Proc. Natl. Acad. Sci. USAVol. 90, pp. 7632-7636, August 1993Biochemistry

Tat protein of human immunodeficiency virus type 1 repressesexpression of manganese superoxide dismutase in HeLa cells

(oxidative stress/free radicals/gene regulation)

SONIA C. FLORES*, JOHN C. MARECKI, KIMBERLY P. HARPER, SWAPAN K. BOSE, SALLY K. NELSON,AND JOE M. MCCORDWebb-Waring Institute for Biomedical Research, University of Colorado Health Sciences Center, 4200 East Ninth Avenue, Denver, CO 80262

Communicated by David W. Talmage, April 19, 1993

ABSTRACT Using a HeLa cell line stably transfected withthe tat gene from human immunodeficiency virus type 1, wehave found that the expression of the regulatory Tat proteinsuppresses the expression ofcellular Mn-containing superoxidedismutase (Mn-SOD). This enzyme is one of the cell's primarydefenses against oxygen-derived free radicals and is vital formaintaining a healthy balance between oxidants and antioxi-dants. The parental HeLa cells expressed nearly equivalentamounts of Cu,Zn- and Mn-SOD isozymes. Those cels ex-pressing the Tat protein, however, contained 52% less Mn-SOD activity than parental cells, whereas that of the Cu,Znenzyme was essentially unchanged. The steady-state levels ofMn-SOD-specific RNAs were also lower in the HeLa-tatcell linethan in the parental line. No difference was seen in thesteady-state levels of Cu,Zn-SOD-specific RNAs. In addition tothe decreased Mn-SOD activity, HeLa-tatceDls showed evidenceof increased oxidative stress. Carbonyl proteins were markedlyhigher, and total cellular sulfhydryl content decreased in ceDlextracts at a faster rate, probably reflecting ongoing lipidperoxidation. HeLa and HeLa-tat extracts were incubated withradiolabeled Mn-SOD transcripts, and the reaction productswere subjected to UV crosslinking, digestion with ribonucleaseA, and electrophoretic analysis. The results suggest a directinteraction between Tat protein and Mn-SOD gene transcripts.

Oxidative stress is a condition characterized by increasedproduction of cellular oxidants (including superoxide radical,hydrogen peroxide, and hypochlorous acid) and/or de-creased concentrations of antioxidants and antioxidant en-zymes [including glutathione, vitamin E, ascorbate, glu-tathione peroxidase, superoxide dismutases (SODs), andcatalase]. Oxidative stress is now recognized to be associatedwith nearly all pathological states, especially those involvingthe inflammatory process (1). Human immunodeficiencyvirus type 1 (HIV) infection is associated with oxidativestress. Glutathione levels are decreased in both infectedindividuals and cell cultures (2). Exogenous reducing agentssuch as N-acetylcysteine and glutathione suppress HIV ex-pression in chronically infected monocytes (3). Conversely,ultraviolet irradiation (4) or cytokine treatment (5), which areknown to increase oxidative stress, activate HIV expression.Cells acutely infected with HIV have been reported toexpress less Mn-SOD and to lose their ability to induce thisantioxidant enzyme in response to tumor necrosis factor (6).

Several HIV gene products are important in the regulationof viral gene expression (for reviews, see refs. 7 and 8). Ofthese, the Tat (trans-acting transcriptional activator) and Revproteins are essential for virus replication. Tat acts bybinding to a sequence, termed the transactivation responseelement (TAR), located downstream from the site of tran-

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scriptional initiation in the viral long terminal repeat (9).Transcripts containing the TAR RNA sequence adopt astable stem-loop structure to which Tat binds tightly andspecifically (10). The mechanism of action of Tat is stillcontroversial. Some investigators have suggested that Tatacts by increasing the rate of viral transcription rather than bymodulating mRNA stability (11), but others have proposedthat Tat acts at both the transcriptional and the posttran-scriptional level (12). In addition to regulating HIV geneexpression and replication, Tat has been reported to influ-ence cellular phenotype by affecting the expression of cel-lular genes (13-18).We have found that HeLa cells which produce the HIV

regulatory protein Tat have depressed levels ofMn-SOD andshow other evidence of oxidative stress. This deficiency inthe antioxidant enzyme Mn-SOD may contribute to theestablishment and maintenance of oxidative stress and poisethe cell for active proliferation (19) and/or transcription (20),conditions which may alter the cellular phenotype and mayfavor viral replication.

