IOMMENT

New insights into the mechanism of HIV-1 trans-activation JONATHAN KARN AND MARIA A. GRAEBLE MRC LABORATORYOF MOLECULARBIOLOGY, HILLSROAD, CAMBRIDGE,UK CB2 2QH. In 1985 Haseltine, Sodroski and their colleagues discovered that replication of human immunodeficiency virus type 1 (HIV-1) was strictly dependent upon activation of transcription from the viral long terminal repeat (LTR) by the transactivator protein, Tat. Similar transacting factors were found subsequently in all other lentiviruses, including HIV-2 and simian immunodeficiency virus (SIV). These findings stimulated intensive research into the mechanism of action of Tat and a search for antiviral compounds 1. Now, nearly seven years later, a coherent picture of how Tat works is emerging. Transcription from the HIV LTR is initiated by the binding of cellular DNA-transcription factors and RNA polymerase to the proviral LTR. In the absence of Tat, most of the viral transcripts produced are short RNA molecules of 60-80 nucleotides, and most of the RNA polymerases engaged in transcription appear to stall near the promoter 2-4. In the presence of Tat, there is a large increase in the efficiency of elongation, and the density of RNA polymerases downstream of the promoter is markedly increased2,3.

Recognition of TARRNAby Tat Tat interacts with the trans-activation-responsive region (TAR) 5,6, a regulatory element located downstream of the initiation site for transcription, between residues +1 and +79. Unlike most DNA regulatory elements, TAR is only functional when it is placed 3' to the HIV promoter and in the correct orientation and position e. These observations suggested that TAR does not act as an ordinary DNA element, but instead encodes a functionally important RNA sequence. Highly purified recombinant Tat protein expressed in Escherichia coli is able to bind TAR RNA specifically in vitro TM and forms a one-to-one complex 7. As shown in Fig. 1, the TAR RNA sequence folds

into a highly nuclease-resistant stem-loop structure. One structural element of TAR RNA, which is important for Tat recognition, is a U-rich 'bulge' sequence located six residues below the apex of the TAR RNA stem-loop 7,8. Chemical probing experiments indicate that these bulged uridine residues bend the TAR RNA stem and introduce local distortions that widen the major groove 9. Several bases in TAR RNA that are directly recognized by the Tat protein have now been mapped. These include U23, the first base in the U-rich bulge, and 026 and A27, two bases found immediately above the bulge 7,9. Recently, we have extended these studies using synthetic chemistry (F. Hamy et al., unpublished). Removal of single hydrogen bond acceptors by substitution of G26 by 7-deaza-dG or A27by 7-deaza-dA completely abolishes specific Tat binding. Similarly, preventing the N3 position of U23 from serving as a hydrogen donor blocks Tat binding. The simplest interpretation of these results is that hydrogen bonds are formed between the Tat protein and functional groups exposed in the distorted major groove of TAR RNA9. Two phosphates located between positions 22, 23 and 24 also appear to be involved in Tat binding 1°. In addition to the hydrogen bonds formed at these specific sites, numerous nonspecific contacts between Tat protein and TAR RNA contribute to the overall binding energy.

Trans-activation mechanism Mutagenesis experiments confirm that at least one function of TAR RNA is to act as a 'loading site' that brings Tat close to the transcription machinery. When mutations that inhibit the binding of TAR RNA to Tat protein are introduced into the viral LTR, Tatstimulated transcription is dramatically reduced 7,8. Furthermore, fusion proteins composed of Tat and the TIG NOVEMBER1992 VOI,. 8 NO. 11

@1992ElsevierSciencePublishersLtd (UK)

