Control of mRNA stability by the virion host shutoff function of herpes simplex virus.

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0022-538X/89/051897-10$02.00/0

Control of mRNA Stability

by the Virion Host Shutoff Function

of

Herpes

Simplex

Virus

ASHA A. OROSKAR ANDG. SULLIVAN READ*

Department of Microbiology and Program in Molecular Biology, Loyola University of Chicago, Stritch School

of

Medicine,

2160

Soluth

FirstAvenue,

Maywood,

Illinois 60153

Received 3 October 1988/Accepted 25 January 1989

vhslisamutantofherpessimplex virustype1thatis defective in the virion hostshutoff function responsible forthedegradation ofcellular mRNAs and the concomitant shutoffofhostprotein synthesis.In thisstudy,the effect ofthe vhsl mutationonthe metabolismof viralmRNAswasexamined by measuringthe half-lives and

patternsof accumulation of10 different viralmRNAsrepresenting all kinetic classes. The vhsl mutationhad theeffect ofdramatically lengtheningthecytoplasmic half-lives ofall10mRNAs. Inwild-typevirusinfections, the 10 mRNAshad similar half-lives,suggesting that little,ifany,targetmRNAselectivitywasexhibitedby the

vhs function. The vhsl mutation caused overaccumulation of a number of mRNAs. The effect was most

dramatic for thealpha (immediate-early) mRNA for ICP27and the beta(early) mRNAsencoding thymidine kinase, ICP8, andDNApolymerase. Whereasinwild-typeinfections these mRNAs increasedtopeak levels and subsequently declined in abundance, in vhsl infections they continued to accumulate until late times. A significant but less dramatic overaccumulation was observed for several beta-gamma (delayed-early) and

gamma (late) mRNAs. The results suggest that the vhs protein plays an important role indetermining the half-lives of viral mRNAs belonging to all kinetic classes and in so doing is important in the normal

downregulation atlate times ofalphaand betageneexpression.

Incells infected with herpes simplex virustype1(HSV-1), viral gene expression is regulated in a complex cascade

fashion (22). For most viral gene products, the rate of synthesisofaproteinisdirectly proportionaltotheamount of thecorresponding mRNAin thecytoplasm (14, 27). Since mRNAlevelsarenecessarily governed bytherateofmRNA degradation as well as the rate of synthesis, factors that control either process will affect gene expression. Many

studies of the control oftranscriptioninHSV-1-infected cells have been reported; however, to date few have addressed the factors that govern mRNA stability. The experiments reportedin thispaper concerntheregulation of viralmRNA half-livesby the virion host shutoff (vhs) function of HSV-1. One of the earliesteventsduring lyticHSV-1infections is the shutoff ofmost cellular protein synthesis (3-6, 11-13, 16-18, 20, 23, 28-30). This shutoff is initially caused by a

structural component of the infecting virion which causes

disaggregationof cellularpolyribosomes (29, 30) and degra-dation of host mRNAs (24). Mutant virus containing alter-ations in the vhs gene produce virions that are unable to

suppress host protein synthesisordegrade cellular mRNAs

(20, 28). Recently, the mutation carried by themutant vhsl hasbeenmappedtoanopen readingframe abletoencodea

protein with a predicted molecular mass of 55 kilodaltons (13, 15). However, the vhs protein has yet to be identified within virionsor infected cells.

Thefirst demonstration that the vhsl mutation affects the half-lives of viral as well as host mRNAs was provided by

the observation that, following reversal ofacycloheximide

blockpresent fromthe timeof infection, alpha (immediate-early) viral mRNAs decayed much more rapidly in cells

infected with wild-type, HSV-1 than in cells infected with vhsl (18). Subsequently, it was shown thatifvhsl-infected

cells that were already expressing alpha mRNAs were

su-perinfected with wild-typevirus in the presence of

dactino-* Correspondingauthor.

mycin, acomponentof the superinfecting wild-type virions destabilized the alphamRNAsencoded by vhsl (12).

Several studies havesuggestedthat thevhslgeneproduct

may affect the stabilities of beta (early), beta-gamma (de-layed early), and gamma(late) viral mRNAs in addition to alphaand host mRNAs. First, the vhsl mutation has been known forsome timeto affecttheaccumulation of beta and

gamma viral polypeptides within the infected cell (20). Second,atlate times after vhslinfection,thereareincreased

amountsofsomebeta andgammamRNAsinthecytoplasm

(28). Third, Kwong and Frenkel added dactinomycin to infected cultures and then pulse-labeled the cells with [35S]methionineeitherimmediatelyorafteranadditional 6 h

in thepresence ofdactinomycin (12). When they compared the proteins being synthesized before and after the 6 h incubation in thepresence ofdactinomycin, theyfound that

manyviralproteins continuedtobetranslatedlongerincells infected with vhsl than in cells infected withwild-typevirus. While the above dataareconsistent with thehypothesisthat the vhsgeneproductaffects thephysical stabilityof beta and

gamma mRNAs, they are also consistent with other

possi-bilities.

The presentstudy wasundertaken toevaluate the role of the vhs gene product in regulating the half-lives of viral mRNAs belongingto different kinetic classes andto assess

the importance of controls of mRNAstability in the overall scheme ofgeneregulation in HSV-1-infected cells. To this end, the half-lives and patterns ofaccumulation of 10 dif-ferent viral mRNAs were determined incells infected with

wild-type virus or vhsl. The results indicate that the vhs gene product significantly affects the stability of viral

mRNAs belonging to all kinetic classes. In so doing, it affects the levels and kinetics ofaccumulation ofanumber of viral mRNAs. Inparticular, the vhs proteinappearsto play

an important role, in conjunction with transcriptional

con-trols,in the normaldownregulationatlatetimes ofalphaand betageneexpression.

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MATERIALS AND METHODS

Cells and virus. Vero and HeLa S3 cells were purchased

from the American Type Culture Collection and grown in Eagle minimum essential medium (MEM; GIBCO Labora-tories) supplemented with antibiotics and 10%(vol/vol) calf

serum. Stocks of wild-type HSV-1, strain KOS, and the

mutant vhsl were prepared from infected Vero cell

mono-layers asdescribed previously (20). The vhslmutantgrows

well at all temperatures from 34 to 39°C and exhibits a

defective virion host shutoff function at all temperatures (20). All experiments were performedat 34°C. In all

exper-iments, virus was allowed to adsorb for 1 h in MEM containing 1% (vol/vol) calf serum. The inocula were then

aspirated, and the cells were overlaid with fresh MEM containing 1% calfserum.