MATERIALS AND METHODSHeLa-tat-III cells (21, 22), which stably produce the HIV-1Tat III protein under the control of a heterologous promoter,were obtained from the AIDS Research and Reference Rea-gent Program. Parental HeLa cells of the same lineage asHeLa-tat-Ill were used as controls. The presence of biolog-ically active Tat was verified by transfection of the cells withpLUCA41 (23), a plasmid containing the luciferase geneunder control of the HIV long terminal repeat, followed byassay for luciferase as described (24). The HeLa-tat cell lineproduced 1600-fold more luciferase activity than the parentalline. SOD was assayed by the cytochrome c method (25).Differential determination of the Cu,Zn- and Mn-containingisozymes was achieved by first assaying total activity, theninactivating the Cu,Zn-enzyme by a modification of themethod of Iqbal and Whitney (26). Extracts were incubatedwith 1 mM diethyldithiocarbamate in 50 mM potassiumphosphate (pH 7.8) at 37°C for 1 hr. To prevent interferencewith the assay by diethyldithiocarbamate and to furtherinactivate the Cu,Zn-SOD, hydrogen peroxide was added (1mM) and the incubation continued for 15 min. [This concen-tration of hydrogen peroxide does not inactivate the MnSOD(27) or otherwise interfere with the assay.] Thin-film agarosegel electropherograms (Ciba-Coming Diagnostics) werestained for SOD activity (28). Total protein was determinedby the method of Lowry et al. (29). Cell supernatants wereprocessed for sulfhydryl determination (30) with 5,5'-dithiobis(2-nitrobenzoic acid). Carbonyl protein content wasmeasured by the method of Oliver et al. (31).

Abbreviations: HIV, human immunodeficiency virus; SOD, super-oxide dismutase; TAR, transactivation response element.*To whom reprint requests should be addressed.

7632

Proc. Natl. Acad. Sci. USA 90 (1993) 7633

Aliquots from HeLa and HeLa-tat cell lysates were pro-cessed for RNA extraction by the RNAzol method (Cinna/Biotecx Laboratories, Friendswood, TX), which is a modi-fication of the method ofChomczynski and Sacchi (32). TotalRNAs from parental HeLa and HeLa-tat cells were subjectedto Northern blot hybridization analysis (33) using radiola-beled human Mn-SOD cDNA as a probe. Duplicate blotswere hybridized to radiolabeled human Cu,Zn-SOD cDNA as

an internal standard. To create the probes, the coding se-quences from recombinant plasmids containing the cDNAsfor Cu,Zn- and Mn-SOD were released by restriction enzymedigestion, and the appropriate bands were identified byethidium bromide staining of low-melting-point agarose gels(NuSieve, FMC). The bands of interest were cut and radio-labeled in the melted gel by random primer labeling accordingto the manufacturer's specifications (New England Nuclear/DuPont).The complete Mn-SOD cDNA was cloned between the Pst

I and Sma I sites of plasmid pBluescript II KS(+) (Strata-gene), placing the gene under control of the bacteriophage T7promoter. After transformation of competent Escherichiacoli, recombinant bacteria were identified, clones were se-lected and amplified, and the DNA was extracted and puri-fied. This plasmid was then linearized by the appropriaterestriction enzymes and served as template for in vitrotranscription in the presence of T7 RNA polymerase and[a-32P]CTP according to the manufacturer's specifications(Promega). The DNA template was removed by digestionwith RQ1 RNase-free DNase I (Promega). As a control, weprepared radiolabeled transcript of the vector without theMn-SOD insert.

Radiolabeled (106 cpm) transcripts were incubated withand without parental or Tat-containing cell extracts (32, 35,or 40 ,g of protein per assay) in 20-25 jd of a buffercontaining 50 mM Tris-HCl (pH 7.6), 5 mM MgCl2, 2 mMdithiothreitol, tRNA at 0.2 pg/ml, salmon sperm DNA at 0.5,pg/ml, RNasin ribonuclease inhibitor (Promega) at 40units/ml and heparin at 5 mg/ml as a nonspecific competitorfor RNA-binding proteins (9). Mixtures were incubated at15°C for 15 min. To determine the size and identity of thecomplexes formed in vitro, the binding-reaction productswere UV crosslinked (9), treated with ribonuclease A (1mg/ml), and resolved in SDS/15% polyacrylamide gels. Gelswere dried and analyzed by autoradiography.

