Sequence, function, and regulation of the Vmw65 gene of herpes simplex virus type 2.

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Sequence, Function, and Regulation of

the Vmw65 Gene of

Herpes Simplex Virus

Type

2

RICHARD F. GREAVESt ANDPETER O'HARE*

Marie CurieResearchInstitute, The Chart, Oxted, Surrey RH8 OTL, United Kingdom

Received 10 June 1991/Accepted 21 August 1991

We determined thesequenceofthegeneforthe virion transactivator protein Vmw65 of herpes simplex virus

type 2 (HSV-2), strain 333. An analysis of the coding sequence revealed an overall high degreeofprimary

sequence conservation (86%) relative to the HSV-1 protein, although the carboxy-terminal region which

encompassesthepowerfulacidic transactivation domain of the HSV-1 proteinwasslightly less well conserved (70%). One importantchange in this regionwas thepresence ofa proline residue ina regionof the HSV-2 protein which is thoughttoformanamphipathic alpha-helix intheHSV-1homolog.Despitetheoccurrenceof this helix-disruptingresidue, theHSV-2 protein exhibited powerful transactivation properties for

immediate-earlytarget promoters.We also demonstrated that the HSV-2 protein formsatranscriptional complex (TRF.C) with the cellular Oct-i protein and target TAATGARAT elements from immediate-early promoters. A

comparison of upstream sequences from the two Vmw65 genes revealed good conservation of proximal

promoterelementsbut considerable divergenceelsewhere. Specifically, theHSV-2promoteralone carries9.5

copiesofa9-bpdirect repeat(GGGGCGGGA) ending 85 bpupstreamofthe conserved TTAAAT element. An

analysisoftranscription factor bindingsites invitrorevealed thatcellular factorSplboundtothe directrepeat

sequence of the HSV-2 promoter and thatcellular factor USF boundto aproximal element presentinboth

HSV-1 andHSV-2promoters. Mutational analysis of the HSV-2promoterdemonstrated that theintegrity of bothofthesebindingsiteswas importantforthefull activityof thepromoter.

The virion protein Vmw65 (VP16, a-TIF, ICP25) of herpes simplex virus (HSV) performstwo known functions during the virus lifecycle. It isamajor structuralcomponentof the virion (25, 52) and as such has an essential function for

normal virus assembly (1, 41). Vmw65 also specifically

transactivates immediate-early (IE) gene expression after virus infection (9, 47). This function is dispensible for infection in tissue cultures at a high multiplicity but is

essential fornormal virusreplicationatlow multiplicities of infection and for virulence after intracranial or

intraperito-neal inoculation in mice (2).

Although Vmw65 does notindependentlybind DNA (35, 37), it forms a complex with cellular transcription factor

Oct-1, which bindstothe octamerorTAATGARAT motifs presentinHSV IEgenes(37,42, 43,48, 49, 53). Induction of

transcription is mediated by the acidic transactivation

do-main locatedatthecarboxy terminus of Vmw65.

Mutagen-esis of the HSV type 1 (HSV-1) protein has allowed the dissection of these constituent activities (20, 55). Residues 49to388 of HSV-1 Vmw65aresufficient for the formation of

transcriptional complex TRF.C (21), which includes

Vmw65, a TAATGARAT element, Oct-1 protein, and a

second cellular factor (27, 30, 61). The formation of this

complexcorrelates tightlywithtransactivation by wild-type Vmw65 (21, 43), providing a mechanism for selective

loca-tion of the activaloca-tion domain(whichis itselfdispensible for TRF.Cformation)ontotargetgenes. We have demonstrated thatpointmutations in theregion between residues 366 and 387 frequently affect the ability of the Vmw65 protein to

participate in TRF.C and have predictedthat this region is

directlyinvolved atan interface within the TRF.Ccomplex

*Correspondingauthor.

t Presentaddress:DepartmentofMicrobiologyandImmunology,

StanfordUniversity School ofMedicine, Stanford,CA 94305-5402.

(21). Consistent with this proposal is the abolition of

com-plex formation bytheinsertion of short two- tofour-residue peptides at (among others) position 369 or 379 (1, 59).

Furthermore, we have shown that a peptide derived from

this region can specifically interfere with transcriptional

complexformation(23).

The acidic transactivation domain is contained within residues 413 to 490 of Vmw65 and retains function when linked to the DNAbinding domains of other proteins (11, 50). This region is predicted to form amphipathic alpha-helices, consistentwith theproposalthathelix formation isa

requirement foracidic activation function(34). Surprisingly, however, the recent results of Cress and Triezenberg (12)

indicate thatalpha-helixformation maybeunimportant and thatspecific hydrophobic interactions and anoverall nega-tivechargearethe crucial featuresrequiredfor function. The

acidicregionisthoughttoactivatetranscription by

contact-ing TFIID (54), TFIIB (33), or a transcriptional adapter (4,

28).

The Vmw65 protein has two identified homologs, both among the alphaherpesviruses. The Vmw65 protein of

HSV-2 has been shown to activate IE gene transcription (45), although its sequence is presently unknown. In con-trast, the Vmw65 protein of varicella-zoster virus, UL10,

whose sequencehas been determined(15), seemsnot to act as a transcriptional regulator or to form a complex with

cellular transcription factors (21a). Knowledge of the

se-quenceandpropertiesof the HSV-2homologwillclearlybe

useful in advancingourunderstanding of structure-function

relationships withinVmw65.

