Vol. 67, No. 9JOURNAL OF VIROLOGY, Sept. 1993, p. 5617-56220022-538X/93/095617-06$02.00/0Copyright © 1993, American Society for Microbiology

Functional Analysis of Interactions between Tat and thetrans-Activation Response Element of Human

Immunodeficiency Virus Type 1 in CellsYING LUO,1 STEVEN J. MADORE,2 TRISTRAM G. PARSLOW,3 BRYAN R. CULLEN,2

AND B. MATIJA PETERLINl*Howard Hughes Medical Institute and Departments ofMedicine, Microbiology and Immunology' and

Pathology,3 University of California, San Francisco, California 94143, and Howard Hughes Medical Instituteand Section of Genetics, Duke University Medical Center, Durham, North Carolina 277072

Received 12 March 1993/Accepted 15 June 1993

Transcriptional trans-activation of the human immunodeficiency virus type 1 long terminal repeat requiresthat the virally encoded Tat effector interacts with its target trans-activation response element (TAR) RNAstem-loop. Although the arginine-rich region of Tat from amino acids 49 to 59 is sufficient to bind to TAR RNAin vitro, the RNA-binding domain of Tat has not been defined in vivo. Human immunodeficiency virus type 1also encodes the Rev protein, which acts through an RNA stem-loop called the Rev-response element totransport unspliced and singly spliced viral RNA species from the nucleus to the cytoplasm. To map theRNA-binding domain of Tat, we performed assays that relied on Rev function using the heterologousRNA-tethering mechanism of Tat and the TAR. By examining the effects of selected targeted mutations of Taton the abilities of hybrid Tat/Rev proteins to rescue the expression of unspliced mRNA via the TAR, wedemonstrated that residues throughout the N-terminal 59 amino acids of Tat are required for binding of Tatand TAR RNA in vivo.

Tat interacts with the trans-activation response element(TAR) RNA stem-loop, which is located at the 5' end of allviral transcripts, to greatly increase human immunodefi-ciency virus type 1 (HIV-1) gene expression and replication(3, 9, 15, 24). Tat is essential for high levels of virionproduction, cellular cytopathology, and expression of hu-man disease (4, 8, 13). From studies to date, Tat appears tocontain two functional domains. Whereas the N-terminaldomain, from amino acids 1 to 48, comprises the activationdomain of Tat, the adjacent arginine-rich region from aminoacids 49 to 58 is necessary and sufficient for its binding toTAR RNA in vitro (2, 6, 31) and is required for its functionin vivo (10, 16, 26). Furthermore, it is the number of basicamino acids in this domain rather than the precise sequenceof arginines that is important for trans-activation (1, 5).However, if the arginine-rich sequences were indeed the soledeterminant of RNA binding in vivo, then any cellularprotein with a similar basic amino acid motif should interactwith the TAR and compete for trans-activation by Tat.Clearly this does not occur. For example, whereas thearginine-rich regions of Rev and bacteriophage A N proteincan functionally replace the basic domain of Tat, no inhibi-tion by Rev of trans-activation by Tat has been observed(28). Thus, other sequences in Tat must also contribute tospecific interactions between Tat and the TAR.Rev binds to the Rev-response element (RRE), which is

located in the middle of the env gene, to transport unsplicedand singly spliced HIV-1 RNA species from the nucleus tothe cytoplasm (3, 7, 19, 24). Although the precise mechanismof action of neither trans-activator is understood, the func-tions of the TAR and RRE are solely to bring Tat and Rev,respectively, to HIV-1 RNAs. Thus, both Tat and Rev canbe targeted to viral transcripts via heterologous RNA-teth-

* Corresponding author.

ering mechanisms, for example, by the coat protein ofbacteriophage MS2 and its operator RNA stem-loop (22, 26,30). Functional equalities between protein-RNA interactionsof these trans-activators suggested that Rev could be tar-geted to HIV-1 RNAs via Tat and the TAR and that assaysof Rev function in cells could be used to map RNA-bindingdomains of Tat in vivo. To this end, we constructed severalhybrid Tat/Rev proteins and replaced the RRE with theTAR. Whereas Rev by itself could not interact with thismodified target, Tat in the hybrid Tat/Rev protein efficientlytargeted Rev to the TAR, which resulted in the transport ofunspliced viral RNAs from the nucleus to the cytoplasm. Bytesting several mutants of Tat in this context, we demon-strated that not only basic but also activation domains of Tatare required for binding of Tat to TAR RNA in vivo.

