Identification of a transactivating function mapping to the putative immediate-early locus of human herpesvirus 6.

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Vol.65,No. 10

Identification of

a

Transactivating Function Mapping

to

the

Putative

Immediate-Early

Locus

of

Human

Herpesvirus

6t

MICHELLE E. D. MARTIN,* JOHN NICHOLAS, BRIAN J. THOMSON, CAROL NEWMAN,

AND ROBERTW. HONESS

Division of Virology, National Institute for Medical Research, London NW7]AA, United Kingdom Received19 April 1991/Accepted 18 July 1991

Sequencingstudies have indicated that the uniquecomponentof the human herpesvirus 6 (HHV-6)genome and the unique long segmentof the human cytomegalovirus genome are genetically colinear. Of particular interest is theidentification ofaregion oflocalCpG dinucleotidesuppression in thegenomeof HHV-6,afeature

conserved in thegenomes of human cytomegalovirus, murine cytomegalovirus, andsimian cytomegalovirus, and acharacteristic ofthemajor immediate-early loci oftheseviruses. Adjacenttothis region in HHV-6are approximately 30 copiesofa 103-to108-bpsequenceelement, which containsconsensusbinding sites forthe

transcriptionfactors AP2 and NFKB, in additiontoasingleKpnIrecognition site.Together,theseKpnIrepeat

units may compose an immediate-early enhancer, analogous to those found in the cytomegaloviruses. We presentthesequenceof this region of HHV-6 and demonstrate thatatransactivating functionis encoded bythis region.Wehaveused polymerase chain reactiontosynthesizefragments containing openreading framesand 5'sequenceswithorwithout theupstreamKpnIrepeatunits. Effectorplasmids containing these HHV-6 coding

and 5' sequences were able to effect activation of heterologous promoter-chloramphenicol acetyltransferase

(CAT)constructs, includingadenovirus E3-CAT and E4-CAT, human T-cell lymphotropic virus typeI long

terminalrepeat(LTR)-CAT,andhumanimmunodeficiency virus LTR-CAT, in cotransfection experiments in Verocells and peripheral blood lymphocytes. Furthermore, wehaveidentified the majoropenreadingframe

(RF4;2.3kb)asbeing essential for activation, andwehaveshownthat theNFKB,SP1, and TATA boxmotifs in the human immunodeficiency virus LTR are all required for full induction of the promoter by the

HHV-6-encoded transactivator.

Human herpesvirus 6 (HHV-6) is a recently identified herpesvirus, firstisolated following the in vitro cultivation of peripheral blood lymphocytes (PBLs) from six patients with lymphoproliferative disorders, two of whom were also in-fectedwith human immunodeficiency virus(HIV)(48). The

genome ofHHV-6consists ofalargely uniquesequence of 141 kbp with direct repeats ofapproximately 10 kbp at the termini. We haverecently constructed the restriction endo-nuclease map of the genome ofHHV-6 strain U1102 (40), andthis hasprovidedabasis forthefurthercharacterization ofthegenetic organizationoftheHHV-6genome. Previous

sequencing studies have indicated that the organization of

codingfeatures in the genome is colinear with the unique long segment of the genome of human cytomegalovirus (HCMV) (30). Furthermore, this has been confirmed and

extended by subsequent sequencing studies (lla, 39a, 57a, 57b). This sequencing has allowed us to predict the likely

location of theviral immediate-early (IE) regulatorygenes.

The IE genes are the first genes to be transcribed in

productive herpesvirus infections and are essential for the expression of delayed early genes, which are transcribed prior to viral DNA replication, and late gene expression.

Thus, the IEgeneproducts actastransactivators of

expres-sionfromhomologouspromotersand,inaddition,havebeen shown experimentally to transactivate heterologous viral and cellularpromoters (46). One such promoter isthe HIV

long terminal repeat (LTR), which has been shown to be transactivated by the IE genes of several herpesviruses, includingHCMV (9, 39, 41, 45),Epstein-Barrvirus(29, 37),

*Corresponding author.

t Wededicate this papertothememoryof BobHoness.

pseudorabiesvirus(59), and herpes simplexvirus(41, 42) by tat-independent mechanisms.

HHV-6 ishighlyprevalentin thepopulation,and virus has been isolated from the salivaofhealthyadults(4, 18, 32, 44)

andpersistsinperipheralblood ofamajorityofseropositive

individuals (15). Furthermore, HHV-6 has a predominantly CD4+ T-lymphocyte tropismboth in vivo and in vitro (35, 56) which, together with its ability to coinfect CD4+ T

lymphocytes with HIV and to transactivate the HIV en-hancer (12, 22, 34), has implicated HHV-6 as acofactor in the progression of HIV infection to AIDS. More recently

Lussoet al. (33) have shown that HHV-6 infection

upregu-lates the expressionofCD4, the major membrane receptor for HIVtype1 (HIV-1) (8),and thereforehasthepotentialto

expand therange ofHIV-1-susceptible cells. Thus, HHV-6

may actas a cofactor in HIV infection eitherby expanding therangeofsusceptiblecells and/orby regulatingthe

expres-sion of the HIV-1genes.Thegeneproduct(s)involved in this transactivation is yet to be characterized, although the

regulation has been reported tobe mediated either directly or indirectly through the NFKB sites in the HIV enhancer

region (34).

With a view to identifying the HHV-6 IE genes, our

interest has focused on a region near the right end of the

unique component of the HHV-6 genome corresponding preciselywiththelocation of themajorIEgenes inHCMV. This region shares with betaherpesviruses HCMV, murine

cytomegalovirus (MCMV), and simian cytomegalovirus (SCMV) boththe feature of local relative CpGdinucleotide

suppression (21)andacomplexarrayofrepetitivesequences

(5, 14, 26). We havesequencedtheputative IEgene locus of

HHV-6 and have identifiedopenreadingframes(ORFs)and

a putative enhancer of 3 kb, comprising KpnI repeatunits,

5381 JOURNALOF VIROLOGY, Oct. 1991,p. 5381-5390

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5382 MARTIN ET AL.

upstream of these ORFs. We show that effector plasmids containing this region transactivate avarietyof heterologous

promoter-chloramphenicol acetyltransferase (CAT) con-structs, including an HIV LTR-CAT target in transient expression assays, and that the major 2.3-kb

ORF,

RF4, is required for this function. Our studies using HIV LTR mutants have identified those sequences in the HIV LTR enhancer important for transactivation by the HHV-6 IE geneproduct(s).