RNA-binding domain derived from the R17 bacteriophage coat protein can act as efficient trans-activators of HIV transcription when the entire TAR element is replaced by the appropriate exogenous RNA-binding site11. How does Tat regulate gene expression after binding to TAR RNA? Peterlin and his colleagues have made the important suggestion that Tat acts through an antitermination mechanism and has properties similar to the N protein of bacteriophage )v (Refs 2, 6, 11). During replication of bacteriophage ~,, transcription through the ~, n u t site produces an RNA stem-loop structure that is bound directly by £N protein and permits the assembly of a complex composed of the ~,N protein and the host proteins NusA and NusB on the surface of the transcribing RNA polymerase12,13. This specific RNA-protein interaction contributes directly to the stability of the modified transcription complex. In the presence of ribonuclease, ~,N will not associate with RNA polymerase 12,13. The ~,Nmodified RNA polymerase is then able to overcome the termination activity of the p factor at distal sites 12,13. In the Tat-TAR system, TAR clearly functions analogously to the ~, n u t site, but does Tat produce an antitermination effect, and where is the terminator?

Trans-activation in vitro A rigorous test of the antitermination model would be the direct detection of a modified transcription complex in vitro. This may soon be possible because cell-free transcription systems that respond to Tat have recently been described 14-17. The development of these systems has been difficult because the HIV LTR behaves in vitro as a highly active promoter even in the absence of Tat. Fortunately, this high basal level of transcription is short lived. As the transcription reaction proceeds, the basal level declines and

~OMMENT the ability of Tat to stimulate transcription becomes more and more apparent14,15. Marciniak and Sharp 15 have suggested that HeLa cell extracts contain two types of RNA polymerases: a class of highly processive polymerases that do not respond to Tat, and a second class of polymerases that are less processive and therefore able to respond to Tat. Early in the reaction, the processive RNA polymerases account for most of the RNA synthesis and addition of Tat results in only a small increase in transcription. By contrast, late in the transcription reaction, most of the RNA polymerases initiating transcription on the viral LTR are less processive and produce a higher proportion of short transcripts. Only these poorly processive polymerases can be stimulated by Tat. An alternative explanation has been suggested by Laspia et al. (M. Laspia, pers. commun.), w h o have argued that the basal level of transcription is reduced because an inhibitor of elongation accumulates during the transcription reaction. Tat is then able to promote elongation by counteracting the effects of the inhibitor. This antirepressor model fits nicely with the observations by Laspia et al. that preincubation with the anionic detergent sarkosyl can elevate basal transcriptional elongation. Is the preincubation of the transcription extracts merely an operational requirement of the cell-free system, or a reflection of an underlying cellular process? Transcription p e r se does not seem to be required to 'activate' the system since other protocols involving preincubation of the extracts in the presence of ATP 15 or sodium citrate 16, or the use of dilute extracts 17, all reduce basal transcription to levels where trans-activation by Tat is detectable. Is the basal level of transcription high in the absence of such preincubation, and are the RNA polymerase populations heterogeneous, because the enzyme was exposed to high salt during the preparation of the extracts? When bacterial RNA polymerase is extracted under similar conditions, the elongation factor NusA is removed from the actively transcribing polymerases, and only a small fraction of the enzyme is associated

uoG ,C. °G °

.c

•

a

ii.a,a~,.G~c.G "A

U*vG, ~U"

2a'U, .C 21G-C ° 0 A-U FIGil

The structure of the Tat-binding site on TAR RNA. The top of the TAR RNA stem-loop structure is drawn to indicate bending of the stem induced by the bulged nucleotides U?3 to U25 and the structure of the apical loop. Bases in the stem are numbered from the start of the TAR RNA sequence, at the initiation site for transcription. Alterations to the bases and phosphates shown in red reduce Tat binding more than tenfold. Alterations to the bases shown in blue reduce Tat affinity three- to tenfold, but these positions are unlikely to be sites of direct contact to the protein and probably only affect the overall structure of the Tat-binding site.

with the a initiation factor 18,19. The comparable eukaryotic initiation and elongation factors have not yet been identified, but it is clear that the cell-free systems contain ratelimiting amounts of transcription factors. Kato et al. 16 have recently shown that the addition of purified elongation factor TFIIF increases the basal level of transcription from the viral LTR and overcomes the requirement for Tat. By contrast, addition of TFIIS, a second purified elongation factor, can boost transcription from the viral LTR in a cooperative manner with Tat. Obviously a great deal more experimental work is needed, but it seems likely that important clues to understanding h o w Tat functions can be obtained by comparing the structure of the Tat-responsive RNA polymerase to an RNA polymerase that is not responsive to Tat.