MATERIALS AND METHODS

Plasmid constructions. Plasmids which direct the synthesisof wild-type and mutant hybrid Tat/Rev proteins were con-structed as follows. pcTat and pcRev have been previouslydescribed (17, 19, 29). In pcTat, the transcription termina-tion signal AATAGA was replaced by CCATGG, whichforms an NcoI site, by using synthetic oligonucleotides. InpcRev, the translation initiation site CTATGG was mutatedto CCATGG to create an NcoI site. The SalI-NcoI fragment,which contains the entire tat gene, was then ligated into theSalI-NcoI sites in pcRev to form pcTat/Rev (29).pcTat/M1O was created in the same way as pcTat/Rev,

except that mutated pcRev was used in this construction. InpcTat/M10, L and E, which are amino acids at positions 78and 79 in the activation domain of Rev, were changed to Dand L, respectively. This mutant of Rev has no biologicalactivity as Rev (17).pARK/Rev, pAN/Rev, pC22S/Rev, pC37S/Rev, and

pK41A/Rev were created by polymerase chain reaction with

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1 TAT 861 REV 116

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FIG. 1. Effector and target plasmid constructions. (A) Diagrammatic representation of plasmids which code for hybrid Tat/Rev proteins(pcTat/Rev). The immediate-early promoter of cytomegalovirus directs the synthesis of full-length Tat (amino acids 1 to 86) linked tofull-length Rev (amino acids 1 to 116). All mutations in Tat and Rev were introduced into this plasmid construction. (B) Diagrammaticrepresentation of the four plasmids which code for RNA effectors. All direct the synthesis of CAT. pHIVSCAT contains the TAR of HIV-1in its native position next to the site of initiation of transcription. pDM128 contains splice donors (SD) and acceptors (SA) of HIV-1 and theRRE 3' to the CAT reporter gene. pDM12TAR and pDM257 were created from pDM128 and contain 12 TARs (12TAR) or four operators ofbacteriophage MS2 (4MS2) in place of the RRE. Transcription of pDM128, pDM12TAR, and pDM257 intitiates from the simian virus 40 earlypromoter (SV40) and terminates at the SV40 polyadenylation site (pA). See Materials and Methods for more details.

overlapping primers which contained appropriate nucleotidesubstitutions. In pARK/Rev, KKRRQRR sequences in thearginine-rich domain of Tat were replaced by YVQILLY. InpAN/Rev, the N-terminal proline-rich domain of Tat (aminoacids 2 to 12) was deleted. In pC22SRev, the cysteine atposition 22 in Tat was replaced by a serine. In pC37S/Rev,the cysteine at position 33 was replaced by a serine. InpK41A/Rev, the lysine at position 41 was replaced by analanine.pDM128, which contains the chloramphenicol acetyltrans-

ferase (CAT) reporter gene and the RRE, has been previ-ously described (12). pDM257 was created by replacing theRRE in pDM128 with four tandem MS2 operators (22).pDM12TAR was constructed by replacing the RRE with 12tandem TARs from p12TAR (11).

Transient expression assays. CV1 cells were transfected bythe CaPO4 method as described previously (22). In assays ofTat function, 5 ,ug of pHIVSCAT and 5 ,ug of various hybridTat/Rev plasmids were used in each transfection (12).pCMVA, a derivative of pcTat/Rev with a deletion of boththe tat and rev genes, was used as a control plasmid in allexperiments. In assays of Rev function, 20 ,ug of hybridTat/Rev effector plasmid and 1 ,ug of target plasmid (pDM257or pDM12TAR) were used in each transfection. ForpDM128, only 1 ,ug of hybrid Tat/Rev plasmid was used incotransfection experiments. Cells were harvested 40 to 48 h

after transfection. After transfection, medium was changedafter 18 h. Cells were harvested 40 to 48 h later and lysed inTriton lysis buffer. CAT assays were performed as previ-ously described (12). Protein concentrations were used tonormalize CAT assays by using a Bio-Rad kit. Experimentswere performed several times in triplicate when standarderrors of the mean were less than 30%.