Parental and HeLa-tat cells were grown to --80%o conflu-ency in 175-cm2 tissue culture flasks in Opti-MEM (GIBCO/BRL) supplemented with 3.75% fetal bovine serum. Themedium was then replaced with serum-free Opti-MEM. Afterincubation for 24 hr, the medium was decanted and the cellswere detached by trypsinization. Vital-dye exclusion re-vealed that cell viability and numbers among the recoveredcells varied between flasks by <1%. The cells were sedi-mented and washed by resuspension in Ca2+/Mg2+-freeHanks' balanced salt solution. Cell suspensions were thenlysed by sonication and cleared by centrifugation. The su-

pernatants were divided into two equal portions: one aliquotwas processed for biochemical assays and the other aliquotwas processed for RNA extraction. Biochemical determina-tions were performed on 4-10 separate flasks of cells, as

indicated in the figure legends.Statistical analyses, where indicated, were by one-way

analysis of variance. Data are presented as the mean +

standard error. Differences between groups were determinedby the Newman-Keuls test.

RESULTS

Sonicated extracts ofHeLa and HeLa-tat cells were assayedfor Cu,Zn- and Mn-SOD. Fig. 1 shows that the level of totalSOD activity expressed in the cells producing Tat was

8 _ Total Cu,Zn Mnp <O.05 N.S. p <O.OO1

E40

HeLa HeLa-tat

FIG. 1. Total SOD, Cu,Zn-SOD, and Mn-SOD specific activities[units (U)/mg of protein] in sonicated HeLa and HeLa-tat cellextracts. The total SOD content of Tat-producing cells was 82% ofcontrols (n = 10; P < 0.05). The contents of Cu,Zn-SOD were notsignificantly different (N.S.). The Mn-SOD content of HeLa-tatcells, however, was only 48% that of controls (n = 10; P < 0.001).

decreased to 82% (P < 0.05) of that in the parental cell line.The level of Cu,Zn-SOD was 115% of the parental level andnot significantly different by statistical analysis, whereas thelevel of Mn-SOD was only 48% of parental (P < 0.001).Thin-layer agarose gels stained for SOD activity confirmedthe spectrophotometric results (Fig. 2) and, further, showedno qualitative changes in electrophoretic behavior of either ofthe two isozymes. To determine whether the decreasedMn-SOD activity observed in HeLa-tat cels correlated withdecreased Mn-SOD RNA levels, total RNAs were isolatedfrom HeLa and HeLa-tat cells. Duplicate Northern blots ofthese RNAs were hybridized with a radiolabeled humanMn-SOD cDNA probe or a radiolabeled human Cu,Zn-SODcDNA probe (Fig. 3). The steady-state levels of Mn-SODRNA are much lower in HeLa-tat cells than in the parentalcells. The multiple transcripts seen have been ascribed toalternative polyadenylylation signals (34). Cu,Zn-SOD RNAlevels were the same in both cell types. Northern blots ofpoly(A)+ RNA isolated from both HeLa parental and HeLa-tat cells showed the same patterns of expression (data notshown).A likely consequence of decreased SOD activity is an

increase in lipid peroxidation (35). Lipid peroxidation prod-ucts, which include many a,4-unsaturated aldehydes anddialdehydes (such as malondialdehyde) are reactive withprotein sulflhydryl groups, yielding products collectivelytermed "carbonyl proteins" (36). Accordingly, we measuredcarbonyl proteins in HeLa and HeLa-tat cells and found thatHeLa-tat cells had =75% more carbonyl proteins than theparental cells (P < 0.0002) (Fig. 4).As lipid hydroperoxides are produced in oxidatively

stressed cells, they are reduced to alcohols by the actions ofglutathione peroxidase and phospholipid hydroperoxide:glu-

Mn-SOD

C.).5

0

0CO

Cu,Zn-SOD

-.- +

Distance migrated

FIG. 2. Scans of thin-film agarose gel electrophoretogramsstained for SOD activity. Samples were aliquots of HeLa andHeLa-tat cell extracts containing 10 Ag of protein.

HeLa

HeLa-tat

Biochemistry: Flores et al.

