In vitro mRNA degradation system to study the virion host shutoff function of herpes simplex virus.

CopyrightX 1991,American Society forMicrobiology

In

Vitro mRNA Degradation System To Study

the

Virion

Host

Shutoff

Function of Herpes Simplex Virus

CHARLES R.

KRIKORIAN'

ANDG. SULLIVAN READ2*

DepartmentofMicrobiology andProgram in Molecular Biology, Stritch School of Medicine, Loyola Universityof

Chicago, Maywood, Illinois

60153,1

and Divisionof Cell Biology and Biophysics, School of BasicLife Sciences, University of Missouri-Kansas City, KansasCity, Missouri 64110_24992

Received 15May 1990/Accepted 25 September 1990

The virion host shutoff (vhs) gene of herpes simplex virus encodes a virion polypeptide that induces degradation of host mRNAs at early times and rapidturnover of viralmRNAsthroughout infection. Tobetter

investigate the vhs function, an in vitro mRNA degradation system was developed, consisting of cytoplasmic

extracts

fromHeLacells infected with wild-type herpes simplex virus type 1ora mutantencodingadefective vhspolypeptide. Host and viral mRNAs were degraded rapidlyinextracts from cellsproductively infectedwith

wild-type

herpes simplex virus type 1 but notinextracts from mock-infected cells orcells infected with the

mutant vhsl. Incontrast, 28S rRNA was stable in all three kinds ofextract. Accelerated turnover ofhost

mRNAs was alsoobserved in extracts from cells infected with wild-type virusinthepresenceofdactinomycin, indicating that the activity was induced by a structural componentofthe infecting virions. The in vitro vhs

activity was inactivated by heat orproteinaseKdigestion butwas insensitive to brief treatmentoftheextracts

withmicrococcal nuclease.Itwas notinhibitedbyplacental RNase inhibitor,it exhibited astrongdependence

upon addedMg2+,it wasactive at concentrations ofK+ up to200mM,and itdid notrequire the components of anenergy-generatingsystem. Insummary, the in vitro mRNA degradation systemappearstoaccurately reproduce the vhs-mediated decay of host and viralmRNAsandshould be useful forstudiesof themechanism

of vhs action.

In cells infected with herpes simplex virus (HSV), the

shutoff ofhost macromolecular synthesis and the cascade

regulation of viral gene expression are achieved through a

complex set oftranscriptional and posttranscriptional

con-trols (26, 45, 59, 66, 74, 76, 77). Ofthe posttranscriptional

regulatorymechanisms,the most thoroughly studied to date has been theregulationof host and viral mRNAstabilitiesby theproduct ofthe HSV virion hostshutoff(vhs) gene. The

vhs gene encodes apolypeptidethatis astructural compo-nentof virionsand atearly times afterinfectioncauses the

shutoff of most host cell protein synthesis by inducing degradation of cellularmRNAs(16-22,28, 42, 43, 55, 58, 66, 69, 71,72). Since copies ofthe vhsproteinarepresent within the infecting virion, virion host shutoff is not dependent uponpriorde novoviral protein synthesis (16-22, 42, 43, 55,

66, 69). The activity of the vhs protein is not limited, however, topreexistingcellular mRNAs. After the onset of

viraltranscription, the vhs protein induces rapidturnoverof

viralmRNAsbelonging to all kinetic classes (32, 44, 45, 69).

Thus, virus mutants that encode a defective vhs protein are

defective in virion host shutoff and produce viral mRNAs with significantly longer half-lives than those of mRNAs produced by the wild-type virus (32, 44, 45, 69). The vhs

polypeptide, therefore, appears to provide a nonselective mRNA degradation function that, when coupled with

spe-cific transcriptional controls, plays an important role in

regulatingthe steady-state levels and patterns of

accumula-tion ofboth viral and cellular mRNAs (32, 44, 45).

An ever-increasing body of data has made it clear that controlofmRNA stability plays an important role in

regu-lating the expression of many mammalian genes (5, 60).

Exposure of cellsto avariety of hormones and other stimuli

*Correspondingauthor.

increases the levels ofspecific mRNAs, at least in part,by increasing their cytoplasmic half-lives. Glucocorticoids

en-hance the stability of human growth hormone mRNA (46),

whereas estrogen specifically stabilizes vitellogenin mRNA

(8)andprolactininduces anincreasein thehalf-life ofcasein

message(25).Conversely,stimuli thatincreasethe cytoplas-mic concentration of iron destabilize transferrin receptor mRNA, apparently by reducing the affinity of a specific

mRNA binding protein for cis-acting sequences located

within the 3' untranslatedregion ofthe message(40,41,64).

Thestabilityof

3-tubulin

mRNAisregulated bythe concen-tration ofunpolymerized

P-tubulin

in the cytoplasm. Thus,

treatment of cells with cytochalasin B, which disrupts mi-crotubules and raises the concentration of

unpolymerized

tubulins,induces rapiddegradation of

,3-tubulin

mRNA(23,

78). Similarly, in vivo and in vitro studies suggest that an

increase in the level of freehistones,whichaccompaniesthe

cessation of DNA synthesis at the end of the S phase,

induces rapiddestabilization ofhistonemRNAs(24, 27, 38,

47,51, 52, 61-63).Finally,the presenceofspecificcis-acting

sequences within the 3', and sometimes 5', untranslated regions ofthe mRNAs encoding a number oflymphokines

andproto-oncogene products causes these messages to be very short lived (11,29, 53,54, 67, 68, 75).Abrogationof the normal controlofthestabilities of these mRNAscanleadto

celltransformation, altered growthproperties, or abnormal

differentiation (1, 10, 12, 13, 15, 36, 37, 50, 67).

A number of studies suggest that the degradation of mRNAs

normally

occurs by 3'-to-5' exonuclease digestion.

Studies from J. Ross's laboratory with extracts from unin-fected K562 cells have shown that the in vitrodegradationof c-myc mRNAinvolvesprogressive shorteningof thepoly(A) tail, followed by 3'-to-5' degradation of the body of the message(6,7). That this mechanismisgenerally followedfor

112

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the decay of polyadenylated messages in vivo is suggested by the finding of Wilson and Treisman that sequences located in the 3'

untranslated

region of c-fos mRNA, which accelerate its rate of

turnover,

also cause more rapid short-ening of the poly(A) tails of c-fos messages (75). 3'-to-5'

degradation also occurs for nonpolyadenylated messages. Ross and co-workers have shown, both in vitro and in

vivo,

that the decay of nonpolyadenylated histone mRNAs pro-ceeds by cleavage of the last 4 to 13 nucleotides from the 3' end of the mRNA, followed by rapid3'-to-5' degradation of the remainder of the message (51, 52, 61-63).

At present, only a limited amount has been reported

concerning

the development of in vitro mRNA degradation systems. The most extensive studies to date have come from Ross's laboratory involving extracts from uninfected K562 erythroleukemia cells (3, 4, 6, 7, 51, 52, 60-63). In this system, in vitro degradation of histone mRNAs is mediated by a polysome-bound exonuclease that can be isolated in an active, soluble form by washing the polysomes with buffers containing 0.3 M KCl (61, 62). Although the RNase that degrades histone messages is normally polysome associated, in vitro regulation of the rate of histone mRNA decay by the concentration of free histones also requires a factor that is present in the S130 high-speed supernatant obtained after pelleting the polysomes from a cytoplasmic extract (51). Similar results have been obtained for the decay of some polyadenylated messages. In vitro poly(A) shortening and

3'-to-5'

degradation of c-myc message can be observed upon incubation of isolated polysomes, indicating that the relevant nucleases are polysome associated (6). However, c-myc decay is greatly accelerated by a labile factor that is found in theS130postpolysomal supernatant fraction from cytoplas-mic extracts (7). This factor specifically accelerates the decay rates of c-myc and c-myb mRNAs and is inactivated by treatment with micrococcal nuclease, suggesting that it may be a ribonucleoprotein (7). However, in spite of this impressive progress, as yet little is known concerning the identity of the nucleases involved in mRNAturnover and the factors that control their activities.

The vhs-mediated control of mRNA stabilities in HSV-infected cells provides a particularly attractive model system for studying the factors that control message half-lives in mammalian cells. In particular, the vhs protein is one of the few trans-acting regulators of mRNA stability that have been identified to date. In addition, mutants encoding defective vhs proteins have been isolated and characterized (17, 55), allowing a combination of genetic and biochemical tech-niques to be applied to the problem. Mapping of the mutation carried by the mutant vhsl to the UL41 open reading frame of HSV type 1 (HSV-1) has allowed identification of the vhs gene (34, 39), and recent characterization of the structural polypeptides encoded by a vhs deletion mutant has allowed identification of the vhs polypeptide within purified virions (55a). Clearly, elucidation of the mechanism of vhs-mediated control of mRNA half-lives would be greatly facilitated by the availability of an in vitro mRNA degradation system that accurately reflects the vhs activity observed in vivo. In this paper we report the development and preliminary character-ization of such a system from HSV-1-infected HeLa cells.

