Further characterization of the replicative complex of vesicular stomatitis virus.

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0022-538X/79/08-0494/12$02.00/0

Further

Characterization

of

the

Replicative Complex

of

Vesicular Stomatitis Virus

CHRISTIAN C. SIMONSEN,* VIRGINIA M. HILL,AND DONALDF. SUMMERS

Departmentof Cellular, Viraland MolecularBiology, Universityof Utah Medical Center, Salt LakeCity,

Utah 84132

Receivedfor publication 23 March 1979

Replicating vesicular stomatitis virus ribonucleoprotein (RNP) complexeswere

isolated in nonequilibrium Renografin density gradients. These nascent RNPs

had the same buoyant densityas virionnucleocapsids in both isopycnic

Renq-grafm and CsClgradients. Both transcribing and replicating RNP complexeswere

showntobestable insucrosegradients,whereasonly replicatingRNPcomplexes

werestable inRenografin gradients.Sizeanalysisof the5-min-pulse-labeledRNA

speciesfrom the replicatingRNPs using methylmercury gels revealed that the

nascentstrandswere primarily less thanfull-lengthmolecules. Longertimesof

radiolabeling demonstrated that the nascent RNA accumulated as 42S RNA,

whichwasprimarily ofthesamesense asthe virion strand when itwas

radiola-beledat5hpostinfection.The percentage of this radiolabeled RNA whichwas

plusstrandedwashigherat 2.5hpostinfection, reflective of the shift inplus-to

minus-strandfull-length 42SRNAsynthesiswhichoccursinthecell. Addition of

cycloheximidetotheinfectedcellsbefore the addition of the radiolabelprevented

the formation of these RNP complexes. Both the changein the percentage of

minus strands found in the RNPcomplexesatthe different timespostinfection

and the sensitivitytocycloheximide indicate that the RNP complexwhichwas

isolatedwasindeed thereplicative complex.

During the infection of HeLa cells by vesicular

stomatitis virus(VSV),both virion minus-strand

andcomplementary plus-strand full-length42S

RNAcanbe isolated from intracellular

ribonu-cleoprotein (RNP) complexes (34, 36). These

RNPs are composed of 42S RNA and three

VSV-specific proteins: the N, or nucleocapsid,

protein; the NSprotein,aphosphorylated

poly-peptide;and the Lprotein(17, 18, 36). The virion

nucleocapsid is able todirect the in vitro

syn-thesis of the five VSV-specific mRNA's which,

likethe in vivoVSV mRNA's,arecappedatthe

5'end, methylated,andpolyadenylated(2, 7, 20,

25-27,32).

Reconstitution experimentshave shown that

both the L and NSproteins are required to form

thevirion-associatedRNA-dependentRNA

po-lymerase (17,18,28). Thetemplate for

transcrip-tion has been demonstratedtobecomposed of

minus-strand 42SRNA and Nprotein (11, 17).

Therequirements forreplication, however, are

notwellknown. Thetemperature-sensitive

mu-tant tsG114, which possesses a thermolabile L

protein (21), has been usedtoprovide indirect

evidence that theLproteinisinvolvedin

repli-cation (30). Several othermutants suggest arole

inreplication forthe N andNSproteins aswell

(15, 16). For thesereasonsit has been assumed

that replication occurs on an RNP template

similartothetranscriptiveRNP.

Fractionation techniques previously used to

isolate VSV RNPs do notseparate replicating

RNPs from transcribing RNPs. Sedimentation

velocity centrifugation analyses have indicated

thatintracellular VSV RNPs sedimentas 140S

molecules, whether or not nascent mRNA is

associated with the RNP complexes (31, 35).

The lack of resolution is furthercomplicated by

the fact thatreplicationaccounts for only 10%

of the total VSV-specific RNA synthesized in

infectedcells (38);thus,sucrosegradients have

notbeenabletoseparatereplicatingRNPsfrom

transcribing RNPs. CsCl gradients have been

used toisolate glutaraldehyde-fixed messenger

RNPs (19) and intracellularnucleocapsids(34);

however, themessengerRNPsare notstablein

CsCl withoutprior fixation withglutaraldehyde.

RNPsisolated with CsClgradientsdo notretain

the L and NS proteins, which are known to

comprisepart of thefunctionalRNPs inthecell

(17, 18).Inaddition,allof the intracellularRNPs

band at the same density in CsCl. For these

reasons, CsCl gradients have not been used to

separate the RNPcomplexes.

lodinateddensitygradientmedia suchas

me-trizamide and Renografm have beenemployed

494

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toobtainedresolution of proteins, nucleic acids, and RNPs in the same gradients (6, 22). These

media arehypertonic and minimize nonspecific

RNA-protein interactions, yet do not dissociate

mostRNPs. Severalinvestigatorshave used

me-trizamnide and Renografin to examine chromatin

(12),influenza RNPs (10), and proteins (8). We

haverecently described the isolation in

Reno-grafingradients of RNP complexes from

VSV-infected HeLa cells (V.M.Hill, C. C.Simonsen,

and D. F. Summers,submitted for publication).

Several peaks of radioactivity were observed

when an infected cell extract radiolabeled from

3 to5hpostinfection (p.i.) wascentrifuged in a

Renografin gradient. One of those peaks

con-tained predominantly minus-strand 42S RNA

and was termed peak I. A similar pattern of

radioactivitywasseenafteraninfectedcell

ex-tract pulse-labeled for 5 min at 5 h p.i. was

analyzed, except that peak I exhibited a

pro-nounced trailing edge. Virion nucleocapsids

formedonlyahomogeneouspeakIwhichlacked

atrailingedge.

