Coronavirus JHM: Cell-Free Synthesis of Structural Protein p60

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Coronavirus

JHM:

Cell-Free

Synthesis of Structural

Protein

p60

STUART G.SIDDELL,* HELMUTWEGE,ANDREABARTHEL, ANDVOLKERTER MEULEN

Institutfuir Virologie und Immunbiologie derUniversitatWurzburg,8700Wurzburg, West Germany

Sac(-) cells infected with murine coronavirus strain JHM shut off host

cell

protein synthesis and synthesized polypeptideswith molecular weights of 150,000,

60,000,and23,000. The60,000- and 23,000-molecular-weight polypeptides

comi-grated with virion structural proteins p60 and p23, and the 60,000-molecular-weightproteinwasidentifiedasp60 by tryptic peptide fingerprinting. Polyade-nylate-containing RNA

[poly(A)

RNA] extracted from the cytoplasmof infected cellsdirected thesynthesis of both 60,000- and 23,000-molecular-weight polypep-tides in

messenger-dependent

cell-free systemsderived frommouse

L-cells

and rabbitreticulocytes. The reticulocytesystemalsosynthesized a 120,000-molecu-lar-weight polypeptide that was specifically immunoprecipitated by antiserum raised against JHM virions. The identity ofthe 60,000- and 23,000-molecular-weight in vitro products was established by comigration with virion proteins, immunoprecipitation, andinthecaseofp60, tryptic peptide fingerprinting. The cytoplasmic poly(A) RNAs whichencoded p60 andp23sedimented in sucrose-formamide gradients at 17S and 19S, respectively, and were clearly separable. These RNAs were among the major poly(A) RNA species synthesized in the cytoplasm of actinomycin D-treated cells late in infection, and the in vitro translation of size-fractionated RNA released from polysomes confirmed that

theyrepresent

physiological

mRNA's. These resultssuggest that theexpression

of the coronavirus JHMgenomeinvolvesmorethanonesubgenomic mRNA.

Coronaviruses cause a

variety

ofdiseases in both animals and humans.

They

areassociated withrespiratory and enteric diseases inhumans, bronchitis in

birds,

transmissible

gastroenteritis

and

encephalitis

in

pigs,

and

demyelinating

en-cephalitis

and

hepatitis

inrodents

(11, 20;

J. A. Robb and C. W.

Bond,

inH. Fraenkel-Conrat and R. R.Wagner,

ed., Comprehensive

Virology,

vol. 14, inpress).

The

neurotropic

murine coronavirus JHM (JHMV) induces a

variety

of central nervous

system disorders in mice and rats and canbe

usedas amodel for virus-induced

demyelination

(4, 8, 12, 13). JHM virions are

pleomorphic

spherical

particles

about 100 nmi in diameter

with characteristic club-shaped surface

projec-tions (11).The virusmatures,asdo other

coro-naviruses, by budding from internal cellular

membranes and not from the

plasma

mem-branes (11). JHM virions have been

character-izedrecently.Thegenome isaninfectious

single-stranded RNA withamolecular

weight

ofabout

5.4 x

10c'

(7, 21). The RNA is a continuous

strand, and atleast one-third of the molecules

contain

polyadenylate

[poly(A)]

sequences

(7,

21).The vironcontains six

major proteins,

four

of which are

glycosylated (2, 22).

Subviral

par-ticles produced bytreatment of the virus with

NonidetP-40include RNAand oneof the

major

structural

proteins

of the

virus,

p60.

Incontrast,thereis verylittleinformationon

the

replication

of JHMV

(2,

16; Robb and

Bond,

in press). We

have,

therefore,

initiated studies onthe

synthesis

ofviral RNAs and

proteins

in infected tissue culture cells. In this paper we

reporton

expe

imtwhich demonstrate

the in

vitro

translation

and

size-oT-the

-m7NA

which

encodes the

major

nuCle`capsicFprotei

of

t

virus,60. Th m pxneriments also

provi!ke

miiformaion

on the synthesisof another

viriol

protein

J.1

ancd

mciicate tne mvoivement ot

more one

cation

7rategy

of

coronaviruses.

MATERIALS AND METHODS

Cells and virus. Sac(-) cells are nermant

murine

M21oney

Iar

omc

are

grown at 37°C either m suspension with the Joklik modificationofminimal

een'tialmedYum

containing 5%fetal bovineserum(Spinnermedium)or as a

mono-biu m ). We obtained JHMV

from L. P.Weiner, JohnsHop University,

Balti-more,Md.,inanhomogenateofsucklingmousebrain.

The virus waspropagated twice in suckling mouse

brain, isolated, plaque purified four times on L929

cells,andthen grown inSac(-)cellsatlow

multiplic-10

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mRNA

itiesofinfection (MOI).Virusstockswereprepared by infecting monolayersofSac(-) cells at an MOI of 0.2

50% tissue culture infectivedose(TCID50) percelland

harvesting the medium20 hlaterwhen 75 to 100%of thecells hadformedsyncytia.Thetiterof virus in the

clarifiedmedium was between107and108TCID5o/ml. Radioactive labelingandpurificationofvirus.

