Regulation of herpes simplex virus true late gene expression: sequences downstream from the US11 TATA box inhibit expression from an unreplicated template.

(1)

0022-538X/91/126749-12$02.00/0

Regulation of

Herpes

Simplex

Virus

True Late

Gene Expression:

Sequences Downstream from the US11 TATA Box Inhibit

Expression from

an

Unreplicated Template

PHILIP K. KIBLER, JOANNE DUNCAN, BRUCE D. KEITH, THOMAS HUPEL,

ANDJAMES R. SMILEY*

Molecular Virology and ImmunologyProgram, PathologyDepartment,

McMaster

University,

Hamilton, Ontario,

Canada L8N

3Z5

Received 11March 1991/Accepted 18 September 1991

Thetruelategenesof herpes simplex virus type1 (HSV-1)areexpressed only after theonsetof viral DNA replication. Previous studies demonstrated that latepromoters lack elementsupstreamof the TATA box and suggested that only a subset of TATA elements can function in the context of true late promoters. We determined which structural features oftruelatepromotersareresponsible for the stringent requirement for viral DNA replication by insertingaseries of simple modelconstructsinto the HSV-1genome in place ofone of the two promoters ofthe UL24gene.Anoligonucleotide consisting of 19 nucleotides spanning the TATA box of theHSV-1 true late US)) genedrove barely detectable levels of expression; bycontrast,the corresponding regions of the Adenovirus type2major latepromoterand the HSV-1 truelate glycoprotein Cpromoterwere much moreactive. Transcripts driven from all of these minimal TATA box promotersaccumulated without viral DNA replication. The activityof the US)) TATA boxwasstimulated by addingupstream Spl-binding sitesorplacing the US)) orrabbit

13-globin

cap/leader region (-11to+39) downstream. TheSpl-TATA and TATA-j8-globin cap/leader constructs remained replication independent, while the TATA-USII cap/leader promoter displayedtrue late regulation. These results demonstrate thatsequences located within the US)) cap/leader region imposeastrict requirement for viral DNA replicationon a minimal TATA boxpromoter.

DNA replication is thought to play a central role in the

activation and repression ofgene expression during

devel-opment (7, 83), yet little is known about the mechanisms

involved.DNAviruses provide useful models for

investigat-ingthe link between transcription and replication, because

expression ofaspecificsubsetofviral genes (the late genes)

requires viral DNA replication. In adenovirus and herpes

simplexvirus type 1 (HSV-1), replication appears to induce a cis-acting modification of the template that renders it

permissive for late gene expression (14, 49, 76). To gain

further insight into the mechanisms that control late gene

expression, we examined the cis-acting requirements for regulated late promoter activity during HSV-1 infection.

HSV-1 is a large enveloped virus with a 152-kb

double-stranded DNA genome

bearing

atleast 70 genes(51).Shortly

afterinfection, theviralgenomeis delivered to the nucleus,

where it is transcribed by the host RNA polymerase II. HSV-1genes fallinto atleastfour distinct temporal classes

that are regulated in a cascade fashion (35; reviewed in

references 21 and 80). Fiveimmediate-early (IE or a) genes aretranscribed first, and thesearetheonly HSV genes that are expressed in the absence of de novo viral protein synthesis. The IE proteins, in turn, activate the early (E or

,B)geneswhichencode the viral DNAreplicative machinery.

Theleaky late (L or yl) genes are induced soon after the E

genes; they are first expressed

prior

to the onset of viral

DNAreplication,andexpression increasesatlatertimes ina

replication-dependent fashion. Finally, the true L (or -y2)

genes are activated only after DNA replication has begun.

Each HSV gene bears itsown promoter-regulatory region,

and promoter transplant and nuclear run-on transcription

*Correspondingauthor.

experimentsindicate thattemporal regulationoccurslargely atthe transcriptionallevel(27, 59, 66, 82).

Coordinate activation of HSV IE gene expression is

mediated through a class-specific consensus sequence, TA

ATGARAT, thatis found in one or morecopies ineach of

the IEcontrolregions (13, 25, 46, 47, 60, 78). IE

transcrip-tionis activatedbyanabundant structural componentofthe

virion, Vmw65 (also known as VP16, oa-TIF, and ICP25),

thatis delivered into cells upon infection(2, 10, 59).Vmw65

formsacomplex with cellularfactors, including OCT-1,and

theresultingactivatorcomplex bindstothe TAATGARAT

sequence, in part through the DNA-binding domain of

OCT-1(3, 26, 43, 44,52, 56, 61). Fourof thefiveIEproteins (ICPO, ICP4, ICP22, and ICP27), in turn, contribute to

activation oftheviral E and L genes(reviewedin reference

21).

The mechanisms of activation of E and L promoters

remain unclear.EandLpromotersdonotcontainobviously

conserved class-specific sequences analogous to TAATGA

RAT,andextensive mutationalanalyseshavenotuncovered

cis-actingsequencesthataresolely and specifically

required

for activation by IE

polypeptides.

Rather, the E and L

promoter elements that are required for activation by IE

proteinsarethesame asthose needed for basal-levelactivity

in uninfected cells (12, 15, 17, 18, 33, 39, 65; reviewed in

references 21 and 54). These data have been

interpreted

to

suggest that IEproteinsinduceEandLgene

expression by

alteringtheactivity ofone or morecomponentsof thecore

transcriptionapparatus(21,54). Consistent with this hypoth-esis, the HSV IE proteins display relatively relaxed target

specificityandare abletoactivateavarietyof

heterologous

genes that have been newly introduced into cells

by

trans-fection or as part ofan infecting viral genome

(18-20, 22,

55-57, 73, 77). Some evidence suggests that TATA box

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factor TFIID serves as a critical target of the IE proteins:

linker scanning mutations of the TATA box of the E

thymi-dine kinasegene(tk) result in a more severephenotype when

assayed under conditions ofIE trans induction than when

assayed under basal conditions (12), and changes in the

TATAbox sequence can markedlyaffect the responseofthe

simianvirus 40 early promoter to HSV IEpolypeptides (22).

Inaddition,certain constructs containing only aTATAbox

cap site region are efficiently induced by IE proteins in

transfection assays (65). Direct evidence for a functional

interaction between IE proteins and TFIID comes from

studies showing that the pseudorabies virus IE protein (a

homolog of HSV-1 ICP4) activates transcription invitro by

increasing the rate ofbinding of TFIID to the TATA

se-quence(1).

