Characterization of a temperature-sensitive defect of enterovirus type 70.

0022-538X/82/100098-09$02.00/0

Copyright©1982, American Society for Microbiology

Vol. 44, No. 1

Characterization of

a

Temperature-Sensitive

Defect of

Enterovirus

Type 70

NAOKAZUTAKEDA,*KIKUKOMIYAMURA, REISAKU KONO,ANDSHUDOYAMAZAKI

Central Virus Diagnostic Laboratory, National Institute of Health, Musashimurayama, Tokyo 190-12,Japan

Received 8March 1982/Accepted8 June 1982

The

mechanism of the failure of enterovirus

type70toreplicateata

nonpermis-sive temperature

(39°C)

was

investigated,

and the following resultswereobtained.

(i) Viral RNA synthesis

was not

observed

at

39°C

in LLC-MK2

cells,

in

accordance

withour

previous findings with primary

monkey kidney cells

(Miya-muraet

al.,

Intervirology

9:206-213,

1978). (ii)

Shutoff of hostcell

macromolecu-lar

synthesis by virus infection

was as

efficient

at

39°C

as at a permissive

temperature

(33°C). This inhibitory effect similarly

occurredevenin thepresence

of

guanidine hydrochloride. (iii) Viral protein synthesis

proceeded

in

vivoatthe

nonpermissive

temperature,

and the

rate

of

the protein synthesiswashigherthan

that

at

the

permissive

temperature

under the conditions in which

sufficient viral

mRNA

had been accumulated. This

was

also confirmed

by analyzing the

intracellular proteins synthesized

at

the nonpermissive

temperature by sodium

dodecyl sulfate-polyacrylamide gel electrophoresis, which identified

them as

virus-specific proteins. (iv) When infected cells

were

incubated

at

39°C

and then

transferred

to

33°C, viral RNA synthesis took place

even

in the

presence of

cycloheximide. (v) Furthermore, in experiments performed with

an

in vitro

cell-free

assay system,

viral

polymerase activity

was

found in the membrane-bound

preparation extracted from infected cells which had been incubated

at

39°C

in the

presence or

absence of

guanidine hydrochloride. These results indicate that

early

translation of mRNA proceeds normally

at

the nonpermissive

temperature and

that the

temperature-sensitive defect resides in the transcriptional

stage.

Enterovirus type 70

(EV70), a causative agent

of acute

haemorrhagic

conjunctivitis

(14), has

a

temperature-sensitive

nature,

as

has been shown

by the

fact that the wild strains grow best

at

33°C

and

do not grow

at

39°C

(21,

22).

This

character-istic of

the

virus is

particularly

interesting

be-cause

the

conjunctiva,

the

temperature

of which

is

relatively low,

is the

primary

site for

replica-tion of this virus in natural

infections.

Moreover,

the

virus

seems not to

grow

extensively

in

the

intestinal

tract

and is

not

easily

isolated from it

(29). These

findings

distinguish

the

virus

from

many other

enteroviruses; thus,

EV70 is

consid-ered

as a

naturally

occurring

temperature-sensi-tive virus (22).

In

previous studies,

the

sedimen-tation

profile

of

the RNA

extracted from

EV70-infected

primary monkey

kidney

cells

showed

that

no

virus-specific

RNA

replication

occurred

at

the

nonpermissive

temperature

(23).

In

the

present

study,

we

attempted

to

investi-gate

more

precise

mechanisms

to

regulate

viral

growth at the

nonpermissive

temperature. The

location of

the

site

of

the

temperature-sensitive

lesion in viral

replication

will also be

discussed.

MATERIALSANDMETHODS

Chemicals. Guanidine hydrochloridewas obtained

from Research PlusLaboratories, Inc., Denville, N.J.

Actinomycin D (AMD) was purchased from Makor

ChemicalsLtd.,Jerusalem,Israel. Bovineserum

albu-min fraction V, ATP,CTP,andGTPwerefromSigma

ChemicalCo., St. Louis, Mo.; agarwas from Difco

Laboratories, Detroit, Mich.; andNonidet P-40 was

from Shell ChemicalCo., Houston,Tex.Polyethylene

glycol (Carbowax 6000), trichloroacetic acid,

cyclo-heximide, acrylamide,N,N'-methylenebisacrylamide,

sodiumdodecyl sulfate(SDS),

N,N,N',N'-tetrameth-ylethylenediamine, ammonium persulfate, Tris, and

glycine were purchased from Wako Pure Chemical

Industries, Ltd., Osaka, Japan.Phosphoenolpyruvate

and pyruvate kinasewerepurchasedfromBoehringer

MannheimBiochemicals,Mannheim,West

Germany.

Dithiothreitol wasfrom Seikagaku Kogyo

Co., Ltd.,

Tokyo, Japan.

L-[355]methionine

(1,080 to 1,350 Ci/

mmol), 14C-methylated

protein mixture, and

[5,6-3H]uridine

(40Ci/mmol) wereobtained from the

Ra-diochemicalCentre, Amersham, England.

"4C-amino

acid mixture (50

mCilmatom)

and [3H]UTP (45 Ci/

mmol) were purchased from New England Nuclear

Corp.,Boston, Mass.

Cells, virus,and infection.LLC-MK2 cells,an

estab-98

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VOL.44, 1982

lished cell line derived from rhesus monkey kidney

cells(12), and Bristol-HeLa cellswere propagatedat

37°C in Eagle minimal essentialmedium (MEM)

sup-plementedwith5% calf serum. To prepare seed virus,

cells grown in roller bottles (growth area,

approxi-mately 800cm2)wereinfected with the standard strain

of EV70, J670/71, and incubated at33°C (22). Virus

fluid wasroutinely concentrated by8% polyethylene

glycolin 0.5 MNaCl, and a multiplicity of infection of

about 500 PFU was employed. In all experiments,

virusadsorption was performed for 30 min at 4°C and

another 30 min at37°C. "Hoursafter infection" used

in thefigures refers to the elapsed times after the end

of the adsorption period. MEM without serum was

used for the culture medium after infection unless

otherwise specifically described. Culture bottleswere

immersed in a waterbath, and temperature was

main-tained within an errorof less than +0.05°C.

