Temperature-sensitive mouse cell factors for strand-specific initiation of poliovirus RNA synthesis.

JOURNALOFVIROLOGY,

JUlY

1993, P.3989-3996 0022-538X/93/073989-08$02.00/0

Copyright X 1993, American Society for Microbiology

Temperature-Sensitive

Mouse

Cell

Factors

for

Strand-Specific

Initiation

of

Poliovirus

RNA

Synthesis

KAZUKOSHIROKI,1* HIROYUKI KATO,'SATOSHI KOIKE,2 TAKESHI ODAKA,3

ANDAKIONOMOTO"2

Departmentof Microbiology, Instituteof Medical Science, The University of Tokyo, 4-6-1, Shirokanedai Minato-ku, Tokyo 108,1 Department of Microbiology, Tokyo MetropolitanInstituteof MedicalScience, Honkomagome, Bunkyo-ku, Tokyo113,2andDepartmentof Microbiology, Saitama Medical College, 38,

Morohongo, Moroyamacho, Irumagun, Saitama350-04,3Japan Received 19 January 1993/Accepted2April1993

Twocelllines,TgSVAandTgSVB,wereestablished fromthekidneys of transgenicmice carrying the human geneencodingpoliovirusreceptor.The cellswerehighlysusceptibletopoliovirus infection, andalargeamount

ofinfectiousparticleswasproduced in the infected cellsat37°C.However,thevirus yieldwasgreatlyreduced at40°C.Thisphenomenonwas commontoallmousecells tested. To identify the temperature-sensitive step(s) of thevirusinfectioncycle, differentstepsofthe infection cyclewereexaminedfortemperaturesensitivity.The results strongly suggested that the growth restriction observed at40°C wasdue toreduced efficiency of the initiationprocessofvirus-specificRNA synthesis. Furthermore, this restriction appearedtooccuronlyonthe synthesisofpositive-strandRNA.Virus-specificRNA synthesis in crudereplicationcomplexeswasnotaffected bythenonpermissive temperature of40°C. Invitrouridylylation of VPg seemed tobetemperaturesensitive

onlyafterprolonged incubationat40°C. Theseresultsindicatethataspecific hostfactor(s)is involvedin the

efficient initiation process of positive-strand RNA synthesis of poliovirus and that the host factor(s) is temperaturesensitive inTgSVA and TgSVB cells.

The genome of poliovirus is a single-stranded, positive-senseRNAcomposedofapproximately 7,500 nucleotidesto whichare attacheda small protein VPgatthe 5' end (4, 11, 20,24)andpoly(A)atthe 3' end(40).ThisRNAfunctionsas

mRNAtoproduceasingle largeprecursorpolyprotein of 247

kDaafterentryinto the host cellcytoplasm.Thepolyprotein iscotranslationallycleavedbyproteases toformviralcapsid proteins (VP1, VP2, VP3, andVP4) andnoncapsidproteins (2A, 2B, 2C, 3A, 3B, 3C, and 3D). Oftheviral noncapsid proteins, 2A, 3C,and3CDareviralproteasesinvolvedin the processingofpolioviruspolyprotein (19), 3BisVPg(20, 24, 28, 29, 39), 3D is viral RNA-dependent RNA polymerase (26, 28, 39),2Band 2Careconsideredtoplay importantroles inviralRNAsynthesis (3, 5, 21, 25, 28, 29, 39),and 3Acould have an important role(s) in uridylylation of VPg in the initiation process of RNA synthesis (12, 28, 35). Thus, of biologically significant knowledgeofmanyviralproteinshas

been accumulated.

In additiontoviralproteins, poliovirus requireshostcell factors for itsreplicationcycle.Poliovirusstrains,exceptfor type2virulentstrains, infectonly primates. Recentsuccess

inmolecularcloningofgenomicandcomplementaryDNAs encoding human poliovirus receptor (PVR) allowed us to rendernonprimatecellspermissivenessforpoliovirus infec-tionby introducingthe human DNAs into the cells. Indeed, mouse L cells transformed with the PVR DNAs are very

susceptible to poliovirus (17, 22). Furthermore, transgenic mice carrying the human PVR gene were found to be susceptible to poliovirus (18, 27). These observations indi-cate that any kind of cells can be permissive for viral adsorption andpenetration byintroduction of PVR DNAs. This, in turn, makes it possible to findcells ofnonprimate

*Correspondingauthor.

originthathave defects in host cell factors other than PVR supportingpoliovirusinfection.

PoliovirusRNAreplicationoccurs onmembranes induced

by virus infection(5, 6, 8, 9,35, 36). Indeed, themembrane fraction, called the crude replication complex (CRC), iso-lated from poliovirus-infected cells can synthesize virus-specific RNA (34, 35). This in vitro reaction represents mostly chain elongation of the positive-strand RNA of poliovirus (10). Takegamietal.(35), however,demonstrated that the CRC is able to synthesize nucleotidyl proteins, VPg-pU andVPg-pUpU, thatarethe5'-terminal structures of all virus-specific RNA molecules except for the viral mRNA (24). This nucleotidyl protein was shown to be incorporated into VPg-pUUAAAACAGp, an RNase T1-resistant oligonucleotide derived from the 5' end of the poliovirus genome (11, 24, 34). Although neither the uridy-lylation of VPg nor the subsequent chain initiation of the

RNA is efficient, theseresults suggest that VPg-pU(pU) is utilizedas aprimerforpoliovirus RNAsynthesis.

The amount of positive-strand RNA synthesized in in-fected cells is muchlargerthanthatofnegative-strandRNA. This fact suggests that there is a difference(s) in initiation mechanisms forpositive- andnegative-strand RNA synthe-ses if only the viral RNA-dependent RNA polymerase (3DP"') is involved in chain elongation of both strands. In fact, a template-priming mechanism has been proposedfor synthesis of a negative-strand RNA molecule by using a soluble, membrane-free system including 3DP'1 and a host cell factor (2, 37). According to the model, VPg is not involved in the initiation step ofnegative-strand RNA

syn-thesis(37).

Here we report that poliovirus replication is generally temperature sensitive in mouse cells. Using cell lines

(TgSVA andTgSVB) establishedfromtransgenicmice sus-ceptible to poliovirus (18), we show that the temperature-sensitive step resides in the initiation process of positive-3989

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strand RNA

synthesis

of

poliovirus, indicating

that a host

cell

factor(s)

is involved in some

step(s)

of the initiation process.