Isolation of cytoplasmicRNA. TotalcytoplasmicRNAwas

prepared from monolayer culturesof 1 x 107to2 x 107Vero

or HeLa cells as described previously (18). The cellswere

first scraped into ice-cold phosphate-buffered saline and collected by low-speed centrifugation. They werethen

sus-pended in 2.2 ml ofresuspension buffer (10 mM Tris [pH 7.9], 0.15 MNaCl, 1.5 mMMgCl2) andlysed byaddition of Nonidet P-40(Calbiochem-Behring)to0.5%. After 10minon

ice, the nuclei were pelleted by low-speed centrifugation,

and thesupernatantwasremovedtoafresh tube. The nuclei

were washed with 1.2 ml of resuspension buffer containing

0.65% Nonidet P-40, and the wash was added to the first supernatant. The combined cytoplasmic supernatants were

further clarified by centrifugation ina Sorvall SS34 rotorat 10,000rpmfor10minand then addedtoanequalvolume of

ureabuffercontaining10 mM Tris(pH 7.9),7 Murea,0.35 M NaCl, 10mMEDTA,and 1% sodiumdodecylsulfate(SDS). The mixture was extracted twice with phenol-chloroform (1:1) and twice with chloroform, and the RNA was precip-itated byaddition of 2.5 volumes of ethanol and storage of the sample at -20°C overnight. After precipitation from ethanolasecondtime, the RNAsamplesweredissolvedina

small volume of 10 mM Tris (pH 7.9)-i mM EDTA and stored frozen at -90°C.

Gel electrophoresis and Northern (RNA) blot transfer of RNAs. Samples (5 ,ug) of total cytoplasmic RNA were

denatured with glyoxal and electrophoresed through 1.2%

agarose gels cast in 10 mM sodium phosphate, pH 6.8, as

describedpreviously (21).The RNAs werethen electroblot-tedontoGeneScreenPlus membranes(DuPont NEN Prod-ucts)withatransblotapparatus (Bio-Rad Laboratories)and

a transfer buffer of12 mM Tris-6 mM sodium acetate-0.3 mM EDTA, pH 7.5. Following electrophoretic transfer of the RNA, the membranes were rinsed for 30 s in 50 mM

NaOHtoremovetheglyoxal and then neutralizedbya30-s

rinsein0.2MTris(pH 7.5)-i x SSC(0.15MNaCl,0.015 M sodium citrate). The filters were then air dried at room

temperature.

DNAprobes. DNAprobesforuseinNorthernanalysisor

pulse-chase experiments were derived from recombinant pBR325 plasmidsorM13phages containingasinserts

appro-priate fragmentsof the HSV-1genome. Theplasmids pSG28 and pSG87, which were used for Northern blot

hybridiza-tion, werekindly provided by Myron Levine and Rozanne Sandri-Goldin(8). pSG28 wasusedas aprobe todetect the mRNAs forICP4, ICPO, andICP27, whilepSG87 wasused

todetectthymidinekinase andglycoproteinH(gH)mRNAs. Bothplasmids were maintained in Escherichia coliHB101.

Theplasmid pXlrll contained a4.6-kilobase (kb) fragment

of Xenopus laevis rDNA inserted into the EcoRI site of

colicin El

(2).

The cloned

fragment

contained most of the 28S rRNA

coding

region

and a small

portion

of the 18S rRNA

region. pXlrll

was

kindly provided by

Jeff

Doering.

PlasmidDNAswere

prepared

by

standard

protocols

(1).

The

probes

thatwereused forNorthern

analysis

of

ICP8,

DNA

polymerase,

glycoprotein

B

(gB),

ICP5,

and

glycopro-tein C

(gC)

mRNAs were

prepared

from recombinant M13

phages

containing

asinserts

intragenic fragments

ofthe viral genes. These

phages

were

kindly

provided by

Steven Wein-heimerand have beendescribed

previously

(31).

The

phages

contained the

following

inserts of HSV-1 DNA:

ICP8,

a

1.6-kb SalI-BamHI

fragment;

DNA

polymerase,

a 0.6-kb

BglII-EcoRI

fragment; gB,

a 0.6-kb PstI-SaIl

fragment;

ICP5,

a0.6-kbBamHI-HindIII

fragment;

and

gC,

a 1.6-kb EcoRI-BamHI

fragment.

To prepare

probes

forNorthernblot

analysis,

the inserts wereexcised from

appropriate

plasmids

orM13

replicative

forms. DNA

fragments

were

separated

by

agarose

gel

elec-trophoresis,

and the desired inserts were

purified

from

low-melting-temperature

agarose

(21).

The

probes

werethen labeled with 32P

by

nicktranslation withanicktranslation kit fromBethesda Research Laboratories

(Rockville, Md.).

For

preparation

of

probes

to be used in the

pulse-chase

experiment,

we found it convenient to reclone the inserts fromrecombinantM13

phages

intothe

equivalent

sites ofthe

polylinker

of the

plasmid Bluescript

SK

(Stratagene).

The recombinant

phages

that were used for the

pulse-chase

experiment

contained the

following

inserts: for

ICPO,

a 1.6-kbSstI-BamHI

fragment;

for

TK,

a1.1-kb

BglII-HindIII

fragment

from the 3' tkdeletion mutantA3'-1.6

(31).

These

phage

were also

provided by

StevenWeinheimer.

Hybridizations.

Membranes

containing

transferred RNAs were

hybridized

tonick-translated

probes by

the

protocol

of Graham and co-workers

(9).

The filterswerefirst

prehybrid-ized at

42°C

for 24 h in

prehybridization

buffer

containing

50% formamide

(Mallinkrodt, Inc.),

10% dextran sulfate

(Pharmacia Inc.),

10%Denhardt solution

(1),

Sx SSPE

(1x

SSPEis 180 mM

NaCl,

10mMsodium

phosphate [pH

6.8],

1 mM

EDTA),

1% sodium

lauryl

sulfate,

and 250 ,ug of denatured salmon sperm DNA per ml.