MATERIALSANDMETHODS

Plasmids, mutagenesis, and sequencing. Plasmids pRG90,

pRG91, and pRG131 were used for double-stranded

se-quencing of the HSV-2 Vmw65 gene from strain 333 and 6705

CopyrightC) 1991, American Society for Microbiology

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contain, respectively, 1.6-kbp KpnI, 0.75-kbp XhoI-KpnI,

and 1.2-kbp SstI-KpnI fragments from plasmid pGR135 (45) inserted into pUC19. Expression vector pRG1 contains HSV-2 strain 333 sequences from an XhoI site in the untranslated leader of Vmw65 to anSstI site in the next open reading frame downstream, cloned via linkers between the HindIII and EcoRI sites of plasmid pCMV-IL2 (13). This procedure places the HSV-2 Vmw65 coding sequence under the control of the powerful human cytomegalovirus IE promoter in a vector which contains the simian virus 40 origin of replication and which can therefore replicate in COS cells. The analogous expression vector pRG70 ex-presses Vmw65 from HSV-1 strain MP and has already been described (21).

Plasmids pRG154, pRG155, pRG163, and pRG164 (see Fig. 7 for a summary) all contain Vmw65 promoter se-quences cloned (via linkers) in the appropriate orientations upstream of the chloramphenicol acetyltransferase (CAT) coding sequence ofplasmid pCATB' (58). The HSV-1 pro-moter sequence was numbered by designating thecentral T ofthe sequence GCTGT +1 (seehighlighting in Fig. 2). This site has been mapped as the mRNA cap site (+2bases) by

Dalrymple et al. (14) and Pellet et al. (46). The HSV-2 promoter sequence was numbered by naming the homolo-gous T residue in the HSV-2 promoter +1 also (see Fig. 2).

Plasmid pRG154, the parent constructfor promoter studies,

contains a 418-bpXhoIfragment from the Vmw65 promoter ofHSV-2 strain 333, stretching from -288 (in the

penulti-mate codon of the HSV-2 UL49 homolog) to +131, 34 bp

upstream of the predictedinitiator codonforVmw65. Mutations were introduced into the promoter sequences by subcloning the sequences into pTZ vectors (39) and performing oligonucleotide-directed mutagenesis on

single-stranded derivatives (31) witha Bio-Rad Mutagene kit. All mutations were verified by double-stranded sequencing of plasmids. Plasmid pRG164 isidenticaltopRG154exceptfor

a single point mutation resulting in an XhoI site at -57. Plasmid pRG174 is identical to pRG154 except for two

transversions which generateanSphI siteat -109. Plasmid pRG178 is similar to pRG174 except that sequences up-streamof theMluI site at -211 have been deleted. Plasmid

pRG163issimilar to

pRG174

exceptthat the viral sequences upstream of the novelSphI site(-109) have been deleted. SeeFig. 7 for a summary ofthese

plasmids.

Plasmid pBB5 encodes the IEllOK gene and contains a

4,365-bp BstXIfragment of HSV-1strain MP cloned into the

SmaI site of pUC19. This

fragment

contains IEllOK pro-moter and coding sequences but lacks LAT promoter

se-quences (3). Plasmid pBB37 (2a) encodes the IE175K gene under the control of the human

cytomegalovirus

IE pro-moterandcontains an approximately 5-kbp SalI-DraI

frag-mentof HSV-1strain MPclonedintovector

pCMV-IL2

(13).

Reporter plasmid pAB5 (42) contains the HSV-1 IEllOK

promotercloned upstreamof thecoding sequencefor CAT. Plasmid pPOH3 (44)containsthe

thymidine

kinase promoter

of HSV-1clonedupstreamofthe

coding

sequencefor CAT.

Plasmid sequences were determinedbychain termination sequencing with a Sequenase 2 kit

(USB).

Plasmids were

prepared for analysis by alkaline denaturation. For the HSV-2 Vmw65 gene sequence,

plasmids

pRG1,

pRG90,

pRG91, and pRG131wereusedas

templates.

For compara-tive purposes,wealsosequencedthe promoter

region

of the Vmw65 gene from HSV-1 strain MP. This sequence was

identical to thepublished sequence(46) for HSV-1 strain

F,

with the singleexception that it lackeda G residueat -68. There are only two additional changes between the

se-quences of these two strains and the sequence of HSV-1

strain

17,

which containsaTresidue instead ofaGresidue

at

position

-17 and lacksa Tresidue at

position

+78

(14).

Universal

primers

were usedtocommenceHSV-2

sequenc-ing;

thereafter, 15-bp

primers

derived from the determined

sequencewere

synthesized

and usedtoextend the sequence until the

complete

gene was

sequenced

on bothstrands.

All

oligonucleotides

were

synthesized

on an

Applied

Bio-systems 381A

synthesizer.

Complementary

oligonucleotide

pairs

for

gel

retardation studies were annealed

by

being

heated

together

to

70°C

and then

slowly

cooled to

30°C.

Oligonucleotide

pairs

containing

binding

sites for

transcrip-tion factors

PHO4,

USF,

AP-1,

and

Spl

were

kindly

pro-vided

by Colin

Goding.

Gel retardation

analysis.

Cell extracts were incubated with

probes

in 20 ,ul of

binding

buffer,

which contained 25 mM

N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic

acid

(HEPES) (pH 7.9),

50 mM

KCl,

5 mM

dithiothreitol,

1 mM

sodium

EDTA,

and0.05% NonidetP-40. A

5-min

incubation

at

20°C

with

nonspecific

DNA

(0.5

,ug of sonicated salmon spermDNA per

,ul

of nuclearextract; 5

,ug/l,

of whole-cell

extract)

was followed

by

a20-min incubation at

20°C

with

specific

unlabelled

competitor

oligonucleotides

(when

appro-priate)

and then

by

a30-min incubationat

20°C

with

approx-imately

0.5 ng of end-labelled

oligonucleotide probe.

Gel

retardation

analysis (18)

was

performed

on 4%

nondenatur-ing

polyacrylamide gels

with a 19:1

acrylamide/bisacryl-amide ratio as described

previously

(21).

Quantitative

esti-mates of labelled

probe

bound were made

by

liquid

scintillation

counting

of retarded bands excised fromdried

polyacrylamide gels.