MATERUILSANDMETHODS

Cells and viruses. HeLa S3 cell

monolayers

and African green

monkey kidney

cells were maintained in Dulbecco

modified

Eagle

medium

(DME) supplemented

with5%

new-born calf serum

(NCS)

and used for virus

preparation

and

plaque

assay.

Suspension-cultured

HeLaS3 cellswere

main-tained in RPMI medium

supplemented

with 5% NCS. La

cells are L cells

expressing

cDNA

encoding

human

PVRao

and maintained in DME

supplemented

with 10% fetal calf

serum

(FCS) (17). TgSVA

and

TgSVB

cellswereestablished from

kidney

tissues of

transgenic

mice

(ICR-PVRTgl)

car-rying

the human gene for PVR

(18),

taking advantage

of immortalization with simian virus 40

(SV40)

T

antigen

as

described in

Results,

andmaintained in DME

supplemented

with5% FCS. Poliovirustype1

Mahoney

strainwas

propa-gated

in

suspension-cultured

HeLa S3 cells as

previously

described

(15).

SV40

(strain 777),

herpes simplex

virustype 1

(strain Mt),

and Sindbis viruswerealso used.

Virusinfectionand

growth.

Toexaminevirus

growth,

cells in 6- or 12-well

plastic

culture

plate

were infected with

light-sensitive poliovirus

prepared

as

previously

described

(17)

at a

multiplicity

ofinfection

(MOI)

of 20. After

incuba-tion for1hatindicatedtemperatures,the cellcultureswere

exposed

to

light

for 1 h in ordertoinactivate residual virus.

Virus

yields

at 24 h

postinfection

were measured

by

virus titrationonAfrican green

monkey kidney

cells.

Analysis

of

proteins by

PAGE. HeLa

S3, TgSVA,

and

TgSVB

cells in 12-well

plates

wereinfected with

poliovirus

atanMOI of 50. The cellswereincubated in methionine-free

DMEat 37 and

40°C

and labeled with 2

,uCi

of

[35S]methio-nine

(1.45 Ci/mmol; Amersham)

per ml for 30 min

prior

to harvest. At intervals of 1

h,

theradiolabeled cell

monolayers

were collected and dissolved in

sample

buffer

(62.5

mM Tris-HCl

[pH 6.8],

2.3%sodium

dodecyl

sulfate

[SDS],

100 mM

dithiothreitol,

10%

glycerol,

with0.024%

bromophenol

blue as an

indicator). Samples

were heated at

100°C

for 5

min,

applied

toa12.5%

polyacrylamide

gel

(SDS-polyacryl-amide

gel electrophoresis [PAGE]

plate 12.5;

Daiichi Pure

Chemicals

Co.,

Ltd.),

and run at 60 mA. After treatment with

Amplify (Amersham),

the

gels

weredried and

exposed

to

Fuji X-ray

films.

Labeling

ofinfectedcellswith

[3H]uridine.

Cells in24-well

plates

wereinfected with

poliovirus

atanMOIof20. Aftera

30-min

adsorption

period,

DME

supplemented

with5%NCS or FCSwas

added,

and the cellswere incubated at 37 and

40°C.

At 2 and 2.5 h

postinfection,

actinomycin

D

(5 ,ug/ml,

final

concentration)

and

[3Hjuridine

(1 ,uCi/ml,

final

concen-tration), respectively,

were added. At indicated

times,

the mediumwas discarded and the cellswerecollected in cold

phosphate-buffered

saline and treated with trichloroacetic

acid

(TCA).

TCA-insolubleradioactivitiesweremeasuredin a universal scintillater

(DOTITE;

Scintisol

EX-H) by

a scintillationcounter.

RNase

protection

assay. An RNase

protection

assay was

performed

asdescribed

by

Katoetal.

(16),

with the

modifi-cation described below. RNAs were

prepared

from HeLa

S3,

TgSVA,

and

TgSVB

cells

(4

x 10

each)

that had been

infected with poliovirusat anMOIof 20andincubatedfor 5 h. The sense

[32P]RNA

probe

was

synthesized

in the pres-ence of

[a-32P]CTP

with T7

polymerase (Promega)

from

Spel-linearized

pBL-PVKB.

The antisense

[32P]RNA

probe

was synthesized in the presence of [a-32P]CTP with T3 polymerase (Promega) from BglI-linearized pBL-PVKB(see

Fig. SC). To reduce the specific activities of RNAprobes,

the reactionwasperformed with 12.5 ,uCi of

[a-32P]CTP

per mmol. PlasmidpBL-PVKB is a derivative of plasmidvector pBluescript SK that has an insert of poliovirus cDNA

corresponding to the genome region from nucleotides 3665

(KpnI

site) to5600 (BglII site) at

KjpnI

andBamHI sites of thevector(see Fig. SC). The cytoplasmic RNAs (1 ,ug), after heating at 100°C for 5 min, were hybridized with excess amountsof the [32P]RNAprobes(5 x 104 cpm[Cerenkov])

at 37°C for 3 h in 20 ,u of 40 mM

piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES; pH 6.7)-i mM EDTA-0.4 M NaCl-80% formamide. The sampleswerethen diluted with 15 volumes (300 ,ul) of an ice-cold buffer containing 10 mM Tris-HCl (pH 7.4), 5 mM EDTA, 300 mM NaCl, 6 ,ug of RNase A per ml, and 0.3 ,ug of RNase T1 per ml and incubated for 30 min at 37°C. The reaction mixtures were further incubated after addition of0.5% SDS and 1 mg of proteinase K per ml. The products, after extractions with phenol-chloroform (1:1) and chloroform, were recoveredby

ethanolprecipitation and analyzed by electrophoresis on 5%

polyacrylamide gelsin the presence of 8M urea.

UridylylationofVPgand RNAsynthesis in vitro. CRCwas

prepared from poliovirus-infected HeLa S3, TgSVA, or

TgSVB cells as described by Takeda et al. (34), with the modifications described below. Cells(8 x 108) infected with

poliovirus were incubated at 37°C and harvested at 5 h

postinfection.Each CRCwassuspended in 1.2 ml of buffer

(10 mM Tris-HCl [pH 8.0], 10 mM NaCl, 15% glycerol, 1 mMdithiothreitol, 1%Trasylol [aprotinin,Bayer; 50,000U/5 ml]), divided into 20-pA portions, and storedat -70°C.