RESULTS

Wild-type and mutant Tat and Rev in the hybrid Tat/Revproteins are expressed as functional proteins. To characterizeinteractions between Tat and the TAR in vivo, we deter-mined RNA-binding properties of Tat independently of itsability to trans-activate transcription. For this purpose, weconstructed hybrid Tat/Rev proteins and examined Revactivities in the context of the heterologous RNA-tetheringmechanism provided by Tat and the TAR. Since Rev has tobind to and multimerize on the RRE, which consists ofseveral RNA stem-loops, to transport unspliced and singlyspliced viral RNA species from the nucleus to the cytoplasm(7, 19), we reasoned that several TARs in place of the RREshould allow Tat in hybrid Tat/Rev proteins to perform thesame function. Thus, we only had to examine the Revactivity to determine the binding of Tat to TAR RNA in vivo.However, we first had to demonstrate that wild-type and

A. EFFECTORS

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ANALYSIS OF INTERACTIONS BETWEEN Tat AND THE HIV-1 TAR

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pfHVSCAT pDM128FIG. 2. Activities of hybrid Tat/Rev proteins on the HIV-1 LTR and RRE RNA targets. (A) Diagrammatic representation of deletions and

mutations in Tat or Rev, which were expressed as hybrid Tat/Rev proteins. Deletions and mutations in Tat (hatched bar) and Rev (shadedbar) are depicted above the full-length fusion protein. Locations of activation and basic domains of Tat and Rev are given below the bardiagram. (B) In the left and right panels are CAT activities from cotransfections with target plasmids, which contained wild-type TAR(pHIVSCAT) and RRE (pDM128) sequences in their native configurations, respectively, with plasmids that coded for seven hybrid effectors,one of which contained a mutant of Rev (pcTat/MlO) and the others of which contained mutants of Tat (pAN/Rev, pC22S/Rev, pC37S/Rev,pK41AlRev, and pARK/Rev). pcTat/Rev contained wild-type Tat and Rev sequences. Shown are wild-type Tat and Rev sequences (solidbars), wild-type Tat sequences with mutations in Rev (hatched bars), and wild-type Rev sequences with mutations in Tat (shaded bars).Results are representative of several experiments performed in triplicate. Standard errors of the means were less than 30%.

mutated hybrid Tat/Rev proteins were expressed and func-tioned in cells. To this end, effector plasmids, which ex-pressed mutants of Tat or Rev in the context of hybridTat/Rev proteins, were cotransfected with target plasmids,which contained either HIV-1 long terminal repeat (LTR)targets (pHIVSCAT) for Tat assays or RRE targets(pDM128) for Rev assays, into CV1 cells (Fig. 1). In pHIVS-CAT, the HIV-1 LTR directs the synthesis of the CATreporter gene. In pDM128, the CAT reporter gene wasinserted into the env intron of HIV-1 such that increasedCAT activities were observed only if unspliced RNA wastransported from the nucleus to the cytoplasm by Rev.Representative plasmid constructions are diagrammed inFig. 1.

Results of these cotransfections are presented in Fig. 2B.Consistent with previous observations and in contrast topcTat/Rev, pAN/Rev, pARK/Rev, pC22S/Rev, pC37S/Rev,and pK41A/Rev, which contained deletions or mutations inactivation and basic domains of Tat (Fig. 2A), all eliminatedtrans-activation by Tat (Fig. 2B, left panel). pcTat/Rev andpcTat/M10, which contained wild-type Tat, trans-activatedpHIVSCAT four- to fivefold (Fig. 2B, left panel). It shouldbe noted that much higher levels of trans-activation, which

were equivalent to those with Tat expressed as a singleprotein, were observed with these plasmids in HeLa cells(data not presented). However, Rev functioned normally inthese mutants of the hybrid Tat/Rev protein (Fig. 2B, rightpanel). The sole exception was pcTat/MlO, which lacked afunctional activation domain of Rev and was inactive (Fig.2B, right panel). Thus, pcTat/Rev, pAN/Rev, pARK/Rev,pC22S/Rev, pC37S/Rev, and pK41A/Rev increased CATactivities from pDM128 from 15- to 30-fold in CV1 cells (Fig.2B, right panel). These results confirmed that all hybridTat/Rev proteins were expressed and functioned in CV1cells.Hybrid Tat/Rev proteins function as Rev via the binding of

