Proc. Nati. Acad. Sci. USAVol. 85, pp. 2733-2737, April 1988Immunology

rpt-1, an intracellular protein from helper/inducer T cells thatregulates gene expression of interleukin 2 receptor andhuman immunodeficiency virus type 1

(T-cell activation/acquired immunodeficiency syndrome/mouse lymphocytes)

ROBERTO PATARCA*, JOEL SCHWARTZ*, RAJESH P. SINGH*, QuI-TONG KONG*, ELIZABETH MURPHY*,YVONNE ANDERSON*, FENG-YI WEI SHENG*, PRATIMA SINGH*, KAREN A. JOHNSON*,SHEILA M. GUARNAGIA*, TIMOTHY DURFEEt, FRED BLATTNERt, AND HARVEY CANTOR**Laboratory of Immunopathology, Dana-Farber Cancer Institute, Department of Pathology, Harvard Medical School, Boston, MA; and tUniversity ofWisconsin, Madison, WI

Communicated by Frank Lilly, December 9, 1987

ABSTRACT The Rpt-1 (for regulatory protein, T-lymph-ocyte, 1) gene, selectively expressed by resting but not byactivated CD4+ inducer T cells, encodes an intracellularprotein (rpt-1, Mr 41,000) that down-regulates gene expressiondirected by the promoter region of the gene encoding interleu-kin 2 receptor a chain and by the long terminal repeat ofhuman immunodeficiency virus type 1. The data reported heresuggest that rpt-1 levels may be inversely correlated withactivation of CD4 + T cells and human immunodeficiency virusreplication leading to clinical symptoms of the acquired im-munodeficiency syndrome.

Analysis of cellular and viral proteins produced by clones ofinducer, cytotoxic, and suppressor T lymphocytes hasshown that each T-cell subset is genetically programmed tospecify particular patterns of protein synthesis before andafter activation by antigen (1-3). For instance, the levels ofexpression of human immunodeficiency virus type 1 (HIV-1), the retrovirus associated with the acquired immunodefi-ciency syndrome, are markedly increased upon activation ofinducer T cells (3-6). Insight into the molecular basis ofcellular and retroviral gene expression requires a descriptionof the intracellular proteins that regulate genes expressed inresting and activated T cells (e.g., ref. 5). The studiesreported here define a recombinant intracellular product,termed rpt-1,f that is selectively expressed by resting in-ducer T cells and affects gene expression directed by thelong terminal repeat (LTR) promoter region of HIV-1 or bythe promoter region of the gene encoding the a chain of theinterleukin 2 receptor (IL-2Ra).

MATERIALS AND METHODSCells. All cells were of murine origin unless otherwise

indicated. Cl.Lyl-T1 and CI.Lyl-N5 are helper/inducerT-cell clones (7, 8), Cl.NK-11 is a natural killer cell clone (9),and Cl.Ly23.4 is a suppressor T-cell clone (10). Ar-5 is anarsonate-reactive helper/inducer T-cell clone (11). Ar-5v is avariant of Ar-5 that constitutively expresses high levels ofIL-2Ra. Ar-5v, but not Ar-5, is activated by recombinantIL-2 (Genzyme, Norwalk, CT) in the absence of antigen, andthis activation is completely blocked by anti-IL-2R mono-clonal antibody AMT-13 (Boehringer Mannheim). Ar-5vdoes not produce detectable levels of IL-2 unless activatedby IL-2 or antigen and I-Ad macrophages. Jurkat (5) andEL-4 are human and murine T-cell lines, and COS-7m6 is a

simian virus 40 (SV40)-transformed monkey kidney epithe-lial cell line (12).

Production of a T-Cell Probe. Poly(A)+ RNA from L cells(a fibroblast tumor) and from 2PK3 (a B-cell lymphoma) wasprepared and hybridized to 32P-labeled cDNA obtained fromCl.Lyl-T1 cells (22 hr after activation) as described (13, 14).The remaining single-stranded cDNA, purified by hydroxy-apatite chromatography, was hybridized with poly(A) +RNA from MOPC 315 (a B-cell myeloma) and the single-stranded fraction was again isolated by hydroxyapatite chro-matography.

Construction of a T-Celi cDNA Library. Poly(A)+ RNAfrom Cl.Lyl-T1 (22 hr after activation) was used to preparea cDNA library of 3.8 x 105 independent clones in the pcDvector (12, 15). Approximately 11,300 colonies from a size-selected [0.5- to 20-kilobase (kb)] cDNA-insert sublibrary(19) were sparsely plated on nitrocellulose and then probedwith the T-cell-specific probe described above (16). Posi-tively hybridizing colonies were picked for further analysis.

Nucleic Acid Blot Hybridization and Protein Immunoblot-ting. RNA slot and gel blots and DNA (Southern) blots wereprepared as described (15, 17). Extracts of 107-10' mousespleen cells were analyzed by electrophoresis and immuno-blotting as described (18).