Genomelengthand smaller RNAswerefound

in thepeak fractions of thepulse-labeled peak I,

whereas onlythe smaller RNAswere found in

the trailing fractions. This RNA, pulse-labeled

for 5 min at 5 hp.i., was 80%minus stranded,

the same percentage observed with

pulse-la-beled42S RNA obtained from intracellular

nu-cleocapsidsat 5hp.i.,whenreplicationis maxi-mal (34). The nascent minus-strand RNA was

shown tobe identicaltothe 5'end of the VSV

genome RNA. We also observed that thenascent

RNAwasRNase resistant,wassinglestranded,

andwasisolatedat adensity of 1.240

g/cm3

in

Renografin. All of this suggested that peak I

contained nascent replicating VSV RNA

asso-ciated withprotein as areplicating RNP

com-plex. Herein we extend these observations and

further characterize the complexes to show by

several different criteria that these RNPs are

indeed VSVreplicativecomplexes.

MATERIALS AND MErHODS

Cells,virusinfection,andradioactivelabeling.

Suspension culturesofHeLa S3 cellsweregrownin

Joklikmodifiedminimal essential medium(Flow

Lab-oratories) supplementedwith 2 mM glutamine plus

5%fetal calfserum(FlowLaboratories) at a concen-tration of 2 x 105 to 8 x 10' cells per ml. Stock

preparationsof VSV(Indiana serotype) were grown in

HeLacells, purified, and assayed asdescribed previ-ously (3, 4). Cells were collected by centrifugation, resuspended in growth medium minus serum at a

concentration of6 x106cellsper ml, and then infected with 10PFUofVSVper cell. At 1hp.i. serum was addedto5% and actinomycin D (a gift fromMerck, Sharp & Dohme) was added to 1 yg/ml. At times

ranging from 2.5 h to 5 h p.i.

['H]uridine

or

['H]-Corp., Boston, Mass.) or both were added to a concen-trationof 200,tCi/mlfor the indicated labeling periods, after which crushed, frozen medium was added and

thecellswerepelletedbycentrifugation for 3min at

900xg.Thecell pelletwaslysed by the addition of 1

mlof 1% Nonidet P-40 in NT buffer (0.1 M NaCl, 0.01 MTris,pH7.5)and kept on ice for 10 min. The nuclei were removed by centrifugation as described above andwerethenresuspended in 1 ml of 0.2% deoxycho-late-0.3% Nonidet P-40 in NT buffer and recentri-fuged. Thesupernatants from the 1% Nonidet P-40 treatmentand the deoxycholate-Nonidet P-40 wash werethencombined andlayered onto preformedCsCl

orRenografingradients.

Isolationof RNPcomplexes.RNPs were isolated

by using eitherRenografin,CsCl, or sucrose gradients. Forisolation of the replicativeRNPs,the cytoplasmic extractsfrom above were layered onto preformed

36-ml, 15to50%or20 to60%Renografin (E. R. Squibb & Sons) gradients in NT buffer and centrifuged at 23,000 rpmand4°C in an SW27 rotor for 16 h (for

nonequilibriumgradients) or for 80 h (for equilibrium

gradients). Thegradients were fractionated by pump-ingfrom the bottom, and radioactivity was determined bydirectly assaying50-1lportions in7mlof

scintilla-tion cocktail in aBeckman liquidscintillation spec-trometer.

Preformed 20 to 40% (wt/wt) CsCl (Varlacoid

ChemicalCo.) gradientsin TNEbuffer (0.025 M

Tris-hydrochloride, pH7.5, 0.05 MNaCl, 0.002 M EDTA)

were overlayed with fractions from the Renografin

gradient which had been diluted 1:3 in NT buffer. Thesegradientswerecentrifugedin anSW41 rotor at 33,000 rpmfor16hat4°C.Aftercentrifugation, the

gradientswerefractionated,andacid-precipitable

ra-dioactivitywasdeterminedasdescribedpreviously(3, 4). Thedensitiesof the fractionsweredeterminedby measuring the refractive indexes withaBausch and Lombrefractometer.

Cytoplasmic extractswerealso layeredonto 15 to

30% sucrosegradientsin NTbuffer,whichwere

cen-trifugedinanSW27rotorfor16h at16,000 rpm and

4°Cand fractionatedasdescribedabove.

Phenol extractionof RNA. Anequalvolume of

phenolsaturated with NETS buffer(0.1MNaCl,0.001

MEDTA,0.01 MTris, pH7.5,0.2% sodiumdodecyl

sulfate) wasaddedto the samplesin 3 ml of NETS

buffer andmixed,and thetwo layerswereseparated

by centrifugation. The aqueous layerwas collected,

and thephenol layerwasextractedoncemorewith 3 ml of NETS buffer. The aqueous fractionswere

com-bined,sodiumacetate wasaddedto afinal

concentra-tionof 0.2 M,2volumes of ethanolwereadded,and

theRNAwasprecipitatedat-70°C overnight. RNA

waspelletedbycentrifugationat16,000xgfor 30min

at40C.

Purification ofVSV-specificRNA. Minus-strand 42S RNAwasprepared frompurifiedvirionsby dis-rupting the virus in NETS buffer andcentrifugingthe

samplesin 15to30% sucrose-sodiumdodecylsulfate

gradients inanSW41rotor. The 42SRNApeakwas

ethanolprecipitatedanddissolved in2xAbuffer(0.3

M NaCl,0.02MTris,pH 7.4,0.002MEDTA). VSV

mRNAwaspreparedfrom infectedcellsat4.5hp.i.A

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cytoplasmicextract waspreparedasdescribed above

and was phenol extracted, ethanolprecipitated,and

chromatographed on an oligodeoxythymidylic

acid-cellulose column.