MATERIALS AND METHODS

Cells and virus. HeLa S3 and Vero cells were purchased from the American Type Culture Collection and grown at

37°C

in Eagle minimum essential medium (MEM; GIBCO) supplemented with antibiotics and 10% (vol/vol) calf serum

(45). Stocks of wild-typeHSV-1 strainKOSand the mutant vhsl were prepared by infectionofVero cellmonolayers as previously described (45). vhsl grows well at all

tempera-tures from 34 to 39°C and exhibits adefective virion host shutoff function at alltemperatures(55). In these

studies,

all infections wereperformedat34°C.HeLa S3 cellswereused

for the preparation of all invitro mRNAdegradationextracts and were infected and maintained as described

previously

forVerocells (31, 45). Inallexperiments, virus wasallowed to adsorb for 1 hin MEMcontaining 5%(vol/vol) calfserum. The inocula werethenaspirated,and thecellswereoverlaid

with fresh MEM plus 2% calfserum. Mock-infected cells

were treated inthe same way asinfected cells, except that they were exposed to lysates of uninfected Vero cells prepared in the samewayasthe virus stocks were

prepared

from infected cells.

Plasmids. The plasmid pHcGAP contains a1.2-kb cDNA

insert encoding a portion ofhuman

glyceraldehyde-3-phos-phate dehydrogenase (GAPD) (73) and was obtained from

the American Type Culture Collection. The

plasmid,

pHSV106, whichcontains a 3.4-kb BamHI

fragment

encod-ing the HSV-1 thymidine kinase, was purchased from Be-thesdaResearchLaboratories. Theplasmid pXlrll contains

a 4.6-kb fragmentofXenopus laevis rDNAinsertedintothe EcoRI site of colicin El (14) and was provided by Jeff

Doering. The inserted fragment in pXlrll hybridizes with

28S rRNA. pXlrll was maintained in Escherichia coli

HB101, whereas pHcGAPandpHSV106weremaintained in E. coli DH5 alpha (Bethesda Research

Laboratories).

Plas-midDNAs were prepared by CsCl density gradient

centrif-ugation asdescribed previously (45, 56, 57).

In vitro mRNAdegradation extracts. Cytoplasmic extracts

for studyinginvitromRNAdegradation were

prepared

from infectedormock-infected HeLa S3 cellsbyamodification of

theprocedureofBrown andcolleagues forthe

preparation

of

in vitro translation extracts (9). At various times after

infection ormock infection, HeLacells werewashed twice with ice-cold wash buffer consisting of0.15 M sucrose, 33 mM NH4Cl, 7 mM KCl, 4.5 mM magnesium acetate

[Mg(OAc)2], and 30mM HEPES

(N-2-hydroxyethylpipera-zine-N'-2-ethanesulfonic

acid) (pH7.4). Thecellswere then

permeabilized by the addition of 300

jxg

of

lysolecithin

(L-a-lysophosphatidyl

choline; Sigma)per mlin wash buffer

directly tothe monolayers for 60 s. After the

permeabiliza-tion buffer was removed, the cells from one 100-mm dish

were scraped into 200,ul ofstandard reaction buffer

[0.1

M HEPES (pH 7.4), 0.2 M NH4Cl, 20 mM

Mg(OAc)2,

7 mM

KCl, 1 mM dithiothreitol, 1 mM ATP

(dipotassium

salt),

1 mM GTP (sodium salt), 40 ,uM eachofthe 20 amino

acids,

0.1 mM

S-adenosylmethionine,

1 mM

spermidine,

10 mM creatine phosphate (dipotassium salt), 40 U of creatine

kinase per ml, and 100 U of placental RNase inhibitor (RNasin; Promega Corp, Madison,

Wis.)].

The cells were

then disrupted by 10 passages through a

25-gauge

needle.

The nuclei were removed by low-speed

centrifugation,

and the supernatant was stored on ice.

To initiate mRNA degradation reactions,

cytoplasmic

supernatants were transferred to a 30°C water bath. At

various times after the start of incubation,

samples

were removed, addedto an equal volume ofureabuffer

(7

Murea, 10 mM Tris hydrochloride [pH 7.9], 0.35 M

NaCl,

10 mM

EDTA, and 1% sodium dodecyl

sulfate),

extracted twice

with phenol-chloroform (1:1,

vol/vol)

andtwicewith chloro-form, and precipitated from

ethanol,

all as described

previ-ously (45, 56, 57). Samples taken

immediately

before trans-ferring the reaction mixtures from

4°C

to

30°C

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time points. The sampleswere subsequently analyzed forin

vitro decay of host and viral mRNAs and 28S rRNA by

Northern RNA blotting and hybridization as described

be-low.

In experimentsto optimize the concentrationof

Mg2+

ion needed for efficient in vitro mRNA degradation, extracts

were prepared in the standard reaction buffer modified to

contain 2.5 mM Mg(OAc)2 and then supplemented with concentrated Mg(OAc)2 tobring the

Mg2+

concentration to

the desired values. To optimize the

K+

ion concentration,

standard extracts were prepared containing 7 mM KCl and

then supplemented with concentrated KCl to bring the

K+

concentration tothe values described in the figure legends. To testthe requirement for energy-generating components,

extracts were prepared in standard reaction buffer lacking

ATP, GTP, creatine phosphate, and creatine kinase. The extractswereall incubatedand analyzedasdescribedabove.

Pretreatment ofextracts with heat,proteinase K,or

micro-coccal nuclease.Totestthesensitivity of theextracts tobrief heattreatment, standard in vitrodegradation reactions were

heated at90°Cfor 10 min, cooled to

4°C,

and then analyzed

for in vitro decay of mRNAs according to the standard

protocol. To test the sensitivity ofthe extracts to

pretreat-ment with protease, standard extracts were supplemented

with proteinase K (Sigma; molecular biology grade) to a

concentration of1 mg/ml and incubated at

30°C

for 30 min.

Theextractswerecooledbrieflyto

4°C

and thenincubatedat

30°C andanalyzed forin vitromRNAdegradation according

to the standard protocol.

Totest the effect of pretreating the extracts with

micro-coccal nuclease, standardin vitro degradationextracts were

supplemented with micrococcal nuclease (Pharmacia) to

1,000 U/mlandCaCl2to 1 mMand thenincubatedat

30°C

for 10min.Ethyleneglycol-bis(,3-aminoethyl

ether)-N,N,N',N'-tetraacetic acid (EGTA) was added to 2 mM, and the extracts were chilled on ice for 10 min. To each reaction

mixture was added deproteinized total cytoplasmic RNA

fromanequivalentnumber of uninfected HeLa cells, and the

mixtures were incubated at 30°C. Samples were taken at

various times and analyzed by Northern blotting for the in

vitro decay ofhost mRNAs and 28S rRNA.

Agarosegel electrophoresis, Northern blotting, and

hybrid-ization. RNA samplesweredenaturedwith glyoxal,

electro-phoresed through 1% agarose gels cast in 10 mM sodium

phosphate (pH 7.0), and transferred to Nytran membranes

(Schleicher and Schuell) by capillary blotting, all as

de-scribed previously (45). Filters containing immobilized

RNAswereprehybridizedfor 1to2h and thenhybridizedto

nick-translated probes as described previously (44, 45). In

someexperimentsprobes werestripped from the filters, and

the membranes were rehybridized with a second probe as

previously described (45). Nick-translated pHcGAP and

pHSV106were used as probes to detect the cellular GAPD

and the HSV-1 thymidine kinase mRNAs, respectively.

Nick-translated pXlrll was used to detect28S rRNA.

Quantitation ofmRNA levels. To quantitate the levels of

host and viralmRNAs and 28S rRNA, autoradiograms from

the Northern blots were scanned with a Hoeffer model

GS300scanningdensitometer. Since 28S rRNA provedtobe

stable in all types ofextracts, the levels of specific mRNAs

were normalizedtotheamountof28S rRNA in each sample

before mRNA decaycurves were plotted in all experiments

exceptthatshownin Fig.7. In Fig. 7the relativeamountsof

28S rRNA and GAPD mRNA were plotted without prior

normalization. Nick-translated pHcGAP hybridized to

GAPD mRNA;it alsocross-hybridized atalow levelto28S

rRNA. Equivalent results were obtained for the decay

curvesof GAPD mRNAregardlessofwhether theamountof

GAPD mRNA was normalized to the level of

28S

rRNA detected by cross-hybridization with pHcGAP or

by

strip-ping the pHcGAPprobe from the blot and thenrehybridizing

with nick-translated pXlrll.