Virus diluted with Pucksaline wasabsorbed in

sus-pension for20minat37°C to 5 x108Sac(-) cellsat an MOI of 0.2 TCID5o/cell. Unabsorbed virus was

removed bycentrifuging the celsapnsth

m Spinnermeu . Mismeum sharvested20 h

latiina

claiied

Sy

cen-t-rifu

aition,and t eviu a

purifiedasdescnibed1byWegeetal.(22).Purified virus

forgelelectrophoresiswasstoredat-20°Cin 50 mM

Tris-hydrochloride, pH 6.8, containing2% sodium do-decyl sulfate (SDS)and 10%glycerol.

Virus labeledwithl3iS]methioninewasprepared by

comommg

the purinication ofunlabeled virus as

de-scied above with clarified medium taken from

mono-layerculturesfinfected 12-hpreviouslywithJHMVat anMOI of6TUFL½,o/cell.Atbetween 7andfn10har

infection,themiueumonthemonolaversywa

renlacId

bymedium

containing7Ttheu-sual

amount of

methi-nie2%di-alyzecatetalbovine serum,and4~~Q

L J

tr_Mnine

(SZJ20am

ucer,

Braun-schweig, WestGermany) per ml.

Labeling of infected-cellproteins. (i) Pulse-la-beling. JHMV, ormedium in the case of mock-in-MTcte8-cells, was absorbed to monolayers of Sac(-) cellsgrownin 5-cmpetri dishes for45minat37°C at

an MOIof6 TCID5o/cell. Unabsorbed virus was re-movedby washingwithmedium,and theinfected or mock-infected cells were maintained in medium at 370C. At the times indicated in Fig. 1, the mediumwas

replacedby mediumwith l/otheusualconcentration

ofeIMiitd aiiiino acidsandwithoutserum(ioamino ai TMeTiu).AlTer 15min,thi mediumwasreplaced by l/io amino acid medium conta 0 aCi oa

mixtureof15'H-IaneeaIMmoacids(TRK440;

Amer-sham?uchler) and alvzed fetal bovine serum per

ml.

After

another 15mi hi mium wasremoved.

and the monolayer was with ice-cold

phosphate-buffered saline and at room

temperaurewit .lo 50lmMrTris-hdchloride,

pH

6.8,' tamn

-D,4M

ra 2

Iercaptethanolperpetrih. e ysate waspassed throughasyringeneedle10times, heated to 100°C for

2min, andstoredat-20°C.

(ii) Tptic peptide fnp . Cells were

infected ofinfection,

the medium wasreplaced by ediumwithout

methip-onmne or serum (minus methionine medium),

apd

15

m a this mediumwasreplaced withminus me-thionine medium contaCining 100 ,iCi of

rf.S]methio-nm e -=lad izeaterul

Antisera andimmunoprecipitation.Antiserum

against JHMV (anti-JHM serum) was produced in

ranuits- y three intramuscular injectionso-Fpurified

virusat4-weeKintervais.Antiserumaguinst

vMalTpro-temp60(anti-pWserurM wasprepared by two

intra-pen=toneal

to e ya-myiam`de

get cp

cc-patnogen-free NMRI mice. Preimmune sera were taken from

anflwr cTwere shown to lack JHM-neutralizing antibodies. All sera were adsorbed to a powdered acetoneextract-ofuninfected Sac(-) cells before use.

Immunoprecipttion of-the prnfuets of in vitro translationwasperformed essentiallyas

des-cribed

y'

Pau_ca etal. (14),usinga1/100 utionof antiserum orpreixnmune serumand adso- tinr tona uispPnniq

f oteinAbearingStaphylococcusaureus(5

rearationofRNA.-Poly RNAwas isolated fromthe cytoplasm of JHMV-infected or uninfected

cellsessentially as described by Wheeler et al. (25). Briefly, 5 x 108 Sac(-) cells in suspension were

in-fected or mockinfMeddle uescnBec above for virus

paMktion.

After 20 h of infection, the cells were

relleted andw a he-r

me,pH7.4. The cellpeletwassuspendedin 7volumes

of ice-colc 2U-mM Tris-hydrochloride (pH 7.5)-100

mMNaCI5mMgMgCl2.Resuspens in d cells

resulted i-ncel

lysis,

and the lysatewas immediatel

centrifuged at- 1 50/ x g for a min at 4"C. With

c _ _ h

uninfected cells, it wasnecessary to add TritonX-100 to a final concentration of 0.1% to lyse the cells.

Polyvinyl sulfate and SDS were added to the

cyto-plamiclyste ofnal concentrationsot100~I.g/mland

05,respectively,

and te KNA .was extracted

by

cjohol

( 50:5U1)anap-recipit-atedwithacohol. Poly(A)

RN asselecteca trtom tnis- matenialby

chromatosz-rahyonpovuidlicacid-Sepharose (10).

