Ability of nonpermissive mouse cells to express a simian virus 40 late function(s).

0022-538X/81/060940-12$02.00/0

Ability

of

Nonpermissive

Mouse

Cells

to

Express

a

Simian

Virus

40

Late

Function(s)

MICHELE LANGE, EVELYNE MAY, AND PIERRE MAY*

Institut deRecherchesScientifiques sur le Cancer, 94 800Villejuif,France Received11December1980/Accepted10March 1981

Mousecellsare

fully

nonpermissive

forsimianvirus40

(SV40).

Infection does not lead to detectable virusreplication. In this report, itwas

shown, first,

that spliced16S and 19SSV40late mRNA werepresent incytoplasmicandpolysomal polyadenylated acid+RNApreparationsfromSV40-infected

baby

mousekidney cells. The 16S and 19S SV40 late mRNA's produced in infected

baby

mouse kidney cells were identical to or similar to the 16S and 19SSV40 late mRNA's produced in permissive monkey cells as judged by their Si

mapping

patterns performed with the late strand of

HpaII-BamHI

fragment

B and

by

their sedimentation patterns in a sucrosegradient.Itwasalso shown that the 16Slate mRNA frominfectedbabymousekidneycellscouldbetranslatedintoa

polypep-tide which was identical to or similar to virion protein VP1 in every aspect examined, including the pattern of peptide

mapping

by

limited proteolysis. Second, wereportedthat mousekidneycellsproduced

detectable, although

low, levels ofSV40 virion protein VP1, as shown by the sodium dodecyl sulfate-polyacrylamide gelautoradiogram of[35S]methionine-labeled proteins immuno-precipitated byarabbitantiserumdirected against SV40virionproteins. Third, it was reported that infected baby mouse kidney cells produced late mRNA's either (i) when the infection was done at a restrictive temperature with the nonleaky tsA58mutant or (ii) in cells treatedwith 100yg ofcycloheximide per ml, inwhichlarge Tantigen synthesiswasinhibited bymore than 99.9%. This suggested thatlargeTantigenwas notrequiredforthe synthesis of latemRNA in mousecells.

During the lytic cycle of simian virus 40 (SV40), SV40 mRNA is synthesized in two phases.During theearly phase,whichlasts until thebeginningof viral DNAreplication, thereis asynthesis of twoearly (19S) mRNA's coding forthe SV40 smallt andlarge T antigens, re-spectively.Inthe latephase,beginningwith the commencement of viral DNA replication, two latemRNA's(19Sand16S) aresynthesized;the late 16SmRNA codes for VP1 protein, and the late19SmRNAcodes forVP2and VP3 proteins. Until recently, it was usually assumed that only"early"mRNAwasproduced early in pro-ductivelyinfectedmonkey cellsand inabortively infectedmouse cells where SV40 DNA replica-tioncould not bedetected.Nevertheless, exper-imental evidenceisaccumulatingthatSV40(or polyoma) latetranscriptionand viral DNA rep-lication are not as closely linked as previously believed (forreviews, see references 1 and 2).

Astudyof the SV40transcription during pro-ductive infection of BSC-1 monkey cells with early temperature-sensitive mutants of SV40 grown at

410C

(a nonpermissive temperature) indicatedthepresence of a

small

but

reproduc-ible fraction of late virus-specific cytoplasmic RNA (22). A late viraltranscription was shown tobe initiated in thelyticcycleintheabsence of viral DNA replication, by characterizing the SV40 transcriptional complexes isolatedbythe Sarkosyl extraction procedure (6, 12). The cy-toplasm of CV1monkeycells atearly periodsof productive infection with SV40 was shown to containlatemRNA's (16).Inaddition, the pres-ence oflate mRNA's wasobservedinthe cyto-plasm of CVI infected with anSV40tsA mutant whichhad beenmaintained at41°C and contin-uously cultured in the presence of cyclohexi-mide, suggesting that the late transcription did notrequirethe Ageneproduct(16). In the case ofpolyomavirus, itwas shownthat viral tran-scription during productive infection ofmouse 3T6 cellsrevealed theappearanceof RNA com-plementaryto bothDNA strands in the nuclei and thecytoplasm of thecellsbefore viralDNA synthesis was detectable (31).

The question of whether a late transcription occurs during an abortive infection with SV40 was alsoinvestigated by Khouryetal. (20, 21); these authors

consistently

foundtheappearance

940

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of late transcripts in nonpermissivemousecells infected with this virus.Whether the late

tran-scripts detected in infected (nonpermissive)

mouse cells weredestined to be processed and

to become functional mRNA(s) remained an

open question. In this report, we show that

SV40-infected mouse cells do produce spliced

and functional latemRNA(s) and that they

syn-thesize a small amount ofSV40 virion protein VP1. Moreover, we report that SV40 late

mRNA's are produced by infected mousecells either wheninfection is performed at a

nonper-missive temperature with the nonleaky tsA58 mutant or when large T antigen synthesis is inhibited more than 99.9% by cycloheximide

(100,ug/rml).

MATERLALS AND METHODS

Cels and virus. Primary mouse kidney (BMK)

cell cultures were prepared from 10-day old Swiss

albino CR1 mice. The BMK cells weregrown in

10-cm-diameter plastic petri dishesin Dulbecco modified

Eagle medium (DMEM) supplemented with 10% calf

serum(GIBCO Laboratories)and were used for

infec-tion 3days after they had reached confluence; they wereinfectedat amultiplicity of 50 PFU/cell. After 1

hof adsorption (at 37°C, unlessspecified otherwise),

the BMK cellswererefedwith DMEM without added

serum. The established monkey cell line CV1 was

cultivated in Eagle minimum essential medium

(MEM)plus 10% calfserum.Confluent monolayers of

CV1 cellswereinfected at amultiplicity of25PFU/

cell; after1hofadsorptionat37°C, cellswererefed with MEMplus 2% calfserum.