HSV true L promoters appear to haveasimpler structure

than E promoters, and some evidence suggests that this

difference playsanimportant regulatory roleduring infection

(33, 34, 39). The E promoters that have been examined

consist ofanear upstreamregioncomposedofbinding sites for cellular transcriptionfactors linked to a TATA

box/cap

site (18, 53, 54). In contrast, the promoters ofthe true L

US]], glycoproteinC (gC), and UL38 genes lack elements

upstreamof the TATAbox (24, 33, 34, 39). Inthe gCgene,

a 15-nucleotide (nt) segment spanning the TATA box was

reported to be sufficient for true L temporal regulation

during lytic virus infection (33). Johnson and Everett (39) first noted that these results suggest a correlation between

promoter complexity and temporal regulation during

infec-tion. Oneinterpretation of these observationsis that true L

regulation represents the default state of a minimal core

promoter. According to this view, the upstream promoter

elements found in E promoters allow expression from an

unreplicated template while DNAreplication is required to

activateaminimalpromoterconsisting ofonlyaTATAbox.

Consistent with this hypothesis, Johnson and Everett (39)

found that a transfected construct bearing the intact E

glycoproteinD(gD) promoter was expressedin the absence

of DNA replication, while a derivative lacking sequences

upstream of the TATA box was active only when the

template was allowed to replicate. Conversely, Homaetal.

(33) were able to relieve the requirementfor DNA

replica-tionforexpressionof a true L gene byaddingthe upstream

regionof the Etkpromoter tothe gC TATA box. However,

severalobservationssuggest that thesituationismore

com-plex and provide evidence that true L promoters contain

specialized cis-acting sequences that actively contribute to

true Lcontrol. (i) Imbalzano et al. (37) found thata mutant

tk genelackingallknown promoter elements upstreamofthe

TATA box remained under E control, implying that

up-streamelementsare notalwaysrequiredfor expression from

anunreplicatedtemplate. (ii)Homaetal. (33)foundthat the

tk TATA sequence was unable to function when it was

transplanted to the gC locus, leading to the hypothesis that

true L promoter activity requires a specific type of TATA

box sequence. (iii) Mavromara-Nazos and Roizman (50)

observed that sequences downstream of the TATA box of

thetrueL-y42gene canconfer some aspects of L regulation

on anE promoter.

We investigated the structural features that contribute to

temporalregulation of HSV-1 true L promoters bystudying

aseriesofsimple model promoters placed in the intact viral

genome. We confirmed that a short region spanning the gC

TATA box was highly active when removed from the

contextof the intactgC promoter, while thecorresponding region of the tk promoter was inert. However, the US]]

TATA boxwasbarely

active, indicating

that the functional

differences between TATA elements revealed

by

this type of

assaydonotstrictlycorrelate with thetemporalclass of the

promoters from which

they

are derived. In addition,

tran-scripts driven fromall of the active minimal TATA promot-ersaccumulatedin the absenceof viral DNAreplication.We

found that sequences located downstream of the US]]

TATA boxcontributed topromoter activity and imposed a

strict requirement for viral DNA replication on minimal

TATA box promoters that were otherwise relatively

repli-cation independent. These data demonstrate that sequences

downstream ofthe US]] TATA box play an active role in

specifying

true L

temporal

regulation.

MATERIALS ANDMETHODS

Viruses and cells. HSV-1 KOS PAAr5 (29) was used

throughoutthese studies. Virus stocks weregrown and their

titersweredeterminedonVerocells maintained inaminimal

essential medium (GIBCO) supplemented with5% fetal calf

serum.

RNAextraction. Verocells wereinfected at amultiplicity

of infection of 10 PFU per cell, and where indicated,

aphidicolin was added at 10,ug/ml. Cytoplasmic RNA from

infected cellswaspreparedbythemethod of Berk andSharp

(4).

Primer extension analysis. Primer extension analysis was

performed as previously described (73). The following

syn-thetic primers were purchased from the Central Facility of the Institute for Molecular Biology and Biotechnology,

McMaster University: (i) gD, 5'-CCCCATACCGGAACG

CACCACACAA-3'; (ii) HSV-1 UL24, 5'-CGAGACAATC

GCGAACATCTACACC-3'; and (iii) 7SL RNA, 5'-AACT

TAGTGCGGACACCCGATCGGC-3'.

Construction of model promoters. Simplemodel promoters

were assembled from synthetic oligonucleotides purchased

from the Central Facility of the Institute for Molecular

Biology and Biotechnology, McMaster University, byusing

standard methods (48). All of the promoter constructs were

inserted into the unique BamHI site of pTKSB (79) in the

UL24 orientation. pTKSB contains the HSV-1 tk gene

bearing a 200-nt deletion extending from +480 to +680; the

deletion is spanned by a BamHI linker.

Promoters consisting of the TATA boxes of the

adenovi-rus type 2 (Ad2) major late promoter (MLP) and the HSV-1

US]] and tk promoters were assembled from

double-stranded 25-mers containing 19 nts spanning the TATA box

flanked by linker sequences and 5' GATC protruding ends.

Theseduplexes regenerateaunique BamHI site downstream

of theinsert following insertion into pTKSB. The sequences

of the top strands of the oligonucleotides used were as

follows:

Ad2 MLP: 5'-gatctcgagctcGGGGGGCTATAAAAGGGG-3' HSV-1 US1: 5'-gatctcgagctcTGAGATCAATAAAAGGGG-3' HSV-1 tk: 5'-gatctcgagctcACTTCGCATATTAAGGTG-3'

Uppercase type indicates sequences derived from TATA

regions, and lowercase type indicates flanking linker DNA.

A promoter bearing the HSV-1 gC TATA box was

gener-ated as follows. A pTKSB derivative bearing the Ad2 MLP

TATA insert was digested with SstI and BamHI to release

the Ad2 sequence. Adouble-stranded 19-mer bearing the gC

sequence and terminating in SstI and BamHI cohesive ends

was then cloned in its place. Thesequence of the topstrands

of the oligonucleotides used to generate the gC TATA

sequence was5'-cGCCCGGGTATAAATTCCG-3'.

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ers bearing point mutations in the Ad2 and USJI TATA

sequences were constructed by the same strategy. The

sequencesof the upperstrands of the oligonucleotides used to generate thepoint mutations were as follows:

Ad2 MLP: 5'-cGGGGGGCAATAAAAGGGG-3'

USJJ: 5'-cTGAGATCTATAAAAGGGG-3' A promoter bearing two tandem Spl sites linked to the

USJJ TATA sequence was constructed by cleaving the

pTKSB derivative bearing the USJI TATA sequence with

SstI and BamHI andthen ligating adouble-stranded 47-mer

bearing the Spl-TATA construct and SstI and BamHI

pro-truding ends. The sequence of the upper strand of the

inserted oligonucleotide was 5'-gatctcgGGGGCGGGGCTG

GGGCGGGGCCTGAGATCAATAAAAGGGG-3'. A

pro-moterbearing mutated Spl sites linkedtothe USJJ TATA

box was constructed in the same manner, aside from the

changes in the Spl sequences noted (see Fig. 6).