Plasmids. pcD-rptl was obtained from the T-cell cDNAsublibrary. pcD-rptlfs is a frameshift mutant obtained bydigesting pcD-rptl with the restriction enzyme BstEII, blunt-ending the restriction fragments with bacteriophage T4 poly-nucleotide kinase, and religating. pSV2cat contains the SV40enhancer/promoter region upstream of the bacterial chlor-amphenicol acetyltransferase (CAT) gene (20). The plasmidIL2RpCAT, containing the human IL-2Ra promoter regionupstream of CAT was kindly provided by W. Leonard (21).The plasmid pLTR-1CAT, containing the U3 and R regionsof the HIV-1 LTR (nucleotides -463 to +80) (6, 22), andpSV7fdtat, an expression vector for the tat-encoded proteinof HIV-1 (6, 22), were kindly provided by P. Luciw.

Transfection and CAT Assays. Adherent cells (106 pertransfection) and cells grown in suspension (107 per trans-fection) were transfected by the DEAE-dextran technique(23). CAT assays were performed 48 hr after transfection(20). Experiments were done in the linear range of the assay

Abbreviations: CAT, chloramphenicol acetyltransferase; HIV-1,human immunodeficiency virus type 1; IL-2, interleukin 2; IL-2Ra,a chain (p55/Tac) of the IL-2 receptor; LTR, long terminal repeat;SV40, simian virus 40.tThe rpt-1 sequence reported in this paper has been deposited in theEMBL/GenBank data base (Bolt, Beranek, and Newman Labora-tories, Cambridge, MA, and Eur. Mol. Biol. Lab., Heidelberg)(accession no. J03776).

2733

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Proc. Natl. Acad. Sci. USA 85 (1988)

FIG. 1. (A) Expression of Rpt-J in lymphocyte clones. Five A B Cmicrograms of poly(A) + RNA from each cell type was analyzed by rpt -1electrophoresis in a 1.5% agarose gel, transferred to nitrocellulose, a b C d e f g h a b 0 4 8 12 16 20 24and hybridized with a 2P-labeled probe prepared by nick-translationof the 3.7-kb cDNA insert of pcD-rptl. Cell types were as follows:MOPC 315, a murine myeloma (lane a); Cl.NK1.1, a Thy-i+ natural -killer cell clone (lane b); Cl.Ly23.4, a suppressor T-cell clone with(lane c) or without (lane d) Con A; Cl.Lyl-T1, a helper T-cell clone _without (lane e) or with (lane f) Con A; and Cl.Lyl-N5, a helperT-cell clone without (lane g) or with (lane h) Con A. RNA washelpeobtained 15 hr after activation with Con A at 5 ,ug/ml. When I - IFnormalized against actin mRNA, the intensity of the radioactive - 0 4 8 12 16 2024signal in lanes f and h is -50% of that in lanes e and g, respectively.Size markers at left correspond to 6000, 1765, 1426, and 920 bases.(B) Expression of Rpt-1 in heterogeneous lymphocyte populations. _ 3Five micrograms of poly(A)+ RNA from splenocytes (lane a) orthymocytes (lane b) was hybridized with the Rpt-1 insert as de-scribed above. (C) Time course of Rpt-1 expression upon activationof the T-cell clone Cl.Lyl-T1. Five micrograms of total RNA wasobtained from Cl.Lyl-T1 at the indicated times (hours) after stimulation by antigen (trinitrophenylated bovine gamma globulin) and splenicadherent cells (7-10). After electrophoresis and transfer, blots were hybridized with a nick-translated probe corresponding to the 5' 1.3-kb RsaI-Xba I fragment (coding region) of Rpt-I (Upper) or to the coding region of y-interferon (Lower).

and were repeated 3-5 times with different batches ofpurified DNA.

Immunofluorescence. To detect rpt-1, cells were placed oncoverslips and incubated consecutively with glutaraldehyde(0.5%; 30 sec), the IgG fraction of a rabbit anti-Lys-Lys-Glu-Lys-Lys-Glu (anti-KKEKKE) antiserum (1:200 final dilu-tion; 4°C, 40 min), and a rhodamine-conjugated goat anti-rabbit IgG (1:100; Cooper Biomedicals, Malvern, PA). Todetect cell surface antigens, Thy-1+ and Ig+ spleen cells (2,8, 10) were incubated with either AMT-13, L3T4, or Ly-2monoclonal antibodies (Becton Dickinson), washed thrice,and incubated with fluorescein isothiocyanate-conjugatedgoat anti-rat IgG (1:200; Cooper Biomedicals). For each cellpopulation analyzed by flow cytofluorimetry with the fluo-rescence-activated cell sorter (FACS), the intensity of fluo-rescence is presented on a base-10 logarithmic scale (Figs. 4

0 1000-= = =_-- x-

,L4~, -a -ab-v 0O

2000

O. 0

_ 0 =

I I I I I I I

ORF 353 AA-~~~~~~~~~~~~ and 6). Where indicated, cells were visualized by a fluores-

cence photomicroscope (Olympus IMT2) with barrier filtersof 420-490 nm for fluorescein and 500-590 nm for rho-damine.

