at the same position during transcription of the 3% templates, the interfering antisense sequences will not be completely synthesized until pausing is relieved. In the antitermination model (Kao et al., 1987), this transcriptional elongation block is functionally removed by Tat. Thus our 3% mutant data, although not an independent proof, are compatible with this model. In the process of testing the 3% mutants with increased hairpin stability, we found a discrepancy between the level of CAT mRNA and the level of measured CAT enzyme. In follow-up in vivo and in vitro experiments, we explained this discrepancy by the efficiency of mRNA translation. Our experiments conformed to the correlation between leader RNA hairpin stability and translational inhibition, which is presumably caused by the reduced ability of the 40s ribosomal subunit to scan the leader RNA (Pelletier and Sonenberg, 1985; Kozak, 1988; Parkin et al., 1988). Complete abolition of translation was seen for a structure with AG=-57.8 kcallmol (see Figure 4, 3C5). Therefore, a precaution learned from our experiments is that quanti-

tation of trans-activation of TAR constructs should not be solely based on protein (i.e., CAT) analysis. We may cautiously suggest some generalities for the regulation of transcription by other lentiviruses. We note a considerable conservation in the nucleotide sequences of the TAR elements and in the amino acid composition of the Tat proteins from HIV-i, HIV-2, and SIV (Guyader et al., 1987; Chakrabarti et al., 1987). In fact, cross-tmns-activation of heterologous genomes by the different Tat proteins has been reported (Arya et al., 1987; Guyader et al., 1987; Emerman et al., 1987; Viglianti and Mullins, 1988; Arya and Gallo, 1988). It is reasonable to suppose that these viruses may share a related mechanism(s) for Wansactivation. One aspect of this may be the utilization of a nascent RNA target. If true, this aspect may be unique to the HIV and SIV group of retroviruses, since other primate retroviral &ens-activator proteins function in apparently different ways. For example, the HTLVI and HTLVII viruses encode the Tax protein, which induces transcription through cellular factors that bind to LTR DNA elements (Sodroski et al., 1984; Nyborg et al., 1988; Altman et al., 1988; Jeang et al., 198813). Our starting objective was to investigate the question of whether the TAR sequence functions as a DNA or RNA molecule. Using an antisense mutagenesis analysis, we conclude that TAR functions as an RNA. We cannot presently offer a molecularly detailed answer for the role of Tat in activating a TARcontaining target. However, we do not believe it likely that Tat by itself directly activates a nascent TAR RNA target. Instead, it is more likely that cellular factors are involved. This possibility is consistent with the finding that mammalian cells contain specific TAR RNA binding proteins (Gatignol et al., 1989) and with the recent report that trans-activation cannot efficiently occur in prokaryotic cells (Kashanchi and Wood, 1989) or in eukaryotic insect cells (Jeang et al., 1988a). How Tat interacts with TAR and its subsequent effects on transcriptional initiation and elongation await further study. That a nascent TAR RNA structure forms the basis for this interaction is our current conclusion. Experlmental

P~~ceduns

Plasmid Constructions All plasmids were constructed by standard techniques. Nucleotide numbers refer to the positions on the HIV-1 transcript with +l being the capped G residue. LTR-CAT is as previously described (Gendelman et al., 1988; pBennCAT) with the exception of a deletion of pBR322 sequences between the Accl sites (nucleotides 851 and 2246; pBR322 coordinates). LTR-CAT contains HIV-1 LTR sequences (LAV strain) up to the Hindlll site at position +77, which is fused to the CAT reading frame. TAR mutants were generated by cloning synthetic double-stranded DNA oligomers into the LTR-CAT vector between the unique sites Sac1 (+33)-Hindlll(+77) or between an introduced Xhol site (-9) and Bglll (+19). All mutations were verified by dideoxy sequencing. SPG-IFN contains the SP6 promoter fused to the HIV-l TAR region (+l to +232) and directs transcripts starting at position +l (Muesing et al., 1987). A full-length interferon? cDNA (Devos et al., 1982) was cloned as a Sau3Al fragment into the BamHl site of plasmid pSP64. A Sall-EcoRI fragment was then subcloned in plasmid pSP6 HIV+1 (Muesing et al., 1987) cut with Xhol and EcoRl to generate SPG-IFN. For in vitro transcriptions, the plasmid was linearized with EcoRI. SPG-TAR was used for the structural analysis of TAR RNA. It con-