SV40 virus strains were the wild-type SVi (also

designatedSVLP)(44), and the temperature-sensitive

mutanttsA58(41).

Cellfractionation. Cells in 2010-cm-diameter

pe-tridisheswerewashed twice with5ml perpetri dish of buffer I (10 mM triethanolamine (pH 8)-25 mM

NaCl-5 mMMgCl2-250 mM sucrose). After the

me-diumwasremoved from thecells,2.5mlper 10petri

dishes of cold bufferI plus 1% Nonidet P-40 (Shell

ChemicalCo.)wasadded; wherenecessary(in

prepa-rationofpolysomes),2mgofyeastRNAperml(type XI;Sigma Chemical Co.), which had been repurified by phenol extraction, was added as a carrier. The

mixture wasleft for 5min, and thelysate wasthen

carefully scraped offthesurfaceandcollected intoa glasscentrifuge tube(SorvallHB4rotor). Nuclear and cytoplasmic fractionswereseparatedby centrifugation

at 2,500rpmfor 5minat4°C and recovered in the

pellet and the supernatant, respectively. The

cyto-plasmicfraction eitherwasused forextractingRNA

or wasfurther fractionatedtopreparepolyribosomes.

Preparationofpolyribosomes.Thecytoplasmic

fraction (from 20 petri dishes) was centrifuged at

10,000 rpm (Sorvall SS 34 rotor) for 20 min. The

resultingpost-mitochondrial supernatant wasplaced

intoatubecontaining0.25nilof 10%sodium

deoxy-cholate (final concentration ofsodium deoxycholate

was0.5%).The mixturewascentrifugedthrougha0.5

M/2M sucrosedouble layer (3 ml of0.5M sucrose

over2.5ml of2Msucrose)inaSpinco fixed-angle65

rotor at38,000 rpmfor2.5 h at4°C. The pelletwas

then suspended carefully in 0.75 ml ofbuffer I. (As judgedfrom an analytical sucrose gradient centrifu-gation,morethan80%ofthesuspension consisted of polyribosomes). Weadded to thesuspension 0.1mlof

0.1MEDTA,thusobtaining thepolyribosomal

prep-arationtobe usedfor extractingRNA(seebelow). Preparation of cytoplasmic and polysomal RNAs. The fluids designated asthe cytoplasmic or thepolysomal fractions,respectively (see above),were

diluted with 10 volumes of0.01 M triethanolamine buffer (pH 7.4) (4°C) containing50mMNaCl,6mM EDTA, and 1.1% sodiumdodecyl sulfate (35).Anequal

volume of a mixture of phenol-chloroform-isoamyl

alcohol in the ratio50:50:1wasadded, the extraction mixturewasblended inaVortex mixerat room

tem-perature for5minand thencentrifugedat8,500rpm

for5minin the Sorvall HB4rotor.Theaqueousphase and the interphase were reextracted with an equal volume ofphenol-chloroform-isoamyl alcohol mixture andrecentrifuged. After this, theaqueousphase and the interphasewere extracted withanequal volume of chloroform at room temperature for 5 min and centrifuged as described above, and the RNA was precipitated fromthe aqueousphase with2 volumes of ethanol after addition of sodiumacetate(pH 5) to afinal concentration of0.15M.

Preparation of cytoplasmic (or polysomal)

poly(A)+ RNA. The cytoplasmic (or polysomal)

RNA was passed through an oligodeoxythymidylic

acid-cellulose column to recoverthe cytoplasmic (or

polysomal) polyadenylated [poly(A)+]RNAand then precipitatedwithethanol(35).

Preparation of early and late strands of SV40 DNAHpaH-BamHIrestrictionfragments A and

B. Closed circular SV40 DNA labeled in vivo with

32P04 (specific activity: 5 x 105 to 5 x 106 cpm/,ug)

wasdigestedtocompletionwithsingle-siterestriction

endonucleases HpaII (0.725 mapunits) and BamHI (0.14 mapunits). These enzymes cleave SV40 DNA

neartheearly and late SV40 junctions (0.66and 0.17 mapunits) (22). The individual vidualstrandsofthe

fragmentswereseparatedby the method of Hayward (17). Theseparated strandswerepurifiedasdescribed by Alwine and Khoury (3). The resulting purified

separated strandsweredialyzedagainst 0.1x SSC. RNAmapping by gelelectrophoresisof endo-nucleaseSi-resistantRNA-DNAhybrid.The S1 mapping method usedwasthat of Berk andSharp(5), slightly modifiedaccordingtoMayetal.(26). Forthe

hybridization reaction cytoplasmic or polysomal

poly(A)+ RNAwasannealed with10-to20-fold molar

excess of

'P[DNA]

probe (20to30ng) consistingof

theearly strand (AE)ofHpaII-BamHI fragmentAor

the late strand(BL)ofHpaII-BamHI fragmentB. The

conditions ofDNAexcess wereestablishedbytitration

ofdifferentsamples. RNAamountsused in

hybridi-zationmixtureswere asindicated in thelegendstothe

figures.

Antiserum.Antiserumdirected againstthe SV40

virionproteins(predominantlyVP1)waspreparedby

intravenousinjection ofpurifiedSV40virionproteins

intorabbits,asdescribedbyTevethia(42).This anti-serum wasdesignatedasanti-VPserum.Normal

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MAY,

bitserumwasusedasacontrol.