RESULTSAfter activation by antigen or by the T-cell mitogen conca-navalin A (Con A), the T-cell clone Cl.Lyl-T1 undergoesone or two rounds of division and secretes inducer-specificproteins (1, 8). Of the 11,300 colonies from a size-selectedCl.Lyl-T1 pcD cDNA library, 230 hybridized to a cDNAprobe enriched for genes expressed in T cells (see Materialsand Methods). One of these inserts, termed Rpt-l (regula-tory protein, T-cell), hybridized to a 3.7-kb mRNA presentin helper/inducer T-cell clones (Fig. 1A, lanes e-h), but not

3000 3800

*p

** polyAI

: ,I:-,'-.'',' : ' '.o. --4--- ------ F----- ----- -- --

RTR7- F2 TR7-D

CTGCAGGGGGGGGGGGGGTCTTCACAGATCCTGAGTCTCTTGGTTGGTATCTGAAGATGTGAGCCAATTTCCAACGCAGA

GTTTGGAGGAATTTAGAAAAAGGCACAGTCTTTGCTCTGCAGTGTGAGGAGTCAGGACAGGCAGACACAGGATCACAGCAG V R T G R H R I T A

ACTATGGCCTCATCAGTCCTGGAGATGATAAAGGAGGAAGTAACCTGTCCTATCTGTTTGGAGCTCCTGAAGGAACCTGTT ( A S S V L E M I K E E V T © P I © L E L L K E P VGAGTGCTGATTGTAACCACAGCTTCTGCAGAGCCTGCATCACACTGAATTATGAGTCCAACAGAAACACAGACGGGAAGG

27 S A D C N H S F ©) R A ©) I T L N Y E S N R N T D G K GGCAACTGCCCTGTATGCCGAGTTCCTTACCCATTTGGGAATCTGAGGCCTAATCTACATGTGGCCAACATAGTAGAGAGG

54 N ) P V © R V P Y P F G N L R P N L H V A N I V E RCTCAAGGGATTCAAGTCCATTCCAGAGGAGGAGCAGAAGGTGAATATCTGTGCACAACATGGAGAGAAACTCCGGCTCTT

80 L K G F K S I P E E E 0 K V N I C A Q H G E K L R L FCTGTAGGAAGGACATGATGGTCATCTGCTGGCTTTGTGAGCGATCTCAGGAGCACCGTGGTCACCAAACAGCTCTCATTG

107 C R K D M H V ' C W L © E R S Q E 9 R G 9 Q T A L I EAAGAGGTTGACCAAGAATACAAGGAGAAGCTGCAGGGAGCTCTGTGGAAGCTGATGAAAAAGGCAAAAATATGTGATGAA

134 E V D Q E Y K E K L 0 G A L W K L H K K A K I C D ETGGCAGGATGACCTTCAACTGCAGAGAGTTGACTGGGAGAACCAAATACAGATCAATGTAGAAAATGTTCAGAGACAGTT

160 W Q D D L Q L 0 R V D W E N 0 I Q I N V E N V 0 R 0 FTAAAGGACTAAGAGACCTCCTGGACTCCAAGGAGAATGAGGAGCTGCAGAAGCTGAAGAAAGAGAAGAAAGAGGTTATGG

187 K Q L R D L L D S K E N E E L 0 K L IK K E K K E V H EAAAAGCTGGAAGAGTCTGAAAATGAGCTGGAGGATCAGACAGAGTTGGTGAGAGACCTCATCTCAGATGTGGAACATCAT

214 K L E E S E N E L E D 0 T E L V R D L I S D V E H HTTGGAGCTCTCAACCTTAGAAATGCTGCAGGGTGCAAATTGTGTCCTGAGAAGGAGTCAGTCCTTAAGCCTGCAACAGCC

240 L E L S T L E H L 0 G A N C V L R R S 4 S L S L Q 0 PCCAAACTGTCCCCCAAAAGAGAAAAAGAACATTCCAAGCTCCAGATCTGAAAGGCATGCTGCAAGTGTATCAAGGACTCA

267 Q UT V P Q K R K R Tl F 0 A P D L K G M L Q V Y 0 G L MTGGATATCCAGCAATACTGGGTGCATATGACTCTACATGCAAGGAACAATGCAGTCATTGCCATTAACAAAGAAAAAGAC

294 D I Q Q Y W V H H T L H A R N N A V I A I N K E K DAAATACAGTATAGAAGTTACAATACGGTTCCAGTTTCTGAGATCTACCATTTGGGTGTCCTGGGATATCCAGCTCTTTCC