In vitro RNAsyntheseswerecarriedoutbyincubating 50

,ulof the standard reactionmixturecontaining20pul of CRC

(10).

Atindicatedtimes,0.2puCiof[a-32P]UTPwasadded to each in vitro reaction mixture. After indicated periods of incubation ateither 36or40°C, thereactionswere stopped

by

addition of10% TCA. TCA-insolublecounts were deter-mined in ascintillationcounter.

In vitro formation ofVPg-pU(pU)wascarriedoutin 50pul

of reaction mixturecontaining20 ,ul of CRCasdescribed by Takedaetal.

(34).

Atindicatedtimes, 10 ,uCiof [aL-32P]UTP wasaddedtothe in vitroreactionmixtures. After incubation at37 or40°C, the reactionswerestopped by addition ofan

equalvolume of thesamplebuffer. Thesampleswereheated for5 min and dilutedbytheaddition of 0.5 volume of H20 and 5 volumes of

radioimmunoprecipitation

assay buffer. The

VPg-pU(pU)

formedwasreacted againstacomplex of

protein A-Sepharose and anti-VPg antibody (Cl and N10)

(30).

The

immunoprecipitates

were then subjected to

elec-trophoresis

on anSDS-12.5%polyacrylamide minigel (Dai-ichi Pure Chemicals), usinga Laemmligelbuffer system.

RESULTS

Establishment of cell lines susceptible to poliovirus. To establishmousecelllinessusceptibletopoliovirus infection,

kidneys, spleens, brains,

bone marrow, and wholeembryos of

transgenic

mice

(ICR-PVRTgl)

expressing the human genefor PVR(17)wereminced, dispersedby trypsinization, and cultured. Aportionofthesecultured cells was infected with SV40or transfected with adenovirus ElA DNA (32). The cells

expressing

SV40T antigen or ElA protein were tested for

sensitivity

topoliovirusinfection. Although many cell lineswere

susceptible

to infection, mostsensitive were those

expressing

theSV40Tantigenderived fromthe kidney

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HOST FACTORS FOR INITIATION OF POLIOVIRUS RNA SYNTHESIS TABLE 1. Sensitivitytopoliovirus infection and

production of poliovirus

Virus titerrequired Virusyield

forcytopathiceffect

(loglo

PFU/ml)

Cells

(logl0

PFU)

37°C 40°C 37°C 400C

Mouse

TgSVA lfOf 5.0 8.0a 5.0

TgSVB 1.0 4.5 8.5 6.0

TgN-5 3.0 5.5

Tg-emb-7 2.5 5.5

La 2.5 5.0

Human

HeLa 1.5 1.5 8.5 8.0

TIG-7 1.5 1.0

aMeasured inHeLaS3 monolayer cells.

(18). Among them, two clones were selected and named TgSVAand TgSVB.

Temperature-sensitive growth ofpoliovirus inmouse cells. InTgSVA and TgSVB cells, the plaque assay of poliovirus wascarried out at either 37 or 40°C. Plaques were efficiently

formed in both cell lines at 37°C but not at 40°C, whereas

plaque numbers on HeLa cells at 40°C were the same as thoseat37°C. Asimilarobservationwasobtained when the

cytopathiceffect ofpolioviruswasinvestigatedat37 or40°C

(Table 1).VirusyieldsinTgSVA and TgSVB cells in a single cycleof infectionwere examined at 37 and 40°C(Table 1). The amountsofinfectious particles produced in these cells at 40°Cwerereducedto1/1,000 (TgSVA cells)or1/300(TgSVB cells)of thoseat37°C. The resultsindicatethat theinefficient

plaque formation observed at 40°C is a result of reduced

efficiencyinsomereplication step(s)ofpoliovirus.

To examine whether the inefficient plaque formation at

40°C is common to all mouse cells, La (Lfibroblast cells, derived from mouse strain C3H/He, that express PVRa) (17), Tg-emb-7 (secondary cultures from an embryo of a

transgenicmouse),andTgN-5(acell line established without theimmortalization processfrom thekidneyofatransgenic mouse)wereinfected withpoliovirusand incubatedateither 37or40°C(Table 1).These mouse cells alsowere tempera-turesensitive for thedevelopmentofplaques andcytopathic effectsinducedbypoliovirus.These resultsstronglysuggest that the restricted growth of poliovirus in TgSVA and

TgSVB cells at 40°C is not due to expression of SV40 T

antigen, specific tissues, or specific strains ofmice. As to humancells,TIG-7 cells (human fibroblasts), in additionto HeLacells, weresusceptibletopoliovirusinfection atboth 37 and40°C (Table 1). Thus, temperature-sensitive

replica-tion ofpoliovirus seems to becommon to all mouse cells, suggestingthata cellularfactor(s) forpoliovirus replication

is temperature sensitive in murine cells.

Herpes simplexvirus (strain Mt) and Sindbisvirus grew wellin TgSVA and TgSVB cells at both 37 and 40°C (data not shown). Therefore, temperature-sensitive host factors are notutilized in replication of these viruses.

Temperature-sensitive step of poliovirus replication. To identify the temperature-sensitive steps in the poliovirus infection cycle in TgSVA and TgSVB cells, temperature shiftexperimentswerecarriedout onthe infected cells(Fig. 1). Virus yields were determined in HeLa, TgSVA, and TgSVB cells which had been shifted from the permissive temperature of 37°C to the nonpermissive temperature of

40°Corshifted from 40to37°Catintervals of 1 hduringan

A

- 8

2

U.

a7

*56

'5

B

,% U.

5

Shift-down Exp. (40(C-37TC)

- -

--.--

.'

-v

e C x~~~~~~~~~~~~~~~~;;---.--.---O

O0 1 2 3 4 5 6 7 8

Time at temperature shift(h)

Shift-up Exp. (37°C-40t)

8~**~ - @

6

5

012345678

4 ,

.

