Temperature-sensitive defect of vesicular stomatitis virus in complementation group II.

CopyrightC1977 American Society forMicrobiology Printed inU.S.A.

Temperature-Sensitive Defect

of Vesicular Stomatitis Virus

in

Complementation Group II

ARLETTE COMBARD,* CHRISTIANE PRINTZ-ANE, CLAIRE MARTINET, ANDPIERRE PRINTZ Institut deMicrobiologie, Universitg de Paris-Sud, Centre d'Orsay, 91405 Orsay, France

Received for publication5August1976

The prototype member of the complementation groupIItemperature-sensitive

(ts)mutantsofvesicularstomatitisvirus, tsII052,has been investigated.In ts

II052-infectedHeLacellsatthe restrictive temperature (39.50C), reduced viral

RNA synthesis was observed by comparison with infections conducted at the

permissivetemperature (30'C). It wasfound that foran infection conductedat

39.50C,no38S RNA or intracytoplasmic nucleocapsids were present. For

nucleo-capsids isolated from ts II052purified virions or fromts II052-infected cells at

300C, the RNAwassensitive to pancreaticRNaseafter an exposure at 39.50Cin

contrast totheresistance observed for wild-typevirus. The nucleocapsid

stabil-ityof wild-typeviruswhenheatedto 63°Corsubmittedto varyingpHwas not

foundinnucleocapsids extractedfrom tsII 052purifiedvirions. The datasuggest

that fortsII052there isanaltered relationship between the viral 38S RNA and

the nucleocapsid protein(s) by comparison with wild-type virus. Such results

arguefor thecomplementation groupIIgene product being Nprotein, so that

thetsdefectints II 052representsanalteredN protein.

Early analyses of the temperature-sensitive

(ts) mutants of vesicular stomatitis virus

(VSV), Indiana serotype, demonstrated that

mutants belonging to complementation group

II could not be classified clearly as RNA+ or

RNA-. Inourhands,atthe nonpermissive

tem-peraturethesemutants neversynthesizedmore

than 50% of their normal RNA yield (9). For

those belonging to the Glasgow ts collection,

the prototype ts II G22 has a complete

RNA-phenotypeand ts G21induces a poor and

varia-ble amount of RNA synthesis (16). Studies of thetsmutantsof group IIhave beenhampered

by certain characteristicsregularly associated

with preparation of mutant stocks, i.e., low

yield, leakiness, and high reversionrate.

Nev-ertheless, the results obtainedin two laborato-ries(9, 23)haveledtothe conclusion that thets

defect in group II mutants might concern the

replication process of the virus. To date, this

aspect of the VSV infectioncycle hasnot been

defined. On the assumption that studies of the

group II ts mutants might help to elucidate

some stepsof the replication process, we have

designed experimentstodetermine the nature

of thets II052defect. Toobviatetheproblemof

leakiness andmultiplicationofrevertants, both

invitroandinvivostudieshave beenused.The

results obtainedrevealthatthenucleocapsid of

the ts II 052 mutant is unstable andprobably

due to some defective association between the

protein and the genome RNA.

(This work istakeninpartfrom athesisby

C.M. submittedtothe Universitkde Paris-Sud

in partial fulfillment of the requirements for

thePh.D. degree.)

MATERIALS AND METHODS

Cells andviruses. All analyses ofin vivo RNA syntheses used HeLa cell monolayers grownas de-scribedpreviously (18) or, for experimentsinwhich alow level ofresidual cellular protein synthesis was required,inchicken embryo cultures (3).

VSVs (Indiana serotype) investigated were: a heat-resistant wild type with equivalent growth ca-pabilitiesat 39.5and 30°C;a tsmutantbelongingto complementation groupIV,tsIV0111 (8);a ts mu-tant belonging to complementation group II, ts II 052 (8);andtwoindependent revertants of wild-type phenotype obtained from subclones of ts II052. All virusstocksweregrown onchicken embryo cells and contained only standard B particles (18). Virus stocks were verified by suitable complementation tests and checked for their inability to grow at 39.5°Corlowcontentofrevertants. Usually progeny virusproduced by thetsII052mutant atthe nonper-missive temperature represented 1%that obtained atthepermissive temperature. Sincetheyieldsof ts II 052 werefrequently of low titer, a concentration stepwasusually used, which involved pelletingthe virions(22,000 rpmfor1h in theSpinco SW27 rotor). Analysesofuridine-labeled products. Aftera 45-minadsorptionperiodat roomtemperature,infected cellsweretreated with 10,ug ofactinomycin Dper ml. Whenprimarytranscriptionwasstudied,

cyclo-heximide (100 jg/ml) was also added. Cells were

labeled byincorporating [3H]uridine (20 ,uCi/ml)in 913

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ET AL.

the culturemedium. Cytoplasmic extractswere pre-pared as described previously (3). Nucleocapsids wereobtained from these extractsby successive ad-dition of deoxycholate and Brij-58, both to a final concentrationof 0.5%,and centrifugationin alinear sucrose gradient (15 to 30%)ineither NEB(0.01 M Tris, pH 7.4, 0.01 M NaCl, 0.02 M EDTA) or HSB (0.01MTris, pH 7.4, 0.5MNaCl, 0.05MMgCl2) at 27,000 rpm for 3 h at4VC in the Spinco SW27.1 rotor. RNA species wereisolated from the cytoplasmic ex-tract by addition of sodium dodecyl sulfate (SDS) (1%, wt/vol, final concentration). The RNA species were fractionated at 21,000 rpm for 15 h in the SW27.1 rotor, usinga 15 to30% sucrosegradientin SDS-TENbuffer (18).

Analysis of viralproteins. Chicken embryocells werepreferred for viral protein studies because the shutoff of cellular protein synthesis is both rapid and total (3).

