The repressing and enhancing functions of the herpes simplex virus regulatory protein ICP27 map to C-terminal regions and are required to modulate viral gene expression very early in infection.

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0022-538X/90/073471-15$02.00/0

The Repressing and

Enhancing Functions of the

Herpes

Simplex

Virus Regulatory Protein ICP27

Map

to

C-Terminal

Regions

and

Are Required

To

Modulate Viral Gene

Expression Very Early in Infection

LINDAMcMAHAN AND PRISCILLA A. SCHAFFER*

Laboratory of TumorVirus Genetics, Dana-Farber Cancer Institute and Department of Microbiology and

MolecularGenetics, Harvard Medical School, Boston, Massachusetts 02115 Received 7 February 1990/Accepted 12 April1990

The phenotypic properties ofICP27temperature-sensitiveanddeletionmutantsand the results of transient expression assayshave demonstratedthatICP27hasamodulatory effectonviralgeneexpression induced by ICPs0and 4.Inordertoidentify the regions of theICP27molecule thatareresponsible for itsenhancing and repressing activities, 10nonsenseand 3 in-frame deletion mutationswereintroduced into the codingsequence

ofthe cloned ICP27 gene. Thesemutantgenes were tested in transientexpressionassaysfor their ability to complementanICP27 nullmutantandtoenhance andrepressexpression fromaspectrumof herpes simplex virus type 1promotersinreporterCATgeneswhen expressionwasinducedbyICPOorICP4. Theresultsof assayswithclonedmutantgenesdemonstrate that theICP27polypeptide containstworegions, located between

amino acidresidues 327and 407 and residues 465 and 511, that contributetoitsrepressing activity. The amino acid region located betweenthetworepressing regions (residues 407to465) is ableto interfere withICP27 repressingactivity. None of themutantgenesexhibitedefficient enhancing activity foranyof the herpes simplex type 1 promoterstested, demonstrating that amino acidscomprising thecarboxy-terminal half of theICP27 molecule, including theterminal phenylalanine residue,arerequired for wild-type enhancementaswellasfor efficient complementation of an ICP27 null mutant. Phenotypic characterization of an in-frame deletion mutant, vd3, and a previously isolated null mutant, 5dl 1.2 (A. M. McCarthy, L. McMahan, and P. A. Schaffer, J.Virol. 63:18-27, 1989), demonstratedthatICP27 is requiredtoinducetheexpression ofall classes of viralgenesveryearlyininfection and confirmedtherequirementfor ICP27laterin infection(i)torepress earlygeneexpression, (ii)toinducewild-type levels of delayed-earlyor-ylgeneexpression, and (iii)toinduce truelateory2geneexpression. The vd3mutant,whichspecifiesanICP27 peptidelacking the repressing region

betweenresidues 327and407, isableto(i)repressearlygeneexpression, consistent with the repressing ability

of thed3 mutation in transient expressionassays, (ii) inducethesynthesisofsignificant but reduced levels of delayed-early(yl)proteinsandnoy2 proteins (thus vd3 exhibitsalateprotein phenotype intermediatebetween

thatofthewild-type virusand 5dl1.2),and(iii)confer alteredelectrophoretic mobilityonICP4,demonstrating arole forICP27in theposttranslationalmodification of this essentialregulatory protein.Takentogether,these observations support a model in whichICP27 performsits regulatoryactivities differentiallyovertime and mediates these activities indirectly via interactions with, and modifications of, ICP4 andperhapsother viral and cellularproteins.

The genome ofherpes simplex virus type 1 (HSV-1) has

beencompletely sequenced(33-35, 42)and encodes 72 viral

proteins. These proteinsare divided into fourmajorkinetic classesonthebasisofrequirementsfor their

expression

and the times of their maximum rates of

synthesis:

immediate

early, early, delayed early

(sometime

referred to as

"leaky

late" or

yl

proteins), andtruelate (-y2) (5, 25, 27,reviewed in reference 56). Viral genes of all four kinetic classes are

transcribedbythecellularRNA

polymerase

II(1, 6)andare

expressed inacoordinate and

sequential

fashion

during

the course of

productive

infection

(26).

Immediate-early

genes are the first to be expressed

during

the

replicative cycle,

followed by the early genes, whose

expression requires

functionalimmediate-early

proteins (13, 26, 43).

Finally,

the

delayed-early andtrue late genes are

expressed.

Maximum

expression

of these lattertwo classes of genes

requires

the

activitiesof both

immediate-early

and

early

gene

products

as

wellasviral DNAsynthesis (24, 38).The

primary

difference

*

Corresponding

author.

betweendelayed-early (-yl)andtruelate(-y2)genesis that

-yl

gene

expression

does notrequire viral DNA

synthesis

(al-though DNAsynthesisisrequired for maximumexpression of -yl genes),whereas

expression

of-y2genes is

stringently

dependentonviral DNAsynthesis (24, 38).

Immediate-early

genes encode the major HSV regulatory

proteins,

early

genescode forproteinsinvolved in viralDNA

synthesis,

and

delayed-early and late genes specify virion structural pro-teins.

Immediate-early genes are

expressed

in the absence of

prior viral

protein

synthesis (5).

Thesegenes are

transcrip-tionally activated by a

protein

(VP16) present in

incoming

virus particles (2, 4). VP16, together with cellular factors

including the

octomer-binding protein,

forms

protein-DNA

complexes with the consensus sequence

TAATGARAT,

located in the 5'

promoter-regulatory

regions

of the five

immediate-early genes (4, 34, 42). The

immediate-early

genesencode infected-cell

polypeptides (ICPs) 0, 4, 22,

27,

and47(41). Thelocationsof the genes

encoding

immediate-early proteinsin the HSV-1 genome are indicated in

Fig.

1

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(top line). All immediate-early proteins except ICP47 are

nuclearphosphoproteins (57) and act toregulate their own

synthesisaswellasthe synthesisofproteinsof later kinetic

classes (9, 10, 13, 19, 31, 38, 39, 44).

Insight intothe functional roles of each of the immediate-early proteinsintheregulationof viralgeneexpressioncame initially from studies of viral mutants. Characterization of temperature-sensitive (ts) and deletion mutants in the five immediate-early geneshas shownthatonly ICP4 andICP27

are absolutely essential for virus replication (9, 10, 13, 29, 32, 43, 47, 48, 50, 54). Studies ofmutantshave also shown that ICP4 is involved in the down regulation of its own

expression as well as that of other immediate-early genes and isrequired throughoutthe viral replicative cycle to up regulate the expression of early, delayed-early, and late genesatthe level oftranscription (9, 10, 12, 13, 43). Studies ofmutantsin thegeneencoding ICP27have shown that this

protein is requiredtorepressexpression of certain

immedi-ate-early andearly genes and is absolutely requiredfor the

transcriptional activation of late genes, a function that is

independent of early protein and viral DNA synthesis (32, 46, 47). Compared with the wild-type virus, ICPO deletion mutants grow well and induce the synthesis ofwild-type

amounts of viral proteins at high multiplicities of infection but they grow poorly and induce reduced levels of viral

proteins atlow multiplicities (48, 54). Thus, ICPO playsan

important although nonessentialrole inproductiveinfection. ICP22appears tobe involved in the activation of lategene

expression and has been shownto be essential for efficient virus replicationinsomecell types but notinothers (50;C. Bogard, unpublished observations). ICP47isapparentlynot essentialfor viral growth, atleast in cell culture (29).

Transient assays that measure the regulatory effects of

individual immediate-early proteins on the expression ofa

reporter gene (chloramphenicol acetyltransferase [CAT]) controlled by various HSV promoters have confirmed the findingsobtained with viralmutants. Thus,ICP4was shown toactivateCATexpressioncontrolledby earlyand late viral promotersandtorepressexpressionfrom its ownpromoter (i.e., ICP4 is autoregulatory) by bindingto a specific hexa-nucleotide (ATCGTC) near the ICP4 transcriptional start site(10, 15, 19, 31, 39, 44). Although phenotypic analysisof ICPO deletion mutants has shown that this protein is not

absolutely requiredfor viralgeneexpression during produc-tive infection, transientexpression assayshave shown that (i)ICPO isapotenttransactivator of all four kinetic classes of HSVpromoters, as wellas a variety of cellularpromoters, and(ii)thetransactivating activityof ICPOforthese

promot-ersis increasedsynergistically inthepresence of ICP4(15, 19, 31, 38, 39, 44, 51, 52). The mechanism of ICPO-mediated transactivation is unknownat present.

