Herpes simplex virus IE63 acts at the posttranscriptional level to stimulate viral mRNA 3' processing.

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Copyright X 1992, American Society for Microbiology

Herpes Simplex Virus IE63

Acts

at

the Posttranscriptional

Level To Stimulate Viral

mRNA

3' Processing

J. McLAUCHLAN,' A. PHELAN,1 C. LONEY,1 R. M. SANDRI-GOLDIN,2 ANDJ. B.

CLEMENTS"*

Medical Research Council Virology Unit, Instituteof Virology, University of Glasgow, Church Street,

Glasgow Gil SJR, Scotland,1andDepartmentofMicrobiology and MolecularGenetics,

College of Medicine, University of California, Irvine, California 927172

Received 9 July 1992/Accepted 21August 1992

We have shownpreviouslythatanovel herpes simplex virus-induced activity, LPF, selectivelyincreasesRNA 3'-endprocessingatthepoly(A)site ofalate virusgene(J. McLauchlan, S. Simpson, and J. B. Clements, Cell

59:1093-1105, 1989). Here,ourin vivo and in vitroanalysesboth demonstrate that LPF is induced duringearly stages ofvirus infection. Studies of virusmutantsindicate that expression ofthe immediate-earlyIE63geneis

required for induction ofthis activity. The selective effectson3' processing displayed inthepresenceofIE63

provide direct evidence that IE63 caninfluence this posttranscription process.This extends previous studies

whichreported increases inreportergeneactivity with certainpoly(A)sites byIE63 (R. M. Sandri-Goldinand G. E. Mendoza, Genes Dev. 6:848-863, 1992).

Transcription of herpes simplex virus (HSV) DNA can be

separatedinto three broadtemporal phases, immediate early

(IE), early, and late (L). To achieve regulated expression, HSV encodes polypeptides with transactivating functions which mediate control through sequences present within virus gene promoters (reviewed in reference 9). However,

there isno reason to assumethat suchregulationoccursonly

at the level oftranscription initiation, since modulation of

gene expression could be mediated also by

posttranscrip-tionalevents. Such regulation operates inanumberof virus systems through various mechanisms such as differential

splicing and polyadenylation (21), RNA transport (6), and

RNAstability (12).

Recent transfection studies on the function of the HSV type 1 (HSV-1) IE63 (UL54) geneproduct suggest that this

polypeptide influences the efficiency of mRNA 3'-end

for-mation at selectedpoly(A) sites and reducesexpression of

splicedgenes (30, 32).Virus recombinants containing

alter-ationsto IE63 display a

variety

ofphenotypes; however, a common feature is the

dependence

on IE63

expression

for

thesynthesisof late geneproducts

(16,

19, 26,

29).

Instudies

ofonetemperature-sensitive (ts)mutant, lack of correlation

between levels of transcription and accumulation of gC

mRNA, a late-gene product

(UL44),

suggested that IE63

mayaffect

posttranscriptional processing

ofRNA

(33).

Mu-tational

analysis

hasrevealed thatIE63canbothrepressand

activate geneexpression,and thesefunctional domains have

been mapped to the C-terminal

portion

of the

protein

(11,

19). IE63 also stimulates

expression

from

regulatory

se-quencesof retroviral

origin;

this stimulationwas

dependent

onsequencesattheretrovirus

poly(A)

site butwas

indepen-dent of the retroviral promoter

(5).

Fromin vitro

polyadenylation studies,

wehave identified

a virus-induced factor, LPF, which

selectively

increases

processingatthe

poly(A)

site of the HSV-2 UL38 gene

(18),

a late gene

encoding

a

capsid protein

(27). By

contrast, a second

poly(A)

sitecommon tothe

US10, US11,

andUS12

genes failedto respond toLPF. In accord withour invitro

results,analysisof mRNAs

produced by

virusrecombinants

* Correspondingauthor.

provided evidence that LPF exerted this selective effect during lytic growth in tissue culture cells (18).

Here, we extend our in vitro analysis of LPF and show that LPFactivitycanbedetected not only ininfected HeLa cells but also in infected BHK cells. Moreover, during productive infection, selective increases in poly(A) site usage can be detected from early stages of lytic growth.