1H-labeled R2NA was prepare-d from Sac(-) cells

infected in suspension at an MOI of 6 TCID5o/cell.

MUt

er 4 h ofinfection, the Spinner medium was

re-plcdby

inn aum

otungE-c,

h

M

amount of

vitaamis

and amino

acids,_

10% dialyzed

fetalIbovine serum,and 5 utg ofactinomyci per ml,

follo-wed 30mnlater

by

30 Cof['H]uridin-e

(TRK178;7mrhmBche)e

land2.25hlater

by 100

.ug

of cycloheximide per h after

infection,thecellswerewashedonce inice-coldlTKM butter~1u

m~Iris-y_dro_hlor~ide

~[pH7.41. 150 m

KCfI, 10 mm MgCI2), resuspended in- TKM buffe_r

co-ntaining 100

tig

-of polyvinyl sulfatepe ml, and disruein a ounce Te lsate wI

centrifuged at 1,000 xg

-for

5 minat 0C, and SDS

wasddedto te supernatant to a concentration of

0.5%. NA

wasextrGa.cted

by repeated shaking with7

phenol-chloroform-isoamyl alcohol

adpei-pitated-with etha-nol. PolFy(A) RNA wasselected bpolyuri-dycacid-Sep-harosechromatography.

Polyso-mal RNA was prepared fro-m Sac(- cells

infected and treated as described above, except the

[3H]uridine was omitted and the washed cell pellet

wassuspendedinTKM buffercontaining0.2%

Noni-detP-40,3.6%sucrose,100jigofcycloheximideperml,

100/ig ofpolyvinylsulfate perml, and 0.05%

83-mer-captoethanol. The 1,000 x g supernatant from the

disruptedcelllysatewasmade 1% with sodium

deox-ycholate, layered onto a linear 15 to 40% sucrose

gradientin TKMI buffer (TKM buffercontaining 30

tLg each of polyvinyl sulfate and cycloheximide per

ml),andcentrifugedfor 2.5 hat25,000rpm at40C in

anSW27rotor.Theregionof thegradientcontaining

polysomes sedimentingbetween about 100Sand300S,

asjudged byabsorbanceat260 nm,waspooled,diluted

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toaconcentration of 15%sucrose with TKMIbuffer, and overlaid ontoa 5-ml cushion of 2 M sucrose in TKMI buffer. The polysomeswerepelletedat25,000 rpmfor12hat4°CinanSW27rotor.The pellet was suspended in RSB buffer (10 mMTris-hydrochloride [pH 7.4], 10 mM NaCl, 3 mM MgCl2), RNA was released from thepolysomesby puromycin treatment, andribosomal subunitswereseparatedon a5 to20%o sucrosegradientasdescribedbyBlobelandSabatini (1).Material sedimenting slower than 50S was pooled and diluted withwater to a sucroseconcentrationof 7.5%, anda l/io volume of 10%SDS, a l/io volumeof

lOx RNA extraction buffer (500 mM NaCH3COOH [pH 5.0],1MNaCl,25mMEDTA),and ascites tRNA

to aconcentration of10,ug/mlwereadded before RNA

wasextracted byrepeated shakingwith

phenol-chlo-roform-isoamyl alcohol. The RNAwasrecovered by

alcohol precipitation, re-precipitatedthree times, and

stored inwaterat-700C.

Cell-free protein-synthesizing systems. The

rabbitreticulocvte lysatewas prepared as

described

byPelliamandJackson (15), excepttheaminoacids

m-eahand2-aminopurinewasaledto afnl

.runcentrationof6imm.lncubationw asfoIh at 2.

The efficiency ofincorporation was determined as describedby Dahl and Dickson(3). Sam for elec-trophoresis on SDS-polyacry were

pre-pae ymixing 1,do? the-reactionmxuewith 9 uI

otwater an 11) od electrnnnrSig

samnle

bufe

floebyheating

to100'C for 2 min.

The

messenger-diependn

L-elS0wspeared

as described

by.

Paucha et -al.

IF

7nd

Dahl and Dickson

(3).

The determination ofincorpo6ration

effi-ciencyand thepreparationofsamplesfor electropho-.resiswereasdescribedbyPauchaetal. (14).

Gel electrophoresis. Samples were

electropho-resedon15%discontinuousSDS-polyacrylamide gels

asdescribedby'Laemmli (6).Electrophoresis sample buffer contained 2% SDS, 10% glycerol, 0.001%

bro-mnophenol

blue, 0.1 M dithiothreitol, and 62.5 mM Tris-hydrochloride, pH6.8.Theproceduresfor

stain-mganddryingthepolyacrylamide gelsand the

expo-sureofautoradiographshave been describedbySmith

etal. (19).Insomecases,gelswereimpregnatedwith

PPO (2,5-diphenyloxazole) andsubjected to fluorog-raphyat-80'C (9).

