Control of protein synthesis by a temperature-sensitive mutant of reovirus 3. I. Temperature-sensitive function of ts261-b mutant.

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Control

of

Protein

Synthesis by

a

Temperature-Sensitive

Mutant of

Reovirus 3

I. Temperature-Sensitive Function of ts261-b Mutant

NOBUKO IKEGAMI

Division ofVirology, Memorial Sloan-Kettering Cancer Center, New York, New York 10021 Received for publication 25 May 1976

The ability of a temperature-sensitive (ts) mutant of reovirus, ts261-b, to synthesize virus-specificRNAsandproteinsduringinfectionatthe nonpermis-sive temperature (3700) was investigated. The relative amounts of the mutant virus-specificsingle-stranded (ss) RNAs anddouble-stranded(ds) RNAs synthe-sized in cells at 37C were 20 to 25% as much as those synthesynthe-sized in the wild-type virus-infected cells. The 10 segments of the mutant ds RNAs and the three size classes of the ss RNAs were synthesized in the usual proportions. The methyla-tion ofthe mutant viral mRNA's (ss RNAs) was not blocked at

3700

ininfected cells. A striking temperature-sensitive restricted function of the ts261-b mutant wasexpressed in the synthesis of the viral proteins. This study, which uses an in vitroprotein-synthesizing system reconstituted with an endogenous polysomal fraction andapostribosomal supernatant from reovirus-infected cells, has dem-onstrated that the endogenous polysomes obtained from ts261-b mutant-infected cellsat

3700

are not active inthesynthesisof theviral polypeptides ofknown molecular weights, and the amounts ofthemutant viral polypeptides synthe-sized in vitro by these polysomes are 5 to 9% of those synthesized by the corresponding fraction from wild-type-infected cells. The impaired protein-synthesizing capacity ofthe mutant virus-specific polysomes can be restored during maintenance of the infected cellsat

300C

after shift-down from

370C.

The invitrosynthesis of viralpolypeptides ofknownsizeby theactiveendogenous polysomes derived from cells infectedatthepermissivetemperatureis acceler-ated by the addition of the postribosomal supernatant obtained from cells infected at the permissive temperature. The postribosomal supernatant from mutant-infected cells at

370C

did nothave a stimulatory effect, but rather, it inhibited in vitro viral protein synthesis.

The viralgenome of reovirus consists ofthe 10 segments ofdouble-stranded (ds) RNA, and eachsegmenttranscribesamonocistronic viral mRNA (5, 25). From this genomic structure, the existence of at least 10 genetic classes of temperature-sensitive (ts) conditional-lethal mutants ofreovirus can beanticipated. Thets mutants of reovirus type 3 (Dearing strain) have beenisolated in twolaboratories (12, 21). Those isolated by Fields andJoklikhavebeen classified into seven classes, A through G, by genetic recombination tests (12). Their investi-gations demonstrated that the ts mutants in

thedifferent classes expressed thedifferent re-stricted

functions

during the infective cycle at the nonpermissive temperature,

390C.

The re-strictedfunctionsoftheir ts mutants were: (i) the virion-associated transcriptase (groupG; 7);

(ii) the abilityto synthesize progenyds RNAs (groupsC, D, andE; 7, 23, 33); (iii)the assem-blyormaturation of progeny subviralparticles into the completed virus particles (groups B andG; 14, 34). All of thesetsmutants areable tosynthesize incells at 3900 the 10segments ofreovirus-specific

single-stranded

(ss) RNAs and all of themajor

virus-specific

polypeptides (7, 8, 13).

The isolation and characterization of the ts

mutants that have been carried out in this laboratoryhave beenreported (21,22).The ge-netic class of thesets mutants isdifferent from each of the five classes A

through

E (unpub-lished data). Inthisreport, Idescribe the

tem-perature-sensitive restricted function of a ts

mutant,ts261-b,isolatedinthislaboratoryand discusssomeaspectsrelatedtothe

regulation

of

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reovirus proteinsynthesisthat have been

stud-iedinanin vitrosystemusing theendogenous virus-specific polysomes.

MATERIALS AND METHODS

Cells and media. L cells, strain 929, were used for all experiments. The media forgrowth of cellsand

the proceduresfor cultureofthe cellsweredescribed

previously (21, 22).

Viruses.Reovirus type 3, Dearing strain, cloned

(19), was used as thestock of wild-type virusafter

two further passages in L cells in suspension at

370C. The clonedts261-b mutants (22) were grown up

to astockin Lcellsonmonolayersafterone passage

at30'C. Fortheproductionof the mutant virus, cells

were infected at the low multiplicity of infection

(MOI) of 0.1 PFU/cell. When the complete

cyto-pathiceffect was observed, the infected cells were

frozen and thawed, and the lysate wascentrifuged

at alowspeedto removecelldebris. The virus

par-ticles in the clarified supernatant werecollected in

apellet by centrifugationat72,000 xg for3h,and

thepellet wasresuspendedinfresh mediumat1/20 of the original volume. This material was usedas the stockof ts mutant virus.

Isotopes and chemicals. [U-14C]uridine (447mCi/

mmol), L-[methyl-3H]methionine (11 Ci/mmol),

S-adenosyl-L-[methyl-3H]methionine (12.6 Ci/mmol),

L-[35S]methionine (150 to 380 Ci/mmol), 3H-labeled

L-aminoacids mixture,andGTPlabeledwith 32p in the a position werepurchasedfrom New England Nuclear Corp. (Boston, Mass.). Unlabeled ribonu-cleosidetriphosphateswereobtained from P-L Bio-chemicals (Milwaukee, Wis.). Goat antiserum to

rabbit 7S gammaglobulin was purchased from

Hy-land Laboratories(CostaMesa,Calif.).