Tat to TAR RNA. To determine whether interactions be-tween Tat and the TAR could replace those between Revand the RRE, we cotransfected pcTat/Rev with pDM12TARinto CV1 cells. In pDM12TAR, 12 tandemly repeated TARsreplaced the RRE in pDM128 (Fig. 1B). As demonstrated inthe left panel of Fig. 3, pcTat/Rev increased CAT activitiesfrom pDM12TAR 14.5-fold. To further prove that this effectwas due to the activation domain of Rev, a mutant of Rev,pcTat/M10, was also cotransfected with pDM12TAR intoCV1 cells. The activity of Rev via the RRE was abolished in

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pcTatlRev

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pDM12TAR pDM257FIG. 3. Activities of hybrid Tat/Rev proteins on TAR and operator RNA targets. In the left panel are CAT activities from cotransfections

of the target plasmid, which contained 12 TARs (pDM12TAR), with plasmids which coded for hybrid effectors. pRev/MS2 contained thefull-length Rev linked to the coat protein of bacteriophage MS2. In the right panel are CAT activities from cotransfections of a control targetplasmid, which contained four operators of MS2 (pDM257), with the same effector plasmids. pRev/MS2 served as the positive control fortransfections with pDM257 (open bars). ND (not done) refers to experiments which were not performed. Other designations are as in Fig. 2.Results are representative of several experiments performed in triplicate. Standard errors of the means were less than 30%.

this mutant (Fig. 3, left panel). This result confirmed thatincreased CAT activities were due solely to the mechanismof action of Rev. However, Tat, by interacting with theTAR, brought Rev in these hybrid Tat/Rev proteins to theunspliced RNA.To test the specificity of the binding of Tat to TAR RNA,

multiple TARs were also replaced by four operators frombacteriophage MS2 (pDM257) (Fig. 1B). Cotransfections ofpcTat/Rev with pDM257 resulted in no trans-activation inCV1 cells (Fig. 3, right panel). We conclude that the bindingof Tat to TAR RNA in pDM12TAR is specific and is requiredfor the effects of Rev.The activation domain of Tat is required for the binding of

Tat to TAR RNA in vivo. In vitro, the basic nine amino acidsfrom positions 49 to 58 are sufficient for binding of Tat toTAR RNA (1). This basic domain is also absolutely requiredfor trans-activation in vivo (10, 16, 26). However, theN-terminal 48 amino acids represent the activation domainof Tat that can function independently of the RNA-bindingdomain when presented to the transcription complex viaheterologous RNA- and DNA-tethering mechanisms (14, 26,29). To determine whether these arginine-rich sequences arealso sufficient for binding of Tat to TAR RNA in vivo,various deletions and mutations of Tat in hybrid Tat/Revproteins were examined. In pAN/Rev, pARK/Rev, pC22S/Rev, pC37S/Rev, and pK41A/Rev, N-terminal sequences ofTat were deleted, the basic domain of Tat was replaced,cysteines at positions 22 and 37 in Tat were replaced byserines, and the lysine at position 41 in Tat was replaced byan alanine. Except in the case of pARK/Rev, all of thesemutations were in the activation domain of Tat.As presented in the left panel of Fig. 3, none of these

mutants of Tat trans-activated pDM12TAR. Among theseplasmid effectors, pARK/Rev and pAN/Rev increased CATactivities only 1.29- and 2-fold, or 9 and 14% of levelsobserved with pcTat/Rev, respectively, and other mutationsin the core and cysteine-rich domains of Tat resulted in nodetectable trans-activation (Fig. 3, left panel, pK41A/Rev,

pC37S/Rev, and pC22S/Rev). These data are consistent withthe requirement of nine basic residues for binding of Tat toTAR RNA in vitro. Moreover, they indicate that core andcysteine-rich domains are also absolutely required for inter-actions between Tat and the TAR in cells. Given the greatlyreduced activity of Tat with even the deletion of N-terminalamino acids (pAN/Rev), we conclude that the entire activa-tion domain of Tat is required for binding of Tat to TARRNA in vivo.

DISCUSSION

By using hybrid Tat/Rev proteins, we studied the require-ments for binding of Tat to TAR RNA in vivo. Since theoriginal target plasmid pDM128 contained the CAT reportergene in the intron of the env gene, CAT activity increasedonly if unspliced transcripts were transported from thenucleus to the cytoplasm by Rev (12). However, the modi-fied target plasmid pDM12TAR contained the TAR in placeof the RRE. To increase the CAT activities of this plasmid,unspliced transcripts had to be transported from the nucleusto the cytoplasm via the binding of the hybrid Tat/Revprotein to TAR RNA. Thus, interactions between Tat andthe TAR replaced those between Rev and the RRE in ourassay. However, the activation domain of Rev still per-formed the function of RNA transport. By mutating Tat andRev in these hybrid proteins, we demonstrated that basicand activation domains of Tat are required for the binding ofTat to TAR RNA in vivo.