Conditions for labeling viral proteins within in-fected cells werethe same aspreviously described (3). After the recovery from reticulocyte standard buffer (RSB)-disrupted cells, cytoplasmic extracts were separated into soluble and insoluble compo-nents bycentrifugation at 42,000 rpmfor90 minin the Spinco SW65rotor.Proteinswereextracted from the supernatantand from thepellet by boiling sam-plesin 1%SDS, 1%mercaptoethanol, and1Murea, andwereanalyzedon 7.5%neutralSDS-acrylamide

gels. Electrophoresiswasperformedand radioactiv-ity wasestimated asdescribed elsewhere (3).

Isolation of nucleocapsidsfrom virions. Pellets of concentrated virionswereresuspendedin avolume of RSB such that theprotein concentrationdidnot exceed0.2mg/ml, andnucleocapsidswereextracted following the proceduredescribedby Emerson and Wagner (5), except that the 1-h solubilization of viruses was conducted at room temperature. Nu-cleocapsids were separated from the solubilized viral componentsbycentrifugationat45,000 rpm(Spinco SW65 rotor) for90 min. Thepelleted nucleocapsids

wererinsed several times withasaline buffer and thenhomogenized inthemedium required for fur-theranalysis.

Chemicals. Mostcompoundswerepurchasedfrom sourcesmentioned elsewhere (3, 18). ActinomycinD was agift from Merck, Sharp & Dohme. Triton X-100and Brij-58 were obtained from Sigma Chemical Co., St. Louis, Mo., and sodium deoxycholate was purchased from BDH, Poole, England. Radioiso-topes were obtained from CEA, Saclay, France. Pancreatic RNase(crystalline 5x)wasfrom Nutri-tionalBiochemicals Co., Cleveland, Ohio, and Reno-grafin-76wasfromSquibb, Princeton, N.J.

RESULTS

Characteristics of ts II 052 infections. (i)

Viral RNAsynthesized at39.5°C. Sucrose

gra-dient separations of [3H]uridine-labeled viral

RNAinducedat39.5°C by ts II 052 in HeLa cells

were found to produce an unvarying pattern. Peaks of 28S and 13 to158mRNA were always

observed, but not the peak of388 RNA (Fig.

1A). No qualitative variation was observed

0

10 20 30 40

FRACTIONS

FIG. 1. In vivo RNA synthesis directed by ts II 052. HeLa cells were infected for 45 min at room temperature by tsII 052 at a multiplicityof infection

of -40PFU/cell. Theythen receivedcomplete Eagle

minimumessential medium with 10pgof actinomy-cinDper ml and[3H]uridine(20,uilml) from1to4 hpostinfection. Cells were washed, scraped off the plates, pelleted, and disrupted in RSB. SDS was added (1%, wt/vol,final concentration) to solubilize cytoplasmic RNAs, which were thenanalyzed by cen-trifugation in 15 to30% sucrose gradients (21,000 rpm for 15 h in the Spinco SW27.1 rotor at 17°C). Arrows onthe figure show the peak positions of 18S and 28S ribosomal markers from HeLa cells. (A)

CompleteRNAsynthesis; (B)restricted RNA

synthe-sisafteradditionof100 pg of cycloheximideper ml justafteradsorption.Symbols: (@) 30°C; (0)39.5°C.

after changing the lengthortime of the labeling

period(1h or3hbetween 0to7 hpostinfection)

or the input multiplicity (10 to 100 PFU/cell), eventhough these factors do affect the apparent quantities of mRNA synthesized (unpublished

observations). First, along with the timeof

in-fection, a slight amplification was demon-stratedinthequantityof mRNAprogressively

produced at 39.5°C. Nevertheless, at all times

transcription at the nonpermissive

tempera-ture was severely depressed compared with

thatat30°C; this was noticed also for the early stages ofprimary transcription (Fig. 1B). In

these features, ts II 052 withstood a high

tem-perature less well inHeLacells than in BHK cells (1) andslightly differed fromtsIIG22 (23). In addition, in our hands, after blocking by

cycloheximide less and less RNA was

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sized at39.50C, whereas at300C alinear

accu-mulationoccurred for several hours, asit does

normally (11).

Aneffect of themultiplicityof infectionupon

theamountof RNAsynthesis obtained withts II052-infected chicken embryo cells(9)wasalso

observed in the HeLa cell system. For HeLa

cells, the quantity ofviral RNA produced at

39.50C byts II052wasdirectly proportional to

the multiplicity of infection used (.100 PFU/

cell). At higher multiplicities, the amount of

RNA synthesis plateaued (50% of the 300C

yield).

(ii) Intracellular nucleocapsids. To

deter-mine whether the absence of 3&S virion-like

RNA at39.50Cwasduetothe absence of

prog-eny nucleocapsids, cytoplasmic extracts

ob-tained 4hpostinfection from ts II 052-infected cells were separated in 15 to30% sucrose

gra-dients, and radioactivitywassoughtinthe

re-gionof thegradient where VSV nucleocapsids

should berecovered (Fig. 2). A120S nucleocap-sid peak was obtained from cytoplasmic ex-tracts made from cells infected at 300C, but

none wasdetected fromextractsobtained from cells incubated atthe nonpermissive

tempera-ture. Inaddition, nucleocapsids have notbeen

seenby electronmicroscopy of cells infected at

39.50C(V. Deutsch, unpublished observations).

Our results corroborate those of Unger and Reichmann (23), who also observed no

virus-i. 6

NC

I.