Usingconditions selective for theexpression of certain virus

genes, our results indicate that the IE63 gene product is required for the increase in utilization of the UL38 poly(A) site. Data derived from transfection studies reinforce the view that IE63 is a component of the LPF-mediated effect.

MATERIALSANDMETHODS

Cells andviruses. BHK-21C13 cellsweregrownas mono-layers in Glasgow minimum essential medium supplemented with 10% newborn calf serum. Spinner cultures of HeLa cellsweremaintained in Dulbecco's mediumsupplemented

with5% newborn calf serum. Vero 2-2cells, which express

IE63 (33), and Vero cells were maintained in Glasgow

minimumessential medium supplemented with5%newborn calf serum and 5% fetal calfserum. Monolayers of HeLa

(WS)cellsweregrowninDulbecco's mediumsupplemented with5%newborn calfserumand5%fetal calfserum.

Stocks of wild-type (wt) HSV-1 17, vFJ7, and vSAU3 weregrownat37°C,whileHSV-1ts mutants weregrownat

31°C.Virus271acZ,inwhich the IE63 gene is inactivatedby

insertion of lacZ(33),wasgrownonVero 2-2cells. Viruses

d11403,

which contains a 2-kbp deletion in both copies of

IE110(34),and

HSV-1(F)A325,

which containsadeletion in IE68 (23; a gift from I. Halliburton) were grown on BHK

cells.

UV inactivation of viruswasperformed by

treating

virus

preparationswith 240,000 ,uJ ofUVlight at 260nm with a

Stratalinker model 1800

(Stratagene).

Construction of virus recombinants. Details of the con-struction ofvFJ7,which contains the promoter for the small subunit of theribonucleotide reductase gene

(R2),

have been described previously (28). To generate

vSAU3,

plasmid

pSAU3Cwasconstructed. pSAU3Cwasmade

by

replacing

a 100-bp DNA fragment

containing

the HSV-2 mRNA

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6940 McLAUCHLAN ET AL.

poly(A)sitesequences inplasmid pFJ7 (28)with a HindlIl-SmaIfragmentfrompSAU3which carries the HSV-2 UL38 poly(A) site sequences. pSAU3C was digested withXbaI,

and the DNA fragment containing the chloramphenicol acetyltransferase (CAT) and lacZ gene cassetteswas

puri-fied and ligated into the unique XbaI site in HSV-1 1802 DNA (28). Ligation products were transfected into BHK cells and overlaid with 2 ml of 0.5% agarin medium. After incubation at 37°C for 3 days, cells were overlaid with a

further 2 ml of 0.5% agar in medium containing 750 ,ug of 5-bromo-4-chloro-3-indolyl-,-D-galactopyranoside per ml. Blue plaqueswere isolated and purifiedto homogeneity by furtherplaque selection. One isolatewaschosen and named

vSAU3. Stocksof vSAU3weregrownand storedat -70°C. In vitro polyadenylation reactions. Spinner cultures of HeLa cells(1x 109)ormonolayersof BHKorHeLa cells(6

x 107) were infected for up to 16 h at a multiplicity of

infection of 10attheappropriatetemperature. For infections carried out in the presence of cycloheximide, cells were

incubatedin mediumcontaining50 ,ugofcycloheximideper mlfor 40 min priortovirus inoculation, andcycloheximide

was maintained atthis concentration in the medium for the durationofthe infection.

Nuclearextractswerepreparedeitherasdescribed

previ-ously (18)orby usingasmall-scale extraction method (14).

For the small-scale method,cells grownto 70% confluence

wereharvestedinphosphate-bufferedsaline (PBS) and

pel-leted by centrifugation at 1,400 x g at 4°C. Cells were

washed with 30 packed-cell volumes of PBS and pelleted. Aftersuspensionin 1packed-cellvolume of buffer A(10mM HEPES [N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid; pH 8.0],1.5 mMMgCl2, 10 mMKCl,1 mM dithiothre-itol),cellswereswollenonice for 15minand thenlysed by passing them through a narrow-gauge needle five to eight times. A crude nuclear pellet wasrecovered by

centrifuga-tionat12,000x gfor 20s.Nucleiweresuspendedin2/3vol

of theoriginal packed-cellvolumeby usingbufferC(20mM HEPES[pH 8.0], 1.5 mMMgCl2,25%[vol/vol] glycerol,420 mM NaCl, 0.2 mM EDTA, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride) and incubated on ice for 30 minwith continuous agitation. Following centrifugation at

12,000 x g for 5 min at 4°C, the supernatant, designated nuclearextract,was storedat -70°C.