In this study, Rev could not bind to TAR RNA (Fig. 3,pRev/MS2 and pDM12TAR). Previously, basic amino acidswere exchanged between Rev and Tat, and this substitutedTat still trans-activated the HIV-1 LTR, which might suggestthat the full-length Rev could interact with the TAR (28).However, hybrid Tat/Rev proteins, which contained mu-tants of Tat, did not function via the TAR in pDM12TAR(Fig. 2), nor did Rev in the hybrid Tat/Rev protein interferewith trans-activation of the HIV-1 LTR (Fig. 2). Moreover,

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FIG. 4. Model for interactions between Tat and the TAR in vivo. Although the basic nine amino acids from positions 49 to 58 are sufficientfor binding of Tat to TAR RNA in vitro, the activation domain of Tat from amino acids 1 to 48 is also required for productive interactionsbetween Tat and the TAR in vivo. Thus, the nine basic residues contact the 5' bulge in the stem-loop, whereas the N-terminal half of Tatinteracts with a cellular protein that not only increases the binding to RNA but conveys activation by Tat to the transcription complex. Thissingle protein or complex of proteins most likely interacts with the central loop in the TAR and with a component ofRNA polymerase II. Theactivation domain of Tat (48 amino acids) and the minimal functional Tat of HIV-1 (60 amino acids) are represented by two ellipses, the largerofwhich not only contains the smaller activation domain of Tat but also encircles the 3' bulged nucleotides. Tat-binding protein is representedby a large gray sphere that contacts the transcription complex (dashed ellipse).

Tat did not block the activity of Rev, and neither trans-activator alone or together had any effect via the operator ofMS2 in the absence of the coat protein (Fig. 3, pcTat/Revand pDM257). Together, these data demonstrate that inter-actions between all three proteins and their RNA targets arevery precise and specific in vivo.However, to observe the effects of Rev with the hybrid

Tat/Rev protein, multiple TARs in place of the RRE wererequired. Reducing the number of TARs from 12 to 2completely eliminated the Rev effect (data not presented).Since this mirrors the requirement for Rev to multimerize onthe RRE (18), we postulate that multiple TARs are necessaryto achieve sufficient density of Rev for their transport fromthe nucleus to the cytoplasm. Similar results were obtainedwhen the coat protein of bacteriophage MS2 was used totarget Rev to the RNA, i.e., up to four operators wereneeded for high levels of trans-activation (22). However, wecannot absolutely exclude the possibility that multiple TARsare only required for the proper folding of one or a few TARsin the middle of a long primary transcript, especially since

the TAR (AG. = -35 kcal) (1 cal = 4.184 J) (25) has a higherpredicted free energy than the RRE (AG = -115 kcal) (19).These data lead to two possible models for interactions

between Tat and the TAR. The first is that Tat is a verycompact protein and that any changes in the activationdomain disrupt the secondary structure of the entire protein(23). In this scenario, basic residues would assume newconformations with different mutations in the activationdomain, which could interfere with the binding of Tat toTAR RNA. However, given all the in vitro binding studieswith different lengths and compositions of these basic aminoacids, this possibility appears unlikely (1, 2, 6, 31, 32).Alternatively, a cellular protein or complex of proteins thatinteracts with the activation domain of Tat is also requiredfor the specific binding of Tat to TAR RNA (Fig. 4). This"coactivator" could perform two functions, activating tran-scription and binding to TAR RNA. It is very attractive toconsider the possibility that this protein binds to the loop ofthe TAR, which is absolutely required for trans-activationby Tat in vivo but is dispensable for binding of Tat to TAR

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RNA in vitro. This protein is unlikely to be TRP-1/TRP-185,which by binding to the loop displaces Tat from the TAR invitro (27, 33). Furthermore, there is precedence for such aprotein in Escherichia coli. NusA, which is a bacterial proteinthat is required for antitermination of bacteriophage X tran-scription by the N protein, not only increases the binding tothe N utilization site (Nut B), which forms an RNA stem-loop, but also conveys the activation by N to core RNApolymerase (20, 21). Thus, these studies support the existenceof a protein or a complex of proteins that interact with Tat, theTAR, and the transcription complex (Fig. 4). The identifica-tion, cloning, characterization, and expression of this proteinare necessary next steps in the research of Tat.