10 20 30 40 sI

Bottom FRACTIONS

FIG. 2. Intracellularnucleocapsidsproc II052. HeLa cells were infected in the r

actinomycin D,labeled with[3H]uridinefc processedtoobtaincytoplasmicextracts as Thedetergents deoxycholateandBrij-58u

totheextracts (finalconcentrationsof0.5 for each) just before layeringtheextractson

sucrosegradientsin NEBbuffer.Centrifuh

performedin theSpincoSW27.1 rotor at4 at27,000 rpm. Asa marker, unlabeled

sides extractedfrom wild-typevirions were

in the samegradient; theirpeak position

minedby optical densityisrepresentedby inthefigure. Symbols: (-)30°C; (0)39.5

like RNAornucleocapsidsintsIIG22-infected

cells incubated at the nonpermissive

tem-perature.

(iii) Cytoplasmic compartmentalization of

the N protein. Cells infected with ts II 052 at

either 30 or 39.50C were labeled for 3 h with

[3H]leucine. Cytoplasmic extracts were

re-solved accordingtothe procedure of Wagneret

al. (26) intoasedimentable anda

nonsediment-able fraction.

At300Cthe distribution of viralproteins

pro-ducedints II052-infected cells wassimilarto

that obtained from thewild-typevirusinfection

(Fig. 3B). The pellet contained both viral

pro-teins that were inserted into cellular

mem-branes (G and M) and those thatwere

associ-ated with genome RNA in nucleocapsids (N,

FRACTIONS

FIG. 3. Separation intosedimentable and

nonse-dimentable components of the cytoplasmicproteins

produced bytsII052.Afteranadsorption period of

Top 40 min at room temperature (multiplicity of

infec-Top tion, -50PFU/cell), plates(100mmindiameter) of

duced byts chicken embryo cells werecovered with5ml of1/10

presence of (vollvol)-dilutedminimumessential medium and

in-vr4h,and cubatedatthe indicatedtemperature;[3H]leucine (20 sinFig.1. uXCi/ml)wasaddedfrom1to4hpostinfection.At the

vereadded end ofthe labeling period,cytoplasmicextracts were

%, wt/vol, obtained by disrupting cells inRSB witha Dounce 215to30% homogenizerand separating them into sedimentable nationwas and nonsedimentable fractions by centrifugation at £°C for3 h 42,000 rpmfor 90 min in the Spinco SW65 rotor. nucleocap- Proteins were extracted from thesupernatantfluid alsospun (0)and thepellet(*)andanalyzedon a7.5%neutral as deter- SDS-acrylamide gelat5mA/gel for6 h.(A) Infected

thearrow cellsincubatedat39.5°C;(B)infected cells incubated

F0C. at30°C.

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916 COMBARD ET AL.

NS, and L). In the supernatant, the proteins present asfree molecules within thecytoplasm

wereevident. Itwasfound that theNSprotein

remained essentially in the nonsedimentable

fractiontogether with about30%of theN

mole-cules.

For ts II052-infected cells incubated at39.50C

(Fig. 3A), 50%of theN proteinremainedinthe

soluble fraction and the amount of labeled N

proteinthereinwas greaterthan theamountof

labeled NS protein (compare Fig. 3A and B).

These observations, together with the lack of

nucleocapsids observedinthets II052-infected

cells described above, suggest that possibly N

proteins were present in the pellet fraction

(Fig. 3A) notbecausetheywereassociated with

RNA, but because they were associated with

some other structural entity in the infected

cells (e.g., polysomes, membrane components,

etc.). Other possible explanations could be

en-tertained;however,itisclearthat(i)N, G, NS,

andMproteinsweresynthesized at39.50Cin ts

II052-infected cells and (ii) the distribution of

N protein wasabnormal.

Studyofts II 052nucleocapsidsinvitro. The

data obtained from the in vivo experiments

reported above suggest that ts II 052 may be

defectivein someaspectofitsreplicative

capac-ity. One possibility, as suggested previously

(9), is that the nucleocapsids synthesized at

39.50Carerapidly destroyed duetoanunstable

featuretheypossess atthattemperature. This

possibilitycanbe tested duetotheproperty of

VSVnucleocapsidsto befullyresistantto

pan-creaticRNasedigestion(19, 25)and other

dena-turing agents. Therefore, we examined the

ef-fect ofsome of thesefactors onthe stability of

nucleocapsids from ts II 052 virions and those

from wild-type virus. Three of these

experi-ments are reported, which demonstrate that

thereare infact significant differences between

thenucleocapsids ofts II 052and those of

wild-typevirus.

(i) Preparation of virionnucleocapsids.

Nu-cleocapsids labeled with suitableradioisotopes

wereobtained by treating a dilutedsuspension of virions in RSB by the high-salt solubilizer cocktail of Emerson and Wagner (4). It was

found that this treatment gave nucleocapsids

containing 38S RNA and Nprotein (no L pro-tein). The amountofNS protein stillassociated

withthepurifiednucleocapsidswasdetermined

by labeling the virion stock with

[32P]phos-phoric acid and determining their content of

NSphosphoprotein (14, 20)bycomparisonwith

the original virus preparation. Only5% of the

initialNSprotein wasrecoveredinthe

nucleo-capsid pellet (eitherofwild-type virus orofts

II052), whichsuggests that each nucleocapsid

containing some 700 to 2,300 molecules of N protein probably possessed no more than 2 to

11 NS molecules (2, 24). For one specified

ex-periment, the recently described extraction

procedureofEmersonand Yu (6) wasused; in

thiscase, virtuallyno NS phosphoprotein was detected in thenucleocapsid cores.