SAU5 precursor RNA was transcribed in vitro as

de-scribed previously (18). In vitro polyadenylation reactions

wereperformedwith 1x 104to5x 104cpmof RNA in 1 mM

3' dATP-5 mM creatine phosphate-264 mM KCl-0.7 mM MgCl2-8.8% glycerol-8.8 mM HEPES (pH 7.6)-0.1 mM EDTA-0.2 mM dithiothreitol-2.5% polyethylene glycol at

30°Cfor 2 h(18). Inreaction mixturescontaining amixture

ofnuclearextracts, 7,ul of HeLa cellextractwasmixed with 4 ,ulof BHKcellextract. Reactionproductswere analyzed

asdescribed previously (20).

CATassays.Atappropriatetimes after eithertransfection

orinfection, cellswerewashed with PBS andsuspended in

250 mM Tris-HCl (pH 8.0). Cellswere frozen and thawed

threetimes, andcell debriswasremoved by centrifugation.

ExtractswereassayedforCAT activity by using the solvent

extraction procedure (31), and protein concentrations were

determinedby Bradford assays

(3).

CAT activities are ex-pressed as the percentage of [1 C]chloramphenicol

con-vertedtothebutyrylated formpermicrogram of proteinper

hour.Forsampleswithmorethan30% substrate conversion,

activities were determined from sample dilutions. Thus,

activities could bemorethan 100%.

Transfections. Transfections were performed on

50-mm-diameterpetridishescontainingsubconfluentmonolayersof HeLa cells with DOTAP transfection reagent (Boehringer

Mannheim) accordingto the manufacturer's instructions. A

3-p,g

portionofplasmid carryingthe CAT reporter gene and

theappropriateamountof effectorplasmidweretransfected

foreach dish. At 24 h aftertransfection,cellswereincubated infreshmedium; theywereharvested afterafurther 24 h.

Plasmids. The construction of plasmids pLW2 (10),

pSAU5

(18),

and

pSG130B/S (32)

have been described

previously. pSAU2was constructed by inserting a 265-bp

Sau3A1 fragment

containing

the HSV-2 UL38

poly(A)

site

into pUC9,generating plasmid pSAU1.HindIII-XmnI

frag-ments from

pSAUl

[containing

the HSV-2 UL38

poly(A)

site] and pLW1

(containing

the HSV-2

IE-4/-5

promoter

linkedto the CATcoding sequences;

10)

were isolated and

ligatedtogive plasmid pSAU2.

RESULTS

LPF is active from

early

stages of virus infection.

Previ-ously, we showed thatan

activity

which alters

poly(A)

site

usage could be detected in nuclear extracts made from

HSV-1-infected HeLa cells. This

activity,

termed

LPF,

selectivelyincreased

processing

in vitroatthe HSV-2 UL38

poly(A)

site

(termed

the L

site),

whereas

poly(A)

site

se-quencesfromHSV-2US10-US11-US12

(termed

theIE

site)

failed to respond. To detect LPF

activity

in extracts, we

constructed a

plasmid,

pSAU5, in which the L site was

linked to the IE site

(Fig. 1B). Using

precursor RNA

synthesized from pSAU5, we

developed

an assay system

whereby increased levels of LPF arereflected by a rise in

processing at the L site. As shown in

Fig.

1A, nuclear

extractsfrommock-infected HeLacellsprocess

efficiently

at

theIEsite,while theamountof RNA

processed

atthe L site

is either low

(Fig.

1A, lane

1)

or notdetected

(Fig. 3,

lane

2).

By contrast, infected-cell extracts process considerably

more efficiently at the L site (Fig. 1A, lane

2),

while no

increase in processing at the IE site is routinely detected

(18).

Although

ourdata hadindicatedthat LPFcouldinfluence

processingatthepoly(A)site ofalate virus gene, thephase

of the virus

lytic cycle

at which the

activity

could be detected was not known. Therefore, HeLa cells were in-fected with wt HSV-1 virus at 37°C, and nuclear extracts

were prepared at 2-h intervals

following

infection. The

presenceof LPF activity in theseextracts was determined

by comparingthe relative levels ofprocessingat theIE and

Lsites in SAU5 precursor RNA.