Transient

expression

assays.COScellsand Verocellswere

routinely

cultured in Dulbecco modified

Eagle

minimal

es-sential medium

containing

10% newborncalfserum. Trans-fections and CAT assays werecarried out in COS orVero

cells as

previously

described

(21).

When

appropriate,

cells were infected at 18 h

postinfection

with 5 PFU of HSV-1 strain MP per cell and harvested 22 h later. A

quantitative

estimate of

chloramphenicol

acetylation

was obtained

by

excision ofthe substrate and

products

from

thin-layer

chro-matography

plates

and

subsequent

measurement

by

liquid

scintillation

counting

in an LKB 216 scintillation counter.

Activity

is

expressed

ascounts perminute

appearing

in the

acetylated

products.

Cell extract

preparation

and

protein analysis.

For

large-scale nuclearextract

preparation,

HeLacells

(1

x

1010

to 2

x 10l°

cells)

were grown in

suspension

in Joklik modified

Eagle

minimal essential medium

containing

10% newborn

calfserum.Nuclearextractswere

prepared

by

themethodof

Dignam

et al.

(16).

For

preparation

of transfected-cell

ex-tracts, 2.5 x

107

COScells weretransfected with 20 ,ug of

expression

vector

by

themethod of Chen and

Okayama

(10)

with

N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic

acid-buffered saline. Whole-cellextractswere

prepared

after40 h

asdescribed

by

Wu

(60).

For the

comparison

of

transactiva-tion and

complex

formationincellstransfected with Vmw65 from HSV-1 or

HSV-2,

equal aliquots

of both total cellular material and solubilized extract were

analyzed by

sodium

dodecyl

sulfate-polyacrylamide

gel

electrophoresis

(32)

fol-lowed

by

Western

immunoblotting

(7),

using

monoclonal

antibody

LP1

(38)

as

previously

described

(20,

21).

Nucleotide sequence accession number. The

2,228-bp

se-quence determined in this work was submitted to the Gen-Bank data base andcanberetrieved underaccessionnumber M75098.

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1- MDLLVDELFADMNADGASPPPPRPAGGPKNTPAAPPLYATGRLSQAQLMP HSV-1

111111 MIii III

1- MDLLVDDLFAD--ADGVSPPPPRPAGGPKNTPAAPPLYATGRLSQAQLMP HSV-2

51- SPPMPVPPAALFNRLLDDLGFSAGPALCTMLDTWNEDLFSALPTNADLYR

49SPPMPVPPAlAl lLDlll GlMDTNFSAGPALC EDl P::lTADl

49- SPPMPVPPAALFNRLLDDLGFSAGPALCTMLDTWNEDLFSGFPTNADLYR

101- ECKFLSTLPSDVVEWGDAYVPERTQIDIRAHGDVAFPTLPATRDGLGLYY

99- ECKFLSTLPSDVIDWGDAHVPERSPIDIRAHGDVAFPTLPATRDELPSYY 151- EALSRFFHAELRAREESYRTVLANFCSALYRYLRASVRQLHRQAHMRGRD

11:: ll::Iliii111111 11111 111111111111111111111111 149- EAMAQFFRGELRAREESYRTVLANFCSALYRYLRASVRQLHRQAHMRGRD

201- RDLGEMLRATIADRYYRETARLARVLFLHLYLFLTREILWAAYAEQMMRP

111 1111 1111111111111111111111111:11111111 1111111 199- RDLREMLRTTIADRYYRETARLARVLFLHLYLFLSREILWAAYAEQMMRP

251- DLFDCLCCDLESWRQLAGLFQPFMFVNGALTVRGVPIEARRLRELNHIRE

1111 111111111111l 1111:11:11:1111111:1111111111111

249- DLFDGLCCDLESWRQLACLFQPLMFINGSLTVRGVPVEARRLRELNHIRE

301- HLNLPLVRSAATEEPGAPLTTPPTLHGNQARASGYFMVLIRAKLDSYSSF 299- HLNLPLVRSAAAEEPGAPLTTPPVLQGNQARSSGYFMLLIRAKLDSYSSV

351-

349-region required for TRF.C formation

TTSPSEAVMREHAYS T1fl~tIZb~?D1~D+APEEAGLAAPRL

ATSEGESVMREHAYSRGRTRNNYGSTIEGLLDLPDDDDAPAEAGLVAPRM

I>> acidic actvation domain

400- SFLPAGH-TRRLSTAPP-TDVSLGDELHLDGEDVAMAHADALDDFDLDML

111 11 11111111111111111111:1

399- SFLSAGQRPRRLSTTAPITDVSLGDELRLDGEEVDIMMWALDDFDLEML

448- GDGDSPGPGFTPHDSAPYGALDMADFEFEQMFTDALGIDEYGG -490 HSV-1 11 :11 11:1 11 111 111111:1:1

449- GDVESPSPGMT-HDPVSYGALDVDDFEFEQMFTDAMGIDDFGG -490 HSV-2 FIG. 1. Deduced primary sequence of the HSV-2 strain 333

Vmw65protein, aligned with theprimary sequenceofthe Vmw65

protein from HSV-1 strain 17 (14). Identical residue pairs are

indicated by a vertical line, and semiconserved residue pairs are

indicated by a colon. Residues in the HSV-1 proteinrequired for TRF.C formationareshaded,asis the prolineresidue atposition436

in the HSV-2 activation domain.

RESULTS

HSV-2 Vmw65 protein sequence. We obtained the

se-quence(Fig. 1andFig.2) of 2,228bpoftheHSV-2 strain 333 genomeknowntocontainthe genefor Vmw65(UL48in the nomenclature of McGeoch et al. [36] for HSV-1). Our determined sequence begins with the last 10 codons ofthe

presumptive HSV-2UL49homolog. The stopcodon forthe UL49readingframe is followed after 29 bp bya consensus

polyadenylation signal (AATAAA) (Fig. 2). On the basis of the overall alignment of the HSV-2 sequence with the

correspondingsequenceof HSV-1 (14, 46)and the

assump-tion that the primary sequence homologies, including, for

example, the TATA box sequence, are indicative of

tran-script homology, the cap site and initiator codon of the HSV-2 Vmw65 genecanbepredictedwith virtualcertainty (Fig. 2),particularly since the resultant mRNA would have the capacity to encode a 490-residue protein with 86%

primary homology to the HSV-1 Vmw65 protein (Fig. 1).

