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of HIV-1-Tat Protein Mediates PathogenesisCutting Edge: A Short Polypeptide Domain

Yamada and Subhash DhawanM. Wahl, Hynda K. Kleinman, John N. Brady, Kenneth M.Yong Song Gho, Sherwin F. Lee, Indira K. Hewlett, Larry Robert A. Boykins, Renaud Mahieux, Uma T. Shankavaram,

http://www.jimmunol.org/content/163/1/151999; 163:15-20; ;J Immunol 

Referenceshttp://www.jimmunol.org/content/163/1/15.full#ref-list-1

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 1999 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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Cutting Edge: A Short PolypeptideDomain of HIV-1-Tat Protein MediatesPathogenesis

Robert A. Boykins,* Renaud Mahieux,‡

Uma T. Shankavaram,§ Yong Song Gho,¶

Sherwin F. Lee,† Indira K. Hewlett,† Larry M. Wahl,§

Hynda K. Kleinman,¶ John N. Brady,‡

Kenneth M. Yamada,¶ and Subhash Dhawan1†

HIV-1 encodes the transactivating protein Tat, which is essen-tial for virus replication and progression of HIV disease. How-ever, Tat has multiple domains, and consequently the molec-ular mechanisms by which it acts remain unclear. In thisreport, we provide evidence that cellular activation by Tat in-volves a short core domain, Tat21–40, containing only 20 aaincluding seven cysteine residues highly conserved in mostHIV-1 subtypes. Effective induction by Tat21–40 of both NF-kB-mediated HIV replication and TAR-dependent transacti-vation of HIV-long terminal repeat indicates that this shortsequence is sufficient to promote HIV infection. Moreover,Tat21–40 possesses potent angiogenic activity, further under-scoring its role in HIV pathogenesis. These data provide thefirst demonstration that a 20-residue core domain sequence ofTat is sufficient to transactivate, induce HIV replication, andtrigger angiogenesis. This short peptide sequence provides apotential novel therapeutic target for disrupting the functionsof Tat and inhibiting progression of HIV disease. The Journalof Immunology,1999, 163: 15–20.

T he transactivator Tat of HIV-1 (1) is an 86-aa proteinreleased by infected cells and plays a critical role in theprogression of HIV disease (1, 2). Transactivation of the

HIV-long terminal repeat (LTR)2 promotor by the Tat protein is

essential for both viral gene expression and virus replication. Ex-tracellular Tat released by infected cells during the acute phase ofinfection enters noninfected cells and disrupts many host immunefunctions by activating a wide variety of genes regulated by spe-cific viral and endogeneous cellular promotors (3, 4). We and oth-ers have previously shown that Tat mimics many of the effects ofHIV infection of monocytes including increased matrix metallo-proteinase-9 and cytokine production, and collagen expression inglioblastoma cells (5–7). These observations correlate with highlevels of cytokines such as IL-1, IL-6, and TNF found in sera fromHIV-infected individuals that leads to an increase of the level ofHIV replication. These reports suggest a role of extracellular Tat inpromoting viral patogenesis. However, Tat has multiple domains,and consequently how Tat induces these diverse effects is notclearly understood. In the present study, we have dissected thesequence of Tat and identified a domain that mediates the cellularand viral effects of extracellular Tat protein. These findings arepotentially important for understanding the progression of HIVpathogenesis and in the development of potential therapeuticapplications.

Materials and MethodsTat protein

The HIV-1-Tat protein used in these experiments was obtained through theAIDS Research and Reference Reagent Program, Division of AIDS, Na-tional Institute of Allergies and Infectious Disease, National Institutes ofHealth, from Dr. Andrew Rice or Dr. John N. Brady (6). HIV-Tat wasdissolved at 10mg/ml in treatment buffer (PBS containing 1 mg/ml BSAand 0.1 mM DTT) and frozen in aliquots at280°C. Tat preparations werescreened and found to be negative for endotoxin contamination.