Immunoprecipitation and electrophoresis of

proteins extracted from infected cells.

SV40-in-fected BMK and SV40-inSV40-in-fectedCV1cellswerelabeled

atthe timesand for theperiodsindicated belowwith

100,iCi ofL-[35S]methionine(750 to 930 Ci/mmol; The

Radiochemical Centre, Amersham, England) in

me-thionine-free medium. After labeling, the proteins

wereextractedfrom thecells asdescribedbyKresset

al. (23).

The solubleextract(from107CV1cellsor108BMK

cells) wasincubated with 2 1lof rabbit anti-VPserum

ornormal rabbitserum.The immunecomplexeswere

isolated by the methodofSchwyzeretal.(37)slightly

modified by Kressetal. (23)with thenewmodification

introduced byIto (18) to reduce the background in

final autoradiograms. Immune complexes were

de-sorbed fromaproteinA-Sepharosecolumn with 100

pd of 7 MureainTBS buffer(25mM

Tris-hydrochlo-ride[pH9]-137mMNaCl-5mMKCl-1mM

CaC12-0.5mMMgCl2-0.7 mMNa2HPO4-10% [wt/vol]

glyc-erol).

Theeluted sampleswerediluted 10times with TBS

buffer andreincubatedovemightwith 20,ld ofsettled

protein A-Sepharose. The immune complexes were

then reeluted from the protein A-Sepharose beads

with 50,Alof electrophoresis buffer containing 0.08M

Tris-hydrochloride (pH 6.8), 2% sodiumdodecyl

sul-fate(SDS), 5%2-mercaptoethanol,15%(wt/vol)

glyc-erol, and 0.001%bromophenolblue andincubated at

100°C for 10 minbefore analysis by

SDS-polyacryl-amidegelelectrophoresis (PAGE) (24).Conditions for

SDS-PAGEhave beenpreviouslydescribed(23).

Translation of gradient-fractionated mRNA.

Thecytoplasmic poly(A)+RNAsample from infected

BMK cells (250 Ag of RNA) in 400

Al

of a buffer

containing 10 mM triethanolamine (pH 7.4),50 mM

NaCl, and 1mMEDTA (used for preparingsucrose

gradients)wascentrifuged in 16 ml of 15to30%(wt/

vol) sucrose gradients in a Spinco SW27.1 rotor at

23,000rpmfor 22 hat20°C. Fractions (0.4 ml) were

collected fromthetopof the tubes withanISCO640

density gradient fractionator, and absorbance at 254

nm wasmonitored simultaneously withanUA-4

ab-sorbancemonitor. Eachfractionwasprecipitated with

2volumes of ethanolafter addition of sodiumacetate

(pH 5)toafinal concentration of 0.15 M. After ethanol

precipitation, the RNA from each fraction was

pel-leted, dissolved in 301,lofwater,andstoredat-20°C

(this RNA solution was also used in Si mapping

analyses). Thenuclease-treated rabbit reticulocyte

ly-satesystem (30) wasprogrammed with 20

pi

of the

RNAsolutionfrom eachfraction (200 ,l of reaction

mixturecontaining60,Ci of L-[nS]methionine,750to

930Ci/mmol; the Radiochemical Centre). Incubation

was for 1 h at 30°C. The reaction was stopped by

adding Nonidet P-40to afinal concentrationof2%.

The translation products were immunoprecipitated

withcontrol andanti-VPsera(1,lA ofserum per100

,lIoflysate).Theimmunecomplexeswereisolated by

themethod of Schwyzeretal. (37), and analysiswas

carriedoutby gel electrophoresis,asdescribed above.

Peptide mapping. Peptide mapping by limited

proteolysiswasperformedby the digestionprocedure

forproteinsin gel slicesasdescribed by Clevelandet

al.(9). The protease usedwasStaphylococcusaureus

V8 protease(MilesLaboratories, Inc.).

RESULTS

Production of early and late SV40

mRNA's in(nonpermissive)BMKcells. We first wantedtodetermine whether thecytoplasm of BMK cells abortively infected with SV40 containedspliced late mRNA's.Poly(A)+ cyto-plasmic RNA extracted at various times after SV40infectionfromBMK cells wasanalyzedby Si gel mapping. The 32P-labeled SV40 DNA probes usedin thepresentstudyweretheearly

strand (AE) of HpaII-BamHI fragment A and

the late strand (BL) ofHpaII-BamHI-B. Frag-mentB contains essentially all of the lategene region except for the terminal portionslocated between 0.144 and 0.170 map units and between 0.66and 0.725 mapunits,respectively.Fragment A contains all of the early region plus those terminal portions of the late region lacking in fragment B.

Figure 1A shows the nuclease S1 analysisof poly(A)+ cytoplasmic RNAsamplesfrom BMK cells at 12 h and fromCV1 at48 h afterSV40 infection, respectively. The analysis of RNA from infected BMK cells with the AE single-strandprobeshows thepredictedbands for the mRNA's whichencodelarge T andsmall t

an-tigens (3, 5, 11, 27, 32). We observed a band migrating at2,050nucleotidescorrespondingto

the 3'-exon (RNA segment distalto the single splice)which iscommon tobothearly mRNA's;

a600-nucleotideband of 5'-exonof smallt anti-genmRNA (theRNA segmentproximaltothe single splice); and a 300-nucleotide band of 5'-exonoflargeTantigen mRNA. Using thesame DNA probe to examine the early RNA in the poly(A)+ cytoplasmic RNA from CV1 infected for 48 h, we observedthe same bands, except that the2,050-nucleotide bandwasreplaced by

a1,945-nucleotideband.AlwineandKhoury(3) have demonstrated that this 1,945-nucleotide bandiscolinearwiththe2,050-nucleotideband, but is shortenedatthe 3' end. Theshorteningof this band could be due to RNA-RNA hybridi-zationbetweenlateandearly SV40 RNA, which coulddisplace the last 100bases of early RNA from thelabeled probe.