Infectivity assay. Toquantitatevirusyields in

infect-ed cells, onlycell-associated virus wasmeasured by

plaque assay on confluent monolayers of LLC-MK2

cells. Two or three dishes(Falcon tissue culture dish

3002) were employed for each serial 10-fold virus

dilution. The inoculated cultures were incubated in

MEM containingO.9o agar(Difco) at33°C ina

hu-midified atmosphere of 5%CO2.After 2days, the agar

overlay was removed, the cell monolayers were

stained with0.1% crystal violet in

20%o

ethanol, and

plaques were counted.

Measurements of RNA and protein syntheses. (i) Viral RNAsynthesis.Toestimate therateof viral RNA

synthesis, infected LLC-MK2 cell monolayers (5x106

cells)wereincubated in 5.0 ml of MEMcontaining5%

fetal calfserumand 10 ,ug of AMD per mltoblock host

cellular RNAsynthesis (32). At therequiredtime, the

cells were pulse-labeled with 2 to 10 ,uCi of

[5,6-3H]uridine per0.5 mlfor 30 min. The incorporation

wasterminated byadding 0.5 ml of cold MEM

contain-ing1,OOOx concentrations of unlabeled uridine. After

three cycles of washing with the samemedium, the

cellsweresolubilized with 1.5 mlof cold TNE (0.01 M

Tris[pH7.4], 0.1 MNaCl,0.001 MEDTA)containing

0.5% SDS. Radiolabeled viral RNAwas precipitated

withanequalvolumeof cold10%trichloroacetic acid

onice for 60 min and thenwascollectedonglass fiber

filters(WhatmanGF/C). After drying, the filterswere

submerged in toluene-based fluors, andradioactivity

wascounted with aPackard scintillation

spectropho-tometer.

(il)

Viralprotein synthesis. Tomeasureviralprotein

synthesis, infected HeLa cell monolayers (3 x

106

cells)werecovered with 5.0 mlof MEMcontaining 2.5

,ugof AMD per ml and5%fetal calfserumandwere

incubated in the presence of 2 mMguanidine

hydro-chloride during the first 3 h after infection (1, 30).

During this period, host cellular protein synthesiswas

shutoff by virusinfection, andnewviral RNA

synthe-sis was inhibited. After the removal of guanidine

hydrochloride, the incubation was continued in the

absence ofguanidine hydrochloride to allow newly

synthesized viralRNA toaccumulate in the cells.At

therequired time, the cellswerewashed three times

withMEMwithoutmethionine and labeled with 5 ,Ci

of

["S]methionine

per ml. At the end of 30 minof

pulse-labeling, 1.5 mlof coldMEMcontaining 300x

concentrations of unlabeled methioninewasaddedto

the cultures toterminate theincorporation. The cells

99

wereimmediatelychilled onice andsubjectedtothree

cycles offreezing and thawing. Theradioactivity

in-corporatedintotrichloroaceticacid-insoluble fractions

was collected and measured in a liquid scintillation

counter. ForSDS-polyacrylamidegelelectrophoresis

(SDS-PAGE), infected HeLa cell monolayers were

similarly pulse-labeled for 30 min with 20 ,uCi of

[355]methionine. Inpulse-chaseexperiments, infected

HeLa cells were incubated and pulse-labeled in a

suspension cultureasdescribed in thelegendtoFig.5.

(iii)CellularRNA andprotein syntheses. Toestimate

the rates of cellular RNA and protein syntheses,

infected LLC-MK2 cell monolayers (5 x 106 cells)

were incubated with 5 ml of MEM. At therequired

time, the cells were washed three times with cold

Earle balanced saltsolution andweregiven 0.5 mlof

Earle balanced salt solutioncontaining0.4p.Ciof

[5,6-3H]uridineand 0.1 ,uCi of

"4C-amino

acidmixture. At

the end of15 min ofpulse-labeling, the cells were

quickly chilled inanice water bath, and 1.5 ml of cold

MEMcontaining 1,000x concentrations of unlabeled

uridine was added to terminate the incorporation.

After three cyclesof freezing and thawing,

trichloro-aceticacid-precipitableradioactivitywasdetermined.

Mock-infected control culturesweresimilarly treated,

exceptthatviruswasomitted.

Preparation ofcytoplasmicextracts and acetone

pre-cipitation ofradiolabeled polypeptides. Monolayersor

suspensionsofradiolabeled HeLa cellswerechilled in an icewaterbath and washed three times with cold isotonic salt solution (0.01 M Tris [pH 7.4], 0.14 M

NaCl, 0.015 MMgCl2).The solution containing 0.5%

Nonidet P-40 wasadded, and the cellswereplacedon

ice for 20 minto besolubilized (26). Theextract was

thencentrifuged at 800 xgfor 5 minat4°C to remove

nuclei, and thesupernatantwasstoredat-70°C until

it was to be used. Seven to eight volumes of cold

acetone was added to the radiolabeled cytoplasmic

extracts, and the mixtureswerekeptat -20°C

over-night. The precipitatewascollectedbycentrifugation

at10,000x gfor 20 min, air dried, and then dissolved

inthesample buffer. Thelysatewas boiledfor 3 min

before electrophoresis (5).