--nlI a a a a

O 1 2 3 4 5 6 7 8 Time at temperature shift( h )

FIG. 1. Growth of poliovirus at 37 and 40°C. HeLa

(@----@),

TgSVA (-4), and TgSVB (0) cells in 12-well plates were infected withlight-sensitive poliovirus Mahoney strainat anMOIof 20 andculturedat40or37°CasdescribedinMaterialsand Methods. At1-hintervalspostinfection, portions of cellswereshifted from40

to

370

(A)or37to40°C(B), and all cultureswerefrozenat24 h after infection.Virusyieldsweremeasured by plaqueassayandplottedat theindicated timesoftemperature shifts.

infection cycle. As shown in Fig. 1A, the temperature shiftdowncarriedoutupto5 hpostinfection appearednot to affect the virus yield. Alonger incubation at 40°C (6 h or longer postinfection), however, resulted in a reduction of virusyield.Thisfinding may indicate that thenonpermissive

temperature leadsto abortive infection ofpoliovirusby 6 h

postinfection. When the temperaturewasshifted from 37to

40°C up to 3 h postinfection, the virus yield was reduced (Fig. 1B). However, the temperatureshiftupat4 h(or later) postinfection provided the normal levels of virus yield.

Theseresults indicate that thetemperature-sensitive stepof

poliovirus replication is at some point between 3 and 4 h

postinfection in TgSVA and TgSVB cells.

Adsorption and penetration of poliovirus. Poliovirus

ad-sorption andpenetrationinTgSVAandTgSVBcellsat40°C

were further examined by using light-sensitive poliovirus. TgSVAandTgSVBcellswereincubated withlight-sensitive poliovirusat37or40°Cin the darkasdescribed in Materials and Methods. These cultures were exposed to light 1 h

postinfection and further incubatedat37or40°C. The virus titersobtainedat 24hpostinfectionin culturesof HeLaand TgSVAcellsareshown in Table 2. Incubationat40°Cfor the first 1 h did not affect virus yields in either

poliovirus-infected HeLa or TgSVA cells. Further incubationat40°C

caused a reduction of the virusyield onlyin TgSVAcells,

notinHeLacells(Table2andFig. 1).Asimilar reduction of virusyieldwasobtained in thecaseofTgSVBcells

(data

not shown andFig. 1).These results indicatethat human

polio-virus receptorsonTgSVAandTgSVBcellsarefunctionalat the nonpermissive temperature of40°C and that very

early

stepsof virusinfection,including

adsorption

and

penetration

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3992 SHIROKI ET AL.

TABLE 2. Virus titers obtained at 24 hpostinfection

Temp

(°C)

Virus titerat 24 h

postinfection

0-1hpostinfection 1-24 hpostinfection HeLa TgSVA

37 1.2x102 1.2x102

37 37 1.8 x 106 3.6x 105

40 37 3.9 x 106 2.3 x 106

40 40 2.1 x 106 7.5x103

ofvirus, arenottemperaturesensitive inTgSVAandTgSVB

cells.

Translationofpoliovirus RNAinTgSVAandTgSVBcells. Translation of infected virion RNAwasanalyzed. A sensi-tive assay used forpoliovirusproteinstranslated

early

inthe infection cycle is to monitor the inhibition of hostprotein

synthesis(31,33).HeLaandTgSVAcellswereinfectedwith poliovirus at anMOIof 50 and incubated at 37 and40°Cin the absence or presence of 1 mMguanidine

hydrochloride

(Gua-HCl), an inhibitor for the initiation of

virus-specific

RNA synthesis (7). At 1-h intervals, a portionof the cells

106-80-p

49-.

32-.

I0

6-

B0-49-.

32-p

C 1 2 ;3 4 5 6 7 8 9 n 11

:5:: _

\ .,,t, t wt' *:

L

Fs

...

._

1

i,

-.,

_

^_

s

._

_?=

...:_

!?._ _ ss

,^j;,i,,

'_?,

..

..._

* .

:" ws''''' *_

[

§§

....

Z

??..;X::;.,. r.',,. :

*' '?: w w

*t: ... '. §X

:..,a,''j

*a§}F

-'?'' .:'

iabJl ( - ) ..G.ua-INCI ( + ) !

*_ _

C 1 2 3 4 5 6 7 8 9 10 11

Gua-HCI( HCI(+)

B

4mm.

t

t

7

was pulse-labeled with [35S]methionine for 30 min priorto harvest in order to examine the relative amount ofprotein

synthesis. Poliovirus infectionateither 37or40°Cresulted in the accumulation of labeledpoliovirusproteinsinHeLacells and theinhibition of host cellproteinsynthesis

(Fig.

2Aand B, lanes 1 to 5). When poliovirus-infected cellswere

incu-bated in the presence ofGua-HCl, the virus proteinswere not detected by autoradiography because

virus-specific

RNAsynthesiswasblockedbythe inhibitor

(Fig.

2A andB,

lanes 6 to10). However,agradualshutoff ofhost cellprotein

synthesis was observed, since a small amount of viral products was synthesizedfrom the introduced virion RNA

(8).

InTgSVAcellsincubatedat37°C,synthesisofpoliovirus

proteins as wellasshutoff of host cellproteinsynthesiswere observedasin HeLacells

(Fig.

2C,lanes 1to

5). However,

thesynthesisofpoliovirus proteinswas

suppressed

at

40°C

(Fig. 2D, lanes 1 to 5), and the pattern of labeled proteins

was nearly identical to that observed in Gua-HCl-treated

cells incubatedateither 37or40°C

(Fig.

2C andD,lanes 6to

10). Thedata indicated that the input

poliovirus

RNAwas translated in TgSVA cells at 40°C, resulting in a gradual

shutoff of host protein synthesis as in cells treated with

,.ij,

C 1 2 3 4 5 6 7 8 9 10 11

-0

47.

_ _ 0_

449

GuaE HCI

ii0

I

C 1 2 3 4 5 6 7 8 9 10 Al

D

4..

*

fi..

.*...,

...

-!06 _80

Gua-HCI.

- U ---~~~~~~~.-

-)

Gua-HG

,--.