Cell 280

tains the HIV-1 TAR region as a Pvull-Hindlll fragment (-22 to +77) placed downstream of the SP6 promoter into the Smal-Hindlll opened polylinker of pGEM-4. The resulting plasmid carries a unique Pvull site (downstream of the TAR region and a T7 promoter element) that was used for linearization. Primer extension with reverse transcriptase was performed with the T7 promoter primer. pSVtat contains a full-length cDNA of Tat (strain SF2) under the transcriptional control of the SV40 early promoter (Kao et al., 1987). Cell Cultum, Transfection, and RNA and Protein Analysis COS and CV1 cell lines were maintained in Dulbeccos minimal essential medium containing 10% fetal bovine serum. Transfections were carried out with DEAE-dextran in the presence of chloroquine and Nuserum as described (Seed and Aruffo, 1987). We used 0.5 wg of both LTR-CAT and psV-tat plasmid per transfection onto a 60 m m tissue culture dish. Total cellular RNA was isolated by the guanidine-HCI method. A singlestranded DNA probe was generated from an M13CAT construct (Jeang et al., 1988a) for use in Sl nuclease protection assays. Northern blot analysis was done using a 32P-nick-translated DNA probe specific for the CAT gene. Synthesis of radiolabeled run-on RNAs in isolated nuclei was performed according to Treisman and Maniatis (1985). CAT assays were performed as previously described (Gorman et al., 1982). Usually, 5% of a COS cell lysate was incubated for 30 min, while 75% of a CV-1 lysate was assayed in a 2 hr incubation. For quantitation, the percentage of acetylation was determined in a scintillation counter by cutting the acetylated and nonacetylated products from the chromatograph. All transfections were performed on at least three separate occasions, and quantitations were on CAT assays that were in the linear range. In Vitro Transcription and Translatlon SP6 transcripts were made from EcoRI-linearized SPG-IFN or Pvulllinearized SPG-TAR DNA constructs in the presence of cap analog according to standard procedures (Melton et al., 1984). Quantitation of the RNA samples was done by ODsm measurements after DNAase I treatment to remove DNA. RNAs used for structural analysis were purified over a 8% acrylamide-8 M urea gel after visualization by ethidium bromide staining and were eluted with high-salt buffer. For in vitro translation equal amounts of RNA (1.5 ug) were heated at 68OC for 30 s immediately before translation in the rabbit reticulocyte cell-free system (Stratagene). RNA (3 ul) was mixed with 2 ul of [35S]methionine (1200 Cilmmole) and 20 PI of lysate. and was incubated at 30°C for 45 min. Upon the addition of 25 pl of (2x concentrated) SDSsample buffer, the samples were boiled and analyzed on a 15% acrylamide-SDS gel. RNA Secondary Structure Analysis Gel-purified SP6 transcripts were dissolved in water and modified with the following single-strand-specific reagents: DER ribonuclease Tl, and nuclease Sl. For DEP modification, 20 ul of DEP was added to 1 ttg of TAR RNA in 200 ul of 5 m M sodium cacodylate (pH 7.5). 10 m M MgCls, and incubated at 3pc for 30 min. Upon the addition of 10 ug of carrier tANA, the RNA was recovered by ethanol precipitation. For RNAase Tl modification, 1 J of RNA (1 pg) was mixed with 4 ul of Tl buffer (10 m M Tns-HCI [pH 7.51, 200 m M NaCI, 2 m M MgCIs, 0.25 uglul tRNA) and 0.1 U of Tl enzyme. After a 5 min incubation at 3pC, the RNA was recovered by ethanol precipitation. For modification by nuclease Sl. 1 J of RNA (1 ~9) was mixed with 4 ul of Sl buffer (30 m M sodium acetate [pH 4.51, 280 m M NaCI, 4.5 m M zinc acetate, 0.25 ug/uI tRNA) and 0.05 U of Sl enzyme. A 5 min incubation at 3PC was followed by ethanol precipitation. Primer extension was used to identify the sites of modification. The T7 promoter primer oligonucleotide was end-labeled with [$sP]ATP and T4 polynucleotide kinase. The labeled oligonucleotide (2.5 ng) was mixed with 1 ug of modified RNA in a total volume of 5 VI of annealingbuffer (40 m M Ths-HCI [pH 7.51, 25 m M MgCls, 50 m M NaCI), heated at 65°C for 2 min, and slowly cooled down to 25OC. One unit of AMV reverse transcriptase in 10 ul of RT-buffer (50 m M Tris-HCI [pH 7.5], 75 m M KCI, 10 m M [XT, 100 uglml BSA, 05 m M dNTPs) was added, followed by an incubation at 3pc for 5 min. An equal volume of formamide sample buffer was added to the RNA sample, which was denatured at 90°C and analyzed in a 8% acrylamide-8 M urea gel.