MATERIALS AND METHODS

Cells. Vero cell monolayers (Flow Laboratories, Inc., Irvine, United Kingdom) were grown at

37°C

in Dulbecco modified Eagle medium containing antibiotics (penicillin and streptomycin; 10 and 20 pLg/ml, respectively) and 10% new-born calfserum. PBLs were isolated by centrifugation on

Ficoll-Paquegradients (Pharmacia) and grown at

37°C

in5% CO2 in RPMI 1640 medium supplemented with 10% fetal calf serum and antibiotics (penicillin and streptomycin). PBLs, for use in transfection, were stimulated with both phytohe-magglutinin (5

jLg/ml)

and human recombinant interleukin-2 (20U/ml; Boehringer Mannheim).

Growth of virus and viral DNA preparation. All experi-mentsreported here employed a Ugandan isolate (U1102) of HHV-6 isolated by R. G. Downing (11). The propagation of the virus and subsequent purification of viral DNA for use in polymerase chain reactions (PCRs) were as previously de-scribed (40).

DNAsequencing. The isolation of the HHV-6 strain U1102 clonepHD12 (HindIII-C) and the PCR product from which the sequence presented in this report was obtained have been described elsewhere (40). The nucleotide sequence of sonicated DNA was determined from single-stranded tem-plate by using the dideoxynucleotide chain termination method (2, 49) or from the cloned DNA by using double-stranded sequencing methods (60). Sequence data were assembled by using the computer programs DBAUTO and DBUTIL (52, 53) and analyzed for the presence ofORFs

longer than 300 nucleotides by using the program ANALY-SEQ (54).

PCR. Oligonucleotides for use as primers in PCR were synthesized with an automated DNA synthesis machine (ABI 380B). The sequences (capital letters represent viral

sequences) of the primers used were as follows: RPT3, 5'

acacgaaTTCCAAAATTGGGAATCATGTGT

3'; RK1, 5'

acacgaaTTCTCGGAAAATTAAAGGTCA

3'; RK2, 5' acac

gaaTTCCTACCCAAGCGGGTTAGA

3'; RF1A, 5' acacgg

atccACATCTAGGTTTCATCTAGC

3'; RF1B, 5' acacaagc

ttTTAAACATGTGACATATAAC

3'. The positions of the

primers used in PCR, with the exception of RK2, which lies in the unique sequence at the right end of the KpnI repeat units, are annotatedin Fig. 2. PCR was performed with 200

ng ofU1102 DNA as the template and 1,uM (final

concen-tration) of each primer in a reaction mix containing 50 mM

KCl, 10 mM Tris-HCl (pH 8.8), 1.5 mM MgCI2, 0.01%

gelatin, 250 ,uM each deoxynucleoside triphosphate, and S U of Taqpolymerase (Amplitaq; Perkin Elmer Cetus) in a final volume of 100

pul.

Initial denaturation at94°C for 6 minwas followed by 25 cycles of denaturation

(94°C;

1 min), anneal-ing (55°C; 1min),extension(65°C; 12, 10, or 5 min for 8.6- to 7.5-, 5.4-, or 2.6-kb products, respectively). These primers

allowedthe specific synthesis of the required products with a yield ofapproximately 500 ng to 1 p.g per reaction. The products were extracted with phenol-chloroform and chlo-roform. Fragments were then digested with the appropriate

restriction enzyme, gel purified (57), and cloned into the appropriate vectors.

HHV-6 U1102effector gene constructs. Genetic

manipula-tions described below were performed as described

by

Maniatis et al. (38). Vectors used in cloning

experiments

were pUC19 and pBC12/CMV/IL-2 (7). The primer pair

products of RPT3/RK2 (7.5 to 8.6 kb) and RPT3/RK1 (5.4

kb) were designed with terminal

EcoRI

sites andcloned into pUC19. The RPT3/RK1 product was cloned into

pUC19

to

create pK28. The products synthesized with primers RPT3 and RK2 showed some size variation. Two different

clones,

pR56

and pR47, were isolated from the RPT3/RK2 product

and differ only in the number of KpnI repeat units present

(pR56,

7 copies; pR47, 28 copies), as confirmed by

partial

digestion with

KpnI.

The primer pair RF1A/RF1B product

(2.6 kb) was cloned as a

HindIII-BamHI

fragment into pBC/CMV/IL-2 to give pBC-ORF.

pR56.ml

was created by

inserting an EcoRV linker (8-mer) at the unique

BstEII

site following

BstEII

digestion of

pR56

and end repair. Linker insertion was confirmed by digestion and double-stranded sequencing with a USB Sequencing Kit (United States Biochemical Corporation, Cleveland, Ohio)according tothe manufacturer's instructions.

Heterologous target and effector genes. pLTR-CAT and pTAT were gifts from A. Akrigg. The construct pLTR-CAT contains the entire LTR of HIV-1, the cat gene from

SV2-CAT (16), and the polyadenylation-termination se-quences from the HCMV major

IE

gene (1) cloned into a modified pUC18 vector. The HIV LTR-CAT plasmids

con-taining mutations in the KB, TATA, KB+TATA, or

Spl

sites (gifts from G. Nabel) have previously been described (42).

pU3R-I,

which contains the LTR (nucleotides 1 to 670) of

human T-celllymphotropic virus type I

(HTLV-I)

linked to CAT, has been described by Sodroski et al. (51). The plasmid pE4-CAT1 contains the adenovirus early E4 pro-moter (nucleotides -224 to +33 relative to the transcription initiation site) and pE4-CAT2 is identical except for muta-tions in each of the three ATF binding sites (43). The adenovirus early E3 promoter-CAT plasmid, pE3-CAT, has been described by Weeks and Jones (58). pMLP-CAT

con-tains adenovirus major late promoter sequences between -260 and +33 (relative to transcription initiationsite) cloned

upstream of the cat gene and was a gift from C. Goding. DNA transfection and transient CAT expression assays.

Plasmid DNA was purified on cesium chloride-ethidium bromide gradients (3). Monolayer cultures were transfected essentially as described by Gorman et al. (16). Vero cell monolayers were split 1 day prior to transfection. Calcium phosphate precipitates were applied for 10 h prior to chang-ing the medium, and cells were harvested 48 h after changchang-ing the medium. The cell extracts were quantitated for protein concentration, and equal amounts of protein were assayed for CAT activity as described by Gorman et al. (16).