Following

prehybrid-ization,

the

probe

wasdenatured

by being placed

in

boiling

waterfor10 minandwasthenaddedtothe

prehybridization

buffer. The final

probe

concentrationwas

generally

5 x

107

to 10 x 107

cpm/ml. Hybridization

was then continued at

42°C

for 24 h. At the end of

hybridization,

the blots were

washed twice for 45 mineach at room temperature in 2x

SSPE-0.4% SDS and then twice more for 15 min each in 0.1x SSPE at 600C. The still moist blots were then sealed inside freezer

bags

and

exposed

to Kodak XAR-5 film for

autoradiography.

For

quantitation

of mRNA

levels,

appropriate

exposures of the

autoradiograms

were scanned with a

Gilford-Re-sponse

spectrophotometer

equipped

with a

gel-scanning

apparatus, and theareasunder the

peaks

were

integrated.

In

Fig.

2

through 5,

showing

the kinetics of mRNA

accumula-tion,

the amount of any

given

mRNA is

expressed

in

arbitrary

units. The

specific

activities of the

probes

for different mRNAs

varied,

and exposures ofdifferent

length

were scanned for different mRNAs.

Thus,

while the data allowed

comparison

of the levels ofa

particular

mRNA in mutant and

wild-type

infections or the levels at different times after

infection,

itwasnot

possible

to

directly

compare the level ofone typeofmRNA withthat of another.

After

appropriate

exposures of the

autoradiogram

had been

obtained,

the

probe

was

stripped

from the filters

by

boiling

the membranesfor 20to 30min in 10mMTris

(pH

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7.9)-i mM EDTA-0.1% SDS. Complete stripping of the probe was monitored by autoradiography. Following strip-ping, the filters were ready for hybridization to another probe. This procedure allowed at least five rounds of hybrid-ization to be performed with each filter.

Measurement of mRNA half-lives with a dactinomycin chase. Vero cell monolayers were infected with 20 PFU of wild-type virus or vhsl per cell. At 6 h postinfection 5

pig

of dactinomycin per ml was added to the culture medium, and incubation was continued. Total cytoplasmic RNA was extracted at 45-min intervals, and the decay of specific mRNAs in the presence of dactinomycin was analyzed by Northern hybridization. To control for small variations in the amount of mRNA that was loaded onto different gel lanes, the results were normalized to the amount of 28S rRNAdetected by hybridization to the plasmid pXlrll.

Measurement of mRNA half-lives with a pulse-chase proto-col. Measurement of mRNA half-lives by pulse-chase anal-ysis was performed as described by Pilder and co-workers (19) with minor modifications. HeLa cells infected with 20 PFU of wild-type virus per cell were pulse-labeled with MEM containing 200 ,uCi of [3H]uridine perml for 30 min beginning at 5.5 h postinfection. The label-containing me-dium was then removed, and thecellswerewashedand then overlaid with MEMcontaining 5 mMeachcold uridine and cytidine and 35 mMglucosamine. Total cytoplasmic RNAs were prepared at 45-min intervals and then subjected to exhaustivedigestionwithRQ1DNase(RNasefree;Promega Biotec), followed by phenol-chloroform extraction and eth-anol precipitation. The samples oflabeled RNA were then hybridized to specific fragments ofthe viral genome which had beenfixedto nitrocellulose filterdisks. For pulse-chase experiments, HeLa cells were used rather than Vero cells becausewe wereable toobtainamorecomplete chaseof the label with HeLa cells. Nevertheless, the patterns of viral gene expression are very similar in HSV-1-infected HeLa and Vero cells (datanot shown).

To fix the recombinant plasmid DNAs to nitrocellulose filters, the plasmids were linearized by restriction enzyme digestion, and 15-,ug portions were denatured and fixed to 25-mmnitrocellulose filterdisksasdescribedpreviously(1). Labeled RNA samples from approximately 5 x 106 cells werehybridized to the filter disks as described by Zhang and co-workers(32). Briefly,thediskswereprehybridized over-nightat 68°C in 2x SSC(lx SSC is 0.15 NaCl and 0.015 M trisodium citrate) containing 5x Denhardt solution. The labeled RNA samples werethen added to the prehybridiza-tionbuffer,andhybridizationwascontinuedat68°Cfor 48 h. The filters werethen washedtwice for30 min eachat room temperaturewith 2x SET(lx SET is 0.15 MNaCl, 20mM Tris [pH 7.8], 1 mM EDTA)containing0.1%SDS andtwice for30 mineachat60°Cwith0.1x SETcontaining0.1% SDS. They were then digested for30 min with 5 ,ug of RNase A

(Sigma Chemical Co.) perml and then washed twice more for 30 min each at 600C with 0.1x SET-0.1% SDS. The filters were then air dried and counted with a toluene-based fluor.

RESULTS

Effect ofvhsl mutation on accumulation ofdifferent viral mRNAs. In an earlier study, we showed that, following reversal ofacycloheximide block present from the time of infection and addition of dactinomycin, alpha mRNAs de-cayed more rapidly in cells infected with wild-type HSV-1 than in cells infected with the vhsl mutant (18). This

sug-A

WT VHS1

IcP0 *~

ICP27 6

*

* - *o m

it

TK * a

gH - is..

1 2 3 4 5 6

7* 914011w

7 8 9 10 1112

B

WT

DNA Pol

ICP 8

gB ICP 5

gC w...

1 2 3 4 5 6 7 8

VHS 1

,._..sw..