SDS-PAGE.Electrophoresis wascarriedoutwitha

discontinuous gel system by the method of Laemmli

(15). For fluorography, gel slabs were fixed in 30%

methanol-10%acetic

acid-60o

water, treatedby the

methodof Bonner and Laskey (3)orimpregnated with

EN3HANCE

(NewEngland NuclearCorp.), dried in

vacuo, and exposed to Sakura X-ray film for an

appropriateperiodat-70°C.

Preparation ofthe viral polymerase complex. The

crude viral replication complexwas prepared by the

method of Yin and Knight (33). Cells were removed by

scrapingfrom the culture flasks and thenwere

collect-edby low-speedcentrifugation(600xg)for5min, and

the cell pelletwasfrozenat-70°C. Approximately4x

108

frozencellsweresuspended in 10 ml of cold buffer

containing0.05 MTris (pH 7.2),0.002MMgCI2, and 0.1 MNaCl. Thecellswereallowedtoswell for 10 min

onice and were ruptured with 15 strokes in a

tight-fittedglassDouncehomogenizer. Unbroken cells and

nucleiwereremovedbycentrifugation at 800x gfor 5

min, and the supernatantfraction was centrifugedat

30,000 xgfor 20 min. The pellet was suspended ina

buffer containing 0.05 M Tris (pH 8.0) and 0.01 M

NaClat afinal concentration of50,ugoftotalprotein

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per 50

RI

and served as the viral polymerase complex.

The protein concentration was determined by the

method of Lowry et al. (18).

Assay for polymerase activity in theceil-freesystem.

A50-ilsample of the polymerase complex was mixed with an equal volume of the reaction mixture contain-ing the followcontain-ing constituents: 100 mM Tris buffer (pH

8.0); 40 mM KCI; 10mM

MgC92;

2.5 mM

phosphoenol-pyruvate; 13 mMdithiothreitol; 10 ,ug of AMD per ml;

50,ugof pyruvate kinase per ml; 0.3 mM each of

ATP,

CTP, and GTP; and 10,Ciof[3H]UTPper ml (cold

UTP was notincluded in the reaction mixture because

the addition of 10 ,uM UTP did not affect the results substantially). The mixtures were incubated at 33°C in

a water bath. After the desired time, the incubation

was terminated by spotting the mixture onto

DEAE-cellulose disks (Whatman DE 81) which were then

soaked for 5 min at room temperature in 5%Na2HPO4.

The wash was repeated four times more inNa2HPO4

and twice each in water and ethanol, and the dried

disks were counted in a toluene-based scintillation fluid.

RESULTS

Effect of temperature shift on viral growth and

viral RNA synthesis. To investigate the effect of

temperature on

EV70 replication, temperature

shift

experiments were carried out. The infected

cells

were

shifted from 33

to

39°C

or

vice

versa

at

various times after

infection, and the virus

yields

and

viral RNA

synthesis

were

determined

(Fig.

1). When the

infected

cells

were

incubated

at

33°C, virus grew

exponentially

after

a

2-h

latent

period, and one cycle

of

viral growth

was

accomplished within 7 h after infection. No viral

replication

occurred

if the

shift-up

was

done

during the first 2 h after

infection,

and

if the

shift-up

was

carried

out

later than 2

h after

infection, some viral growth was observed

dur-ing the next

1 h

after the shift but soon leveled

off

(Fig.

1A). The RNA

synthesis rapidly

de-creased

and ceased within

1

h

after the shift

(Fig.

1C).

In

shift-down

experiments,

the cultures

were

transferred from 39 to 33°C. The viral

growth

immediately

started and reached

a

maxi-mum.

The virus continued

to

grow

at a

similar

rate when

the

temperature

was

shifted down

within

5

h after infection

(Fig. 1B).

When the

shift-down

was

made

within 3 h after

infection,

viral RNA synthesis started at a

similar

rate

after

a 1- to

2-h

lag and reached

a

maximum

6 to

7 h

after

infection,

irrespective of

the

time

of

the

temperature shift

(Fig.

1D).

Shutoff effect on host

cell

macromolecular syn-thesis.

Circumstantial evidence suggests

that the

inhibition of

host

protein and

RNA

syntheses

in

picomavirus-infected

cells

results

from

a

viral

gene-specific

effect(s), although its molecular

mechanism is

not

completely understood (4, 8,

11, 19).

To

examine whether this viral function

could

occur at

the

early stage of

infection,

the

shutoff

effect

on

macromolecular

synthesis

of

infected cells was investigated. EV70-infected

and

mock-infected cells were pulse-labeled with

[3H]uridine and

14C-amino

acid

mixture at 1-h

intervals after infection to monitor cellular RNA

and

protein syntheses. The shutoff effect was

evaluated by the rate of

inhibition estimated as

the

incorporated radioactivities in infected cells,

expressed as a percentage

of

those of

mock-infected cells (Fig. 2). The RNA inhibition was

50 to

60%

at

6 to 7 h

after infection, although the

kinetic curves of the inhibition were not exactly

the same at

39 and at

33°C. The inhibition of

cellular

protein

synthesis

reached 70 to

80%6

at 6

to 7 h

after infection at both temperatures. In

one

experiment

on

cellular

protein

synthesis,

guanidine

hydrochloride, a selective inhibitor of

picornavirus RNA synthesis, was added to the

culture medium after infection

(Fig.

2B).

The

shutoff

effect was similarly observed in the

pres-ence

of the drug. These results suggest that

some

of the early events preceding viral RNA

synthesis

could occur

even at

39°C.

Translation of

viral mRNA

at the

nonpermis-sive

temperature. It is

generally supposed that

one

of the processes

preceding

RNA

replication

in

picornaviruses is early

translation (17). If this

process is

blocked at 39°C, then subsequent viral

replication may not occur.