Analysis

of the levels of

processingateach timepointrevealed that increasedrelative

amounts of L-site product can be detected by 2 h after infection comparedwith the amounts in extracts of

mock-infected cells

(Fig.

1A, lane

3).

Later, the relative levels of

processing at the two sites remained essentially the same

(Fig.

1A,lanes 4 through 6).

Parallel studies with virus recombinantshad indicated that

LPFwascapableofincreasingusageof the Lpoly(A) sitein

vivo

(18).

To investigate whether LPF activity could be

detected in vivo atearly times, assuggested by the in vitro

data, the levels of CAT activity produced by two virus

recombinants,vFJ7andvSAU3,weremeasured.Virus vFJ7

contains the CAT polypeptide coding sequences linked to the R2 promoter and has the IE site downstream from the CAT gene (Fig. 2B). Virus vSAU3 alsocontains the CAT

coding sequences linked to the R2 promoter; however,the

IE site isreplaced bythe L site(Fig. 2B). Spinnercultures of HeLacellswereinfected with eithervirusat amultiplicityof

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A

1 2 3 4 5 6

Pre

L

I-₀

C.)

co

IE *4

_.

_

B

IE L

nt) 259nt

-73nt-FIG. 1. Analysisof LPFactivity atvarious times after HSV-1 infection. (A) SAU5 RNAreactionproducts generated by nuclear extracts prepared at different times afterinfection. Reaction mix-tures contained nuclear extracts prepared at the following times postinfection:lane2, 16h;lane3,2h;lane4,4h;lane5, 6 h; lane 6,8 h. Reactionproductsin lane 1weregenerated bymock-infected

nuclearextract.BandscorrespondingtounreactedprecursorRNA

(Pre)and the IE and Lcleavage productsareindicated.(B)Sizes of SAU5precursorRNA andproducts generated by cleavageatthe IE and Lpoly(A)sites. The size of SAU5 RNA is shown in

parenthe-ses,and the sizes ofcleavage products (in nucleotides)withrespect

tothe RNA 5' terminusareindicated.

infection of 5at37°C, and CATactivitywasdetermined for each virusatdifferent times after infection.

The CAT activitiesproduced byvFJ7inspinner cultures of HeLa cellsatvarious timesduringinfection(Fig. 2A)are

comparabletothose measured in BHK cells(28). However, CATactivityissignificantly higherin vSAU3-infected cells 3 h after infectioncomparedwith that invFJ7-infectedcells, andby6h,the level of CATactivity produced byvSAU3 is

approximately 10-fold greater than that produced byvFJ7.

At 9 and 12 hpostinfection, CATactivityinvSAU3-infected cells remains significantly higherthan that in vFJ7-infected cells. Since the promoter sequences linked to the CAT

codingsequences areidentical in vFJ7 andvSAU3,the data

suggestthat CATmRNA ismoreefficiently processedatthe

L site thanattheIEsitefromearlystagesof virusinfection.

Similar increasesin the amount of CAT mRNAprocessedat

the L sitecomparedwith thatprocessed atthe IE sitewere

observed whenBHK and HeLa cellmonolayerswere used

Prom CAT IE Prom CAT L

FIG. 2. CAT activities produced by virus recombinantsvFJ7 and

vSAU3. (A) HeLa cellswereinfectedat37°C, and CAT activities

weredeterminedat3-h intervals following infection. Activitieswere

expressedasthepercentageof["4C]chloramphenicolconvertedper

microgram of protein per hour. *, vFJ7 activities; 0, vSAU3

activities. (B) Description of CAT mRNAs produced by vFJ7 and vSAU3. Prom, R2promotersequencesusedtoinitiate synthesisof

CAT mRNAs.

(data not shown). These results are consistent with our in vitro results and indicate that LPF is active fromearlystages of infection. Thus, agene productmadeearly during infec-tionapparently induces the selective increase inprocessing

at the L site.