Promoters bearing the USJJ cap/leader sequence

down-stream of the TATA box were constructed by cloning a

double-stranded 55-mer bearing the USJJ cap/leader and

BamHI cohesive ends into the BamHI site immediately

downstream of the TATA box. The sequence of the top

strand of the USII cap/leadersequenceoligonucleotidewas

5'gatcGAGGACCGGGAGGCGGCCAGAACCGCCGTG

CACGACCCGGAGCGTCCCCTG-3'. The promoter

bear-ing the rabbit,-globin cap/leader sequence wasconstructed

in the same manner. The sequence of the upper strands of

the oligonucleotides use to generate the rabbit ,-globin

cap/leader sequencewas 5'gatcAGGGCAGCTGCTGCTTA

CACTTGCTTTTGACACAACTGTGTTTACTTGCAAT

CCG-3'.

All of the constructs were verified by DNA sequence

analysis using thedideoxy-chain termination method (63).

Marker rescue. Viralrecombinants bearing inserts of the

promoterconstructs were produced by in vivo

recombina-tion following cotransfection of Vero cells with infectious

viral DNA and plasmids carrying the appropriate promoter

sequences (71, 72). The resulting tk-deficient viral mutants

wereisolated by plaque purification in thepresenceof 100 ,ug

of5-bromodeoxycytidine perml. Recombinants bearing the

desired insertions were identified by Southern blot

hybrid-ization(74).

RESULTS

Functional heterogeneity of TATA box elements during

HSV-1 infection.Previous work suggested that thepromoters

of the HSV-1 true L USJJ and gC genes lack required

upstream regions, leading to the hypothesis that a TATA

box/cap site regioncanserve as anefficientpromoterduring

thelateststagesof infection (33, 34, 39). Inaddition, the data

of Homaetal. (33) indicated thata 15-nt segment spanning

the TATAregion of the gCpromoterwassufficient fortrue

L regulation while the TATA box of the E tk gene was

unable to substitute. As afirst step in exploring structural

features thatcontribute to true L regulation during HSV-1

infection, wecomparedthe activities ofavariety of TATA

box elements all placed precisely in the same sequence

context within the HSV-1 genome. In this assay, we

re-placed thepromoterforatranscript of the UL24gene with

oligonucleotides bearingtestTATA sequences.

Twotranscripts of the UL24geneoverlap the tkgeneand

are transcribed from the opposite DNA strand (32, 51, 62,

85). We took advantage ofapreviously described deletion of

tk coding sequences (ASB; reference 79) to link various

TATA box elements to UL24 transcribed sequences. The

ASB deletion extends from -172 to +24 relative to the initiationsite of thelarger UL24

transcript

(UL24b; Fig. 1A)

and is

bridged by

aBamHI linker. As shown in

Fig. 1C,

a

viralmutant bearingtheASB mutation failedtoaccumulate

detectable levels of UL24b

RNA,

indicating

that this dele-tioninactivatesthe UL24bpromoter. Weannealed

comple-mentary

pairs

of

oligonucleotides

to

produce duplexes

con-taining

various TATA box sequences

(Fig. 1B).

These

duplexes, bearing

5' GATC

protruding ends,

wereinserted

intothe BamHI sitethat spans the ASB deletion

endpoints,

in effect

replacing

the natural UL24bpromoterwith thetest

TATA element. The

resulting

tk-deficient

deletion-substitu-tion mutadeletion-substitu-tions were thentransferred into theintact HSV-1

genome

by homologous

in vivo recombination.

Following

selection of tk-deficientprogeny

by plaque

purification

in the

presence of 100 ,ug of

5-bromodeoxycytidine

per

ml,

virus

isolates

bearing

the TATA insertswereidentified

by

South-ern blot

analysis (74).

The activities ofthe various TATA

boxes were then assessed

by

scoring

for

production

of

UL24b RNAs

during

lytic

infection of Vero cells. On the

basis of the structures of the constructs,

transcripts

pro-moted

by

the inserted TATAboxeswere

predicted

toinitiate

atca. +39relativeto the

wild-type

UL24b cap

site,

within

sequences that

ordinarily

comprise

thenontranslatedleader

ofthe UL24b

transcript.

Firstwe studiedthe

activity

ofan

oligonucleotide bearing

19 residues

spanning

the consensus TATA box ofthe Ad2

MLP. Verocellswereinfected with

wild-type HSV-1,

ASB,

and a recombinant virus

bearing

the MLP TATA box

in-sertedacrosstheASB

endpoints,

and

cytoplasmic

RNAwas

examined

by

primer

extension

using

a

primer

complemen-tarytoresidues +116to +92 of thenative UL24b

transcript.

As

expected, wild-type

transcripts

gave rise to a ca. 116-nt

primer

extension

product

while ASB gave no

signal

(Fig.

1C). In contrast, RNAobtained fromcells infectedwiththe

construct

bearing

the TATA insert gave rise to a novel

extension

product

ofca. 78nts

(Fig.

1C).Thesedataindicate

that the Ad2 MLP TATA box induced the formation ofa

novel UL24

transcript

thatinitiated atthe

predicted

siteca.

30 nts downstream of the first T of the TATA sequence.

Confirming

this

interpretation, Si

nuclease

analysis

using

a

5'-labelled

single-stranded fragment extending

between the NruI and

Hinfl

sites

diagrammed

in

Fig.

1A

(labelled

atthe NruI

site)

gave rise to the

predicted

ca. 70-nt

protected

fragment

(data

not

shown).

This novel

transcript

accumu-lated to

higher

levels than

wild-type

UL24b RNA and

increased in abundance between 6 and 12 h

postinfection

(Fig.

1C). Accumulation of

transcripts

was

completely

pre-vented when

synthesis

of IE

proteins

was blocked with

cycloheximide

(data

not

shown).

These data are consistent

with the

suggestion

ofHomaet al.

(33)

that certain TATA

boxes are able to serve as

relatively

efficient promoters

during

the late

phase

of HSV

infection; however,

the results

leave open the

possibility

that the

activity

ofthe inserted

TATA sequence

depends

on elements

provided by

the

flanking

HSV

sequences.

We next examined

analogous

viral strains

bearing

the TATA box

regions

of the HSV-1 true L

USJJ

and

gC

promoters, and the HSV-1 E tk promoter embedded in

precisely

thesamesequencecontextasthe Ad2 MLP TATA

box

(Fig. 2).