Oligodeoxythymidylic acid-cellulose

chroma-tography ofRNA.Oligodeoxythymidylic

acid-cellu-lose (Collaborative Research, Inc., Waltham, Mass.)

columns werepreparedandelutedby themethod of

Banerjeeand Rhodes(7).Thehigh-saltelution buffer

contained 0.01 M Tris-hydroxychloride, pH 7.4, and 0.5 MNaCl,and thelow-saltbuffer contained 0.01 M

Tris-hydrochloride,pH7.4.RNA wasloadedontothe

columninhigh-salt buffer.Fractionscontaining bound

materialwerepooled andethanolprecipitatedas

de-scribed above.

Hybridization of VSVRNA.Hybridizationswere

performedin sealedcapillarypipettes in0.02-mIl

vol-umesbyusingthe conditionsofKolakofsky (24).To

eachsampleof[3H]RNAisolated fromnucleocapsids

were added increasing amounts of unlabeled VSV

virion 42S RNA or mRNA. The sampleswere then heat denatured by heating to 115°C for 3 min and incubated for 3 h at730C. Eachsamplewastreated with a 30-,ug/ml solution of RNase A (Worthington

Biochemicals Corp.)in2xAbuffer for 30 min at250C

and wasthenassayed foracid-precipitable

radioactiv-ity asdescribed above. The self-annealing reactions

were identical, except that no unlabeled RNA was

added to thesamples.

Methylmercury-agarose gel electrophoresis.

Methylmercury-agarose gel electrophoresis was

per-formed byamodificationof the method ofBaileyand

Davidson (5). Seakemagarose powder was obtained from Marine-Colloids, Inc., and methylmercury (II)

hydroxidewasobtainedas a 1 Msolution from Alfa

Products, Danvers, Mass. Because of the toxic and

volatile nature of the mercury compound, operations

wereperformedunder ahood. Horizontal slab gels (19

by13.3by0.4cm; 100-mlbedvolume)were1%agarose inborate buffer (0.05 M boric acid,0.005 M sodium

borate, 0.01 M sodium sulfate, 0.001 M trisodium

EDTA, pH 8.0) and contained 0.005 Mmethylmercury

hydroxide.Sampleswereappliedin 40,ulof 1:1

sam-ple-samplebuffer(boratebuffercontaining50%

glyc-eroland 0.1%bromophenol blue),withmethylmercury

hydroxide addedto a concentration of 0.005 M just

beforeloading onto the gel. Thegel reservoirs con-tained boratebuffer,and this buffer was recirculated

atapproximately80ml/h throughoutthe

electropho-resisperiod.Gels were run at 75 to 100 V (50 mA) for

6h(dyefrontmigrated 14cm).

Fluorographyofmethylmercurygels. Gels

con-taiing [3H]RNAwerefixedfor 10 min in 10% acetic

acid containing 0.01 M cysteine. They were

dehy-drated in 100% methanolfor two successive 1-h pe-riods.Afterthey were dried to paper thinness under a vacuum, the gels were soaked in a 10%(wt/vol)

solu-tion of 2,5-diphenyloxazole (New England Nuclear

Corp.).inmethanolfor3h.Gelswere soaked inwater

for10mintoprecipitatethe2,5-diphenyloxazole,

blot-ted dry, and mounted on a, piece of 3 MM paper (Whatman, Ltd., England).Afterbeingcovered with SaranWrap, gelswereexposed to Kodak SB-5 X-ray filmat-70°C anddevelopedafterappropriateperiods of time.

J. VIROL.

RESULTS

Kinetics of

labeling

of

peak

I. We wished

todetermine whether 42S RNA would

accumu-late in the Renografin-isolated peak I if the

length of the pulse-labeling period was

in-creased. We also wished to learn whether the

material in thetrailing fractions ofpeakI would

also accumulate duringalongerpulse-labeling

period. VSV-infected HeLa cells were

radiola-beled for 5, 10, 15, and 30 min at 4.5 h p.i.

Cytoplasmic extracts wereprepared from each

sample and centrifuged in identical 15 to 50%

Renografin gradients. The results (Fig. 1)

dem-onstratethat thetrailing fractions of the 5-min

sample (fractions26 to31) were not as apparent

in thel0-minsample and wereevenless

notice-able in the 15-and30-min samples. All samples

werecontinuously labeled; therefore, the

mate-rial in the trailing fractionswas still presentin

the longer-labeled fractions, but comprised a

minorproportionof the totalradiolabeled

ma-terial duetotheincrease in the amount of

radi-olabel inthe peakfractions (fractions 26 to 31,

allsamples).

We nextpooledthe fractions of thereplicative

complexfromFig.1.Thetrailing fractions of the

5-min-pulse-labeled sample were included

be-causetheradiolabeled RNAcontainedinthese

fractionsrepresentedanappreciable amountof

thetotal radiolabeledRNA.Only the peak

frac-tions from the remaining samples were pooled

becausethematerial inthetrailing fractions did

notrepresentasignificant proportionof the total

radiolabeled RNA. The RNA was extracted

from these pools and analyzed in

methylmer-cury-agarose gels.Thedensitometertracingsof

theautoradiographsfrom these gels are shown

inFig.2.The RNAfromthe5-min-pulse-labeled

sampleswasheterogeneous insize,rangingfrom

less than18Sto42S,althoughthe average size

of thissamplewasapproximately26S. The size

distribution ofthe 10-minsample was also

het-erogeneous; however, the average size of the

labeled RNA increased to a value of

approxi-mately 35S,andtherewas anobvious42S band.