The analysis of RNA from infected CV1 cells with theBLsingle-strandprobe (Fig. 1A) shows the predicted bands for the 19S and 16S late SV40 mRNA's. Two bands migrating at 1,100 nucleotides and 2,000 nucleotides correspond to the3'-exon of 16S and 19SlatemRNA's, respec-tively.The bandof180nucleotides(Fig. 1) cor-respondstothe 5'-exonof 16S (and possibly of 19S)latemRNA, since

Si

mapping analysis with

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MOUSE CELLS EXPRESS SV40 LATE FUNCTION(S)

_- Y

m

_ co

A oC

V) V)

I I

r''l

_I (A

--~~l

48h 12h AE

BL

AE BL

B

I

6h 9h

AE BL AE

BL

-lr

0n

12h 21h

AE BL AE BL

-3040

2147-

2147--11540- -1100il;200

1150--*80

21067-- -- 200

350--1150--~ ~ ~ ~

20-XD!-110 SOW;- 0;.;;XS , ; 18

FIG. 1. NucleaseSl analysis of SV40 cytoplasmic poly(A)+ RNAs Cytoplasmic poly(A)+ RNA from CVJ or from BMKcells infected for the indicated time withSV40 was hybridized to the early strand(AE~)ofthe largerfragment (A) of32P-labeled SV40DNAgenerated by digestion with the restriction endonucleases HpaII and BamHI (0.144 to 0.725 mapunit), or to the late strand (BL) of the smaller (B) HpaII-BamHI fragment. Samples were hybridized, nucleaseSi treated, electrophoresed, andautoradiographed as described in the text.Input RNA amounts were as follows: 2.5 and0.3pg for hybridizingCVIcell RNA to AE and BL probes, respectively; 5 and 10pg for hybridizing BMK cell RNA toAEand BLprobes, respectively. Kodirex X-ray films wereexposed to gels for 2 days (A) or 8 days (B). Numbers at the left of thegelsare marker sizes in nucleotides.

HpaII-BamHI BL probe essentially allows the detection of that leader sequence

(5'-exon)

whichiscommon to16Sand19S mRNAspecies and mapsbetween nucleotides243and444(13). This leader sequence indeed generates a 180-nucleotide band corresponding to a DNA

seg-mentmappingbetween nucleotides 267and444, since the point ofcleavage of SV40 late DNA strand by HpaII restriction enzyme isbetween nucleotides266and 267. Itshouldbe noted that both the bodies

(3'-exons)

of 16S and 19S late mRNA's, respectively, maybe attachedto

sev-eral other leader sequences (13, 15) which are

probablynotdetectablebythisS1

mapping.

Using the same DNA

probe

to examine the late RNA in poly(A)+ cytoplasmic RNA from infected BMK cells (Fig. 1), it is

possible

to

detect the 1,100 nucleotide- and 180-nucleotide bands characteristic of 16S late mRNA. The 2,000-nucleotide band characteristic of 19S late

mRNA 3'-exon is alsodetectable, thoughrather faint. (Fig. 1B,9-h and 12-hRNAs; Fig. 2, cyto-plasmic RNA). The same band appears much darker inthe S1pattern of RNA from infected BMK cells treated with 100 ,ig of cycloheximide perml, due to drug-inducedoverproduction of latemRNA's (see Fig. 8).

When the same S1 analysis was done with polysomalpoly(A)+RNA extracted from SV40-infected BMKcells(Fig. 2), the 1,100-nucleotide band, corresponding tothe 3'-exon oflate 16S mRNA,wasfound. The amount of 16S mRNA used in this analysis is probably too low to

permit the detection of the corresponding

5'-exon. These results indicate that mousekidney cellsproduce detectableamountsofspliced late 16S and19S mRNA's.Asin thelytic cycle, the 16S mRNA appearstobe the majorspecies of late mRNA.

The intensity of the bands in the autoradi-943

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944 LANGE, MAY, MAY

CL (A

*

E%

o 0

_1 '

O

C

r

U

. .

AE

BL

AE

BL

-2000

-1100

-600

-300

ti:r:

|:00'.E':DE.' 0:

X- 0-00; a0ES?,00 t, ;.?, E;

C:: <:

--0 -0:f- 10-j-t0000 00: SX -180

0:;: :: CE :ajE: ;:: :: ::

-0:0 -t ::;. fffiSX i;0:0:: :: i:-:

-0-:X f.

00

FIG. 2. Nuclease Si analysis of SV40polysomal

poly(A)+RNAs.Polysomalpoly(A)+RNAfromBMK

cellsinfected for12h withSV40washybridizedto

HpaII-BamHI AEandBLprobes. Sampleswere hy-bridized, S1 nuclease treated, electrophoresed, and

autoradiographedasdescribed in thetext.For

com-parison, cytoplasmic poly(A)+ RNA extracted from SV40-infectedBMKcell culturesafter12hof

infec-tionwashybridizedinparalleltothesameprobes,

S1nucleasetreated,andelectrophoresedonthesame

gel.Input RNA amountswere2.5 and5pgfor

hy-bridizations toAE andBL probes, respectively.

Ko-direxX-ray filmswere exposedtogelsfor10days.

Numbers atthe left ofthegels aremarker sizes in

nucleotides.

ograms can be considered as an approximate measure ofthe relative amountsof the various RNA species, since thehybridizations with the labeled DNAprobeswereperformed under

con-ditions of DNA excess.