PBLs were transfected by using the DEAE-dextran method essentially as described by

Hammarskjold

et al. (17). PBLs were taken directly from 3-day-old

phytohemaggluti-nin-interleukin-2-stimulated cultures. Briefly, PBLs were washed twice with 10 ml of TD (140 mM NaCl, 25 mM Tris,

5 mM KCI, 0.5 mM

Na2HPO4

[pH 7.5]) and resuspended in 1.5 ml of TD containing 50 p.g of DNA and DEAE-dextran (500

p.g/ml/107

cells). After a

40-min

incubation at

37°C,

cells were suspended in 10 ml of TD, washed once with medium, and incubated at

37°C

in 10 ml of growth medium until harvest. Cells were harvested 48 h posttransfection and assayed for CAT activity as described by Gorman et al. (16).

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HHV-6-ENCODED TRANSACTIVATOR 5383

Kpnl

NN Mr

Kpni IJ A B D C ILIGIKI F Jjfl Ej77TR161.5kbp

I I~~~~~~~~~~~KKH

RF3

RF4 RF2 RFI

----

7

c

a

1 kb

4 6 8 10 12 kbP

FIG. 1. The KpnI restriction endonucleasemap of the genome of HHV-6 strain U1102.

Ml,

a fragment in thedirect left repeat; Mr, a fragment inthedirect right repeat. Below the genome is an expanded restriction map of the regionsequenced. B,BamHI; Bs,BstEIl; E, EcoRV; H,HindlIl; K,KpnI. KpnIrepeat units,describedpreviously (40), are indicated. Positions of fourleftwardORFs are illustrated in shadedboxes and are designated RF1, RF2, RF3, and RF4.

The percentage ofchloramphenicol converted to acetylated

forms wasquantitated by liquid scintillation spectroscopy. Nucleotide sequence accession number. The sequence of theputativeIEregion of the HHV-6 genome reported in this

article willappearin EMBL, GenBank, and DDBJ nucleo-tide sequence data bases under accession number M73681.

RESULTS

Sequence analysis of the putative IE locus. The position of theregionsequenced at theright end of the unique segment

ofthe HHV-6 genome strain U1102 is illustrated in Fig. 1. Theprinciple features of the DNA sequence of 5,900

nucle-otides within this region are illustrated in Fig. 2. The translations of the ORFs RF1 (1668 to 2045), RF2 (2203 to 2574), RF3 (2547 to 2879), and RF4 (2846 to 5146) are shown above the nucleotide sequence. The deduced translation products of the ORFs shared no significant amino acid sequencesimilaritieswith the IE products of the betaherpes-viruses HCMV, MCMV, and SCMV. The IE proteins en-coded by these previously sequenced IE genes do not themselves share extensive similarity, although large

glu-tamicacid-rich domains intheircarboxy-terminalregions, a

feature not shared by RF4, have been reported (28, 55). However, this region in HHV-6 exhibited local CpG

dinu-cleotide suppression (Fig. 3), which is a characteristic

fea-tureofthe betaherpesvirusmajorIE geneloci(21). TheCpG dinucleotide suppression is accompanied by an increase in

TpG and CpA dinucleotides, indicating exposure of this

regiontoDNAmethylation. Thisfeatureis not shared by the sequencesimmediately upstreamofthereadingframes (Fig. 3) orby anyothersequenced regionsof the HHV-6 genome

(30, 39a, 57b).

The positions of two overlapping putative AP1 binding

sites between nucleotide positions 593 to 603 (TGACTGA

and TGAGTCA) are indicated on the sequence (Fig. 2). Upstream ofthefirstORFare25to30

copies

ofarepeatunit containinga single KpnI site. The sequences of three

com-plete repeat units are presented (Fig. 2, nucleotides 64 to

375). Repeat units vary in length from 103 to 108

bp

and

containaperfectconsensussequence forthe

binding

siteof thecellulartranscriptionfactor AP2(25)anda9of10match

(GGGTCTTTCC) ofthe consensus sequencerecognized by

the NFKBfamily oftranscription factors

(GGGRATYYCC)

(31).There areonly minor sequence variations in eachofthe

copies of the repeats. By analogy with the presence of multiply repeated transcription binding sites in the HCMV

andSCMVmajor IE enhancers (5, 24, 26, 36), it is possible thatthe HHV-6KpnIrepeatunits perform a similar function. Becauseoftheposition of ORFs colinear with HCMV IE genes, the relative local CpG dinucleotide suppression and the presence of upstream sequences with enhancerlike fea-tures, we investigated the possibility that trans-regulatory functions were encoded by this region.

Transactivation of heterologous promoters. Effector plas-mid pR56, which contains the four ORFs (Fig. 1), the 5' sequences, includingseven copies oftheKpnI repeats, and

800 bp of

3'

sequences, was used ininitial experiments. To

determine the effects of gene products encoded by this plasmidonHIV-I LTR-directedtranscription, pR56(1to30 ,ug) wascotransfectedinto Vero cellswith 1 ,ug of the target

plasmid pLTR-CAT. The resultsoftheseexperiments

dem-onstrated that pR56 transactivated pLTR-CAT, with an

optimal induction of eightfold observed with 20 ,ig of ef-fectorplasmid (Fig. 4a).

Effector construct pR56 (40 ,Ig) was cotransfected with

pLTR-CAT (20 jig) into PBLs by using DEAE-dextran.

Expression was stimulated approximately eightfold over

basalexpression (Fig. 4b). Therefore, pR56 encodesa

trans-activator(s) which is functional in both epithelial and lym-phoid cells. Because of the relative ease ofcalcium phos-phate transfectionsand thehigherbasal levelsofexpression, furtherexperiments wereall performedin Vero cell

mono-layers.

To determine whether pR56 could activate

transcription

from other heterologous promoters, effectorplasmid pR56

was cotransfected into Vero cells with the target plasmids pLTR-CAT, pE4-CAT1, pE4-CAT2, pE3-CAT, pMLP-CAT, orpU3R-I(Fig. 5). pR56 induced levelsof

activity

of between2.5- and9.5-foldfor all theseheterologous promot-ers,andsimilar levels of activationwereobtained in numer-ous other experiments (not shown). pE4-CAT1 and pE4-CAT2, which has a mutation in each of the three ATF

binding sites, wereboth responsive topR56,

demonstrating

that the ATFsite is notrequired for induction.