Preparation ofcytoplasmicextracts forthe

ex-tractionofvirus-specificRNAs. The L cells in

sus-pensionculture were infected witheitherwild-type

virusorthets261-bmutant at an MOI of 20. After an

adsorption of2 h, the suspension ofinfected cells

wasmadeataconcentration of about106 cells/mlin

complete spinner medium containing 5% fetal

bo-vine serum. Whereindicated, the suspension of

in-fected cells was made in methionine-free spinner

medium containing 5% fetal bovine serum. Actino-mycin D (0.15 ,ug/ml) was present throughout the infective cycle. The cells were collected by

centrifu-gationand werewashedoncewithRSB-Na+ buffer

(0.01 M Tris-hydrochloride [pH7.4], 0.01 MNaCl, 0.0015 MMgCl2), andthen the infected cells were disrupted in RSB-Na+ buffer containing 2 mM dithi-othreitol, 0.1% Nonidet P-40, 20 ,ug of polyvinyl sulfate perml, and10% glycerol in a Dounce homog-enizer.The cytoplasmic extract, free ofnuclei, was obtained as described by Penman (37).

Extraction of RNA from cytoplasmic extract, separation ofreovirus-specificss anddsRNAs, and velocity sedimentation analysis. To the total

cyto-plasmicextract, sodium dodecyl sulfate (SDS) and

polyvinylsulfate wereaddedtofinal concentrations

of2% and 200j.g/ml,respectively;then the cytoplas-mic extract was diluted threefold in TNE buffer (0.01MTris-hydrochloride [pH7.4],0.001MEDTA,

0.1 M NaCl) containing 0.6% SDS. Extraction and purification ofcytoplasmic RNAs were carried out according totheproceduredescribedby Moyeretal. (35). The purified RNAs were precipitated by the addition of 2 volumes ofethanol, and the RNA pre-cipitate wasdissolved in TNE buffer. Anequal vol-ume of 4 M LiCl was added to the RNA solution to separate ss RNA from ds RNA. The mixture was kept overnight at 0°C and then centrifuged. The resulting precipitatecontained ss RNA. The ds RNA present in the supernatantsolution was precipitated once morebytheaddition of2volumesof ethanol. The RNA in each precipitate wasdissolvedinTNE

buffercontaining 0.5%SDS. TheRNAsamplewas

layeredonto alinear 15 to 30% sucrosegradientin

TNE buffer andcentrifugedat 30,000 rpm for 17 h in an SW41 rotor at 15°C. Fractions of 0.35 ml were

collectedfrombelow andassayedfor acid-insoluble

materiallabeled with theradioactivityasdescribed by Gomatos (18). The sedimentation values of the

radiolabeled RNAs weredeterminedrelative to the

positionsof 28S and 18SrRNA's,which sedimented ina companion sucrosegradient.

In vitro transcription. (i) RNA synthesis by

wild-typeviral cores. Theassayprocedures for in vitro

RNA synthesis by the wild-type virion-associated

transcriptase were the same as those described by

Shatkin (40). The complete reaction mixture,

con-taining the chymotrypsin-treated wild-type virion, was incubated for 60 min at 45°C. At the end of incubation, the reactionmixture was centrifuged at 30,000 xgfor 30 min. The supernatant, which con-tained the RNA products, was saved, and the pel-leted viral cores were resuspended in the fresh, com-plete reaction mixture. Incubation was carried out

for another 60 min at45°C. The reaction mixture

wascentrifuged as described above, and the super-natant obtained was combined with the previous

one. The RNA products in the combined

superna-tant werepurified by extracting three times with an

equal volume ofamixture (1:1) of phenol and

chloro-form containing isoamyl alcohol. The RNA

mole-cules in theaqueous phasewere chromatographed

through SephadexG-50. Fractions containing

radio-active RNA were pooled and precipitated with 2 volumes of ethanol in the presence of 0.3 M NaCl.

(ii) RNA synthesis by ts261-b mutant subviral

particlespresent inthe cytoplasmic pellet. The

cy-toplasmic extract wascentrifuged at4°Cfor 20min at 35,000 x g. The pelleted material was

resus-pended by homogenization in0.01 M Tris (pH 8.1)

and was used as a source of the mutant subviral

particleswithout furtherpurification.The

cytoplas-mic pellet containing 900 ,ug of protein was incu-bated at 37°C for 30 min in the presence of chymo-trypsin (450 ,ug). Atthe end of incubation, all of the components necessary for in vitro transcription wereadded, andthe final concentration ofeach com-ponent in the reaction mixture was the same as described by Shatkin (40). The incubation was con-tinued at 37°C for 90 min. The reaction mixture was centrifuged at 35,000 x g for 30 min. The RNA product in the supernatant waspurifiedby the pro-cedure described above.Theproductassociated with the pelleted material was extracted andpurified by

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the proceduresdescribed for purification ofssRNAs fromthe cytoplasmic extract.