Alloftheconstructs

displayed

similarlevels of

gD mRNA

(Fig. 2B),

confirming

that the

multiplicities

of

infection used were

comparable.

In contrast,

large

differ-ences were observed between the levels of UL24b RNA

(Fig. 2A):

the

gC

TATA sequencewas almost as activeas

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A

UL24b

WT

ASB

tk

UL24a

tk

TATAinsert

- primer %. TATA

inserts

Hinft BamHl Nrul

-.,,-- Novel UL24b ' RNA

ASB WT TATA

M 6 12 6 12 6 12

149 _

124

-112 q.p

92 _

78 _

69 _

gatctcgagctcGGGGGGCTATAAAAGGGG agctcgagCCCCCCGATATTTTCCCCCtag

FIG. 1. (A) Experimental design. Oligonucleotides bearingtestTATAboxsequences wereinserted intothe HSV-1genomeacrossthe

ASBdeletion inthe UL24 orientation, and then infected cell RNAwasscored for thepresenceof the predicted novel UL24btranscript by primer extension and Si nuclease analysis. The transcriptionmapof the HSV-1 tk locus isdisplayed, andtheextentof the ASB deletion is

indicated. ASBremoves200ntsof tk codingsequences, aswellasthe UL24bpromoter,and is spanned byaBamHI linker.(B) Sequence

ofthe Ad2 MLPTATA boxinsert.Boldfacetype,TATAsequence;lowercasetype,flanking linker DNA. (C) Analysis of UL24b transcripts induced by the Ad2 MLP TATA box. A 20-,ug sample of cytoplasmic RNA harvested 6or12 hpostinfection with 10 PFUof the indicated

virus strainpercellwasanalyzed byprimer extension usingaprimer complementarytoresidues 116to92 of the native UL24b transcript (A). Thewild-type(WT) strainwasHSV-1 KOS PAAr5. Markers (M)were3'-labelled HpaII fragments of pBR322DNA. The numberstotheleft

aresizes in nucleotides.

the Ad2 MLP sequence, while the USJJ TATA box was

muchless active and the tk TATA boxwascompletelyinert

inthisassay.Each of the active TATA elementsgaveriseto

a transcript that initiated at the same position within the

flanking UL24 sequences, so that the resulting RNAs all

have the same primary nucleotide sequence. It therefore

seems likely that the large differences in UL24b transcript

abundance reflect relative promoter strength rather than

differences in RNAprocessing, transport,orstability. These

results confirmed previous suggestions that the gC and tk

TATA sequences differ markedly in the ability to drive

transcription when they are removed from the context of

theirintactpromoters(33). However, the low activity of the

USJJ TATAelement suggested that these functional

differ-encesdonotstrictly correlate with the temporal class of the

promotersfrom which the TATA elementswere derived.

TheintactHSV-1 US]] andgCgenes aresubjecttotrueL

regulation, and their expression is almostcompletely

depen-dent on viral DNA replication (30, 40). To determine

whether the US]J and gC TATA regions residing at the

UL24 locusdisplayedthesameregulatory propertiesastheir

parentalpromoters,weassayed UL24b RNA levelsat6and

12 h postinfection with and without a block of viral DNA

replication imposed by aphidicolin. Transcriptsdrivenfrom

the Ad2 MLP, USJJ, andgC TATA box regions

accumu-lated without viral DNA replication, althoughthe amounts

were somewhatreduced (Fig. 2A).Thisresponseis

charac-teristic ofleakyrather thantrueLregulation. One interpre-tation of this result isthat the USJJ andgCTATAregions fall under theinfluence oftemporalcontrolsignalspresent in

the flanking DNA sequences when they are placed at the

UL24 locus. Consistentwiththis hypothesis, the wild-type UL24btranscript is also subjecttoleakyL regulation (data

notshown). Anotherpossibilityis that theseconstructslack

true Lregulatory elements thatare present in theintactgC

and USJJ promoters. Data supportingthis latterpossibility for the USJJ promoterarepresentedbelow.

Structural basis forthe lowactivityof the USJJ TATAbox. We examined the Ad2 MLP and HSV-1 USJJ TATA

se-quences for features that might account for their very

B

UL24a

I

A

'k

I I

I

I I

I

I I

r---

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[image:4.612.65.547.84.426.2]

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A

IK

z a- CL nsCL~~~~~~~~~~~O C!LC

Mca s a m a +

M

92 am

78 __ _

69 W

47 26 100 26 7.4 4.7 6.5 3.8 49 17 65 16 - %max.

abs.

Ad2 T -> A:

Ad2 MLP:

US 11 TATA:

US11 A -> T:

GGGGGGCAATAAAAGGGG 4

GGGGGGCTATAAAAGGGG

* * ** *

TGAGATCAATAAAAGGGG TGAGATCTATAAAAGGGG

B

Ad2 Usll Ad2-A Usll-T

M 6 9 12 6 9 12 6 912 6 9 12

124 _

I12 4

92 m

78 _ _

B s

C. Ad2; MLP GGGGGGCTATAAAAGGG ESV1 USL1 TG.AGATCAATAAAAGGGG

HSV1 gO

22 86100 988.15.0 16 14 15 24 36 30 %mox abs.

[image:5.612.351.543.78.392.2]

GCCCGGGTATAAATTCCG HSV1 tk ACTTCGC.ATATTAAGGTG

FIG. 2. Activities of various TATA elements. (A) Vero cells

wereinfected with recombinant viruses containing the Ad2 MLP,

USII, gC, or tk TATA sequence with and without 10 ju.g of

aphidocolin (aph)perml, and 10-,ug aliquots of cytoplasmic RNA

harvested at 6 and 12 h postinfection were scored for UL24

transcripts by primer extensionasdescribed in the legendtoFig. 1. Relativesignal intensitieswerequantified by microdensitometry and are indicated below the lanes (strongest signal, 100%). Molecular weight markers (lane M) were 3'-labelled HpaII fragments of

pBR322 DNA. Marker fragment sizes (in nucleotides)areshownon

the left. max. abs., maximum absorbance normalized to100% for the Ad2 MLP 12-h timepoint. (B) Levels of HSV-1 glycoproteinD

(gD) transcripts. Twenty-microgram aliquots of RNA from thesame

time points used for panel A were assayed for levels of gD

transcripts by primer extension. (C) Sequences of the Ad2 MLP, HSV-1 USIH, HSV-1 gC, and HSV-1 tk TATA inserts.

different activities. The oligonucleotides bearing the MLP and USJJ TATAregionsdiffer in sequence atfivepositions

(Fig. 3A): the first residueof theAT-rich TATAcore is aT

in the MLP sequence and an A in the USJI element; in

addition,four G-C base pairspresentin the MLP sequence

immediatelyupstreamoftheTATAboxareA-Tpairsatthe

corresponding positionsof the USJJ sequence. Wechanged

the first residue of eachTATA core to the sequence ofthe other(Fig. 3A) and assayedthe resulting constructs during lytic infection of Vero cells (Fig. 3B). A mutation that

changedthe first Tof the MLP TATAcoretothe Afound in

the USJJ sequence reduced its activity six- to sevenfold;

however, the resulting promoter was still approximately

threefoldmoreactive thanthe USJJ construct. Conversely,

changingthe first Aofthe USJJ TATAcoretoaTincreased

promoter strength sixfold but this construct was still less active than the Ad2sequence. These resultssuggestthat the

AT-richTATAcoreand5'-flankingresiduesboth contribute

to the differences observed between the Ad2 and USJJ

TATAregions.