Therefore,

viral

pro-teins

synthesized

in

vivo

were

monitored

to

examine whether viral RNA

can

function

as

mRNA at

39°C. First, infected cells were

prein-cubated

at

33°C

in the

presence

of

guanidine

hydrochloride to eliminate cellular

protein

syn-thesis, and then the guanidine hydrochloride

was

washed

off, after which the cells

were

incubated in the absence of

guanidine

hydro-chloride

to

accumulate sufficient viral mRNA

and

were

transferred

again

to

39°C.

Viral

protein

synthesis

was

then

compared by measuring

[

5S]methionine

taken

up

by

cells

at

39 and

33°C

(Fig.

3). Viral

protein synthesis proceeded

at

39°C

as

well

as, or even

better

than,

it did

at

33°C

regardless

of the

presence or

absence of

guanidine

hydrochloride (Fig. 3).

The

subse-quent

decrease in

therate

of

protein synthesis

at

39°C

was

probably

due

to

defects in viral

RNA

synthesis

at

this

temperature

since the kinetic

curve

followed the

same

pattern

as

that

ob-served

at

33°C

in

the presence

of

guanidine

hydrochloride.

These results

indicate

that

the

translation of

viral

mRNA

proceeds

at

the

non-permissive

temperature.

To

make

certain

that

the translation

products

formed

at

39°C

were

virus-specific proteins,

extracts

from infected cells

were

analyzed

by

SDS-PAGE. Infected cells

were

incubated

at

33°C for

3

h in

the presence

of

guanidine

hydro-chloride and continued

to

incubate

at

33°C

in the

absence

of

guanidine hydrochloride.

During

that

time,

large

amounts

of viral

mRNA

might

have

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Hours

after Infection

FIG. 1. Effectof temperature shift on viral growth and viral RNAsynthesis. LLC-MK2cellmonolayers(106

cells)grownin a tissue culture flask

(5-cm2

growth area) were infected with theJ670n1strain and incubated for

various times before they were transferred from the permissive temperature (33°C) to the nonpermissive

temperature

(39°C) orvice versa. The temperature was shifted up (A andC)ordown(B and D) at thetimes

indicatedbythearrows.Tomeasurethe virusyield, duplicatecellcultures from eachtemperature groupwere

collectedat1-hintervals,washed threetimes,andsubjectedtothreecyclesoffreezingandthawing.Virusyields

weredeterminedby plaqueassay onLLC-MK2cell monolayersasdescribed in the text (A and B). Viral RNA

synthesiswasmonitoredby 30-min pulse-labelingof infected cells with

[5,6_3H]uridine

atintervals of 0.5or1has

describedinthetext

(C

andD). Thetotalradioactivity incorporatedintoan

acid-precipitable

fraction from

SDS-solubilized cells wasplotted.Eachcircle represents the middle of thelabeling

period.

Symbols:

0,

incubatedat

33'C; 0,

incubatedat39°C.

been accumulated in the

cells,

so

that

a

maximal

rate

of viral

protein synthesis

could be

expected.

The

cultures

were

then

transferred

to

39°C

at4

or5

h

after the removal of

guanidine

hydrochlo-ride.

Subsequently,

virus-specific proteins

were

pulse-labeled for 30

minat

30,

60, and 120

min

after the shift and

were

analyzed by SDS-PAGE

(Fig.

4). Protein bands from both temperatures

were

identical, and almost all cleavage

products

of the viral

proteins

were

detectable

except

the

two

capsid proteins

VP2

and

VP4.

These data

not

only confirm the

occurrence

of

virus-specific

protein synthesis

at

39°C

but

also

suggest

that

some

cleavages

even

proceed

at

the

nonpermis-sive

temperature. The latter

possibility

was

fur-ther

investigated

(see

below).

44,

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0

1

2 3 4 5 6

7

0

1 2

3 4

5

6

7

Hours

after

Infection

FIG. 2. Inhibitory effect of EV70 infection on cellularRNA and protein syntheses. The J670/71

strain-infectedormock-infectedLLC-MK2 cell monolayerswereincubated with MEMateither 33or39°C.Atvarious

timesafterinfection, the cultureswerepulse-labeled for15min withamixture of[5,6-3H]uridine and

"4C-amino

acidmixture. One culturegroupwasincubatedat39°C in thepresenceofguanidinehydrochloride (2 mM). The

radioactivitiesincorporated into cellular RNAandproteins ininfected cellswereplottedaspercentagesof the

correspondingvalues inmock-infectedcells. Each circle indicates the middle of thelabeling period. (A) Cellular

RNAsynthesis at39°C(0)and33°C (X); (B)cellularprotein synthesisat39°C (0), 33°C (0),and39°C inthe

presenceofguanidine hydrochloride (O).

Cleavage of viral polypeptides at the

nonper-missive temperature. To examine the cleavage

process at 39°C in detail, viral proteins synthe-sized at 33°C in HeLa cells were pulse-labeled for 5

min

with [35S]methionine at5 h after the removal of guanidine hydrochloride and then

were chased in the presence of an excess amount of the unlabeled amino acid atboth 33 and39°C for the periods indicatedinthelegend

to Fig. 5. Figure 5 shows comparative gel

pat-terns ofvirus-specific proteins after the chase.

At bothtemperatures, theintensity of

nonstruc-tural viral proteins of high molecular weight larger than NCVP2 decreased gradually. Con-versely, NCVP7c increased obviously at both

temperatures. Since the nonstructural protein NCVP4,acandidate for the viral RNA

polymer-ase, wasalready detectable before the chase, the

cleavage to NCVP4 at 39°C could not be

ex-plained from this experiment.

However,

it seemed that the cleavage to NCVP4 at 39°C proceeded normally because the decrease in NCVP2, a precursor peptide of NCVP4 and

NCVP7c (13,28), and the concomitant increase inNCVP7cwereobvious. Thedifference is clear

inthe band ofa virion structural protein, VP2,

whichwasevidentat33°C butnotat39°C. The absence of VP2at39°C would be explainedbya

lack of RNA synthesis at thistemperature,

be-causedenovoviralRNAsynthesiswasrequired

forits encapsidation into progenyvirus,

result-ing in the cleavage of VPO to VP2and VP4 (2, 25).