320 K Y S I E V T I R F Q F L R S T I W V S W D I Q L F PTCAGGGAAGCATTACTGGGAAGTAGACATATCTAGAAGTGATGCCTGGCTCCTCGGATTAAATGACGGAAAGTGTGCTCA

347 Q G S I T G K *

ACCCCAACTTCACTCAAAGGAAGAAATGGGCATCAAAAAAAACCTTCATTCTCAGATCAAACAAAATGTATTGTTTCAGC

FIG. 2. Restriction map of the insert of80 pcD-rptl and sequence of the coding re-

160 gion. The sequence was determined by themethod of Maxam and Gilbert (24) and

240 was confirmed in its entirety on both320 strands. The restriction map includes ar-

400 rows to indicate the extent and directionof sequence determined and asterisks to

480 denote potential polyadenylylation sites.560 The position of the open reading frame640 encoding the 353 amino acid rpt-1 protein

(ORF 353 AA) is indicated. TR7-D and720 TR7-F2 are partial cDNA clones and800 TR7-X is a full-length cDNA clone. The880 predicted amino acid sequence is given in

one-letter symbols below the nucleotide960 sequence. Cysteine and histidine residues1040 that may be involved in metal-finger for-1120 mation are circled. The first methionine,

the predicted most hydrophilic region(amino acids 205-210), and the putative

1280 nuclear localization signal (amino acids1360 268-276) are boxed.

- TR7-X

250bp

L--

2734 Immunology: Patarca et al.

*

Proc. Natl. Acad. Sci. USA 85 (1988) 2735

_ | A~~~COS-rpt-l+

- 58,000

-48,500

-36,500Mean Fluorescence Intensity

C

C-

FIG. 3. (A) Immunoblot of mouse spleen-cell extracts. Blot wasprobed with a rabbit IgG (1:200 dilution) obtained after immuniza-tion with keyhole limpet hemocyanin conjugated to a syntheticpeptide corresponding to the most hydrophilic portion of the rpt-1protein (KKEKKE; see Fig. 4). Bound IgG was visualized byincubation with 1251I-labeled goat anti-rabbit antibody and autoradi-ography. Molecular weight markers (Sigma) correspond to Mr58,000, 48,500, and 36,500. Background bands are nonspecific, sincethey also appeared in the marker lane. (B) COS-7m6 cells weretransfected with 6 jig of either pcD-rptl (COS-rpt-1 +) or pcD-rptlfs(COS-rpt-lfs). The FACS profile of an aliquot of each cell popula-tion after incubation with the anti-KKEKKE antibody is shown.Slot blot analysis of RNA from both groups of COS-7m6 cellsindicated equal amounts of mRNA that hybridized to the Rpt-1probe. (C) Immunofluorescence of a COS-rpt-1 + transfectant. Pre-dominantly nuclear fluorescence was seen in the COS-rpt-1 popu-lation. Immunofluorescence was not detectable in the COS-rpt-lfscells.

in a suppressor T-cell clone (lanes c and d), a natural killercell clone (lane b), or a murine myeloma cell line (lane a).Expression was not dependent on long-term growth of T-cellclones, since the Rpt-J probe hybridized to a 3.7-kb RNAfrom freshly explanted lymphoid cells in spleen and thymus(Fig. 1B).

Resting inducer T cells (Fig. 1A, lanes e and g) showedhigher levels of Rpt-1 RNA than activated inducer T cells(lanes f and h). Analysis of the time course of Rpt-1expression during activation of Cl.Lyl-T1 showed that Rpt-1transcription was not detectable 4-8 hr after activation (Fig.1C Upper). Expression of other genes we have studied,including T-cell-specific genes, is either unchanged (data notshown) or, more commonly, increased after activation (Fig.1C Lower).The complete nucleotide sequence of the full-length Rpt-1

clone cDNA insert [3700 base pairs (bp)] was determined

cc cc ccI I I

CCHHII

I

- ulLo Co a) _M m a W- _ 2~cxi cuj oj N 0N N~Cj CNJ F4 to) tq) m cx F

'IW.WO'T§ .;f ..

FIG. 5. Southern blot analysis. Genomic DNA from liver ofC57Bl/6 (B) and DBA (D) mice and of the indicated recombinantinbred strains derived from B and D parental strains (The JacksonLaboratory) (32) was digested with HindIll. Genomic DNA fromCl.Lyl-T1 (Ti), the helper T-cell clone (derived from a BALB/cmouse) from which the rpt-1 cDNA was isolated, was treated in thesame way. The digested DNA was electrophoresed in a 0.7%agarose gel, blotted onto a Zeta-Probe membrane (Bio-Rad), andwas hybridized with a 32P-labeled probe prepared by nick-translation of the 5' 1.3-kb Rsa I-Xba I fragment (coding region) ofpcD-rptl. The B-strain DNA digest shows a major band at 10 kb andminor bands at 9.5, 4, 2, 1, and 0.8 kb, whereas the D strain DNAshows a major band at 9 kb and minor bands at 12, 10, and 1 kb.