Incontrast totheactivitiesof ICPOandICP4,ICP27 alone has littleregulatory activityfor HSVpromotersin transient expressionassays(9, 15, 51). Anexceptiontothisrule is the observation by Rice and Knipe that ICP27 by itself can

transactivate the delayed-early glycoprotein B (gB)

pro-moter (45). Despite its limited regulatory activity alone, ICP27 can both repress and enhance expression from a varietyof HSV-1promoters whoseexpression is induced by ICPOorICP4orboth (16, 45,51, 55).Thus, ICP27 actsas a modulator ofthe regulatory functions of ICPO and ICP4. Availabledata havenotestablished whether ICP27mediates its modulatory effects by direct interaction with ICPO and ICP4, indirectly through interaction withormodificationof

viral or cellularproteins, orby direct orindirectbindingto DNA. Althoughless wellstudied,neitherICP22 norICP47,

aloneorin combination with otherimmediate-early proteins,

has been shown to exert

regulatory

effects on viral gene

expression in transientexpressionassays.

Thus, of the five HSV-1 immediate-early proteins, only ICPO, ICP4, and ICP27 havebeen demonstrated to exhibit

significant trans-regulatory activity. Systematic

mutational

analyses of the

coding

sequences for ICP4 and ICP0 have

identifiedspecific regionsof theseproteinsthatareinvolved in their

respective

transcriptional regulatory

and

DNA-binding activities,

sites of

phosphorylation,

and nuclear and

intranuclear localization (3, 12, 17, 40). As noted above, ICP27 has at least two

major

regulatory

functions. It acts both as a repressor of selected immediate-early and early

gene

expression

andas anenhancer of late geneexpression.

LikeICP27, the

immediate-early protein

ElA of adenovirus

actsbothas a

transcriptional

repressorand activator of viral

gene expression (37). Moreover, these activities are

speci-fiedby different

regions

of the

polypeptide (28).

In order to

assign

the functional activities of

repression

and enhancementto

specific regions

of the ICP27molecule and to

gain

further

insight

into the

significance

of these activities in the cascade of viral gene expression, we have

introduced nonsense and in-frame deletion mutations into

the

coding region

of the cloned ICP27 gene. We have

compared

mutantICP27

proteins

with the

wild-type protein

fortheir

ability

to

complement

anICP27 nullmutantandto repress and enhance

expression

ofreporter genes

coopera-tively

with ICPs 0 and 4. We have also characterized a

mutant

virus,

vd3,

containing

an in-frame deletion that has lost its

ability

to enhancebut retained its

ability

to repress HSV-1 gene

expression.

The results ofstudies ofmutant

plasmidsin transient assays have

(i)

confirmedthe

ability

of

ICP27 to trans repress and trans enhance

expression

from HSV-1 promotersactivated

by

ICPOand

ICP4, (ii)

localized

twofunctionaldomains

responsible

for the

repressing

activ-ity ofICP27 within the

hydrophobic carboxy-terminal

one-third of theICP27

molecule,

(iii)

demonstrateda

requirement

for the

carboxy

half of the ICP27 molecule for

enhancing

activity,

and

(iv)

shown that the

integrity

ofthe

carboxy-terminal

phenylalanine

residueofICP27is

required

forboth

enhancing activity

and efficient

complementation

of an

ICP27 null mutant. Studies of the mutant virus vd3 have confirmed

findings

that ICP27is

responsible

for a

distinct shift in the

mobility

of ICP4 in sodium

dodecyl

sulfate

(SDS) gels, implicating

ICP27in the

posttranslational

modification of ICP4. The

ability

ofICP27 to

modify

ICP4 was also shown to be distinctfrom its repressing activity.

Finally,

studies of vd3 have revealed thatICP27performsits

repressing

and

enhancing

functions in a manner that is

dependent

upon the time course of viral infection. Thus,

ICP27 functionsas anenhancerofviral geneexpressionat a veryearly stage in infection and as repressorof early gene

expression

andan enhancer of late geneexpression atlater

stagesin the HSV-1 replication cycle.

MATERIALSANDMETHODS

Cells and viruses. African green monkey kidney cells

(Vero; ATCCCCL81)andICP27-expressing Verocells (3-3 cells[32])werepropagatedaspreviouslydescribed (47). The

wild-type

KOS strain of HSV-1, the KOS-derived ICP27

deletionmutant5dl1.2(32),and amutant, vd3, generated in these studies were propagated and assayed as described

previously

(49).

Plasmids. (i) Immediate-early

effector

plasmids. Effector

plasmids

containingtheimmediate-earlygenesforICPs0, 4,

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and 27 of strain KOS were used in these studies. Plasmid pW3, which contains the ICP0 gene, was obtained from

Wendy Sacks (Dana-Farber Cancer Institute, Boston, Mass.) (48), andpnllND, which contains the gene for ICP4, wasobtained from Neal DeLuca (Dana-Farber Cancer Insti-tute) (11). (Please note that we have designated the ICP4-containing plasmid pnll, originally described by Neal De-Luca,as pnllND to distinguish it from theICP27-containing

plasmid pnll generated in these studies.) Plasmids pW3 and pnllND have been shown to transactivate HSV-1 early genes in transient expression assays (11). Plasmid pKHX-BH, which contains the ICP27 gene, was provided by

Stanley Person (Pennsylvania State University, University Park, Pa.).

(ii) CAT reporter plasmids. Chimeric CAT plasmids

pICP4CAT (also know as pIE3CAT [45]), ptkCAT, pVP5

CAT, and pL42CAT, which contain the CAT gene under controlof promoters for the genes encoding ICP4 (an

imme-diate-early protein), thymidine kinase (an early protein), VP5 (a delayed-early or -yl protein), and L42 (a true late or -y2 protein), respectively, were obtained from Neal DeLuca (10). pgBCAT, which contains the promoter of the g B gene (adelayed-early gene), wasprovided by Steven Rice (Har-vardMedical School, Boston, Mass.) (45). All enzymes and

restrictionenzymelinkers used to construct ICP27 nonsense and deletion mutants were obtained from New England

BioLabs,Inc. (Beverly, Mass.)and used as suggested by the

manufacturer. The promoters inserted into all of these

plasmidswerederived from the KOS strain of HSV-1. DNA isolation. Bacterial plasmid DNAs were isolated and

purifiedasdescribedpreviously (3). CsCl-banded KOS- and vd3-infected cell DNAs were isolated as described by De-Lucaetal. (8, 48).

Transfection.Fortransient expressionassays,transfection

of Vero cells was carried out by the calcium phosphate precipitation procedure followed by glycerol shock,as

pre-viously described (3). The amounts and types ofplasmid DNAsused in thesetests areindicatedin thetextandfigure legends. Contransfection ofICP27-expressing 3-3 cellswith 2 ,ug ofinfectious KOS DNA and 4

jg

oflinearized pd3

mutant plasmid during efforts to generate the vd3 mutant virus was carried out as described previously for marker rescue (47).

Construction of ICP27 mutant plasmids. A series of 10 nonsense and 3 deletion mutations were introduced into

coding sequences of the cloned ICP27 gene in

plasmid

pKHX-BH. The locations of HSV DNA sequences in

pKHX-BH relative totheviral genome and the locations of the mutationsin pKHX-BH are shown in

Fig.

1. Nonsense mutations in the ICP27 gene were generated as described

previously by

inserting

a

synthetic

oligonucleotide

contain-ing a HpaI site and translational termination codons in all three

reading

frames

5'GGCTAGTTAACTAGCC3'

HpaI

3'CCGATCAATTGATCGG5'

into five NaeI sites, four SmaI

sites,

and the

unique Sall,

StuI, NsiI,EcoNI, andSacl sites in the cloned ICP27gene

(Fig. 1) (11). Since the

ICP27-containing

plasmid pKHX-BH

has no HpaI

cleavage

sites, HpaI

digestion

of

plasmids

containing

the

synthetic

oligonucleotide

results in linear

molecules,

demonstrating

the presence of the

oligonucleo-tide in theseplasmids.