In vivo synthesis of viral RNA polymerase at

the nonpermissive temperature. Viral protein synthesis was observed at 39°C if the viral

mRNAhad been accumulatedinthe cells (Fig. 4). This result suggests that input virion RNA

canalsofunctionasmRNAat39°Csothat viral

RNApolymerase is produced at this tempera-ture. Intracellular synthesis of viralpolymerase

at the nonpermissive temperature during the

first 2 hafter infection was monitoredby

mea-suring the subsequent synthesis of viral RNA

after the shift-down in infected cells in the presenceof

cycloheximide,

which should block

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TEMPERATURE SENSITIVITY OF EV70 103

10

0 1 2 3 4 5 Hirs after Remval of6Oaaidi,e-Il

FIG. 3. Translation of viral mRNA. HeLa cell

monolayerswereinfected with theJ670M1 strain and

were incubated in the presence ofguanidine

hydro-chloride (2 mM) at33°C for 3 h. The five groups of

cultures weresubsequentlywashedwellto removethe

guanidine hydrochloride (0 h after the removal of

guanidinehydrochloride) andincubated at33°C with-out guanidine hydrochloride, except for group E, which was similarly treated but was further incubated

with guanidinehydrochloride after being washed and

which served as a control. After 2 h (arrow), each

group received thefollowingtreatment: A,shifted up

to 39°C and incubated without guanidine

hydrochlo-ride; B, incubated at33°C without guanidine

hydro-chloride; C, guanidine hydrochloride addedagain to

the culture medium and incubated at 39°C; D,

guani-dinehydrochlorideaddedagaintothe culturemedium

and incubated at 33°C; E, incubated at 33°C in the

presence ofguanidine hydrochloridefrom 0 to 5 h. At

various times indicated in the figure, the cultures were

pulse-labeledwith[35S]methioninefor 30min,and the

radioactivity incorporated into an acid-precipitable

fraction was measured as described in the text.

de

novo

synthesis

of viral

polymerase

at

33°C

(Fig.

6). Immediate viral RNA

synthesis

was

observed if

the temperature

was

shifted down

at

2

h after

infection,

even

in the

presence

of

cycloheximide,

but

no

viral

RNA

synthesis

was

observed if the

temperature was

maintained

at

39°C.

This indicates that the viral RNA

polymer-ase was

synthesized

at

39°C and could function

at

33°C but

not at

39°C.

In

vitro

assay of viral polymerase

activity

of

infected cells. The

synthesis of viral polymerase

in

the early

stage

of infection

at

the

nonpermis-sive

temperature was

demonstrated in

vivo in

the

previous

experiment

(Fig.

6). To

confirm it

further,

we attempted to use a cell-free assay

system

in which the viral

polymerase

activity in

various

cellextracts was compared in vitro by

the

kinetic

curves

of

incorporation

of

[3H]UMP

into

RNA

products. The

J670/71

virus-infected

cells

were

incubated

for

1.5 h at

the

permissive

or

the

nonpermissive

temperature

before they

were

harvested

to

prepare

the crude

replication

complex. Guanidine hydrochloride

was

added

to

some

cultures

to

inhibit de

novo

synthesis of

viral

mRNA so thatthere might exist only

input

virion RNA which can function as mRNA to

translate viral polymerase.

A

warmth-adapted

clone, which had been

artificially induced by serial passages of the prototypeJ670/71 strain at an elevated tempera-ture to grow at 39°C, was used as a positive control for theproduction of viral polymerase at

390C.

Thereplication complex extracted from cells infected with the J670/71 strain at 330C (Fig.

7A)

and that from cells infected with the

warmth-adapted clone

at

390C (Fig. 7C)

revealed

compa-rable kinetics of polymerase

activity.

It should

be noted that the

polymerase activity

of the complex extracted from cells

similarly

infected but incubated in the presence of guanidine

hy-A

_{_}

B

C

D E

F G H I

J

1-_.

___

_. 49_

~VP]

VP

2

VP4

FIG. 4. SDS-PAGE of virus-specific proteins

formed in EV70-infected cells at the nonpermissive

temperature. Infected HeLa cell monolayers were

incubated at33°Cinthe presence ofguanidine

hydro-chloride for 3 h, and the cultures were washed to

removeguanidinehydrochloride and were again

incu-bated with MEM withoutguanidine hydrochloride at

33°C.Then, some groups were shifted up to39°Cat 4h

(lanes E, F, andG)and 5 h(lanes H andI)after the

removalofguanidine hydrochloride. The cultures of

each temperature group werepulse-labeledfor 30 min

with [35S]methionine at intervals of 0.5 or 1 h. The

cytoplasmic extracts were analyzed forvirus-specific

proteins by SDS-PAGE. Electrophoresis was

per-formed onresolving gel slabs (170 by 150 by 1.2 mm)

for 6 h at 140 V. The gels were fluorographed as

described inthe text. Lanes A to D, Incubated and

pulse-labeled at 33°C at 4, 5, 6, and 7 h after the

removal ofguanidine hydrochloride; lanes E to G,

shiftedup to39°C at 4 h after the removal of guanidine

hydrochloride and pulse-labeled at 30, 60, and 120 min

after the shift; lanes H to I, shifted up to 39°C at 5 h

after the removal of guanidine hydrochloride and

pulse-labeled at 30 and 60 min after the shift; lane J,

"4C-labeled

virions oftheJ670/71 strain.

VOL.44,1982

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[image:6.496.52.194.56.197.2] [image:6.496.261.436.258.449.2]

A B C D E F G H J K L M K .1 ...