.(

+)

491-FIG. 2. Proteinsynthesis in HeLa and TgSVA cells infected withpoliovirus.HeLa(AandB)andTgSVA(CandD)cells in 12-wellplates

wereinfectedwithpoliovirusat anMOI of 50 and grownat37°C(AandC)or40°C (BandD)in the presence(lanes6to10)orabsence(lanes 1to5)of 1 mMGua-HCl.The cellswerelabeled with[35S]methioninebya30-minpulsepriortoharvest. At1(lanes1and6),2(lanes2and 7),3(lanes3 and8),4(lanes4and9),and5(lanes5 and10)hafterinfection,extracts werepreparedandanalyzedasdescribed in Materials and Methods.Mock-infectedcellswerelabeled in30-minpulseat5 h after mock infection(lane11).Forlanes11inpanelsBandD, 1/5volume oftheextractfrom mock-infected cellswasloadedontothegel.Thesamplesforlanes Care extractsfrom infected HeLa cells andarethe

sameasthat for lane 5 inpanelA.Sizes(kilodaltons)ofproteinmarkers areindicatedonboth sides.

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HOST FACTORS FOR INITIATION OF POLIOVIRUS RNA SYNTHESIS 3993

A

B

C

10

9

32

i t)

CL

6

c-c11 V

xI

(1)

3 10 50 0 3 10 50 0 3 10 50

Log,PFU per ceN Log.,PFU per cell Log,PFU per cell

FIG. 3. PoliovirusRNAsynthesis. HeLa (A), TgSVA (B), and TgSVB (C) cells in 12-well plateswereinfected with poliovirusatan

MOI of 3, 10,or50 and incubated at37°C (closed circles)or40°C (closed squares).At 2and 2.5 h afterinfection, actinomycinD(final concentrationof5 ,ug/ml)and0.1 1Ci of[3H]uridine, respectively,

were added to the cultures. The cellswerecollected at 6 h after infection, andTCA-insoluble countsweremeasured bya

scintilla-tioncounter.

Gua-HCl. Thus, thetemperature-sensitive steps of poliovi-rusreplication in TgSVA cells appearsnot to reside in the virus-specific protein synthesis. Aslight synthesis of polio-virusproteins observedinTgSVA cellsat40°Cmaybe due

to ahighMOIofpoliovirus (Fig. 2D,lanes 3 and4).

RNA synthesis in TgSVA and TgSVB cells. To examine viral RNA synthesis at the nonpermissive temperature of 40°C, HeLa, TgSVA, and TgSVB cells were infected with poliovirusatMOIs of3,10, and 50. At 6 h after the infection, incorporation of [3H]uridine inpoliovirus RNAs was

mea-sured.IncorporationinTgSVAandTgSVBcells cultured at

40°Cwasmarkedlyreducedcomparedwith thatat37°C(Fig. 3B andC). However,theincorporation occurredatthesame

level in HeLa cellsatbothtemperatureswhenthe cellswere infected with poliovirus at an MOI of 10 (Fig. 3A). When

HeLa cells were infected with a high dose (MOI of50) of virus, the cellsweredamaged,andmanycellsweredetached

fromdishesat6 h after the infection at40°C, resultingin an

apparent difference in radioactivity incorporated at 37 and 40°C. Theseresults indicate that someprocess(es)of polio-virusRNAsynthesisisdefective inTgSVA and TgSVB cells

at 400C.

Toexamine whether the defect is in the initiationorinthe elongationprocessof viral RNAsynthesis,temperatureshift experiments were carried out. TgSVA and TgSVB cells infected with poliovirus were incubated at the permissive temperature of 370C. Portions of the infected TgSVA and TgSVB cellswerethenshiftedtothenonpermissive

temper-ature of 40°C at 3.5 and 4.5 h postinfection, while the remaining cells were kept at 37°C with or without 1 mM

Gua-HCl. Incorporation of [3H]uridine ceased immediately in cellculturesshiftedto400Cat3.5h,and theincorporation patterns were found to be similar to those in cell cultures treated with Gua-HCl at 37°C (Fig. 4). In TgSVA cells treated withGua-HClat4.5hpostinfection,by which timea largenumber ofviralreplication complexesmustbeformed, normal incorporation of [3H]uridine was observed tempo-rally and ceased shortly thereafter(Fig. 4A). This delayin cessation of the incorporation may be due to continued elongation of viral RNA synthesis. Avery similar incorpo-ration pattern was observed in the TgSVB (Fig. 4B) cell

U

6

4

2 A

S S

I)

D

9"2 3 4 5 6 7 * 0 2 3 4 5 6 7 8

nciubation time(h ) hkcbation tine(h )

FIG. 4. Effects ofincubationat40°C andGua-HCIonpoliovirus RNAsynthesis. TgSVA(A)and TgSVB(B)cells in 12-well plates wereinfected withpoliovirusat anMOIof 20 and incubated at37°C (closed circles).At2 h and 2.5 hafterinfection, actinomycinD(final concentration of5 ,ug/ml) and0.1 .Ciof[3H]uridine, respectively, wereadded. At 3.5 and 4.5 hafter infection, portionsofcultures wereshifted to 40°C (closed triangles) orsupplementedwith Gua-HCl(Gua;final concentrationof 1mM) (closedsquares).At3,3.5, 4, 4.5, 5, 6, and 7 h postinfection, cells were collected, and TCA-insoluble counts weremeasuredbyascintillationcounter.

culturesshiftedto40°C at4.5 hpostinfection. The

incorpo-ration patterns obtained after the temperature shiftupwere always very similarto those after the addition of Gua-HCI. Thus,poliovirus RNA synthesis in TgSVA and TgSVB cells at 40°C may be blocked at a step(s) other than the chain

elongation of RNA. These results may suggest that the

step(s) detected in mouse cells has some relation to those affectedbyGua-HCl, although the mechanism for the effect ofGua-HClonthe initiation ofpoliovirusRNAsynthesisis notclearatpresent.

Quantitative RNase protection experiments were then carried out to determine the relative amounts of positive-and negative-strand RNAs present in the infected cells as described in Materials and Methods(Fig. 5).Alargeamount of positive-strand RNA was detected in both TgSVA and TgSVB cells incubatedat37°C(Fig. 5A,lanes4and7),but theamount wasgreatly reducedat40°C (Fig.SA,lanes5 and

8). On the otherhand,similar levels ofpositive-strandRNA weredetected in HeLa cells at 37and 40°C(Fig.5A,lanes1 and 2), whereas the amount of negative-strand RNA

ap-peared not to be reduced at 40°C in HeLa, TgSVA, and TgSVB cells (Fig.SB). Slight differences in the intensities of radioactive bands are observed in Fig. SB. This relative

intensity

pattern

was not reproducible in repeated experi-ments. These results suggest that the positive-strand RNA

synthesis of poliovirus was specificallyblocked in TgSVA andTgSVB cells atthe nonpermissive temperature.