Both the15-and30-minsamples contained

pri-marily42SRNA, althoughthereweresignificant

amountsofheterogeneous material less than 42S

RNA in size. This suggests that the radiolabel

accumulatedasgenomelengthRNAasthepulse

timewasincreased.Figure2alsoimplies that it

tooklonger than5min for 42SRNA tobegin to

accumulate.It is notclearwhythis was thecase,

sinceonewouldexpect thata5-minpulse-label

woulduniformly label all nascent RNA. Perhaps

thesynthesis of VSV 42SRNA does not occur

at a uniform rate throughoutthe template; as

wehave notedpreviously, thereisanapparent

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T 0

FRACTION NUMBER

FIG. 1. Renografin gradient centrifugationof cytoplasmic extractsfrompulse-labeled VSV-infected cells.

Infectedcells at 4.5 hp.i.were labeled with 100,uCi each of[3H]adenosineand[3H]uridineperml.Samples

wereremovedat5, 10, 15, and 30 minaftertheadditionofthe labels. Cytoplasmicextracts wereprepared,

layeredonto 15 to50%Renografin gradients, andcentrifuged for17h at 23,000 rpmand 4°C in an SW27

rotor.Fractions (0.5ml)werecollected by pumping out from the bottom, and the radioactivity in each fraction

wasdeterminedbycounting50-,iuportions in a toluene-based scintillation fluid.

pause site near the juncture of the L- and

G-proteincistrons (Hilletal.,submitted for publi-cation).

Hybridizationof pulse-labeledRNAfrom RenografinRNPs. We have recently reported

that the ratio ofplus-tominus-strand 42S RNA

synthesisin infected cellsisgreaterearly in the

infection (2.5 h p.i.) than when replication is

maxinal at 5 h p.i., at which time 80% ofthe

newly synthesized 42S molecules are minus

stranded (34). Wehave also shown that

pulse-labelednascentRNAspecieswhich areisolated

from putative replication complexesat 5 hp.i.

are approximately80% minus stranded (Hillet

al., submitted forpublication).

Todetermine whether pulse-labelednascent

RNA extractedfrom thereplicationcomplexes

wouldreflect the in vivofindings,infected HeLa

cellswerelabeled for5minwith[3H]uridineat

2.5 or 5 h p.i. Cytoplasmic extracts were

pre-pared from these cellsand were centrifugedin

15 to.50% Renografin gradients. The nascent

RNA contained in the RNP complexes (at a

peakdensityof1.240g/cm3)washybridizedwith

unlabeled VSV 42SRNAandVSVmRNA.The

RNPslabeledat2.5hp.i.containedsignificantly

more plus-strand RNA than the RNPslabeled

at5hp.i. (Table1). The changeinthe ratio of

plus- tominus-strand RNA contained in these

RNPcomplexesfromapproximately35:65at2.5

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498 SIMONSEN, HILL, AND SUMMERS

origin 42S 28S

I I I

185

E

0

to

cli

.o

cn

2 4 6 E

DISTANCE MIGRATED(cm)

FIG. 2. Densitometer tracings of fluo methylmercury-agarose gels. The RNA co

thepulse-labeled RNPswasextractedfrom tionspooledas indicated in Fig. 1. Appi 50,000cpmofpurified RNA from eachst

electrophoresed inamethylmercury-agaro

fluorographedasdescribed in thetext. T

diograph was analyzed with an Ortec n

densitometer. Thescale isidenticalfor a,

hp.i.to 15:85at5hp.i.is similarto ti

in the ratio ofplus-to minus-strand

synthesiswe have previously observe

indicatingthat theplus-strandRNAsl

lated inpeakIwereproductsofreplic

Effectofcycloheximideonthere

RNA. A further means of differenti

replicative processfrom thetranscrip

ess is by using the protein synthesis

cycloheximide. The synthesis of

mRNA's is notaffected appreciably t

dition ofcycloheximide, whereasrepl

42S RNA israpidlyinhibited (30, 38)

shows that the synthesis of the pea

complex from Renografinwasinhibit

additionofcycloheximide, whereas t:

tion of the slower-sedimenting peak

containsexclusivelyplus-strandseque

etal.,submitted forpublication),wasi

by the addition of the drug. This result also

demonstrates that the cycloheximide-sensitive

5min RNPcomplex isareplicative complex.

Isopycnic gradient centrifugation of the

replicative complexes.Wenextexamined the

RNP complexes in isopycnic Renografin

gra-_ _ dientstostudythebasisforthe isolation of the

heterogeneouspeak I.A5-min-pulse-labeled

cy-10min toplasmicextractwaspreparedfromcellsat5h

p.i. asdescribedaboveandwascentrifugedina

15to50%Renografm gradient for17hat23,000

rpm in an SW27 rotor. Fractions from three

portions of the RNPpeakwerepooledas

indi-cated(Fig. 4A)andcombined, and sampleswere

recentrifuged on identical 20 to 60% gradients

15min for 17, 40, and 80 h (Fig. 4B, C, and D). The

replicative RNP peak, which was very

hetero-geneousafter beingrecentrifuged for 17h(Fig.