(ii) Action of pancreatic RNase on nucleo-capsids. Labeled nucleocapsids obtained from wild-type or ts II 052virionswerediluted in 2 x SSC (SSC = 0.15 M NaCl + 0.015 M sodium

citrate), pH 7.4, in order to obtain the same

final RNAconcentration (5 ,ug/ml). Theywere

incubated at 30 or 39.5°C for varying periods

ranging from 10 to 240 min. At the indicated

time, aliquots were removed and divided into

two samples. One was immediately cooled in

ice and trichloroacetic acid precipitated; the

second was digested with pancreatic RNase

(100 ,ug/ml, final concentration) for 30 min at

370Cand assayed for acid-precipitable

radioac-tivity.The RNase resistance obtainedwas plot-tedagainstthetimeof incubation(Fig. 4).

Nu-cleocapsids from the wildtyperemained

essen-100

0 0

so o -_

0

4-1

1 2 3 4

Time (h)

FIG. 4. RNase resistance ofts II 052 and wild-type nucleocapsids upon incubation at 30 and 39.50C. [3H]uridine-labeled nucleocapsids were

ex-tracted from ts II 052 or wild-type virions (=101°

startingPFU)asdescribedinMaterials and Meth-ods.Theywerehomogenizedin 2xSSC, pH 7.4, and divided into two batches, one incubated at 300C and the other incubatedat39.50C. At theprescribed

time, aliquots were cooled and assayed for their trichloroaceticacid-precipitable radioactivity, either before or after30 min of subsequent incubation at

37'C with100pgofpancreatic RNase per ml.

Sym-bols: *, wild-type nucleocapsids (the same curves were observed upon incubationat39.5 and30°C);

*, ts II 052 nucleocapsids incubated at 30°C; 0, tsII 052nucleocapsids incubatedat39.5°C.

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tially completely resistant to RNase at both 30

andat39.50C. Thiswas also the case for ts II 052

nucleocapsids heated at3000; however, the ts II

052nucleocapsids were much more labile

dur-ing their incubation at 39.50C. Nevertheless,

total degradation of the RNA was not obtained

and a plateau (20 to 60%) of residual RNase

resistance was observed (dependingon the

ex-periment). Anexplanation for this plateau was

obtained byexamining thesedimentation

prop-erties ofts II 052 or wild-type nucleocapsids

either in their native form or after in vitro

incubation at 39.50C with or without RNase

treatment (Fig. 5). Freshly isolated

nucleocap-sids from ts II 052sedimented only at 120S in sucrose gradients, as did nucleocapsids from

K'

a

standard wild-type particles. Radioactivity in

this peak was insensitive to pancreatic RNase

(Fig. 5A). After incubation for 2 h at 39.50C,

nucleocapsids from wild-type virus also were

recovered in the form of a120S peak resistant to RNase (Fig. 5B). However, nucleocapsids

from ts II 052 gave a more heterogeneous

pat-tern with a bimodal distribution around two

peaks (Fig. 5C), which were estimated to

pos-sess sedimentation coefficients of 120S and

140S.The120S peakwasessentiallyeliminated

bypretreatment withRNase,whereas the140S peakwas not affectedbysuch treatment. These

results suggest that the explanation for the

residual RNase resistance seeninthe previous

experiment (Fig. 4) was associated with the

BOTTOM- FRACTNS _TOP

FIG. 5. Sucrosegradient separation oftsII 052 orwild-typenucleocapsids exposedor notexposedto39.5°C andthensubmittedor notsubmittedtopancreaticRNase.Nucleocapsidswereextractedfrom [3H]uridine-labeled virionsofts1 052mutant orrevertants orofthewild-typestrainand dilutedin 2xSSC.For each virus, two equivalent samples (-5 Pg of RNA) were analyzed: one sample consistedof freshly isolated nucleocapsids (A, D), and the other was incubatedat39.5°C for2h(B, C, E, F). Eachsample wasthen dividedin twoaliquots. Onewasimmediately cooledinice(0);the otherwastreatedwith100pgofpancreatic RNaseper mland incubatedat37°C for30min(0),after which the aliquotwascooled andreceived40p1 of

diethylpyrocarbonate to inhibit the RNase activity. All samples were centrifuged on15 to 30% sucrose

gradientsinHSBat 27,000 rpmfor3h (SW27.1 rotor).Exogenous wild-typenucleocapsidswereaddedto

eachsampletolocate the 120Speak position of the normal VSVnucleocapsid(arrow).In AandD,the wild-typenucleocapsid sedimentationpatterns werenotreportedbecause oftheirsimilaritytothoseoftsII052;

andfor thesamereason,onlyone revertantcontrolprofileisgiven.

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918 COMBARD ET AL.

occurrence and amountofthe 140Speak.

With various preparations of ts II 052, we

haveregularly observed the RNase sensitivity

of the 120S nucleocapsid and the occurrenceof

the 140Speakuponincubation at39.50C;

how-ever, the relativeproportion of 140S and 120S

varied with the preparation (compare Fig. 5C

andFig. 6). It varied also for each preparation

with the time of incubation: the radioactive

material was first mainly in the 120S region,

and then the140S peakprogressively appeared

and was often thehighestpeakafter 2 to 4 h of

incubation at39.50C.

Other minorpeaks have also been

occasion-ally observed; they are believed to be

aggre-gates of one formoranother.

It hasbeendetermined that thenearly total

degradation of the 120Sts II 052nucleocapsids

by RNase doesnotleadtosmallderivativesin

the top half of the gradient but only to

acid-soluble material. The RNase-resistant 120S

peak has been found to contain complete

nu-cleocapsids possessing 38S RNA andNprotein.

Possibly these resistant structures represent revertantnucleocapsids.