Proc. Natl. Acad. Sci. USA 90 (1993)

&

~- MnSOD

*_ Cu,ZnSOD

FIG. 3. Northern blot hybridization ofHeLa and HeLa-tat RNAsto radiolabeled Mn- and Cu,Zn-SOD cDNA probes. Samples con-tained 10 Mg of total RNA. Note the decreased amount of Mn-SODRNA in HeLa-tat relative to HeLa, while the amounts ofCu,Zn-SODRNA appear equivalent.

tathione peroxidase. This drain on cellular reducing equiva-lents can ultimately appear as a loss of total cellular sulfhy-dryl content. We quantified the total sulfhydryl content ofboth HeLa and HeLa-tat cells in freshly prepared extracts,and Tat-producing cells contained 93% as much as parentalcells (difference not significant). When extracts were assayedagain after storage for 2 weeks at -20°C, the sulfhydrylcontent of Tat-producing cells had decreased to 68% ofparental (P < 0.0001) (Fig. 4B).Because all known mechanisms of action of Tat are man-

ifested via binding to RNA stem-loop structures (9, 37), wesought evidence for the binding of Tat to Mn-SOD tran-scripts. Radiolabeled Mn-SOD transcripts and control tran-scripts of plasmid alone were synthesized in vitro and incu-bated in the presence of HeLa or HeLa-tat cell extract.Proteins were crosslinked to the RNA by UV irradiation.After digestion of the unprotected regions of RNA by ribo-nuclease A, the protein-RNA adducts were subjected toSDS/PAGE (Fig. 5). Heparin was added as a nonspecificpolyanionic competitor for RNA-binding proteins. Two pre-dominant crosslinked products were observed from the in-cubations that contained HeLa-tat cell extracts. These spe-cies migrated with apparent molecular masses of 14 and 9kDa, based on comparison with protein standards. (The9-kDa band did not separate completely from the third large,fully digested band seen at the bottom of each lane.) The twospecies protected from ribonuclease digestion were not ob-served with the incubations containing parental HeLa ex-tracts. No protected species were observed when the controlradiolabeled plasmid-alone transcript was substituted for the

c

.-C

0

0

as 0.90

E 0.60

EC

.

804-.

cnX*- C

0 E'-

FIG. 4. Additional indices of oxidative stress in HeLa-tat cells.(A) Carbonyl proteins in extracts of HeLa and HeLa-tat cells (n =

4; P < 0.0002). (B) Total sulfhydryls reactive with Ellman's reagent[5,5'-dithiobis(2-nitrobenzoic acid)] in cell extracts after storage at-20°C for 2 weeks (n = 9 for HeLa-tat; n = 10 for HeLa; P < 0.0001).

Putativetat/RNAcomplexes

Fully digestedtranscript

FIG. 5. Protection of Mn-SOD transcript from ribonuclease Adigestion. Cell extracts were incubated with radiolabeled Mn-SODtranscripts and treated as described in Materials and Methods. Theautoradiogram shows two crosslinked products indicated by arrows(and observed only in the incubation that contained HeLa-tat cellextracts), as well as a third large band in each lane representing fullydigested transcript. The two protected species migrated with appar-ent molecular masses of 14 kDa and 9 kDa.

Mn-SOD transcripts, indicating the specificity of the inter-action between Tat and the Mn-SOD transcript.

DISCUSSIONThe relationship between viral infection and oxidative stresshas not been well understood. It has been shown thatHIV-infected HUT-78 cells have decreased levels of Mn-SOD and lose their ability to induce this enzyme in responseto tumor necrosis factor (6), but no mechanism was delin-eated. The loss of SOD might simply reflect the greatlyperturbed metabolism of a dying, virus-infected cell, or itmight reflect a specific event brought about by the virus inorder to ensure conditions appropriate for replication andsurvival. If the latter case were true, one might ask how thevirus could bring about specific repression ofa host gene. TheHIV genome contains several genes which encode regulatoryproteins, one of which is tat. Thus, the tat gene productseemed a likely candidate to examine further. Experimenta-tion was facilitated by the availability of HeLa cells stablytransfected with this single HIV gene, eliminating the cyto-pathic effects of viral infection. We therefore examined levelsof Mn-SOD expression in HeLa and HeLa-tat cells.We found that Tat-producing cells had significantly less

total SOD activity than parental cells. The difference wasattributable to their production ofonly half as much Mn-SODactivity per milligram of protein, while their content of theCu,Zn enzyme was unchanged (Fig. 1). The identical elec-trophoretic behavior ofMn-SOD from the two cell types (Fig.2) eliminates the possibility that the Mn-SOD gene wasdisrupted by the transfection process. Further, the promoter-proximal regions responsible for regulation of the Mn-SODgene appeared to be intact, as both cell lines responded totumor necrosis factor by induction of Mn-SOD, albeit to alesser extent in the Tat-producing cells (data not shown).Wong et al. (6) saw no response ofMn-SOD to tumor necrosisfactor in acutely infected HUT-78 cells, which may reflect theinvolvement of other, Tat-independent events in the regula-tion of this gene in HIV-infected cells.Humans have two genes encoding intracellular SODs, but

only the Mn-SOD gene is known to be inducible and regu-lated. In humans, this gene product may account for >70%of the cell's SOD activity (38). The inability of the constitu-tively expressed Cu,Zn-SOD gene to respond to changes inoxidative status ensures that repression of Mn-SOD will