Tryptic peptide fingerprinting. Tryptic peptide fingerprintingwasperformedasdescribedbySiddell

(17).The oiiet -dgsd etideswere se

-arated

oncluoeae0eco

oreass

in H21--uferand ascending chromatography in Sucrose-formmiai7e7jididenit centrifugation. Cytoplasmic poly(A) RNA and RNA released from

polysomeswerefractionatedon5 to20%sucrose

gra-dients containing 50% formamide as described by Smithetal. (18), butwithcentrifugation inanSW41

rotor at30,000 rpmfor 18 to 20 hat 1800. Samples

weredenatured in 60% formamide-6 mM Tris-hydro-chloride (pH 7.5)-0.6mM EDTAat37CCfor 10 min

beforecentrifugation.

RNA markers. 'H-labeled rRNA was prepared from uninfected b-cells, and 32P-labeled 18S rRNA

waspreparedfromuninfectedCVicellsandwas agift

from J. R. Stephenson. Poly(A) RNA was removed fromrRNA by two cycles of polyuridylic

acid-Sepha-rosechromatography.

RESULTS

Protein synthesis in infected cells. The

[35S]methionine-labeled

polypeptides ofJHMV areshownin Fig. 1(track V). Thenomenclature used is basedon thepreviously described molec-ular weights (hereafterKwillbe used todenote molecular weight x 1,000) of the proteins and whether theyareglycosylated(22). Thepattern is

essentially

asdescribed by Wege et al.,with

some minor differences (S. G. Siddell,

manu-script in preparation). The

majoreglyc

of the virus are

gpl7

g

2k

sylatedn e major

nucleocapsid

pro-n

The-Wect nof

Sac(-) cells

with a high

mul-tpjici

of JHMV led to an almost

corn_e

I.In hstcel

roems

g

esis

wt 8 to

.V (Fg 1)

Piilse-labeling

thecells witha

iiiix-tureof15

3H-amino

acids showed that from 5 h postinfection onwardthe

morLroein

te-sized in infected

cells

a 6

olspeptide

wEhich

comiated

with the viron tein

60.

At 7 to 8h

specic

afesr

infection,

the

synthesis

HOURS AFTER iNtCTION

M 5 b 7 8 9 10 11 150KF

60Kb

30K 23K.

- c

V

s qp170

(1)98

-4 j1) 65

_ldomm 1)6f

g1)25

FIG. 1. Time courseofJHMV-specific protein

syn-thesis. Sac(-) cells were infected with JHMVand

pulse-labeledwithamixtureof3H-amino acidsatthe

times indicated, and lysates wereprepared as

de-scribedinthetext.Samplesofeachlysateweremixed withequalvolumesofelectrophoresis sample buffer, heated to

100°C for

2 min, and

electrophoresed

in

parallelwithasampleofpurifiedvirus.JHMVwas

labeled with

[35S]methionine

andpurified as

de-scribedin thetext.Labeledviruswasmixed withan

equal volume of electrophoresis sample buffer and

heatedto37°Cfor2minbefore electrophoresis. The

gelwasfluorographed.

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13

ininfected cells oftwo more

major

poyRpeDtides

(150K and23K) andafourth minor

polypentidf

(30,as

also detected. Of

these,

o h

protein

comigrated

with a virion

protein,

i.e.,

Confirmationthat the 60K intracellular

pro-tein hadaprimarystructure

similar,

ifnot

iden-tical, to p60 was provided by tryptic peptide

fingeprinting.

Figure2Aand D showsthatp60

from the virus and theputative p60 fromthecell

A

both had six major

[35S]methionine-contamning

tryptic peptides, five of whic

comigrated

in

chromatography

and

electrophrsi.Although

t

migratededifferently

beledX when

the samplesderived fom thevron orthe cell wereelectrophoresed separately, asample

con-taining

equalradioactive

amountsofdigestfrom eachprotein

produced

a

ingeprm

inwuc six

peptides

comlgated . e-cann

exFpl-ai

-the apparent

variable

-electrophoreti"c

B

9,

;gi

C

D

ax

FIG. 2. TrypticpeptidefingerprintsofJH.MVp6Omade invitro, purified virionp60, andp60synthesized

in infected cells. Samples of [35S]methionine-labeled virus, [35S]methionine-pulse-labeled cell lysate, and

[35S]methionine-labeledL-cell translationproductswerepreparedandelectrophoresed, theareaofthegel

containing p60 was cut out, and the protein was eluted and processed all as described in the text.

Electrophoresiswasfrom lefttoright, andchromatography wasfrom bottom to top. (A)p60from purified JHMV;(B) p60made invitro; (C) amixtureof equalradioactiveamounts (25,000 cpm)of (A), (B), and(D); (D)p60purified from infectedcells.