(24) and found to contain an open reading frame 353 aminoacids long (nucleotides 165-1226; Fig. 2), followed by a verylong 3' untranslated region (2466 bp; sequence to be pub-lished elsewhere) with several potential polyadenylylationsignals. The subsequence that includes the first methioninecodon fits the consensus for eukaryotic translation initiationsignals (25). The predicted protein (rpt-1) has a molecularweight of 41,330, is relatively neutral in charge (pI of 6.29),and includes a potential N-linked glycosylation site (aminoacid 31). However, the lack of an obvious signal sequence ormembrane-spanning region (26) makes it unlikely to be amembrane-bound protein.Immunoblot analysis of mouse spleen cell extracts re-

vealed a protein of the expected molecular weight (Fig. 3A).Immunofluorescence analysis of different lymphoid lineagesshowed that <0.3% of splenic Ig+ cells (B cells), <2% ofLy-2+ T cells, and 78-91% of L3T4+ cells were positive forrpt-1.The carboxyl-terminal half of rpt-1 has a subsequence

(Thr-Val-Pro-Gln-Lys-Arg-Lys-Arg-Thr, amino acids 268-

I I

VS NRP A S NE D E TK C Y DL N N GL H L KE S T GL F I N

IC\MeR A uMe' PP\ A R \ VPC/c c c

,l

I

IIlIIl

II

RSQRO0E E

LC' HRWC/M H G2l

N L

Lo

KKEKKE [VQK R K R|T rpt-lKKEKKE TP KK KR|V SV40

FIG. 4. Hydrophilicity plot of the pre-dicted rpt-1 protein (25). Above the hori-zontal line, hydrophilic; below, hydropho-bic. Potential finger structures includingthe pairs of cysteine (C) and histidine (H)residues marked above the plot (circled inFig. 2) are schematized in the lower panel(Me, metal ion). The location of the pre-dicted most hydrophilic region in rpt-1(KKEKKE) is shown (black box), as wellas the subsequence similar to the nuclearlocalization (NL) signal of the SV40 largetumor (T) antigen (26).

rpt-1*rpt-1~~~~~~~~~~~~

Immunology: Patarca et A

Proc. Natl. Acad. Sci. USA 85 (1988)

276; Fig. 2) similar to the nuclear localization signal of theSV40 large tumor antigen (Fig. 4 and ref. 27), which may

interact with a receptor in the nuclear envelope (28). COS-7m6 cells transfected with the expression vector pcD-rptl butnot pcD-rptlfs (a frameshift mutant of rpt-1) were positive forrpt-1 (Fig. 3B) and microscopic observation showed predom-inant nuclear staining (Fig. 3C).The amino-terminal half of the rpt-1 protein contains pairs

of cysteine residues (Figs. 2 and 4) in the pattern Cys-Xaa2-Cys-Xaa16-Cys-Xaa2-Cys-Xaal6-Cys-Xaa2-Cys, which issimilar to those found in proteins involved in metal bindingand regulation of gene expression (reviewed in ref. 29). Thecysteine residues are believed to bind metal ions such asZn2 +, while the intervening amino acids protrude in a

fingerlike projection. The rpt-1 protein has the potential toform two alternative fingers (Fig. 4). Secondary-structurealgorithms (30, 31) predict that the regions containing thecysteine pairs are hydrophobic and in a 8-sheet configura-tion, whereas the intervening segments representing finger-like projections are hydrophilic and include a 13-turn (Fig. 4).There is an additional pair of cysteine residues in the rpt-1protein followed by a pair of histidines with the potential toform a metal-binding finger (Fig. 4).

A

Ez

C.,

To determine whether the Rpt-J gene maps to any of theloci that are thought to affect T-cell function, we determinedthe genetic linkage of Rpt-J by using restriction fragmentlength polymorphisms present in recombinant inbred strainsderived from C57B1/6 (B) and DBA (D) mouse strains. EachB xD strain could be unambiguously assigned a parentalorigin and correlated with the genetic maps of the 26 B x Dstrains. This analysis indicated linkage to Hbb, the hemo-globin 3-chain locus on chromosome 7 (Fig. 5).A polymorphism that affects expression of IL-2Ra on

L3T4+ inducer T cells (but not Ly-2+ T cells) has also beenlinked to the Hbb locus by mapping using B x D lines (33).Analysis of the relationship between expression of rpt-1 andIL-2Ra on various T-cell clones showed an inverse correla-tion (Fig. 6A). Moreover, T cells (Ar-5v, see Materials andMethods) transfected with pcD-rptl, but not pcD-rptlfscDNA, pcD, or pcD-lacZ (data for latter two not shown),showed reduced levels of IL-2Ra (FACS analysis, Fig. 6A).The following experiments were performed to determinewhether these results reflected a direct effect of rpt-1 on