Expression ofIEgenes is sufficient for detection ofLPFin

vitro. Furtheranalysis of the temporalclass ofgene which mediates LPF activity was done in vitro by performing

infections under conditions that limited virusgene expres-sion. In vitro analysis of LPFwas facilitatedby the devel-opment ofanassay method which allowed detection of the

activity in BHK cell monolayers. This was necessary

be-cause the phenotypes of the ts mutants had been fully characterized in these cells. Moreover, for technical

rea-sons, the ability to detect LPF in extracts made from cell monolayers would permit experiments to be performed undermoretightlycontrolledconditions. Anexampleofthe assay method is shown in Fig. 3. BHK cells grown on

90-mm-diameter petri disheswere infected with HSV-1 at 37°C for 16h, and the abilities of thenuclear extracts from these cellsto process SAU5 RNAweretested. Analysis of the reaction products indicated that the extract from BHK cells failedtoprocessateither the IEorthe L sites(datanot shown);this failureis duetothe inherentinabilityto prepare

active processing extracts from this cell type. However,

A

120

B

Time(h)

vFJ7 vSAU3

All-1-11

9%, 1?1..

M ,,:-,-.z.

4.0",_W-1,

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1 2 3 4 5 6 7 Pre5i. ....

L

IE * Aw

FIG. 3. LPF activity produced in BHK cells infected under conditions which limit virus gene expression. SAU5 RNA was

incubated with the following quantities of HeLa and BHK cell nuclear extracts;lane2, 11 p.lofmock-infectedHeLa cell extract; lane 3, 11 p.1 of infected HeLa cell extract; lane 4, 7 ,ul of mock-infected HeLa cellextractplus4p.1ofextractfromBHKcells infected with wt HSV-1 for 16 h at 37°C; lane 5, 7 ,ul of mock-infected HeLa cell extract plus 4 p.1 of extract from BHK cells infected with wtHSV-1in the presence of50

pLg

ofcycloheximide perml; lane 6,7p.lofmock-infected HeLa cellextractplus4pl. of extract fromBHKcells infectedatNPTwithtsK;lane 7, 7

p.l

of mock-infected HeLa cellextractplus4p.1ofextractfromBHK cells infected atPTwithtsK. Lane 1contains unreacted SAU5 RNA. Bandscorrespondingtounreacted precursor RNA(Pre)andtheIE and Lcleavage productsareindicated.

combinationof theextract frominfected BHK cells withan

extract from mock-infected HeLa cells generatedproducts

processed at both the IE and the L sites (Fig. 3, lane 4). Incubation of SAU5 RNA with onlythe HeLa cell extract

gaveriseto aproductprocessed atthe IE site butfailed to

generate anyL-site product (Fig. 3, lane 2). Therefore, in this assay,the components for RNAprocessingaresupplied

bythe HeLa cell nuclear extract. This allows detection of LPF activity in the extract from infectedBHKcells,which stimulates processing at the L site. Nuclear extracts from

mock-infected BHK cells also failed to process RNA at

eitherpoly(A) site and, on combination with extracts from mock-infected HeLa cells, did not stimulate processing at the L site (data not shown).

Withthis assay method, the presence of LPF in extracts prepared from cells infected under various conditions was examined.Todetermine whether de novo synthesis of virus

polypeptides was necessary for LPF induction, BHK cells

were infected with HSV-1 in the presence of 50

p.g

of

cycloheximide perml, and a nuclear extract was prepared 8

h after infection. Combination of this extract with

mock-infected HeLa cell nuclear extract did not produce RNA

processed at the L site; however, efficient processing did occur at the IE site (Fig. 3, lane 5). Similar results were obtained with extracts from BHK cells infected with

UV-TABLE

1.

Correlation ofLPFactivitywithvirus geneexpression

Virus Virusgene(s) expressed LPFactivity

wtHSV-1 IE, E,L +

wtHSV-1 + None

cycloheximide

UV-inactivated wt None HSV-1

ts1204 None

tsK IE +

ts1207 IE, E +

d11403 IE(exceptIEllO), E, L +

271acZ IE(except IE63), E, L

HSV-1(F)A325 IE(truncatedIE68), E, L +

inactivated virus (Table 1). Thus, the inability of virion components to stimulate LPF indicates that virus gene expression is required for activity.