Is Tat a generalized elongation factor or a specific antitermination factor? Marciniak and Sharp 15 have also reported that the distribution of RNA polymerases that are not associated "FIG NOVEMBER 1 9 9 2 VOL. 8 NO, 11

;6(

with Tat downstream of the viral LTR follows the pattern expected if there were an equal probability of termination at each nucleotide. This suggests that Tat can act as a generalized elongation factor. On the other hand, recent biochemical evidence suggests that TAR itself can act as a terminator both in vitro 2° and in vivo 4. Furthermore, in the absence of Tat, RNA polymerase II initiating at a viral LTR stalls after encountering an exogenous terminator sequence derived from the U2 snRNA. After stimulation by Tat the ability of RNA polymerase to read through the terminator element is enhanced 4. Our o w n experiments suggest that the Tat-stimulated RNA polymerase has a genuine antitermination activity. We have found that under conditions of Tatqndependent transcription, a significant fraction of the transcribing RNA polymerases pause and terminate transcription within three to five nucleotides downstream of TAR. There is also termination five to ten nucleotides downstream of a second stable stem-loop structure located immediately downstream of TAR. Nucleases present in the extract subsequently remove the single-stranded 3' extensions to produce a series of discrete transcripts. When plasmids carrying artificial terminator sequences (i.e. a stable RNA stem-loop structure followed by a tract of nine uridine residues) were used as templates, recombinant Tat stimulated the synthesis of fulllength transcripts but did not increase the number of transcripts ending at the terminator sequences. A similar change in the ratio of fulllength to prematurely terminated transcripts is seen when extracts prepared from HeLa-Tat cells are used. As outlined in Fig. 2, transcription from the viral LTR can n o w be thought of as a reaction involving at least six distinct phases. Initiation by RNA polymerase is dependent u p o n the binding of cellular transcription factors to the proviral 5' LTR. Soon after initiation of transcription, probably after the polymerization of the initial 10-12 nucleotides, the RNA polymerase changes its conformation and releases its initiation factors. In the model drawn in Fig. 2, the initiation factors are not immediately replaced by elongation factors, and the RNA

L~ONIMENT

Step 1

~

Step 2

:,~

Step 3 Initiationfactor(s)

Initiationfactor(s)

/

-1 TF]]D ~ ° ~ (/LBP-1

TAR

~------Cellular factors

TAR RNA Step 4

Tat ~

Step 5

~

Step 6

FIG[] Model for transcriptional control by HIV-1 Tat. Step 1: Cellular transcription factors bind to the proviral LTR. Step 2: RNA polymerase II together with its initiation factors is engaged by the promoter. Step 3: As the RNA polymerase initiates transcription, it undergoes a conformational change and the initiation factors dissociate. Step 4: After transcription through TAR RNA, the RNA polymerase pauses. Step 5: Tat, cellular TAR-binding factors and possibly other cofactors form a complex together with the RNA polymerase and the TAR RNA present on the nascent chain. In the absence of Tat, these factors are not recruited and the unstable RNA polymerase usually disengages from the template within several hundred nucleotides of the end of TAR. Step 6: The modified transcription complex elongates efficiently. Once the polymerase has moved away from the TAR region, the promoter becomes accessible and a new RNA polymerase is able to initiate transcription.

polymerase becomes unstable. After transcription of TAR RNA, the polymerase pauses while Tat and cellular elongation factors assemble on the surface of the enzyme to create a modified transcription complex that is then able to transcribe the remainder of the HIV genome efficiently. In the absence of Tat, the RNA polymerase never acquires a suitable complement of elongation factors and usually disengages from the template within a short distance of TAR.