7634 Biochemistry: Flores et al.

N,

Proc. Natl. Acad. Sci. USA 90 (1993) 7635

result in an imbalance of antioxidant defenses that cannot becompensated.Down-regulation of cellular Mn-SOD by the HIV Tat

protein may lead to oxidative stress by the sequence ofeventsshown in Fig. 6. The rise in superoxide radical concentrationleads to mobilization of iron from tissue ferritin (39) or fromproteins containing the 4Fe/4S chromophore, such as aconi-tase (40). The liberated iron catalyzes lipid peroxidation (41),seriously affecting the structural integrity and function of cellmembranes. The accumulating phospholipid hydroperoxidesare reduced by phospholipid hydroperoxide:glutathione per-oxidase (42), or the peroxidized fatty acids are liberated byphospholipases and subsequently reduced by glutathioneperoxidase. These enzymes deplete reduced glutathione, andcytosolic NADPH is in turn drained by glutathione reductasein an attempt to maintain reduced glutathione levels. Glu-tathione, which is present in the cell in millimolar amounts,provides cells with their reducing environment and serves asthe major cellular antioxidant. It acts in conjunction withglutathione peroxidase to eliminate hydrogen peroxide aswell as organic peroxides and in synergy with SOD as animportant antioxidant defense system (43). Because of thedrain in cellular glutathione and its link to the reducedpyridine nucleotide pool, a state of generalized oxidativestress results.The data shown in Fig. 4A reflect an end result ofoxidative

stress. Peroxidation of polyunsaturated fatty acids producesa variety of toxic and reactive products which include a,,-unsaturated aldehydes such as 4-hydroxynonenal. Thesealdehydes may react with protein sulfhydryl groups via aMichael-type addition reaction, resulting in covalently boundaldehyde groups and loss ofprotein sulfhydryls (36). The lossof reduced glutathione resulting from oxidative stress, how-ever, is much greater than can be accounted for by thismechanism alone and may be as great as S or 6 mM (44). Thisprobably reflects formation of mixed disulfides betweenoxidized glutathione and protein sulfhydryls. Living cellsstrive to maintain steady-state levels of reductants via normalmetabolic pathways, so that a sizable drain may produce onlya small perturbation in the steady state. We believe thatHeLa-tat cells have elevated levels of lipid peroxidationgoing on, but that the loss of sulfhydryls is within the cells'ability to repair it by maintaining the level of reducedglutathione. Upon cell death, the ability to repair comes to anend, but the lipid peroxidation continues to drain reductants,which can no longer be replenished. Thus, we observed acontinuing loss of sulflhydryls in stored cell extracts, but witha significantly higher rate of loss in Tat-producing cell lysates(Fig. 4B). This mechanistic link between lipid peroxidation

Decreased MnSOD

Increased superoxideFerritin i Fe/S proteins

Liberation of iron

Iron-mediated lipid peroxidation

Elevated lipid hydroperoxidesPLHPGPx I GPx

Glutathione depletionI GR

NADPH depletion

Oxidative stress

FIG. 6. How suppression of Mn-SOD might lead to oxidativestress. PLHPGPx, phospholipid hydroperoxide:glutathione peroxi-dase; GPx, glutathione peroxidase; GR, glutathione reductase.

and sulfhydryl consumption may account for the decreasedglutathione levels observed clinically in HIV-infected pa-tients (2) as well as in HIV-infected monocytes (3).The Tat protein has been reported to interact with the TAR

region contained at the 5' end of all HIV transcripts, but themechanisms by which this interaction regulates HIV geneexpression are controversial. The fact that Tat has beenshown to bind only to RNA makes it somewhat difficult toenvision a mechanism for transcriptional regulation, yet ourdata (Fig. 3) are consistent with this view. Clearly, thesteady-state levels of Mn-SOD mRNAs are lower in Tat-producing cells, probably reflecting a net decrease in tran-scription. On the other hand, our data support an interactionbetween Tat and Mn-SOD mRNA (Fig. 5). It seems highlyunlikely that this interaction is coincidental and independentof the down-regulation of SOD.