Identification ofthoseregions necessaryfortransactivation

incoding andnoncoding sequences. To establish whether the

largest ORFwas essential for the

transactivating

function,

wecreatedaplasmid,pR56.ml,whichcontainsaframe-shift

mutation in RF4. Thiswascreated

by

inserting

alinker in the

BstEIIsite(Fig. 1)inpR56, and the mutationwasverified

by

sequencing. CAT

activity

was

dramatically

reduced when

pR56.ml was cotransfected with the HIV LTR construct,

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J. VIROL. 5384 MARTIN ET AL.

Kpnl k8 Ap2

GCGA.ACGAGAACT,CTAAAAGCTGCTAACGCGGCAACCGCAGTTCCTGTTTTTCTTCATAAAG AGATCAGTGGGTACCGCGGT7OIGGGGTCTTTCCTGCACAGGCGAACGAGAACTCT

10 20 30 40 50 60 70 80 90 100 110 120

Kpn1 kB Ap2

v

AAAAGCTGCTAACGCGGCAACCGCAGTTTCTOTTTTTCT7CIGGAAA AAGGTCAGTOGGGTACCGCGGTTr.AGG7rTGTCCTACCCAGGCG)AACGAGAACTCTKACATCTGCTA.ACGCG

030 040 150 060 70 180 190 200 200 220 230 240

TKvnl kB Ap2

250 260 270.L 280 290 300 310 320 330 340 350 360

1 Ap2

370 J 380 390 400 410 420 430 440 450 460 470 480

Apl

490 500 500 520 530 540 550 560 570 580 590 600

610 620 630 640 650 660 670 680 690 700 700 720

730 740 750 760 770 780 790 800 800 820 830 840

850 860 870 880 890 900 910 920 930 940 950 960

970 980 990 1000 1010 1020 0030 1040 1050 0060 1070 0080

1090 1100 1110 1020 0130 0040 1050 0160 0070 0180 0190 1200

0210 1220 0230 0240 1250 0260 1270 0280 1290 0300 1310 1320

0330 1340 0350 1360 1370 0380 1390 1400 1400 0420 1430 1440

0450 1460 1470 0480 1490 0500 0500 1520 1530 0540 0550 0560

K Q TOIO

1570 0580 0590 1600 0610 1620 0630 1 640 1650 166 0 1670 1680

DI F F F IL 60E C KO IOYN 0 010100X TOY P s L T 8500S F 06A V GO 8 C

1690 1700 1710 0720 1730 1740 0750 1760 1770 1780 1790 1800

A Y3M L QOR R608 TOSE 00 L 60Y LW P o I 100156810I A 16818068SR

1800 1820 1830 1840 0850 1860 0870 1880 0890 0900 1910 1920

A S I F R LV F POVP 000DFF P1I8MV00 TOD V 608E L C 66800080GV

1930 1940 0950 1960 1970 1980 1990 2000 2010 2020 2030 2040

2050 2060 2070 2080 2090 2100 2000 2120 2130 2140 2150 2060

BOooHI S L L0L00L8 L 0KF N I 0Y

v

2170 2180 2090 2200 2210 2220 2230 2240 2250 2260 2270 2280

HindIII

8 P o P886M 6E0 I 66E I L E 6 S L MC 0 TOS F 00 C I P G L 6VS F 0 L I P

2290 2300 2310 2320 2330 2340 2350 2360 2370 2380 2390 2400

60088160I8E 806600006S G LOKT L AG 008801Y I C P6K1IE8N66K6

2400 2420 2430 2440 2450 2460 2470 2480 2490 2500 2510 2520

80S F 680 TO 81N L 8000S F TOS

8 TQNI F FOYIL A600608G C 10 010G 6006668606MQ

25r30- 2540 2550 2560 2570 2580 2590 2600 26010 2620 2630 2640

RFlB

Q0008606D L I K C AOG LCT 00008S F IG L O LE Q L 00Q L I 808 L L

26 50 2660 267 0 2680 2690 2700 2710 2720 2730 27 40 2750 2760

180 F A C 00K FOOGL

060 TOO 0600068SK L 8066K L D0FF N88 COEP 6 FM F L H0 I N80MG

2770 2780 2790 2800 2810 2820 2830 2840 2850 2860 2870 2880

0000606008ER L L L D L R86888801N I 060001D F 006 TOVT COD C

2890 2900 2910 2920 2930 2940 2950 2960 2970 2980 2990 3000

00K CN8006 T M LHMNACM 86 HNKE80 80 P0D 10000K F C 6018I T COO0T C

3000 3020 3030 3040 3050 3060 3070 3080 3090 3000 3110 3120

C 60686106IV TNAN608801I LOQT F 6100K MN 01I F MOO COO A F COK 3030 3140 3050 3060 3170 3180 3090 3200 3210 3220 3230 3240

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VOL. 65, 1991 HHV-6-ENCODED TRANSACTIVATOR 5385

L N V N C K N L I T A A K N L A N T I L 0 S I V I C S N E F S W 0 N L X L L R R

ACTTAATGTGAATTGTAAAAATTTAATTACTGCCGCCAAAAATCTTGCCAATACTATTCSCAGAGTASTGTTATATCCAGCAATGAA7TSATGGCAACACCTCAAACTTCTGAGACG

3250 3260 3270 3280 3290 3300 3310 3320 3330 3340 3350 3360

G F K V T M L N H I T O A C E C L E S D Y D D T G L I K P L T P L Q I N D G Y I

GGGATTTAAAGTTACCATGCTGAATATCATTACCCAGGCATGTCAATGCTTAGAA7CAGATTATrATGATACTCGTTTAATTAAACCSTrAACTCCCCTTCAAATAASr.ACGGCTACAT

3370 3380 3390 3400 3410 3420 3430 3440 3450 3460 3470 3480

N M N K N R 0 S S I C D G N T D P S D IS I L D L A D F D D H G R Y S E E S S I

TkATATGAATAAAAATAGACAGTCTTCCATCTGTGATGGAAACACAGACCCATCAGACTCAATGATATTACACTSAGC7SCATTTTGA7CACCAGGA,AGATATTCAGAGGAA7CCTTCTAT