In-framedeletion mutations in theICP27

coding

sequence were constructed

by

restricting pKHX-BH

with the

indi-cated enzymes(Fig. 1), filling in or trimming the protruding ends, and adding appropriate synthetic linkers at the indi-cated sites. Forexample, the 5' protruding end of plasmid pdlwastrimmedand the 3' protruding ends of plasmids pd2 andpd3 were filled in with Klenow, and 12-base-pair (pdl), 8-base-pair (pd2), and 10-base-pair (pd3) BglII linkers were blunt end ligated to the respective plasmids. The ligated plasmids were then cleaved with excess BglII to ensure that each plasmid contained only one copy of the linker (pKHX-BH hasno BglII cleavage sites). The cleaved plasmids were religated with T4 ligase. The locations of the HpaI insertion mutations and of the limits of deletion mutations (Fig. 1) were confirmed by restriction enzyme analysis.

Complementation. Complementation of theICP27 deletion mutant 5dl 1.2 by peptides expressed transiently from cloned, mutated ICP27 genes was conducted in a manner similar to that described by Sacks et al. (47), except that Vero cells wereused instead of CV-1 cells and thefollowing proceduralmodifications were employed: 0.5 ,ug (instead of 0.3 ,ug) ofplasmid DNA and 4.5 ,ug (instead of 4.1 ,ug) of salmon testis DNA in 0.3 ml (instead of 0.2 ml) ofHEPES (N- 2-hydroxyethylpiperazine-N' -2-ethanesulfonic acid)-buffered saline was used to transfect -2 x 105 cells per 35-mm dish. Mutant 5dl 1.2 is an ICP27 deletion mutant that fails to replicate in Vero cells and expresses no detectable ICP27polypeptide (32).Transfected cells were infected with 5dl 1.2 24 h after glycerol treatment. After 18 h postinfection, infected cellswereharvested and progeny viruswasassayed on 3-3 cells toquantify total virus (i.e., virus generated by

complementationaswellasanyrecombinantsthat may have beengenerated) and on Vero cells to quantify any

replica-tion-competent recombinants. 5dl 1.2 DNA provides no sequence homology to the left of the BamHI site in the

ICP27-containing BamHI-HpaIinsert inplasmidpKHX-BH (32). As expected, no replication-competent recombinants between 5dl 1.2 and any of the plasmids tested were de-tected.

CAT assays. The in vitro assay for CAT activity was performed byamodification(10) of the method of Gorman et al. (21).

Southern blot analysis. DNA restriction fragments sepa-rated by agarose gel electrophoresis were transferred to

nitrocellulose and hybridized to labeled probes by the method ofSouthern(53). Theprobeused forSouthern blots wastheplasmid pKHX-BH

(Fig.

1).Theprobewaslabeled with [32P]dCTPand -dGTP (Dupont, NEN Research Prod-ucts, Boston,Mass.) by nicktranslation(30).

Analysis of infected-cell polypeptides. Infected Vero cells werelabeled with [35S]methionine (Dupont, NEN Research

Products)asindicated infigurelegends. Lysatesof

radioac-tivelylabeledinfected-cell cultureswere

prepared

and

elec-trophoresed on

SDS-polyacrylamide gels

as described

pre-viously (48).

RESULTS

ICP27mutantplasmids. Ten nonsenseand three deletion mutationswere introduced into the codingsequenceof the clonedICP27genein

plasmid pKHX-BH (Fig.

1).The genes

containing nonsense mutations

specified

ICP27

peptides

composedof from 26(pn2)to511(pnll)amino acid residues of the 512-residue

wild-type protein.

The

synthetic

linker used to generate nonsense mutations was also introduced into sequences downstream of the 3'

transcription

termina-tion site of the ICP27 gene. The

resulting plasmid,

pnl4, specifies the wild-type ICP27 protein. The three deletion

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ICPO ICP4 ICP47

IC PO _____---- ICP27 -. P22 ICP4

N N SNS BSI N BpSt NS EN S ScH

100bp, N N Nl- 8 N Na E S Sc

ICP27mRNA

ICP27codingsequence

UMMM/ 7/77777 /"//"/ 77 /"//

COMPLEMENTATION

INDEX 161

97

1.2 1.2 1.1 0.8 1 .o 1.5 1.9

9.2

9.7

X

*

[

c

0

z

I II II 1

345 6 7 8 910 1112 13

2

n14

iKHX-BH _,512

n2 - 26

n3 153

n4 163

nS 178

n. 261

n7 327

nS 407

ng nlO nIl

1.5 r dl

1.1 o d2

1.1 L d3

.460

-465

- 511

St Ns

I 512

407 460

Bp St

- I- 512

375 407

as St

-_ - 512

258 407

FIG. 1. Physical mapofICP27nonsense(n)anddeletion(d)mutationsandcomplementation of5dl1.2byICP27mutantplasmids. The locations of theimmediate-earlygenesencoding ICPs 0, 4, 22, 27, and 47areshownas arrowsontheHSV-1genomeatthe top of thefigure. Below thegenomeis anexpanded restrictionmap of theBamHI-HpaIviralDNAinsertinplasmid pKHX-BHusedtogeneratemutations in theICP27gene. Beneath theexpandedmap areshownthe limits ofthesequencesspecifyingICP27mRNA,thedirectionoftranscriptionof this mRNA, andthelocations of theICP27coding sequence. The numbered vertical linesbelow theICP27codingsequence indicate the locations ofnonsensemutations introduced into theICP27geneinpKHX-BHtoyield thenonsenseinsertionmutantplasmidsnlthroughn14. Theheavylines in diagrams ofnonsense mutantsindicatetheICP27 amino acidsequenceencodedbymutantplasmids. Theheavylines in deletionmutantdiagramsindicate the DNA sequences that encodein-frameICP27 amino acids.To the left ofmutantdiagramsareshownthe complementationindices(CI) ofmutantpeptides for5dl1.2.(CIequalstheratio oftheyieldof5dl1.2inVerocells transfectedwithamutant plasmidtotheyield of5dl 1.2inVero cellstransfectedwithpBR322.)

mutations removed viral DNA sequences that encode 53 (pdl)-, 32 (pd2)-, and 149 (pd3)-amino-acid residues in the

carboxy-terminalhalfof the ICP27 molecule.

ComplementationofanICP27 deletion mutant (5d11.2)by mutantplasmids. Inordertodeterminewhetherthe mutated ICP27 genes specify peptides with ICP27-associated activi-ties, mutant plasmids were first tested for their ability to

supply these activities by complementation of the ICP27

deletion mutant 5dl 1.2 (32). As shown in Fig. 1 (far left),

with the exception of the wild-type ICP27 protein, which produced complementation indices of 161 and 97 in two

independent tests, peptides specified by nonsense mutant plasmids pn2 through pnlO and deletion mutant plasmids

pdl, pd2, and pd3 failed to complement 5dl 1.2. Mutant plasmid pnll specifies a peptide in which the

carboxy-terminal

aminoacid

residue,

phenylalanine, was replaced by

three amino acid residues (Leu, Ala, and Ser) following

linker insertion. Plasmid pnll was able to complement 5dl 1.2 to only 6% of the wild-type level (complementation index, 9.7). These results demonstrate that one or more essential

ICP27-associated

activities were affected by each

ofthe mutations in our series of mutant plasmids and that

replacementofthecarboxy-terminal-most amino acid,

phen-ylalanine, significantly reduced complementing activity for 5dl 1.2.

Repressing and enhancing activities of mutant plasmids. In order to identify regions of the ICP27 molecule that are

requiredfor its repressing and enhancing activities, mutant plasmids were tested for their ability to modulate the up

regulatory effects of ICPO or ICP4 on CAT reporter genes driven by various HSV-1 promoters. Reporter plasmids tested included ptkCAT, pgBCAT, pICP4CAT, pVP5CAT,

and pL42CAT. The mutant effector plasmids tested were

pn2, pn4, pn6, pn7, pn8, pn9, pnlO, pnll, pnl4, pdl,

pd2,

and pd3. pKHX-BH, which contains the wild-type ICP27 gene, andpnl4wereusedaspositivecontrols. Plasmid

pnl4

contains a nonsense insertion mutation at the Sacl site outside the ICP27 coding sequence and, therefore, like pKHX-BH, specifies the wild-type ICP27 protein (see Sacl sitedownstream of the transcription termination site, Fig.1).