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mobility

of e tide X butnevertheless we con-cleromthesedata thatthetwoproteinsare

closely

related,

ifnotidentical. TIhen Of

the 23K intracellular rotein as 23 has also

been~~~~~~~~~~

cnrmd-v

e-cf

imnrepitation

with- anti-JHM serum and

tryptic

peptide

fin-gerprinting (S. G. Siddell andH. Wege, manu-script in preparation).

In vitro translation of infected-cell

poly(A) RNA.Poly(A) RNA isolated from the cytoplasm of infectedormock-infected cellswas translated in cell-free systems derived from both

mouseL-cells and rabbit reticulocytes. In both

systems,a major

produt

specific tothe trans-lation of poly(A) _NA from infected cells was a polypeptide of f6UK which comiated with p60

=wig.

3A, tracks 1 and 4, and

Fig.

3B,

tracks 1 and 8). Nocounterpart wasfound in the trans-lation of poly(A) RNA from uninfected cells (Fig. 3A, track3,andFig. 3B, track5). The 60K product of the L-cell system was

specifly

imunoprecipia

or

anti-'pD

a(iig.

cA,

tracks5

and-6-an -tracks

7

and 8), and no such protein was immunoprecip-itated from the translation of uninfected-cell

poly(A)

RNA

(data

not

shown).

The 60K product of the reticulocvte

system

was

alspecific

m ciitatedby

anti-JHmserum Fi-g.73B, tracks 9 and

10).

The

immunoprecipitate

of the

reticulocyte

transla-band ig.,trackI, Clu o-,,aie a major

7hich

was

not-present-

in the

-translation.

We

assumed"

tmat tin Protein a

degradain

rduct

which

arose during

immunoprecipita-tion. In the reticulocyte system a protein of

120mawhich

wasnotmade orwas

mein aralamouns m

Mehrel

smse.

fhis protein was

specifically

-ta i

(Fig.

3B, tracks9and

X,

andone

possibility

was that itwas related

to the 150K virus-specific glycoprotein in the infected cell.

Confirmation that the 60K protein

synthe-sized intheL-cell

system

wasclrosel relatedto

iOwas

again provided

by

tryptic

peptidef-A 1 2 3 4 5 6 7 8

gp17VO

gp98> gp65.

p60i _

gp25o

B 1 2 31 4 5 6 ; 8 9 In

gp170.

gp98, gp65 >

p60 . _

gp25 o

p23k #.:

FIG. 3. Polyacrylamide gelanalysis ofproteinsmade in vitro inresponsetoinfected-anduninfected-cell poly(A) RNA andimmunoprecipitatedwith anti-JHM andanti-p60sera.Poly(A)RNAwasisolatedfromthe

cytoplasmofinfected and uninfected cells, translated, andimmunoprecipitated with anti-JHMoranti-p60

serumallasdescribed in the thetext.In track 1, theautoradiographs ofthepolyacrylamide gelsshow inall

casespurified virus preparedasdescribedin thelegendtoFig.1. (A) Proteins made in the L-cellsystemin

response to:(2)noaddedRNA; (3) the equivalent of0.4pgofuninfected-cell poly(A) RNA;(4) theequivalent

of0.4pgofinfected-cell poly(A)RNA.Immunoprecipitation oftheproductsmade inresponsetotheequivalent

of0.4pgof infected-ceUpoly(A) RNA with: (5) rabbitpreimmune serum; (6) anti-JHMserum; (7) mouse

preimmuneserum; (8) anti-p60serum. (B) Proteins made in thereticulocytesystem in responseto: (2) no

addedRNA; (5)theequivalent of40ngofuninfected-cellpoly(A) RNA; (8)theequivalentof40ngof infected-cellpoly(A) RNA.Immunoprecipitation withrabbitpreimmuneserumoftheproducts made inresponseto:

(4)noaddedRNA; (7) theequivalent of200ngofuninfected-cellpoly(A) RNA; (10)theequivalent of200ng

of infected-cell poly(A) RNA.Immunoprecipitationwithanti-JHMserumoftheproductsmade in responseto:

(3)noaddedRNA; (6)theequivalentof200ngof uninfected-cell poly(A) RNA;(9)theequivalent of200ngof

infected-cell poly(A)RNA.

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eiting. Figure2A,B, andC shows that the

["S]methionine-containing

tryptic peptides of the putative p60 made in vitro were identical to those of virion p60 when electrophoresed and chromatographed separately or in combination.

By the criteria of comigration, specific

immu-noprecipitation, and peptide fingerprinting, we concluded that the protein made in vitro was

p60.