___

"-3,aw W

9 1Q 1 11 2 1 3 14 1516

FIG. 1. Accumulation of HSV-1mRNAsduring wild-type (WT) and vhsl infections. (A) Verocells infectedwith20PFU of either wild-type virus (lanes1 to 6) or vhsl(lanes7 to12)percell.Total cytoplasmicRNA wasextracted at 2.5 h(lanes1and7),4.5h(lanes 2and8),6.5h(lanes3and 9),8.5 h(lanes4and10),10.5(lanes5and 11),and 12.5 h(lanes6and12). (B)Vero cellswereinfectedwith 20 PFUof either wild-typevirus(lanes1to8)orvhsl (lanes9 to16) per cell. TotalcytoplasmicRNA wasextractedat2 h(lanes 1and9),4 h(lanes 2 and 10), 6 h(lanes3and 11), 8.25 h(lanes4and12), 10 h (lanes 5 and 13), 12 h(lanes6and14), 14h(lanes 7and15),and 16 h (lanes 7 and 16). Samples (5 ,ug) of RNA were analyzed by Northernblotting. DNAPol,DNApolymerase.

gested that,atleast under the conditions ofacycloheximide reversal experiment, the vhs gene product negatively regu-lates the stabilityofalpha mRNAs. If the Mhs gene product also regulates the half-lives of alpha mRNAs during lytic infections in the absence ofdrugs,onewould expect the vhsl mutationtoaffect theaccumulationofalphamRNAswithin infected cells. To test this prediction, Vero cells were infected with 20 PFU of eitherwild-type or vhsl virus per cell andcytoplasmicmRNAswerepreparedat2-hintervals. The levelsof selectedalpha mRNAswere thenanalyzedby Northernblottingandquantitated bydensitometricscanning ofappropriately exposed autoradiograms. Atthesametime, theaccumulation kinetics of selectedbeta, beta-gamma,and gammamRNAswerealsodetermined. The results shown in Fig. 1through 5 indicate that the vhsl mutation affected the accumulationkinetics ofanumberofdifferent viral mRNAs belonging todifferent kinetic classes.

Alpha mRNAs.The vhslmutation affected the accumula-tion kinetics of thealphamRNAsfor both ICP27 and ICPO. In cells infected with wild-type virus, ICP27 mRNA ap-peared earlyininfection, peakedin abundanceatabout 6h, and then declined (Fig. 1 and 2). By late times, only 40 to

50% of the peakamount ofmRNA was still present in the cytoplasm. Similar accumulationprofileshave beenreported previously for the alphamRNAsencodingICP22 and ICP47 (31) and ICP4 (10,31) with theexceptionthat in thesestudies the time of peak alpha mRNA accumulation was slightly earlierthan wasobservedin ourexperiments. Itislikelythat this difference wasdue to the fact that the present

experi-ments wereperformedat34°Cwhile the earlier studieswere

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z

E

0 6

0 ~ ~

0

Z~~~~~~~

0 2 4 6 8 10 12 14 16

[image:4.612.325.561.74.534.2]

Hourspost infection

FIG. 2. Accumulation ofICP27mRNAduring wild-type (wt)and

'h*sl infections. Autoradiograms of the gel shown in Fig. 1 were

scanned in ordertoquantitate the levels ofICP27mRNA.

performed at 37°C. As expected, the vhsl mutation led to significant overaccumulation ofICP27mRNA.Thus,incells infected withvhsl,the level of ICP27mRNA increased until 8 h and then remained high until at least 12.5 h after infection. At late times there was at least twice as much

ICP27mRNA in cells infected with vhsl as in cells infected

withwild-type virus.

In wild-type infections ICPO mRNA appeared in the cytoplasm at the same time as ICP27 mRNA and initially increased in abundance with similar kinetics (Fig. 1).

How-ever,althoughthe diffusenatureof the band for ICPO mRNA madeprecise quantitation difficult,it isapparentthatat late times the level of ICPO mRNA didnot decline tothe same

extent as did the level of ICP27 mRNA. Similarly, both Harris-Hamilton and Bachenheimer (10) and Weinheimer (31) have reported that at late times ICPO mRNA did not

decline in abundance as much as did the mRNAs encoding ICP4, (10, 31), ICP22, and ICP47 (31). In contrast to wild-type infections,in cells infected with i'hsl the level of ICPO mRNAcontinued to increase throughoutthe first 12.5 h of infection. Thus,atlatetimes therewastwo tothree timesas

much ICPOmRNA in vhsl-infected cells asin cellsinfected

with wild-type virus.

Beta mRNAs. In wild-type virus infections, the beta mRNAs encoding ICP8, thymidine kinase (TK), and DNA polymeraseall accumulated with similar kinetics(Fig. 1 and 3). Eachappeared slightly later than the alphamRNAs and increased topeak levels by 6to 8 h after infection. mRNA levels then declined sharply, so that by 12.5 h very little mRNAremained inthecytoplasm. In ourhands the decline

inabundance ofthe beta mRNAswasnoticeably sharperand morecompletethan itwasforICP27mRNA. Thispatternof accumulation was similar to those reported previously for various beta mRNAs byother investigators (10, 25, 31).

The vhsl mutation had a striking effecton the

accumula-tion of all three beta mRNAs (Fig. 1 and3). ICP8, TK, and DNA polymerase mRNAs all overaccumulated in mutant

infections. In contrast to the wild-type accumulation pat-terns, characterized by a peak and subsequent decline, in

vhslinfections the betamRNAsreachedpeak levelsat 10to

12 hafterinfection and thenleveled toaplateaurather than

declining. Thus,vhsl is defective inafunction thatplaysan

important role in the normal turning-down of beta gene expression.

Beta-gamma mRNAs. In wild-type virus infections, the beta-gamma mRNAs encoding ICP5, glycoprotein B (gB),

E

0

c

t:3

4,

0

E

0

c

-a5

E

0

0 0

*45

tp

0-Owt

5- A. TK mRNA *- *vhs-1

4-.

3

/-/

2-

/

1 -/0 0

10

1 to\°\

/

iii

0 2 4 6 8 10 12 14 16

Hours postinfection

8

6

4

2

2 4 6 8 10 12 14 16

Hours postinfection

_ _ I a I

0 2 4 6 8 10 12 14 16

Hourspostinfection

FIG. 3. Accumulation of beta mRNAsduringwild-type (wt)and

i'hsl infections. Autoradiograms of thegels shown in Fig. 1 were

scanned inordertoquantitate the levels of beta mRNAs.(A) TK; (B) ICP8; (C)DNA polymerase.

and glycoprotein H (gH) all appeared in the cytoplasm at

about the sametime asthe beta mRNAs (Fig. 1, 3, and 4). However, unlike the betamRNAs,thebeta-gammamRNAs did not decline in abundance at late times. Instead,

signifi-cant amountsof thesemRNAsremainedin thecytoplasmas

late as 12to 16 h after infection.