3490 3500 3510 3520 3530 3540 3550 3560 3570 3580 3590 3600

E S I H E D D D N M YV P C T P S P E V P G K S X Y V G T F T E N S R Q S G D E

CGkATCTATCATGAAGATGATIGACAATAAkAATTATCCATGCACTCCTTCTCCAGAAGTGCCAG.GAAATCCAAATATGTAGGAACATTCACTGAAAACAGCAGACAGTCAGGGGACGA

3610 3620 3630 3640 3650 3660 3670 3680 3690 3700 3710 3720

Q T N P N C V G T A SV T D L G G P D N L N S I S G L 0 S C K N M L L E R L L D

ACAAACAAACCCTAACTGTGTAGGTACAGCCTCAGTGACAGATCTGGGCGGCCCTGATAACTTGAACTCCATCAGCGGCCTCCAGAGTSCTAAAA,ACATGSTCTAGAAAGACTACT7GA

3730 3740 3750 3760 3770 3780 3790 3800 3810 3820 3830 3840

T Q C D S V V E G T E Q D G S Y G N T L I S E N N N F G Y E T D H S A P Y E S E

CAC TIC ATIGCGATTCAGTAGTTIGAAGGGACAGAACAAGATGGTTCCTATCCCAACACTTTAATATCAGAAATGATGATCGTTTrCGTTATGAAACTGATSCACTCGGCACCCTATSGAGAGTGA

3850 3860 3870 3880 3890 3900 3910 3920 3930 3940 3950 3960

S D N N D E I D Y I A N S D S A KR T N N I H M N S T D E N T P F IS S I C S P

AAGCGATAACAATGA7GAAATTCArrACATTCCAAACTCTGATTCAGCTGCGAGAACTAATAACATTCATATCAATAGCACTGATCAGAATACACCATTTAGCAATTCTATATGSSCACC

3970 3980 3990 4000 4010 4020 4030 4040 4050 4060 4070 4080

P E V T P S K K N V K P E S MT P G S K P K 9 R V A K R K H V S S K S P K 8K K

ACCAGAGGTCACCCCTTCTAAGAAAAATGTCAAACCTAAGTICTATGACTCCG4GGAAGCAAACCTAAAAAGAGGGTAGCTAAAAGGAAACArTTTCAAGTAAATCACCCAAAAATAAAAA

4090 4100 4110 4120 4130 4140 4150 4160 4170 4180 4190 4200

1 K T D Q L P K A A D V I V I S S E S E D E E D G D N I I G N S I L I K A I K S

GATTAAGACAGATCAGTTACCTAAGGCTGCAGACGTAATTGTTATAAGTTCAGAGAGTGAAGATGAAGAGGATGGTCACAATATTATTGGTAACTCAATCTTAATAAAAGCTATTAAATC

4210 4220 4230 4240 4250 4260 4270 4280 4290 4300 4310 4320

E S D S E S S S E S N D C T S E H0 0 L H L S D Y D E V T N N G H C P S Y G F P

AGAATCAGATTCTGAATICGAGCAGCGAGTCAAATGACTGTACATCTGAACATAA,ACAGTTACATTTGAGTGATTATGATIGAAGTTACCAATAATGGTCATTGSCCAAGTTATGGSTTCC

4330 4340 4350 4360 4370 4380 4390 4400 4410 4420 4430 4440

T P V F T I P I R S Q GI T G G I S K F I P K0 N W I W Y H K K H80 V D N C

AACTCCAGTGTTTACCATCCCAATAAGGTC AATCAGGGTAC CGGTGGTATCAAATCCAA.ATTTATCCCAAAAAAAAITGATTTr.GATATGAAGAAAACTCACCAAGTAGACAATTG

4450 4460 4470 4480 4490 4500 4510 4520 4530 4540 4550 4560

P I H N S E N V D A K D D S D G T E AK H C F M N H F V P I K T D D E D Y D K N

TCCTATTCACAATTCTGAAAATGTGGCATCCAAAAGATCACAGTGATGGGACAGAAGCCAAACACTCCTTTAATCACTTTGTCC CCACAAAACGGATGATGAAGACTATGATAAGAA

4570 4580 4590 4600 4610 4620 4630 4640 4650 4660 4670 4680

N V S Y I Y N K I N S1 I D S G0D I I P T K R L I I D M V 0D N F H D L 9 D I

CAATGTCTCATATASTTATAATAAAATACAGAATAGCAAAATAGAC7CGGGAGACATAATACCAACTAAGAAACTTA7CATAGATATrGGTATGGATAATTTTATGATCTAkAAGATAT

4690 4700 4710 4720 4730 4740 4750 4760 4770 4780 4790 4800

1 0 Q G I T O H C Q D L C N K Y N V V T P T T C E D D L N9 T N S Q T F A T T A

CATTAAACAGGGCATAACCAAACATTGCCAGGA7TCrTGlCAATAAATATAATGSTCSGCACCCCTACTACATCSGAGCACGATTrAAATATGACAAATTCCCAAACSTTTCCAACAACCGC

4810 4820 4830 4840 4850 4860 4870 4880 4890 4900 4910 4920

T Q V F D P P V T G N N S S I L N I I N D T T S Q0 D 0 N R C T E GI S N S N E

ALACTCAAGTGSTTTGACCCACCGGTAACAGGAAATAATT,CCTCCATACTAAATATTATCAATr.ACACAACAAGTCAAAA7TGATGAAAACAGATGCACSC.AAGGTACAAGTAATTCTAA7TGA

4930 4940 4950 4960 4970 4980 4990 5000 5010 5020 5030 5040

OC T N I S D C N9 S D T E A F O L D G Y P S D Y D P F v E N A Q I Y

AAAATGTACCAATATAAGTGASSGCAACAGTGATGGTACAGAGGCCTAAATTAGAST.CTACCCTTCTSATTATGASCCTTTCGTAGAAAASCTCAAATCTATSAOCTAGATGAAAC

5050 5060 5070 5080 5090 5100 5110 5120 5130 5140 5150 RF1A 5160

CTAGATGTAAATTGATTAGCAATICATTAGCATACAGASGTGTATTATGTAAATATGCTrATACTGTTGTAATATAGACTTTCCCCATAr.GTr.TAACT CT,r.CTATAGATTA

5170 5180 5190 5200 5210 5220 5230 5240 5250 5260 5270 5280

Po11(A)