Others have reported alterations in cellular gene expres-sion in cells transfected with the tat gene, and certain oftheseobservations may be secondary to repression ofMn-SOD andthe resulting rise in superoxide, or to the oxidative stress thatfollows. tat expression leads to transformation of primarykeratinocyte cultures (13), increased collagen expression inglioblastoma cells (14), and decreased interleukin 2 receptorexpression in Jurkat cells (15). The tumorigenic potential oftat is further demonstrated by the induction ofdermal lesionsresembling Kaposi sarcoma in transgenic mice (16), alter-ations in the expression of putative angiogenesis factors afterintroduction of Tat-producing cells into nude mice (17), andgrowth promotion of cells derived from Kaposi sarcomalesions of AIDS patients (18). The observed increase incollagen synthesis may, in particular, reflect the increasedconcentration of superoxide in Mn-SOD-repressed cells.Chojkier et al. (45) found that iron plus ascorbate, an oxidant-generating system, stimulated collagen production in cul-tured human fibroblasts. Those authors also found stimula-tion by the lipid peroxidation product malondialdehyde.Chandrakasan and Bhatnagar (46) found stimulation of col-lagen synthesis by superoxide exposure in cultured fibro-blasts. Hence, this example of a Tat-dependent phenomenonis likely a consequence of Tat's repression of Mn-SOD.Similarly, transformation of keratinocytes (13) may be areflection ofrepressed Mn-SOD. Exposure to superoxide andsecondary oxidants led to the induction of the oncogenesc-jun and c-fos (47, 48) or c-myc (49), to increased cellularproliferation (19), and to lung hyperplasia and fibrosis inhamsters (50). On the other hand, Mn-SOD expression wasshown to correlate with the differentiation state. Activity wasincreased in differentiating trophoblasts (51) and Frienderythroleukemia cells (52). Thus, inhibition of SOD activitycould potentially cause neoplastic transformation.Some viruses appear to have evolved mechanisms to

control cellular oxidant status. Influenza virus infection leadsto increased production of superoxide by lung epithelial cells(53), partly due to increased activity of the superoxide-generating enzyme xanthine oxidase (54). Our data suggestthat HIV similarly takes control of the cell's redox status.Why would increased oxidative stress be advantageous to thevirus? Exposure of cells to oxidants results in activation oftranscriptional factors and/or increased proliferation-conditions necessary for successful viral replication. One ofthese cellular transcriptional factors is nuclear factor KBB,whose activity is modulated by the cellular redox status (20).Interestingly, this factor also modulates HIV-1 gene expres-sion (55). The effect of oxidant exposure on cell proliferationhas been discussed above.The recognition that Tat may be instrumental in viral

regulation of host oxidant status underscores the potentialimportance of this HIV protein. A fascinating property of Tatis that it appears to be secreted by infected cells and can betaken up by noninfected cells (56, 57). That is, Tat can effect

Biochemistry: Flores et al.

Proc. Natl. Acad. Sci. USA 90 (1993)

transcellular transactivation. Hence, host tissues not actu-ally infected may nevertheless have their oxidant statusmodified at a distance, possibly rendering those tissues moreamenable to infection by other organisms. Transceilulartransactivation probably accounts for the aforementionedability ofTat protein to promote growth of cells derived fromKaposi sarcoma lesions of AIDS patients (18) and for thealterations in the expression of putative angiogenesis factorsafter introduction of Tat-producing ceils into nude mice (17).The recent description of a drug (Ro 5-3335) that acts as a

potent Tat antagonist (58) raises the possibility that Tat maybe a vulnerable target for therapeutic intervention. Thecompound inhibits HIV replication in infected cells (59) andrestores cell surface CD4 expression (60).

This work was supported in part by a Cardiovascular DiscoveryGrant from Glaxo. S.C.F. was supported in part by Grant 5P50HL40784-SCOR-MIRS from the National Institutes of Health.S.K.N. was supported in part by the Institutional Training Grant forNutrition T32 DK07658 from the National Institutes of Health. TheHeLa-tat-III cell line was provided by the National Institutes ofHealth AIDS Research and Reference Reagent Program and wasoriginally contributed by Drs. W. Haseltine and E. Terwilliger.

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