Some remaining p.zzles Unfortunately the model outlined in Fig. 2 is still very incomplete. First, TAR RNA cannot be simply acting as a Tat-regulated terminator, Mutations in TAR that abolish trans-activation do not result in constitutively high levels of LTR expression5, 6. Similarly, TAR deletion mutants appear to have

abnormally low transcriptional activities in vitro. Is the TAR element required to stabilize the initiation complex? Are cellular DNA-binding proteins that bind to sites in the TAR region required for efficient

initiation21,227 Sequences in the TAR RNA loop are not required for Tat bindingT, 8, although an intact loop is required for maximal trans-activation 7,23,24. These observations indicate that trans-activation requires cellular cofactor(s) that bind to sequences in the TAR RNA loop, and a number of cellular proteins capable of binding to the loop have n o w been identified 17,25,26. Surprisingly, the loop sequence is dispensable when fusion proteins such as Tat-R17 coat protein are used to introduce Tat to the transcription machinery n. Do the cellular factors only act to stabilize Tat binding to TAR RNA, or do they play another TIG NOVEMBER1992 VOL. 8 NO. 11

role in trans-activation? The bestcharacterized TAR RNA loop binding protein, the TRP-185 protein 26 or TRP-1 protein 17, does not seem to be able to form a stable ternary complex with Tat and TAR RNA. Is another factor required? Can Tat stimulate initiation as well as elongation? When Tat is fused to a DNA-binding domain it can activate promoters carrying the appropriate DNA-binding site 27,28. Mathews and his colleagues have argued that Tat stimulates initiation several fold when the basal transcription rate is low, but has little or no effect on initiation when the basal transcription rate is high 3,29. It seems likely that Tat forms a stable complex with TAR RNA that remains with the elongating RNA polymerase. In order to stimulate initiation, Tat would have to dissociate from this complex and then reassociate with another RNA

~]OMMENT polymerase initiating a subsequent round of transcription. Are the experimental measurements of initiation and elongation linked? Does the pausing of RNA polymerase near TAR block the access of the next enzyme to the promoter? There is still m u c h more work to be done on this challenging - and sometimes contentious - problem!

Acknowledgements Special thanks are due to our colleagues in the HIV group at LMB: Mike Gait, Jo Butler, Mark Churcher, Christina Lamont, Derek Mann, Tony Lowe, Sheila Green, Mohinder Singh, Frangois Hamy, Tominori Kimura, Shigenori Iwai, Clare Pritchard and Graeme Hacking.

References 1 Sodroski, J.G. et al. (1985) Science 227, 171-173 2 Kao, S-Y., Caiman, A.F., Luciw, P.A. and Peterlin, B.M. (1987) Nature 330, 489-493 3 Laspia, M.F., Rice, A.P. and Mathews, M.B. (1990) Genes Dev. 4, 2397-2408

4 Ramasabapathy, R., Sheldon, M., Johal, L. and Hemandez, N. (1990) Genes Dev. 4, 2061-2074 5 Muesing, M.A., Smith, D.H. and Capon, D.J. (1987) Cell 48, 691-701 6 Selby, M.J., Bain, E.S., Luciw, P. and Peterlin, B.M. (1989) Genes Dev. 3, 547-558 7 Dingwall, C. et al. (1990) EMBOJ. 9, 4145--4153 8 Roy, S. etal. (1990) GenesDev. 4, 1365-1373 9 Weeks, K.M. and Crothers, D.M. (1991) Cell 66, 577-588 10 Tao, J. and Frankel, A.D. (1992) Proc. Natl Acad. Sci. USA 89, 2723-2726 11 Selby, M.J. and Peterlin, B.M. (1990) Cell 62, 769-776 12 Barik, S. et al. (1987) Cell 50, 885-899 13 Nodwell, J.R. and Greenblatt, J. (1991) GenesDev. 5, 2141-2151 14 Marciniak, R.A., Calnan, B.J., Frankel, A.D. and Sharp, P.A. (1990) Cell 63, 791-802 15 Marciniak, R.A. and Sharp, P.A. (1991) EMBOJ. 10, 4189-4196 16 Kato, H. et al. (1992) Genes Dev. 6, 655466 17 Sheline, C.T., Milocco, L.H. and