Acknowledgments We thank Dr. D. J. Capon and Dr. M. A. Muesing for the gift of plasmid pSPGHIV+l and Dr. Dibyendu N. SenGupta for generously providing SP6-IFN. We thank Alicia Buckler-White for oligonucleotide synthesis and John Semmes and Chuck Buckler for computer advice. We are grateful to Malcolm A. Martin for support and encouragement. We thank Keith Peden, Francois Clavel, and Anne Gatignol for critical review 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 and grant C074BK from Uniformed Services University of the Health Sciences. B. 8. is a Fogarty Visiting Associate. 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 June 22, 1989; revised August 7, 1989 References Ahlquist, P.. Dasgupta, R., and Kaesberg, P (1981). Near identity of 3 RNA secondary structure of bromoviruses and cucumber mosaic virus. Cell 23, 183-189. Altman. R.. Harrick, D.. Garcia, J. A., and Gaynor, R. 8. (1988). Human T-cell leukemia virus types I and II exhibit different DNase I protection patterns. J. Virol. 62, 1339-1348. Arya, S. K., and Gallo, R. C. (1988). Human immunodeficiency virus type 2 long terminal repeat: analysis of regulatory elements. Proc. Natl. Acad. Sci. USA 85,9753-9757. Arya, S. K., Guo, C.. Josephs, S. F., and Wong-Staal, F. (1985). Transactivator gene of human T-lymphotropic virus type Ill (HTLV-III). Science 229, 69-73. Arya, S. K., Beaver, B., Jagodzinski, L.. Ensoli, B., Kanki, P J., Albert, J., Fenyo, E.-M., Biberfeld, G., Zagury, J. F., Laure, F., Essex, M., Norrby, E., Wong-Staal, F., and Gallo. R. C. (1987). New human and simian HIV-related retroviruses possess functional transactivator (tat) gene. Nature 328, 546-550. Chakrabarti. L., Guyader, M., Alizon, M., Daniel, M. D., Desrosiers. R. C., Tiollais, P., and Sonigo, P. (1987). Sequence of simian immunodeficiency virus from macaque and its relationship to other human and simian retroviruses. Nature 328, 543-547. Cullen, 8. R. (1986). Tins-activation occurs via a bimodal mechanism.

of human immunodeficiency Cell 46, 973-982.

virus

Devos. R., Cheroutre, H., Taya, Y., Degrave, W., van Heuverswijn. H., and Fiers, W. (1982). Molecular cloning of a human immune interferon cDNA and its expression in eucaryotic cells. Nucl. Acids Res. 10, 2487-2501. Dinter, H., Chiu, R.. Imagawa, M., Karin. M., and Jones, K. A. (1987). In vitro activation of the HIV-1 enhancer in extracts from cells treated with a phorbol ester tumor promoter. EMBO J. 6, 40674071. Dayton, A. I., Sodroski, J. G., Rosen, C. A., Goh. W. C., and Haseltine. W. A. (1986). The trans-activator gene of the human T cell lymphotropic virus type Ill is required for replication. Cell 44, 941-947. Emerman, M., Guyader, M., Montagnier, L., Baltimore, D., and Muesing, M. A. (1987). The specificity of the human immunodeficiency virus type 2 trans-activator is different from that of human immunodeficiency virus type 1. EMBO J. 6, 3755-3760. Fauci, A. S. (1988). The human immunodeficiency virus: infectivity and mechanisms of pathogenesis. Science 239, 617-622. Feinberg, M. B., Jarrett, R. F., Aldovini, A., Gallo, R. C., and WongStaal, F. (1986). HTLV-III expression and production involve complex regulation at the levels of splicing and translation of viral RNA. Cell 46, 807-817. Felber, 8. K., Hadzopoulon-Cladaras, M., Cladaras, C., Copeland, T., and Pavlakis, G. N. (1989). Rev protein of human immunodeficiency virus type 1 affects the stability and transport of the viral RNA. Proc. Natl. Acad. Sci. USA 66, 1495-1499.