Therefore,

the

experi-mentin Fig. 1B allowsus to comparethe time coursesofappearance of

spliced early

versuslate SV40 mRNA's in infected BMK cells

and,

in fact, both time courses appear to be

virtually

parallel.

Considering the intensities of the

major

bands observedinthe

Si mapping patterns

ob-tained with AE and BL DNA probes,

i.e.,

the 2,050-nucleotide band and the

1,100-nucleotide

band,

respectively,

we can see

(i)

that boththese bandsaredetectableasearlyas6 hafter infec-tion; (ii) that theirintensitiesbecomemaximum ataround9 to 12hafterinfection;and

(iii)

that they

virtually

disappearat21 h after infection. This

time

courseissimilartothatof the

synthe-sis of

hybridizable

SV40RNA inthesame sys-tem, as

reported by May

et al.

(28).

The fact that both timecoursesofSV40mRNA

syntheses

are

parallel

suggests that thesame SV40 DNA moleculesareavailableas

templates

for the syn-thesis of late and

early transcripts.

Cell-free translation

product

of the late 16S mRNA

produced

in mouse cells. The next experiment shows that the

cytoplasmic

fractionisolatedfromSV40-infectedBMKcells containedlatemRNA(s)

functionally

competent in directing the

synthesis

of late

protein(s).

Poly(A)+ cytoplasmic RNA extracted at 12 h postinfection fromSV40-infected cellswas frac-tionatedthroughasucrosedensity

gradient.

Thefractionated RNAsweretranslated in the reticulocyte system, and the translation

prod-uctswerereacted with normal rabbitserumand rabbit anti-VPserum.The

immunoprecipitates

were analyzed by SDS-PAGE followed by

au-toradiography.For

comparison,

the

cytoplasmic

poly(A)+ RNA extractedat48 h

postinfection

fromSV40-infected CV1 cellswastranslatedin thereticulocytesystem,the translationproducts were immunoprecipitated with rabbit anti-VP serum,and theimmunoprecipitatewasanalyzed by SDS-PAGEinthe identical

gel.

The results (Fig. 3) show that the poly(A)+ cytoplasmic RNAcontainsaclass ofRNAencodingaprotein whichcomigrateswith themajor virionprotein VP1, whosesynthesisisdirectedbythe predom-inant late SV40 mRNA from CV1

cells.

As ex-pected,the sedimentationprofileof late messen-gertranslationalactivitydetected in BMK

cells

(Fig. 3) is very similar to that of the 16S late mRNA observedbyS1 mapping ofthe fraction-ated RNAs along the gradient (Fig. 4). More-over, the protein whose in vitro synthesis is directedby the predominant late mRNA from BMK cells appears to be identical to VP1, as judged by peptide mapping analysis of both proteins, performed according to Cleveland et al. (9)(Fig. 5).

Production of the major virion protein

2147-

-1810-

-808-

-526-

-447-

-350-

-250-

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MOUSE CELLS EXPRESS

SV40 inf. BMKcells

liU

1s8

94-

-68-

-60-

-53-

-43-- P-VP1

40

-V V N V N V N V N V N V N V N V V

23 24 25 26 27 28 29 30 31

FIG. 3. PAGE of proteins made in response to gradient-fractionated cytoplasmic mRNA's from SV40-infected BMK cells. Cytoplasmic poly(A)+ RNAfrom SV40-infected BMK cells was fractionated on a16-ml linear (15to30%[wt/vol])sucrose gradient bycentrifugation in a Spinco27.1rotor at23,000 rpm for 22 h at 20°C. Forty0.4-mlfractionswerecollected. TheRNA from each fraction was precipitated with 2 volumesof

ethanolafter addition of sodium acetate (pH 5) to a final concentration of 0.15 M, and it was then redissolved

in30,ulofwater.Thereticulocytelysatewasprogrammed with 20p1of the RNA solution from each fraction,

andthetranslationproductswereimmunoprecipitated with rabbit anti- VP serum (V) and with normal rabbit

serum(N)andanalyzed by SDS-PAGE. Sedimentation was from right to left. Fractions 15 and 25 correspond

topositionsof 28S and 18S rRNA's, respectively, sedimented in a parallel gradient. The fraction numbers are indicated above eachpair of tracks. For comparison, cytoplasmic poly(A)+ RNA was extracted at 48h

postinfection fromSV40-infectedCVJ cells and then translated in the reticulocyte system. The translation products were immunoprecipitated with rabbit anti-VP serum, and the immunoprecipitate was analyzed by SDS-PAGE in the identicalgel (7.5% polyacrylamide). The numbers on the left indicate the positions of the molecular-weight markers(Mrx10-3).KodakKodirex film was exposed to the gel for 4 days.

VP1bySV40-infected BMKcells. The

pres-ence in infectedBMK cells oftranslatable 16S latemRNA,atleastpartof which isapparently polyribosome-associated,

prompted

us to inves-tigate whether these cells

produce

detectable levels ofvirionproteinVP1.

BMK cells (15 plates) were labeledwith 100

t,Ci

of

[35S]methionine

per ml from 14to 15 h after SV40 infection. After the

labeling period,

soluble extracts were

prepared

from the cells andwerereacted with normal rabbitserumand anti-VP serum. The immune

complexes

were

isolated bythe method ofSchwyzer et al. (37) modified by Ito (18) as described above, and theywere analyzed bySDS-PAGEfollowed

by

autoradiography.