The end of the HSV-2 openreadingframe isfollowed after 91bp byaconsensuspolyadenylation signal (AATAAA)and

subsequently by aTATAA signal, which may be the TATA box site of the HSV-2 UL47homolog. The completeDNA sequence oftheregion canberetrieved from the GenBank database underaccession number M75098.

UL49 UL49

TaqI stop poly A

-233 GTCGA CTTC---CGTACC---CAGAC CACCAA HSV-1 I-28ICTCAG1C1 C1C1T1A 1A1G1 1A1 11 H1S1

-288 CTCGAG aGACCCCGGCCGCGTTCAGACGACAGAC~ CATCAC HSV-2 poly A

-193 CAGGGGTTCA---TTCGGTGTTGGC----GTT----GCGTG---CCTT

I11 1iii III 11 III III -238 GGTCGATTTAACCACTTCGCTGTCAGCACGCGTTTGTGGCGAGGGGCGGG

MluI

-159 TGTTTCCCAATCCGACGG-GGACCGGGACTGG---GTGGCGGG--GGG

11 1 11111 11 1 111111 III

-188 AGGGGCGGGAGGGGCGGGAGGGGCGGGAGGGGCGGGAGGGGCGGGAGGGG

9.5 perfect direct 9bp repeats - SP1 sites

-117 TGGGTTGGAC---AGCCGCCCTCGGTTCG---CCTT---CAC

III 11I 11 11 11 11 III

-138 CGGGAGGGGCGGGAGGGGCGGGAG=GGCGACACGCCTCCCTTCCGAGCGC

USF? CTF?

-84 GTGACAGGAGCCAAT -88

-38

USF SPi?

-GTG-GGGGAAGTCACGAGGTACGGGGCGGCC 1111 1I1111111111111111 11111111

GGGGGACGGGCCGCCCGGAGCGTGGGGAAGTCACGAGGTTTGGGGCGGCA

USF SPi?

'TATA'box mRNA cap

CGTGCGGGTTGCTTAAATGCGGGGTGGCGACCACGGGCT?TCATTCCTCGl -38 CTGGGGTGATACTTAAATGCGGGGTGGTGGACGCGAGATGTCAGTCCTCG

'TATA'box

+13 GGAACGGACGGGGTTCCCGCTGCCCACTTCCCCCCATAAGGTCCGTCCGG

111111111 1111 11111 I111 III

+13 GGGACGCACGGCACCCCCGGCGA----TTCCCTTCGCGAGGGCC--CCGG

111111II11 11I111

+57 -CCTTTTTC---TGTCGCGGGTCC-CGGATCCCTCCCCCCC---TCTC

EcoRV

+113 ACACTCTCTGGGCGGGCGGGGACGATCGCATCAAAA

111111111 Ill III 11III

+97 -CGC-CGCCGGGCGCTCGGGCACG-TCTCATTCGCC

Met

+163 TTCCCGTATCAACCCCACCCASM +185 HSV-1

11111 11 I111 111

+145 TTCCCGGACCCAACCGCCCCC2I +168 HSV-2 +63

AGCCCGATATCGTCT 1IIIH11111

CTCTCGAGATCGTTA XhoI

Met

FIG. 2. Alignment of nucleotide sequences upstream of the reading frames for Vmw65 inHSV-1 strainMPand HSV-2 strain

333. The stop codons and polyadenylation signals for UL49 are

shaded,as aretheinitiator codonsfor Vmw65.Restrictionsites used

for reporter constructs are marked, and the positions of point

mutationsused to createSphI sitesareindicatedby asterisks. Also

shadedaretheputativeTATAboxes,theHSV-1transcriptionstart

site(14, 46), thebinding site for USF demonstrated inthis paper,

andtheSpl binding directrepeatsof the HSV-2 promoter.

There is very good alignment between the HSV-1 and HSV-2 proteins

(86%

homology), and the greatest diver-gence in the primary sequence occurs in the

carboxyl-terminal acidic domain

(70%

homology). Theoverall strong homology isconsistentwith conservation of the dual roles of Vmw65 as an essential structural component and as the viriontransactivatorofIE genes. As formostregionsof the genome, the

region

encoding

Vmw65 can be contained

within viable intertypic recombinants(40).

The primarysequenceof the HSV-2

protein

in the

region

fromresidues 366to388

(which

wehavedemonstratedtobe crucial for Vmw65-Oct-1 complex

formation)

showed two fairly conservativechanges

(Ala-367

to

Gly; Lys-370

to

Arg)

(Fig. 1). Although we have determined that

lysine

370 is critical forcomplex formationin the HSV-1

protein,

wehave also shown that its mutation to

arginine

is

tolerated,

consis-tent with the presence of

arginine

at this

position

in the HSV-2protein (21b).

Ananalysis oftheprimarysequenceof the acidicdomain

of the HSV-2

protein

revealed a more

unexpected

feature: the presenceofa

proline

residueat

position

436

(Fig.

1).