ACKNOWLEDGMENTS

We thank Michael Armanini for expert secretarial assistance,David McDonald for gifts of plasmids, and members of our labora-tories for instructive criticism.Ying Luo is supported by a fellowship grant from the California

Universitywide AIDS Research Program.

REFERENCES1. Calnan, B. J., S. Biancalana, D. Hudson, and A. D. Frankel.

1991. Analysis of arginine-rich peptides from the HIV Tatprotein reveals unusual features of RNA-protein recognition.Genes Dev. 5:201-210.

2. Calnan, B. J., B. Tidor, S. Biancalana, D. Hudson, and A. D.Frankel. 1991. Arginine-mediated RNA recognition: the argi-nine fork. Science 252:1167-1171.

3. Cullen, B. R. 1991. Regulation of human immunodeficiencyvirus replication. Annu. Rev. Microbiol. 45:219-250.

4. Dayton, A. I., J. G. Sodroski, C. A. Rosen, W. C. Goh, andW. A. Haseltine. 1986. The trans-activator gene of the human Tcell lymphotropic virus type III is required for replication. Cell44:941-947.

5. Delling, U., S. Roy, S. M. Sumner, R. Barnett, L. Reid, C. A.Rosen, and N. Sonenberg. 1991. The number of positivelycharged amino acids in the basic domain of Tat is critical fortrans-activation and complex formation with TAR RNA. Proc.Natl. Acad. Sci. USA 88:6234-6238.

6. Dingwall, C., I. Ernberg, M. J. Gait, S. M. Green, S. Heaphy, J.Karn, A. D. Lowe, M. Singh, M. A. Skinner, and R. Valerio.1989. Human immunodeficiency virus 1 Tat protein binds trans-activation-responsive region (TAR) RNA in vitro. Proc. Natl.Acad. Sci. USA 86:6925-6929.

7. Felber, B. K., C. M. Hadzopoulou, C. Cladaras, T. Copeland,and G. N. Pavlakis. 1989. Rev protein of human immunodefi-ciency virus type 1 affects the stability and transport of the viralmRNA. Proc. Natl. Acad. Sci. USA 86:1495-1499.

8. Fisher, A. G., M. B. Feinberg, S. F. Josephs, M. E. Harper,L. M. Marselle, G. Reyes, M. A. Gonda, A. Aldovini, C. Debouk,R. C. Gallo, and F. Wong-Staal. 1986. The trans-activator geneof HTLV-III is essential for virus replication Nature (London)320:367-371.

9. Frankel, A. D. 1992. Activation of HIV transcription by Tat.Curr. Opin. Genet. Dev. 2:293-298.

10. Frankel, A. D., S. Biancalana, and D. Hudson. 1989. Activity ofsynthetic peptides from the Tat protein of human immunodefi-ciency virus type 1. Proc. Natl. Acad. Sci. USA 86:7397-7401.

11. Graham, G. J., and J. J. Maio. 1990. RNA transcripts of thehuman immunodeficiency virus trans-activation response ele-ment can inhibit action of the viral trans-activator. Proc. Natl.Acad. Sci. USA 87:5817-5821.

12. Hope, T. J., X. J. Huang, D. McDonald, and T. G. Parslow.1990. Steroid-receptor fusion of the human immunodeficiencyvirus type 1 Rev transactivator: mapping cryptic functions of thearginine-rich motif. Proc. Natl. Acad. Sci. USA 87:7787-7791.

13. Huet, T., M. C. Dazza, V. F. Brun, G. E. Roelants, and S.Wain-Hobson. 1989. A highly defective HIV-1 strain isolatedfrom a healthy Gabonese individual presenting an atypical

Western blot. AIDS 3:707-715.14. Kamine, J., T. Subramanian, and G. Chinnadurai. 1991. Spl-

dependent activation of a synthetic promoter by human immu-nodeficiency virus type 1 Tat protein. Proc. Natl. Acad. Sci.USA 88:8510-8514.