The 120S and 140S entities were shown to

possessthe sameratio of RNA to protein

con-tent, asjudged bydouble-labeling experiments

using 14C-labeled amino acids and [3H]uridine

(Fig. 6)and by theircommonbuoyant densities

inCsCl (1.32g/ml).The sedimentationinto two

distinct nucleocapsid categories could not be

related to the length of their isolated

compo-nents: both yielded only 388 RNA and N

pro-tein, withnodetectablechangeintheir

respec-06

5

0

a --I,

FRACTIONS

FIG. 6. Protein and nucleic acid content ofts II

052 nucleocapsids incubatedat39.50C for2 h. Nu-cleocapsids wereprepared from tsII052virions

la-beled with both '4C-amino acids and [3H]uridine. They were incubated at 39.5°C for 2 h and then centrifugedina15to30%sucrosegradientinHSB

as in Fig. 5. Symbols: (@)14C-labeledaminoacids;

(0)[3H]uridine.

tive electrophoretic mobilities (data not

shown); precise evaluation ofthe NS protein

contamination couldnotbe done. However,the

same experiment as in Fig. 5 was conducted

with ts II 052 nucleocapsids that were

com-pletely NS depleted by the purification

proce-dure of Emerson and Yu (6). Nucleocapsids fromtsIV 0111 werechosen as controls (Fig.7).

Nucleocapsids just recovered after the final

treatmentof both mutants sedimented as two

peaks, 120S and 140S, which were RNase

re-sistant; somepreferential loss of 120S material

wasevident withts II 052. Nochange occurred

after a 2-h incubation at 39.5°C ofts IV 0111

nucleocapsids, whereas the 120S peakwas

re-ducedtoasmallRNase-resistantamount inthe

ts II 052 pattern.

Itwasof interest todeterminewhether

cyto-plasmicts II 052nucleocapsids obtained at the

permissive temperature possess the same

sen-sitivity to RNase as do the virion

nucleo-capsids. Therefore, ts II 052-infected cells

la-beled with [3H]uridineat300Cfor3hwereused

topreparecytoplasmicextracts inRSBasusual.

Half of eachextract received anequal volume

ofa 4x SSC solution and then was incubated

with pancreatic RNase(finalconcentration, 100

,ug/ml)

similarly digested after a prior incubation at

39.5° for2h. Parallel analysesonHSB-sucrose

gradients of the remaining acid-insoluble

radio-active material demonstrated that the invivo

nucleocapsids fromts II052-infected cells were

sensitive to the nuclease digestion when, and

only when, they were exposed to 39.5°C. Both

120S and 140S peaks were still generated, the

radioactivity of the 140S peak beingRNase

re-sistant and the 120S material being 60%

de-graded (data notshown).

(iii) Melting of the nucleocapsidcomplex at

high temperature. "Melting" curve analyses

canbe used for studying interactions between

proteinsand nucleic acids. Byfollowing the fate

of RNA and protein usingwild-type

nucleocap-sidsheated for30min atvarioustemperatures,

M. Soria (Ph.D. thesis, Harvard University,

Cambridge, Mass., 1974)demonstratedarapid

dissociationofVSVnucleocapsids at 60 to650C

and a subsequent degradation of the nucleic

acid.

Wetherefore compared thestability of

wild-type and ts II 052nucleocapsids after30 minof

incubation in RSB (pH 7.4) at 37, 40, 50, 60, 65, 70, and 1000C. For both viruses, exactly the

same narrow temperature range for

dissocia-tion,60 to650C,wasobtained(Fig. 8).To study

the differentialthermolability of the twoviral

nucleocapsids, 630Cwaschosen. Nucleocapsids

extracted from 14C-amino acid-labeledts II 052

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C)

U0

a.

FIG. 7. Analysisofts II052andtsIV0111 nucleocapsidsafter complete removal of the NS protein. The procedureofEmerson and Yu (6) was used to completely remove the NS protein from nucleocapsids ofts 1 052 andtsIV 0111.As inFig. 5, for each mutant, the nucleocapsid suspension in 2xSSCwasdividedin two samples;one wasmaintained inice(top),and theotherwasincubatedat39.50C for2h(bottom).One-half of

eachsample wasthen treated by pancreatic RNase (0), and the other halfwas untreated (-).

100

50_

z

35 51060 7001WC

FIG. 8. RNase resistance ofts II 052 and wild-typevirionnucleocapsidsincubated atdifferent tem-peratures. [3H]uridine-labeled nucleocapsids were obtainedfromts1 052andwild-type virions. After

suspension inRSB,pH 7.4, they were dividedinto aliquots of100 p1(-15,000 cpm), which were incu-batedfor30 min at the indicatedtemperatures. At the endof the incubation, all samplesweredigested

with25pgofpancreatic RNase permlat37 Cfor30 min before acid-precipitable radioactivity was

as-sayed. Symbols: (0)wild-type nucleocapsids; (0)ts

1 052nucleocapsids.

and [3H]leucine-labeled wild type were mixed

and incubated at 63TC. After incubation, the

remaining nucleocapsid structures were

puri-fiedfrom released radioactive material by

su-crosegradientcentrifugation;all fractions

sedi-mentingfasterthanorwith the 120Sexogenous

marker werepooled. The amount of each

iso-tope recovered in acid-precipitable material

versusthe time of incubationat630Cwas

deter-mined (Fig. 9). Other thanacommon 20%loss

inradioactivity ofbothlabels duringthe first 20

min of incubation at 630C (which could have

been duetodamageduringtheextraction

pro-cedure), itwasapparentthat the wild-type

nu-cleocapsidswere moreresistanttodegradation

than were those of ts II 052. We concluded,

therefore, that theinteractionsbetween the

ge-nome RNAand thestructuralprotein(s) of ts II

052 nucleocapsids were weaker than those

in-volvedinthe wild-type nucleocapsids.

(iv) Influence of pH on the nucleocapsid

configuration.Thechemical bonds involvedin

the stabilization of VSVnucleocapsids are not

known. However, ithasbeen found thatacidic

pH hampers nucleocapsid formation (7, 12).