IL-2Ra gene expression.Cotransfection of pcD-rptl, compared with pcD-rptlfs,

pcD, or pcD-lacZ (data for latter two not shown), resulted in

Mean Fluorescence Intensity

Cos TEL-4 JURKATRResting Activated Resting Activoted

2 31 4 5 6 7 8 12 13 14 16

*~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~IO

0 0

0 1 0 3 1 1 1 03 1 11

0 1 1 1

FIG. 6. (A) FACS analysis of rpt-1 and IL-2Ra expression by clones Ar-5 (Left) and Ar-5v (Center). The IL-2Ra fluorescence profile ofAr-5 shown was indistinguishable from the FACS profile obtained using an irrelevant first antibody (data not shown). Twenty-four hours afterstimulation with recombinant IL-2, aliquots of Ar-5v were transfected with 6 Mg of pcD, pcD-rptlfs, or pcD-rptl before measurement of surfaceIL-2Ra by FACS analysis 48 hr later (Right). Approximately one-third of the cells transfected with pcD-rptl (hatched bars), but not withpcD-rptlfs, did not express significant levels of IL-2Ra (compare with IL-2Ra FACS profile of Ar-5, Left). The levels of IL-2Ra expressed bycells after transfection with pcD-rptlfs (Right) did not differ significantly from those of cells after transfection with pcD or pcD-lacZ (data notshown). Slot blot analysis of these transfectants showed that they had similar levels of interleukin 3 mRNA and confirmed the reduction inIL-2Ra levels. (B) CAT assays of COS-7m6 cells and of resting and activated EL4 and Jurkat cells. Jurkat cells were activated 20 hr beforethe CAT assays by the addition of phorbol 12-myristate 13-acetate (40 ng/ml) and phytohemagglutinin (10 ug/ml). In addition to these, calciumionophore A23187 (5 ,g/ml) was added for activation of EL4 cells. CAT reaction mixtures were incubated for 20 min, and the conversion ofradioactive chloramphenicol to its acetylated derivatives was assessed by thin-layer chromatography followed by autoradiography. Results areshown for cells transfected with the following plasmids (see Materials and Methods). COS7-m6 cells: 4 ,ug of IL2RpCAT cotransfected with6 ug of either pcD-rptl (lane 1) or pcD-rptlfs (lane 2); 3 ug of pLTR-1CAT cotransfected with 6 ,ug of either pcD-rptl (lane 3) or pcD-rptlfs(lane 4); 2 ug of pSV2cat cotransfected with 6 ,ug of either pcD-rptl (lane 5) or pcD-rptlfs (lane 6). EL-4 cells: 5 ,ug of pLTR-1CAT and 4 ,ugof pSV7fdtat cotransfected alone (lanes 7 and 10), or with 6 Mg of either pcD-rptl (lanes 8 and 11) or pcD-rptlfs (lanes 9 and 12). Jurkat cells:5 ,ug of pLTR-1CAT and 2 Mg of pSV7fdtat cotransfected alone (lanes 13 and 16) or with 3 Mug of either pcD-rptl (lanes 14 and 17) or pcD-rptlfs(lanes 15 and 18). Varying the ratio of reporter gene to Rpt-l gene (from 2:1 to 1:2) in the transfections described above resulted in inhibitionthat ranged between 50% and 209%o. Chloramphenicol conversions (percent) obtained with pcD-rptlfs cotransfections were assigned a value of1 and those obtained after cotransfection with pcD-rptl are expressed as a proportion thereof. Lanes 7, 10, 13, and 16 are included as controlsfor T-cell activation. Standard errors of absolute chloramphenicol conversion percentages obtained in each experiment were <30% of the mean.As a transfection control, expression of the Rpt-1 cDNA insert from pcD-rptl and pcD-rptlfs were confirmed with respect to protein (FACS)and RNA (slot blots using the SV40 polyadenylylation signal region from the pcD vector, to avoid hybridization with endogenous Rpt-l RNAin T cells).