Preparation ofsubcellular fraction for invitro

protein synthesis. Cells were infected with

wild-type virus at anMOI of20orwiththets261-b mu-tant at anMOI of 50.Afteradsorption of virus for 2 h, the cells were resuspended in complete spinner mediumcontaining 5%fetalbovine serum,and acti-nomycin D (0.15

Ag/ml)

wasadded to the infected cellsuspension(106/ml) andwaspresentthroughout

the infective cycle. The infected cellswerecollected

atthe time indicated and were washedonce with

RSB-K+buffer(pH 7.8).Thewashedcellswere

dis-rupted in a Dounce homogenizer in the RSB-K+

buffer containing 2 mM dithiothreitol and 0.2%

NonidetP-40.Thecellhomogenatewascentrifuged

at250 xgfor8min. The postnuclear supernatant was collected and mixed with glycerol in a 9:1 (vol/ vol)proportion.The nuclear sedimentswere

resus-pendedinbufferA(RSB-K+ containing2mM

dithi-othreitol and10%glycerol); then the suspensionwas

centrifuged at 600 x g for 8 min. The resulting

supernatant fluidwascombinedwiththe

superna-tantcollectedpreviously. Thetreatmentofthe

nu-clear sediments wasrepeated once ortwice in the

same way,andthesupernatantfluidscollected after

each centrifugation were combined with the

pre-vious ones. The total postnuclear supernatant fluid was then centrifuged at 10,000 x g for 20 min. The resulting supernatant fluid was designated S-10.TheS-10 wasfurthercentrifugedat150,000x

gfor2h.The clearsupernatantfluid inthemiddle

part of the centrifuge tube was collected, and it wasdesignated S-150. The surface of the pellet was washed once with buffer A, and the material in the

pelletwasresuspended inbuffer Aby

homogeniza-tion, to give a protein concentration of 13 to 15 mg/ml. This suspension wasdesignated P-150.

Assay forin vitrosynthesis of viral polypeptides.

In a total volume of600 ,l, thefollowing

compo-nents were added to give the final concentrations

indicated:Tris-hydrochloride (pH 7.8),44mM;KCl,

40mM;NH4Cl,44mM;MgCl2,4.5mM;

2-mercapto-ethanol, 11 mM; ATP, 1.1 mM; GTP, 0.5 mM;

K-phosphoenolpyruvate, 5 mM; pyruvate kinase, 160

,g/ml; 19unlabeledamino acids(except

L-methio-nine), 2.2

AM;

L-[35S]methionine (150 to 380 Ci/

mmol), 100 ,Ci; subcellular fraction, 420 ,l. The

reaction mixture wasincubatedat30°C.Toanalyze

kinetics of in vitro incorporation of -[35S]methio-nine, asampleof20,ul was removed and mixed with 2mlof5%trichloroaceticacidatthe timeindicated. After 30 min at 0°C, the samples were heated at 95°Cfor20minand then cooledat0°C.Theresulting

precipitates were collected on membrane filters

(Millipore Corp.), and the amount of radioactive

material on the filter was counted.

Immuneprecipitation of viralpolypeptides syn-thesized in vitro. Tworabbitserahyperimmune to reovirus werepreparedinthislaboratory.To

immu-nizerabbits,virusstockwaspreparedinthe

follow-ing way. Wild-typevirus wasgrowninmonkey

kid-ney cells, and the ts mutant virus was grown in

BHK-21 cells. Thelysateofcell infected with each viruswascentrifugedto removecelldebris,and the

clarified supernatant was used for immunization. One antiserum is hyperimmune to the wild-type virusand the otherishyperimmune to the ts mu-tant. The procedurefor immune precipitation was essentially as describedbyFields et al. (13). A rab-bit immune serum (20 ul) was added to the reaction mixtureof 480

tMl.

The mixture wasincubated at4°C

overnight, andanadditional 20,ulof thesame

rab-bit immune serum was added to this mixture and incubated overnight at 4°C; then 0.2 ml of goat anti-rabbit serum was added. Incubationwas continued for an additional 24 h at 4°C.The immune precipi-tate wascollected by centrifugation andwaswashed three times with cold phosphate-buffered saline. Withthisimmune precipitationmethod,tworabbit immune sera were equally capable of precipitating over 90% ofradiolabeled virion structural proteins of aknown amount, as judged by the total radioactive counts precipitated and bythe polyacrylamide gel

electrophoresis (PAGE)patternof thelabeled

poly-peptides ofX-, ,u-, and a-size classes. When the

immune precipitation method was applied to the reaction mixture at the end of the in vitro assay, each ofthe reovirus antiseraprecipitated

approxi-mately15%ofthe total acid-precipitable

radioactiv-ity,whereas normal rabbit serumprecipitated less

than 1%oftheacid-precipitablecounts.

PAGE. Analysis of viral proteins. The im-mune precipitate was dissolved by heating at 100°C for 2 min in 100 ,ulof buffer containing 0.1 M

phos-phate(pH 7.4), 2%SDS,4%2-mercaptoethanol, and

0.5 Murea.Thetotaldissolvedsample was subjected

toPAGEanalysis.The proceduresforpreparationof

polyacrylamide gelcontainingSDS,urea,and

phos-phatebuffer and for electrophoresis of gel in a

phos-phate buffersystem were as described previously

(22). The gel was frozen and thawed once and then cut into 1-mm slices. Two slices were placed into

eachvial and solubilized by heating at 55°C

over-nightin0.5ml of hydrogen peroxide.Ten milliliters

ofatoluene-TritonX-100 (2:1)scintillationmixture

was added, and the amount ofradioactivity was

determined. Toserve as internal protein markers,

3H-aminoacid-labeledpurified reovirus, after

solu-bilization, was subjected to coelectrophoresis with

each immune precipitate.