The

XhoI stop

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[image:3.612.322.552.76.415.2]

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a)

A\k (A\Mi

( AMP

b)

ISV'

I(SV-V

0-I|.i_{11:~~~~~~~~~~~~~J}K(.\ r_ _

IWA

FIG. 3. (a) Transactivation of the IE 110K-CAT construct by

cotransfection with Vmw65 expression vectors. COS cells were

transfected with 20 ng oftarget plasmid and 10 ng ofpRG50 for

HSV-1Vmw65 expression, pRG1 for HSV-2 Vmw65 expression, or

controlvectorpCMV19Aforbasal activity.CAT activity in soluble

extracts was measured at40hposttransfection. Ac-CAMP, acety-lated chloramphenicol products; CAMP, chloramphenicol

sub-strate. (b) Formation of the TRF.C transcriptional complex on a

TAATGARAT element by HSV-1 Vmw65 and HSV-2 Vmw65.

End-labelledoligonucleotideTAAT24 (1 ng)wasincubated together

withaHeLa cell nuclearextract (1,ul)and extracts of transfected

COScells (1,ul). Bound oligonucleotide wasresolved by

nondena-turing electrophoresis; unbound probe isnot shown in this figure.

Vmw65 was expressed in COS cells by transfection with vector pRG50 (HSV-1)orpRG1 (HSV-2). Thebindingprofile ofanextract

madefrom cells transfected with the control vector is shown in

parallel(pCMV19A).Approximatelyequalamountsof Vmw65were

detected in pRG1- and PRG50-transfected cells by Western blot

analysis of the extracts with type-common monoclonal antibody

LP1(datanot shown).

acidic activation domain has been predicted to form two

amphipathic alpha-helices, but the presence of a proline

residueinthe HSV-2 proteinmay preclude theformation of

atleastoneof these structures. Alternatively, ifahelix does

forminthis region, it wouldbe restricted toaround residues

438 to 448. This region of the HSV-2 protein is clearly

functional intranscriptional activation, as we show below;

we and others have also demonstrated that an Ala-to-Pro

switchatanadjacent residuein the HSV-1 activation domain

has no detectable effects on its function (12, 41a). Despite

the slightly lower levelofsequence homology between the

acidic domains, key features are clearly well conserved.

Negative charge and its distribution are very similar in the

two proteins (all acidic residues present in the HSV-1

activation domain are present in its HSV-2 equivalent),

althoughtherearethreeAsp-to-Glu changes. The carboxyl-terminalregionof theHSV-2protein also hastwoadditional

acidic residuesas theresult of Ala-to-Asp changes. HSV-2 Vmw65 protein function. An expression vector (pRG1) which expresses Vmw65 of HSV-2 strain 333 was

constructed. The vectorused has asimian virus40origin of replication andthe IEpromoterfrom human

[image:4.612.58.296.69.253.2]

cytomegalovi-rus and can be used toproduce large quantities offoreign protein intransfected COS cells. We previously described similarvectors whichexpressthe Vmw65proteinof HSV-1 strainMP (20). Assays fortransactivationofanIE promoter-CATconstruct byHSV-1 and HSV-2 proteinsareshown in

Fig. 3a. Cotransfection ofvectors expressing Vmw65 from

HSV-1 or HSV-2 stimulated CAT expression to similar

extents, indicating that the proline residueat position436 in

the HSV-2 acidic domain has no substantial effect on the

activation function. Figure 3b shows that HSV-2 Vmw65 overexpressed in COS cells is also fully functional for the

formation of transcriptional complex TRF.C with cellular

proteins and target promoter sequences. This result was as we would predict, since TRF.C formation seems essential fortransactivation (1, 21, 43, 59).

The HSV-2 Vmw65 promoter contains a repeat structure. The intergenic region between the UL49and Vmw65(UL48) reading frames from HSV-1 strain MP andHSV-2strain333 is shown in Fig. 2. The homologywithinthis region isclearly

not as great as that within the protein coding region. How-ever, there is very clear homology just upstream of the mapped cap site of the HSV-1 transcript. This homology

includes a conserved CTTAAATGCG sequence at -27, presumably the functional TATA box (14, 46), aGGGGCG

GC sequence at -47, and a GGGGAAGTCACGAGGT

sequence at -65. On the basis of the conservation of the

nucleotide sequence and spacing, it would seem likely that

the latter two elements may representcis-acting sites shared

between the two promoters and may be binding sites for transcription factors. Upstream of the -65 element and downstream of the UL49 polyadenylation signal there is considerably less homology between the two promoters. The HSV-1 promoter carries only unique sequence in this region which contains potential binding sites for transcription fac-tors USF (CACGTG) at -87 and CTF (CCAAT) at -74. In contrast, the HSV-2 promoter has 9.5copiesof a 9-bp direct repeat (GGGGCGGGA) flanked by a unique sequence. These repeated elements closely resemble the binding site consensus sequence for cellular transcription factor

Spl

(6). On the basis of these observations, it seems that the two promoters share similar structures proximal to the transcrip-tion start site but may have different distal elements.

Expression and transactivation of the HSV-2 Vmw65

pro-moter. We constructed plasmids which linked the promoter

region to the coding sequence for CAT. The fragment used spanned from the

XhoI

site at position -288 in the penulti-mate codon of UL49 to the

XhoI

site at position +131 in the

5'-untranslated leader region of the Vmw65 gene. The

activ-ities of the promoter and mutant versions were assessed by

determining CAT activity in transfected Vero cells. A measure of basal activity in a dose-response experiment is shown in Fig. 4a. The HSV-2 promoter construct (pRG154) showed good basal activity, approximately twice that of the delayed-early thymidine kinase promoter con-struct (pPOH3). The results of virus superinfection of trans-fected cells are shown in Fig. 4b. The HSV-2 promoter was clearly induced by virus superinfection; the induction ratio

relative to mock-infected controls was in the range of 4- to 12-fold. Superinfection by HSV-1 or HSV-2 induced

pro-moter activity to similar levels (data not shown). HSV-1 IE

transactivator proteins were introduced separately in cotransfection experiments, and typical results from these experiments are shown in Fig. 4b. Cotransfection with a vector expressing the

IEllOK

protein induced expression from the HSV-2 promoter by 25-fold, and cotransfection with a vector expressing theIE175K (ICP4) protein induced expression by approximately 15-fold.