GAALATTACATAAGCAALATGTAAC71'TATTA7TGTAATACATCAACAAACACGTSGATTCC TCAAAr-TCAAATTTTCAATA7SCACCTTACAAATAATAAAALAGTAATATAGACATTG

5290 5300 5310 5320 5330 5340 5350 5360 5370 5380 5390 5400

CrrCTCIILLAATCCAAGAC ATGTACATICAACTGACTATA.AGTCATCATATAATATT7CCATAGTTAATTCCAGCTTTCTTAGAGACG7SATACTATAACAAST7GTATCT7

5410 5420 5430 5440 5450 5460 5470 5480 5490 5500 5510 5520

AAACCTTATAC AGAMTG AAGTATllCAGAITT AC CTCCCAACTAAACCAGTTGATTAJGAC T"AACCCCCGAGACATCTATTCTATAAATCATAGTACTGTTA

5530 5540 5550 5560 5570 5580 5590 5600 5610 5620 5630 5640

A.ATATATTACAlTAGC TATAGCTAGATCATAACACTC TACAALA/TATTT,TTlCTTTAGGTATATTAA7TCTGTTTT- GCTT-ACATCACTTATCATTATGAAXSACACATT

5650 5660 5670 5680 5690 5700 5710 5720 5730 5740 5750 5760

C AACCATTAGACTTTNGATGTCACACCAACACTThGAAGTATATAl:TCAATACCAAATICCATTTTTATTGCAACATClTATTAAiCTGATCACTTTs-GAAGGTAAGATGCTACACA

5770 5780 5790 5800 5810 5820 5830 5840 5850 5860 5870 5880

T7ArTTCCAATTT GAAAC

[image:5.612.135.478.94.661.2]

RPT3 5890 5900

FIG. 2. Sequenceof5,900bpacrosstheputativeIEregion, readingfromrighttoleft

(approximately,

nucleotide

positions

9400to3500) as shown inFig. 1, withthetranslationproductsofthemajorORF RF4 and minor ORFsRF3, RF2,and RF1. The DNAsequenceincludes thefragments KpnI-J, 1.4kbofKpnI-K,and threecompletecopiesof theKpnIrepeatunits. Positions of the

recognition

sites forselected restrictionendonucleases;thepolyadenylation signal; AP1, NFKB,and AP2consensus sequences;and the

oligonucleotide primers

used in PCRareannotated. Thepositionof the PCRprimerRK2(notshown)is

approximately

3kbupstreamof the

position

of RK1. Therepeatunits, withinternalKpnI sites, areinbrackets.

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5386 MARTIN ET AL.

a

Lane 2 3 4 5 6 7 Lane

b

luur%G+C

0

C1)

X

*-x

(n)

0

%00

C.)

0

25 CpG

-25

-25 GpC

0 .k& A~.. A,L, .

--25 t25

-TpG

O~~~~~~~1

-

_

-25 +25 -GpT

i

o%L ""

O

n f-MNENEWW "nmm

-2 +2

I

4 5 6 7 8 9 10 11 12

[image:6.612.90.267.84.490.2]

(Kbp)

FIG. 3. Frequencyofoccurrenceofselected dinucleotide pairs correspondingtotheregion of thegenomeof HHV-6 expanded in

Fig. 1. Themajor ORFs within this regionare indicated, together

with aplot of the mean percent G+C content. The other panels show thedifferencebetween theobserved andexpected number of

occurrences (observed-expected) of the dinucleotides CpG, GpC,

TpG, GpT, CpA, and ApC ina401-nucleotide sliding window with

datumpoints plottedevery5nucleotides and with plotsscaled from +25 to -25. A significant deficiency of CpG doublets occurs

between 4 and 9 kband isaccompanied byan excess of TpG and

CpA.

demonstratingthatthelargest ORFwasessential for efficient transactivation (Fig. 6a).

The regionencoding the transactivatorwasdefined more clearly by using pBC-ORF, which contains readingframes RF3 and RF4, under the control of the HCMV major IE

promoter/enhancer (see Materials and Methods). The amount of pBC-ORF required for optimal activation of

pLTR-CAT was determined from the results of

dose-re-sponseexperiments (1 ,ug; datanotshown). pBC-ORF (1 ,ug)

was cotransfected with pLTR-CAT (1

jig)

into Vero cells, andcellextractswere assayed for CAT activity. pBC-ORF

inducedexpression of CAT significantly above basallevels

0:

*

**

* *@

*

effector 0

/ug 5

10 15 20 30

°J6Ac 3.8 45 67 200 287 361 285

pUC pR56

%K.Ac 0.3 2.5

FIG. 4. (a) CAT activity induced in cotransfections of pLTR-CAT(1 jig)withincreasingamountsof the effectorplasmid, pR56 (as indicated), which contains the putativeIE sequences. Percent

acetylations (% Ac) are indicated below. Transfections were

per-formed in Vero cell monolayers as described in Materials and Methods. AmountsofDNAused in transfectionsweremadeupto

atotal of 31,ugwithpUC18. Fold inductionswere1.2, 1.8, 5.3, 7.5, 9.5, and7.5forlanes 2to 7, respectively. (b) Induction of pLTR-CAT (20 ,ug) expression by pR56 (40 jig) in PBLs. Cells were

transfectedbytheDEAE-dextranmethod,asdescribedinMaterials and Methods.

(Fig. 6a). Therefore, the sequences spanningRF3 andRF4

are sufficientfor the transactivating function.

Toexamine thepotentialeffectof theKpnIrepeatunitson theexpressionfrom the effectorplasmids,levels of

transac-tivation bypR47 (28 repeats),pR56 (7 repeats), andpK28 (0 repeats) were compared. All plasmids contain the same

sequences, including the AP1 sites, downstream of the repeats. The results shown in Fig. 6b demonstrate that at

approximately equimolaramounts of effectors pR56, pR47,

andpK28 (0.35, 0.28, and 0.28pmol, respectively),

compa-rablelevels ofexpression wereinduced in transient

expres-sionassaysin Veromonolayers, suggestingthat the minimal promoterelements are present in pK28 and that the KpnI

repeat units are notessential for expression in these cells. Since the level of activation, when equimolar amounts of effectorplasmidwereused,wasgreater withpK28than that induced by pR47, the KpnI repeats may in fact have a modest negative effecton expression. However, this repet-itiveregion containing multipleNFKB and AP2bindingsites

may actas an enhancer of HHV-6IEgene expressionin T cellspermissive for HHV-6productiveinfections.