C

_m

_ii

_

gD

FIG. 3. Effects ofpoint mutations onthe activities ofthe Ad2

MLP andHSV-1USIITATAsequences. (A)Structures of mutant

TATA elements. Asterisks denote differences between the Ad2

MLP and HSV-1 USII TATAsequences. (B) Activities ofmutant

TATA elements. Cellswereinfected with viralstrainsbearingthe

indicated TATA insertsattheUL24 locus. RNAwasharvestedat6, 9, and 12 h postinfection, and 10-sg aliquotswereassayed by primer

extensionanalysisasdescribed in thelegendtoFig. 1.Thearrowto

the right indicates the location of the expected primer extension product. Relative signal intensities are shown below the lanes

(strongest signal, 100%). Molecularweight markers(lane M) were

3'-labelledHpaII fragments of pBR322 DNA. Marker fragment sizes (in nucleotides)areshowntothe left.max. abs., maximum absorb-ance normalized to 100% for the Ad2 MLP 12-h time point. (C) LevelsofgDmRNA.Ten-microgram aliquotsof the RNAsamples used forpanel BwerescoredforgD transcripts by primerextension.

Although we have not systematically defined the

struc-tural requirements for efficient TATA activity in the

se-quencesettingofourassay,the datapresentedinFig. 2and 3 are consistent with the hypothesis that aclear T-A-T-A motifflanked byGC-richsequences is required. In support of thisinterpretation, wefound that several otherconstructs

bearing these features were as active as the Ad2 MLP

sequence (datanot shown).

Effect of theUSIHcap/leader region.The resultspresented above indicated that the USJJ TATAregion extendingfrom -33 to -14 served as a relatively inefficientpromoterthat

was subjectto leakyLregulationwhen itwasplacedatthe

UL24locus in the intact viralgenome. Incontrast, Johnson and Everett(39)found thataDNAsegmentextendingfrom -31 to +40 relative to the USJJ cap sites drove efficient

replication-dependent expression in a transienttransfection

assay,inafashion thatcloselymimicked true Lregulationof A.

Ad2 MLP US1' gC

go

69 _

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HSV genes. Together, theirresults andourdatasuggestthat

sequences downstream of the USJJ TATA box play an

important role in regulating USJJ expression. We tested this

possibility by adding sequences extending from -11 to +40

relativeto the USJJ capsitestoconstructsbearing the USJJ

and Ad2 MLP TATA boxes. A double-stranded 55-mer

bearing the USJJ cap/leader region and BamHI cohesive ends was cloned into the BamHI site at the 3' end of the TATA box sequences, and the resulting constructs were transferred into the UL24 gene in the intact viralgenomeas described above. With the construct bearing the USJJ TATA box, this manipulation regenerated the correct se-quence of the HSV-1 USJJ promoter region from -33 to +40, asidefrom three changes at -14 to -12. Addition of the USJJ cap/leader region is predicted to increase the length of the UL24 transcript driven from the test TATA sequences by ca. 55 nts relative to that produced by con-structsbearing only aTATA region (Fig. 4B).

Vero cells were infected with viral strains bearing the Ad2 MLP and USJJ TATAboxes linkedto the USJJ cap/leader region, and RNAwas harvested at6and 12 h postinfection with and withoutaphidicolin blockadeof viral DNA synthe-sis (Fig. 4). Controlexperiments(Fig. 4B) demonstrated that the RNA samplesusedcontained equivalentamountsof7SL RNA, a cellular cytoplasmic RNA that does not change in abundance afterinfection (57a), and that eachconstructgave rise to similar levels of gD mRNA. Addition of the USIJ

cap/leaderregionto the USJJ TATA box had three effects.

(i) UL24b-related transcripts derived from the cap/leader promoter displayedthecharacteristic spread oftranscription start sites over a ca. 6-nt interval previously described for the native USJJ gene (38, 40; data not shown), whereas transcripts arising from the USJJ TATA box alone displayed a single start site. Further analysis revealed that transcripts driven from the USJJ cap/leader promoter initiated at pre-cisely the same sites as those arising from the native USJJ

promoter and that the largest primer extension product

correspondedtothe residuedesignated +3onthebasis ofSi

nuclease analysis (39; data not shown). (ii) The levels of

RNA arisingfrom the USJJ TATAboxcap/leaderpromoter

were boostedeightfold by 12h postinfection relative tothe levels of RNA arising from the construct bearing only the

USJJ TATA box. Lastly, in contrast to the promoter

con-sistingof only the USJJ TATA box, accumulation of UL24

RNA was prevented by blocking viral DNA replication. Thus, the construct bearing the USJJ cap/leader region was

regulated like the intact USJJ gene. Addition of the USJJ

cap/leader did not increase the levels of RNA arising from

the Ad2MLP TATAbox; however, in this case as well,the

resultingconstruct displayed increaseddependence on viral

DNA replication. These datasuggestthat sequenceslocated

downstream of theTATA box contribute to USJJ promoter activity and play a role in the temporal regulation of USJJ gene expression.

Effect of a heterologous cap/leader sequence. The

con-structs bearing isolated TATAelements linkedto the UL24

gene initiate transcription within sequences that do not

ordinarily serveasatranscription startsite. Incontrast, the

TATA-USJJ cap/leaderpromoters drive initiation from the normal USJJ cap sites. To determine whether theincreased activity and replication dependence of the TATA-USIJ cap/leaderconstructsresulted from provision ofabona fide

initiator element, we joined the USJJ TATA box to an

oligonucleotide bearing the corresponding portion of the

rabbit f3-globin gene (-16 to +40), such that transcription initiatedat theglobin RNAcapsite (Fig.SC).Addition of the

A. MLPlAlA

MLP TATA US.-CAP

CL < n

a u aL 0

u t1 CI 4

M c cD C2 N + N N

162 _

I49 _

a

124 _ 1-12

-92 _

78 _

69 _

US11TATA

US1 TATA US! 1 CAP aL a a Cl

S0 - CZ L

-U_tD CO

*E -uA.- b

B.