Inboth cell-free systems, many more

polypep-tides were

synthesized

in response to

infected-cell

poly(A)

RNA. Among these was a 23K

poly-ptide

whichwas amajor roductof the L-cell Vse,cmgae -t viin roteinp23,wa-s

specificallyv

immunoprecipitated by

anti-JHM serum, and had a I 'S

lmethiiionine-containing

trypticpeptide fmgerprintsimilar, if not

identi-cal t that of virion 23

Fig. 3A,

tracks 1, 5,

and

unpublis

e data). We concluded that

thisproduct was p23. The apparent discrepancy

between the amountsof p23 synthesized in the

infected cell and those synthesized in response tocytoplasmic poly(A) RNA in vitro (compare

Fig. 1 and 3A) hasnotbeenexplained, but may

beaccountedfor byalarger number of methio-nine residues in p23 than is normal. We are currentlyinvestigating this possibility.

Toestablish the size of the poly(A) RNAs that

encode p60 and p23, we first investigated the

poly(A) RNA synthesized in the cytoplasm of actinomycin D-treated cells late in infection.

Fiaure4shows the sedimentation in a

sucrose-formamide

gradie-nt

of

cyoplasmic

polyIA'

RN pulse-labeled in the

2resence

of

actino-mycm

l c ominationwith "P-labeled 18 rRNA.Thema orspecies

synthesized

within the

sizerange

analyze

in sgradient sedimented

at 17S. A

poly(A)-containing

RNA with the

samesedimentation valuewasalso a

major

spe-specically r esd

by

puromycin orEDA

treatment of polysomes

isolated

at the same

time of infection (unpublished data). With

su-crose-formamideiigradients,

it was not possible

toclearly resolve additional species in this size

range, althoughwebelieve that the shoul

19S indicates anotherR secies

Second, we sedimented

poly(A)

RNA from

the

cytoplasm

offinfectg

cellsonsucros-

-.1th-U:e-~adients.

fractionatedthe

gradient.-covered the RNA from eachfraction,and

trans-la-ted it in vitro.

J.igure

5A

sfio-WTTh7

prdcts

made with unfractionated RNA (tr d

selected fractions o-f the same ma-terial after

cent_ion

_ 1 The

translation

products

of the portion of the gradient including

RNA sedimenting at 15S to 25S, as judged

by

the sedimentation ofL-cell [ HrN ina

par-allel

gradient, are shown. 'l'he RNA encodirg

th-e

6K

product

sediment_ed

aT7about

1:79An

FIG 4. Seietto5fctolsiIoyAN

2-0

I I

a.

10,~~~~~~~~~

FRACTION NUMBER

FIG. 4. Sedimentationofcytoplasmicpoly(A)RNA synthesized in actinomycin D-treated cells late in infection.Atotalof 75,000 cpm of[3H]uridine-labeled cytoplasmicpoly(A) RNAwaspreparedasdescribed in the text andcosedimented inasucrose-formamide gradient with 25,000 cpmof 32P-labeled 18S Vero cell rRNA.Symbols:(0) 3H-labeled RNA; (0) 32P-labeled 18S rRNA.Sedimentationwasfromrighttoleft.

the protein could be identified as p60 both

by

comigrtinwith

virionp60O

(Fig.

5Aand B)

and

by immunoprecipitation with anti-p60 serum

(Fig. 5B). There was no immunoprecipitation

with preimmune serum (data not shown). A

more accuratesedimentation value of 17S was obtained from the translation of poly(A) RNA fractionatedincombination with rRNA markers (data not shown). We have analyzed gradients wihwould

include-

RN

sJenting

up to

'50S and have not found another

cvtoplasmic

0 RNAspecieswhich directs thesynthesis

of

p6

invitro.

F'gure

5

also

shows that size-fractionated

poly(A) RNA directed the

synthesis

3T ther

proteins

inVitro. A serieS O: these was

specifi-cay

immunoprecipitated by

anti-p60

serum,

nd th RNAwhic encode them had thesame

size distribution as that encoding p60. These

pTrductsmav beirematureterminations of the

invitro system whichwererelated top-60. It is

u1nllkV.lythat

they

were

cleavage

products

of

p60.

a.i

theyweresynthesized

in-vitro

in thepresence

of600Kallikrein units of

aprotia

protease

inhibitor,per ml (datanot

shown).

Another

majortrans product of fraction-atedpoly(A) RNA of this size classwas a 23K

protein

which

comigrated

with virion protein

p23;

EhiDroductkwas not

immunoprecipiva

ed

with

anti-p60

serum

(fi1g.

_i5B;

although-a

prec-ip-'is

t5'-blfleiitr-ak

10,compare therelative VOL. 33, 1980

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1 2 .'4 *J 6 8

18S

9 10 11 12 13 14

:..~:e .:

si;

:a.

...

.:...

FIG. 5. In vitro translationoffractionated poly(A)

RNAfrom JHMV-infectedcellsand

immunoprecipi-tation oftheproductswith anti-p60serum. Poly(A)

RNAwasisolatedfrom JHMV-infectedcellsas de-scribed in thetext.RNA (20pg)wasdenatured and sedimentedon asucrose-formamide gradient.