Thetemporal class of gene which generates LPFactivity

was analyzed by using a series of virus mutants with

restrictedphenotypes.One mutant,tsK,hasanalteration in Vmwl75,aprotein which iscontinuously requiredfor prog-ressof virus infectiontotheearlyandlatephasesof thelytic

cycle (7, 35). At the nonpermissive temperature (NPT;

38.5°C),tsKsynthesizesIEpolypeptidesbut failstoproduce

earlyand lategene products. BHK cells were infected with

tsKat 31°C,thepermissivetemperature (PT), andat38.5°C

for 16 h, at which point nuclear extracts were prepared. Addition of either of these extracts to mock-infected HeLa cell nuclearextractstimulatedprocessingatthe L site(Fig. 3, lanes 6 and 7). Indeed, processing at the L site was

consistently greater with extracts prepared from cells

in-fected at the NPT than with those infected at the PT. Another ts mutant, ts1207, which contains a mutation in

UL39 and synthesizes IE and early but not late virus

products(22, 24),also stimulated LPFactivityatthe PT and

NPT (Table 1). By contrast, a third ts mutant, ts1204,

generates LPFatthe PT butnot atthe NPT(Table 1).ts1204

virionsbind to cells but donotrelease their contents because

of an alteration in gene UL25 (1, 17); hence, they fail to express anyvirus genes. These results suggest that an IE component is involved ininduction of LPF activity.

Inactivation of IE63 reduces LPFactivity. Since IE175 is not functional in tsK-infected cells at the NPT, it appears reasonable toconclude thateitheroneofthe four other IE

products or a combination of them is involved in LPF

activity.tsmutations ineachof these four IE genesare not

available. However, recombinants which fail to express

IE110 (d11403; 34), IE68 [HSV-1(F)A325; 23], and IE63

(271acZ; 33) have been constructed. Extracts made from

HeLacellsinfected with these recombinantsweretested for

LPF activity. Figure 4 shows that extracts made from

d11403-infectedcells contain LPFactivity(lane4), whereas

extracts from 271acZ-infected cells fail to induce any com-parable increase inprocessingatthe Lsite(compare lanes 1 and 2with lane3).The apparent smallincreaseinprocessing at the L site between lanes 1 and3 representsvariability in the intrinsic processing activities in individual extracts. In commonwithd11403,extractsfromHSV-1(F)A325-infected

cells produced LPF activity (Table 1). These data identify IE63 as acomponentrequired for the induction of LPF.

Expression of IE63 intransient assays

selectively

increases processingattheLpoly(A) site.Previous results from trans-fections had indicated that plasmids expressing IE63 were J. VIROL.

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1 2 3 4

Pre

IE

FIG. 4. LPF activity produced by virus recombinants with inac-tivatedIEgenes.HeLa cellswereeithermockinfectedorinfected with different virusesat37°C for 8 h,atwhich pointnuclearextracts

were prepared. SAU5 RNA was incubated with the following

extracts: lane 1, mockinfected; lane2,wtHSV-1; lane 3,271acZ; lane 4, d11403. Bands corresponding tounreactedprecursor RNA (Pre) and the IE and L cleavage products areindicated.

A

0)

(U C1) U

o

LL.

1 2 3 4 5

Molar

equivalents

IE63

B

pLW2

capableofstimulatingCAT reportergeneactivity(5, 30, 32). This stimulation was dependent on the polyadenylation signalsused butindependentofthepromotersequences.To determine whether IE63 could increase expression from plasmids containing the Lpoly(A) site, transfections were

performedwithpLW2 andpSAU2. Both oftheseplasmids contain the CAT coding sequences linked to promoter

sequences from the HSV-2 IE-4/-5 genes; however, pLW2 contains the IE site downstream from the CATsequences,

whilepSAU2possessesthe L site(Fig.5B). HeLa cellswere

transfected with either of theseplasmids along with increas-ingamounts ofpSG130B/S,aplasmid whichproduces IE63

(32). Results revealed that CAT activity from pSAU2 was

stimulated up to fivefold by increasing amounts of pSG130B/S (Fig. 5A).In contrast, the level ofstimulationof CAT activity from pLW2 was maximally twofold at the highest ratio of pSG130B/S to pLW2. In parallel experi-ments, aplasmid expressing HSV-1 IE175 stimulated CAT activity from both plasmids to the same extent (data not

shown). Therefore, in agreement with previous results ob-tained by using other poly(A) sites, expression of IE63 is reflected in increased reportergeneactivitywhich ispoly(A) sitedependent.Takentogether, ourin vitro andtransfection data indicate that IE63 isrequired for increases in 3'

proc-essing atthe L site.