Chemical analysis. Protein concentration was

determined by theLowryprocedure (30), using

crys-talline bovineserum albumin as the standard.

RESULTS

Synthesis of virus-specific RNAs by the

ts261-b mutant at the nonpermissive

tempera-ture, 37°C. Since the level of the yield of the infectiousts261-b mutantat37°Cis 0.01%of the maximalyieldproducedat30°C, 37°C has been chosen as the nonpermissive temperature for the ts261-b mutant. To compare the ability of the ts261-bmutant tosynthesizethe virus-spe-cific ds andssRNAsinvivo at37°Cwiththat of the wild-type virus, cells were infected with either ts261-b mutantor wild-typevirusat an

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MOI of20 and were labeled with ['4C]uridine

for the respective period, from2 to 6h(the early time) and from 6 to 11 h (the latetime) after infectionat370C. The RNAswereextracted and

purified from the cytoplasmic extract of each

set of the infected cells. The ss RNAs were

separated from the ds RNAs by2MLiCl precip-itation. After velocity gradient centrifugation of the RNAsamples, theamountsof the

reovi-rus ds RNAs that sedimented between 10.5S

and14S (1) and those of the reovirusssRNAsof

the three size classes1, m,and s (17, 25) were

determined. The resultsareshown inTables 1

and 2.

Theamountof themutantdsRNAs

synthe-sized andlabeled in vivoat370C relativetothat of thewild-type viralds RNAswas15%earlyin

the infective cycle and8%late inthe infective cycle (Table 1). In experiment 2, when cells

were infectedat 370Cwith the ts261-b mutant

at an MOI of 250, the increase in the total

synthesis of themutantviraldsRNAswasless

than twofold, whichwasnotproportionaltothe increase in MOI. These data indicate that the maximal level of ts261-bmutantds RNAs

syn-thesizedin vivoat370C is,atmost,20 to25%as

much as that of the wild-type viral ds RNAs

synthesizedat370C. PAGEanalysis of the

mu-tant ds RNAs synthesized in vivo at370C

re-vealed that the 10 segments ofthe mutantds RNAhad been synthesized (Fig. 1). The ts261-b

mutant, thus neither is adeletionmutant (43)

TABLE 1. Synthesis ofreovirus ds RNAs in vivoat 37°C

Period of 14CCpM

labeling inds Ratio

Expt Infectiona postinfec- RN

tionb X103

(h)

1 ts261-b 2-6 15 15.3

Wildtype 2-6 98 100

ts261-b 6-11 26 8.4

Wildtype 6-11 307 100

2 ts261-b 2-11 68

aAtotal of108cellswereinfected with each virus atanMOI of 20 inexperiment 1and 250 in experi-ment2. ActinomycinD (15jig)wasadded2h after theonsetofinfection andwaspresentuntil the end ofthelabeling period.

bInfected cellswerelabeled with['4C]uridine (50

,uCi)for theperiodindicated.

cRNAs were extracted and purified from the wholecytoplasmicextractof eachset,and ds RNAs

wereseparated fromssRNAsasdescribed in

Mate-rials and Methods. 14C cpm represents the total

counts incorporated intoreovirus-specific ds RNAs that sedimentedbetween 10.58 and14S(1)andwere

resistanttohydrolysis bypancreatic RNase(3 jug/

[image:4.508.264.456.74.184.2]

ml).

TABLE 2. Comparison ofinvivotranscription at 370C

Period of '4Ccpm(x10-3) inssRNAs8 labeling

Infection

postin-fection 1 m 8 Total

(h)

ts261-b 2-6 17 20 18 55

Wild type 2-6 77 88 69 234

ts261-b 6-11 86 113 84 283

Wild type 6-11 438 499 388 1,325

aProceduresfor infection ofcells at an MOI of 20

and for labeling ofinfected cells are the same as

thosedescribedinTable1.

b RNAs were extracted and purified from the

whole cytoplasmicextractof each set,andssRNAs

wereprecipitatedwith2MLiCl. Threesizeclasses

ofvirus-specific ss RNAs (1, m, and s) were

sepa-rated by velocity gradient centrifugation as

de-scribed in Materials and Methods. The

sedimenta-tionvalues of the three sizeclasses,24S for1,19Sfor m, and 14S for s (17), weredeterminedrelative to the position of 28S and 18S rRNA's, which sedi-mented in a companion sucrose gradient. 14C cpm

represents the total counts incorporated into the respective ssRNAclass.

nordoes it seem to belong to the C, D, and E classes (23),butitcanbeplacedintothe cate-gory ofds RNA-positivemutantsbyusingthe criteriathathave been applied for the ts mu-tantsof classes A,B, F, and G (25).

The ability of the ts261-b mutant to tran-scribe the reovirus-specific ss RNAs invivo at

370C

is also20to 25%of the level of transcrip-tionexpressed by the wild-typevirus

through-outthe infective cycle (Table2).Whenthe mu-tant subviral particles obtained from cells in-fectedat37 or300C and the wild-typesubviral particles obtained from the infected cells at 37°C were assayed invitrofor their transcrip-tase activities,theratesofthe invitro synthe-sis of the virus-specific ss RNAs were two- to threefoldgreater at

370C

astheassay tempera-turethan at

300C

inall cases. It is not clearly understood at present what mechanism(s) is responsible for thereduced level of in vivo tran-scriptionintheearly time after infection with ts261-b mutant at

370C.