Fifty-eight fractionswerecollected,and the RNA in each

fraction wasrecovered, precipitated twice, and

sus-pendedin15 ulofwater.Samplesweretranslated in the L-cell system. Purified virus prepared as

de-scribed in thelegendtoFig.1is shown intrack 1.(A)

Products made inresponse to: (2) the equivalent of

0.33yg of unfractionated RNA; (3to14)2ul ofRNA recoveredfromfractions19to31,respectively. (B) (2

to14) Translationproductsidenticalto(2)to(14)in

(A) which were immunoprecipitated with anti-p60 serum. Sedimentation isfrom right to left, and

3H-labeled L-cell rRNAwasanalyzedinaparallel

gra-dient. The 4S, 18S, and 28S rRNA markers sedi-mented infractions correspondingto49, 28, and12, respectively.

immunoprecipitation ofp60and the

precipita-tion of the23Kproductintracks2, 10, and 13),

hu

bUtLNA iimMunoprecipitated by nti-JH

se-rum (datanotshohwn

W

'he JtNAactivity

a5

=Cgthe

23Kproduct

sedUiien

_W1Tm

19S and wasclearlyseparable from that

encod-In

vitro translation of infected-cell

mRNA.

We have

presented

dataonthe in vitro translation products and the sizes of poly(A) RNA isolated from the cytoplasm of infected cells.Toconfirm thatthese RNAswere mRNA's

in the

F--

efr-

netgatcfi

ie n

translation

products

of

RNAs

which were

spe-cificaly

released frommected-cell polvs in

such a way as to exclude

contnatiowith

RNA

deriveed

rom

nucleocapsids

which

might

havebeen presentmtMepolysome

mateial (see

abov'e).

Th>eresulIts thatweobtainedwere

essen-tially

ideetola

nIpJyA)

ItiNA.

VInedcettxl

TiniLtNArce

thei sinthe L-cell system ofa similar

spectrun

of

proteins.

asdidcytoplasmic poly(A) RNA, and among the products were proteins whichcomigrated with p60 andp23. Both

prod-ucts were

specifically

immunoprecipitated with

anti-JHMserum, whereas onlythe 60K product was specifically immunoprecipitated with

anti-p60serum.

We also confirmed the sizes of the mRNA's whichencoded theseproteins, again by fraction-ation on sucrose-formamide gradients. The in

vitro translation of fractionated mRNA again

indicated that the mRNA's encoding p60 and p23 sedimented at 17S and 19S, respectively, andwereclearly

separable.

DISCUSSION

The data

presented

in thispaper

provide

some of thefirst information on coronavirus

replica-tion.Atlatetimes ofinfection, the major protein synthesized inJHMV-infected cellswasshown

to be one of the virion nucleocapsid proteins,

p60.

Also

synthesized

in infected

cells, although

inlesseramounts,was a23K

polypeptide

which wasidentifiedasvirion

protein

p23.The

synthe-sisof these

proteins

was

easily

detectedashost

&ell nprotein

synthesis

was

efficiently

shu

f n HMV-infected Sac- cells an apparent

dis-crepancywith earlierreports

(2)

that is

probabby

accounted for by the

higher

MOI usedin

our

,udy-The

poly(A)RNAs

encodingp60

andp23were isolated, translated in

vitro,

and fouid to

sedi-ment insucrose-formamidegradientsat17nd

l9S.

resectivelv.

Both of the RNAs

co5ildbe

specifically

released from infected-cell

poly-somesandare

thprPfrephysiological

Also, the 17S

and,

we

believe,

the 19S RNA

constitute a

large

proportnion T tne-vira synthesizedlate in infection and are therefore unlikelyto resultfrom

degradation.

Our inter-pretationof these data is that these RNAs

rep-resent two

subgenomic

coronavirus JHM

A

c-1l70 qp98 a

gpO65

-t)60 s

gp253

B

gpl10IOF

gp98

-gp650

p60 *

J. VIROL.

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(8)

mRNA's. each of which encodes s diffiaront structural protein of the virus.

The data donot however exclude number ofmore

complex

inte retations. For example, the mRNA's which sediment at 17S and 19S _beconforma forms oneofwhichhas

an

indi-rect,evidence for internal initiation of

positive-stranded virus mRNA has beenreported (24), and thesizing of mRNAonsucrose-formamide gradients

probably

dependsto a degreeon the hydrodynamic properties of theRNA. Possibil-ities suchasthiscanonly be excluded by analysis of themRNA's themselves.