(i) Repression of ptkCAT activity. Results of cotransfec-tions ofptkCATtogether with the various ICP27 alleles and

plasmids expressing either ICPO (pW3) or ICP4

(pnllND)

areshown inFig. 2A. Consistent with previous findings

(10,

19, 31, 38, 44), both ICPO and ICP4 induced ptkCATactivity significantly above(-70-fold) the uninduced level, whereas wild-type ICP27 (pKHX-BH) exhibited no significant

en-hancingactivity for the thymidine kinase (tk) promoter. Also consistent withprevious findingswastheability ofwild-type ICP27 (specified by pnl4) to repress ptkCAT activity in-duced by ICPO or ICP4 (51). The pn8 and pnll forms of ICP27 also exhibited significant repression, but repression

by pn8 was less efficient than that observed with the wild-type protein. The ICP27peptides encoded by pn6 and pn7

exhibited modestrepression of both ICPO- andICP4-induced ptkCAT activity, whereas neither the pn2 nor the pn4 form of ICP27 exhibited repression of either ICPO- or ICP4-induced activity. Interestingly, the pn9 and pnlO forms of ICP27 reproducibly exhibited no repression ofICP4-induced

activity, butthey repressed -30 and -40% ofICPO-induced ptkCAT activity, respectively.

These observations indicate that the 80 amino acids present in the pn8 protein but absent from the pn7 protein (Fig. 1) specify significant repressing activity for the tk

14

B BamHI

Bp BspMI

Bs BasHII

N NaeI S SmaI

Si SalI

St Stul

Ns Nail

E EcoNI

Sc SacI

H HpaI

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A. ptk-CAT

0 80 -i

~0

.E 40 o 20

U-0t CYCY t*CD r.- Q0a0a0oo -- t,*x

cc cc cc cc cc cc -_--m

--- Ot ow ov0) ovov ++ + +++ x

ov OVov x 9

QL

pgB-CAT

C.

C 0 ~0

r--I

0 LA.

-OV CM NV VZD IDf.- N octlD X O0 -IVV x

COCL

C cc

4c

cccc---- --m

20++ +0+++ ++ 0+0+ +C CC C

0V 0v X

ot0

tIC

OL

150

80

60

40

20

0

pgB-CAT

22-ox*0

l+

+0

I+

1m

m m

SII

+

O1*

FIG. 2. Effect of wild-typeandmutant ICP27 peptides on tkCAT and gBCAT expression induced byICP0orICP4.Monolayers of Vero cellswerecotransfectedwith 2 ,ug of ptkCAT or 4 ,ug of the pgBCAT reporter plasmids and with 2 ,ug of pBR322, 3 ,ug of either pW3 (encoding

ICP0)orpnllND (encoding ICP4), 2

jig

of the indicatedICP27mutant plasmid, or 2

jig

ofpKHX-BH orpnl4.(Note: ThepnllNDplasmid encoding ICP4isdistinct from thepnllplasmid encoding theICP27genemutated in the C-terminal phenylaline residue.) All transfections were brought up to 25 ,ug with salmon testis DNA. Transfected cells were harvested 2 days posttransfection, and cell extracts were assayed forCAT activity.Theuninduced level of ptkCAT or pgBCAT activity was determined bycotransfectionwithpBR322 alone with no effector plasmidadded (not shown). This uninduced level was arbitrarily set at 1.0. The "fold" induction of CAT activity in cotransfections with effector plasmidswascalculated relative to the uninduced value.

promoter. Notably, addition to the pn8 peptide of 53 (pn9) and 58 (pnlO) amino acids interfered with the ability of the pn8 peptide to repress. Likewise, the addition of 46 amino

acids to the pnlO peptide to produce the pnll peptide

relievedthe interference with repression exhibited by pnlO

such that the pnll peptide exhibited wild-type repressing

activity. Thus, studies with nonsense mutations have iden-tified an 80-amino-acid repressing region (327 to 407), a

58-amino-acid region (407 to 465) that interferes with the repressingactivity of the 80-residue region, and a

46-amino-acid repressing region (465 to 511). Results with pnll also

demonstrate thatinsertion of the synthetic nonsense linker

into the codon specifying the carboxy-terminal

phenylala-nine residue did not affect the ability of ICP27 to repress

ICPO- orICP4-induced activity.

Deletion plasmids pdl, pd2, and pd3 all specify the

46-amino-acid repressing region of ICP27 (Fig. 1). The pdl plasmid also specifies the 80-amino-acid repressing region.

Bycontrast,pd2 lacks48(357to407) ofthese 80(327to407) amino acids and pd3 lacks all 80 residues. Notably, the

protein specified by pdl was more efficient than

wild-type

ICP27inrepressing ICPO-inducedptkCATactivity,whereas thepd2 protein was asefficientasthewild-typeproteinand

pd3waslessefficientthan

wild-type

ICP27in this

regard

(see Fig. 4, right-hand panel). The increased

repressing activity

ofpdl relativetowild-type ICP27

is,

by

definition,

duetothe absence of residues 407 to 460. Such an effect is not

unexpected whenone recalls thatthese residues interfered with therepressingactivity ofthepn8protein. The absence of48 residues of the 80-amino-acid

repressing region

in

pd2

hadnoeffectonthe

repressing

activity

ofICP27(see

Fig. 4,

right-hand panel),implying that the activity associated with this region is likely specifiedby residues 327 to 375. Sup-porting this view, the pd3 protein, which lacks the entire 80-residue region, was significantly less efficient than wild-typeICP27 inrepressingICPO-inducedptkCAT activity.The

residual repressing activity retained by the pd3 protein is

likelyduetothe fact that itcontains 45 of the 46 residues in the carboxy-terminal-most region that specifies ICP27 re-pressing activity. Taken together, these data suggest that repression of ICPO- and ICP4-induced ptkCAT activity in-volves residues 327to 375 and 465to 511.

(ii) Enhancement of pgBCAT activity. Previous studies haveshown thatICP27is abletofurther enhanceexpression ofpgBCATactivityinducedbyeither ICPOorICP4(45).We therefore attempted to identify the ICP27 amino acid se-quences responsible for this enhancement. The results of transient expression assays in which the gBCAT reporter gene was cotransfected with

wild-type

and mutant ICP27

plasmidsandplasmidsexpressingICPOorICP4areshown in

Fig. 2B and C. In these tests, the wild-type form of ICP27 alone (specified by pKHX-BH) had no enhancing effect on thegB promoter, whereas

pgBCAT

activity

wasinduced 20-to25-fold by ICPO.ICP4 alone exhibited minimal

enhancing

activity for the gB promoterunder the conditions used in thesetests. Althoughthe

wild-type

form ofICP27 specified by pnl4 enhanced both ICPO- and ICP4-induced pgBCAT activity

significantly,

none of the nonsense

(Fig.

2B)

or deletion (Fig. 2C) mutant forms of the

protein

enhanced ICP4-induced

activity

and

only

pn2,

pn4,

and

pn6

enhanced

ICPO-induced

activity

(-twofold

above the control level).

The failure ofpnlltoenhance

pgBCAT

activity

mustbe due

150j

80 -B.

C

0

C

0I

0 LiL

60

-40

-20

-L

--

l

ttStE

E

tERI

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A.

c 0

0

r-10

IL

B.

C

0

r-70 UI.

C.

C 0

~0

U.

plCP4-CAT 30

-20

10

0 N v 0 N 0 a 0

0. c c c c c c -

-+ + + + + c c

0 0

I I

- CD

C x

0 X

°lI

O0 NVt VWDNCDCD0e C**00-- Vtt I

CL.0 c c c CC CC CC C C- --- -- m

22.+++t++ + c

__ 0t0t 0t 0t 0t 0t X

0C

FIG. 3. Effect of wild-type and mutant ICP27 peptides on

pICP4CAT,pVP5CAT, and pL42CATexpression induced byICPO

orICP4. Forexperiments withpICP4CAT, 2jigofpICP4CATwas

coprecipitated with either1

,ug

of pBR322, 1 pLgof pW3 (encoding ICPO),1jigof the indicatedmutantICP27plasmid inthepresenceof 1 pig of pW3, or 1

Fg

of cloned pKHX-BH. Experiments with

pVP5CAT and pL42CAT were performed and quantified in a

manneridenticaltothatdescribedfor ptkCAT andpgBCATinthe

legendtoFig. 2.

toeither the absence oftheterminalphenylalanine residueor

to its replacement by three amino acids brought about by linker insertion.