C usil

TATA

US11 capleader

Ad2MLP

TATA

JS1 capleader

FIG. 4. Effect of the USJI cap/leader region on the Ad2 MLP andUSHJTATAsequences.(A) Verocellswereinfectedat 10 PFU

percellwith virusescontaining eitherthe Ad2MLPorUSJI TATA sequencealoneorincombination withthe USJHcap/leaderregion.

RNAwas harvestedat6and 12hpostinfection, and 10-,ug aliquots

were assayed for UL24-related transcripts by primer extension as described in thelegendtoFig. 1.Duplicateinfections wereassayed

in thepresenceof 10

,pg

ofaphidicolinperml (lanes labelled +aph).

Molecular weight markers (lane M) were 3'-labelled HpaII

frag-ments of pBR322 DNA. Markerfragment sizes (innucleotides) are shownontheleft. (B) Levelsof 7SLandglycoprotein D(gD)RNAs.

Aliquotsof the RNA samples usedfor panel Awere scored for7SL and gDtranscripts by primer extension. Theassaysfor 7SLandgD

RNAs used 0.5 and 10

,ug

ofRNA, respectively. (C) Structures of the

TATA-USII

cap/leader promoters. The Ad2 MLP and USJI

TATAsequences were ligatedtoadouble-strandedoligonucleotide

bearing the US11 sequence extending from -11 to +39(see Mate-rials andMethods).

globin cap/leader region also increased the levels of RNA arising from the USJJ TATA box; however, the resulting

transcript accumulatedto high levels intheabsence of viral DNA replication (Fig. 5A). Thus, although a heterologous

initiator element increased the activity of the USJJ TATA box, only the USJJ sequence imposed a strict requirement forviral DNA replication. The slight differencebetweenthe sizes of the primer extension products arising from the globin and USJJ cap/leader promoters reflects the fact that thelongest USJJ extension product corresponds to residue +3 in thenumbering scheme of Johnson and Everett (39).

7SL

gD

do. woma i& -- -.

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US-

-ATA--S'' TATA usI CAP A

UjS'- TATA-,3CAP

H _

H < _

I<

H D- CL) < FL

_cn D Cn)

E *-- E

Mv L/) ( 0

c-m Cl) Z

-l)

0L C/

92

78

7SL

- a

69

z- -= a

a

doo 4

-m-

-C. usl1

TATA

B.

SP1 S!te GGGGCGGGGCT

Dp- SP s,e:GGTTCGGGGCT

-SA-A.A

[image:7.612.81.295.77.418.2]

SP, SP, 7A-A F_ 1

FIG. 5. Effect of the rabbit ,-globin cap/leader region on the

USJI TATA box. (A) RNAwasharvestedfrom Vero cellsat6and 12 h postinfection with viruses containing the USHI TATA

se-quence,the US]I TATAsequencelinkedtothe USJI cap/leader,or

the USII TATA sequencelinkedtothe rabbit ,B-globin cap/leader insertedatthe UL24 locus. Duplicate infectionswereassayed in the

presence of 10 ,ug of aphidicolin per ml (lanes marked +aph).

UL24-relatedtranscript levelswereassayed by primerextension of

10 j±gof RNAasdescribed in the legendtoFig. 1. Molecular weight

markers (lane M) were 3'-labelled HpaII fragments of pBR322

DNA. Markerfragment sizes (innucleotides)areshownontheleft. (B) Levels of 7SL and glycoprotein D(gD) RNAs. Aliquots (0.5 and 10 ,ug) of the RNA samples used for panel Awere scoredfor 7SL andgD RNAs, respectively, by primer extension. (C) Structures of thepromotersusedfor panel A.

Effect of theUSIH cap-leaderregiononthe regulationofan

Spl-TATApromoter.Theresultspresentedinthepreceding

sectionssuggested that the USJJ cap/leader region imposed

an increased requirement for viral DNA replication on a minimal TATA box promoter. We tested whether the cap/ leader region was able to exert a similar effect on a more

complexpromoterinwhich the TATA boxwasactivatedby

an upstream promoterelement.

First we constructed a promoter bearing an artificial upstream region consisting of two tandem consensus

Spl-bindingsites linkedtothe USJJ TATA box. TheSpl-binding

sites hadthe samesequence asthe singlesitepresentinthe Ad2Elbpromoter andwerearrangedrelativetoeach other

toreproducethe spacingfound in the simian virus 40early

promoter. This upstream region was joined to the USJJ

FIG. 6. Effect of tandem Spl-binding sites on levels of RNA

driven fromtheUSI1 TATAsequence.(A)Vero cellswereinfected

at 10 PFU per cell with viruses containing the USJI TATA

se-quence, the USHI TATA sequence linked to two tandem Spl-binding sites, orthe USII TATA sequencelinked to two tandem mutated Spl-binding sites inserted at the UL24 locus. RNA was

harvested at 6 and 12 h postinfection, and 10-,ug samples were

assayed by primer extension analysisasdescribed in thelegendto

Fig. 1. Molecular weightmarkers(lane M) were3'-labelled HpaII fragmentsofpBR322DNA. Markerfragmentsizes(in nucleotides)

areshownonthe left.(B)Structures of the promoters used forpanel

A. Thesequencesof thewild-typeandmutantSpl-bindingsitesare

displayed.

TATA box such that the distance between the TATA se-quenceand theproximalSplsitewasthesame asin the Ad2

Elb promoter. As a control, we constructed another

pro-moterin which theSpl-bindingsiteswereinactivatedbytwo

pointmutations previously shown toeliminate Spl binding

(41; Fig. 6B). These two promoters were then fused to

UL24b transcribed sequences, transferred into the viral

genome, and examined during lytic infection of Vero cells. Addition ofSpl-binding sites greatly stimulated accumula-tion of RNA driven from the USJJ TATA box (averageof 17-foldoverthree experiments), whereas the mutated

Spl-bindingsites had no effect(Fig. 6A).

We thencompared the regulation of the Spl-TATA

pro-moterwith that ofaderivativebearingthe USJJ cap/leader A.