For comparison,parallel extractsof SV40-in-fected CV1cellslabeledat47 to 48h

postinfec-tion were analyzed in the identical

SDS-poly-acrylamide gel for the presence of

protein(s)

precipitable with anti-VP serum. The

SDS-PAGE pattern of immunoprecipitable proteins from infected BMKcellsreveals the presence of

abandcorresponding to a molecular weightof approximately45,000and comigratingwith the major SV40 virion protein VP1 from SV40-in-fected CV1cells (Fig. 6).From thisexperiment,

we infer that infected BMK cells do contain detectable levelsofnewly synthesized SV40 vir-ionproteinVP1.However,theselevelsmustbe very low since we were unable to detect the presenceofvirionproteinVP1 inthesecellsby the indirect immunofluorescence technique

us-ing the sameanti-VP serum (data not shown). Thus, the question of whether the synthesisof proteinVP1occursinall theinfected BMK cell populationor onlyin afractionof this

popula-tionremains open.

Production of early and late SV40 mRNA's in BMK cellsin the absence ofA gene activity.Wenextexamined whether the synthesis of SV40 late mRNA's

occurring

in 945

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[image:6.495.127.367.73.312.2]

946 MAY

18S

3020-

-2200-

1810

-1150-

-1067

-808-

-526-

-447-

-350-

-250-

--2000

-1100

-180

22 23 24 25 26 27 28 29 30

FIG. 4. Sl mapping ofthe sucrose

gradient-frac-tionatedcytoplasmicRNAsfromSV40-infectedBMK cells byhybridization to the late strandof

HpaII-BamHIfragment B. A10-,ulportion ofeach

fraction-ated mRNA ofthe sucrosegradient represented in

Fig. 3 washybridizedtothe late strand(BJ) of

32p-labeledHpaII-BamHI fragmentB. Thehybrid mol-eculeswerenucleaseSltreated,electrophoresed,and autoradiographedasdescribed in thetext.The

frac-tion number isindicated below each track. Numbers

ontheleftaremarker sizes in nucleotides.

nonpermissive cells required the synthesis of a functionallarge T antigen.

BMK cells were first infected with the non-leaky SV40temperature-sensitive mutant tsA58. Itshould be noted that tsA mutants are defective inthe production of a functional large T antigen required for viral DNA synthesis (36, 38, 40). tsA58 mutants fail to produce any detectable progeny virus at temperatures above

390C

(39). Moreover, we have verified thatno newly syn-thesized radioactive viral DNA is detectable in theHirtsupernatantfromCV1cellsinfected at

410C

with this mutant. After a 1-h adsorption period at 33 or410C,respectively, the cells were incubatedat330C for 17 h or at 410C for 12h, respectively. At this point, the cytoplasmic poly(A)+ RNA was isolated from the cells and analyzed by S1 mapping

performed

with the HpaII-BamHI BL probe. Figure 7 shows that the BMKcellsinfected at

410C

(nonpermissive temperature) contained a detectable level of spliced 16S late mRNA, asjudged by the pres-ence, in the

Si

mapping

pattern,

of the typical

1,100-nucleotide band corresponding to the 3'-exonof16S late mRNA.

Wethenstudied the effects ofacycloheximide treatment of infected BMK cellsonthe synthe-sisofearlyand late SV40 mRNA's. Cyclohexi-mide (0, 15, 50, or 100 ,ug/ml) was added to infected BMK cells immediately after the 1-h period of virus adsorption.

Cytoplasmic

poly(A)+ RNA was isolated from infected cells at 14 h postinfection and analyzed by Si gel mapping

performed

withHpaII-BamHI AEand BL probes. The Si mapping patterns (Fig.

8)

revealthat cycloheximide treatment results in anoverproductionof SV40earlyRNAs and that late mRNA's are produced in cycloheximide-treated cells and that the latter are, in

fact,

overproducedin the presence of 100 ,ug of

cyclo-heximnide per ml. The overproduction ofearly SV40 mRNA's in infected BMK cells treated with

cycloheximide

issimilartothat observed in

SV40-infected

CV1

monkey

cells

exposed

tothe same drug (16). This overproduction probably results from two distinct effects.

First,

an in-creasedrateof

early transcription

canbe dueto

CVI BMK

A B_

o ° N en o - CD pgprotease

I

Li

40- --ii

29

-18.4- 4.

14

-FIG. 5. Digestionduring re-electrophoresis ofthe translation products observed in the gelshown in Fig.3.The bandofVP1(from infected CVI cells)and the bandscomigratingwith itandcorrespondingto

gradientfractions24-28were cutfrom theSDS gel (Fig. 3). The band of VPI was divided into five samples, which wereplaced respectively in thefive

sample wells labeled CVI (A). Thegel slices

corre-spondingtofractions24to 28 wereplacedfrom leftto

right, respectively, in the five sample wellslabeled BMK(B). Each slicewasoverlaid with S.aureusV8 protease, accordingtoCleveland etal. (9). The

num-bers on theleftindicate the molecularweights(Mrx

lo-)ofthe markers. Theprotease concentrationsare

givenaboveeachtrack.Thegel(15%polyacrylamide)

was fluorographed using the autoradiography

en-hancerEN3HANCE (New EnglandNuclearCorp.).

Kodak SB5filmwasexposedtothegelat-70°C for

21days.

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[image:7.495.53.242.53.307.2] [image:7.495.260.451.332.479.2]

MOUSE CELLS SV40 LATE

the decreasedproduction of large T antigen, on the basis ofthe autoregulation model of Tegt-meyer (33, 41). Secondly, cycloheximide seems to produce a rather generalized increase in mRNA content, with the drugprobablyacting

> ax

_ "c

C_.