The sedimentation properties ofnucleocapsids

fromwildtype or ts II 052 have been studied at

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920 COMBARD ET AL.

u

q, 100

IV

.0

X 50

G

0

4

0

v

b ~ ~~~~~9

10 20 30 40 50 60 Time (min)

FIG. 9. Stability oftsII052or wild-type

nucleo-capsids incubated at 63°C. Nucleocapsids were

ex-tracted from[3H]leucine-labeled wild-type virus and from 14C-amino acid-labeled ts II 052 virus. The preparations weremixed andhomogenizedinRSB,

pH 7.4. The suspension was incubated at 63°C 0.1°C, and at intervals aliquots were removed,

re-solvedon sucrosegradients, andassayed for label

remaining in the nucleocapsids. Symbols: (V) 3H-labeled wildtype;(V) "4C-labeledtsII052.

40CinsucrosegradientscontainingHSB buffer

adjusted tovariouspH values. ChangesinpH

wereobservedto exert two maineffectsonthe distribution of nucleocapsid material in the gradient: (i) achange in sedimentation veloc-ity; and (ii)greaterheterogeneityasjudged by

the spread of the radioactivity (Fig. 10). The variation obtainedversusthepHexposurewas

determined for both viruses and is plotted in reference to (i) the exact position in the

gra-dient of the maximum of thenucleocapsid peak and (ii) the width of this symmetrical peak at

half-height value (Fig. 11). For the wild typeno

particularchangeswereobservedinS valueor

peak width atpH values greater than 7.0. At lowerpHvalues, the nucleocapsids exhibiteda

lower S value. FortsII052nucleocapsids, both

anincreaseinpeakwidth(30%)andadecrease

inthe sedimentation coefficient (10%) were

ob-served around pH 7.0. These results indicate that thets II 052 nucleocapsids at neutral pH

possessabnormal features. DISCUSSION

Inagreementwith thestudies by Unger and Reichmann (23)on ts II G22, we have

demon-strated thatthe ts II 052 mutant at 39.5°C in

HeLa cells doesnot accumulate either nucleo-capsids or new 388 molecules. This

prop-erty, together with other characteristics of the viral RNA synthesis obtained at the

non-permissive temperature bytsII 052, makes the

group II mutants unique among the RNA+ ts mutants ofVSV; i.e., group III and group V mutants are clearly different (23; in

prepara-tion).

Theprevious data obtained withtsII052 (9)

suggested two alternative explanations for its failure to grow at39.50C: (i)there is no

synthe-sis of new genome RNA; or (ii) 388 molecules

arepolymerized but rapidly broken for lack of

stabilization. This latter possibility wasbased

onthe observation that for wild-type virus

in-tracellular 38S RNAisprincipally recoveredin

ribonucleoprotein structures involving the N

protein (10, 22). Calculations by Bishop and Roy (2) suggested that thereis atight packing

of the Nprotein along the RNAstrand, and this

probablyaccountsfor the remarkable stability

ofthe VSV nucleocapsid complextopancreatic

RNase and various other agents (10, 21, 25).

In theseanalyseswehavetested the stability

of the ts II 052 nucleocapsids under various

physical or denaturing conditions. We have

demonstrated that nucleocapsids isolated from thismutant are moresensitive toRNase when

preincubated invitro at39.50C by comparison

to those preincubated at 300C. This is in

con-trast tothe resultsobtainedat 30and39.50Cfor

wild-typevirusand for representativemutants

of all othercomplementation groups. The

nu-cleocapsids ofanother group II mutant, ts II

FIG. 10. Effect ofpHonthesedimentation

proper-tiesofnucleocapsids from wild-typeorts 052

viri-ons. [3H]uridine-labeled nucleocapsids from

wild-typeandts 052 virionsweredivided in equivalent

batches andhomogenizedinHSB buffered with

var-iousTris-maleate bufferstogiveapHrangefrom6.6 to 7.4 or with Tris-hydrochloride to give pH 9.0.

They were then layered on15 to30% sucrose

gra-dients inHSB of thesamepHand spunat27,000

rpmfor3h in theSpincoSW27.1rotor at4°C.The profileradioactivity ofdistributionwasobtainedin

eachcase.

I.

J. VIROL.

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bottom

(A

Is

(U

._

x 20 C 16

zE12 8

OW

3a

v

a -I I I f

of pH

I I I I I I 1

6.6 6. 7.0 72 7A ' 9.0

FIG. 11. Demonstration from Fig.10. This

sum-marizesthemaineffects ofpHonthe sedimentation

propertiesof nucleocapsids from wild-typeorts 052 viruses.(A)Position inthe gradient where thetopof thenucleocapsid peak occurred is plotted against the pH used. (B) Width of the nucleocapsid peak (repre-senting the half-height value fromagraphin whicha

gradient fraction of0.3mlwasrepresented by5mm)

is plotted for each pH value. Symbols:(V)wild-type nucleocapsids; (V)tsII052 nucleocapsids.

063, were also observedto be RNase sensitive whenpreincubatedat 39.50C.

In addition, the fact that RNase-resistant 140S structures were derived for the ts II 052 nucleocapsids upon preheating at 39.5°C has been documented, but its significance is not

known. Any attempt to prevent its formation by using high-ionic-strength EDTA, dimethyl sulfoxide, mercaptoethanol, or Mg2+ ions was

unsuccessful. However, the unavoidable

con-version ofts II 052 120S nucleocapsidsto 140S

structureshasoccasionally been observed with the wild type or other mutant groups,

espe-cially with old stocksorwithpreparations

han-dled foralong time (see, for instance, in Fig. 7

the control pattern obtained with ts IV 0111 nucleocapsids extracted by the procedure of Emerson and Yu [6]). Unger and Reichmann (23) have reported the faster migration of the nucleocapsids ofone ts III mutant in sucrose

gradientsat39°0. Whether the140S form rep-resents aparticular physiological state of

nor-mal VSV nucleocapsidsisunclear. Inany case,

the fact that this change in the nucleocapsid

structure wasregularlyseenonly for mutants

ofgroup II (ts II052 and tsII 063) shows that

the ts II nucleocapsids are unstable in some way.