62 _ *,49_

241

112

92 IN

78 a

69 _

B.

gD

US,

TATA

tif

US''caD

eace-US, TATA

we

a Z::

ML zl: C- -a

7: ct

M co L-D S. -_, LO :.r-l 1-- 11 G --

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A

co-infection (CD

oo

H-EL i

on or,

149

TATA-CAP -_

n-TATA-iii- a

TATA --w

1.24

112

92

78

69

B.

us,

P. SP! TATA 1

--- 1F'1 2us_

UlS ce:.DCi:.c-'

FIG. 7. Effect ofthe USI1 cap/leader regiononan Spl-TATA promoter. (A)Vero cells werecoinfected with viral strainsbearing Spl-TATA and Spl-TATA-USII cap/leaderpromoters inserted at

the UL24locus(5 PFUof each virus strainpercell).Samples (10 ,ug) ofRNA harvested at 3, 6, 9, and 12 h postinfection were then assayed for UL24-related transcripts by primer extension as de-scribed in thelegendtoFig.1.Whereindicated,10,ugofaphidicolin (aph)per ml wasincluded.The twolanesontheright containedcells singly infected witheach virus strain(MOI, 10PFU percell).The arrows on theleftindicate the expected sizes ofprimer extension products for promoters with and without the

USJJ

cap/leader region. Molecular weightmarkers (lane M)were3'-labelledHpaII fragmentsof pBR322 DNA. Markerfragment sizes(in nucleotides) are shownon the right. (B) Structures ofthe promoters used for panelA.

region downstream (Fig. 7B). Vero cells were coinfected

with viral strains bearing the Spl-TATA and

Spl-TATA-USJJ cap/leader constructs, and RNA was harvested at 6

and 12 h postinfection with and without an aphidicolin

blockade of viral DNAreplication. The UL24-related

tran-scripts arising from the Spl-TATA and Spl-TATA-USII cap/leaderpromoters gave rise to distinct primer extension

products, allowing visualization of each transcript in mix-tures containing both. Addition of the

USJJ

cap/leader region to the Spl-TATA promoter resulted in increased

dependence on viral DNA replication: in the experiment depicted in Fig. 7, the levels of RNA arising from the

Spl-TATApromoter werereduced twofold by blockade of DNA replication while the RNA levels resulting from the

Spl-TATA-USII cap/leader

construct were inhibited

six-fold.These data indicate that the USI Icap/leaderaltered the

regulation of the Spl-TATApromoter; however, the

result-ing construct was significantly less dependent on DNA

replication than was theTATA-USIJ cap/leaderpromoters

lackingupstreamSplsitesdescribedearlier. Thus,upstream

Spl sites partially alleviated the requirementforDNA

rep-licationthatisimposed bythe USJJ cap/leader regionon a

minimal TATApromoter.

DISCUSSION

We examined the structural features required for the

characteristic DNA replication dependence of the HSV-1

trueL US] Ipromoterby comparing the activitiesofaseries

of simple model promoters in the intact HSV-1 genome.

Previous work had led to the hypothesis that HSV true L

geneexpression requires viralDNAreplication becausetrue

Lpromoters lack elements upstreamof the TATA box(33,

39). Accordingtothisview,upstreamelements suchasthose

foundin Epromotersallowexpressionfromanunreplicated

template while DNA replication is necessary for activation

ofaminimal TATA box promoter. In addition, Homaetal.

(33)found that only asubset of TATA elements are able to

serveas trueLpromoters. On the basisofthesedata, itwas

proposed that true L promoters are characterized by a

specializedtype of TATA sequence that canfunction in the

absence of other promoter elements. Our results confirm

that certainpromoterslackingupstreamelementsaresubject

to trueLcontrolduring HSV-1 infection; however, wefound

that thisregulatory responserequires specialized sequences

located downstream of the TATAbox.

In agreement with earlier findings, ourdata indicate that

TATA elements differ markedly in the ability to drive

transcription when they are removed from the context of

their native promoters: the Ad2 MLP and gC TATA

se-quences werehighly active, the US]] sequencedrovebarely

detectable levels of expression, and the tk element was

completely inert. The results obtained with the gC and tk

TATA elements paralleled those obtained by Homa et al.

(33) in an entirely different sequence setting (the gClocus),

reinforcing the conclusion that these elements are

function-ally distinct. However, the low activity of the US]J TATA sequence demonstrated that the behavior of a TATA

ele-mentin thistype ofassaydoesnotstrictly correlate withthe

temporal class of the promoter from which it was derived.

Taken in combination, our data are consistent with the

hypothesis that efficient activity in the sequence setting of

our assay requires a clear T-A-T-A motif embedded in

GC-rich flanking sequences. Similar conclusions have

emerged from saturation mutagenesis of the gC TATA

element (62a). It is interesting that these features are

char-acteristic of the "consensus" TATA sequence derived by

comparing avariety of cellular and viral elements (6, 8).

The TATA box binds transcription factor TFIID as the

firststepintheassemblyof the RNA polymerase II initiation

complex (9, 23). Therefore, the heterogeneity of TATA

elements revealed by our assay presumably reflects

func-tionally distinct interactions with TFIID. Functional

differ-ences between TATA elements have been previously

ob-served in other systems (67, 68, 75, 81): forexample, onlya

subset ofTATAboxes can serve astargets for transactiva-tion by the adenovirus Ela protein (67, 68). Inasmuch as current evidence suggests that yeast and mammalian cells

haveasingle geneencoding TFIID (11, 16, 28, 31, 36, 42, 58,

64),the differences thatwe haveobserved can be explained

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in two ways. The TATA elements may bind TFIID with

different affinities, directly leading to the observed

differ-encesin promoteractivity. Accordingtothishypothesis, the

Ad2 MLP TATA sequence would have the highest affinity

while the tk TATA box would have the lowest.

Alterna-tively, as previously suggested (81), TFIID may bind the

various TATA boxes with similar affinities but adopt

func-tionally distinctstates,dependingontheprecisesequenceof

the TATA element. Indeed, recent data demonstrate that

TFIIDundergoes aconformational change upon bindingto

the TATA element (45). Accordingtothis latterhypothesis,

only a subset of TATA boxes would result in a TFIID

conformation that is susceptible to transactivationby HSV

IEproteinsin the absence of other promoter elements.

We found that TATA elements were subject to leaky

rather thantrue Lregulation whenthey wereplaced atthe

UL24 locus. In

principle,

this

finding

may either reflect a

regulatory influence ofthe UL24 sequences that flank the

TATAinsert orindicate that theintactgC and USJJ genes

contain regulatory elements in addition to the TATA box

thatarerequiredfortrueLcontrol. While the firstpossibility

cannot be

entirely

discounted, our data strongly argue for

the second hypothesis in the case ofthe

USJJ

promoter.