RESULTS

Strategy. TheHSVvirion host shutoffproteinis known to

induce rapid degradation of host and viral mRNAs in the cytoplasm (32, 44, 45, 66, 69). In an effort to develop anin vitro mRNA degradation system to study the vhs function,

we decided to compare the rates of mRNAdegradation in in vitro translation extracts from mock-infected HeLa cells and extracts from cells infected with either wild-type HSV-1 or the mutant vhsl, which has been shown to encode a

defec-tive vhs polypeptide (55). HeLacells were chosen because they are readily infected with HSV and because numerous studies have proven them to be a particularly suitable cell line for the preparation of in vitro translation extracts. Although in vivo studies indicated that vhs-induced mRNA degradation does not require efficient ongoing translation of

the mRNAs (66, 69), we decided to prepare invitro transla-tion extracts because we reasoned that they would be the best initial approximation to a functional cytoplasm.

Extracts were prepared by a procedure shown by Brown and co-workers to be suitable for the preparation of highly active in vitro translation extracts from a variety ofcultured

cells (9). This procedure involves briefly exposing the mono-layers to lysolecithin to permeabilize the cells, harvesting the cells directly into buffer containing the components required for in vitro translation, disrupting the cells by repeated passage through a 25-gauge needle, and removing the nuclei by low-speed centrifugation. This protocol was chosen because it is simple and rapid, making itpossible to prepare and analyze the activity of extracts on the same day. This enabled us to perform all experiments with freshly prepared lysates and eliminated the need to freeze in vitro degradation extracts before analysis, an obvious advantage in view of the possible detrimental effects that freezing and thawing could have upon the activities of as yet uncharac-terized proteins.

To optimize the concentration of lysolecithin needed to permeabilize HeLa cells and to verify that this procedure yielded active in vitro translation extracts, standard reaction mixtures were prepared by using a variety of lysolecithin concentrations in the permeabilization buffer. Unlabeled methionine was omitted from the reaction buffer and

[35S]methionine

was added to a concentration of 75

,uCi/ml.

At various times after the start of incubation, samples were withdrawn and the amount of

[35S]methionine

incorporated into trichloroacetic acid-precipitable material was deter-mined as described previously (55). Exposure of the HeLa cells to 300

pug

oflysolecithin per ml was found to be optimal for in vitro translation (data not shown). For extracts pre-pared with this concentration of lysolecithin, efficient in vitro translation continued for approximately 40

min,

after which the amount of incorporated label leveled to a plateau (Fig. 1).

In vitro degradation of host mRNAs. The vhs function was originally identified on the basis of its ability to induce rapid degradation of host mRNAs and the concomitant shutoff of host polypeptide synthesis (16, 17, 19, 20, 55, 66). To determine whether HeLa celllysateswould be suitable for in vitro studies of the vhs function, standard in vitro translation

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1.0

-0 20 40 60 TIMEOFINCUBATION (MINUTES)

FIG. 1. In vitro translation by HeLacell extracts. An in vitro

mRNAdegradationextractwasprepared from mock-infectedHeLa

cells in standard reaction buffer containing 2.5 mM Mg(OAc)2, lacking unlabeled methionine, and supplemented with

[3S]methio-nineto75 uCi/ml.Thereaction mixturewasincubatedat30°C.At

varioustimessampleswerewithdrawn, and theamountof radioac-tivity incorporated into trichloroacetic acid-precipitable material

wasdeterminedasdescribed previously (55).

extracts were prepared from mock-infected cells and from

cells 5 h after infection with 20 PFU of either wild-type

HSV-1 or the mutant vhsl per cell. The extracts were

incubatedat30°C;atvarious timessampleswerewithdrawn

andextracted withphenol and chloroform, and the decay of specific cellular mRNAswasanalyzed by Northern blotting.

Tocontrolfor the totalamount ofcytoplasmic RNA loaded

ontoeachlaneofthegel,theblotswerealsoprobed for 28S rRNA, and the amount of mRNA was normalized to the

amount of28S rRNA before the mRNA decay curve was

plotted.

Intheseinitialexperimentswedecidedtostudy the decay

ofendogenous cellular mRNAs rather than that of added

exogenousmRNAsbecause wereasoned that the structure

ofmessengerribonucleoprotein particles(mRNPs)

reconsti-tutedon exogenousmRNAsmight differ from that of

endog-enous mRNPs. mRNP structure could easily affect mRNA

stability; in fact, HSV infection has been shownto induce changes in mRNPstructure that correlate with awild-type

virionhostshutoff function (31). Focusinguponthe decayof

endogenous mRNAs should, therefore, remove one poten-tial variable from the experiments. In addition, the results involving decay of endogenous mRNAs should provide a

base line for later attempts to study the degradation of

exogenous messages.

Thedecay ofthecellularmRNAencoding GAPDisshown inFig.2and3. GAPDmRNAwaschosenforstudybecause

it has along invivo half-life in uninfected cells (73). Thus,

any vhs-induced reduction in message stability should be

moreeasilydetectedforthismRNAthanforamessagethat is inherently unstable. GAPD mRNA was relatively stable foratleast 4 h inextractsfrom mock-infected cells (Fig. 2,

lanes 1through 5; Fig. 3). Incontrast, itdecayed rapidlyin extracts from cells infected with wild-type HSV-1, so that little detectable mRNA remained by 1 h after the start of

incubation(Fig. 2,lanes6through 9; Fig. 3).That consider-ablevhs-induced degradation had occurred invivo in wild-typeinfections before thepreparationofextractsisindicated by the fact that the intensity of the band formed by GAPD mRNA at 0 h of incubation for wild-type extracts was

reduced considerably relative to that observed at 0 h for mockorvhsl infections (compare Fig. 2,lanes1,6,and 10,

FIG. 2. Invitrodegradation of host mRNAs. Standard in vitro mRNA degradation extracts were prepared from HeLa cells S h

aftermockinfection (lanes 1through 5),orinfection with 20 PFU of wild-type HSV-1 (lanes 6 through 9)orvhsl (lanes 10through 14)

percell.Sampleswerewithdrawn fromthereactionsat0 h(lanes 1, 6, and 10), 1 h (lanes 2, 7, and 11), 2 h (lanes 3 and 12), 3 h (lanes 4, 8, and 13), or4h(lanes 5, 9, and 14). Thesampleswereextracted

twice with phenol-chloroform and twice withchloroform, and the RNAswereprecipitatedfromethanol. Samples of totalcytoplasmic

RNA were denatured with glyoxal, electrophoresed through 1% agarose gels, and transferred to Nytran membranes by capillary blottingasdescribed in thetext.Themembraneswerethenprobed

todetectGAPD mRNA and 28S rRNAasdescribed in thetext.

withFig. 3A). Incontrast tothe casefor wildtype-infected

cell extracts, inextractsfromcells infected with vhsl GAPD mRNA was every bit as stable as in extracts from mock-infected cells (Fig. 2, lanes 10 through 14; Fig. 3). In vitro

decaywasspecificformRNAs,asevidencedbythe fact that

28S rRNA was equally stable in mock-, wild type-, and

vhsl-infected cellextracts.Alltold, the rankorder ofinvitro

decayratesof GAPD mRNAwasthe sameasthatobserved in mock,wild-type, and vhsl infections in vivo.

The vhsproteinisastructuralcomponentofvirions and is therefore ableto induce degradation of host mRNAs in the absence ofpriorde novo viral gene expression (17, 22, 55, 66, 69). Thus, virion host shutoff is induced by

UV-inacti-vatedvirusaswellasafterinfection ofcells in thepresence

ofdactinomycin to block viraltranscription (17, 22, 55, 66, 69). To determine whether the accelerated degradation of GAPD mRNAobserved inextractsfromcells infected with

wild-type virus was induced by a virion component or

requireddenovo viralgene expression,invitrodegradation

extracts were prepared from cells 5 h after a productive

wild-type virus infectionor5 hafterinfection with 50 PFU of

wild-type virus or vhsl per cell in the presence of5 ,ug of

dactinomycinperml.Degradationof theGAPD mRNAwas

equally rapid in extractsfrom cells infected withwild-type

virus in thepresenceandabsence ofdactinomycin (Fig. 4).

In contrast, GAPD mRNA was stable for at least 5 h in extractsfromcells infected with vhsl.Thus,theaccelerated

degradation of host mRNAs that was observed in extracts

from cells infected withwild-typeHSV-1wasnotdependent

upon de novo viral gene expression and was therefore

induced bya componentof theinfecting virions.