HIV-1 Tat Tram+Activation 261

Requires

Nascent TAR RNA

Feng, S., and Holland, E. C. (1968). HIV-l tat trans-activation the loop sequence within TAR. Nature 334, 165-167.

requires

Fisher, A. G., Feinberg, S. F., Joseph% S. F., Harper, M. E., Mar&e, L. M., Reyes, G., Gonda. M. A., Aldovini, A., Debouk, C., Gallo, R. C., and Wong-Staal, F. (1966). The trans-activator gene of HTLV-III is essential for virus replication. Nature 320, 367~371. Frankel, A. D., Bredt, D. S., and Pabo, C. 0. (1966). Tat protein from human immunodeficiency virus forms a metal-linked dimer. Science 240, 70-73.

(1966b). Characterization of cellular factors that interact with the human T-cell leukemia virus type I p4&responsive 2%base-pair sequence. J. Virol. 62, 4499-4569. Jones, K. A., Luciw, F! A., and Duchange, N. (1966). Structural arrangements of transcription control domains within the B’untranslated leader regions of the HIV-1 and HIV-2 promoters. Genes Dev. 2, 1101-1114.

Gallo, R. C. (1967). The AIDS virus. Sci. Am. 256, 47-56.

Kao, S. Y., Calman. A. F., Luciw, P A., and Peterlin, B. M. (1967). Antitermination of transcription within the long terminal repeat of HIV-l by tat gene product. Nature 330, 469-493.

Garcia, J. A., Wu, F. K., Mitsuyasu, R., and Gaynor, R. B. (1967). Interactions of cellular proteins involved in the transcriptional regulation of the human immunodeficiency virus, EMBO J. 6, 3761-3770.

Kashanchi, F., and Wood, C. (1969). Human immunodeficiency viral long terminal repeat is functional and can be trans-activated in Escherichia colt Proc. Natl. Acad. Sci. USA 86, 2157-2161.

Garcia, J. A., Harrich, D., Pearson, L., Mitsuyasu, R., and Gaynor, R. B. (1966). Functional domains required for tat-induced transcrip tional activation of the HIV-1 long terminal repeat. EMBO J. 7, 31433147

Kozak, M. (1966). Influences of mRNAsecondarystructureon initiation by eucaryotic ribosomes. Proc. Natl. Acad. Sci. USA 83, 2650-2854.