The v'hsl mutation affected the accumulation of the three beta-gamma mRNAs to different extents (Fig. 1 and 4). However, inno instancewas the overaccumulation as dra-matic asthat observed for the beta mRNAs. At late times,

ICP5 mRNAwasoverproducedtothegreatest extentof the three mRNAs. In wild-type infections, the levels of ICP5 mRNA remainedrelatively constant at8 h postinfection. In contrast, in i'hsl infectionsthelevel of ICP5 mRNA

contin-O-Owt

8- B. ICP 8 mRNA 0- *vhs-1

6--01.

6-.

,@~-

/ *

2

0i a

0- wt

C. DNA PolmRNA 0- *vhs-1

. -

_-

-/

0

'0 /

_

A _ . . . i

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O-Owt

8-- A. ICP 5 mRNA 0- *vhs-1

6

2-- 0

0~~~~

O /~~~~~

0~~~0~

0 2 4 6 8 10 12 14 16

Hours post infection

O-Owt

8 B.g.9mRNA 0- *vhs-1

6

0-0

*

/

4

/

0 0

/O

2

/0

_/-0i

0 2 4 6 8 10 12 14 16

Hours post infection

O-Owt

C. gH mRNA 0- *vhs-1

2

_-0

o

0~~

1 /

/

0 A-0-

1-z-

I

0 2 4 6

8

10 12 14

16

[image:5.612.54.293.75.540.2]

Hourspostinfection

FIG. 4. Accumulation ofbeta-gamma mRNAs during wild-type

(wt) and 'hsl infections. Autoradiograms ofthe gels showninFig.

1 were scanned in order to quantitate the levels of beta-gamma mRNAs. (A) ICP5; (B) gB; (C) gH.

uedto increase until 16 h, by which time therewas atleast 2.5timesasmuchmRNApresentasinwild-type infections.

In addition, although the amounts ofICP5 mRNA in vhsl-infected cells eventually surpassed the wild-type levels, at

early times accumulation ofICP5 mRNA in vhsl-infected cells actually lagged slightly behind that observed in cells infected with wild-type virus.

The mRNA encoding glycoprotein H accumulated with similar kinetics in vhsl and wild-type infections forthefirst 8.5 hofinfection (Fig. 1and 4).However, afterthattimethe levels ofgHmRNAin wild-type infections either plateaued

or decreased slightly, while they continued to gradually increase in vhsl infections. By 12.5 h after infection, there

z :

E

0

c

J

Q) 0

E

0

._:

0

u 2 4 6 a U0 12

Hourspostinfection 14 16 FIG. 5. Accumulation of thegammamRNAencoding glycopro-tein Cduring wild-type(wt)andvhslinfections. Autoradiogramsof the gels shown in Fig. 1 were scanned in orderto quantitate the levels ofgCmRNA.

was approximately twice as much gH mRNA in cells

in-fected with vhslasin cells infected withwild-type virus. The

vhsl mutation had the leasteffectonthe accumulationof the

mRNA forglycoproteinB, causing onlyamarginal increase

in the gB mRNA levels at intermediate and late times after infection (Fig. 1 and4).

Gamma mRNAs. In cells infected withwild-type virus, the

gammamRNA for glycoprotein C begantoaccumulate by 6

hafter infection(Fig. 2 and5). It had reached high levelsby 8 h, and large amounts of gC mRNA persisted in the cytoplasm until at least 16 h afterinfection.

The vhsl mutation led to some overaccumulation ofgC

mRNA, but only at late times afterinfection. By 16 h there

was more than twice as much gC mRNA in vhsl-infected

cellsasincells infected with wild-type virus. However,prior to 12 h, the levels of gC mRNA in vhsl infections lagged behind the levels in cells infected withwild-type virus.

Measurement of mRNA half-lives with a dactinomycin

chase. We nextturnedourattentiontothe specificity of the lhs gene product; that is, are some mRNAs intrinsically more sensitive or resistantto vhs-induced degradation than others? In cells infected with wild-type virus, beta-gamma

andgamma mRNAs continued to accumulate at late times

when alpha and beta mRNAs were declining in abundance.

Onepossible explanation was that beta-gammaand gamma

mRNAs areintrinsically more resistantto the effects ofthe vhsgeneproduct than arealpha and betamRNAs.

Alterna-tively, all viral mRNAs may be equally sensitive tothe vhs protein, butsomemaypersistathigh levelsbecause theyare

continuously replenished by new transcription, while

tran-scription of othergenesis turnedoff. Aswasdiscussed in the

preceding section, the vhs mutation affected the accumula-tion of different viral mRNAs to different extents. Thus, whilethebeta mRNAs forICP8, TK, and DNApolymerase

were affected dramatically, accumulation of some

beta-gammamRNAs, suchasgB mRNA,wereaffectedtoamuch

lesser extent. This may reflect differential sensitivities of

different mRNAs to vhs-induced degradation.

To resolve these questions, the half-lives of 10 different viralmRNAsweremeasured beginningat6hafter infection. Wedecided that the best way to determine the question of differentialtarget sensitivitywastomeasurethe half-livesof

differentmRNAsexposedtothesamecytoplasmicfactorsat thesametime.Wetherefore choseatimeatwhich members of allkinetic classes of mRNAwerepresentinthecytoplasm O-Owt

8 j- gC mRNA *- -@vhs-1

6

0

2

/-0

S

.-i----;

ri n A e- O d^ e n AA 4.

t-xE

0

c

I

0 0

a

XI

0

E

0 ;&j

C :3

0

E

0

:3

.