Invitrodegradationof viral mRNAs.Althoughvhs mutants

were originallyisolated on the basis oftheir defects in the

degradation of cellular mRNAs and the shutoff of host

protein synthesis,recentstudies indicatethat the vhsprotein playsacentral roleindeterminingthe half-lives of bothviral

and cellular mRNAs within the infected cell (32, 44, 45).

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FIG. 3. Quantitation of in vitro decay of host mRNAs. The

autoradiogram shown in Fig. 2 was scanned, and the amount of

GAPD mRNAin each lane was normalized tothe amount of 28S

rRNA. In panel A the relative amounts of GAPD mRNA in

mock-infected (0), wild-type virus-infected (A), and vhsl-infected (@) cellextractsareplottedinarbitraryunits. InpanelB theamount

ofGAPDmRNApresentinasample fromanyofthethree typesof

extractisexpressedas afraction of theamountpresentat0hin that

kindofextract.

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Time of Incubation(h)

5

FIG. 4. Invitrodegradationin extractsfrom cellsinfected inthe presence ofdactinomycin. Standard in vitrodegradation extracts

wereprepared from cells 5 h afterinfection with 50 PFU ofwild-type

virus(0)orvhsl(A)percell inthepresenceof 5,ugofdactinomycin

permlor5hafter infection with 50 PFU ofwild-typeviruspercell in theabsenceofanydrugs(0).Theextractswereincubatedat30°C

fortheindicatedtimes,atwhichpointsampleswerewithdrawnand totalRNAs wereextracted andanalyzed for GAPD mRNA and 28S rRNA by Northern blotting as described in the legend to Fig. 2. Autoradiograms were scanned with a Hoeffer model GS300

scan-ningdensitometer, andtheamountofGAPDmRNAin eachsample

wasnormalizedto the amountof 28SrRNA. The amountof GAPD mRNAremainingatvarious timeswas plottedas afractionof the amountpresentat0 h.

mentstocharacterize someofthebiochemical

requirements

ofthein vitro mRNAdegradation system.

A preliminary experiment was undertaken to determine

theeffectof RNasinuponvhs-inducedmRNAdecay. Paral-leldegradation extracts wereprepared fromHeLacells5 h after infection with 20 PFU of either

wild-type

HSV-1 or

vhsl percell. RNasin (100U/ml) wasincluded inhalf ofthe

reactionmixtures and omitted fromthe other half.

Regard-lessofwhether theinhibitorwaspresent,mRNAswerevery

Measurementsof the half-lives of 10 different viral mRNAs in cells infected with wild-type virus or the mutant vhsl

revealed the following: (i) in wild-type infections the

half-lives of all 10 messages, representing all kinetic classes of

viral mRNA, were very similar; (ii) the vhsl mutation

resulted in dramatic increases in the stabilities of all 10

messages (44, 45). Thus, the vhs protein inducesthelargely

nonselectivedegradation of bothviral andcellular mRNAs. To determine whether degradation of viral mRNAs was

alsoacceleratedinourin vitromessagedegradationsystem, in vitro degradation extracts were prepared from cells 5 h afterinfection withwild-typevirusorvhsl andanalyzedfor

the degradation of the mRNA encodingthe viralthymidine

kinase (TK). TK mRNA was degraded rapidly in in vitro

extracts from cells infected withwild-type HSV-1 but was

relatively stable foratleast 5 h inextractsfrom cellsinfected

withvhsl (Fig. 5). Therefore,once againthein vitroresults

paralleledthose observed invivo.

Characterizationof the invitromRNAdegradationsystem. Inallrespectsexaminedtothis point, in vitro degradation of

hostandviralmRNAsobserved in HSV-infected HeLa cell

extracts paralleled vhs-induced mRNA degradation

ob-served in vivo. We therefore undertook a series of

experi-'E

.0 E

E 0.1

0

9--- Vhs 1

0.05-4

0 1 2 3 4 5

Time ofIncubation(h)

FIG. 5. In vitrodegradation ofviral mRNAs. Standard in vitro mRNA degradation extracts were prepared from cells 5 h after infection with 20 PFU ofwild-typeHSV-1(0)orvhsl(-)percell.

The extracts were incubated at 30°C for the indicated times, at

which point samples were withdrawn and total RNAs were

ex-tracted andanalyzed for TK mRNA and 28SrRNA by Northern

blottingasdescribed in thelegendtoFig.2. Autoradiogramswere

scannedwitha Hoeffer model GS300scanning densitometer, and

theamount of TK mRNA in each sample was normalized to the

amount of 28S rRNA. The amount of TK mRNA remaining at

varioustimeswasplottedas afraction oftheamountpresentat0 h. Errorbars indicate the standard errors of the means determined

fromreplicate experiments. B

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0-A..

O-O Mock A'.

a@

-AWT

0- -OVhs1

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e

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0 U

a

L,

1.00'

0.50

0.10*

n nr..

1 2 3 4 5

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TimeofIncubation(h)

FIG. 6. Effect of heat and proteinase K pretreatment upon

vhs-induced in vitro degradation. Standard in vitromRNA

degrada-tion extracts were prepared from cells 5 h after infection with 20

PFU ofwild-type HSV-1percell. Extractswerepretreatedeither by

the addition ofproteinase K and digestion for 30minorby heating

to90°C for 10min(0),after which theextractswerereturnedto4°C. Control unpretreated extracts were left at 4°C until the start of incubation (0). The extracts were incubated at30°C for the indi-cated times. Samples were withdrawn and analyzed for GAPD

mRNA and 28S rRNA by Northern blotting, and the amount of GAPDmiRNAin each samplewasnormalizedtotheamountof 28S rRNA.

stableinextractsfrom cellsinfected with vhsl, whereas they decayed rapidly in extracts from cells infected with

wild-type virus (datanotshown). In vhsl-infected cells extracts,

there was slightly more mRNA decay in the absence of RNasin than when itwasincluded in the reaction mix. We

attribute this to nonspecific RNases that are inhibited by

RNasin. Because it didnotinhibit vhs-induced degradation and itsinclusion yielded slightly cleaner results, RNasinwas

included as a component in the standard in vitro reaction

bufferandwasused inall of theotherexperiments described

in thisreport.

Thenext setof experimentswasundertakentodetermine whether the in vitro vhs activity could be inactivated by pretreating the extracts with heat or proteinase K. Three

parallel in vitro degradation extracts were prepared from

HeLa cells 5 h after infection with 20 PFU ofwild-type

HSV-1 percell. One extract was heatedto 90°Cfor 10min

and then chilledonice. A secondextractwas supplemented

withproteinase K and digested at30°C for 30min, and the

third was left untreated. All three extracts werethen

ana-lyzed for invitro vhsactivity. Pretreatment of the extracts

by either heating or proteinase K digestion completely abolished vhs-mediatedinvitro degradation(Fig. 6). These results areconsistent with the involvement ofone or more

heat-labile proteinsinvhs-mediated message turnover.

Recently Brewer and Ross showed that a factor that is

presentinthe postpolysomal supernatant fraction from the

cytoplasmof K562 cells and thatspecificallyaccelerates the

decay ofc-myc and c-myb mRNAs is inactivated by brief

digestion with micrococcal nuclease (7). To determine whether similar micrococcal nuclease pretreatment of

ex-tracts from HSV-1-infected cells would inactivate the in

vitro vhs activity, standard in vitro degradation extracts

were prepared from HeLacells 5 h after mock infection or

infection with 20 PFU of wild-type HSV-1 per cell. The extracts were supplemented with micrococcal nuclease and

CaCl2and thenpreincubated at30°Cfor10min. EGTAwas

thenaddedtochelate theCa2+and inactivate the micrococ-cal nuclease, and the extracts were chilled briefly on ice.

1.001

I

0

0.10

c

0 .

0-1 2 3

TimeofIncubation (h)

[image:6.612.325.546.79.214.2]

4 5

FIG. 7. Decay ofexogenous GAPD mRNA and 28S rRNA in

micrococcal nuclease-treated in vitro degradation extracts. Stan-dard in vitromRNA degradationextractswerepreparedfrom cells 5 h after mock infection or infection with 20 PFU ofwild-type

HSV-1percell.Theextractswerepretreatedasdescribed in thetext

with micrococcal nuclease in the presenceofaddedCa2". EGTA wasaddedtochelate the Ca2+,and deproteinized total cytoplasmic RNAfromanequivalent number of cellswasaddedtoeachextract.

The extracts were incubated at 30°C for the indicated times.