4B), formedahomogeneous peakatadensityof

1.248g/cm3 in the isopycnic gradients (Fig. 4C

andD). Under thesameconditions of

centrifu-30min gation, VSV42SRNAwasfoundat 1.19g/cm3

(Fig. 4E). Poliovirustop component

(nucleocap-sidwhich lacksRNA) andpoliovirus infectious

particleswerefoundintheequilibrium gradients

at 1.325and 1.218 g/cm3,respectively (Fig. 4F).

Theresult indicated that theratio ofproteinto

RNA in the nascent RNPs was the same

throughout the peak and trailing fractions of

peakI,sincethesmallerRNPscontained in the

,rographed

trailing

edge

of

peak

I had the same

isopycnic

intained in densityinRenografinastheRNPs in thepeak

mthefrac- fractions.

roximately

ample was TABLE 1. Hybridization analysis of the RNA

ge

getoand

isolated

from replicative

RNPs

from cytoplasmic

'he autora- extractspulse-labeledat2.5and5hp.i.a

Moael 4MlU

11samples. he change 42S RNA

d invivo,

pecies

iso-ation. bplicative

iating the

itive

proc-inhibitor

the VSV

bythe

ad-lication of

I.Figure3

tk I RNA

;edbythe

he

forma-II, which

-nces(Hill

anaffected

Self-annealed Unlabeled RNA added Tixme Total material

p.i. mRNA 42S RNA

(h) cpm %

cpm % cpm %

2.5 776 298 38 545 70 266 34

2.5 707 277 39 488 69 247 35

5 1,247 407 33 1,175 94 225 18

5 572 192 33 544 95 60 10

a Infected cells werepulse-labeled at either 2.5 or 5 h p.i. as described in thelegendtoFig.1.The replicative RNP complex

wasisolated fromnonequilibriumRenografingradients, and the RNA contained in the RNP was isolated byphenol ex-traction. Thepulse-labeled RNA was mixed with increasing

amounts of unlabeled VSV mRNA or 42S RNA in 20-Il reactionvolumesby using the conditions ofKolakofaky(24). Thesampleswereheatedto115°Cfor1min, annealedfor3

h at730C,and then treated with30ygof RNaseAperml in

2xAbuffer for 30 min at 220C. Plateau levels of RNase resistanceareexpressed after being corrected forthe RNase-resistantmaterial whichwas present when thesamplewas

heatdenatured butnotannealed. Thisfigure varied from2 to

4%oftheinput radioactivity.Self-annealingof thematerial

wasdeterminedasdescribedabove, exceptthatnounlabeled RNAwasaddedtothesamples.

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Pe E

[image:6.504.102.425.56.278.2]

n Number

FIG. 3. Effect of cycloheximideontheproductionofreplicative RNP complex. A culture of infected cells

wassplitinhalfat 4.5hp.i.,and toone-half ofthe culture cycloheximide was added to a final concentration

of100

tLg/ml.

After 15min in thepresence ofthe drug, [3H]uridine wasadded to each culture to afinal concentration of 100 ,aCi/ml. After a 5-min incubation, the cells were removed by centrifugation and

cytoplasmicextractswerepreparedasdescribed in the text.The extracts were layered onto separate identical

15 to50%Renografin gradientsand werecentrifuged for16 hat23,00( rpm and 4°C in anSW27 rotor. The

radioactivity from the fractionated gradients was analyzed as described previously. Symbols: -4,

untreatedcontrol;0---S cycloheximide-treated sample.

ThenascentRNPswerefurther analyzed by

centrifugingfractions from thepeakandtrailing

fractions of peak I in CsCl gradients (Fig. 5).

The nascent RNPs had identical equilibrium

densities inCsCl. This result alsosuggeststhat

the nascent RNPs have a protein-RNA ratio

sunilartothat of virionnucleocapsids, since all

of the radioactivity was found at a density of

1.31

g/cm3

(19). The nascent RNA was then

removed from the RNPs and analyzed in

su-crose-sodium dodecyl sulfate gradients, which

showed that the RNA contained in the

CsCl-banded RNPswasshorter than fulllength and

notjust42S RNA (Fig. 6). The demonstration

thatnascentRNPsarestable inCsCl is further

evidence suggesting that these nascent RNPs

arederived fromareplicativeintermediate since

transcriptive productsdonotband inCsCl (19).

Itthusappearslikelythat theheterogeneity of

the 5-min-pulse-labeled RNP complex which

hasbeen isolated is not due to adifference in

densitybetween the RNPs in the peakand the

trailingfractions.

Examination of the trailing fractions of

peak I. The results shown in Fig. 1

demon-strated that the pulse-labeled material in the

trailingfractions didnotaccumulateduring long

pulse-labeling periods. Furthermore,it hasbeen

shown thatnascent RNA is found in both the

peak and the trailing fractions of peak I, but

detectable newly replicated 42S molecules are

found in the peak fractions only (Hill et al.,

submitted for publication). These results

per-haps suggest that the material in the trailing

fractions isnascentRNA which has dissociated

from thetemplateRNA. We have observed that

thenonequilibrium Renografin gradientsresolve

RNA molecules on the basis ofsize; thus, it is

possible that the trailing fractions contain

smaller RNPsseparatedonthe basis of size.

At the present time there are two possible

waysthat thisproblemcanbestudieddirectly.