Taken together, these observations suggest that the

car-boxy terminus ofICP27isrequired for the efficient

enhance-ment of ICPO- and ICP4-induced transactivation of the gB

promoter.

(iii) Repression of pICP4CAT activity. ICPO is able to

induce expression from the ICP4 promoter (10, 39), and

ICP27

is able to repress this activity (38). Similarly, in our

handsICPO enhanced pICP4CAT activity byapproximately

25-fold (Fig. 3A) and the wild-typeICP27protein specified

by pnl4 as well as the mutant peptide specified by pnll

repressed this activity with nearly equal efficiency.

Signifi-cant repressive activity was also seen with pn8. The other

mutantforms ofICP27 (pn2, pn4, pn6, pn7, pn9, andpnlO) exhibited no significant repression of ICPO-induced

pICP4CAT activity. These results are similar to those

de-scribed above for repression of the tkpromoterand support

the existence ofan80-amino-acid region between residues

327(pn7) and 407(pn8) anda46-amino-acid

region

between residues 465 (pnlO) and 511

(pnll)

that

specify

repressing

activity.

(iv) Repression of pVP5CAT and

pL42CAT

activity.

In order toidentify the

region(s)

of the ICP27

peptide

respon-sible for the modulatory effectsofICP27 onthe

expression

ofrepresentative-ylandy2genes

(51),

mutant

peptides

were tested for their ability to affect the

expression

of a

-yl

(pVP5CAT) and a -y2

(pL42CAT)

reporter gene.

In these studies, ICP27 alone

(pKHX-BH)

exhibited no

enhancing orrepressing

activity

for either gene, ICPO exhib-ited a strong enhancing effect on the

expression

of both reporter genes, and ICP4exhibitedmodest

enhancing

activ-ity for pVP5CAT but not for

pL42CAT

(Fig.

3B and

C).

Wild-type ICP27, specified

by

pnl4,

and the

pnll

mutant

peptide repressed the

enhancing

activities induced

by

both ICPO (for pVP5CAT and

pL42CAT)

and ICP4

(for

pVP5CAT) to uninduced control levels. The

ability

to

re-press ICPO- and ICP4-induced activation of

pVP5CAT

was

also a property of the pn8 mutant

peptide

and to a lesser

extent of thepeptides

specified

by

pn7

and

pnlO

(for

ICPO-induced activity). The relative

strengths

ofthe

repressing

activities of the various ICP27 mutant

peptides

for ICPO-induced pL42CAT activity was

nearly

identical tothat seen

with ptkCAT and pICP4CAT.

(v) Verification ofthe repressing activities ofmutant pep-tides. In order todetermine

definitively

whether the repress-ingactivity exhibitedby

wild-type

plasmids

pKHX-BH

and pnl4 and mutant plasmidspn8,pnll

(Fig.

2A),

pdl,

pd2,

and pd3 is aconsequence oftheactivities ofthe

corresponding

ICP27 peptides, additional tests were conducted with

ptk-CAT (Fig. 4). Inthesetests, effector

plasmids

werecleaved withinICP27 coding sequences

(Sall

for

pnl4,

pKHX-BH,

pn8, pnll, pdl, and pd2; NsiI for

pd3)

or in

plasmid

sequences with PvuII, before cotransfection of cells with ptkCAT and pW3 (see Fig. 1 for relevant

cleavage

sites).

Cotransfection of SalI- or

NsiI-digested

plasmids

with the ICPO plasmid did not result in

repression

ofICPO-induced

ptkCATactivity (i.e., CAT

activity

similar tothat

produced

incontrol cellstransfected withthe ICPO

plasmid

alonewas observed). Therefore,

interruption

of

ICP27

coding

se-quences in the wild-type and mutant

plasmids

impaired

the ability of the peptides they encode torepress ICPO-induced

ptkCAT activity. In contrast, cotransfection of

PvuII-di-gestedplasmids with the ICPO

plasmid

resulted in

repression

of ICPO-induced ptkCAT

activity (i.e.,

CAT

signals

were similar to those of the

undigested

plasmids).

Thus,

the repressing ability of the ICP27protein

specified

by

wild-type

plasmids pKHX-BH and n14 and mutant

plasmids

pn8,

pnll, pdl, pd2,andpd3requiresthe

expression

ofthe

ICP27

proteins they encode.

One may argue that the

repressing

activity

of the mutant

forms of ICP27 is not the residual

activity

ofICP27 but a

trans-dominant negative phenotype converted from a

posi-tive activity. If this were the case, this trans-dominant negative activity should have a negative effect on the

gB

promoter, in contrast to wild-type

ICP27,

which

displays

an

enhancing effect. We didnot observe sucha

negative

effect,

in that mutants which lost the ability to activate

pgBCAT

(e.g., pn8) did not acquire the

ability

to repress

pgBCAT

(Fig. 2B). On the other hand, the mutants resembled wild-typeICP27 in that theyrepressed

expression

from the ICP4, tk, VP5, and L42 promoters.

Therefore,

the mutant

pheno-types reflect the repressing activity ofthe

wild-type

ICP27

molecule and can be used legitimately to map the

region

involved inrepression.

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TKIEI-CAT

F----ICPO

pBR322 nB8 nil n14 fnB nil n14 n8 nll M4 n14

Sa Pvuli

IIIf*lag

₀ 0 it

0

1p0

3-Ac

1-Ac

It

* * *t *t

it

t

9

Cm

le " I.. - -I 0 0e e e .

%Ac 09 83 93 75 83 45 5-9 12 39 3-4 5-0 0-6 0-4 R 1 92 103 83 92 50 6-6 13 43 368 56 0 7 0 4

ii'

i'

i

_322 dt d2 d3 1 dl d2 d3 I dl d2 d3 1 I

Soli 1 WSI PWII

*--3

,

?

Con

# .- .*e 4 a * . * * *0 Cv *

%Ac03 219713 34260-7 2 44 23 04244517 0403 R 1 7032 43 113 87 2 t-7 157-7 1-3 t51557 1-31

ICP27inRNA ICP27coding

Sall NTsl Psull

FIG. 4. Effectof intra- and extragenic cleavage of ICP27 mutant plasmids on ptkCAT expression induced byICP0.Prior to transfection, plasmids eitherwere cleaved withSall,NsiI,or PvuII or were not cleaved. Shown below the autoradiogram is the ICP27coding sequence andthe locations ofthe relevantrestrictionenzyme cleavage sites. This experiment wasperformed in a manner identical to that described inthelegend to Fig. 2 for ptkCAT.

Collectively, the results of transient expression assays

demonstrate that ICP27, which alone had little or no effect ontheexpression ofanyof the HSV-1 reporter genes tested, acts as arepressorofICPO- and/orICP4-inducedexpression

ofpICP4CAT, ptkCAT,pVP5CAT,andpL42CATandas an

enhancer ofpgBCAT activity induced by ICPOorICP4. The resultsof transient expressionassayswith theICP27mutant

plasmids are summarized in Fig. 5. The following

conclu-sionscanbedrawnfromthesestudies: (i)tworegionsof the

ICP27 molecule exhibit repressing activity-one region be-tweenamino acid residues327 and 407 andasecond between

residues 465 and 511, (ii) residues 407 to 460 are able to

interfere with the repressing activity specified by residues 327to407,(iii)manyregionsof thecarboxy-terminalhalfof the ICP27 protein are critical for the enhancing effect of

ICP27onthe gB promoter andforefficientcomplementation ofanICP27 deletionmutant, 5dl 1.2,and(iv) substitution of

a nonsense codon specifying three amino acids for the

carboxy-terminal phenylalanine codon destroyed the en-hancing activity of ICP27 but did not affect its repressing

activity.