The results of analyses ofnucleocapsids

in-volvingtwoother physicalparameters,pH and

high temperature, suggest that the

interac-tionsbetweenthegenome anditssurrounding

proteins are quitedifferent forts II 052

nucleo-capsids by comparison with those ofwild-type

virus, and possibly thesedifferent interactions account for the weak stability of the mutant nucleocapsids.

Therefore,all intracellularfeatures observed

with ts II 052 at 39.50C are explained by the

incorrectpacking andprotection of ts II 052 new genomesissued froman enzymatically normal

replicationprocess, which isevidenced by the

progressive amplification oftranscription with

timeand by the depressive effect of added

cyclo-heximide.

Inrelationtothequestion of what component

isresponsible for thetsII052nucleocapsid

de-fects,somedata havetobediscussed. We have

demonstrated that the nucleocapsid coresthat

weanalyzedcontainonlytwoviralproteins: N

and NS. The general beliefisthat the N

pro-tein, present as thousands of units strongly

boundtothe genomeRNA, is endowed with the

ability to normally stabilizethe RNA. On the

otherhand, although95%of the NSprotein was

removed during the nucleocapsid extraction,

any possible role of the approximately 10

re-mainingNS molecules couldnot berejected: a

fewspecialfoldingpoints fastenedthrough the

NS protein mightsufficeto stabilize N

protein-RNA interactions. Thecomplete removal of the

NS protein from ts II 052 nucleocapsids by

Emersonand Yu'streatment (6) wasthen

car-riedout to determine whether the NS protein

must be taken into account. Insufficient

ma-terialwas available atthe end of thets II 052

nucleocapsid extraction to allow numerous

analyses, butthe effect of RNasewasthought

tobe themostconvincingtest.Two factsargue

for the preservation of characteristicanomalies

ofts II 052 nucleocapsids at 39.5° under these

conditions:occurrenceof the 140Speak and

in-stability of the 120S material. However, this

latter aspect did not involve the exogenous

RNasetreatment:the120S material

spontane-ously disappeared upon incubation at

39.50C.

Thiswas in contrast tothecompleteresistance

observedwith ts IV 0111nucleocapsids. Inthis

case, a 140S structure was also presentaswell as ts II 052 nucleocapsids notyet incubated at

39.5°C. But we have already mentioned that

the 140S form was occasionally observed with

differentmutants,especiallyafter along

prepa-ration procedure. The mutant ts IV 0111 was

chosenforcomparisonbecauseNganetal. (15)

havesuggestedthat thetemperature-sensitive

defect ofgroup IV mutants is relatedto the N

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922 COMBARD ET AL.

protein. However, asreported by Hunt et al.

(13),the negativeresultsobtainedinthe

exper-imentsthat had ledtothis suggestiontell little about the actual lesion in group IV mutants.

The physiological behavior ofts IV mutants

reported previously (3, 18) would be more

ap-propriatelyrelated to a defect ina regulatory

protein implied in the transcription process,

which appearsto be thecase (6) for the

phos-phorylated NS protein. Similarly, our

stud-ieswithts IImutantsstronglysuggestalesion

intheir Nprotein. The defective glycosylation

of the Gprotein ints II 052-infected cells has

alsobeenreported (17). In HeLa cellswedidnot

usually encounter this defective event.

How-ever, the possible simultaneousness of

abnor-mal phenomena at both the N and G protein

levels-also reported by Wunner and Pringle

(28) on the New Jersey strain-would be

sig-nificant.

Inanycase, thepresentresults show thatts mutants belonging to complementation group

IIcould be of special interest in regardtoVSV nucleocapsid functions. Since several

independ-ent mutants have been selected (8), one may suppose that the different ts mutations have caused various changes in the nucleocapsid

structure undernonpermissive conditions. For instance, in the case of ts II 052, the defect interfering with the nucleocapsid allows the viral polymerase to act, although this enzyme

requires for its template thenucleocapsidcore

(4).Various degrees ofrelaxation in the ribonu-cleoprotein might be expected in other

mu-tants,whichin turnwouldmodifyinsome way

the template capacity of the nucleocapsid and thusleadtotheRNA-phenotype. Therefore, a

largersurveyofmutants ingroupIIwould help

to unravel thisaspectof the VSV cycle.

ACKNOWLEDGMENTS

This workwas partially supported bygrantsfrom the CentreNational de la Recherche Scientifique (L.A. 136and ATP4999.2.1),theCommissariat a l'Energie Atomique, and theFondationpourlaRechercheMedicaleFrangaise.

Wethank Annick Friedmann for her excellent technical assistanceandA. Berkaloff forcriticaladvice andreading ofthemanuscript.

C.M.isapredoctoral fellow.

LITERATURE CITED

1. Bishop,D.H.L., and A.Flamand.1975. The transcrip-tion ofvesicularstomatitisvirusandits mutants in vivoandin vitro,p.327-352. In B. W. J.Mahy andR. D. Barry (ed.), Negative strand viruses. Academic PressInc., London.

2. Bishop, D. H. L., and P. Roy. 1972. Dissociationof vesicular stomatitis virus and relationofthe viral proteinstotheviraltranscriptase. J. Virol. 10:234-243.