Inhibition of protein synthesis in ts261-b mutant-infected cells at

370C.

Incells infected with the ts261-b mutant at the nonpermissive temperature,

370C,

there occurs dissolution of pre-existing host cell polysomes, and the inhibi-tion ofsynthesis of host cell protein is evident by 7 h after infection. When such cells are pulse-labeled for 15 min with the radioactive aminoacidsatany time after7hpostinfection

at

370C,

the incorporation of the radiolabeled amino acids into the polysomal fraction is

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BY MUTANT

I IV

ts261-bdsRNAs

M S

i-_

WildtypedsRNAs

L S

4-

2-20 40 60 80

Mobility(mm)

FIG. 1. Polyacrylamide gel electropherogram of reovirus ds RNAssynthesizedin vivoat37°C. Virus-specific ds RNAs from infected cells labeled with [14C]uridine for 9 h (2 to11 h after infection) were

obtained and purifiedasdescribedinMaterialsand

Methods and in Table1. ThedsRNA sample was

analyzed in 7.5% gel. Theprocedure forpreparation ofgel and for electrophoresiswere asdescribedby Ito

andJoklik (23). Electrophoresiswascarriedout at4 mA/gel for 30 h. The gel wascutinto1-mmslices,

andtheradioactivityineachslicewasdeterminedas

described in Materials andMethods.

markedly inhibited. When the cytoplasmic

ex-tract of ts261-b mutant-infected cells at 370C

wasanalyzed byPAGE,itwasdifficult to

iden-tify any virus-specific polypeptides of known

molecular weights, because the amount of the

newly synthesizedand labeled proteins inthe

cytoplasmic extract ofts261-bmutant-infected

cellswas too little to resolve into

distinguish-able proteinbands. However, aswas reported previously(22), newly synthesizedmutant

sub-viral particles at a density of 1.43 g/ml were

detectable inCsCldensity gradient analysisof

a concentrated cytoplasmic pellet, but no

ma-ture mutant progeny particles at adensity of

1.36 g/ml were detectable. To determine

whether all ofthe viralproteinsaresynthesized

in a markedly reducedamount not detectable

in a conventional PAGE analysis or whether

the synthesis of a particular viral protein is

selectively inhibited in ts261-b mutant-infected cells at 3700, the present experiments have been carried outby analysis of in vitroproducts synthesized in a cell-free system reconstituted with a concentrated polysomal fraction and a

postribosomal supernatant from infected cells. Formationof polysomes in vitro byfraction S-10 obtained from reovirus-infected cells. Thepostmitochondrial supernatantfraction (S-10), obtained from a cytoplasmic extract of wild-type virus- or ts mutant-infected cells at the permissive temperature, containsaquantityof virus-specific ss RNA of the threesizeclasses, anamountequal to 50% of the total present in the original cytoplasmic extract. These virus-specific ss RNAs are already bound to ribo-somesandpolysomes. If all components neces-sary for protein synthesis are present and func-tionallyactive intheS-10fraction, these endog-enous viral mRNA's are translated during in vitro incubation of the S-10 fraction with the protein-synthesizing assay mixture, and the subsequent analysis of the reaction mixtureby velocity gradient centrifugation will demon-stratethe incorporation of35Slabelintothe80S monosomes and polysomes of various sizes. Three kinds of S-10 fractions were prepared to compare theirprotein-synthesizing abilities: (i) the wild-type S-10, which was obtained from cells infected with wild-type virus for 8 h at

370C

(the permissive temperature); (ii) the ts261-bS-10 (370C),fromcellsinfectedwiththe mutantvirusfor8.25h at370C (the nonpermis-sive temperature); (iii)the ts261-b S-10 (300C), fromcells infected with themutantvirusfor19 h at 300C (the permissive temperature). The length of infection atthe permissive tempera-ture chosen in this experiment wasoptimal to obtaintheS-10fractions, whose protein-synthe-sizing activitywasinthe range of themaximal level. Each of these S-10 fractions wasmixed with the the assay mixture and incubated in vitro at 30 or37°Cimmediatelyafter addition of

[35S]methionine.

Atthe end ofincubation, each

reactionmixture wasanalyzedbysucrose gra-dientcentrifugation. Theresults are shownin Fig. 2. The incorporation of 35Slabelproceeded duringincubation at 30 or370C, but not at00C (Fig. 2a). The 35S-labeled 80S monosomes and polysomes were formed in the reaction mix-turesthatcontained theS-10fractionfromcells infected at the permissive temperature with either wild-type virus (Fig. 2b) or the ts261-b mutant (Fig. 2e and f). The increase in 35S

counts inthepelletedmaterials suggestedthat

the larger polysomes were formed and sedi-mentedduringvelocity gradientcentrifugation. In contrast tothese results, the datainFig. 2c and ddemonstrated that the ts261-b S-10

(3700)

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36

Wild-typeS-10 fromcells infected at37° for 8 hrs. a)0min.atO' 12Fb)370

ls261-b S-10 fromcells infected at370 for81/4hrs,

4 c)300 4 d)37°

E tP;12xi06 80S P06.106 80S

Qo

ts 261-bS-10 from cells infected at 30°for19 hrs. iz-e)30°1 l'' f) 370

80S 480S

8

8-4 4

tp; e8 loetp2 X0

0 10 20 30 0( 10 20 30

[image:6.508.67.244.65.326.2]

Fraction number

FIG. 2. Formation of 80S monosomes and poly-somesduring in vitro incubation ofS-10 in theassay

mixture for protein synthesis. The reaction mixture (2.4ml) contained S-10 andL-[35S]methionine (250

,uCi); itwasincubatedfor 20 minat30°Corfor 30

min at37°Cand then treated with detergents (so-diumdeoxycholate,Brij-58,and EDTAatfinal

con-centrationsof0.7%, 0.5%, and 0.5 mM, respectively). Thedetergent-treated samplewaslayeredon alinear

15to30%sucrosegradient inRSB-Na+ buffer.