Thus, sequences spanning the

USJJ

transcription initiation

sites andaportionof the nontranslated leader increased the

levelsof RNA driven from the

USJJ

TATAbox andimposed

a

stringent

requirementfor viral DNAreplicationonminimal

TATAbox promoters. The latterregulatoryeffectappeared

to be specific in that the corresponding region ofthe rabbit

,-globin

gene did not impose a replication requirement.

These data confirm Johnson and Everett's conclusion (39)

that sequences extending from -31 to +40 relative to the

USJJ capsitesaresufficientfortrueLregulationand further

suggest that sequences downstream ofthe TATA boxplay

anactiveregulatoryrole. The simplest interpretation isthat

the

USJJ

downstream sequences block expression from a

minimal TATA box promoter and DNA replication over-comes this

negative

effect. Several previous reports have

suggested

a role for sequences

directly

downstreamof the

TATA sequenceinregulatinglevelsofHSV-1transcripts(5,

12, 24, 84).Inaddition,Mavromara-Nazos andRoizman (50)

found that the nontranslated leader of the true L-y42 gene

enhanced tk expression at late times postinfection when it

was placed downstream of the tk TATA box. While these

studies suggest that sequences downstream of the TATA box can

play

a

regulatory

role

during infection,

sequences

responsible

forthereplication dependenceoftruelate genes have notbeen

previously

identified.

Because the

USJJ

downstream sequence rendered

expres-sion fromaminimal TATA promoter stringently dependent

onDNA

replication,

wetested the

ability

of this sequenceto

impose

a replication requirement on a more complex

pro-moter in which the

USJJ

TATA box was activated by

upstream Spl-bindingsites. Whilean

Spl-TATA-USJJ

cap/

leader promoter was moredependentonviral DNA

replica-tion thanan

Spl-TATA

promoter,

significant expression

was

observed without viral DNA

replication.

Thus, upstream

Spl

sites were able to relieve the replication dependence

imposed by

the

USJJ

downstream sequences

partially.

These dataarein agreement withpreviouswork

demonstrat-ing

that upstream promoter elementscaninduce

replication-independent

expressionfrom TATA

box/cap

regionsthatare

otherwise

subject

to true L control

(33, 39).

In

addition,

these results illustrate howa range of

regulatory

responses

canbeachieved

by

various combinatorial arrangements ofa

limited numberof

cis-acting

elements.

The USJJ cap/leader region increased the levels of RNA

arisingfrom the USJJ TATA box. We consider itlikelythat

this effect results fromprovision of bonafide transcription

start sites at the

appropriate

distance from the TATA ele-ment. Consistentwiththisview,theanalogous regionof the

rabbit

P-globin

genehadasimilareffect;inaddition,

prelim-inary data indicate that a linker-scanning mutation of the

US]] cap sites

strongly

inhibits promoter activity

(unpub-lished data). ManyRNApolymeraseII promotersdisplay a

conserved sequence atthe

transcription

start site

(6).

This

initiator elementplaysa role in promoter function(69) and,

in some cases, can support

TFIID-dependent transcription

in the absence ofa TATA box (70). The sequence of the

USIJ start sites bears little resemblance to the initiator

consensus,and USJJ

transcription

initiatesat

multiple

sites

spread over a ca. 6-nt interval (38, 40). However, the

characteristic

spread

of US]] initiation sites is

preserved

when the

USJJ cap/leader

region is linkedtothe Ad2 MLP

TATAbox

(Fig.

4 anddatanot

shown),

suggesting

that the

USII cap

region plays

a role in start site selection. It

therefore seems

likely

that the USJJ promoter bears a

variant initiator element. TheUSJI

cap/leader region

didnot

increase RNA levels when it was linked to the Ad2 MLP TATAbox.

Perhaps

the

highly

active Ad2 element doesnot

benefit from

linkage

tothe US]]initiator because this TATA

core sequence is sufficient to form a

productive complex

with TFIID.

How does the US]I

cap/leader region impose

anincreased

requirement

for DNA

replication

on aminimal TATA

pro-moter? The

ability

of upstream

Spl-binding

sitestoalleviate

the effect

partially

argues that the block

imposed

by

the

USJJ

sequence is

probably

at the level of

transcription.

Assuming

that this is the case, two distinct classes of

mechanisms can be

proposed.

It is

possible

that the USJJ cap/leader region binds viral orcellularproteins that block

transcription

initiationor

elongation. According

tothis

view,

replication

forks

passing through

the

region might dislodge

the repressor(s),

relieving

the

inhibitory

effect.

Alterna-tively,

itis

possible

that the sequencesatthe USJJ

transcrip-tion start sites are

permissive

for initiation driven from a

minimal TATA box

only

after conformational

changes

are

induced byDNA

replication.

This

hypothesis

proposesthat

the US]]

cap/leader region

cannot interact with the basal

transcription

machinery

toforma

productive

initiation

com-plex

on an

unreplicated template.

The available evidence doesnot

distinguish

between these

possibilities,

and bothare

consistent with the observation that DNA

replication

is

required

incis for

expression

oftrue L genes

(49).

We are

trying

tomapmore

precisely

the

regulatory

sequencesin the

US]J

cap/leader region

asthefirst step towards

distinguish-ing

between these alternatives.

It will be of considerable interest to determine what

proportion

of HSV-1 true L promoters bear

regulatory

sequences downstreamofthe TATAbox. Previous studies

suggestthat thetrue L

gC

gene doesnotcontain

regulatory

sequencesinthe

cap/leader region (33), implying

thatnotall

true L genes are controlled in the same way as USJJ.

Consistent with this

view,

the

gC

and US]] promoters

behave very

differently

when

they

are linked to reporter

genesand

assayed

in transienttransfection

experiments:

the

USJJ

promoter

stringently

requires replication

in cis

(38),

while the gC promoter drives efficient

expression

from an

unreplicated template (65).

These observations raise the

possibility

that sequences located outside the

gC

promoter

region

maybe

required

fortrueL

regulation

ofthis gene. The

experiments

presented

inthis report demonstrate that

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asegmentoftheUSJIJ promoterextendingfrom-11to +40

is sufficient toconfer true Lregulationon aminimalTATA

boxpromoter. Further study of the mechanismsinvolved is

likely to increase our understanding of the link between

DNAreplication and transcription.

ACKNOWLEDGMENTS

We thank Margaret Howes for technical assistance and F. L.

Graham for critical reviews of the manuscript.

Thisworkwassupported bya grantfrom theMedical Research Council of Canada. J.R.S. is aTerry Fox Senior Scientist of the

NationalCancer Institute of Canada, andT.H.wastherecipient of

astudentship from the National Science and Engineering Research

Council of Canada.

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