If our interpretation is correct, the

repli-cation strategy ofJHMV contrasts with other well-characterized

nontransforming

positive-stranded RNA animal viruses. Unlike

picorna-viruses, alphaviruses, and flaviviruses, the expression of the JHMV genome appears to involve more than one

subgenomic mRNA,

at leastone of which is

apparently

monocistronic (i.e., the 17S RNA encodesa 60K

protein)

and

one ofwhich

clearly

has a

potential coding

ca-pacity in excess of that

required

to encode its translationproduct (i.e., the 19S RNA encodes a23Kprotein). Our studieson

protein synthesis

inthe infectedcell andonthein vitrotranslation and characterization ofmRNA's which encode virus-specific proteinsindicate that in addition tothesetwo

mRNA's,

atleast anotherone,and possiblymore

subgenomic mRNA's,

isinvolved in the replication strategy ofcoronaviruses, in additiontothe assumed mRNA

activity

of the genomic RNA. Our future

experiments

will be aimed at

defining

these

RNAs,

their

products,

and their

relationships

toeach

other, eventually

arriving

at an

understanding

of the molecular biology of JHMV.

ACKNOWLEDGMENTS

We thank HannaWege for technical assistance,J. Ste-phenson forhelpfulcriticism,andHelgaSchneider fortyping ingthemanuscript.

Thisresearchwassupported byDeutsche Forschungsge-meinschaft grant Mz 270/18.

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9. Laskey, R.A., and A. D. Mills. 1975. Quantitative film detection of 3H and"4Cinpolyacrylamide gels by fluo-rography. Eur. J. Biochem. 56:335-341.

10. Lindberg,U., and T. Persson.1974.Isolation of mRNA fromKB-cellsby affinitychromatographyon polyuri-dylic acid covalently linked to Sepharose. Methods Enzymol.34:496-499.

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13. Nagashima,K., H.Wege,R.Meyermann,and V. ter Meulen. 1979. Demyelinating encephalomyelitis in-ducedbyalong-termcoronavirusinfection inrats.Acta Neuropathol. 45:205-213.

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15. Pelham, H. R.B.,and R. J. Jackson.1976. Anefficient mRNAdependent translation system from reticulocyte lysates. Eur. J. Biochem. 67:247-256.

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17.Siddell, S. G.1978.RNA hybridization to DNA coupled withcyanogen-bromide-activated sephadex: the purifi-cation ofpolyomamessengerRNA.Eur. J. Biochem. 92:621-629.

18. Smith,A.E.,R.Kamen,W. F.Mangel,H.Shure, and T.Wheeler.1976.Location of the sequencescodingfor capsidproteins VP1 and VP2. Cell 9:481-487. 19.Smith, A. E., T. Wheeler, N. Glanville, and L.

Kaivainen.1974.Translation ofSemlikiForest Virus 42S RNA ina mousecell-free system to give virus coat proteins. Eur. J. Biochem. 49:101-110.

20. Tyrrell,D. A.J.,D.J.Alexander, J. D. Almeida, C. H.Cunningham,B.C.Easterday, D. J. Garwes, J. C.Hierholzer,A.Kapikian, M. R. MacNaughton, and K. McIntosh.1978.Coronaviridae: second report. Intervirology10:321-328.

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VOL. 33,1980

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Figure

FIG.1.pulse-labeledparallelgelscribedthesis.timeswithheatedequalscribedlabeledheated Time course ofJHMV-specific protein syn- Sac(-) cells were infected with JHMV and with a mixture of3H-amino acids at the indicated, and lysates were prepared as de- in the
FIG.1.pulse-labeledparallelgelscribedthesis.timeswithheatedequalscribedlabeledheated Time course ofJHMV-specific protein syn- Sac(-) cells were infected with JHMV and with a mixture of3H-amino acids at the indicated, and lysates were prepared as de- in the p.3
FIG. 2.[35S]methionine-labeledJHMV;Electrophoresisincontaining(D) infected Tryptic peptide fingerprints of JH.MV p6O made in vitro, purified virion p60, and p60 synthesized cells
FIG. 2.[35S]methionine-labeledJHMV;Electrophoresisincontaining(D) infected Tryptic peptide fingerprints of JH.MV p6O made in vitro, purified virion p60, and p60 synthesized cells p.4
FIG. 3.poly(A)preimmunecytoplasmserumresponseofofcasesaddedofcell(4)(3)infected-cell 0.4 infected-cell Polyacrylamide gel analysis ofproteins made in vitro in response to infected- and uninfected-cell RNA and immunoprecipitated with anti-JHM and anti-p60 s
FIG. 3.poly(A)preimmunecytoplasmserumresponseofofcasesaddedofcell(4)(3)infected-cell 0.4 infected-cell Polyacrylamide gel analysis ofproteins made in vitro in response to infected- and uninfected-cell RNA and immunoprecipitated with anti-JHM and anti-p60 s p.5
FIG. 5.pendedfractionRNAscribedsedimentedeightRNAtationProductsrecoveredscribedtheserum.labeled0.33todient.mented(A) 14) In vitro translation offractionated poly(A) from JHMV-infected cells and immunoprecipi- of the products with anti-p60 serum
FIG. 5.pendedfractionRNAscribedsedimentedeightRNAtationProductsrecoveredscribedtheserum.labeled0.33todient.mented(A) 14) In vitro translation offractionated poly(A) from JHMV-infected cells and immunoprecipi- of the products with anti-p60 serum p.7