A conserved binding site for cellular transcription factor USF is required for full activity of the HSV-2 Vmw65 pro-moter in transient expression assays. The 16-bp sequence GGGGAAGTCACGAGGT is located approximately 65 bp

ob

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a) b)

a) CDef

x

I5

b)

basal nf basal -Ell OK +1E175K

pRG1S4 (HSV-2 promoter)

FIG. 4. (a) Dose-responseassays of CAT expression from HSV Vmw65 promoter-CAT constructs. Vero cells(106cells) were

trans-fected with various amounts of the indicated plasmids, and CAT

activity insoluble cell extracts was measured at 40 h

posttransfec-tion. (b) Induction of expression from Vmw65 promoter-CAT con-structsbysuperinfection with HSV-1 strain MP or by cotransfection with genesfor HSVIEproteins. Vero cells were transfected with

pRG154(1 ,ug) andinfectedat 18 haftertransfection with 5 PFU of

HSV-1percell. CAT activity insolubleextracts was measured at 20

h postinfection. For the cotransfection experiments, target con-structpRG154(1 ,ug) wastransfected intoVero cellstogether with

IEllOK expression vector pBB5 (50 ng) or IE175K expression vectorpBB37 (20 ng). CAT activity in soluble cell extracts was measured at 40 hposttransfection. inf, infected.

upstream ofthe transcription start site for the HSV-1

pro-moterand isperfectly conserved in both HSV-1 and HSV-2 promoters. To test whether this sequence was a

transcrip-tion factor binding site, we synthesized 29-bp

oligonucleo-tides spanning the conserved region and examined the

protein binding profile in gel retardation assays. Using a

nuclear extractofHeLacells (16)as asourceoftranscription

factors andoligonucleotides from either the HSV-1 orthe HSV-2 promoter, weobserved identicalcomplexes

consist-ing ofa single major species and a minor, faster-migrating species (Fig. Sa).Wenoted similaritieswithin theprobesto

the binding sites for cellular factors AP-1 and USF and

binding experiments were therefore conducted with unla-belled competing oligonucleotide pairs containing known

bindingsitesforAP-1,USF,Spl,and yeastfactor PHO4. Of these, the USF site competed strongly for the specific

complex,thePH04 sitecompetedweakly,andtheAP-1and

Spl sites did not compete significantly at any of the doses tested (datanot shown). We therefore tentatively assigned

the binding activity from HeLa cell nuclei as USF. Weak

competition bythe PH04site is consistent with this

conclu-AtNII.1' iS%62 VZV HSIVI

F

- c h |- c h |- c h - c h

c)

=60

N l.SFl 0

4 n.s. >

AdMILP HS V2 ('tIA%(t

hi

i

12 3 1 2 3s1 1 x

pRGil64

hl;sE: site poitTl IttulEtitit

ha,al -110K --?'K

FIG. 5. (a) Binding ofHeLa cellnuclear factorUSF to similar elements in AdMLP (Ad MLP), the HSV-2 Vmw65 promoter

(HSV2), the varicella-zoster virusUL10 promoter(VZV),and the HSV-1Vmw65 promoter(HSV1). Bound end-labelled oligonucleo-tideprobeswereresolvedby nondenaturing gel electrophoresis.For purposes of increased resolution, unbound probe was

electro-phoresed offthe gel but was shown to be in excess in parallel experiments. Lanes: -, competition by nonspecific competitor

DNAonly;c,specific competition by AdMLP USF site oligonucle-otides (100-fold molar excess); h, formation ofthe complex by a

heat-treated HeLa cell nuclearextract. (b) Binding ofaHeLacell nuclear factor to USF sites in AdMLP (Ad MLP), the HSV-2

Vmw65 promoter(HSV2),and a mutantHSV-2 Vmw65 promoter

(CTCGAG). Bound end-labelled oligonucleotide probes were

re-solvedbynondenaturing gel electrophoresis;unboundprobe isnot

shown. Lanes: 1, competition by nonspecific competitor DNA at

100ng;2,competitionbynonspecific competitorDNAat500 ng;3, competition bynonspecific competitorDNAat500 ng and AdMLP

oligonucleotides (100-foldmolarexcess). USF,USFcomplex;n.s.,

nonspecific complex. (c) Inductionofexpressionfromwild-typeand

mutant Vmw65 promoter-CAT constructs by viral IE proteins

supplied by cotransfection. Target promoters (1 ,ug) were

trans-fectedinto 106Vero cellstogether with IEllOK expressionvector

pBB5 (50 ng) or IE175K expression vector pBB37 (20 ng). CAT

activity in solublecellextracts wasmeasuredat40h

posttransfec-tion. w/t, wild type.

sion, as the PH04 protein is, like USF, a helix-loop-helix protein and binds to a related motif(18a). To confirmour

conclusion,weend labelledanoligonucleotide pair

contain-ingthe USFsite from the adenovirus 2

major

late promoter (AdMLP) and used it inparallel gelretardation assays. The

W

to

W

i

4a

db

"b

40 4D 4D

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result (Fig. Sa) shows that the mobilities of complexes formed on the AdMLP USF probe and the HSV element probes were identical (lanes marked -) and that all of the complexeswere specificallycompetedforbythe unlabelled AdMLP USFsite (lanes marked c). Furthermore, in recip-rocal cross-competition experiments, the HSV-2 site

com-peted effectively for USF binding to the AdMLP site. In

addition,lanes marked hshowthat thebinding activityfrom HeLa cell nuclear extracts was relatively resistant to heat treatment (10 min at 80°C), a characteristic of the USF protein (51).