Electronmicroscopic examinationof the

mate-rial contained in thepeak andtrailingfractions

wasperformed; however, duetothelarge

num-ber of nonradiolabeledstructurespresentin the

same area aspeakI,wecouldnotprovethat the

trailing edge representednascentstrands

disso-ciated fromtheir templates (C. Naeve,

unpub-lisheddata). We didobservepossiblereplication

formsinthepeak fractions (Naeve, manuscript

inpreparation),but duetothehigh background

of nonreplicating RNPs, it was impossible to

deal conclusively with the problem of nascent

strand attachment to template RNP. Another

wayofdirectly showingthat thepeakfractions

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500 SIMONSEN, HILL,

7d - E

5 3 5 20 25 30 35 5 1015 20 25 30 35

E FF C

4-

~~~~~~~~~~~~8-- -1.20

2- ..

4--1.10

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25 30 35 40 45 50 55 5 10 '15 20 25 30 '35 FRACTION NUMBER

FIG. 4. EquilibriumRenografindensitygradientcentrifugation ofthe 5-min-labeled RNP complex. Infected cells at 5 hp.i. were labeled bythe addition of100,LCi each of[3H]uridine and [3H]adenosineper ml.

Crushed,frozenmedium was added to thesampleafter5min in thepresenceofthelabels,and the cells were

removed bycentrifugation. A cytoplasmic extract wasprepared andlayered onto a 15 to50%Renografin gradientwhich wascentrifuged for16h at 23,000 rpm and4°C in an SW27 rotor. The radioactivity was

quantitated asdescribed in thetext.Thefractionsindicatedfiomthepeakandtrailingfractionsin(A) were

combined and diluted. Samples were layered onto identical 20 to 60% Renografin gradients and were

centrifugedat23,000 rpm and4°CinanSW27rotorfor17h(B)40h(C),or80h(D).3H-labeled42S RNA (E)

andpoliovirusnucleocapsidand top component(F) werecentrifugedin identicalRenografin gradientsat

23,000 rpm and4°C for80h.

8-A PeokI PeakH2-B C D

g |~12"23':~ 5 1"

6- *140

E

.4 , , , . , 2 . , 5 130

2- .120

5 10 152025 30 35 5 10 15 5 8 15 5 10 15

FRACTION NUMBER

FIG. 5. CsClgradient centrifugation of peak Ifrom a Renografin gradient. Acytoplasmic extractfrom

infected cells pulse-labeled for 5 min at 5hp.i. was layered onto a 15 to50% Renografin gradientand

centrifugedasfor Fig.1. The RNPpeakwassplit into three poolsasindicated in(A).Thepoolswerethen

layered onto 20 to40%(wt/wt)CsClgradients in TNEbufferandcentrifuged for16hat33,000 rpm and4°C

in an SW41 rotor. Fractions (0.5 ml) were collected from the bottom, trichloroacetic acid-precipitable

radioactivity was determinedfor250itlofeachfraction, and thedensitywasrecorded bymeasuring the

refractiveindexesof thefractionswith a Bausch and Lombrefractometer. (B)CsClgradientofpool1from

(A).(C) Pool 2. (D) Pool 3.

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[image:7.504.87.428.448.582.2]

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[image:8.504.103.412.66.218.2]

FRATIONNUMBER

FIG. 6. Size analysis of the pulse-labeled RNA contained in CsCl-bandedreplicative RNPs. The pulse-labeled RNPs which were banded inCsClinFig.5B, C,and D werephenolextracted,and the RNAcontained inthesecomplexeswascentrifugedin 15 to30%sucrose-sodiumdodecylsulfategradientsfor16 hat20,000

rpm in an SW41 rotor. Trichloroacetic acid-precipitable radioactivity was determined for each of the

fractions,which were collectedby drippingfromthe bottomofthe tube.Symbols:0,['4C]uridine-labeled42S,

28S, 18S, and 4S markers; *, [3H]uridine-labeled RNA from Fig. 5B, C, and D (panels A, B, and C,

respectively).

contain nascent strands attached to their

tem-plate is tocompletethe nascent strands invitro;

however, at the present time this has not been

possible.

It wastherefore necessary to study this

prob-lemindirectly.Asizeanalysisof the RNPs

con-tained in the peakandtrailing fractions of the

Renografin gradient was attempted by using

sucrosegradients,but due to aggregation of the

RNPs when removed from Renografin, the

re-sultswereinconclusive.Thesizingof the RNPs

was nextattempted by comparing the

sedimen-tation characteristics of RNPs from the peak

andtrailing fractions ofpeakI tothose of RNPs

derived from a strain of defective-interfering

(DI)particles containingagenomewhich isonly

15%of thewild-typegenome. We reasoned that

the DI RNPs , which also band inCsClat 1.31

g/cm3 (M. Leppert, personal communication),

should be found in the sameregion of a

none-quilibrium Renografin gradientasnascentRDYPs

similar in sizetothe DI RNPs iftheRenografin

gradients could separate RNPs on the basis of

size. Figure 7 shows that the nucleocapsids

de-rived fromthe DIparticles are found in exactly

the sameregionofanonequilibriumRenografin

gradientas nascentRNPs, which are about 15%

replicated (Fig.7A and B; density, 1.18g/cm3).

Virion nucleocapsids cosedimented with the

peak fractions ofpeak I at a density of 1.22 g/

cm3 (data not shown). TheDIRNPspresumably

have the same structure as the nascentRNPs,

sinceDI RNPsandnascent RNPs have identical

densitiesin both isopycnic Renografin (Fig. 7C)

andCsCl gradients. This suggests, but does not

prove, that the RNPs contained in the trailing

fractions are dissociated from their templates

andarebeingseparatedfrom thepeakfractions

onthe basis of size.However,we cannotexclude

thepossibility that conformational factors cause