Sampleswere withdrawnandanalyzed for GAPD mRNA and 28S rRNA by Northern blotting. The relativeamountof 28S rRNA in mock-infected(0) and wild-type virus-infected (0) cellextractsis plottedas afraction of theamountpresent at0 h. Incontrast toother figures in thispaper,therelativeamountof GAPDmRNAdetected in mock-infected(A) and wild-type virus-infected(A) cellextractsis plotted without being normalized to the amount of 28S rRNA

presentin thesample.

Micrococcal nuclease treatmentisroutinelyusedto deplete

in vitro translation extracts ofendogenous mRNAs andto rendertranslation dependent uponexogenouslyadded

mes-sages(49). Therefore,toprovideatargetfor thevhs-induced

degradative activity, after micrococcal nuclease pretreat-ment the extracts were supplemented with deproteinized

total cytoplasmic RNA fromanequivalent numberof

unin-fected HeLa cells. Theextractswerethen incubatedat30°C

andanalyzedfor in vitrodecayofexogenous GAPDmRNA and 28S rRNA.

Figure7shows thedecayofexogenousGAPD mRNAand total 28S rRNA. Three conclusions canbe drawn from the

resultsofthisexperiment. First,pretreatmentof theextract from wild-type virus-infected cells with micrococcal

nucle-asedidnotinhibittherapiddegradationofexogenousGAPD mRNA. That thisdegradationwasduetothe vhsactivityand

wasnotthe resultsof residual micrococcal nucleaseactivity

is indicatedbythe fact GAPD mRNAwasrelativelystablein the micrococcal nuclease-treated extracts from mock-in-fected cells. Second, the fact thatexogenousGAPD mRNA

wasdegradedin the wildtype-infectedcellextractssuggests that the invitro mRNAdegradationsystem willbe usefulfor

studying the decay of both exogenous and endogenous

mRNAs. Third, 28S rRNAwasstable in extracts fromboth mock-infected and wild-type virus-infected cells. Since

ini-tiallythe totalamountof 28SrRNA was a50:50 mixtureof

endogenous and deproteinized exogenous 28S rRNA, the results indicate thatinwildtype-infectedcellextracts

depro-teinizedexogenousGAPDmRNAwasdegradedmuchmore

rapidlythandeproteinizedexogenous28S rRNA. Thisresult is an additional indication that the RNase activity seen in wildtype-infectedcellextractswasspecificfor mRNAsand

wasnotthe resultofanonspecific RNase.

The next setofexperimentswere undertaken toexamine

0-0 WT Control

*- - WT + Heat

A. A WT +ProteinaseK

%_---

4

_...,.._....!

9

0-0 WT-: 28SrRNA 0-@Mock: 28S rRNA

A . *W:GAPD mRNA

A -A Mock:GAPD mRNA

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0P

._

.a

E

z

E

0

c

0

0

1.00I

0.50

0.104

1 2 3 4

[image:7.612.333.549.77.207.2]

TimeofIncubation(h)

FIG. 8. Mg2+dependence of vhs-induced mRNA degradation. In vitro mRNAdegradationextractswereprepared from cells 5 h after

infection with 20 PFU ofwild-type HSV-1per cell. The extracts

wereprepared in standard reaction buffer modifiedto contain 2.5 mM (0), 10 mM (0), 20 mM (A), or40 mM (A) Mg(OAc)2. The

extracts were incubated at30°C for the indicated times. Samples

werewithdrawnandanalyzedfor viral TK mRNA and 28S rRNA by

Northernblotting, and theamountof TK mRNA in each samplewas

normalizedtotheamountof28S rRNA.

the Mg2+ dependence of the in vitro degradation system.

Parallel in vitro degradation extracts were prepared from

HeLa cells 5 h after infection with 20 PFU of wild-type

HSV-1 per cell. Individual reaction mixtures were

supple-mented with concentrated Mg(OAc)2 to bring the Mg2+

concentrationtothe desired value.The extracts were

incu-batedfor5 handanalyzed for the decayof theviral mRNA

encodingTK. Invitro degradation of TK mRNA showed a

strongdependenceupontheconcentration ofMg2+ion(Fig. 8). Although a significant amount of mRNA degradation

could beobserved atanMg2+concentration of10mM(Fig. 8) or5mM (datanot shown), increasing the Mg2+

concen-trationto20mMorhighersignificantlyincreased the degra-dation ratein wild-type extracts (Fig. 8). That theeffect of raising theMg2+ concentrationwas not simply the result of inducing nonspecific changes in mRNP structure or the

activation of nonspecific nucleases is indicated by the fact

thatasignificant differenceinthe mRNA decayratesinwild type- and vhsl-infected cell extracts was observed at an

Mg2+ concentration of 20 mM (see Fig. 10); 20 mM was

chosen as the optimal Mg2+ concentration for subsequent experiments.

A similar experiment was undertaken to determine the

effect of varying the K+ concentration upon the rate of vhs-induced decay ofTK mRNA (Fig. 9). Parallel in vitro degradation reactions were prepared from HeLa cells 5 h

afterinfection with 20PFU ofwild-type HSV-1 percell. In

thesereactions theMg2+ concentrationwasheldconstant at

20 mM, whereas the concentration of K+ ion was varied

from 7 to500 mM. Efficient degradation ofTK mRNAwas

observed at K+ concentrations from7to200mM,whereas increasing theK+ concentration to 500 mM severely

inhib-itedthedegradation reaction (Fig.9).Wechose 7 mMK+as

optimal.

The final experimentwas designed to determine whether

vhs-induced in vitro mRNA degradation was dependent

upon the components ofanenergy-generating system. Par-allelinvitro degradationextractswereprepared from HeLa

cells at 5 hafter infection with 20 PFU ofeither wild-type

HSV-1 or vhsl. Half of the reactions contained all of the

components ofthe standard reaction, whereas ATP, GTP,

.' *~~~~~~~-- 100 mM

A...A200 mM

c A--A- 500 mM

E

A A

Z 1.00 .A

E A

0.10

0~~~~~~~~

C

0.10

0 1 2 3 4 5

Timeof Incubation(h)

FIG. 9. K+ dependence ofvhs-induced mRNAdegradation. In vitromRNAdegradationextractswerepreparedfrom cells 5 h after

infection with 20 PFU ofwild-type HSV-1 per cell. The extracts

werepreparedin standardreaction buffer modifiedtocontain 7 mM (0), 100mM (0),200 mM(A), or500 mM(A) KCl.The extracts

were incubated at 30°C for the indicated times. Samples were

withdrawn and analyzed for viral TK mRNA and 28S rRNA by Northernblotting,and theamounitof TK mRNAin eachsamplewas

normalizedtotheamountof 28S rRNA.

creatine phosphate, and creatinephosphokinasewere omit-ted fromthe other half. Efficient vhs-induced degradation of TK mRNA occurred in thepresence(Fig. 10A) and absence

(Fig. 10B) of the components ofanenergy-generating

sys-tem.

DISCUSSION

In this reportwedescribe anin vitro mRNA degradation

system, consisting of cytoplasmic extracts from HSV-1-infected HeLa cells, that appears to accurately reproduce the degradation of host and viral mRNAs induced by the wild-type vhs protein in vivo. This conclusion is supported by several important parallels between the in vitro data and in vivo observations. First, host messages were degraded

rapidly inextractsprepared fromcellsproductively infected

withwild-typeHSV-1 butnotinextractsfrommock-infected cells or cells infected with the mutant vhsl. Second, the

accelerated turnover of host mRNAs occurred in extracts

from cells infected with wild-type virus in the presence of

dactinomycin, indicatingthat itwasinduced byacomponent

ofthe infecting virions andwasnotdependentupondenovo

viral gene expression. Third, accelerated turnover ofviral mRNAs was observed in extracts from cells productively infected with wild-type HSV-1 but not in extracts from

vhsl-infected cells. In each of the above cases, the most

important observation supportingthe fidelityof the invitro

systemwasthestrikingdifference between the mRNAdecay

ratesinextractsfrom cells infected withwild-typevirusand in extracts from vhsl-infected cells. This observation indi-cates that the accelerated in vitro degradation of mRNAs

was dependent upon infection of the cells with virions

containingafunctional vhspolypeptideandwasnot simply

theconsequenceofanonspecificRNaseliberatedduringcell

fractionation or induced as a nonspecific consequence of

viralinfection. Finally, although the wild-type vhsfunction

induced accelerated in vitro turnover of both viral and

cellularmRNAs, endogenous 28S rRNAwasequally stable

in extracts from mock-infected cells and in extracts from cells infected with eitherwild-typevirusorthe vhslmutant. Thislends further supportto the conclusion that the

degra-dative activity seen in wild type-infected cell extracts was

specific for mRNAs and was not due to a contaminating

Mg2+ Effects 0-0 2.5mM

*- -0 10 mm

A/ A 20 mM

A 40 mM

*

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[image:7.612.79.296.79.213.2]

vhs-INDUCED mRNA DECAY IN VITRO 119

z z

I

mE

0

10.50

0.50

0.10~

z

z

E

m

I-0

1 2 3 4 5

TiMEOFINCUBATION(H)

0 1 2 3 4 5

liME OFINCUBAllON (H)

FIG. 10. Dependence of vhs-induced mRNA degradation upon the components ofan energy-generating system. In vitro mRNA

degradation extracts were prepared from cells 5 h after infection

with 20 PFU of wild-type HSV-1 (0) or vhsl (A) per cell. The

extracts were prepared eitherin standard reaction buffer (A) orin

standardreaction buffer fromwhich ATP,GTP,creatinephosphate, and creatine phosphokinase had been omitted (B). The extracts

were incubated at 30°C for the indicated times. Samples were

withdrawn and analyzed for viral TK mRNA and 28S rRNA by

Northernblotting, and theamountofTK mRNAineachsamplewas

normalizedtotheamountof 28S rRNA.

nonspecific RNase that should havebeen presentin allthree kinds ofextract.