We proposed thatUSF was

probably

ableto bindto the sequence CACGAG in the HSV promoters; this sequence

contains a single mismatch from the binding consensus

sequence(CACGTG). To confirmthisproposition, we syn-thesized a short oligonucleotide containing only 16 bases around the proposed HSV-2 motif and a corresponding oligonucleotide containingasinglebase-pairmutation within the site (CACGAG to CTCGAG). The results (Fig. Sb)

demonstrated that the shortwild-typeoligonucleotide bound

the factor as efficiently as the longer probe and with an

efficiency comparabletothatoftheAdMLPprobe (compare

lanes 1 and 2from the AdMLP panel with thecorresponding

lanesfrom the HSV-2 panel). The single point mutation in the HSV-2 sequence virtually abolished binding (Fig. Sb) and,whenassayed bycompetitionassays(datanotshown),

theaffinity of USF for this site was apparently reducedbyat

least 100-fold.

To determine the significance of the USF site, we

intro-duced this point mutation into the HSV-2 promoter-CAT plasmid and measured activity in transient expression

as-says. Typical results are shown in Fig. Sc. The construct

containing the point mutation exhibited basal activity

ap-proximately three- tofourfold lower than that of the wild-type promoter but remained inducible by the IEllOK and IE175K proteins in cotransfection experiments (Fig. Sc).

Over the course ofseveral experiments, the ratio of induc-tion by either the IEllOK or the IE175K protein was

approximately the samefor the wild-type and mutant

pro-moters. These results suggest thatUSFbinding contributes

to the high basal and induced activities of the HSV-2 promoterintransient assays but that it isnotrequiredfor the

mediation of induction by viral IE products. Weanticipate

that the homologous site is involved in the previously

demonstrated constitutive and induced expression of the HSV-1 promoter (5).

The9-bp repeat elements can bind cellularfactorSpl and are required for full activity of the HSV-2 promoter in transient assays. The major difference between the HSV-2 and HSV-1 promoters is the presence of the 9-bp repeat

element,andwethereforeinvestigated whether the element

boundacellular transcription factorand whetherthis bind-ing was involved in the HSV-2 promoter activity. A 26-bp end-labelled probe containing two tandem copies of the repeat wasgenerated and was used togetherwith a HeLa cell nuclear extract in gel retardation assays. The probe bound

specifically to a factor in the nuclear extract (Fig. 6a).

Competition experiments with unlabelled oligonucleotide pairs containing AP-1, USF, yeast PHO4, and Spl sites established that the binding activity seen probably repre-sented cellular transcription factor Spl. An end-labelled

probe containing the Spl site of the adenovirus EIla late promoter(19)was runin a parallel gel retardation assay and bound a complex virtually identical in appearance and

mobilitytothatfound with the HSV-2 repeat probe (Fig. 6a).

Complexes

formed with both probes were also effectively

.-\J1i-.)l.ile'SPI }IISV-SPil a)to probe repeats prtlhe

r II--- 1

I

S11 .,.

PIli D

Ul

b)

Si-,I 0

P.S.D

I

- c.

.I.n

+AdiF.2 latc SPI

i1te cr mpeltpiCtr

c) 150S

120)

A-9 (1 CS

F- 6

3

Ad E2 alae SPI

site probec

i

+AdE2 aleSil1 Site coInipCeitiIr

IrD 7

repcats copiT

S pR(3154

(IISV2 w/t)

D

pR(GI

)63r

OISEV2 no repeats)

FIG. 6. (a) BindingofHeLa cell nuclear factor Spl to similar elements in the adenovirus Ella late promoter and the HSV-2

Vmw65 promoter direct repeats. Bound end-labelled

oligonucleo-tide probes were resolved by nondenaturing gel electrophoresis;

unboundprobe isnotshown. Adenovirus EIIalate promoter Spl

site oligonucleotides were used as specific competitors in the

amounts indicated. (b) Competition for Spl binding to an

end-labelled EIlalate promoter Spl site probe. Adenovirus Ella late promoter Spl site oligonucleotides and unlabelled HSV-2 repeat

oligonucleotides wereusedas specific competitorsin the amounts indicated. Bound end-labelledoligonucleotide probeswereresolved

by nondenaturinggelelectrophoresis;unboundprobeisnotshown.

Spl, Spl complex; n.s., nonspecific complex. (c) Induction of

expressionfromwild-typeandmutantVmw65promoter-CAT

con-structs by viral IE proteins supplied by cotransfection. Target

promoters (1

pLg)

were transfected into Vero cells together with

IEllOK expression vector pBB5 (50 ng) or IE175K expression vector pBB37 (20 ng). CAT activity in soluble cell extracts was

measuredat40 hposttransfection. w/t,wild type.

competed

for

by

a 100-fold molarexcessof unlabelled Ella late promoter

Spl

site

(Fig.

6a).

Figure

6b showsa

compe-titionassaytoexamine the relativeaffinities ofthe Ella late promoter

Spl

site and thetwotandem

9-bp

repeatsfor

Spl.

Unlabelled

oligonucleotides

atdifferentconcentrationswere

used to compete for

Spl

binding

to a labelled Ella late promoter

probe.