Identification of the sequences within the HIV-1 LTR re-quired for HHV-6 transactivation. To identify functional

regionswithintheHIV LTRenhancer likelytobe important

forHHV-6transactivation, wetested thesetof site-directed mutantsthatwerepreviouslyusedtoidentifythelocation of cis-actingregulatorysequencesin the HIVLTRrequired for transactivation by HSV-1 and adenovirus ElA (42). The activities of the mutants were tested in Vero cells by cotransfection with pR56. HIV-1 encodes its own potent

transactivator, tat, and this allowed us to use an effector plasmid, pTAT, as a positive control and for comparative

purposesin theseexperiments. Figure7 illustratesthe levels

of CAT activity induced by pR56 (20 ,ug) or pTAT (5 ,ug) when cotransfected with the wild-type plasmid pLTR-CAT orconstructswithmutations in the KBsites, TATA box,Spl sites,or KB+TATA. Adecrease in theconstitutive

expres-sion ofCAT was observed with the mutants (Fig. 7). The results we obtained are clearly in agreement with those

I

<

RF4

KKK[

L

,,W W.- , _

!5 -CpA

!5 ApC

O

I _,

-J. VIROL.

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[image:6.612.318.562.84.228.2]

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40-

35-

30-

25-0 c

20-0

15-0'I

10-

5-K

c

0

0II

K

pLTR-CAT pMLP-CAT pU3R-I pE3-CAT pE4-CAT1 pE4-CAT2

FIG. 5. Induction of the heterologous promoters pLTR-CAT (1kg),pMLP-CAT(1p.g), pU3R-1(1p.g),pE3-CAT(1rig).pE4-CAT1 (1p.g).

and pE4-CAT2 (1 ,ug) by effector plasmid pR56 (20 ,ug). Cotransfections were performed in Vero cell monolayers as described in Materials andMethods.

(a)

previously published, which demonstrated the importance of the Spl sites and TATA box motifs in tat-induced gene expression in both transient expression assays and by using

provirus constructs (13, 19, 20, 27, 47). The levels of expression induced by gene products encoded by pR56 were reduced in comparison to wild-type responses. pR56 activa-tionof the mutant KB LTR-CAT construct was reduced (4.3-to 2.7-fold), but the most dramatic effects on expression wereobserved with the other mutated target plasmids, with

activity reduced to near basal levels. The effect of the KB mutation on the relative induction of LTR-CAT by pTAT andpR56 isparticularly noteworthysince there is a specific

effect on activation by pR56, suggesting that the HHV-6 transactivator acts, at least in part, by a transcriptional mechanism.

c 0

a) C.)

0-

o-DISCUSSION

10- 9- 8- 7- 6- 5- 4- 3-

2-The conservation of the general organization of gene blocks within the HHV-6 genome has identified HHV-6 as

likely to be a member of the betaherpesvirus subfamily.

Furthermore, currentsequencedata suggest that theunique

segmentof the HHV-6 genome and theunique long

compo-nent ofthe HCMV genome arecolinear (lla, 30, 39a, 57a,

57b), and this has allowedustolocalizetheregionofHHV-6 mostlikelytoencode the IE genes. Sequencingof theregion

near the right end of the HHV-6 genome identified ORFs

corresponding precisely with the location of HCMV major

IEgenes, andthisregionisdepletedinCpGdinucleotides, a

feature sharedbytheIE genes of the

betaherpesviruses

(21). Upstreamof the ORFs isanarrayofrepetitiveelements(103

to108bp) which havepotential bindingsites for the cellular

transcription factors NFKB and AP2, which are capable of

activatingtranscriptionin responsetoraisedlevels ofcyclic

Effector

(b)

12 11

10 9 8 7-6 5 4 3 2 1 0

pUCpR56pR56mlpBC-ORF pUC pK28 pR56 pR47

FIG. 6. Determinationof theregionsnecessaryfor transactivation in either coding (a) or noncoding (b) sequences. (a) Effects on LTR-CATexpressionof effectorplasr,idspR56 (20 ,ug);pUC18(20 ,ug); pR56.ml (20 ,ug), containingaframeshiftmutation in RF4; and pBC-ORF (1 ,ug),containing readingframes RF3 and RF4.(b)Effects of the number ofcopies ofKpnl repeat units on the induction of expressionfrompLTR-CAT. pK28(15 ,ug),pR56 (20 ,ug),and pR47 (20 ,ug), with0,7and 28copies, respectively,of the repeatunits, as

determined bypartialdigestion, were cotransfected into Vero cells withpLTR-CAT (1 ,ug)asdescribed in Materials and Methods. The optimalamountsof each effector shown hereweredeterminedfrom dose-responseexperiments (notshown).TotalamountsofDNAused intransfections wasmade upto21,ug withpUC18.

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5388 MARTIN ET AL.

100-

80-

60-40

c 2

0 co20

(x) 1

8-<

16- 14- 12-

10- 4- 2-

0--/

I

_HIV_LI

wttype FoldInduction 1 4.2 24

13 12 11 10 9

7-6 5 4- 3-

2-

1-Mutant kB Mutant

1 2.738.5 1 1.4 4.3 1 1.4 7.7

[image:8.612.141.481.83.379.2]

Targets

FIG. 7. Stimulation of HIV-CATpromotervariantsby pR56. pR56 (20,ug) orpTAT (5 ,ug)wascotransfected withwild-typeormutant

LTRplasmids (1 ,ug) into Vero cell monolayers, as describedin Materials and Methods. Basal levels ofexpressionweredetermined with

pUC18as acontrol. Total transfected DNAwas madeupto21 jgwithpUC18.Thefoldstimulationsobtainedareindicatedbelow.

AMP(25). TheNFKBelements identified in HHV-6 have a pyrimidinerather thanapurineatposition4of theconsensus sequence (31). The effect which this substitution has on factor binding has not yet been established, but high copy

numbermaycompensateforpotentially low-affinity binding.