Garcia, J. A., Harrich, D., Soultanaki, E.. Wu, F. Mitsuyasu, R., and Gaynor, R. B. (1969). Human immunodeficiency virus type 1 LTR TATA and TAR region sequences required for transcriptional regulation. EMBO J. 8, 765-778. Gatignol, A., Kumar, A., Rabson, A., and Jeang, K.-T. (1969). Identification of cellular protein that bind to the HIV-1 TAR RNA. Proc. Natl. Acad. Sci. USA, in press. Gendelman, H. E., Phelps, W., Feigenbaum, L., O.&rove, J. M., Adachi, A., Hawley, P M., Khoury, G., Ginsberg, H. S., and Martin, M. (1966). Trans-activation of the human immunodeficiency virus long terminal repeat sequence by DNA viruses. Proc. Natl. Acad. Sci. USA 83, 97594763. Gorman. C., Moffat, L., and Howard, B. (1962). Recombinant genomes which expresss chloramphenicol acetyltransferase in mammalian cells. Mol. Cell. Biol. 2, 1044-1051. Guyader, M., Emerman. M., Sonigo, f?, Clavel, F., Montagnier, L., and Alizon, M. (1967). Genome organization and transactivation of the human immunodeficiency virus type 2. Nature 326, 662-669. Hammarskjold, M.-L., Heimer, J., Hammarskjold, B., Sangwan, I., Albert, L., and Rekosh, D. (1989). Regulation of human immunodeficiency virus env expression by the rev gene product. J. Virol. 63, 1959-1966. Hauber, J.. and Cullen, B. R. (1966). Mutational analysis of the transactivation-responsive region of the human immunodeficiency virus type I long terminal repeat. J. Virol. 62, 673-679. Hauber, J., Perkins, A., Heimer, E. P, and Cullen, 8. R. (1967). Transactivation of human immunodeficiency virus gene expression is mediated by nuclear events. Proc. Natl. Acad. Sci. USA 84, 6364-6366. Hauber, J., Malim, M. H., Fenrich, R., and Cullen. B. R. (1968). Regulation of HIV-l gene expression: the role of the viral tat gene product. In The control of Human Retrovirus Gene Expression, B. R. Franza, B. R. Cullen and F Wang-Staal, eds. (Cold Spring Harbor, New York: Cold Spring Harbor Laboratory), pp. 11-22. Hauber, J., Malim, M. H., and Cullen, B. R. (1969). Mutational analysis of the COnSeNed basic domain of human immunodeficiency virus tat protein. J. Virol. 63, 1181-1167 Hu Ou, L., Michot, B., and Bachellerie, J.-P (1963). Improved methods for structure probing in large RNAs: a rapid heterologous sequencing approach is coupled to the direct mapping of nuclease accessible sites. Application to the 5’ terminal domain of eukaryotic 26s r RNA. Nucl. Acids Res. 77, 5903-5920. Jakobovits, A., Smith, D. H., Jakobovits, E. B., and Capon, D. J. (1966). A discrete element 3’ of human immunodeficiency virus (HIV-l) and HIV-2 mRNA initiation sites mediates transcriptional activation by an HIV trans-activator. Mol. Cell. Biol. 8, 2555-2561. Jeang, K.-T., Shank, P R., and Kumar, A. (1966a). Transcriptional activation of homologous viral long terminal repeats by the human immunodeficiency virus type 1 or the human T-cell leukemia virus type 1 tat proteins occurs in the absence of de now protein synthesis. Proc. Natl. Acad. Sci. USA 85, 8291-6295. Jeang,

K.-T.., Boros, I., Brady, J., Radonovich,

M., and Khoury, G.