Ar-5

f IL-2RaI, jI

rpt-e1

Mean Fluorescence Intensity

Ar-5v

E rpt-1

E

z

C.) 7,et".IL-2Ra

.A , _)9

Ar-5vIL-2Ra

aEz

0

Mean Fluorescence Intensity

2736 Immunology: Patarca et al.

Proc. Natl. Acad. Sci. USA 85 (1988) 2737

a 70-80% decrease in gene expression directed by thehuman IL-2Ra promoter in COS-7m6 cells (Fig. 6B, lanes 1and 2). In contrast, pcD-rptl transfection had no effect ongene expression directed by the SV40 enhancer/promoter(plasmid pSV2cat) (lanes 5 and 6) or the adenoviral thymi-dine kinase promoter (unpublished data).We investigated the effects of rpt-1 on other inducer-

specific cellular and retroviral genes. Cotransfection ofpcD-rptl, compared with pcD-rptlfs, resulted in a 70%inhibition of HIV-1 LTR-directed gene expression in COS-7m6 cells (Fig. 6B, lanes 3 and 4). Since resting T cellscontain endogenous rpt-1 and the HIV-1 LTR is expressed atlow levels in them (<0.6% chloramphenicol conversion in16-hr CAT assays near the background level due to thesilicon plates; see also refs. 5 and 6), we tested the effect ofrpt-1 on HIV-1 LTR-directed gene expression in the pres-ence of the HIV-1 trans-activator protein, the tat geneproduct. A 70% inhibition was observed in resting EL-4 andJurkat cell lines in the presence of HIV-1 tat (Fig. 6B).However, no inhibition of HIV-1 LTR-directed gene expres-sion, in the presence or absence of HIV-1 tat, was seen afteractivation of these T-cell lines (Fig. 6B).

DISCUSSIONPotential metal-binding fingers in the amino-terminal half(reviewed in ref. 29) and a stretch of predominantly chargedamino acids in the carboxyl-terminal portion (see refs.34-36) are features of several proteins that regulate geneexpression. The marked difference of character between theamino- and carboxyl-terminal halves of the rpt-1 protein maydenote at least two functional domains, as evidenced inXenopus transcription factor IIIA (37): an amino-terminaldomain that targets the protein to a particular set of nucleicacids and a carboxyl-terminal domain that exerts regulatoryactivity. rpt-1 may affect IL-2Ra and HIV-1 expressionthrough cis-acting negative regulatory elements or throughcompetition with proteins that bind to enhancer or activatorsequences.Although gene expression was only decreased by a factor

of 3-5 as assessed by reporter gene assays, preliminary datashow that the decrease is not due to a shift in the transcrip-tional start site. Moreover, this level of inhibition wasphysiologically significant since a substantial fraction (ap-proximately one-third) of the cells transfected with rpt-1 nolonger displayed surface levels of IL-2Ra required for effi-cient activation by IL-2 (Fig. 6A and ref. 38). The resultsshowed that rpt-1, like other trans-acting factors, is biolog-ically active across species. A serologically related protein ispresent in resting Jurkat cells but not detectable 16 hr afteractivation (data not shown).The hallmark of HIV-1-associated diseases is a relatively

long asymptomatic period concurrent with evidence forlow-level persistent infection (4). One explanation of HIV-1latency comes from low levels of transcription factor NF-KBin resting T cells (5, 6). Activated T cells contain increasedlevels of NF-K,3, which induces increases in HIV-1 LTR-directed gene expression (5, 6). The failure of rpt-1 todown-regulate HIV-1 LTR-directed gene expression in stim-ulated T cells suggests that NF-K/3 is dominant in theactivated state.

Expression of the Rpt-J gene in resting CD4+ (L3T4+) Tcells provides an additional basis for the latent state ofHIV-1. The rpt-1 protein directly inhibits HIV-1 geneexpression even in the presence of significant amounts of thetat gene product (Figs. 6B) and prevents expression ofsurface levels of IL-2Ra required for efficient IL-2-mediatedactivation (38) (Fig. 6). A consequence of these activitiesshould be the limitation of HIV-1-mediated destruction toclones of CD4+ cells that are specifically activated byantigenic determinants, after exposure of the immune sys-

tem to bacteria and viruses. Expression of Rpt-1 in theremaining CD4+ clones may preempt secondary activationby IL-2 and efficient expression of HIV-1, which mayaccount for the slow decay of CD4+ T cells despite chronicHIV-1 infection. A prediction of this hypothesis is that rpt-1levels in inducer T cells may be inversely correlated with theincreased HIV-1 replication responsible for the clinicalsymptoms of the acquired immunodeficiency syndrome.

We thank Dr. Gordon Freeman for his help in the initial phase ofthis work and Mrs. Susan Forgione for expert assistance in thepreparation of the manuscript. This work was supported by aCancer Research Institute grant and by National Institutes of HealthGrants A112184, A113600, and CA26695 to H.C. and GM21812 toF.B.

1. Nabel, G., Fresno, M., Chessman, A. & Cantor, H. (1981) Cell 23,19-28.

2. Fresno, M., DerSimonian, H., Nabel, G. & Cantor, H. (1982) Cell 30,707-713.

3. Zagury, D., Bernard, J., Leonard, R., Cheynier, R., Feldman, M.,Sarin, P. S. & Gallo, R. C. (1986) Science 231, 850-853.

4. Klatzmann, D. & Gluckman, J. C. (1986) Immunol. Today 7, 291-2%.5. Nabel, G. & Baltimore, D. (1987) Nature (London) 326, 711-713.6. Tong-Starksen, S. E., Luciw, P. A. & Peterlin, B. M. (1987) Proc. Natl.