CD

(n)

48h

0

U1

18h

94-68-

9m0

60-

53-43- 4

.66-VPI

N V N V

FIG. 6. Virionprotein VP1 inSV40-infectedBMK

cellswaslabeled with 100,pCi of[3S]methionineper

mlfrom 14to 15 h.After labeling, soluble extract

from thecellswasimmunoreactedwith rabbit

anti-VP serum (1< and normal rabbit serum (N). The

immunecomplexeswereisolated andelectrophoresed

in a 7.5%SDS-polyacrylamidegel.Forcomparison, SV40-infectedCV) cellswerelabeled with 100,uCi of

[36S]methioninepermlfrom47to48hafter infection. After labeling, solubleextractfrom these cells was immunoprecipitatedwith anti-VPserum,andthe im-munoprecipitatewasanalyzedby SDS-PAGEin the

identicalgel, revealing essentiallythebandofvirion

protein VP1,asexpected. Thegelwasfluorographed

with the autoradiography enhancer EN3HANCE

(NewEnglandNuclearCorp.). KodakSB5filmwas

[image:8.495.294.414.70.373.2]

exposedtothegelat-70°C for21days.

FIG. 7. SIgel analysis of cytoplasmic RNAs

iso-latedfromtsA-infectedcells.BMK cellswereinfected

with tsA58mutanteitherat33°C for17horat41°C

for12 h. At thispoint,thecytoplasmic poly(A)+ RNA

wasisolated fromthecells andanalyzed by S1 map-pingperformedwith HpaII-BamHI BL probe. Input

RNAamountswere25pgineach hybridization

mix-ture.KodirexX-rayfilmwasexposedtothegel for10

days.Numbersontheleftaremarkersizesin

nucleo-tides.

by promotingthe stability of mRNA's (19, 25, 46). The lattereffectcould also account for the

overproductionof late mRNA's in thepresence

of 100,ug ofcycloheximideperml.

Inaparallel experiment designedtodetermine

the levelofcycloheximide-inducedinhibition of

T antigen synthesis, infected BMK cells were

exposed to cycloheximide with the same drug

concentrationand for thesameperiod.From 12 to13hpostinfection,thecellswereincubatedin

methionine-freeMEM andthenlabeledfrom 13 to14 hpostinfectionwith 100,uCiof [3S]methi-onine per ml added in methionine-free MEM.

Cycloheximide was present in methionine-free MEM and in the medium used for labeling. Treatmentwith15, 50,and 100,ugof cyclohexi-mide per ml resulted in a 95, 98.3, and 99.3%

inhibition ofprotein synthesis, respectively, as

330C

410C

3020-

2200-

1810-

1150-

1067-

808--2000

-1100

-180

526-

447-

350-

250-947

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[image:8.495.77.224.146.504.2]

948 LANGE, MAY, AND MAY

I

AE

BL

E

_- In _

S I

AE BL AE BL AE BL

3020-

-2200

-1810-

1150

808

-526-

-447-

-250-

--2000

--1100

600

-300

-180

FIG. 8. Sl gel analysis ofthecytoplasmicpoly(A)+

RNAs isolatedat14hafter infection fromBMK cells

treated with variousconcentrationsof cycloheximide

(CH).BMK cellswereinfectedwithSV40and treated

with CHat the indicateddosefrom 1 to 14 hafter

infection. At 14 hpostinfection, cytoplasmic poly(A)+

RNAwasextractedfromthe cells. TheseRNAswere

hybridized to 32P-labeled HpaII-BamHI AE or BL

probes (as indicated), nucleaseS1 treated, and

elec-trophoresed as described in the text. Input RNA

amountswere2.5 and 10jLgforhybridizing RNAto

AEandBLprobes,respectively. Thegelwasexposed

toKodirexX-rayfilm for 5 days. Numbersonthe left

aremarkersizes innucleotides.

measuredby[35S]methionineincorporationinto trichloroacetic acid-precipitable material.

Solu-bleextractsfromthecells were

immunoprecipi-tatedwith hamsteranti-SV40tumorserum,and theimmunoprecipitateswereanalyzed by

SDS-PAGE followed by autoradiography, as de-scribed by Kress et al. (23). A relatively long exposure periodwas usedtoenhance detection of large T antigen from drug-treated cells,

re-sultinginanoverexposition of thelarge T anti-gen band from untreated cells. As judged by a

comparison of band intensities made by

densi-tometryofthe autoradiogram (Fig.9), the syn-thesis oflargeTantigenwasinhibitedbymore

than 73, 97.8, and 99.9% after treatment with this antibiotic at concentrations of 15, 50, and 100

jg/ml,

respectively.

The fact thatweobserve aproductionofSV40

-E.

c)

. .

u u

I +

E

EE

fl)

z

o

u

o- o

60--21-

-111

18-

-tu N tu N

t:u

N

tu

FIG. 9. SDS-polyacrylamidegel autoradiography

oflabeled Tantigensfromextractsof SV40-infected

BMK cells treated with cycloheximide (CH).

SV40-infectedBMK cellswere treated with the indicated

doseofCHfrom1 to 14hpostinfection and labeled with [355]methionine (100jiCi/ml) from 13 to 14 h

after infection. After labeling, soluble extractfrom

thecellswasreacted withhamster anti-SV40tumor

serum(tu)and with normal hamsterserum (N). The

immunoprecipitates were analyzed by SDS-PAGE

and autoradiographed. Thegel was at 12.5% poly-acrylamide. KodirexX-ray film was exposedto the

gel for 8 days. Numbers on the left indicate the

molecularweights (Mrx10-3)ofthe markers.Bands

T and tcorrespond tolarge and small Tantigens,

respectively.