Synthesis and purification of Tat peptides

Tat peptides were synthesized by solid phase synthesis on an Applied Bio-systems peptide synthesizer Model 430A (Foster City, CA) (8). After aninitial HPLC purification of the crude cysteine-containing peptides, theywere redissolved in 0.1 M Tris acetate buffer (pH 8.3) and air-oxidizedovernight. Peptides were then subjected to desalting and purification byreverse-phase HPLC, lyophilized, and stored at270°C. Peptide identitieswere confirmed by amino acid compositional analysis and plasma desorp-tion mass spectroscopic analysis.

*Laboratory of Parasitic Biology and Biochemistry and†Immunopathogenesis Sec-tion, Laboratory of Molecular Virology, Center for Biologics Evaluation and Re-search, Food and Drug Administration, Bethesda, MD 20892;‡Laboratory of Recep-tor Biology and Gene Expression, National Cancer Institute, National Institutes ofHealth, Bethesda, MD 20892; and§Immunopathology Section and¶Craniofacial De-velopmental Biology and Regeneration Branch, National Institute of Dental andCraniofacial Research, National Institutes of Health, Bethesda, MD 20892

Received for publication March 12, 1999. Accepted for publication April 30, 1999.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby markedadvertisementin accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 Address correspondence and reprint requests to Dr. Subhash Dhawan, Immuno-pathogenesis Section, Laboratory of Molecular Virology, Center for Biologics Eval-uation and Research, Food and Drug Administration, 1401 Rockville Pike (HFM-315), Rockville, MD 20852. E-mail address: dhawan@cber.fda.gov2 Abbreviations used in this paper: LTR, long terminal repeat; CAM, chorioallantoicmembrane; CAT, chloramphenicol acetyltransferase.

Copyright © 1999 by The American Association of Immunologists 0022-1767/99/$02.00

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Monocyte isolation and infection with HIV

Monocytes were isolated from the PBMC of donors seronegative for HIVand hepatitis after leukapheresis and purification by countercurrent cen-trifugal elutriation (9). Primary monocytes cultured for 5 days were ex-posed to HIV-1Ba-L, a monocytotrophic HIV strain (Advanced Biotechnol-ogies, Columbia, MD), at a multiplicity of infection of 0.01 infectious virusparticles/target cell (10).

Electroporation of cells

Cells were electroporated as previously described (11). CEM cells (12D7)were cultured at a density of 0.5–0.83 106 cells/ml with daily mediaadditions. Typically, 53 106 cells were electroporated with 5mg of eitherpurified plasmid or Tat protein and 5mg of reporter plasmid. Tat peptidesor Tat protein and the reporter HIV LTR-chloramphenicol acetyltrans-ferase (CAT) or the TAR mutant HIV TM26 LTR-CAT were mixed withcells and electroporated using a cell porater apparatus (Life Technologies/BRL, Gaithersburg, MD). Cell mixtures were electroporated at 800mF and240 V in RPMI 1640 medium without serum. Following electroporation,cells were plated in 10 ml complete medium, and samples were collected24 h later for CAT assays.

EMSA for NF-kB

Monocytes (13 107/ml) were treated with rTat protein or Tat peptides at37°C for 15 min. Nuclear extracts were then prepared and analyzed byEMSA as previously described (12).

CAM assay

The chick CAM assay was conducted as described (13) to determine theangiogenic activity of rTat and its derived peptides. Briefly, salt-free aque-ous solution (5ml) containing 5.3 pmol of rTat or its derived peptides(Tat21–40, Tat53–68, or Tat41–52) was loaded onto a 1/4 piece of 15-mmThermonox disk (Nunc, Naperville, IL), and the sample was dried understerile air. The disk loaded with sample was placed on the CAM of a10-day-old chick embryo. After 72 h incubation, negative or positive re-sponses were scored under a microscope. A positive response was char-acterized as the appearance of a typical radiating network (spokewheel)pattern of new blood vessels around the loaded samples. Assays for eachtest sample were conducted in two sets of eggs, and each set contained12–15 eggs.