DISCUSSION

Our studies indicate that the selective increase in

process-ingatthe UL38poly(A)site is mediatedbyexpressionofat least one HSV IE component. This conclusion is derived from both in vivo studies with virus recombinants and in vitro analysis of nuclear extracts made from cells infected with virusmutants. Moreover, the increaseinprocessingat the UL38 poly(A) site is not dependenton either the pro-moterwhich drivestranscriptionorthetemporalclass of the

pSAU2 Prom CAT IE Prom CAT L

FIG. 5. CATactivities producedin cells transfectedwith CAT reporter plasmids together with a plasmid expressing IE63. (A) HeLacellsweretransfected withplasmid pLW2orplasmid pSAU2. Quantities of plasmid pSG130B/S which gave molarratiosin the rangeof1 to5ofIE63 gene to CAT reporter gene were cotrans-fected withpLW2 and pSAU2.At48haftertransfection,cells were harvested and CAT activitiesweredeterminedfor cells transfected with pLW2

(@)

and pSAU2(0). CATactivities are expressed as fold increase relative to those obtained in the absence of pSG130B/S. (B) Description of CATmRNAsproduced by plasmids pLW2 and pSAU2. Prom, promoter sequences derived from the HSV-2IE-4/-5genewhichwereused toinitiate transcription.

promoter. In wild-type DNA, the UL38 poly(A) site is

predominantly usedatlate times ininfection (2, 17a);

how-ever, we have shown that this poly(A) site can respond to

LPFatearly times. Thus, unlike latepromoters, the ability

toincreaseprocessingatviruspoly(A)late sites isnotlinked to additional structural features such as replicating DNA. This conclusion is alsoborneoutby in vitro studies in which

processingis uncoupledfrom activetranscription.

By using in vitro analysis (summarized in Table 1) and

transfection data (Fig. 5), we show that the HSV-1 IE63

polypeptide contributes to the increased processing

ob-servedattheUL38poly(A)site.Expressionof HSV-1IE63

is essential forlyticvirus growth; however,the function of

this polypeptide in virus infection has been unclear. In

transfection

experiments, coexpression

of IE63 with other

IEproducts did enhance transcription fromcertain but not

all HSV promoters (8, 25). More recently, IE63 has been shownto reduce

expression

of genes which contain introns

(30, 32). Moreover,thedata also indicated that the levels of

reporter gene expression from constructs that contained a

synthetic

poly(A) site,

which functioned

inefficiently,

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the IE63 poly(A) site were stimulated in the presence of

IE63; this level of stimulation was similar to that for the UL38poly(A) site used inourstudies. Bycontrast, expres-sion was not increased from constructs containing simian virus early or late poly(A) signals. Thus, the stimulatory effectsweredependent onthe 3' processing signals.

It isnot apparenthow the effect ofIE63onRNA

process-ingis relatedtothephenotypesof virus recombinants which

carry mutations in IE63. These mutants display a range of

restrictions in gene expression, although levels of certain late polypeptides generally arereduced for all mutants (16, 19, 26, 29). Itmaybe thatIE63 isamultifunctionalprotein which could mediate effects on cellular events other than RNAprocessing. Notably,domains whichnotonlyactivate butalsorepress geneexpressionhave been identified within theIE63 gene (11, 19).

The mechanismbywhichIE63influencesRNAprocessing is unknown. HSVinduces alterations in the pattern of small nuclear ribonucleoproteins (snRNPs) in infected cells (15). Our recent analysis has revealed that virus recombinants with mutations in IE63 fail to alter snRNP patterns (22a). Moreover, IE63colocalizes with U2snRNP,which binds to the splicingbranch pointin introns(4, 13).Theseproperties indicate thatatleastonefunction ofIE63istoinfluence the behavior of cellular components which perform posttran-scriptional processing of RNA. The ability of invitro

sys-tems to reproduce changes in levels of processing should facilitate analysis of the mode of action ofIE63.

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