3. Combard, A., C. Martinet, C. Printz-Ane, A.

Fried-mann,and P. Printz. 1974.Transcriptionand

replica-tion ofvesicular stomatitis virus: effects of tempera-ture-sensitive mutations in complementation group IV. J. Virol.13:922-930.

4. Emerson, S. U.,and R. R. Wagner. 1972. Dissociation and reconstitution of the transcriptase andtemplate activities ofvesicular stomatitisvirusBand T viri-ons. J.Virol. 10:297-309.

5. Emerson, S. U., and R. R. Wagner. 1973. L protein requirement forinvitroRNAsynthesis byvesicular stomatitisvirus.J. Virol. 12:1325-1335.

6. Emerson, S. U.,and Y. H. Yu. 1975. Both NS andL proteins arerequiredfor in vitroRNA synthesis by vesicular stomatitis virus. J.Virol,15:1348-1356. 7. Fiszman,M., J. B.Leaute,C.Chany,and M.Girard.

1974.Mode of action of acid pH values on the develop-mentofvesicular stomatitis virus. J. Virol. 13:801-808.

8. Flamand, A.1970.Etudeg6ndtiquedu virus de la sto-matite v6siculaire: classementdes mutants

thermo-sensiblesspontan6sengroupesdecomplementation.

J. Gen. Virol. 8:187-195.

9. Flamand, A.,and F. Lafay. 1973. Etude des mutants

thermosensiblesdu virus de la stomatitev6siculaire

appartenantaugroupe decomplementation.II. Ann. Microbiol. Inst. Pasteur 124:261-269.

10. Huang, A.S., D. Baltimore, and M.Stampfer. 1970. Ribonucleicacid species of vesicular stomatitisvirus. III. Multiplecomplementary messenger RNA mole-cules. Virology 42:946-957.

11. Huang; A. S., and E. K. Manders. 1972. Ribonucleic acidsynthesisof vesicular stomatitis virus. IV. Tran-scriptionby standardvirusinthepresenceof defec-tiveinterferingparticles. J. Virol. 9:909-916. 12. Huang, A.S.,and E. L. Palma. 1974. Vesicular

stoma-titisvirus:defectivenessanddisease,p.87-99.InW. S. Robersonand C. F. Fox(ed.),Mechanisms ofvirus disease. W. A.Benjamin,Inc.,MenloPark, Calif.

13. Hunt, D. M.,S. U. Emerson,and R. R. Wagner. 1976. RNA- temperature-sensitive mutants of vesicular stomatitis virus: L-protein thermosensitivity ac-countsfor transcriptase restriction of group I mu-tants.J. Virol.18:596-603.

14. Imblum, R. L.,and R. R. Wagner. 1974. Protein kinase

andphosphoproteinsofvesicularstomatitis virus. J. Virol. 13:113-124.

15. Ngan,J. S. C., A. F. Holloway, and D. V.Cormack.

1974.Temperature sensitive mutantsof vesicular sto-matitis virus:comparisonof theinvitroRNA polym-erase of group I and group IV mutants. J. Virol.

14:766-.772.

16. Pringle, C. R., and I. B. Duncan. 1971. Preliminary

physiological characterization of

temperature-sensi-tivemutantsof vesicular stomatitis virus. J. Virol. 8:56-61.

17. Printz,P., andR. R.Wagner. 1971. Temperature-sen-sitivemutantsofvesicular stomatitis virus:synthesis ofvirus-specific proteins.J.Virol.7:651-662. 18. Printz-Ane, C., A. Combard, and C. Martinet. 1972.

Studyof thetranscription and thereplicationof vesic-ularstomatitisvirusby usingtemperature-sensitive mutants.J. Virol. 10:889-895.

19. Schincariol,A.L.,andA. F.Howatson.1970. Replica-tion of vesicular stomatitis virus. I. Viral specific RNAand nucleoproteinininfected L-cells. Virology 42:732-743.

20. Sokol, F., and H. F. Clark. 1973. Phosphoproteins, structural components of rhabdoviruses. Virology

52:246-263.

21. Soria,M., S.P.Little, and A. S. Huang.1974. Charac-terization ofvesicular stomatitis nucleocapsids. I. Complementary 40S RNA moleculesin nucleocap-sids.Virology 61:270-280.

22. Szilagyi, J. F., andL.Uryvayev. 1973. Isolation of an

on November 10, 2019 by guest

http://jvi.asm.org/

infectious ribonucleoprotein from vesicular stomati-tis virus containinganactive RNAtranscriptase. J. Virol. 11:279-286.

23. Unger, J.T., and M. E. Reichmann. 1973. RNA synthe-sis intemperature-sensitivemutantsof vesicular sto-matitis virus. J.Virol. 12:570-578.

24. Wagner, R.R.,S. U. Emerson, R. L.Imblum, and J. M. Kelley. 1975. Structure-function relationships of the proteins of vesicular stomatitis virus,p.1-20. In

B. W.J.MahyandR.D. Barry(ed.), Negativestrand viruses.Academic Press Inc., London.

25. Wagner, R. R.,T.C. Schnaitman, R.M. Snyder, and

C. A. Schnaitman. 1969. Structuralproteins of vesic-ular stomatitis virus. J. Virol. 3:611-618.

26. Wagner, R. R., R. M. Snyder, and S. Yamazaki. 1970. Proteins ofvesicular stomatitis virus: kinetics and cellular sites ofsynthesis. J. Virol.5:548-558. 27. Wertz,G. W., and M. Levine.1973.RNA synthesis by

vesicular stomatitisvirusandasmall plaquemutant: effects ofcycloheximide. J. Virol. 12:253-264. 28. Wunner, W. H., andC. R. Pringle. 1974.A

tempera-ture-sensitive mutant of vesicular stomatitis virus with two abnormal virus proteins. J. Gen. Virol. 23:97-106.

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