Cen-trifugation wasat24,000 rpmfor8 h in an SW27

rotor at4C. Fractions of1 ml were collected. The

absorbencyat260nmandacid-insoluble

radioactiv-ity in each fraction were determined as described

previously (22). Theamountofacid-insoluble radio-activity in the pelleted material( P)is shown in the

lowerleftcornerof each panel.

obtained from cells infected at the

nonpermis-sivetemperature(3700)hadessentiallyno

cata-lytic activity for incorporation of35Slabel into

polysomes atbothassaytemperatures, 30 and

370C.The data suggest that theremaybesome

defectsorrestrictioninfunctionofasubcellular

components) presentinthe ts261-bS-10 (370C).

Analysis of viral proteins synthesizedin vi-troby polysomal fraction fromcells infected at the permissive temperature. To examine

whetheradefect(s) responsible for inhibition of

protein synthesis in ts261-b mutant-infected

cellsat370Clies in thepolysomalfractionorin

a components) present in the postribosomal

supernatant, theS-10fractionwasfurther

cen-trifuged at 150,000 x g and separated into a

pellet, P-150, and a postribosomal supernatant, S-150. The P-150 contained free polysomes, monosomes, 60S and 40S ribosomal subunits, and viral mRNA's of the three size classes. The S-150, a clear portion of postribosomal superna-tant, did not contain ribosomes or their sub-units. The viral mRNA's of the three size classes were also undetectable in theS-150.

(i) In vitro products synthesized by the P-150(30CC). The P-150(30'C)wasobtained from cells infected with eitherwild-type virusorthe ts261-b mutant at300C for 21 h, and its protein-synthesizing ability was assayed in vitro in the absence of S-150. Virus-specific proteins syn-thesized by each P-150 (30'C)wereprecipitated with a reovirus-specific antiserum and

ana-lyzed by PAGE. Theelectrophoreticpatterns of in vitroproducts areshown in Fig. 3. Both P-150 (300C)preparationsof the wild type and the ts261-b mutant were capable of synthesizing reovirus polypeptides in vitro at 30and 370C. In the absence ofS-150, the P-150 (300C) synthe-sizedpredominantlythepolypeptidesof the size corresponding to the a- class (43,000 to 33,000 daltons). The 35S-labeled polypeptides, whose mobilities corresponded to the ,-size class (88,000 to 70,000daltons)and theA-size(155,000 to 140,000 daltons), were synthesized in one-third amountsof the35S-labeled polypeptidesof thea-size class. Thus, the relativeproportions of the three size classes (X, ,u, and c-) of 35S-labeled virus-specific polypeptides synthesized bythe P-150 (300C)did notcorrespond to those ofthe three size classes of 3H-labeled virion polypeptides. In addition, 35S-labeled polypep-tides oftwoother size ranges weredetectablein substantial amounts. They migrated faster than the polypeptides ofthe A-size class and those of the ,u-sizeclass, respectively. The ap-parentmolecular weightsofeach are approxi-mately140,000 to 120,000daltonsfor theformer and69,000 to 55,000daltonsfor the latter. Since these polypeptides were precipitated with the reovirus antiserum but did not correspond to thesizes ofthe reovirus structural proteins and to those oftwo nonstructural proteins (88,000 and 36,000daltons),itappears that they may be incomplete polypeptides of the K- and ,-size classes, respectively, that may have arisen from incomplete elongation or from premature termination underthe assay conditions in the absence of S-150. This assumption, however, remains to be proven by identification of the authentic tryptic peptides, which correspond totrypticpeptides from the virion polypeptides ofthe three size classes.

(ii) Effect of postribosomal supernatant (S-150[30°C1).When theS-150(300C)obtained from wild-type virus-infected cells was addedto the

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Protein synthesis in vitro cells infected at 30°

product at 300

D 140,00072,000 34,000

Do H A

by Wild-type P-150 obtained from

product at 370

V740,2000 34,000

4 X

I H H-H

Mobility (mm)

Protein synthesis in vitro by ts 261-b P-150 obtained from cells infected at 30°

20 40 60 80 100 120 0 20 40 60 80 100 120 Mobility (mm)

FIG. 3. Reoviruspolypeptides synthesizedinvitrobywild-type P-150(300C)andbyts261-bP-150(300).