-o

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1.0

0.5

0.2

0.1

Hoursafter Act DAddition

B. ICP0mRNA Owt

0vhs-1

|~~~~~

a;X- - _

0 0

---0 1 2 3 4

Hours after Act D Addition

0 2 3 4

Hoursafter Act DAddition

FIG. 6. Decay ofalpha mRNAs.Verocellswereinfected with 20 PFU ofeither wild-type (wt) virus or vhsl percell. At 6 h after

infection, 5 p.gof dactinomycin (Act D) per mlwas addedto the

medium,andincubationwascontinued.Totalcytoplasmic mRNAs

wereisolatedat45-min intervals, and 5-,ug samples wereanalyzed byNorthernblotting.Theblots wereprobedforviral mRNAs and

for28SrRNA. The 28S rRNAwasusedas anormalization standard

toensurethatequalamountsoftotalcytoplasmicRNAwereloaded

ontoeachlane of thegel. Autoradiogramsof theresulting gelswere

scanned in orderto quantitate the levels ofspecific mRNAs. (A) ICP4; (B) ICPO; (C) ICP27.

toanappreciableextent. Cellswereinfected with 20 PFU of

eitherwild-typeorvhs 1 viruspercell. At6hpostinfection, dactinomycin wasaddedtothe cultures, and total cytoplas-mic mRNAswereprepared at45-minintervals. The levels of different viral mRNAs were analyzed by Northern blotting

andquantitatedbydensitometric scanning of the

autoradio-grams. Following the analysis of viral mRNAs, the blots

were stripped and then rehybridized to aprobe specific for

C

c

.5

E

0

a:

E

0 c 0

0

.

E

0

E

0 c 0

c

10

Lt

CA .__.Ec a:

E

0

c

0

2.0

1.0

0.5

0.2

0.1

1.0

0.5

0.2

0.1

2 3

Hours after Act DAddition

0 1 2 3 4

Hours after Act DAddition

O 1 2 3 4

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Hours after ActD Addition

FIG. 7. Decayof beta mRNAs. Verocellswereinfected with 20 PFU of either wild-type (wt) virus or vhsl per cell. At 6 h after

infection, 5 ,ugofdactinomycin (Act D) perml was addedto the

medium,andincubation wascontinued. Totalcytoplasmic mRNAs

were isolatedat45-min intervals,and 5-p.gsamples wereanalyzed

byNorthern blotting. The blots were probedfor viral mRNAs and for28SrRNA. The28SrRNAwasusedas anormalization standard

toensurethatequalamountsof totalcytoplasmicRNAwereloaded

ontoeach laneof thegel. Autoradiogramsof theresultinggelswere

scannedinordertoquantitatethelevelsofspecificmRNAs.(A) TK; (B) ICP8; (C)DNApolymerase.

28S rRNA. This allowed us to normalize the results to

control for small differences in the amounts ofcytoplasmic RNA that were loaded ontothe different lanes ofthegel.

The results of thisexperiment andasecondsetofhalf-life determinations are shown in Fig. 6 through 9 and

summa-rized in Table 1. The vhsl mutation hadadramatic effecton

the half-lives ofall mRNAs examined. The half-life of each mRNAwasatleastseveralfoldgreater incellsinfected with

cr c

.E

0

E

0

C

0

c

2

to

L.;

0 C

._

E

o

c

._

a:

E

0

C 0

E

0

c0

C

.5 E

0

z

0 C 0

0

LL

B. ICP 8 mRNA Owt

*vhs-1

_- ~- - t-- -

-0

0~~~

b=&__<~~~~

0~

C. DNA Pol mRNA Owt

*vhs-1

- - - - _. _

,

-1

i

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Vb A. IGr aMRNA uwt

c *vhs-1

.0 1

E

0.5

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0

02

0.1eI,...

O1 2 3 4

Hours afterAct D Addition

ot B. gB mRNA Owt

*vhs-1

,C 1.0-- -a-- ____

E

0

a:

1

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o:

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0 c 0

0.2

0.1

0 1 2 3 4

[image:7.612.324.544.75.207.2]

Hours afterAct D Addition

FIG.o8.

Decay gHbeta-gammRNa mRNs.VercllsweeOwt

with 20 FU f ethr wld-ypevius r vsl ercel. A6vhs-1

E

i t 5 o

al:

E

0 C 0

0.2

-HoursafterAct DAdditizon

FIG. 8. Decayofbeta-gammamRNAs.Vero cellswereinfected

with20 PFUof eitherwild-type virusorvhsl per cell. At 6 hafter

infection', 5 p.g ofdactinomycin (Act D) per ml was added to the

medium,and incubationwascontinued. TotalcytoplasmicmRNAs wereisolatedat45-mmn intervals, and5-p.g sampleswere analyzed byNorthern blotting.The blotswereprobedfor viral mRNAs and

for 28S rRNA. The28S rRNAwasusedas anormalization standard

toensurethatequalamountsof totalcytoplasmicRNAwereloaded

ontoeachlane of thegel. Autoradiogramsof theresulting gelswere

scanned in order to quantitate the levels ofspecific mRNAs. (A)

ICP5;(B) gB; (C) gH.

vhsl than incells infected withwild-type virus.Infact,many

mRNAsdecayedsoslowly in vhsl-infected cells that precise

determination of the half-lifewas difficult.

Incellsinfected withwild-typevirus, the mRNA half-lives ranged from approximately 1 to 2.5 h. While it is possible that small actual differences in stability did exist between different mRNAs, the most striking result was that the

half-lives of all 10 mRNAs were remarkably similar. The

0'

c

E

0

z

: E 0

c

0

u

1.0o

0.51

0.2

0.1

Owt

0 *vhs-1

0 0

0 1 2 3

Hours After ActD Addition 4

FIG. 9. Decay of the gamma mRNA encoding glycoprotein C. Vero cells wereinfected with 20 PFU of either wild-type (wt) virus orvhsl per cell.At6hafterinfection,5,gofdactinomycin (Act D) per ml wasadded to the medium, and incubation wascontinued. Totalcytoplasmic mRNAs were isolated at45-min intervals, and 5-,ug sampleswere analyzed byNorthern blotting. The blots were probed for viral mRNAs and for 28S rRNA. The 28S rRNA was used as anormalization standard to ensurethat equal amounts of total cytoplasmic RNA were loaded onto each lane of the gel. Autoradiograms of the resulting gels were scanned in order to quantitate the levels ofgCmRNA.

correlation coefficient relating the fit of the data to acurve with the calculated half-life was determined for each of the decay curves summarized in Table 1. For ICP8 and gC mRNAs in experiment 1, the correlation coefficients were 0.804 and 0.83, respectively. For ICP27 mRNA in experi-ment2, thecorrelation coefficient was 0.84. The correlation coefficients for all of the otherdecay curves exceeded 0.90. In addition, the half-lives of five of the mRNAs were measured in twoseparate expeiiments. The average values for theresulting half-lives were: ICP27, 1.4 h; TK, 1.7 h; gB, 1.6 h; gH, 1.4h; andgC, 1;95 h. In each case the half-life values in each experiment varied from the average by at pnost 0.5 h. This gives an indication of the variability in half-life values that were obtained when half-life measure-ments were performed on different days with different batches ofcells and virus.