Introduction ofthe d3 mutation intotheviral genome. As shown in Fig. 4, the mutant peptides encoded by deletion

plasmids pdl, pd2, and pd3 were able to repress

ICP0-induced ptkCATactivity in transientexpression assays. Of the three mutants, therepressing activity of pdlwasgreater thanthatofthewild-type ICP27protein and that ofpd3was somewhat less than thatofwild-type ICP27 (Fig. 4).Because the d3 mutation exhibited a repressing phenotype midway

between thatofwild-type ICP27and the nullphenotype (Fig.

4,right-handpanel),thed3 mutationwastransferred into the viral genome by marker transfer. Since the pd3 mutant

plasmid failed to complement the ICP27 deletion mutant

5dl1.2 in transient complementation assays (Fig. 1, left

ICP27coding sequence R

I I 2

512

3 407 1 1

repression | |

Iepression

Of tk,ICP4 Activation (P5 andL42 ofgB

promoters promoter

! + +

J5i1 4+

repression

Complementation

of 5dl 1.2

+

(t)

6%

dII~-512 +9

407 460

Deletion d2 1 512 +

375 407

d3 2- 512 +

-L ~~~~258 407

FIG. 5. Locations oftheICP27primaryaminoacid sequencesresponsibleforrepressionof CATactivityfromreportergenes. Beneath the boxrepresentingtheICP27codingsequenceareshown thelocations of thenonsenseinsertion(n)and deletion(d) mutations in mutant plasmidsthatdefinefunctionallyimportantregionsof theICP27molecule. Therepressiveandenhancingactivities of mutantICP27plasmids areshowntotheright ofthefigure.

3-Ac I-AC

Wild-type

n7

n8 Nonsense

nil

nli 1.

-6

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column), it was assumed that replacement of the wild-type

ICP27

gene with the d3 allele would result in a nonviable

virus.ICP27-expressing 3-3 cells used previouslyas

permis-sive hosts for the isolation and propagation of nonviable

ICP27

deletion mutants (32)weretherefore used inefforts to

transfer the d3 mutation into the viral genome. For this purpose, 3-3 cellswerecotransfected with thelinearizedpd3 mutant plasmid and infectious KOS DNA. Progeny virus from the cotransfected culture was plated on 3-3 cells, and

individual plaqueswerepickedand screenedfortheir ability to grow in 3-3 and Vero cells. Plaque isolates that exhibited impaired growthinVerocellsbutgrewwell in 3-3 cellswere

plaque purified three times and amplified in 3-3 cells, and a

single isolate, designated vd3, was chosen for further

anal-ysis.

Figure 6 presents the results of Southern blot analysis of vd3 DNA compared with KOS DNA. Restriction enzyme

digestions with BamHI and EcoNI or BamHI and NsiI showed that vd3 DNA contained BamHI-EcoNI (1.50-kilo-base [kb]) and BamHI-NsiI (1.34-kb) fragments that were

indistinguishable from those of the parental pd3 mutant

plasmid, being 0.45 kb smaller thanthe corresponding wild-type DNA fragments (BamHI-EcoNI, 1.95kb;BamHI-NsiI, 1.79 kb). This analysis demonstrates that viral DNA ofthe vd3 mutant contains the deletion present in the parental plasmid.

Phenotypic analysis of ICP27 deletion mutants vd3 and

5dll.2.(i) Growth properties. The results of single-cycle growth experiments shown in Table 1 demonstrate the following points. (i) Whengrown and assayed inVero cells, no infectious vd3 orSdll.2 was detected, reflecting the nonpermissivenessofVerocellsforthesemutants. Thisisin contrast to KOS, which grew and platedwell in Vero cells. (ii) When grownin Verocells and assayed in 3-3cells, both vd3 and 5dl 1.2 yielded low titers of virus. This virus was likely residual unadsorbed mutant virus, since the plaques were noticeably smaller than KOS plaques on 3-3 cell

monolayers. (iii)TheICP27-transformed cell line, 3-3,

com-plementedboth mutants efficiently, as demonstrated by the

titers and plating efficiencies of the mutants on these cells.

(iv) When vd3 was grown in 3-3 cells and assayed on Vero cells, no detectable wild-type recombinants were observed. This demonstrates that although vd3 DNA contains

se-quences flanking the deletion that are homologous to the ICP27-containing fragment resident in 3-3 cells (32), the

superinfecting mutant virus did not recombine detectably

with residentICP27 sequences.

(ii) Immediate-early polypeptide synthesis. Figure 7

(cyclo+) shows the immediate-early polypeptide profiles of KOS-,5dl 1.2- and vd3-infected Verocellsinthepresenceof

dactinomycinfollowing removal ofcycloheximide.Although

KOS-infected cells contained significant amounts ofICP27

(Fig. 7, arrow), 5dl 1.2 produced no detectableICP27 polypeptide. This isconsistent with thegenotype of5dl 1.2,

whichlacks the transcriptional start site as well as portions

ofthepromoterand codingsequence of theICP27gene(32).

As expected, vd3, which contains an in-frame deletion

mutation inICP27 coding sequences (Fig. 1), did not

pro-duceabandthat migrated in the position of wild-typeICP27

butratherproducedaprominentband of the size expected of thetruncated vd3ICP27 polypeptide.

In HSV-infected cells, ICP4 is often present as multiple

electrophoretic forms, presumably as a consequence of different levels of phosphorylation ofthe ICP4 peptide (13, 41, 57). Recently, Riceand Knipe observed achange inthe

electrophoretic mobility of ICP4 in cells infected with an

BamHI+EcoNNI BamH1+NsII

I I

OU) Y Co

0 ;> Y Ml In > Y CL

._,_ _ 441

-4426

-40 -o:1-95

If 4179

*b

- -c1-S

a -a1.34

-Dl3

_

_---JPC P 2

bP8 St Nm E H

TCP27,mRNA

pKHX BH

B BamHl

BsB ssHTI

StStuI

Ns Nsil

E EcoNI

H HpaI

Bs

d3

-258

512 St

- 5 1 2

407

FIG. 6.

Southern

blot analysis of vd3 and KOS DNAs relative to the viral DNA inserts in plasmids pd3 and pKHX-BH. Viral (vd3 and KOS) and plasmid (pd3 and pKHX-BH) DNAs were digested with either

BamHI

and EcoNI (left-hand lanes) or BamHI andNsiI (right-hand lanes). The digested DNAs were electrophoretically separated on a 0.7% agarose gel, transferred to nitrocellulose, and probed with

32P-labeled

pKHX-BH. Sizes of DNA fragments in kilobases are indicated on the right of the autoradiogram. Shown below the autoradiogram in descending order are a diagram of the KOS genome, an expanded diagram of theBamHI-HpaI viral DNA insert in plasmid pKHX-BH, the locations of relevant restriction sites, the location and direction of transcription ofICP27 mRNA, the location of

ICP27

coding sequences, and the genotypes of the wild-type

ICP27

gene in pKHX-BH and the d3 deletion mutation in plasmid pd3.

ICP27

ts mutant (45). This implies thatICP27 may be involved either directly or indirectly in the posttranslational modification of ICP4. In our hands, the ICP4 species syn-thesized in cells infected with

5dl

1.2 or vd3 migrated more slowly than the ICP4 species synthesized in KOS-infected cells. This difference was also seen in immunoprecipitation experiments in which monoclonal antibody to ICP4 was used (data not shown). Although the d3 mutation is able to repress viral gene expression as described above, it does not confer wild-type-like electrophoretic mobility on ICP4. This indi-cates that the repressive function associated withICP27 is distinct from the function that affects ICP4 electrophoretic mobility. No significant alteration was detected in the quan-tity of ICP4 or in the quantities or electrophoretic mobilities of ICPs 0 or 22 in mutant infected cells.

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[image:9.612.327.552.74.471.2]

TABLE 1. Growth of KOS, vd3, and

5dl

1.2 in Vero and ICP27-transformed cells

Virusyield (PFU/ml) when grownina:

Virus Vero cells 3-3 Cells

Vero 3-3 Vero 3-3

KOS 7.6 x 107 1.0 X 108 1.2 x 108 1.3 x 108 vd3 <101 5.4 x 103 <1o2b 5.1 x 107

5dl 1.2 <10' 4.3 x 101 <102 7.6 x 107

aMonolayers of Vero or 3-3 cells were infected with the indicated virus at a multiplicity of infection of 5 PFU/cell. At 18 h postinfection, infected monolayers wereharvested by scraping cells into medium. Suspensions were thenfrozen, thawed, sonicated, and clarified by low-speed centrifugation. Supernatant fluids were assayed on Vero monolayers.

bWhen vd3 and5dl 1.2 were grown in permissive 3-3 cells and assayed on nonpermissive Vero cells, monlayers of the latter cells exhibited uniform nonspecific cytopathic effects at low dilutions; this was likely due to cell killing at high multiplicity infection.