92.51( Ti

69K_m _ il_f __ -"

46K_W

VDt

VP3

14.3O

FIG. 5. Pulse-chase analysis of the formation of

EV70 viral proteins. HeLacells (3 x 10'cells) were

mixed with 1.0 ml of a suspension of theJ670/71 strain

andwereincubated at 4°C for 30 min, then at 37°C for

30 min, for virus adsorption. The cell suspensions

werecentrifuged to remove unadsorbed virus, and the

cellpellet was suspended in 30 ml of MEM containing

AMD. Incubation wasperformed for 3 h in the

pres-enceof guanidine hydrochloride and another 5 h in the

absence ofguanidine hydrochloride with gentle

stir-ring. The cells were washed by centrifugation with

MEM without methionine, and precipitated cells were

then pulse-labeledfor 5 min in 1.0 ml of MEM

contain-ing100

p.Ci

of

[(5SI

methionine. Afterwards,thecells

were chilled immediately in an ice water bath and

dilutedwith 10 ml of MEMcontaining300x

concen-trationsof unlabeled methionine. At this time, 1.0 ml

of thecell suspension was takenasaprechase sample.

Half (5.0 ml) of the remaining cell suspension was

transferred to39°C,and the other half was held at 33°C

and then both cell suspensionswerechased.

Periodi-cally, 1.0-ml samples were removed to prepare the

cytoplasmic extracts which were then analyzed by

SDS-PAGE and fluorographed as described in the

legend to Fig.4. LaneA, Molecular weight marker

proteinsat200,000 (200K,myosin),92.5K

(phosphor-ylase b),69K (bovine serum albumin), 46K

(ovalbu-min), 30K (carbonic anhydrase), and 14.3K

(lyso-zyme); lane B, 1C-labeled virions of the J670/71

strain; lane C, sample before chase; lanes D to H,

samples chasedat33°C for 10, 30, 60, 90, and 120 min,

respectively; lanes ItoM,samples chasedat39°Cfor

10, 30, 60, 90, and120min, respectively.

that

produced in the

infected cells which

were

incubated

at

the

permissive

temperature

in the

presence

of

guanidine hydrochloride,

an

inhibi-tor

of viral mRNA

synthesis (Fig. 7B). The

results indicate that the

translation proceeds

normally

at

nonpermissive

temperature

by

using

input virion

RNA as

mRNA.

DISCUSSION

EV70 is

a

pathogenic, naturally

occurring,

temperature-sensitive

virus. Since

many

fresh

isolates from the

eyes

of

acute

haemorrhagic

conjunctivitis patients

have

been shown

to pos-sess

the

temperature-sensitive

nature as

their

common

characteristic,

this

property

has

been

considered

to

be

associated

with

a process

through

which the virus

acquires pathogenicity

for human

conjunctivae

(22).

Therefore,

molecu-lar

biological studies of the intracellular

events

associated

with the

temperature-sensitive

defect

in

viral

replication will

provide

an

interesting

experimental model

to

elucidate

a

mechanism

for the

appearance

of

naturally

occurring

tem-perature-sensitive

virus

or a

mechanism

by

which viruses acquire the

ability

to

replicate

better

at

low

temperatures

and become

patho-genic

in

low-temperature

tissues.

In

picomavirus

infection,

viral RNA

synthesis

is

preceded

by

serial

processes

of the

early

replication

steps,

i.e.,

viral

adsorption

to

cells,

subsequent penetration

and

uncoating of

the

virus, and early

translation

of its

input

RNA

(17).

Our

present

study,

as

well

as

the data

A

x

C-.. .\\ B

O

...O...

0 2 3 4 5 6 7

1 2345617

drochloride was low but clearly demonstrated

by the increasing incorporation of [3H]UMP into RNAasafunction of thereaction time in vitro.

The data indicate thatourin vitro assaysystem

is sensitive enough to detect such amounts of viralpolymeraseas weresynthesized in the cells

by utilizing only input virion RNAas the

tem-plate. Thus, the replication complex prepared from cells which had been infected with the J670/71 strain and incubated at the

nonpermis-sive temperature was found to contain viral

polymerase activity inanamountequivalentto

Hours after Infection

FIG. 6. Detectionofvirus-specificRNA

polymer-ase activity by the use ofan in vivo assay system.

LLC-MK2 cell monolayers were infected with the

J670/71 strain and incubated at 39°C. At 2 h after

infection(arrow),somecultureswereshifted downto

33°Cin the absence(A)orpresence(B) of

cyclohexi-mide (10 ,ug/ml) and were pulse-labeled with

[5,6-3H]uridine

for 30 minat1-hintervals. Theremaining

cultures were incubated and labeled at 39°C in the

absence ofcycloheximide(C).Theradioactivity

incor-poratedintoanacid-precipitablefractionwascounted

byscintillation spectrometry.

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[image:7.496.60.254.74.245.2] [image:7.496.307.407.434.565.2]

TEMPERATURE SENSITIVITY OF EV70 105

I

C

G_

c=4

0

30 60

0

30 60

0

30

60

INCORPORATION

TIME

(

MIN

FIG. 7. Detection of RNApolymerase activity ina

cell-freesystemin thecrudereplication complex from

cellsincubated for 1.5 h after infection. LLC-MK2 cell

monolayers grown in flasks were infected with the

J670/71 strainorwithitswarmth-adaptedmutant(see

text)atamultiplicity of infection of approximately 40.