It is possible that this region may constitute an enhancer, sinceit shares manyfeatures with theIE regulatory regions of HCMV, SCMV, and MCMV (5, 6, 10, 26, 36). For, althoughthespecific organization of theseregions differ, all contain multiple binding sites for cellular transcription fac-torswithinreiteratedsequences. HCMVand SCMV contain NFKB andcyclicAMPresponse elements (5, 6, 24, 36).

The usual method for detecting IE viral transcripts in herpesvirus infections employs protein synthesis inhibitors

which allow theaccumulation of IE mRNAs while blocking synthesisofdelayed-earlyand latemessages. However,our

present inability to achieve efficient cell-free infections means thatthis method is notpracticable. Herpesvirus IE

genes encode transactivators of homologous and

heterolo-gous promoters (46), and we have used this property to

provide functional evidence that the right-end sequence encodes an IEgene(s).

By usingPCRwe havesynthesized and cloned fragments containing the ORFs from this region with or without the

repeatunits. The effector plasmids encoded transactivators which enhanced expression from avarietyof heterologous

promoters. Although the adenovirus E4 and E3 promoters

and the HTLV-I LTR have ATF binding sites as common cis-acting regulatory sequences, mutations in these binding

sites in the E4 promoter did not affect induction of CAT activity. Contrarytoother findings (12), thegeneproduct(s)

described here transactivate the HTLV-I LTR.This maybe because of the different cell types used in theseexperiments

and may therefore be a consequence ofthe availability of relevant cellulartranscriptionfactors.

We have found that the carboxy-terminal region of the

largest ORFis requiredfor the transactivation of

heterolo-gous promoters and that reading frames RF3 and RF4

(containedbetween nucleotides 2547 and5146)aresufficient for the transactivating function. The region (KpnI repeats)

upstream of theseORFs containspotential binding sitesfor the cellulartranscription factors NFKB and AP2. Although

thepresenceof repeat units in the effectorconstructsdidnot

appearto influence expression from these plasmids in our

experiments in Verocells, itis still possible thatthey may

confer positive and/or negative regulatory effects on gene

expressioninacell-specificmanner.Since HHV-6replicates onlyinactivated T cells and T-cell activation induces levels of active NFKB (50),the presence ofmultiple binding sites for NFKB implies that this is probably one of the cellular

transcriptionfactors likelytoplay arole inIEgene regula-tion. The role of theKpnIrepeats asregulatory elements in a numberof cell types iscurrently beinginvestigated.

In view ofthepossiblerole of HHV-6as acofactor inthe disruption oflatency and activation of HIV replication, we have investigated the responsiveness ofthe HIV LTR en-hancer to HHV-6-encoded products. In order to identify those sites in the LTR responsive to transactivation by HHV-6, site-specific promoter mutants were used in cotransfections with the HHV-6 effector plasmid, pR56.

Transactivation occursbyatat-independent mechanism, as

has been observed with other herpesviruses (39, 41, 42).

pUC18

pR56

* pTAT

MutantkB+TATA

1 1 2

K

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However, although no single specific cis-acting regulatory sequence has been found to be involved in induction by

HCMV or herpes simplex virus (9, 39, 41, 42, 45), the

adenovirus transactivator, ElA, appears to require the

TATA box element(42),andSpl sites havebeen implicated

as the responsive elements for the pseudorabies virus IE protein (59).We have found that the TATA box,Spl,andKB

sitesall playaroleintheinducibilityof theHIV-1 enhancer

by the HHV-6 putative IEgeneproducts. These datasuggest

thatactivation is mediated, at least inpart, atthe

transcrip-tional level,althoughposttranscriptional effectsmayalso be

involved.

Ensolietal.(12)demonstratedthat the region-103to-48

of the HIV LTRwas critical fortransactivation by HHV-6 andthatHHV-6 induced bindingof NFKB tothe LTR. The apparentdisparity betweenourresults and those of Ensoliet

al. (12) maybe attributedto the fact that these researchers were using mock- and virus-infected cells in transfections andtherefore the possibility that other viral transactivators are playing a role cannot be excluded. We have also used

thesemutantsinsuperinfection studiesand found that

trans-activation with infected cells was NFKB independent (data

notshown). Itis alsotrue,however, that theuseof different cell lines may have a qualitative effect on the ability of HHV-6IE transactivators tofunction.

Although Horvat etal. (23) identified two regions of the

HHV-6 (strain GS) which strongly transactivate the HIV

LTR, these fragments do not map to this locus of HHV-6

(strain U1102) (40). Our studies are the first to identify

reading frames ofan HHV-6 transactivating gene, and our studies have mapped this gene to a region ofthe genome possessing noncoding features characteristic of the IE re-gions of other beta herpesviruses. Our findings linking the

local CpG dinucleotide suppression with a transactivating functionprovide further evidence that the uniquecomponent

of HHV-6 and the unique long component of HCMV are colinearand thatHHV-6 isclosely relatedtomembers of the

beta herpesvirus subfamily. We have shown that a gene product(s) from this locus transactivatestheHIV-1enhancer and therefore may play a role in the regulation of HIV replication. Lusso et al. (33) have recently shown that HHV-6 isinvolved in the upregulation of CD4 expression,

thusexpanding the range ofHIV-1-susceptible cells. It has been suggested that this function is mediated by the IE

genes. Therefore, the IE gene products may have dual functions intheir role as a cofactor in the control of HIV infection.

ACKNOWLEDGMENTS

We thank K. Cameron for technical assistance and Margaret Notleyfor all herpatience andcompetenceinthepreparation ofthe manuscript.

M. E. D.Martinwassupported by aStates ofJersey Education Department grant and B. J. Thomson by a Medical Research

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1989. The promoter-regulatory region of the major immediate-earlygeneof humancytomegalovirus respondstoT-lymphocyte stimulation and contains functional cyclic AMP-response

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25. Imagawa,M.,R.Chiu,andM. Karin.1987.Transcription factor AP-2 mediates induction by two different signal-transduction pathways: protein kinase C and cAMP. Cell 51:251-260. 26. Jeang,K.-T.,D. R. Rawlins, P.J. Rosenfeld, J. H. Shero, T.J.

Kelly, and G. S. Hayward. 1987. Multiple tandemly repeated binding sites for cellular nuclear factor1that surround the major immediate-earlypromotersof simian andhuman

cytomegalovi-rus.J. Virol. 61:1559-1570.

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