Analysis of the crude in vitro system showed thatone or

morefactors necessary forin vitro vhs activity was

inacti-vatedby heating the extracts to90°C or bybriefproteinase Kdigestion.These dataareconsistent with theinvolvement

ofone or moreheat-labile proteins in vhs-induced

degrada-tion. In contrast, pretreatment of wild-type extracts with micrococcal nuclease did notinhibit the subsequent degra-dation of added exogenous mRNA, indicating that the

fac-torsrequiredfor in vitro vhs activity are apparently

insensi-tivetomicrococcal nucleasetreatments knownto inactivate

anumberof small RNAs andribonucleoproteins (30)as well as afactor that accelerates in vitro decay ofc-mycandc-myb

mRNAs (7). Furthermore, the finding that exogenous

mRNAs were rapidly degraded in extracts from cells

in-fected with wild-type virus but were relatively stable in

extracts frommock-infected cells suggests that the in vitro

degradation system will be suitable for studying the

vhs-induced decay ofbothexogenous and endogenousmRNAs.

Itisalso worthnotingthatin theseexperimentsthesourceof

exogenousmRNAwastotaldeproteinizedcytoplasmic RNA

containingamixtureof mRNAand rRNAs.Thus,theresults

shown in Fig. 7 indicate that added deproteinized mRNA

wasdegradedmorerapidly thanaddeddeproteinized rRNAs

in wild-type extracts. This adds further support to the

conclusion that the in vitro vhs activity observed in wild

type-infected cell extracts wasspecific formRNAs anddid

notsimply resultfroma contaminating nonspecific RNase.

Preliminary biochemical characterization ofthe in vitro mRNA degradation system from HSV-infected cells

indi-cates that it is similar in a number of respects to in vitro mRNAdegradationsystemsfromuninfected cells described

previouslybyRossetal. (3, 4, 6, 7, 51, 52, 60-63) and others

(48,

70).

In

particular,

the vhs-induced mRNA

degradation

activity

was not inhibited

by

the placental RNase inhibitor RNasin and was dependent upon added divalent cation.

Efficient vhs-induced degradation occurred at K+ ion

con-centrations ofup to 200 mM but was inhibited by 500 mM

K+,

and mRNA degradative activity was not dependent

upon the addition of ATP, GTP, creatine phosphate, or

creatine phosphokinase. In each of these respects, the

vhs-induced RNase activity was similar to that ofthe

exo-nuclease shownbyRoss and co-workers toinduce degrada-tion of histone mRNAs in extracts fromK562 erythroleuke-miacells (62).

Althoughbothrequiredaddeddivalent cation, the in vitro vhs activity reported here and the exonuclease described by

Ross (62)differ somewhat in the nature of their dependence upon added

Mg2+.

Whereas the exonuclease that degrades

histone mRNAs exhibited abroad optimum ranging from 5 to20mM

Mg2+

(62), the vhsactivityobserved in theextracts

described here was more strongly dependent upon added

Mg2 . Thus, although a striking difference was observed

between mRNA decay rates in extracts from wild-type

virus-infected and vhsl-infected cells at Mg2+ concentra-tions ranging from 2.5 to 20 mM (Fig. 2 through 5, 8, and 10; unpublished data), the rate of mRNA degradation in wild-type extracts increased continuously as the Mg2+

concen-trationwasraised to20or40 mM. At present, the reason for this difference in the Mg2+ dependence of the two in vitro systemsis unclear. It mayreflect anincreased Mg2+

depen-denceof one of the proteins involved in vhs-induced degra-dation. Alternatively, higherMg2+concentrations may favor aconformational change in mRNP structure that renders the mRNA more susceptible to vhs-induced degradation.

Besidesprovidingthe groundwork forfutureexperiments,

the preliminary biochemical characterization of the vhs-induced RNase allows it to be distinguished from several previously characterized RNases that are commonly found in cell extracts. Pancreatic RNase is resistant to boiling but is inhibited by RNasin (48). The fact that the vhs activity is sensitive to heating to 90°C but is not inhibited by RNasin therefore indicates that it does not involve a pancreatic-type RNase. A nucleolar exonuclease has been described that, like the vhs-induced RNase, is dependent uponadded Mg2+ (35). However, unlike the vhs-induced enzyme, the nucleo-lar RNase is inactive at K+ ion concentrations greater than 90 mM (35). A lysosomal acid RNase has been described (65). However, unlike the vhs-induced enzyme, it does not

require added Mg2+.

In several previously characterized in vitro degradation systems, the RNases responsible for mRNA turnover were found to be polysome associated. This was the casefor the nucleasesresponsible for invitro degradation of histone and c-myc mRNAs, although in both cases the decay rate was greatly accelerated by soluble factors present ina postpoly-somal supernatant fraction of the cytoplasm (7, 51). In addition, Brawerman and co-workers haverecentlyreported an RNase that is associated with polysomes as well as free mRNPs in a variety ofmammalian cells (2). At present, the