In vitro RNA synthesis. We further examined RNA syn-thesis in vitrobyusingCRCs isolated fromHeLa, TgSVA, and TgSVB cells infected with poliovirusat thepermissive

temperature of37°C.

[a-32P]UTP

radioactivitywas linearly incorporated in the acid-insoluble fraction up to 15 min at both 36°C (Fig. 6A) and 40°C (Fig. 6B). Since poliovirus

RNAsynthesisin the CRCrepresentsmostlytheelongation reaction (10, 28), these results indicate that the elongation

reaction ofpoliovirus RNAsynthesisinvitro isnotaffected bythenonpermissivetemperature.This result iscompatible

with the observations in the temperature shift experiments

shownin Fig. 4.

Poliovirus RNAsynthesisonmembranes isconsideredto VOL.67, 1993

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A. ;2;3i 4 6 7 86 9

pr bFrA,A

p orube 'i 2 3 4 5) 6 7 8 9

;3?7r

Tpr(.De>B

i

FIG. 5. (A and B) RNaseprotection analysis. HeLa (lanes 1 to

3), TgSVA (lanes 4 to 6), and TgSVB (lanes 7 to 9) cells were

infected withpoliovirusatanMOIof 20and incubatedat37°C (lanes 1, 4, and 7) and 40°C (lanes 2, 5, and 8). At 5hafterinfection,RNAs

were extracted from cells and hybridized with 32P-labeled RNA probesasdescribed in Materialsand Methods. RNAs extracted1h

afterinfection (lanes 3, 6,and9)werealsoexamined. The

RNase-resistantproductswereanalyzed in 5% polyacrylamide gelsinthe

presenceof 8Murea. Positive-strand (A) andnegative-strand (B)

RNAswere detected. Rabeled RNA probes (5 x 104cpm

[Ceren-kov]) alonewereusedintheprobe lanes. Exposure periodswere3.5

(A) and 14 (B)h.Sizesareindicated innucleotides.(C) Structures of probesAandB.

beprimed byanucleotidyl protein, VPg-pU(pU), produced

in the virus-specific replication complex thatconsists of a

number of viral and cellular components (34, 35). Accord-ingly, relative amounts of thenucleotidyl proteinproduced in CRCs incubated at 36 and40°Cwerecompared, and the

ratios of relative amountsofVPg-pU(pU)formedat36°Cto those formed at 40°C were then calculated (Fig. 7). Over successiveexperimental trials, formation of thenucleotidyl proteinin theCRCpreparedfromTgSVAcellswas consis-tentlymoreeffectiveat36°Cthan at40°Cexceptforthe first 30 min (Fig. 7B), while the CRC from HeLa cells was equivalent in synthesis of the nucleotidyl protein at both temperatures(Fig. 7A). Thus, thereplication complexfrom

mousecells becomes inactive with respecttothe formation of VPg-pU(pU) more rapidly at 40°C than at 36°C. The results suggest that the functional replication complexes alreadyformed in the CRCs frommousecells areactive in uridylylationofVPgbutthefunction is somehow less stable at40°Cthanat36°Cincomparisonwith theCRC from HeLa cells.

DISCUSSION

Wehaveshownthatmousecellsaregenerally resistantto

poliovirus infection at 40°C. A similar phenomenon was

observed inourpreliminary experiments involving Chinese

hamster ovary cells. It may be possible that resistance to

poliovirusinfectionatanelevated temperatureis character-istic of rodent cells. It should be noted that these cells of mouseand Chinese hamsteroriginsgrowefficientlyat40°C.

Incubation time( nin) Incubation tine(min)

FIG. 6. Poliovirus RNA synthesis in vitro. HeLa (suspension culture) (closed circles), TgSVA (closed triangles), and TgSVB (opensquares) cellswereinfected withpoliovirus atanMOI of 50. Mock-infected HeLa suspension culture cellswerealso used (open

circles).At 6 hafterinfection, CRCswereprepared fromthesecells

as described in Materials and Methods. In vitro synthesis of

poliovirusRNAwascarriedout at36°C (A)or40°C(B).

Thus, the nonpermissive temperature of 40°C may affect onlythe function of the hostfactor(s)in supporting replica-tion.

Furtheranalysis using cells fromtransgenic mice carrying the human PVRgenerevealed thatthe defect residesonlyin the synthesis ofvirus-specific positive-strand RNA. Thus, thenonpermissivetemperaturemayinactivatesomecellular factor(s)involved inpositive-strandRNAsynthesis.Andino et al. (1) reported that the formation ofaribonucleoprotein complexatthe5' end ofpoliovirusRNAincludesahostcell

factor in additiontoviralproteins (3CPrO and3DP")and that disruptionof theformation of theribonucleoprotein complex resulted in selective reduction ofpositive-strand RNA

syn-A

(1) 30 6i0 -90s 120

Vf'coJa:lplJ . _

-¼/Pg p

¼/Pg pU 1U.Ibk

1.0 0.8 1.0

B

0 30 600 90 120

.4 366C

_00

o.- o .:. -40(4

10 25 32 3.0 (36C/400C)

FIG. 7. In vitrouridylylationofVPg. CRCswereisolated from

poliovirus-infected HeLa(A) andTgSVA (B) cells. Reaction mix-turesincludingthe CRCswereincubated at36or40°C.At incuba-tion times of 0, 30, 60, and 90 min, reaction mixtures were

supplemented with [ot-32P]UTP and incubated for 30 min. VPg-pU(pU) synthesized atdifferentincubation timeswasdetectedby immunoprecipitationfollowedbygelelectrophoresisasdescribed in Materials and Methods. Relative amounts of VPg-pU(pU) were

monitoredbyan image analyzer;ratiosof theamountVPg-pU(pU) synthesized at 36°Cto thatsynthesized at 40°C are shown at the bottom.

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HOST FACTORS FOR INITIATION OF POLIOVIRUS RNA SYNTHESIS 3995 thesis. It is therefore of interest to determine whether the

host factor involvedinthe complex is a molecule equivalent to the mouse cell factor(s) proposed here. In any event,

positive- and negative-strand RNA syntheses appear to differ in fundamental initiation mechanisms.