someof the nascent RNPs to move more slowly

through the Renografingradient, thus forming thetrailingedge.

Effect of Renografin on transcribing

complexes. The total intracellular viral RNPs

consist oftranscribingcomplexes,nonenveloped

virionRNPs to be assembled into virus particles,

andreplicatingcomplexes. Previous studies have shown that intracellular RNPs cosediment with

virion RNPs insucrosegradientsas120S to 140S

molecules. NascentmRNAcanbe detected

as-sociated withsomeof these intracellular RNP

complexes; thus,itis

likely

that theassociation

of nascent product strands to template RNA

doesnotgreatlyaffect the sedimentation

coeffi-cient of the transcribing complex (31, 35). We

wanted to determine whetherreplicative

com-plexeswerefound in the 140Speak,aswellas to

determinethe effect ofRenografinon

transcrib-ingcomplexes.

A 5-min-pulse-labeled cytoplasmic extract waspreparedfrom VSV-infected cellsat5hp.i.

The extractwas divided into twosamples; one

was centrifugedin a 15 to 47%

Renografin

gra-dient (Fig. 8A), and theother samplewas

cen-trifuged in a 15 to 30% sucrose gradient (Fig.

8B). The peak fractions from the Renografm

gradientin Fig. 8A, whichcontained the

repli-cativecomplex,werecombinedasindicatedand

recentrifugedin a 20 to47%Renografin gradient

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502 SIMONSEN, HILL, AND SUMMERS

3

2-aE~~~~~~~~~~~~~~~~~~~~~~~a

C1.246 -1.30

6-61.20

4

[image:9.504.58.246.52.384.2]

FRACTIONNO

FIG. 7. Comparison of RNPsderivedfrom

DIpar-ticdeswith thereplicativeRNPcomplexes.3H-labeled

DIparticles ofthe MS-T strain werepurified byrate

zonal sedimentation insucrosegradients. The

puri-fied preparationwasadjustedto aconcentration of

0.2% deoxycholate-0.3% Nonidet P-40 in NTbuffer

anddivided into twosamples. Onesamplewas

lay-ered ontoa 15to50%Renografin gradient,andthe other was layeredonto a20to60%gradient. Both

werecentrifugedat23,000 rpm and4°Cin anSW27.1

rotor.FractionswereanalyzedasdescribedforFig.

1. (A) Cytoplasmicextractfrom 5-min-pulse-labeled

cellscentrifugedina 15 to50%Renografin gradient for16h at23,000 rpm in theSW27.1 rotor. (B) DI

RNPcentrifugedfor16h in the15to50%Renografin

gradient.(C)DIRNPcentrifuged for80h in the 20 to

60%Renografin gradient. Virionnucleocapsidswere

foundtocosediment withpeak I(fractions 19 to 23)

atadensity of 1.22g/cm3(datanot shown).

(Fig. 8C). All of the radioactivity wasfound in

theareaofpeak I, andno material was found in

the region of the gradient where the nascent

mRNA species have been isolated (peak II).

Fractionsfrom differentregions ofthe 140S peak

in thesucrosegradient (Fig.8B)were also pooled

asindicated andrecentrifugedonidentical20 to

47% Renografin gradients (Fig. 8D, E, and F).

The results of these three gradients were

vir-tually identical; in addition to the material at

peak I, a peak at the position of peak II was

observed. Although transcription accounts for

nearly 90% of the total RNAsynthesis in

VSV-infected cells (38), peak II does not represent

90%of theinputradioactivity because the fully

synthesizedmessages arefoundatthetopof the

nonequilibriumgradients asfree RNA and not

inpeak II (datanotshown). These results

dem-onstratethatthe 140S RNPs isolated insucrose

gradients contain bothtranscribingand

replicat-ing RNP complexes in the same proportions

throughout the 140S peak and that replicating

products can be separated from transcribing

products in Renografin gradients due to some

undetermined compositional differences or

structuraldifferencesorboth.

DISCUSSION

The ability of the VSV RNP core isolated

from virions to transcribe in vivo and in vitro

has beenwell documented (1, 2,7,20, 25-27,32).

Thetranscriptaseenzymehasbeen showntobe

composed of the L and NS proteins, and the

template has been demonstrated to consist of

minus-strand 42S RNA and N protein (17, 18,

28). Theenzyme isinactive with 42Sdevoid of

proteinorin thepresenceofantibody directed

againstthe Nprotein-RNAcomplex (11, 17). It

thus seems well established that the template

fortranscription isanRNPcomplex.Ithasbeen

assumed that thetemplateforreplicationisalso

anRNPcomplexonthe basis ofgeneticstudies with temperature-sensitive mutants. Several

mutantsingroupI(Lprotein)andgroupsIIand

IV (presumablyNand NS proteins) have

tem-perature-sensitive replicative abilities (15, 16,

30). Direct evidence for the role of theseproteins

inreplicationislackingduetothe absence ofan

invitroreplicationsystem. The datapresented

aboveand in anotherreport(Hilletal.,

submit-ted forpublication)supportthehypothesisthat

VSVreplicationoccurs on anRNPtemplate.

The nuclease resistance and density of the

nascentRNPcomplexesareindications that the

nascent RNA species found in the RNPs are

quickly associated with protein. Preliminary

re-sults from this laboratory have indicated that

oneof theproteins whichrapidly associates with

thecomplex is indeed the VSV Nprotein. The

association of thisprotein with thenascentRNA

isintriguing, since thismay betheprocess with

which cycloheximide interferes, and thus

in-hibits, replication. The strong and stable

asso-ciation ofN protein with 42S RNA (9) is not

seenwith theVSVmRNAspecies,eventhough