Acad. Sci. USA 84, 6845-6849.7. Clayberger, C., DeKruyff, R. H., Aisenberg, J. & Cantor, H. (1983) J.

Exp. Med. 157, 1906-1917.8. Clayberger, C., DeKruyff, R. H. & Cantor, H. (1984) J. Exp. Med. 158,

1881-1894.9. Nabel, G., Greenberger, J. J., Sakakeeney, M. A. & Cantor, H. (1981)

Proc. Natl. Acad. Sci. USA 78, 1157-1161.10. Fresno, M., McVay-Boudreau, L., Nabel, G. & Cantor, H. (1981) J.

Exp. Med. 153, 1260-1274.11. Rao, A., Faas, S. & Cantor, H. (1983) J. Exp. Med. 159, 1243-1258.12. Okayama, H. & Berg, P. (1983) Mol. Cell. Biol. 3, 280-289.13. Chirgwin, J., Przybyla, A. E., MacDonald, R. J. & Rutter, W. J. (1979)

Biochemistry 18, 5294-5299.14. Freeman, G. J., Clayberger, C., DeKruyff, R., Rosenblum, D. S. &

Cantor, H. (1983) Proc. Natl. Acad. Sci. USA 80, 4094-4098.15. Okayama, H. & Berg, P. (1982) Mol. Cell. Biol. 2, 161-170.16. Maniatis, T., Fritsch, E. F. & Sambrook, J. (1982) Molecular Cloning: A

Laboratory Manual (Cold Spring Harbor Lab., Cold Spring Harbor,NY), pp. 329-343.

17. Gatti, R. A., Concannon, P. & Salser, W. (1984) Biotechniques 2,148-155.

18. Towbin, H., Staehelin, T. & Gordon, J. (1979) Proc. Natl. Acad. Sci.USA 76, 4350-4354.

19. Shen, F.-W., Saga, Y., Litman, G., Freeman, G., Tung, J.-S., Cantor,H. & Boyse, E. A. (1985) Proc. Natl. Acad. Sci. USA 82, 7360-7363.

20. Gorman, C. M., Moffat, L. F. & Howard, B. H. (1982) Mol. Cell. Biol.2, 1044-1051.

21. Leonard, W. J., Depper, J. M., Kanehisa, M., Kroenke, M., Peffer,N. J., Svetlik, P. B., Sullivan, M. & Greene, W. C. (1985) Science 230,633-639.

22. Sanchez-Pescador, R., Power, M. D., Barr, P. J., Steimer, K. S., Stem-pien, M. M., Brown-Sheimer, S. L., Gee, W. W., Renard, A., Ran-dolph, A., Levy, J. A., Dina, D. & Luciw, P. A. (1985) Science 227,484-492.

23. Queen, C. & Baltimore, D. (1983) Cell 33, 741-748.24. Maxam, A. M. & Gilbert, W. (1977) Proc. Natl. Acad. Sci. USA 74,

560-564.25. Kozak, M. (1984) Nucleic Acids Res. 12, 857-872.26. Sabatini, D. D., Kreibich, G., Monimoto, T. & Adesnik, T. M. (1982) J.

Cell Biol. 92, 1-19.27. Kalderon, D., Roberts, B. L., Richardson, W. D. & Smith, A. E. (1984)

Cell 39, 499-509.28. Goldfarb, D. S., Ganepy, J., Schoolnik, G. & Kornberg, R. D. (1986)

Nature (London) 322, 641-644.29. Berg, J. (1986) Science 232, 485-487.30. Hopp, T. & Woods, K. (1981) Proc. Natl. Acad. Sci. USA 78,

3824-3828.31. Chou, P. Y. & Fasman, G. D. (1978) Annu. Rev. Biochem. 47, 251-276.32. Taylor, B. A. (1981) Genetic Variants and Strains of the Laboratory

Mouse, ed. Green, M. C. (Fischer, New York), pp. 397-407.33. Kawamura, H., Sharrow, S. O., Alling, D. W., Stephany, D., York-

Jolley, J. & Berzofsky, J. A. (1986) J. Exp. Med. 163, 1376-1390.34. Legrain, M., DeWilde, M. & Hilger, F. (1986) Nucleic Acids Res. 14,

3059-3073.35. Keegan, L., Gill, G. & Ptashne, M. (1986) Science 231, 699-704.36. Hope, J. & Struhl, K. (1986) Cell 46, 885-894.37. Miller, J., McLachlan, A. D. & Klug, A. (1985) EMBO J. 4, 1609-1614.38. Bich-Thuy, L., DuKovich, M., Peffer, N. J., Fauci, A. S., Kehrl, J. H.

& Greene, W. C. (1987) J. Immunol. 139, 1550-1556.

Immunology: Patarca et al.