Theeffects of the nonpermissive temperature on poliovi-rus RNA synthesis in mouse cells resemble those of Gua-HCl

(Fig.

4).Thisobservationsuggests that the temperature of40°C inhibitssomestep(s) in the initiation process of RNA

synthesis. Furthermore, poliovirus RNA synthesis

contin-ued to occur at the normal rate immediately after the temperatureshiftat4.5 h postinfection (Fig. 4). Since in vivo

synthesis

of a complete length of poliovirus RNA during virusinfection takes only about 1 min (13), incorporation of

[3H]uridine

in TgSVA cells for approximately 30 min sug-gests that some replication complexes, if not all, are still active in initiating RNA synthesis at the nonpermissive temperature. Thus, the temperature-sensitive step(s) of po-liovirus RNAsynthesismayexist in some step(s) preceding RNA chain initiation, such as uridylylation of VPg and/or formation ofcomplexes required for the uridylylation.

Uridylylation of VPg (Fig. 7B) in the CRC from infected mouse cells was examined for its temperature sensitivity. Thereactionwasnotaffected by the nonpermissive temper-ature of 40°C for the first 30 min. However, prolonged

incubation ofthe CRC at 40°C appeared to result in inacti-vation of the function more rapidly than that at 36°C. The reason for this phenomenon is not clear at present. It is

possible

that formation of the membranous complex

re-quired

foruridylylationis more unstable in CRC from mouse cellsat anelevated temperature than at 36°C in comparison with that from HeLa cells. If so, stabilization of such

complexes

may be one function of the host factor(s) de-scribed here. It is also possible that formation of new membranouscomplexesfor the uridylylation occurs to some extentand is blockedatanelevated temperature in the CRC from mouse cells. In that case, complex formation in vitro may be very inefficient, since the temperature effect was observed only afterprolonged incubation at 40°C.

GuineaandCarrasco(14) reported thatcerulenin, a selec-tive inhibitor oflipid biosynthesis, inhibited positive-strand RNA

synthesis

of poliovirus, suggesting that poliovirus genome

replication

requires continuous synthesis of

phos-pholipids (23).

However, the incorporation of [3H]glycerol into

lipid

fractions of TgSVA and TgSVB cells was not affectedbythe nonpermissive temperature of 40°C (unpub-lished results). Therefore, the defect in the mouse cells appears not tobe in astep(s) oflipid biosynthesis.

Viral and cellular factorspossibly involved in the initiation process ofpoliovirusRNAsynthesis onmembranes are 3AB

(12),

3CPro and3DMIr (1, 28, 38), 2C and 2B (5, 28, 29, 39), and host factors (1, 14). Thus, the replication complex for

poliovirus

RNA synthesisappears to be fairly complicated

(28, 35,

39). Therefore, it may not be easy to elucidate the

precise

interaction among these components involved in the

initiationprocess. The mouse cell factor(s) described here maybe functionally associated with a viral protein(s). To

identify

such a viralprotein(s), a poliovirus mutant that is able to

replicate

efficiently in TgSVA and TgSVB cells at

40°C

is currently being prepared.

ACKNOWLEDGMENTS

We aregratefultoE.Wimmer forproviding anti-VPg antibodies andtoC.Tayaformaintaining poliovirus-sensitive transgenicmice. We are grateful to S. Sugano, H. Toyoda, and H. Shimojo for critical reading of manuscript and to S. Sugano, S. Kuge, H.

Toyoda, H. Iba, and N. Yamaguchi for invaluable advice and discussions. We thank M. Shirato, N. Hashida, Y. Sato, and M. Kobashi fortechnical assistance and E. Suzuki and Y. Kato for help inpreparation of the manuscript.

REFERENCES

1. Andino, R., G. E. Rieckhof, and D. Baltimore. 1990. A func-tional ribonucleoprotein complex forms around the 5' end of poliovirus RNA. Cell 63:369-380.

2. Andrews, N. C., and D. Baltimore. 1986. Purification of a terminal uridyltransferase that acts as host factor in the in vitro poliovirus replicase reaction. Proc. Natl. Acad. Sci. USA 30:745-752.

3. Baltera, R. F., Jr., and D. R.Tershak. 1989. Guanidine-resistant mutants of poliovirus have distinct mutations in peptide 2C. J. Virol. 63:4441-4444.

4. Baron, M. H., and D. Baltimore. 1982. Anti-VPg antibody inhibition of the poliovirus replicase reaction and production of covalent complexes of VPg-related proteins and RNA. Cell 30:745-752.

5. Bienz, K., D. Egger, T. Pfister, and M.Troxler.1990. Structure organization of poliovirus RNA replication is mediated byviral proteins of the P2 genomic region. J. Virol. 64:1156-1163. 6. Bienz, K., D. Egger, T.Pfister,and M.Troxler.1992. Structural

and functional characterization of the poliovirus replication complex. J. Virol. 66:2740-2747.

7. Caliguiri, L. A., and I. Tamm. 1968. Action of guanidine on the replication of poliovirus RNA. Virology 35:408-417.

8. Caliguiri, L. A., and I. Tamm. 1969. Membranous structures associated with translation and transcription of poliovirus RNA. Science 166:885-886.

9. Caliguiri, L., and I. Tamm. 1970. Characterization of poliovirus-specific structures associated with cytoplasmic membranes. Virology 42:112-122.

10. Dorsch-Haesler, K., Y. Yogo, and E. Wimmer. 1975. Replication of picornavirus. I. Evidence from in vitro RNA synthesis that poly(A) of the poliovirus genome is genetically coded. J. Virol. 16:1512-1527.

11. Flanegan, J. B., R. F. Petterson, V. Ambros, N. J. Hewlett, and D. Baltimore. 1977. Covalent linkage of a protein to a defined nucleotide sequence at the 5'-terminus of virion and replicative intermediate RNAs of poliovirus. Proc. Natl. Acad. Sci. USA 74:961-965.

12. Giachetti, C., and B. L. Semler. 1991. Role of a viral membrane polypeptide in strand-specific initiation of poliovirus RNA syn-thesis. J. Virol. 65:2647-2654.