Quantitation

ofthis

experiment by

band

excisionand scintillation

counting

showedthat the

affinity

of

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HSV-2 Vmw65 6711

pRG 154 _{___} _iH~~~~CA

pRG164 CAT

USF

pRG174 ..pAT

pRG178 'T.hM. CATI

Sphl

pRG 163

Sphl

~~~~~S

FIG. 7. Diagrammatic representation ofthe promoter-CAT constructs used, together with relative basal activities in transient assays

(averages ofatleast three independent experiments)andinducibility by viral IE transactivators(averagesofatleastthreeexperiments): ++

= 8-to25-fold. HSV-2promotersequencesareshaded,and HSV-2directrepeats arehatched.

Spl for the HSV-2 site was approximately 10- to 20-fold

lower than its affinity for the EIla latepromoter Spl site.

Toinvestigate whether the 9-bprepeatswere involvedin

the activity of the HSV-2promoter, weintroducedanSphI

siteimmediately downstream of therepeatsby mutagenesis. Nearwild-typelevels of expressionwereseenfrom plasmids intowhich this SphI site was introduced (pRG174) orfrom

which unique sequences 5' to the repeats were deleted (pRG178) (Fig. 7). A plasmid (pRG163) in which repeat sequences 5' tothe introduced SphI site were deleted was made. Typical results of the effects of this deletion on expression from the HSV-2promoter are showninFig. 6c.

Basalexpressionwasreduced by four-tofivefold.However,

expression remainedopento transactivation by the IEllOK

and IE175K proteins, suggesting that the repeats are not

required to mediate either of theseactivities (Fig. 6c). DISCUSSION

Wehavereported thesequence of the coding and regula-toryregions oftheVmw65geneof HSV-2.Acomparison of

theencoded protein sequence with theHSV-1 gene reveals

products ofidentical lengths: 490 residues. Vmw65 peptide

sequence conservation between the two viruses (86%) is high and comparabletothatseen withotheressential HSV

proteins, DNA polymerase (90%), alkaline exonuclease (80%), glycoprotein D(82%), andglycoprotein B (86%) (8, 17, 56, 57). The strong sequence conservation seenis

con-sistent with the dual role of Vmw65 in both viruses as a

majorand essential structural componentand as thevirion

transactivatorofIEgene transcription.

Our aim atthe outset of this work was a comparison of

peptide sequences in regions of the proteins required for transactivation. The region between residues 366 and 388, which we have proposed to form an interactive interface

during transcriptional complex formation (21, 23), as

ex-pected from the overall conservation, showed no major

difference between thetwo viruses. A change in the acidic activation domain wasof greater interest. The HSV-2 pro-teinhas aproline residueatposition436. This helixbreaker ispositioned centrally in a regionpredicted to form a long

amphipathicalpha-helix inthe HSV-1 protein. Ifthe struc-tureof thisregionisconserved between thetwoviruses,the

predicted helix may not be formed in either protein. Our observation(41a)thatanalanine-to-prolineswitchatresidue 436 does not affect the function of the HSV-1 activation domain would seem to confirm this idea. Our data are

consistent with a recent report which demonstrated that

prolinesubstitutionsatposition432or436 hadnosignificant

effect onthetransactivation function of Vmw65 (12). Over-all,theresultssuggesteitherthatalpha-helix formation isnot necessaryfor theactivity of this regionorthat ifahelix does

form, it is a short helix located within the region from positions 438 to 448 (at residue 448, the helix probability significantly declines and the protein sequence enters a

proline- and glycine-rich segment).

The conservation of the sequence between the two

Vmw65promoters isnot extensive and centersaround two

elementsupstreamofaconservedTTAAATelement. One of

the two conserved elements in the HSV-1 and HSV-2

promoters can bind in vitro to cellular protein USF. USF

was characterizedasthe cellulartranscription factor which

bindstothe hexamersequenceCACGTGinAdMLP(51). It

isamember, alongwithMyoD andc-myc,ofthe

helix-loop-helixfamily ofDNAbinding proteins (22).Thecorebinding

site in the HSV promoters(CACGAG)hasasinglebase-pair

mismatch relative to the characterized core site but still

binds USF with a high affinity-about 30% that of the

AdMLP site. Inaddition,ourresultswithaUSFbindingsite

point mutant in transient assays show that USF binding

correlates with efficientexpressionfrom the HSV-2Vmw65 promoter. We have alsodemonstratedthebindingof USFto anelement inaconservedpositionupstreamof thepredicted

TATA box of the varicella-zoster virus ORF10 gene, the

homologof the HSV Vmw65 gene. It isintriguingto specu-late that the preservation of this site may reflect some commonfeature intheregulationof thesehomologousgenes

during the virus life cycle. We have searched the HSV-1 genome for potential USF sites in the upstream regions of other genes. The searches revealed possible USF binding

sites intheregulatory regionsof theUL5, UL33, andUL55 genes. A binding site for USF has also recently been

characterized inanearly gene promoter of another herpes-virus, humancytomegalovirus (29).

Therepeated 9-bpmotifstructureisuniquetotheHSV-2

promoter. We show that this element isan SP1 bindingsite

and isalsorequiredfor efficientexpressionfrom the HSV-2 promoter. However, neither the USF site northe Spl site

accountsfor the inducibilityof thepromoterby IE

transac-tivators. Some other feature of the proximal promoter or

leader sequences must beresponsible.

It is possible thatthe difference in promoter structure is

reflected in a difference in the temporal regulation ofthe

HSV-2 gene compared with the HSV-1 gene. Previous

results demonstrated that CAT constructs containing the

promoter for the HSV-1 gene were inducible by virus

superinfection and that the kinetics of Vmw65 expression during HSV-1 infection followed classical leaky-late (or

Basal activity Induction by Induction by

(% HSV2 wAt) pBB5 (IEIIOK) pBB37(1E175K)

100% ++ ++

27% ++ ++

70% ++ ++

114% ++ ++

15% ++ ++

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P-y)-type

kinetics (5, 62). Earlier results indicated that Vmw65 expression in HSV-1-infected cells was repressed but was still detected in the absence of virus DNA

replica-tion (24, 26).

Our current work indicates that the expression of the

HSV-2Vmw65 genemay be unaffectedbytheinhibition of DNA synthesis (21b), indicating that for HSV-2, Vmw65

expression may follow classical delayed-early (or 3)-type

kinetics. Comparative analysesof these twopromoters and of theregulationof theexpressionof Vmw65 in HSV-1- and HSV-2-infected cells are now in progress to examine this

point.

ACKNOWLEDGMENTS

Thisworkwas fundedbythe MarieCurieMemorial Foundation.

We thank Tony Minson for continued supply of monoclonal antibody LP1 and Colin Goding for oligonucleotides. The typing skillsof Jean Marraregratefully appreciated.

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