A. WITH ENERGYCOMPONENTS

A- -

-A\

°-

-A~-A--A-VHS 1\

O~~~~~~~~

B. WITOUT ENERGYCOMPONENS

~~~~~~A-

--A- -AVHS 1

VOL. 65, 1991

1.00

0.50.

0.10.

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[image:8.612.67.288.75.348.2]

subcellular localization of the proteins required for vhs-inducedmRNA degradation is unknown.

Inpreliminaryexperiments, invitrodegradation extracts

from mock-infected, wild-type HSV-1-infected, or vhsl-infectedHeLacells wereseparated bycentrifugation into a polysome pellet and a postpolysomal supernatant. Polyso-mal mRNAs from extracts ofcells infected with wild-type virus or vhsl were equally stable upon suspension and

incubationof the polysomesin standardreaction buffer or in the postpolysomal supernatant from mock-infected cells

(31a). However, the addition of thehigh-speed supernatant fromextracts ofcellsinfected withwild-typevirus to any of

the three types ofpolysomeresultedinrapid degradation of

thepolysomalmRNAs. These dataindicate thatone or more

factors required for vhs-induced mRNA degradation are found inthepostpolysomal supernatantfraction of extracts fromcells infected with wild-type virus. Whetheradditional

polysome- ormRNP-associatedfactors are alsorequiredfor

vhsactivityis currentlyunderinvestigation.

A central unanswered question concerning the vhs

func-tion is whether the vhsprotein isitself an RNase or,instead, activates a cellular nuclease, perhaps one involved in the normal turnover of mRNAs in uninfected cells. To date,

attempts todemonstrateanRNaseactivityinpreparations of disrupted virions have beenunsuccessful (31a). The reason forthismaysimply be thatmethods ofvirion disruption that

preservetheRNase activity have yet to befound. Alterna-tively,oneor morecellularmacromolecules mayberequired for vhsactivity. Thesimilarity ofthebiochemical character-istics of the vhs-induced activityto those of in vitro degra-dation systems from uninfectedcells (62) is consistent with

this second possibility. Resolution of this question and elucidation of the detailed mechanism ofvhs-induced

mes-sageturnover will require further fractionation and

charac-terization of the in vitro mRNAdegradation system. ACKNOWLEDGMENTS

We thank Annette Schmidt and Kim Knight for many helpful discussions and critical appraisals of the data presented in this report. We are also indebted to Christine Sorenson, Phil Hart, and JeffRoss for helpful discussions and for communicating their data prior to publication.

This work was supported by grant AI21501 from the National Institutes of Health.

REFERENCES

1. Adams, J. M., A. W. Harris, C. A. Pinkert, L. M. Corcoran, W. S.Alexander, S. Cory, R. D. Palmiter, and R. L. Brinster. 1985. The c-myc oncogene driven by immunoglobulin enhancers induces lymphoid malignancy in transgenic mice. Nature (Lon-don) 318:533-538.

2. Bandyopadhyay, R., M. Coutts, A. Krowczynska, and G. Brawerman. 1990. Nuclease activity associated with mamma-lian mRNA in its native state: possible basis for selectivity in mRNA decay. Mol. Cell. Biol. 10:2060-2069.

3. Bernstein, P., S. W. Peltz, and J. Ross. 1989. The poly(A)-poly(A)-binding protein complex is a major determinant of mRNA stability in vitro. Mol. Cell. Biol. 9:659-670.

4. Bernstein, P., and J. Ross. 1989. Poly(A), poly(A) binding protein and the regulation of mRNA stability. Trends Biochem. Sci. 14:373-377.

5. Brawerman, G. 1989. mRNA decay: finding the right targets. Cell 57:9-10.

6. Brewer, G., and J. Ross. 1988. Poly(A) shortening and degrada-tion of the 3' A+U-rich sequences of human c-myc mRNA in a cell-free system. Mol. Cell. Biol. 8:1697-1708.

7. Brewer, G., and J. Ross. 1989. Regulation of c-myc mRNA stability in vitro by a labile destabilizer with an essential nucleic

acid component. Mol.Cell. Biol. 9:1996-2006.

8. Brock, M. L., and D. J. Shapiro. 1983. Estrogen stabilizes vitellogenin mRNA against cytoplasmic degradation. Cell34: 207-214.

9. Brown, G. D., R. W. Peluso, S. A.Moyer, and R. W.Moyer.

1983. A simple method for the preparation ofextracts from animalcellswhich catalyze efficientin vitroproteinsynthesis.J. Biol. Chem. 23:14309-14314.

10. Campisi, J., H. E. Gray, A. B. Pardee, M. Dean, and G. E. Sonenshein. 1984. Cell-cycle control of c-myc but not c-ras

expression is lost following chemical transformation. Cell 36: 241-247.

11. Caput,D., B. Beutler, K. Hartog, R.Thayer, S.Brown-Shimer, and A. Cerami. 1986. Identification ofa common nucleotide sequence in the 3'-untranslated region of mRNA molecules specifyinginflammatorymediators. Proc.Natl. Acad. Sci. USA 83:1670-1674.

12. Coppola, J. A., and M. D. Cole. 1986. Constitutive c-myc oncogene expression blocks mouse erythroleukaemia cell dif-ferentiation butnotcommitment.Nature(London)320:760-763. 13. Dani, C., L. M. Blanchard, M. Piechaczyk, S. ElSabouty, L. Marty, and P. Jeanteur. 1984. Extreme instability of myc mRNA in normal and transformed human cells. Proc. Natl. Acad. Sci. USA 81:7046-7050.

14. Dawid, I. B., and P. K. Wellauer. 1976. Areinvestigationof the 5' to 3' polarity in 40S ribosomalRNA precursor ofXenopus laevis. Cell 8:443-448.

15. Dony, C., M. Kessel, and P. Gruss. 1985. Post-transcriptional

controlofmycand p53expressionduringdifferentiation of the embryonal carcinoma cell line F9. Nature (London) 316:636-639.

16. Fenwick, M. L. 1984. The effects ofherpesviruses oncellular macromolecular synthesis, p. 359-390. In H. Fraenkel-Conrat and R. R. Wagner (ed.), Comprehensive virology, vol. 19. PlenumPublishingCorp., New York.

17. Fenwick, M. L., and J. Clark. 1982. Earlyanddelayedshut-off of hostprotein synthesis in cells infectedwith herpessimplex

virus. J. Gen. Virol. 61:121-125.

18. Fenwick,M.L.,andJ.Clark. 1983.Theeffect ofcycloheximide

ontheaccumulationandstabilityoffunctionalalpha-mRNAin cells infectedwithherpes simplexvirus.J.Gen.Virol. 64:1955-1963.

19. Fenwick, M. L., and M. M. McMenamin. 1984. Early virion-associated suppression of cellular protein synthesis by herpes

simplex virus isaccompanied by inactivation ofmRNA.J.Gen. Virol. 65:1225-1228.

20. Fenwick, M. L., L. S. Morse, and B. Roizman. 1979.Anatomy of herpessimplex virusDNA. XI.Apparentclusteringof functions effecting rapid inhibition of host DNAandprotein synthesis.J. Virol. 29:825-827.

21. Fenwick, M. L., and S. A. Owen. 1988. On the control of immediate early (alpha) mRNA survival in cells infected with herpes simplex virus. J.Gen. Virol. 69:2869-2877.

22. Fenwick, M. L., and M. J. Walker. 1978. Suppression of synthesis of cellular macromoleculesbyherpessimplexvirus.J. Gen. Virol. 41:37-51.

23. Gay,D.A.,T.J.Yen, J.T. Y.Lau,and D. W. Cleveland. 1987. Sequencesthatconferbeta-tubulinautoregulationthrough mod-ulated mRNA stability reside within exon 1 ofa beta-tubulin mRNA. Cell50:671-679.

24. Graves, R. A., N. B.Pandey, N. Chodchoy,andW.F.Marzluff. 1987. Translation is required for regulation of histone mRNA degradation. Cell 48:615-626.

25. Guyette, W. A., R. J.Matusik,andJ.M.Rosen. 1979. Prolactin-mediated transcriptional and post-transcriptional control of casein geneexpression. Cell 17:1013-1023.

26. Harris-Hamilton,E., and S. L. Bachenheimer. 1985. Accumula-tion of herpes simplex virus type 1 RNAs of different kinetic classes in thecytoplasm of infectedcells. J. Virol. 53:144-151. 27. Heintz, N., H. L. Sive, and R. G. Roeder. 1983. Regulation of

human histone gene expression: kinetics ofaccumulation and changesin therateofsynthesisand in thehalf-lives of individual histone mRNAs during the HeLa cell cycle. Mol. Cell. Biol.

on November 10, 2019 by guest

http://jvi.asm.org/

3:539-550.

28.

Hill,

T. M., R. K. Sinden, and J. R. Sadler. 1983. Herpes simplex types 1 and 2 induce shutoff of host protein synthesis in Friend erythroleukemia cells. J. Virol. 45:241-250.

29. Jones, T. R., and M. D. Cole. 1987. Rapid cytoplasmicturnover

of c-myc mRNA: requirement of the 3' untranslated sequences. Mol. Cell. Biol. 7:4513-4521.

30. Kass, S., K. Tyc, J. A. Steitz, and B. Sollner-Webb. 1990. The U3 small nucleolar ribonucleoprotein functions in the first step of preribosomal RNA processing. Cell 60:897-908.

31. Krikorian, C. R., and G. S. Read. 1989. Proteins associated with mRNA in cells infected with herpes simplex virus. Biochem. Biophys. Res. Commun. 164:355-361.

31a.Krikorian, C. R., and G. S. Read. Unpublished data.