15. Karn, J. 1991. Control of human immunodeficiency virus repli-cation by the tat, rev, nef and protease genes. Curr. Opin.Immunol. 3:526-536.

16. Kuppuswamy, M., T. Subramanian, A. Srinivasan, and G.Chinnadurai. 1989. Multiple functional domains of Tat, thetrans-activator of HIV-1, defined by mutational analysis. Nu-cleic Acids Res. 17:3551-3561.

17. Malim, M. H., S. Bohnlein, J. Hauber, and B. R. Cullen. 1989.Functional dissection of the HIV-1 Rev trans-activator--deri-vation of a trans-dominant repressor of Rev function. Cell58:205-214.

18. Malim, M. H., and B. R. Cullen. 1991. HIV-1 structural geneexpression requires the binding of multiple Rev monomers tothe viral RRE: implications for HIV-1 latency. Cell 65:241-248.

19. Malim, M. H., J. Hauber, S.-Y. Le, J. V. Maizel, and B. R.Cullen. 1989. The HIV-1 Rev trans-activator acts through astructured target sequence to activate nuclear export of un-spliced viral mRNA. Nature (London) 338:254-257.

20. Mason, S. W., and J. Greenblatt. 1991. Assembly of transcrip-tion elongation complexes containing the N protein of phagelambda and the Escherichia coli elongation factors NusA,NusB, NusG, and S10. Genes Dev. 5:1504-1512.

21. Mason, S. W., J. Li, and J. Greenblatt. 1992. Host factorrequirements for processive antitermination of transcription andsuppression of pausing by the N protein of bacteriophagelambda. J. Biol. Chem. 267:19418-19426.

22. McDonald, D., T. J. Hope, and T. G. Parslow. 1992. Posttran-scriptional regulation by the human immunodeficiency virus type1 Rev and human T-cell leukemia virus type I Rex proteinsthrough a heterologous RNA binding site. J. Virol. 66:7232-7238.

23. Rice, A. P., and F. Carlotti. 1990. Structural analysis of wild-type and mutant human immunodeficiency virus type 1 Tatproteins. J. Virol. 64:6018-6026.

24. Rosen, C. A., and G. N. Paviakis. 1990. Tat and Rev: positiveregulators of HIV gene expression. AIDS 4:499-509.

25. Selby, M. J., E. S. Bain, P. A. Luciw, and B. M. Peterlin. 1989.Structure, sequence, and position of the stem-loop in TARdetermine transcriptional elongation by Tat through the HIV-1long terminal repeat. Genes Dev. 3:547-558.

26. Selby, M. J., and B. M. Peterlin. 1990. trans-activation by HIV-1Tat via a heterologous RNA binding protein. Cell 62:769-776.

27. Sheline, C. T., L. H. Milocco, and K. A. Jones. 1991. Twodistinct nuclear transcription factors recognize loop and bulgeresidues of the HIV-1 TAR RNA hairpin. Genes Dev. 5:2508-2520.

28. Subramanian, T., R. Govindarajan, and G. Chinnadurai. 1991.Heterologous basic domain substitutions in the HIV-1 Tatprotein reveal and arginine-rich motif required for transactiva-tion. EMBO J. 10:2311-2318.

29. Tiley, L. S., S. J. Madore, M. H. Malim, and B. R. Cullen. 1992.The VP16 transcription activation domain is functional whentargeted to a promoter-proximal RNA sequence. Genes Dev.6:2077-2087.

30. Venkatesan, S., S. M. Gerstberger, H. Park, S. M. Holland, andY.-S. Nam. 1992. Human immunodeficiency virus type 1 Revactivation can be achieved without Rev-responsive elementRNA if Rev is directed to the target as a Rev/MS2 fusion proteinwhich tethers the MS2 operator RNA. J. Virol. 66:7469-7480.

31. Weeks, K. M., C. Ampe, S. C. Schultz, T. A. Steitz, and D. M.Crothers. 1990. Fragments of the HIV-1 Tat protein specificallybind TAR RNA. Science 249:1281-1285.

32. Weeks, K. M., and D. M. Crothers. 1991. RNA recognition byTat-derived peptides: interaction in the major groove? Cell66:577-588.

33. Wu, F., J. Garcia, D. Sigman, and R. Gaynor. 1991. Tatregulates binding of the human immunodeficiency virus trans-activating region RNA loop-binding protein TRP-185. GenesDev. 5:2128-2140.

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