Results and DiscussionTo identify Tat-specific sequences responsible for cellular dys-function, we synthesized overlapping peptides from various do-mains of consensus-B and other HIV-1 subtypes (Fig. 1). Usingthese peptides, we have identified a novel domain that can mediateviral transactivation. We found that, like rTat, the 20-aa core do-

main Tat21–40containing seven cysteine residues, all of which arestrongly conserved in various subtypes, enhanced HIV replicationby greater than 4-fold (Table I). A peptide derived from the basicdomain (Tat53–68) induced a lesser increase in viral replicationcompared with Tat21–40. In contrast, Tat41–52, a peptide sequencelocated between the core and the basic domains, and a variety ofpeptides from other positions in the Tat sequence, had no signif-icant effect on HIV replication.

Consistent with its enhancement of viral replication, Tat21–40

treatment produced a marked increase in HIV-associated cyto-pathic effects in monocytes as indicated by formation of multinu-cleated giant cells (Fig. 2c); the effects were similar to those in-duced by rTat protein itself (Fig. 2b). The effect of Tat53–68 wassubstantially less than that of Tat21–40(Fig. 2d). Tat41–52, the pep-tide between core and basic domains, and peptides from other Tatdomains did not alter HIV-associated cytopathic effects (Fig. 2e,and data not shown). Thus, a major active site for stimulating HIVreplication and monocyte dysfunction can be localized to the 20-residue peptide Tat21–40.

Table I. Effect of various Tat peptides on HIV replication inmonocytesa

ResidueNumbers Tat Sequence

p24 (pg/ml)(mean6 SEM)

Control 4716 10T1–86 rTatb 18866 26Tat1–20 MEPVDPRLEPWKHPGSQPKT 4216 3Tat10–30 PWKHPGSQPKTACTNCYCKKC 3786 4Tat21–40 ACTNCYCKKCCFHCQVCFTT 19586 101Tat31–51 CFHCQVCFTTKGLGISYGRK 4296 24Tat53–68 RRQRRRAHQNSQTHQAS 9696 75Tat25–40 CYCKKCCFHCQVCFTT 8366 123Tat8–19 LDPWNHPGSQPT 3706 2Tat41–52 KGLGISYGRKKR 5696 9Tat8–19 LEPWNHPGSQPK 3766 2

a Monocytes cultured for 5 days were treated with rTat or Tat peptides for 18 h.Cells were then infected with HIVBa-L strain as described inMaterials and Methods.On day 5, cells were harvested, and concentrations of p24 gag Ag in culture super-natants were determined using a DuPont (Wilmington, DE) p24 ELISA test kit. Dataare representative of two separate experiments and expressed as mean6 SEM oftriplicate determinations.

b Recominant full-length Tat.

FIGURE 1. Amino acid sequence of HIV-Tat protein from various subtypes, designated consensus-B, consensus-C, etc. A series of overlapping peptideswere synthesized as indicated by lines and residue numbers. Highly conserved residues are indicated at the bottom, with completely conserved residues inconsensus-B, -C, -D, -F, -O, and -U subtypes marked by shaded boxes, and residues for which there was only one alternative amino acid are indicated byunderlines.

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One of the mechanisms by which HIV-Tat potentiates HIV rep-lication involves transactivation of the HIV-1 LTR via its bindingto the TAR sequence along with other cellular factors, resulting inincreased viral transcription initiation and elongation (14). Tocharacterize further the mechanism of Tat transactivation of theHIV-LTR, CEM lymphoid cells were transfected with wild-typepromoter in the presence of various Tat peptides, and the extent oftransactivation was determined using CAT assays (14). Theseanalyses revealed a 9-fold induction of HIV-LTR by the Tat21–40

peptide (Fig. 3,A andB); full-length rTat produced a 25-fold in-duction. The actual effectiveness of induction by Tat21–40might begreater than observed due to the low solubility of this complexhydrophobic peptide in aqueous buffers.