Each P-150(3090)wasprepared fromcellsinfected withwild-typevirusorthe ts261-bmutant at300C for20 h.The reaction mixture(600,d)contained 100

pi

ofeachP-150(1.3mgofprotein),320

pi

ofbuffer A,and 60

pi

of L-[35S]methionine andwas incubatedfor100 minat300Cor50 minat370C.During incubation,a

sample (20 p1) from each mixture was withdrawn sixtimes, at equal intervals, forkinetic analysis of

L-[35S]methionine incorporation (datanotshown). Afterterminationoftheassay,thereovirus-specificproteins intheremaining reactionmixture(480 d)wereprecipitatedwiththereovirus-specificantiserum. The whole immune precipitate wassolubilized andsubjectedtoPAGEanalysis. Theprocedureswere asdescribedin Materials and Methods. To serve asinternal markerproteins, 3H-labeled polypeptides fromthe wild-type

virionweresubjectedtocoelectrophoresiswitheach 35S labeledsample.Thepositions ofthe three size classes ofreovirus-specific polypeptidesareindicatedbyX(155,000to140,000 daltons), u(88,000to70,000 daltons),

and (43,000to33,000daltons) (46).

wild-type P-150 (300C) and the mixture was

assayed at3000 (see Fig. 4B),thepolypeptides

of the three size classes (X, g, and a-) were

synthesized and labeled with 35S in proportions

similarto thosesynthesized invitrobya

post-mitochondrial supernatant ofreovirus-infected

cells(31). The polypeptides of 140,000to120,000

daltons and those of69,000 to 55,000 daltons

were no longer detectable asthe major species

of theproduct.

Electrophoretic patterns similar to those of

the invitroproductswere seenwhen the

wild-typeP-150(3000)wasassayedinthepresenceof

the S-150 (3000) obtained from cells infected

withthe ts261-bmutant atthepermissive

tem-perature (seeFig. 4C)orinthepresenceof the

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infected-at 300

80- 8

160

6

~40-

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20-0 20 40 60 80 100 120 0 20 40 60 80 100 120 0 20 40 60 80 100 120 Mobility

FIG. 4. Viral polypeptides synthesized in vitro by wild-type P-150(309C)and effect ofS-150 (309C) on this protein synthesis. The wild-type P-150(300C)andthe S-150(300C) were preparedfromcells infected at300C

for20h,andthets261-bS-150(300C)waspreparedfrom mutant-infectedcells at 309Cfor 21 h. The reaction mixturecontained 100,4ofwild-type P-150(300C)(1.4 mgofprotein) and 320piofeitherS-150(300C).Ina

controlassay, theS-150(300C)wasreplaced bybuffer A. Incubation was at300Cfor 100min.Theprocedures

forassay andfor analysis ofthe invitroproducts were as described in thelegendtoFig.3and inMaterials

andMethods.(A)Product in the control assay. (B) Product in the presence of wild-typeS-150(300C).Protein

concentrationof the S-150(300C) added to thereaction mixture was 1.3 mg. (C) Product in the presence of ts261-b S-150(300C).Proteinconcentration of theS-150(300C)added was 1.4 mg. 3H label in each panel

denotes polypeptidesfromthewild-type virion subjected tocoelectrophoresis.

S-150 obtained from mock-infected cells (data notshown). When theS-150 (300C) from virus-infected cells was added to the P-150 from mock-infected cells and the mixture was as-sayed under thesameconditions,no35S-labeled polypeptides were detectable in the immuno-precipitates. ThemutantP-150 (30°C)wasalso analyzed under similar conditions. The re-sponseof themutantP-150

(3000)

tothese S-150 was similar to that seen in the assay of the wild-type P-150.

Protein-synthesizing ability ofthe mutant P-150 (37°C) obtained from cells infected at thenonpermissive temperature

(370C).

To de-termine the level ofprotein-synthesizing abil-ity ofthe P-150 from mutant-infected cells at

370C,

cells were infected at 37°C with either ts261-b mutant or wild-type virus for11h.Each cytoplasmic extract was made from an equal number of the viable infected cells. The P-150 (37°C) was obtained from it and analyzed in

vitro at

300C

for itsprotein-synthesizing activ-ity.

Thekinetics of incorporation of 35Slabelinto hotacid-precipitable material are shown in Fig. 5(left panels). The level of the protein-synthe-sizing ability of the ts261-b P-150

(370C)

was

less than one-tenth that of thewild-typeP-150. PAGE analysis of the virus-specific

polypep-tides that were synthesized by each P-150 is showninFig.5Dand G (note 10-fold difference inscale for35S). Most of themutantviral poly-peptidessynthesizedbythe ts261-bP-150(370C) werepolypeptides of 140,000 to120,000daltons andthose of 43,000to33,000daltons(Fig. 5D). The electrophoreticpatternof theproductmade by the ts261-b P-150 (370C) was qualitatively similar to that of the wild-type product (Fig. 5G).However,theamountsofthemutant poly-peptides synthesized and labeledwith35Swere

only 5 to9%o of thosesynthesized by the

wild-typeP-150 (370C).

Effect ofpostribosomal supernatant(S-150 [370C0). To determine if

synthesis

of the mu-tant viral polypeptides ofthe ,u-size class

in-creasesbythe addition ofS-150to these P-150

(370C),

the S-150 (370C)obtained from cells

in-fected at 370C with either wild-type virus or ts261-bmutant was addedtoeach oftheP-150 preparations, and virus-specific polypeptides synthesized byeachP-150(370C)wereanalyzed by PAGE. When the wild-type P-150 was

as-sayed in the presence of the wild-type S-150 (370C) (Fig. 5H), the synthesis of 35S-labeled wild-type polypeptides of the ,- and a-size classes increased two- to threefold above the levels observedinFig. 5G. It appears that the stimulatory effect of the added

wild-type

S-150

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- Invitro300

[D]-ts261-bP-150 alone

[El---with WT S- 150 [F]-- with 261-b S-150

D

F

20 40 60 80 100

6

1

&2

Invitro 30°

[G]-WTP-150 alone (HI---withWT S-150

[I)- -with261-b S-150

Time (minutes)

ts 261-b P-150 from cells infected for 11 hrsat37°

x t a, [DI Al a, (El 'A ) a, F]