[image:7.612.58.298.76.532.2]

In spite of small differences that were observed in the half-lives for different mRNAs in wild-type infections, it is clear that the differences between theaccumulation kinetics of, for example, DNA polymerase and ICP8 mRNAs on the

TABLE 1. Half-lives of HSV-1 mRNAs inthe presence ofdactinomycina

Half-life(h)

mRNA Wild-type virus vhsl

Expt1 Expt2 Expt1 Expt2

ICP4 1.4 Sb

ICP0 1.1 4.4

ICP27 0.9 1.9 3.3 4.4

TK 1.5 1.9 S 4.4

DNApolymerase 2.5 S

ICP8 2.0 S

lCP5 1.6 5.5

gB 1.3 1.9 S S

gH 1.6 1.2 S S

gC 2.1 1.8 S S

IDetermined fromdatainFig.6through9.

bS,Half-life greater than 8 h.

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one hand and gB or gC mRNAs on the other cannot be explained bydifferential mRNA half-lives. The data indicate that the vhs gene product causes degradation of many different viral mRNAs in a rather nonselective fashion. Differences in the kinetics of accumulation of different mRNAs musttherefore beprimarily duetodifferences in the rates of transcription or processing and transport to the cytoplasm.

Measurement of mRNA half-liveswith apulse-chase proto-col. The procedure of determining mRNA half-lives by measuring their decay in the presenceofdactinomycin has been used widely and produced meaningfulresultsfor many different mRNAs. However, the drug does have secondary effects on processes other than transcription, and these in someinstances may complicate half-life measurements. Fur-thermore, in some instances, turning off transcription may affect the half-lives of mRNAs that already exist in the cytoplasm. Therefore, to confirm the results ofourhalf-life measurements with dactinomycin, we determined the half-lives of selected viral mRNAs with a pulse-chase protocol.

Cellswereinfected with 20 PFU ofwild-typevirus per cell and incubated until 5.5 h postinfection. At this time the infected cells were pulsed for 30 min with

[3H]uridine.

The label was then removed, and the cellswerewashed and then incubated in medium containing an excess of unlabeled uridine as well as cytidine and glucosamine. Cytoplasmic RNA samples were prepared at 45-min intervals after re-moval of the label, and the amount of label remaining in specific viral mRNAs was determined by hybridization to filter disks containing an excess of specific viral DNA fragments. The decay curves for ICPOand TK mRNAsare shown inFig. 10. The resulting half-liveswere:ICPO, 2.4h; TK, 1.5 h. These values, obtained with the pulse-chase procedure, were verysimilar to thoseobtained by measuring mRNAdecayin the presenceofdactinomycinandtherefore confirm the earlier results.

DISCUSSION

The experiments reported here demonstrate that the vhsl mutationdramatically affects thephysical stabilities of many viral mRNAs representing all kinetic classes. The data suggest that the vhs protein plays an important role, in conjunction with transcriptional controls, indeterminingthe abundance levels and kinetics of accumulation ofanumber of viral mRNAs. These studies constitute the firstsystematic measurement,in eitherwild-typeor mutantinfections, ofthe half-lives of HSV-1 mRNAs belongingtoall kinetic classes. The vhs protein is responsible for thedegradation of cellular mRNAs and the concomitant shutoff of host polypeptide synthesis that occurs early during lytic infections. The present experiments suggest that anotherimportant role of theprotein is the regulation of viral mRNA stability.

A central question addressed by these studies was the specificity or selectivity of the vhs gene product. Immedi-ately after infection, copies of the vhs protein thatenterthe cellascomponents of the infecting virioncausedegradation ofavariety of preexisting cellular mRNAs atthe sametime that viral mRNAs begin to accumulate (24, 28). However, cellular mRNAs are not necessarily intrinsically more sen-sitive to

vhs-induced

degradation than are viral mRNAs, as shown by the data of Smiley and co-workers (26). These investigators found that when a rabbit

3-globin

gene was inserted into the viral genome, accumulation oftheresulting

3-globin

mRNA wasregulated in afashion similar to that of members of the beta class of viral mRNAs. In the present

c

a,

a

E

a)

z

wE

0 c 0

aI 4)

1.00o

0.50

0.20

0.100

0.05

0.02

0.01

c

._

E

a)

z

E

4J

0 C: 0

0

U-

1.00-0.50

0.20

0.10+

0.05

0.02

0.01

Hours After Start ofChase

0 0

0

B. ICP 0

1 2 3 4 5 6

Hours After Start of Chase

FIG. 10. Measurementof mRNAhalf-livesby pulse-chase anal-ysis. HeLacellswereinfected with 20 PFU ofwild-typevirus. At 5.5 h postinfection, they were pulse-labeled for 30 min with [3H]uridine,after which the label was removed and thecells were

incubatedin mediumcontainingan excessofcolduridine, cytidine, andglucosamine.TotalcytoplasmicRNAwasextractedatvarious timesafter thestartofthechase,andtheamountof labeledICPO (B)

orTK(A)mRNAwasdeterminedbyhybridizationto an excessof specific viral DNA fragments immobilized on nitrocellulose filter disks.

studies, the vhsl mutationwas found todramatically affect the half-lives of all viral mRNAs thatwereexamined,andin cells infected with wild-type virus the vhs-regulated half-lives of all 10 viral mRNAs were very similar. Thus, the accumulated data strongly suggest that the vhs protein negativelyregulates the stability ofmany,ifnotall, mRNAs with little or no target selectivity.