We found that the presence of Cys22 in core domain Tat21–40

(and three adjacent residues) was critical for viral activation, be-cause deletion of these residues substantially reduced the ability ofTat21–40to activate HIV infection (Tat25–40in Fig. 3 and Table I).Because rTat activation of the HIV-LTR promoter is required forproductive HIV replication (15), our demonstration of induction bythe Tat21–40 sequence conserved in most HIV-1 subtypes furtherconfirms a functional role of Tat21–40in HIV infection. In contrast,transfection of CEM cells with a TAR mutant (HIV TM26 LTR-CAT) construct in the presence of the same peptides failed to in-duce HIV-LTR activation (Fig. 3C), confirming that the HIV-LTRactivation by Tat peptides was TAR-specific. Previous studieshave demonstrated that the arginine-rich basic domain located be-tween residues 49 and 57 constitutes the TAR-binding activity(16–19). Mutation or deletion of the basic domain severely dimin-ishes the ability of Tat to transactivate the LTR. The overlappingpeptide(s) from this region tested in the present study were not asactive as Tat21–40peptide. Studies are underway to investigate theability of the Tat21–40 peptide to induce TAR-dependenttranscription.

Although the precise mechanism of virus regulation by host fac-tors is not clear, it is generally believed that in addition to otherunknown factors, Tat and cytokines play a key role in the patho-genesis of HIV infection. Extracellular HIV-Tat causes activationof intracellular signal transduction pathways that culminate in theproduction of various cytokines (5, 20). Therefore, because of itsability to induce host factors, Tat is believed to be a key factor forviral enhancement. HIV-Tat activates both viral and host cellgenes, and the host NF-kB transcription factor contributes to im-mune dysregulation during HIV infection (21, 22). Because mac-rophages are a well-known reservoir for HIV in vivo, we examinedthe ability of Tat peptides to activate the expression of NF-kB inthese cells. Monocytes were treated with rTat and other peptides,nuclear extracts were prepared, and NF-kB activity was examinedby gel shift assay using an NF-kB consensus oligonucleotide. Ourresults show that the ability of HIV-Tat to activate NF-kB wasretained in core peptide Tat21–40 and to a lesser extent Tat53–68

(Fig. 4). Treatment of monocytes with the Tat21–40peptide rapidlyactivated NF-kB (within 15 min after exposure) by greater than9-fold as compared with 3-fold induction by Tat53–68. Interest-ingly, despite inducing NF-kB activity, Tat53–68had little effect ontransactivation of HIV-LTR as shown in Fig. 3. These observa-tions delineate two distinct mechanisms for viral activation byHIV-Tat: 1) TAR-dependent transactivation of HIV-LTR involv-ing Tat21–40domain, and 2) TAR-independent activation of virusreplication involving the host factor NF-kB by an intracellular sig-nal transduction pathway. Our results are complementary to thoserecently reported by Mayne et al. (23), who have demonstrated theinvolvement of protein kinase A, phospholipase C and protein ty-rosine kinase in Tat-mediated induction of NF-kB and cytokineproduction by monocytes.

Tat is released by HIV-infected cells into the extracellular mi-lieu, and has been implicated as a cofactor in the pathogenesis of

FIGURE 2. Effect of rTat and Tat peptides on HIV-associated cytopathic effects in monocytes. At day 5 postinoculation, cells were washed once withPBS, fixed, and Wright-stained. HIV-associated cytopathic effects were determined by examining for the formation of multinucleated giant cells.a,HIV-infected monocytes.b–e,Monocytes infected in the presence rTat (b), Tat21–40(c), Tat53–68(d), and Tat41–52(e). Morphology of uninfected monocytesis shown inf.

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Kaposi’s sarcoma (24), an angioproliferative disease frequentlyseen in HIV-infected individuals. There is increasing evidence thatHIV-Tat induces endothelial cell migration, invasion, and angio-genic processes in vivo (25). To test for potential angiogenic ac-tivity of the core domain implicated above in viral pathogenesis,we examined the ability of Tat peptides to induce neovasculariza-tion using the CAM assay. Our results indicate that picomol quan-tities (5.2 pmol/egg) of Tat21–40 can induce neovascularization(Fig. 5). Recombinant Tat alone was less effective in inducing anangiogenic response, as reported by others (25). No significantangiogenic response was observed using vehicle alone or the con-trol peptide Tat41–52 containing sequence between the core andbasic domains (Fig. 5). Interestingly, Tat53–68 from the Tat basicdomain also had substantial activity; as noted above, this peptidehad either partial or minimal activities in assays for HIV replica-tion, cytopathic effects, and transactivation of the HIV-LTR pro-motor. The exact mechanism of neovascularization in vivo is notclear. However, one scenario is that Tat-induced cytokines stim-