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[image:9.495.58.258.59.367.2] [image:9.495.225.447.79.514.2]

late mRNA's in (i) infected BMK cells after treatment with the higherdose (100,ug/ml) of cycloheximide resultingin a99.9% inhibition of large T antigen synthesis and (ii) BMK cells infectedwiththe mutant tsA58 at

410C

(restric-tive temperature) suggests thatSV40late tran-scription inBMK cells does not require a func-tionalA geneactivity.

DISCUSSION

Mouse cells are considered as fully nonper-missive for SV40. Infection does not lead to detectable virus replication(2,43). Up to now it has been felt thatSV40-infectedmouse cells did not produce either functional late mRNA or SV40 virion

protein(s).

In the present study, we first found that spliced 16S and 19S SV40 late mRNA's were present incytoplasmicand polysomalpoly(A)+ RNA preparationsfrom SV40-infected (nonper-missive) BMKcells.The16Sand 19SSV40late mRNA'sproducedininfectedBMK

cells

indeed turned out to be identicalto or similar to the 16S and 19S SV40 late mRNA's produced in permissivemonkeycells, respectively,asjudged by their

Si

mapping patterns performed with the late strand of HpaII-BamHI fragment B (Fig. 1, 2, and 4), and by their

sedimentation

patterns (Fig. 3 and 4). Moreover, we showed that the 16S mRNA from infected BMK cells could be translated in the rabbit reticulocyte lysatesystemintoapolypeptidewhich was iden-ticalto orsimilartovirion proteinVP1 inevery aspect we examined, including the pattern of peptide mapping bylimited proteolysis (Fig. 3 and 5). It is noteworthy that the ratio ofthe amountoflate mRNAtothatof early mRNA is of the same order in infected BMK cells (5 to 10%, as determined by hybridization of RNA withclonedregion-specific viralDNAfragments immobilized onfilters [M. Lange,

unpublished

data])as inCV1monkey

cells

attheearly phase oflytic infection (1 to 5%according to Parker andStark[29]).

Secondly,wehavereported evidence that in-fected BMK cells did produce detectable, al-though

low,

levels of virion protein VP1, as

shown

by

the

SDS-polyacrylamide gel

autora-diogram of [3S]methionine-labeled proteins im-munoprecipitated from the cells (Fig. 6). This

confirms

that atleast a part of the late mRNA's produced bythecellswas polyribosome-associ-ated

functional

mRNA.

Ourresults have implications for the nature ofthe factorsdeterminingtheabortiveresponses of mousecellstoSV40infection. Previous stud-ies on the regulatory mechanism ofSV40 late gene expressioninmousecellsledGraessmann andGraessmann (14) to

hypothesize

thata

cel-lularrepressing-typefactor prevents the expres-sion ofthelate SV40 genes in these cells. Our results do not fit wellwith thishypothesis.

On the other hand, Watkins (45) suggested that cells which are nonpermissive for SV40 mightlackfactor(s) specificallyrequired forthe translation of SV40 late mRNA's. From our results, it maybe inferred that ifsuch specific factorsarenecessaryforSV40 late mRNA trans-lation, theyare present innonpermissive mouse cells,althoughwecannot exclude the possibility thatthetranslation efficiency of late mRNA's is lower inmousecellsthan in monkey cells.

Other interesting observationsreported here are that infected BMK cells did produce late mRNA's either (i) when theinfectionwas per-formedat410Cwith thenonleakytsA58mutant or (ii) in cells continuously treated after the virusadsorptionperiod with 100

,ug

of cyclohex-imideperml,inwhich large Tantigensynthesis wasinhibited by more than99.9%.In thelatter case, a drug-induced overproduction of late mRNA'swas, infact, noted.Theseobservations are similar to those reported by Handa and Sharp (16)concerning the synthesis of SV40 late mRNAduring the earlyphase of lytic infection ofmonkeycells. Ourobservations suggest that late mRNA synthesis in mouse cells does not require large T antigen, although we cannot completely rule out the alternative possibility that late mRNAsynthesis requires large T an-tigen but at an

exceedingly

low concentration

(29).

Our suggestion that large T antigen is not required for late mRNA

synthesis

fits well with thehypothesis that the

positive

regulatory

effect oflarge T antigen exertedonlate mRNA syn-thesis inlytic infection ofmonkey cells (4, 7, 10, 22, 29,33) is not dueto a direct interaction of large T

antigen

with the late

promoter(s)

but might be

indirectly

mediated

by

another effect oflarge T

antigen,

suchas

increasing

the number ofSV40 DNA molecules transcribed

(7)

through its role in

initiating

viral DNA

replication (8,

38).Thislatter

hypothesis

isalso consistent with therecent

findings

of Rioetal.

(34), who,

using a cell-free RNA

synthesizing

system, showed that the D2

protein

(biologically equivalent

to SV40

large

T

antigen)

inhibitedSV40

early

tran-scription,

but hadno effect on

transcription

of SV40latesequences,andthat

efficiency

of late

transcription

was enhanced relative to that of early

transcription

asthe concentrations of both earlyand late promotersincreased.

ACKNOWLEDGMENTS

We thankM.Kress forhelpfuldiscussions and for

perform-ingthepeptidemappingofproteins bylimitedproteolysis,J.

Borde and C.Breugnotfor theirexcellentassistance,C.

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sardfor herhelpinpreparingthecellcultures,and M.Maillot forher able assistance inpreparingthemanuscript.

This work wassupported by grantsfrom theDelegation Generale a la Recherche Scientifique etTechnique (ATP 78.2641 and79.7.0665),the Fondationpour la Recherche Med-icaleFrancaise,and from the Associationpourle Developpe-ment de la Recherche sur le Cancer.

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