After virus adsorption for 1 h, half of the cultures

received 20 mlof MEMcontaining5 sLg of AMDper

ml, and the other half received MEM containing the

sameconcentration of AMD and 200,ug ofguanidine

hydrochloride permland all cultures wereincubated

at the temperatures described below. At 1.5 h after

infection, the cells were collected and disrupted to

makethereplication complexatafinalconcentration

of 50 ,ug of totalprotein per50 ±l. Forassaying the

polymerase activity, each100-,ulreaction mixture

con-taining 50 ,u ofreplication complexwasincubatedat

33°Cin awaterbath. Samples weretakenat15-min

intervals, andthe totalradioactivityincorporatedinto

the RNAproductwasdeterminedasdescribedinthe

text. The replication complexes wereprepared from

cells treated under various conditions: infected with

the J670/71strain andincubatedat33°C (A)or39°C (B)

orinfected with thewarmth-adapted clone and

incu-batedat39°C (C). Mock-infected culture controls for

eachtemperaturewereincludedin(A) and (B). Graphs

represent preparations from cells incubated in the presence (0)orabsence (0) ofguanidine

hydrochlo-ride, made with virus-infected(-)ormock-infected

(. )cells.

previously reported (22), confirms that the early

events,

including uncoating, proceed normally

in

EV70 infection

at

nonpermissive

tempera-tures. It has been demonstrated by both the

immediate

rise in the intracellular synthesis of

viral

RNA after the shift-down to

33°C

during

the

course

of viral infection

at

39°C

and more

evidently

by the shutoff of host cell

macromo-lecular

synthesis by viral infectionatthe

nonper-missive

temperaturesinceanexpression of viral

mRNA

function is prerequisite

for the shutoff of

host cell

macromolecular synthesis by

picorna-virus infection

(19).

Furthermore, this

study indicates that

EV70

virion RNA can function as mRNA in vivo at

nonpermissive

temperatures to

synthesize

pre-cursor

proteins,

which

are

subsequently cleaved

into

specific components,

including

an RNA

polymerase.

Despite

intracellular

synthesis

of

the RNA

polymerase, no RNA

synthesis

pro-ceeded unless the

incubation temperature

was

shifted down. Thus, we may reasonably

con-clude that the

early

translation of viral RNA

normally proceeds in

EV70-infected cells at

non-permissive

temperatures and that the

tempera-ture-sensitive

defect is located at the

transcrip-tional level.

Three

possibilities

will be

considered for

the

mechanism to inhibit RNA

synthesis

at

the

transcriptional level. The first

possibility

is that

viral RNA

polymerase could be inactive

intra-cellularly

at

nonpermissive

temperatures. To

investigate

this

possibility,

we

tested the in vitro

polymerase

activity of

the

prototype

strain

J670/

71 at

39°C

and found that it

was

much

lower at

this temperature (data not

shown).

Thus, it

seemed

quite possible that temperature

sensitiv-ity of the prototype strain was due to its

heat-labile polymerase. However, we could not

con-clude that the

temperature-sensitive

defect

at

the

transcriptional level could be

simply

ex-plained

by an in vitro heat-labile property

of

the

replication complex because the

polymerase

complex of

our

warmth-adapted

mutant

also

functioned better at

33°C

than

at

39°C

as

far as it

was

tested in vitro (data not shown). The second

alternative

is that a

hypothetical constituent(s)

of the

replication

complex, other than RNA

polymerase, which

would

participate

in

tran-scription,

might

be

temperature sensitive.

Third,

virion RNA could not function as the template

for the

synthesis

of the progeny

RNA.

It

has

been recently

reported

that

an

isolated

polymerase

of

poliovirus

requires

an

oligouridy-lic acid

sequence as

its

primer

(10, 31) and that

VPg at the 5' end of

picornavirus

RNA

may

play

an

important role in the viral

replication

(9, 16,

24, 27). It has also been suggested that a host

cell

factor(s)

may be

involved in the process of

picornavirus

RNA

replication (6, 7, 20) and act

at

the

initiation step

of poliovirus

RNA

replica-tion

(6). Eventually, a

conclusive

answer

for

the

exact

mechanism of the

transcriptional defect in

EV70 infection will

need

more detailed

analyti-cal

studies on

the

structure

and function

of

viral

as

well

as

cellular

components which

participate

in the

process of RNA

replication.

ACKNOWLEDGMENTS

WearegratefultoRobert R.Wagner,University of Virgin-ia,for his critical reading of the manuscript and for useful discussion.

This work was supported in part by a grant-in-aid for scientific research from theMinistry of Education, Science, andCulture,Japan.

VOL.44,1982

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LITERATURE CITED

1. Adler, R., and D. R. Tershak. 1974. Temperature-sensi-tive defect of type 2 poliovirus. Virology 58:209-218. 2. Baltimore, D., M. Girard, and J. E. Darnell. 1966.

As-pectsof thesynthesis of poliovirus RNA and the forma-tionofvirus particles. Virology 29:179-189.

3. Bonner, W. M., and R. A. Laskey. 1974. A film detection methodfortritium-labeled proteins and nucleic acids in polyacrylamide gels. Eur. J. Biochem. 46:83-88. 4. Crawford, N.,A. Fire,M.Samuel,P.A. Sharp, andD.

Baltimore.1981. Inhibition of transcription factor activity by poliovirus. Cell 27:555-561.

5. Cross, R. K., and B. N. Fields. 1976. Reovirus-specific polypeptides: analysis using discontinuous gel electropho-resis. J.Virol. 19:162-173.

6. Dasgupta,A., P. Zabel, and D.Baltimore. 1980. Depen-dence of theactivity of the poliovirus replicase on a host cellprotein.Cell19:423-429.

7. Dmitrieva, T. M., M. V. Schcheglova, and V. I. Agol. 1979.Inhibition of activityof encephalomyocarditis virus-inducedRNApolymerase by antibodies against cellular components.Virology92:271-277.

8. Ehrenfeld, E. 1982.Poliovirus-induced inhibition of host-cellproteinsynthesis. Cell 28:435436.

9. Flanegan, J.B., R. F. Pettersson,V.Ambros, M. J. Hew-lett, and D.Baltimore.1977.Covalentlinkage of a protein to adefined nucleotide sequence at the 5'-terminus of virion and replicative intermediate RNAs of poliovirus. Proc.Natl. Acad. Sci. U.S.A. 74:961-965.