As would be expected if it lengthened mRNA half-lives, the vhsl mutation affected the abundance levels and accu-mulation kineticsofanumber of viral mRNAsbelongingto different kinetic classes. The effect was most dramatic for the alpha mRNA encodingICP27 and the beta mRNAs for ICP8, TK, and DNA polymerase. Thus, these mRNAs accumulated to higher levels in v,hsl-infected cells than in cells infected with wild-type virus. In wild-type infections the mRNAsencodingICP27, ICP8, TK, andDNA

polymer-aseallappearedatearlytimes,increasedtopeaklevels,and then declined in abundance. The decline at late times was sharper and more complete for the beta mRNAs than for ICP27 mRNA. In contrast, in vhsl infections the levels of these four mRNAs failed to decline after reaching peak values and instead leveled to a plateau. An attractive hy-pothesistoexplaintheseaccumulationcurvesis thatmRNA levels continued to increase for as long as the rates of transcription, processing, and transportof the mRNA tothe cytoplasmremainedhighandthat,followingadecline in any or all ofthese rates, the levels ofalpha and beta mRNAs

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declined in wild-type infections as a result of vhs-induced mRNAturnover.

Whether the normal decline in the rate of appearance of alphaand beta mRNAs in thecytoplasm results exclusively fromtranscriptional controls is unclear. In studies in which transcriptionrates were measured by pulse-labelingcells in vivowith [3H]uridine, Zhang and co-workers observed that the rate of transcription of the beta gene for dUTPase increased from1 to2 hafterinfectionand thendeclined (32). Ifthe transcription patternsforTK, ICP8, and DNA

poly-merase are similar, then the accumulation curvesforthese three mRNAs could be explained as the result of specific transcriptional controls acting in conjunction with the vhs-mediatedturnoverofthecytoplasmic mRNAs. However,in nuclear runoffmeasurementsofthetranscriptionratesofthe TK, ICP8, and DNA polymerase genes, Weinheimer and McKnight were unable todeterminewhether the decline in the steady-state cytoplasmic levels of the mRNAs resulted from transcriptional or posttranscriptional controls (31). Interpretation of theirresultswashampered by the observa-tion that significant amounts ofsymmetric, promoter-inde-pendent transcription ofthese geneswasobservedbeginning

at 4 h after infection and increased thereafter. In other studies

utilizing

thenuclear runofftechnique, Godowskiand

Knipe

obtainedsimilarresultsfortherateofICP8 transcrip-tion(7). Thus,atpresent, one cannotexclude thepossibility that the normalshutoff ofbeta geneexpressionmay involve controlatthe levelofprocessingand transport of the mRNA tothe cytoplasmaswell asregulation of transcription rates and the cytoplasmicstabilities of the mRNAs.

The vhsl mutation ledtooveraccumulation ofsome beta-gammaand gammamRNAs,butnot tonearlytheextentthat was observed for the beta mRNAs. The differences that were observed inwild-type infections between the

accumu-lationpatterns ofthe beta mRNAson theone hand and the

beta-gamma

and gamma mRNAsontheotherwere

particu-larly striking

in view of the fact that the half-lives of the mRNAswere very similar.The datatherefore indicatethat these differences in accumulation kinetics were

primarily

duetocontrolsatthe levels of

transcription, processing,

or mRNA transport ratherthandifferentialmRNA stabilities.

The effect of the vhsl mutation on the accumulation of

some mRNAscould notbe explained by simply postulating that themutation altered mRNA half-lives whileleavingthe rates

of

transcription, processing, andtransportunchanged. This was especiallytrue forthe mRNAforglycoprotein C. In vhsl infections, accumulation of gC mRNA actually lagged behindthat observed in wild-typeinfections until 12

h,

at which point the vhsl mRNA levels finally surpassed thosein

wild-type

infections. Thereasonfor this behavior is notknown. However,overexpressionofone or moreearlier geneproductsmaydelayorreducetranscriptionortransport ofsome mRNAs that are normally expressed later in the viruscycle.

The experiments presented here suggest that inwild-type virusinfections, the vhs gene productcauses rapidturnover

ofmanydifferentviral andcellularmRNAs. Thenetresultof

such a wild-scale decrease in mRNAhalf-lives would be to accentuate the

importance

and

flexibility

oftranscriptional controls. It is

interesting

that HSV apparently encodes a

protein

which isinvolved in the regulationofmRNA stabil-ities and does not rely on unmodified cellular functions for

determining

the rates ofmRNA turnover. A similar obser-vation has recently been made by Kwong and co-workers (12).

The

importance

to virus growth of the vhs-mediated

regulation of mRNA levels is unclear. The fact that vhsl is a viable, nonconditionally lethal mutation indicates that a certain amountofoverexpression of many gene products can be tolerated, at least in cell culture. Nevertheless, the vhs protein is probably important to optimal virus growth, as evidenced by thefacts that the vhsl mutation resulted in a

severalfold reductioninvirus burst size insingle-step growth experiments(20) and wild-type virus rapidlyoutgrows vhsl inmixed infections(13). Furthermore, fine tuning of mRNA levels may be more criticaltogrowth ofthevirus in animals than it is incell culture.

Finally,themechanismof vhs-induced mRNA turnoveris unknown. The vhs protein may be a virus-encoded RNase or,alternatively,mayactivateapreexistingcellular enzyme. Recentdata fromourlaboratorysuggest that at earlytimes afterinfection, changes in the structure of messenger ribo-nucleoprotein particles correlate with a wild-typevhs func-tion (C. R. Krikorian and G. S. Read, unpublished data). Thus, by inducing changes in messenger ribonucleoprotein particle structure, the vhs product might render mRNAs more susceptible to digestion by RNases thatare constitu-tively present within the cell. Elucidation of the exact mechanism of vhs-induced mRNAturnoverwill await exper-iments with in vitro mRNA degradation systems.

ACKNOWLEDGMENTS

We thank Kim Knight for excellent technical assistance and AnnetteSchmidtandCharlie Krikorian forhelpful discussions.We alsothankSteve Weinheimer forprovidingtheM13 clonesandfor helpful discussions.

This workwassupportedby Public Health ServicegrantA121501 fromthe National Institute ofAllergyandInfectious Diseases.

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