the nucleotide sequences ofthe mRNA are a

subset of the plus-strand 42S RNA sequence.

The "leader"RNA,which isidenticaltothefirst

I-I-B

J. VIROL.

,<2q 1,0 L

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[image:10.504.115.404.69.310.2]

FIG. 8. Renografingradientcentrifugation of RNPs obtained from Renografin and sucrose gradients. A

5-min-pulse-labeled cytoplasmicextractwasprepared as described for Fig.1.Thesample was divided; one-half

waslayeredonto a 15 to50%Renografin gradient, and theother halfofthesample was layered onto a 15 to

30% sucrosegradient. TheRenografingradient wascentrifugedfor 16 h at 23,000 rpm and 4°C in an SW27 rotor, and the sucrosegradient was centrifuged for 16 h at 16,000rpm in thesame rotor. Fractions were removedfromboth gradients and recentrifuged in 20 to47%Renografin gradientsfor 16 h at 23,000 rpm in an SW41 rotor. (A) Cyplasmicextractcentrifugedin a 15 to50%Renografin gradient. (B) Cytoplasmicextract

centrifugedin a 15 to30% sucrosegradient. (C)Pooledfractions from Renografingradient in (A) rerun in a

20 to47%Renografin gradient. (D)Fraction18fromthe sucrosegradientin(B)recentrifugedin a 20to47%

Renografin gradient.(E) Fraction 21from (B) recentrifugedin a 20 to47%Renografingradient.(F)Fraction

24from (B) recentrifugedin a 20 to 47%Renografin gradient.The sedimentationpositionsofthe60S and 40S

ribosomalsubunits and the virion RNP are indicated in(B) bythearrows.The materialin(C), fractions5 to

7, which wasfound at agreaterdensitythanpeak I,represents aconformer ofpeakI. The natureofthis materialispresently beingexamined.

48 nucleotides atthe 5' end of the plus-strand

42SRNA (13, 14) isnotfound in themessages

and thus couldcontainasequencewhichacts as

abinding site for the N protein. However, the

leader isnotfound in the infected cellas ashort

RNP, which would be expected if the leader

alone were the signal for the association of N

protein with the nascentRNA (Simonsen,

un-published data). The addition of N protein to

the nascentRNA maywell bean integralpart

ofreplicationwhich is associated with either the

replicase enzyme or the template. This would

accountfor thenuclease resistance anddensity

inCsCl ofthe nascentstrands. Wearepresently

examining

the mechanism by which

cyclohexi-mide inhibitsreplication,aswellasthekinetics

of protein association with the nascent

repli-cated RNA in orderto clarifythe role that N

proteinplaysinreplication.

These datamay alsosuggest that most of the

nascent RNPs remain associated with their

RNPtemplates.We have observed that thevery

homogeneous Renografin peaks from samples pulse-labeledfor15and30min(Fig. 1CandD)

and the peak fraction ofa5-min-pulse-labeled

sample (Fig. 6A) contain RNA ranging in size

from 42S downtoless than18S,but thetrailing

fractions ofpeak I contain primarily less than

full-lengthRNA(Fig.60).Theobservation that

RNPs derived from DIparticles which have a

genomeapproximately 15% of thewild-type

ge-nomecosediment with nascentRNPsfrom the

trailing edge, which are also 15% completed,

leads us to believe that perhaps the trailing

fractions do containnascentRNPs whichare no

longer attachedtotheirtemplates.

Our resultsareconsistentinestablishingthat

nascent replicating RNA is found as part ofa

RNP. Wehavenotfoundsignificantamountsof

double-stranded RNA in thesecomplexes (Hill

et al., submitted for publication). We suspect

that double-stranded RNA molecules which

http://jvi.asm.org/

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504 SIMONSEN, HILL, AND

have been examined (33, 37) as possible

repli-cativeintermediatesareperhaps artifacts of

iso-lation. Since the structures we have isolated

appear tocontaintemplateandnascentstrands,

presumablylinked by thereplicative enzyme or

a small region of base pairs, the addition of

phenolto removeprotein wouldlikelyenhance

thehybridization of thesecomplementaryRNA

molecules which are alreadyincloseproximity

(23, 29). Itthusseemsreasonable toacceptthe

phenol-extracted double-stranded RNA

mole-cules as "collapsed" replicative complexes,

which probably approximate certain aspectsof

replication.

Theisolation ofareplicative complex, coupled

with the recent demonstration of full-length

VSV RNA synthesis in vitro (S.

Batt-Hum-phries,C. C. Simonsen, and E. Ehrenfeld,

Virol-ogy, in press), may now make it feasible to

develop anin vitro replication systemby using

the complexes isolated from Renografin. The

reconstitution ofareplication systemisclearly

required to fully understand the regulation of

replication and the role of host factors inVSV

replication, andwe arecurrentlyattemptingto

studythissystemingreaterdetail.

ACKNOWLEDGMENTS

Wethank Steven Humphries for the gift of labeled polio-viruspreparations, and we are grateful for the kind gift of VSVDIpreparations from Sue Moyer. The helpful discussions of D.Kolakofsky,0. Richards, S. Casjens, C. Georgopoulos, D.Carroll, and A. Larsen are also greatly appreciated.

C.S.is arecipientof aUniversityofUtah Graduate Re-searchFellowship.This work was supported by Public Health Service grant AI-12316-04 from the National Institutes of Health and by National Science Foundation grant PMC77-17867A01.

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