32. Kwong, A. D., and N. Frenkel. 1987. Herpes simplex virus-infected cells contain a function(s) that destabilizes both host and viral mRNAs. Proc. Natl. Acad. Sci. USA 84:1926-1930. 33. Kwong, A. D., and N. Frenkel. 1989. The herpes simplex virus

virion host shutoff function. J. Virol. 63:4834-4839.

34. Kwong, A. D., J. A. Kruper, and N. Frenkel. 1988. Herpes simplex virus virion host shutoff function. J. Virol. 62:912-921. 35. Lasater, L. S., and D. C. Eichler. 1984. Isolation and properties ofa single-strand 5' to 3' exoribonuclease from Ehrlich ascites tumor cell nucleoli. Biochemistry 23:4367-4373.

36. Leder, A., P. K. Pattengale, A. Kuo, T. A. Stewart, and P. Leder. 1986. Consequences of widespread deregulation of the c-myc gene in transgenic mice: multiple neoplasms and normal development. Cell 45:485-495.

37. Lee, W. M., M. Schwab, D. Westaway, and H. E. Varmus. 1985. Augmented expression of normal c-myc is sufficient for cotrans-formation of rat embryocellswitha mutant ras gene. Mol. Cell. Biol. 5:3345-3356.

38. Luscher, B., C. Stauber, R. Schindler, and D. Schumperli. 1985. Faithful cell-cycle regulation of a recombinant mouse histone H4 gene is controlled by sequences in the 3' terminal part of the gene. Proc. Natl. Acad. Sci. USA 82:4389-4393.

39. McGeoch, D. J., M. A. Dalrymple, A. J. Davison, A. Dolan, M. C. Frame, D. McNab, L. J. Perry, J. E. Scott, and P. Taylor. 1988. The complete DNA sequence of the long unique region in the genome of herpes simplex virus type 1. J. Gen. Virol. 69:1531-1574.

40. Mullner, E. W., and L. C. Kuhn. 1988. A stem-loop in the 3' untranslated region mediates iron-dependent regulation of trans-ferrin receptor mRNA stability in the cytoplasm. Cell 53:815-825.

41. Mullner, E. W., B. Neupert, and L. D. Kuhn. 1989. A specific mRNA binding factor regulates the iron-dependent stability of cytoplasmic transferrin receptor mRNA. Cell 58:373-382. 42. Nishioka,Y., and S. Silverstein. 1977. Degradation of cellular

mRNA during infection with herpes simplex virus. Proc. Natl. Acad. Sci. USA74:2370-2374.

43. Nishioka, Y., and S. Silverstein. 1978. Requirements of protein synthesis for the degradation of host mRNA in Friend erythro-leukemia cells infected with herpes simplex virus type 1. J. Virol.27:619-627.

44. Oroskar, A. A., and G. S. Read. 1987. A mutant of herpes simplex virus type 1 exhibits increased stability of immediate-early (alpha) mRNAs. J. Virol. 61:604-606.

45. Oroskar, A. A., and G. S. Read. 1989. Control of mRNA stability by the virion host shutoff function of herpes simplex virus. J. Virol. 63:1897-1906.

46. Paek, I., and R. Axel. 1987. Glucocorticoids enhance stability of human growth hormone mRNA. Mol. Cell. Biol. 7:1496-1507. 47. Pandey, N. B., and W. F. Marzluff. 1987. The stem-loop

structure at the 3' end of histone mRNA is necessary and sufficient for regulation of histone mRNA stability. Mol. Cell. Biol. 7:4557-4559.

48. Pei, R., and K. Calame. 1988. Differential stability of c-myc mRNAs ina cell-free system. Mol. Cell. Biol. 8:2860-2868. 49. Pelham, H. R. B., and R. J. Jackson. 1976. An efficient

mRNA-dependent translation system from reticulocyte lysates. Eur. J. Biochem. 67:247-256.

50.

Pellegrini,

S., and C. Basilico. 1986. Rat fibroblasts expressing

high levels of human c-myc transcripts are anchorage-indepen-dent and tumorigenic. J. Cell. Physiol. 126:107-114.

51. Peltz,S. S., and J. Ross. 1987. Autogenous regulation of histone mRNA decay by histone proteins in a cell-free system. Mol. Cell. Biol. 7:4345-4356.

52. Peltz, S. W., G. Brewer, G. Kobs, and J. Ross. 1987. Substrate specificity of the exonuclease activity that degrades H4 histone mRNA. J. Biol. Chem. 19:9382-9388.

53. Piecharczyk, M., J.-Q. Yang, J.-M.Blanchard, P. Jeanteur,and K. B. Marcu. 1985. Post-transcriptional mechanisms are respon-sible for accumulation of truncated c-myc RNAs in murine plasma celltumors. Cell 42:589-597.

54. Rabbitts, P. H., A. Forster, M. A.Stinson, and T. H. Rabbitts. 1985. Truncation of exon 1 from the c-myc gene results in prolonged c-myc mRNA stability. EMBO J. 4:3727-3733. 55. Read, G. S., and N. Frenkel. 1983. Herpes simplex virus

mutantsdefective in thevirion-associated shutoff of host

poly-peptide synthesis and exhibiting abnormal synthesis ofalpha

(immediate-early) polypeptides. J.Virol. 46:498-512.

55a.Read,G. S., and K.Knight. Submitted forpublication.

56. Read, G.S., J. A.Sharp,and W. C. Summers. 1984. In vitroand in vivo transcription initiation sites on the TK-encoding BamHI Q fragment of HSV-1 DNA. Virology 138:368-372.

57. Read, G. S., and W. C. Summers. 1982. In vitrotranscription of the thymidine kinase gene of herpes simplex virus. Proc. Natl. Acad. Sci. USA 79:5215-5219.

58. Roizman, B., G.S.Borman, andM. Rousta.1965. Macromolec-ular synthesis in cells infected with herpes simplex virus. Nature(London) 206:1374-1375.

59. Roizman, B., and A. E. Sears. 1990. Herpessimplex virusesand their replication, p. 1795-1841. In B. N. Fields and D. M.Knipe (ed.), Fields virology, 2nd ed. Raven Press. New York. 60. Ross, J. 1988. Messenger RNA turnover in eukaryotic cells.

Mol. Biol. Med.5:1-14.

61. Ross, J., and G. Kobs. 1986. H4 histone messenger RNAdecay in cell-free extracts initiates at or near the 3' terminus and proceeds 3' to 5'. J. Mol. Biol. 188:579-593.

62. Ross, J., G. Kobs, G. Brewer, and S. W. Peltz. 1987. Properties oftheexonucleaseactivity that degrades H4 histone mRNA.J. Biol. Chem. 19:9374-9381.

63. Ross, J., S. W. Peltz, G. Kobs, and G. Brewer. 1986. Histone mRNA degradation in vivo: the first detectablestepoccursat or near the 3' terminus. Mol. Cell. Biol. 6:4362-4371.

64. Rouault, T. A., M. W. Hentze, D. J. Haile, J. B. Harford, and R. D. Klausner. 1989. The iron-responsive element binding protein: a method for the affinity purification ofa regulatory RNA-binding protein. Proc. Natl. Acad. Sci. USA 86:5678-5772.

65. Saha, B. K., M. Y. Graham, and D. Schlessinger. 1979. Acid ribonuclease from HeLa cell lysosomes. J. Biol. Chem. 254: 5951-5957.

66. Schek, N., and S. L. Bachenheimer. 1985. Degradation of cellular mRNAs induced by a virion-associated factor during herpes simplex virus infection of Vero cells. J. Virol. 55:601-610.

67. Schuler, G. D., and M. D. Cole. 1988. GM-CSFand oncogene mRNA stabilities are independently regulated in trans in a mouse monocytic tumor. Cell 55:1115-1122.

68. Shaw, G., and R. Kamen. 1986. A conserved AUsequence from the 3' untranslated region of GM-CSF mRNA mediates selec-tive mRNA degradation. Cell 46:659-667.

69. Strom, T., and N. Frenkel. 1987.Effects of herpessimplex virus on mRNA stability. J. Virol. 61:2198-2207.

70. Sunitha, I., and L.I. Slobin. 1987. An in vitro system derived from Friend erythroleukemia cells to study messenger RNA stability. Biochem. Biophys. Res. Commun. 144:560-568. 71. Sydiskis, R. J., and B. Roizman. 1966. Polysomes and protein

synthesis in cells infected with a DNAvirus. Science 153:76-78. 72. Sydiskis, R. J., and B. Roizman. 1967. The disaggregation of

host polysomes in productive and abortiveinfectionwithherpes simplex virus. Virology 32:678-686.

73. Tso, J. Y., X.-H. Sun, T.-H. Kao, K. S. Knight, and R. Wu. 1985. Isolation and characterization of rat andhuman

on November 10, 2019 by guest

http://jvi.asm.org/

dehyde-3-phosphate dehydrogenase cDNAs: genomic complex-ity and molecular evolution of the gene. Nucleic Acids Res.

13:2485-2502.

74. Weinheimer, S. P., and S. L. McKnight. 1987. Transcriptional and post-transcriptional controls establish the cascade of herpes simplex virus proteinsynthesis. J. Mol. Biol. 195:819-833. 75. Wilson, R., and R. Treisman. 1988. Removal of poly(A) and

consequent degradation of c-fos mRNA facilitated by 3'

AU-richsequences.Nature (London) 336:396-399.

76. Yager, D. R., and D. M.Coen. 1988. Analysis of the transcript

of the herpes simplex virus DNA polymerase gene provides

evidence thatpolymeraseexpression is inefficientatthe level of translation. J. Virol. 62:2007-2015.

77. Yager, D. R., A. I. Marcy, and D. M. Coen. 1990. Translational regulation of herpes simplex virus DNA polymerase. J. Virol. 64:2217-2225.

78. Yen, T. J., P. S. Machlin, and D. W. Cleveland. 1988. Autoreg-ulated instability of beta-tubulin mRNAs by recognition of the

nascentamino terminus ofbeta-tubulin. Nature (London) 334: 580-585.

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http://jvi.asm.org/