Malim, M. H., Hauber, J., Fenrick, F, and Cullen, B. R. (1966). Immunodeficiency virus rev tram+activator modulates the expression of the viral regulatory genes. Nature 355, 181-163. Malim, M. H., Fenrick, R.. Ballard, D. W.. Hauber, J., Bohnlein, E., and Cullen, B. R. (1969). Functional characterization of a complex proteinDNA-binding domain located within the human immunodeficiency virus type 1 long terminal repeat leader region. J. Virol. 63, 3213-3219. Melton, D. A., Krieg, l? A., Rebagliati, M. R., Maniatis, T., Zinn, K., and Green, M. R. (1964). Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucl. Acids Res. 72, 7035-7056. Muesing, M. A., Smith, D. H., and Capon, D. J. (1967). Regulation of mRNA accumulation by a human immunodeficiency virus transactivator protein. Cell 48, 691-701. Nyborg, J. K., Dynan, W. S.. Chen, I. S. Y., and Wachsman, W. (1968). Binding of host-cell factor to DNA sequences in the long terminal repeat of T-cell leukemia virus type I: implications for viral gene expression Proc. Natl. Acad. Sci. USA 85, 1457-1461. Okayama, H., and Berg, P (1963). A cDNA cloning vector that permits expression of cDNA inserts in mammalian cells. Mol. Cell. Biol. 3, 260-289. Parkin, N. T., Cohen, E. A., DaNeau, A., Rosen, C., Haseltine, W., and Sonenberg, N. (1986). Mutational analysis of the 5’ noncoding region of human immunodeficiency virus type 1: effects of secondary structure on translation. EMBO J. 7, 2631-2837. Pelletier, J.. and Sonenberg. N. (1965). Insertion mutagenesis to increase secondary structure within the 5’ noncoding region of a eucaryotic mRNA reduces translational efficiency. Cell 40, 515-526. Peterlin, 6. M., Luciw, P A.. Barr, P J., and Walker, M. D. (1966). Elevated levels of mRNA can account for the transactivation of human immunodeficiency virus (HIV). Proc. Natl. Acad. Sci. USA 83, 97349736 Rice, A. P.. and Matthews, M. B. (1968). Transcriptional but not translational regulation of HIV-l by the tat gene product. Nature 332,551-553. Rosen, C. A., Sodroski, J. G., and Haseltine, W. A. (1965). The location of &-acting regulatory sequences in the human T cell lymphotropic virus type Ill (HTLV-IIIILAV) long terminal repeat. Cell 41, 613-623. Rosen, C. A., Sodroski, J. G., Goh, W. C., Dayton, A. I., Lippke, J., and Haseltine, W. A. (1966). Post-transcriptional regulation accounts for the trans-activation of the human T-lymphotropic virus type Ill. Nature 379, 555-559. Ruben, S., Perkins, A., Purcell, R., Joung. K., Sia, R., Burghoff, R., Haseltine, W. A., and Rosen, C. A. (1989). Structural and functional characterization of human immunodeficiency virus tat protein. J. Virol. 63, 1-6. Sadaie, M. R., Rappaport, J.. Benter, T, Josephs, S. F., Willis, R., and Wong-Staal, F. (1966). Missense mutations in an infectious human immunodeficiency viral genome: functional mapping of tat and identification of the rev splice acceptor. Proc. Natl. Acad. Sci. USA 85, 92249226. Seed, B., and Aruffo, A. (1967). Molecular cloning of the CD2 antigen, the T-cell erythrocyte receptor, by a rapid immunoselection procedure. Proc. Natl. Acad. Sci. USA 84, 3365-3369.

Cell 202

Seigel, L. J., Ratner, L.. Josephs, S. F, Derse, D., Feinberg, M. B, Reyes, G. A., O’Brien, S. J., and Wong-Staal, F. (1966). Transactivation induced by human T-lymphotropic virus type III (HTLV-III) maps to a viral sequence encoding 56 amino acids and lacks tissue specificity. Virology 148, 226231. Selby, M. J., Bain, E. S., Luciw, P A., and Peterlin, B. M. (1989). Structure, sequence, and position of the stem-loop in TAR determine transcriptional elongation by tat through the HIV-1 long terminal repeat. Genes Dev. 3, 547-556. Sodroski, J. G., Rosen, C. A., and Haseltine. W. A. (1964). Transacting transcriptional activation of the long terminal repeat of human T-lymphotropic viruses in infected cells. Science 225, 361-365. Sodroski, J. G.. Patarca, R., Rosen, C. A., Wong-Staal, F., and Haseltine, W. A. (1985). Location of the trans-activating region on the genome of human T-cell lymphotropic virus type Ill. Science 229, 74-77. Treisman, R., and Maniatis, T. (1965). Simian virus 40 enhancer increases number of RNA polymerase II molecules on linked DNA. Nature 375, 72-74. Viglianti, G. A., and Mullins, J. I. (1986). Functional comparison of transactivation by simian immunodeficiency virus from rhesus macaques and human immunodeficiency virus type 1. J. Viral. 82. 4523-4532. Wright, C. M., Felber, B. K.. Paskalis, H., and Pavlakis, G. N. (1986). Expression and characterization of the trans-activator of the HTLVIlllLAV virus. Science 234, 966-992. Wu, F K., Garcia, J. A., Harrich, 0.. and Gaynor, R. A. (1966). Purification of the human immunodeficiency virus type 1 enhancer and TAR binding proteins EBP-1 and UBP-1. EMBO J. 7, 2117-2129. Zuker, M., and Stiegler, P (1961). Optimal computer folding of large RNA sequences using thermodynamics and auxiliary information. Nucl. Acids Res. 9, 133-146.