(iii) Viralpolypeptidesynthesis. Previous studies ofICP27

ts anddeletion mutants and the results of transient expres-sion assays have suggested that ICP27 is required to down regulate early gene expression and to enhance late gene

expression. To evaluate the effect of the d3 and 5dl1.2

mutations on the synthesis of early and late viral

polypep-tides,mutantandwild-type infected Vero cells were labeled

fromrr

5 to 17 h postinfection with [35S]methionine (Fig. 7,

cyclo-).

The results of this experiment indicate that ICP27 is

requiredtorepress the expression of some early genes and to

activatetheexpressionof others. For example, cells infected

with the ICP27 null mutant 5dl 1.2 overproduced early

proteins (ICP6 and ICP8) but underproduced the early protein ICP41 relative to wild-type virus. Consistent with

previous observations, 5dl 1.2 induced the synthesis of

markedly reduced levels ofproteins of the -yl class (ICP5,

-25, and -44) and no true late (-y2) proteins (ICP1/2, -15, -19/20, -35, and -43) (32). Like 5dl 1.2, vd3-infected cells underproduced ICP41 and failed to produce true late (y2) proteins. The synthesis of pgB and gB relative to KOS-infected cells was notgreatly affected ineither 5dl 1.2- or

vd3-infected cells in these experiments, and host protein synthesis was not efficiently shut offincells infected with

eithermutant. Althoughvd3 isphenotypicallysimilar to5dl

1.2, the two mutantsdiffer intworegards. vd3 induced the

synthesis of early proteins (ICP6 and -8) and especially ICP6,atalevel lower thanthatof5dl1.2but

slightly

higher

than that of wild-type virus, and it induced some

(yl)

proteins (e.g.,

ICP5

andICP25) atlevelslower than thatof wild-type virusbut slightlyhigherthan thatof5dl 1.2.

Three conclusions canbe drawn with

regard

to the

phe-notype of vd3, based on the

polypeptide profiles

shown in

Fig. 7 (cyclo-). (i)The ability ofvd3torepress

early

gene

expressiontolevelsbetween those of5dl 1.2and

wild-type

virus correlates withtherepressingability ofthepd3mutant

plasmid

for the early target gene tkCAT in transfection

assays (Fig. 4,

right-hand

panel).

(ii)

Unlike

previously

described ICP27 ts and deletion mutants

(32, 47),

vd3 induces the synthesis of

significant

levels of(-yl)

proteins

(i.e., ICP5 and ICP25). (iii) Like theICP27 ts and deletion

mutants (32, 47), vd3 is unable toup

regulate

true late

(-y2)

geneexpressionand isdefectivein theshutoffof host

protein

synthesis.

Inorder toestablishthat the viral

polypeptide phenotype

ofvd3isaconsequence of the absenceofICP27andnotthe

effect of mutations in other

regions

of the genome, the

cyclo+

cyclo-M KOS 5dl12 vd3 PM KOS Sdil-2 vd3

:.- ovo *.s-.

.:....z

:t,'-{Ea

IE22

IE 27

U.-::mm,T

5 L0513

6 E

8 E

96L(31)

pgRL(f1)

15 LO2)

19 20 L.(C2)

25 L(YI)

35L02)

41 E

43

L...

-.

FIG. 7. Polypeptide profilesofKOS-,

5dll.2-,

and vd3-infected cells.Left-handlanes(cyclo+);Verocell

monolayers

were

preincu-batedfor1hat37°Cinthe presenceof75 ,ugof

cycloheximide

per ml. Theywere theninfectedwith 10 PFUoftheindicated viruses percell in thepresenceofthesameconcentration of

cycloheximide.

At 6 h

postinfection,

cycloheximide-containing

medium was re-moved and

monolayers

were washed and overlaid with medium

containing

10 ,ug of

dactinomycin

per ml and 40

,uCi

of [35S]

methionine per 35-mm

plate.

After a further 3 h of incubation,

infectedcellswere harvestedand

analyzed by

SDS-polyacrylamide

gel

electrophoresis.

Right-hand

lanes

(cyclo-);

Verocell

monolay-ers wereinfectedwith 10 PFU percellat

37°C,

labeledwith thesame concentration of

[35S]methionine

indicated above from 5 to 17 h

postinfection,

and

processed

for

SDS-polyacrylamide gel

electro-phoresis

analysis.

The SDS

polypeptides

were

electrophoretically

separated

in 7%

polyacrylamide gels.

Selected

immediate-early

infected cell

polypeptides (ICP4,

-0, -22,and-27)areindicated inthe

left-hand

margin.

Selected

early

(ICP6,-8, and-41);

delayed-early

(yl: ICP5, gB, pgB, -25, and -44); andtrue late

(y2:

ICP1/2, -15,

-19/20, -35,and-43)

polypeptide

bands areindicated onthe

right-hand sideofthe

figure.

mutation in vd3wasrescued

by

standard

procedures

(47)

by

using

the

BamHI-HpaI

DNA

fragment

in

plasmid

pKHX-BH

(Fig.

1).

The rescued

virus, vd3R,

exhibited

wild-type

polypeptide

profiles

in both

cycloheximide

reversal and

22

LC

;.f.';:

.fm...:..z ik- ..;!=-11. xs'I..;f:

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B

27~14

.::..

2h |8 6 h | t M KcZS %,%,2wo 3 M. KO6 5drzIA 3 M -CSSd2'ld 3

5 LI-',)

6e:

8F

pg :01:

pgBL:"rl

25; r: 27It.

39F 43e0e-rI

.7~~~~~~~~~~~~~~L*88361

.0 - ||

IF~5..

FIG. 8. Viral polypeptides synthesizedin KOS-, 5dl 1.2-, and vd3-infectedVerocells intheabsence(A)and presence (B)of PAA(400 ,ug/ml)at2, 6,and12hpostinfection.Verocellmonolayers wereinfectedwith 10 PFUof theindicatedviruses andincubatedat37°Cin the absence (A)orpresence(B) of400

pg

ofPAA per ml. At theindicated timespostinfection,cellswerepulse-labeledfor30minwith100puCi of[35S]methionineper 35-mmplate. Monolayerswerethenwashed, andcelllysateswerepreparedandanalyzed bySDS-polyacrylamide gel electrophoresisasdescribed in thelegend toFig. 7. Polypeptide designations on therightaredescribedin greaterdetail in thelegend to Fig.7.

long-labeling experiments (datanotshown). Hence, the vd3 polypeptide profiles showninFig.7are aconsequenceof the mutationinICP27 codingsequences.

(iv)Kinetics ofviral protein synthesis. Toascertain whether thealterations in gene expression observed in vd3-infected

cells (Fig. 7) are due to the effect of reduced viral DNA synthesisandtoexamine therates of polypeptide synthesis inKOS-,5dl 1.2-, and vd3-infected cells, KOS- and

mutant-infected Vero cellswerepulse-labeled for30minat2, 6,and 12 hpostinfection with [35S]methionine in the presenceand absence of the DNA synthesis inhibitor phosphonoacetic acid(PAA).