ulate endothelial cells, degrade basement membrane matrix by lo-cal enhancement of matrix metalloproteinase-9 secretion and mi-grate into adjacent tissue to form new blood vessel networks.

Detectable levels of Tat have been reported in HIV-infectedindividuals (26), suggesting the presence of extracellular HIV-Tatprotein in certain phases of HIV infection. It has also been shownthat high levels of anti-Tat Abs are directly related to low viralload (27, 28) in seropositive nonprogressor patients. Therefore, astrategy targeting a required site(s) in Tat might provide a noveltherapeutic modality to reduce disease progression in HIV-infectedindividuals. In this paper we have provided evidence that a shortcore domain of the Tat protein, Tat21–40 consisting of 7 cysteineresidues and only 13 other amino acids, is a potent inducer of HIVtransactivation and replication. This domain is highly conserved invarious HIV-1 subtypes, including the newly discovered group 0.Our results suggest that the mechanism by which HIV-Tat acti-vates cellular functions involves primarily the Tat21–40 core do-main, with possible lesser contributions from the Tat53–68domain.Some of these results are complementary to those demonstratingthe involvement of Tat and the core domain in the process ofmonocyte chemotaxis in response to Tat (29, 30), which may con-tribute to altered immunoregulation in HIV-infected individuals. Itis important to note that monocytes differentiate into tissue-resi-dent macrophages, which are nonrecirculating cells. Therefore,HIV-infected macrophages could continue to infect neighboringnormal cells and contribute to the tissue damage typically seenafter HIV infection. Thus, the active domain Tat21–40, possibly incombination with Tat53–68, provides a novel candidate target for apotential therapeutic vaccine or a dominant-negative strategy toreduce Tat-mediated progression of disease in individuals withHIV infection.

AcknowledgmentsWe thank Dr. Hira Nakhasi for constant support, suggestions, and criticalreview of the manuscript.

FIGURE 3. Transactivation of HIV-LTR by rTat and Tat peptides byCAT assay.A, Transfection of CEM cells with wild-type HIV-LTR Tatconstruct.B, Quantitative analysis of HIV-LTR transactivation by Tat andTat peptides.C, Transfection of CEM cells with a TAR mutant constructas a control in the presence of the indicated peptides.

FIGURE 4. Effect of Tat or Tat peptides on activation of NF-kB inmonocytes. Monocytes were treated with Tat or Tat peptides at 37°C for 15min. Nuclear extracts were then prepared, and NF-kB activity was assessedby gel-shift assay.

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FIGURE 5. Induction of angiogenesis byrTat or Tat-derived peptides in the chick CAMassay.A, rTat or its derived peptides (Tat21–40,Tat53–68, or Tat41–52) was loaded (5.3 pmol/egg)on CAMs of 10-day-old embryos. After 72 h in-cubation at 37°C, fat emulsion (whipping cream)was injected under the CAMs to help visualizethe vascular networks. Disks and surroundingCAMs were photographed: vehicle (a), rTat (b),Tat21–40 (c), Tat53–68 (d), or Tat41–52 (e). B, In-cidence of angiogenic response induced by rTator Tat peptides. Each bar represents the averagepercentage of positive eggs from two indepen-dent sets of assays consisting of 12–15 eggs.Positive responses involved eggs which showeda spokewheel pattern of new blood vesselsaround the loaded samples.p values were calcu-lated by using the Student’s pairedt test, basedon comparisons with water control samplestested at the same time,p, p , 0.02.p values vscontrol were as follows: rTat, 0.104; Tat21–40,0.006; Tat53–68, 0.017; and Tat41–52, 0.313.

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