10. Flanegan, J. B.,and T. A.VanDyke. 1979. Isolation of a soluble andtemplate-dependentpoliovirusRNA polymer-asethatcopiesvirion RNA in vitro. J. Virol. 32:155-161. 11. Flores-Otero, G., C. Fernandez-Tomas, andP. Gariglio-Vidal.1982. DNA-bound RNApolymerases during polio-virus infection: reduction in the number of form II enzyme molecules. Virology116:619-628.

12. Hambling, M. H.,and P. M.Davis.1965.Susceptibilityof the LLC-MK2 line of monkey kidney cells to human enteroviruses.J.Hyg. 63:169-174.

13. Kitamura, N., B. L.Semler,P.G.Rothberg, G. R. Lar-seu, C.J. Adler,A.J.Dorner,E. A.Emini,R.Hanecak, J. J. Lee, S. van der Werf, C.W. Anderson, and E. Wimmer. 1981. Primarystructure, geneorganizationand polypeptideexpressionofpoliovirusRNA.Nature (Lon-don) 291:547-553.

14.Kono, R.,A.Sasagawa,K.Ishii,S.Sugiura,M.Ochi,H. Matsumiya,Y.Uchida,K.Kameyama,M.Kaneko,and N. Sakurai. 1972. Pandemic ofnewtype ofconjunctivitis. Lancet i:1191-1194.

15.Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of the head ofbacteriophage T4. Nature(London)227:680-685.

16. Lee,Y.F.,A. Nomoto, B. M. Detjen, and E. Wimmer. 1977. A proteincovalentlylinkedtopoliovirusgenome RNA. Proc. Natl. Acad. Sci. U.S.A. 74:59-63. 17. Levintow, L. 1974. Theproductionofpicornaviruses,p.

109-169. In H.Fraenkel-Conratand R. R.Wagner(ed.), Comprehensive virology, vol. 2. Plenum Publishing Corp.,NewYork.

18. Lowry,0.H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951.Protein measurement with the Folin phenol reagent.J.Biol. Chem. 193:265-275.

19. Lucas-Lenard, J. M. 1979. Inhibition of cellular protein synthesis after virus-infection, p. 73-99. In R. Perz-Bercoff (ed.), The molecularbiology of picornaviruses. Plenum PublishingCorp., New York.

20. Lundquist, R. E., and J. V. Maizel, Jr. 1978. In vitro regulation of the poliovirus RNApolymerase. Virology 89:484-493.

21. Miyamura, K., A. Sasagawa, E. Tajiri, and R. Kono. 1976. Growth characteristics of acutehemorrhagic conjunctivi-tis(AHC) virus in monkey kidney cells.II. Temperature sensitivity of the isolates obtained at various epidemic areas.Intervirology 7:192-200.

22. Miyamura, K., S. Yamazaki, E. Tajirl, and R. Kono. 1974. Growth characteristics ofacutehemorrhagic conjunctivi-tis (AHC) virus in monkey kidney cells. I. Effect of temperature onviralgrowth.Intervirology 4:279-286. 23. Miyamura, K., S. Yamazaki, E. Tajiri, and R. Kono. 1978.

Growthcharacteristics of acute hemorrhagic conjunctivi-tis(AHC) virus in monkey kidney cells. III. ViralRNA synthesisatpermissive and nonpermissivetemperatures. Intervirology 9:206-213.

24. Nomoto, A., B. Detjen, R. Pozzatti, and E.Wimner.1977. Thelocation of thepolio genome protein in viral RNAs andits implication forRNAsynthesis. Nature(London) 268:208-213.

25. Penman, S., Y. Becker, and J.E. Darnell. 1964. A cyto-plasmic structure involved in the synthesis and assembly ofpoliovirus components. J. Mol. Biol. 8:541-555. 26. Penman, S., H. Greenberg, and M. Willems. 1969.

Prepa-ration ofpolyribosomes from cells grown in tissue culture, p.49-58. In K. Habel and N.P. Salzman(ed.), Funda-mentaltechniques invirology. Academic Press, Inc., New York.

27. Pettersson, R. F., V. Ambros, and D. Baltimore. 1978. Identification ofa protein linkedto nascent poliovirus RNAand to thepolyuridylic acid ofnegative-strandRNA. J.Virol. 27:357-365.

28. Rueckert, R. R., T. J. Matthews, 0.M.Kew, M.Pollansh, C. McLean, andD. Omilianowski. 1979. Synthesis and processing of picornaviral polyprotein, p. 113-125.InR. Perz-Bercoff (ed.), The molecularbiologyof picomavirus-es, PlenumPublishing Corp., NewYork.

29. Sasagawa, A.,R.Kono,andK. Konno.1976. Laboratory-acquired infection of the eye with AHC virus. Jpn. J. Med.Sci. Biol. 29:95-97.

30. Tershak,D.R.,W.Mitchell,andB. D.Gerfinkle. 1972. Effect ofguanidineonthegrowthof LScpoliovirus.Can. J.Microbiol. 18:747-755.

31. VanDyke,T.A.,andJ.B.Flanegan. 1980.Identification ofpolioviruspolypeptide p63as asolubleRNA-dependent RNApolymerase.J. Virol. 35:732-740.

32. Yamazaki, S.,K.Natori,andR. Kono.1974. Purification and biophysical properties of acute haemorrhagic con-junctivitisvirus. J. Virol. 14:1357-1360.

33. Yin,F.H.,and E.Knight, Jr.1972. Invivoand in vitro synthesisofhumanrhinovirustype2ribonucleicacid. J. Virol. 10:93-98.

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