The protein profiles shown in Fig. 8A (PAA-) and B (PAA+) demonstrate the following. (i) At 2 hpostinfection, bothvd3 and5dl1.2 failedtoinduce the synthesis of detect-able viralproteins, whereas viral proteins of all classeswere detected inKOS-infected cells (Fig. 8A andB). This obser-vation demonstrates that ICP27 is required to enhance the expression of all viral genes at a very early stage in virus replication. Because both mutantscontain wild-type copies ofICPs0 and 4, the major activators of viral gene expres-sion, the absence of the activating functions of these two proteins at2 h postinfectionimplies thatICP27hasaneffect

on one orboth of these proteins. (ii) At 6 h postinfection, bothvd3and5dl 1.2 induced levels of earlygeneexpression equivalent to those of wild-type virus, whereas -yl gene expression was markedly reduced relative to KOS. These observations indicate that early gene expression, although

slow to beinitiated in mutant-infected cells, reached

wild-typelevelsby6 hpostinfection.This was notthe case for-yl proteins. (iii) At 12hpostinfection, thepolypeptideprofiles of the mutants had changed little from the 6-h profile, whereas in KOS-infected cells, early gene expression was repressed and -yl gene expression was considerably aug-mented. (iv) The mutant profiles were not significantly differentin thepresenceandabsenceofPAA,whereasearly gene expression was repressed more efficiently in the ab-sencethan in thepresenceofPAA,consistent withprevious findings (32). (v)As in thecase oflong-labelingexperiments, themajor difference in thephenotypesof vd3 and5dl1.2 was manifest as aslightly greater repressionofearlygene expres-sionby vd3 than by 5dl 1.2. This differencewasmostnotable at 12h postinfection.

DISCUSSION

ICP27 is a multifunctional regulatory protein that pos-sessesatleasttwodistinctactivities, namely repression and enhancement of ICP0- and ICP4-induced gene expression. Although the mechanism by which ICP27 mediates its regu-latory effects is not clear, the observation that ICP27-stimulated viral geneexpressionoccurs concomitantly with increased rates of the transcriptionstrongly suggests that it exertsits upregulatory effectatthelevel of transcription(16,

32). The ICP27 protein is a nuclear phosphoprotein

com-posedof 512 aminoacids, withanapparentmolecular mass of 63kilodaltons(41). LikeICP4,phosphate has been shown to cycle on andoff the nascent ICP27 protein during

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(11)

tion (57). AlthoughICP27has been reported to bind to DNA (23), it is not known whether this binding is specific or nonspecific or whether it involves interaction with other viral or cellular proteins. The study described herein was designed to begin to relate the specific structural features of

the

ICP27

molecule with its repressing and enhancing

activ-ities.

Functional regions of the ICP27 protein. Although the requirement for thehydrophilic amino portion ofICP27 was not specifically assessed in these studies, Hardwicke et al. have shown that it is not essential for either the repressing or enhancing activity of the protein (22). The importance of the carboxyl portion of the ICP27 molecule to its essential regulatory functions is suggested by the fact that the muta-tions in allICP27 ts mutants mapped to date are located in this region (47) and by the existence of extensive amino acid sequence conservation between this region ofICP27 and the carboxy-terminal halves of the related varicella-zoster virus gene 4 product, the Epstein-Barr virus BMLF1 product, and the IE 52k gene product of herpesvirus saimiri (7, 20; D. J. McGeoch, V. G. Preston, S. K. Weller, and P. A.

Schaffer,

in S. J. O'Brien, ed., Genetic Maps, in press).

In an effort to identify regions of theICP27 primary amino acid sequence required for its repressing and enhancing functions, a series of nonsense and in-frame deletion muta-tions were introduced into the ICP27 coding sequence.

Because

they can be used to delete defined stretches of

amino acids, these two types of mutation are especially useful in assessing the activities specified by individual regions of a protein and in identifying functional domains prior to more detailed analysis by amino acid deletion and substitution mutagenesis.

(i) Repression. The results of transient expression assays with mutant plasmids demonstrated that two regions of the

ICP27 primary amino acid sequence specify repressing ac-tivity for immediate-early, early, and late promoters: an 80-amino-acid region between residues 327 and 407 and a 46-amino-acid region between residues 465 and 511. The 80-amino-acid region is not absolutely required for the repressing activity ofICP27, but it is necessary for wild-type levels of repression, since the pd3 peptide which lacks this

region

was able to repressICPO-or ICP4-induced expression from the early tk promoter significantly.

The pnll peptide but not the pnlO peptide efficiently represses ICPO- or ICP4-induced expression of immediate-early, immediate-early, and late promoters. This suggests that the 46-amino-acid region between residues 465 (pnlO) and 511 (pnll) also contributes to the total repressing activity of

ICP27. Amino acid sequence analysis has shown that the 46-residue region contains a putative zinc finger motif as well as four serine and two threonine residues which may serve as phosphorylation sites. Similar motifs are present in other transcription proteins.

Lying between the 80- and 46-amino-acid repressing re-gions ofICP27is a 58-amino-acid sequence (407 to 465)that is not requiredforrepressing activity but is able to interfere with the repression specified by the 80-residue sequence. Analysis of the primary and secondary structure ofICP27 reveals that the 58-amino-acid region between amino acids 407 (pn8) and 465 (pnlO)is the most hydrophobic and hence the most rigid portion of the molecule. It ispossible that the additional amino acid residues specified by pn9 and pnlObut not by pn8 in the absence of the remainingcarboxyterminus (amino acids 465 to 512) may cause the pn9 and pnlO peptides to assume a conformation that masksthe activity of the 80-amino-acid repressing region. As in the case of the

mutant peptide specified by pdl (Fig. 4, right-hand panel),

mutations in the 58-amino-acid region would likely yield

peptides with greater repressing activity than thewild-type protein. Such mutants might exhibit a trans-dominant phe-notype (i.e., the ability to inhibit viral growth) even in the presence of wild-type ICP27.

(ii) Enhancement and complementation. NoneoftheICP27 mutant peptides tested in these studies was able toenhance ICPO- or ICP4-induced pgBCAT expression. Thus, specific amino acid sequences lying between residues 258 and 407 (pd3), 375 and 407 (pd2), 407 and 460 (pdl), and 460and 512 (pn9) were shown to be required for pgBCAT enhancing activity. Of special interest was the observation that pnll peptide, which contains 511 of the 512 residues in ICP27 (and hence contains the sequences deleted in mutant peptides specified by pdl, pd2, and pd3), was also unable to enhance gB promoter activity or to complement anICP27 null mutant efficiently. These observations have led us toconclude that unlike its repressing function, which appears to be specified by at least two discrete domains of the ICP27 protein, the enhancing and complementingfunctions ofICP27 involve a larger portion of the carboxy half of the ICP27 molecule. Consequently, enhancing activity would be particularly sen-sitive to mutations thatproduce alterations in the conforma-tion of the protein.

Mutant peptides were tested in this study fortheir ability to modulate ICP0- and ICP4-induced gene expression from five viral promoters representing genes of the four major kinetic classes. A comparison of the relative repressing

activities of mutant peptides for the tk, ICP4, VP5, and L42 promoters induced by ICPO revealed a strikingly similar pattern (compare Fig. 1A and Fig. 2B, C, and D). These relative activities were also evident in the repression of ptkCAT activity induced by ICP4. Together, these observa-tions suggest a common mechanism underlying the repres-sion of both ICP0- and ICP4-induced promoter activity by

ICP27. They further suggest that the mechanism of repres-sion is the same for all fourclasses of promoter.

The results of the transient expression assays conducted in this study confirm and extend the results ofother inves-tigators. Thus, the 80-amino-acid region able to repress

ICPO- or ICP4-induced expression from four classes of

promoter identified in this study is a subset of the 143-residue repressing region (263 to 406)described byRiceetal. for the early ICP8 promoter (46). Similarly, the 46-amino-acid repressing region shown for the same four classes of promoter in this study is asubsetofthe 78-residuerepressing

region (434 to 512) described by Hardwicke et al. for the early tkpromoter (22).Together, these three studies suggest that repression ofICPO- orICP4-induced geneexpression is specified by at least two regions of theICP27 molecule.

Our studies agree well with those of Hardwicke et al. concerning the region of the protein required for enhance-ment ofICPO- or ICP4-induced geneexpression in thatmost

ofall of the carboxyl half of the protein is required for this activity (22). Theseresults differ from those of Riceet

al.,

in which a peptide specifying only the amino terminal 263 amino acids exhibited partial enhancing activity (46). The reasons for this discrepancy are currently unclear.

Complexities oftransient expression assays. Investigatorsin severallaboratories haveclearly demonstrated thatICP27 is capable of both enhancing and

repressing

ICPO- or ICP4-induced viral geneexpression in transient

expression

assays. Despite theconsensus with regardtothe activities of

ICP27,

marked discrepancies in the results oftests with individual viral promoters are well documented. Thus, for