13. Girard, M., D. Baltimore, and J. E. Darnell. 1967. The poliovi-rus replication complex: site for synthesis of poliovipoliovi-rus RNA. J. Mol. Biol. 24:59-74.

14. Guinea, R., and L. Carrasco. 1990. Phospholipid biosynthesis and poliovirus genome replication, two coupled phenomenona. EMBO J. 9:2011-2016.

15. Kajigaya, S., H. Arakawa, S. Kuge, T. Koi, N. Imura, and A. Nomoto. 1985. Isolation and characterization of defective-inter-fering particles of poliovirus Sabin I strain. Virology 142:307-316.

16. Kato, H., H. Sumitomo, P. Pognonec, C.-H. Chen, C. A. Rosen, and R. G. Roeder. 1992. HIV-1 Tat acts as a processivity factor in vitro in conjunction with cellular elongation factors. Genes Dev. 6:655-666.

17. Koike, S., H. Horie,I. Ise, A. Okitsu, M. Yoshida, N.lizuka, K. Takeuchi, T. Takegami, and A. Nomoto. 1990. The poliovirus receptor protein is produced both as membrane-bound and secreted forms. EMBO J. 9:3217-3224.

18. Koike, S., C.Taya, T. Kurata, S. Abe, I. Ise, H. Yonekawa, and A. Nomoto. 1991. Transgenic mice susceptible to poliovirus. Proc. Natl. Acad. Sci. USA 88:951-955.

19. Lawson, M. A., and B. L. Semler. 1990. Picornavirus protein processing enzymes, substrates, and genetic regulation. Curr. Top. Microbiol. Immunol.161:49-87.

20. Lee, Y. F., A. Nomoto, B. M. Detjen, and E. Wimmer. 1977. The genome-linked protein of picornaviruses. I. A protein co-valently linked to poliovirus genome RNA. Proc. Natl. Acad. VOL. 67,1993

on November 9, 2019 by guest

http://jvi.asm.org/

Sci.USA74:59-63.

21. Li, J.-P., and D. Baltimore. 1988. Isolation of poliovirus 2C mutants defective in viral RNAsynthesis. J. Virol. 62:4016-4021.

22. Mendelsohn, C. L., E.Wimmer, and V. R. Racaniello. 1989. Cellularreceptor forpoliovirus: molecularcloning, nucleotide sequence,andexpressionofanewmemberof the immunoglob-ulinsuperfamily. Cell 56:855-865.

23. Mosser,A. G., L. A.Caliguiri, and I. Tamm. 1972. Incorpora-tion oflipidprecursorsintocytoplasmic membranes of poliovi-rus-infectedHeLacells. Virology 37:39-47.

24. Nomoto, A., B.Detjen,R. Pazzatti, and E.Wimmer.1977. The location of the polio genome protein in viral RNAs and its implication for RNAsynthesis. Nature(London) 268:208-213. 25. Pincus, S. E., D.C. Diamond, E. A. Emini, and E.Wimmer. 1986. Guanidine-selected mutants of poliovirus: mapping of point mutations ofpolypeptide2C. J.Virol. 57:638-646. 26. Ransone, L. J., and A. Dasgupta. 1989. Multiple isoelectric

forms of poliovirus RNA-dependent RNA polymerase: evi-dence forphosphorylation. J.Virol. 63:4563-4568.

27. Ren, R., F. Constantini, E. G. Gorgacz,J. J. Lee, and V. R. Racaniello. 1990. Transgenic miceexpressingahuman

poliovi-rusreceptor: a newmodel forpoliomyelitis. Cell 63:353-362. 28. Richards,0. C. andE.Ehrenfeld. 1990.Poliovirus RNA

repli-cation.Curr. Top. Microbiol. Immunol. 161:89-119.

29. Sarnow, P., S. J. Jacobson, and L. Najita. 1990. Poliovirus genetics. Curr. Top. Microbiol. Immunol. 161:155-188. 30. Semler,B.L., C. W.Anderson,R.Hanecak, L. F. Dorner, and

E. Wimmer. 1982. Amembrane-associated precursor to polio-virus VPg identified by immunoprecipitation with antibodies directedagainstasynthetic heptapeptide. Cell 28:405-412. 31. Schneider, R. J., and T.Shenk. 1987. Impactofvirus infection

onhost cell protein synthesis. Annu. Rev. Biochem. 56:317-332.

32. Shiroki, K., M.Hamaguchi,andS.Kawai.1992. Highlyefficient focus formationby Rous sarcomavirusonadenovirustype 12 ElA-transformed rat3Y1 cells.J.Virol. 66:1449-1457. 33. Sonenberg, N. 1987. Regulation of translation by poliovirus.

Adv. VirusRes.33:175-204.

34. Takeda, N., R. J. Kuhn, C.-F. Yang, T. Takegami, and E. Wimmer.1986. Initiation ofpoliovirus plus-strand RNA synthe-sis in a membrane complex of infected HeLa cells. J. Virol. 60:43-53.

35. Takegami, T., R. J. Kuhn, C. W. Anderson, and E. Wimmer. 1983. Membrane-dependenturidylylationofthe genome-linked protein VPg of poliovirus.Proc.Natl. Acad.Sci. USA 80:7447-7451.

36. Takegami, T., B. L.Semler, C. W. Anderson, and E. Wimmer. 1983. Membrane fractions active inpoliovirusRNAreplication contain VPgprecursorpolypeptides. Virology 128:33-47. 37. Tobin, G. J., D. C. Young, and J. B. Flanegan. 1989. Self

catalyzed linkage ofpoliovirus terminal protein VPgto poliovi-rusRNA. Cell 59:511-519.

38. Toyoda, H.,C.-F. Yang, N. Takeda, A.Nomoto,and E. Wim-mer.1987. Analysis of RNAsynthesis oftype 1 poliovirus by usinganin vitro moleculargenetic approach. J. Virol. 61:2816-2822.

39. Wimmer, E., R. Kuhn, S. Pincus, C. F. Yang, H. Toyoda, M.J. H.Nicklin,and N. Takeda.1987.Moleculareventsleading topicornavirusgenomereplication. J. CellSci. Suppl. 7:251-276.

40. Yogo, Y., and E. Wimmer. 1972. Polyadenylic acid at the 3'-terminus ofpoliovirus RNA. Proc. Natl. Acad. Sci. USA 69:1877-1882.

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