18

19 20 21 22 23 24 25

26

27

28 29

Jones, K.A. (1991) Genes Dev. 5, 2508-2520 Gill, S.C., Weitzel, S.E. and von Hippel, P.H. (1991) J. Mol. Biol. 220, 307-324 Travers, A.A. and Burgess, R.R. (1969) Nature 222, 537-540 Bengal, E. and Aloni, Y. (1991) J. virol. 65, 4910-4918 Garcia, J.A. et al. (1988) EMBOJ. 7, 3143--3147 Kato, H., Horikoshi, M. and Roeder, R.G. (1991) Science 251, 1476--1479 Feng, S. and Holland, E.C. (1988) Nature 334, 165-168 Roy, S. et al. (1990) J. Virol. 64, 1402-1406 Marciniak, R.A., Garcia-Blanco, M.A. and Sharp, P.A. (1990) Proc. Natl Acad. Sci. USA 87, 3624-3628 Wu, F., Garcia, J., Sigman, D. and Gaynor, R. (1991) Genes Dev. 5, 2128--2140 Southgate, C.D. and Green, M.R. (1991) Genes Dev. 5, 2496-2507 Berkhout, B., Gatignol, A., Rabson, A.B. and Jeang, K-T. (1990) Cell 62, 757-767 Kessler, M. and Mathews, M.B. (1991) Proc. Natl Acad. Sci. USA 88, 10018-10022

Plant genetics flourishes CATHIE MARTIN AND JONATHAN JONES* THEJOHNINNESINSTITUTEAND*THESAINSBURYLABORATORY, JOHNINNESCENTREFORPLANTSCIENCERESEARCH,COLNEYLANE, NORVaCH,UK NR4 7UH. Despite a relatively crowded programme, the FASEB Summer Research Conference on Plant Molecular Genetics (Copper Mountain, Colorado, 9-14 August) allowed good opportunities for discussion, between horse riding, white water rafting and just gasping for breath at the high altitude. The absence of concurrent sessions and the inclusion of communal eating made an intimate and convivial atmosphere. The emphasis was strictly genetical, since much of the most innovative and informative recent progress in plant biology has been in this area. Topics covered included development, plant hormone action, transcription factors, disease resistance, transposon biology, and gene silencing. There was more or less equal and cordial(!) representation from the maize and Arabidopsis plant genetic communities.

Development The meeting began with a challenging keynote address from Mike Freeling (Berkeley) on the genetic analysis of maize leaf development. Many interesting leaf mutations lie in genes of the Knotted h o m e o b o x gene family, but as was made clear, extrapolation from the (usually dominanO mutant phenotypes to the function of the wild-type gene is a difficult and subtle matter. Flower development provides a different kind of challenge, and E. Coen (Norwich), E. Meyerowitz (Pasadena) and M. Yanofsky (San Diego) put more flesh on models for floral morphogenesis. The current model for determination of whorl identity is that three functions - a, b and c - act combinatorially to specify the identity of sepals, petals, stamens and carpels. Although evidence for this model is TIG NOVEMBER1992 VOL. 8 NO. 11

©1992 Elsevier Science Publishers Ltd (UK)

36~"

overwhelming, the nature of the a function in Arabidopsis and A n t i r r h i n u m remains relatively illdefined. In Antirrhinum, the mutant with the predicted phenotype corresponding to a loss of a function (ovulata) turns out to result from ectopic over-expression of the c function (plena), which is proposed to inhibit expression of the a function gene(s) (Coen). Yanofsky reported that, in Arabidopsis, the expression of APETALA 1 (AP1) (which, like the b and c function genes, encodes a MADS box transcription factor) is positively maintained by the putative a function gene APETALA 2 (AP2), implying that some of the phenotypic consequences of loss of AP2 function may in fact be due to loss of AP1 function. This emphasized the role of AP1 in whorl determination as well as in determination of the floral