20 40 60 80 100 120 0 20 40 60 80 100120 0 20 40 60 80 100 120

Wild-typep-150 from cells infected for 11 hrsot370

Mobility (mm)

FIG. 5. Viralpolypeptidessynthesizedin vitroby ts261-b P-150(37'C)and by wild-typeP-150 (37'C), and theeffect of S-150(37°)oninvitroprotein synthesis by P-150(379C).Anequal numberof cellswasinfected

witheither thets261-bmutantorwild-type virus at37'Cfor 11 h. The P-150(37'C) and the S-150 (37'C)were

prepared from these infected cells. Theassayprocedureswere asdescribed in Fig. 3 and 4. Incubation ofeach

reaction mixturewasat309C for 100 min. (Upper panels) Protein synthesis in vitro by ts261-b P-150 (37'C). Proteinconcentration ofts261-b P-150(379C) addedtoeach reaction mixturewas1.3mg.(Leftpanel)Kinetics

of incorporation ofL-[P5S]methionineintohot acid-insolublematerial. A sample of 20p1 fromeachreaction

mixturewaswithdrawnatthe times indicated. Theamountof hot acid-insoluble radioactivityateach time point isexpressedasthe totalamountpresent in 480 y1 ofeach reaction mixtureafter subtraction ofthe corresponding valueat0time:(D) without S-150(37'C);(E)withwild-typeS-150(37'C) (0.9mgofprotein); (F) with ts261-b S-150 (37'C) (0.8mgof protein). (D, E, and F) Electrophoreticanalysis ofmutant viral

polypeptides synthesizedin480piof each reactionmixture. 3Hlabel in eachpaneldenotespolypeptidesfrom

thewild-type virionsubjectedtocoelectrophoresis.(Lowerpanels) Protein synthesis in vitro by wild-type P-150

(37TC).Protein concentration of wild-type P-150 (379C) addedtoeach reaction mixture was1.4 mg. (Left

panel) Kinetics of incorporation of 35S label: (G) without S-150(370C);(H) with wild-typeS-150(37'C)(0.9

mgof protein); (I) withts-261-b S-150 (379C) (0.8mgof protein). (G, H, and I) Electrophoretic analysis of

wild-typeviralpolypeptidessynthesizedin480 ofeach reaction mixture.

results from the supplement ofsome necessary

soluble components for elongation of the

pep-tidechains,sothatsynthesis of thefulllength

ofthe viralpolypeptides has been accelerated.

The same wild-type S-150, however, was not

effective on thesynthesis of the mutant viral

polypeptides (Fig. 5E), suggestingafunctional

defect(s) of themutantP-150(37°C).

Figures5F and I show that themutantS-i150 (3700) obtained fromtsmutant-infected cellsat 3700 has essentially no stimulatory effect on

the synthesis of both wild-type and mutant

viralpolypeptidesof all size classes. This could

mean thatsomenecessarysoluble components are missing, or atype of inhibitors) may be

presentin the mutantS-150 (3700). When the

ts261-b P-150 (370C) was prepared from cells

infected for7.25 hat370Cand assayedat300C

inthesame mannerand under thesame

condi-tions,the results obtainedweresimilarto those

shown inFig. 5D, E, and F. These dataimply

that the mutant virus-specific polysomes

formed in vivo at 37C have alreadybeen

im-pairedasearlyas7 hafterinfection. The levels

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40 IKEGAMI

ofinvivosynthesis at370C ofthe mutant virus-specific ss RNAs of the three size classeswere

20 to 25% of thatsynthesized by the wild-type virusthroughout the infective cycle (Table2), and it is assumed that about one-half of the amounts ofthe mutant ss RNAs synthesized would bethe mutant viral mRNA's,whichare

associated with polysomes and monosomes

present inthe P-150, sothat the observed low levelof theprotein-synthesizing capacity of the ts261-b P-150 (370C) would stem from the re-duced synthesis of themutantviral mRNA's. In addition, the finding that the mutant P-150 (370C) doesnotrespondtothe supplement of the wild-type S-150(3700)suggeststhat themutant viral mRNA's associated with polysomes or monosomes may be nicked or may have an abnormal secondary structure at

370C,

sothat synthesis of thefulllengthoftheviral polypep-tides ispresumably blocked.

Methylationof mutant viral mRNA's. Most viral andcellularmRNA's have been foundto be blocked and methylated at the 5'-ternini (15, 16, 35, 38, 40),andanimportantrole of the blocked and methylated 5'-terminal structure

inefficienttranslationhas been reported(3, 4). Inregard totheinhibitionofviral protein syn-thesisinmutant-infected cellsat

370C,

one pos-sibility was that there might be a defect in methylation of the mutant viral mRNAs syn-thesized during the infective cycleat

370C.

This wasexaminedinthe followingway.

(i)Invivo methylation. The mutant-infected cells were labeled with L-[methyl-3H]methio-nineand [14C]uridineat

370C

for 9h(2to 11h after infection). The virus-specific ss RNAs were isolated from the cytoplasmic extract, purified, and then analyzed by velocity gra-dient centrifugation. Figure 6 (left) shows the sedimentation profile. The mutant virus-specificss RNAs of the three size classes1, m, and s werelabeled with both3H and

'4C.

The complete hydrolysis of the labeled RNAs by pancreatic RNase(3 ug/ml) confirmed that the methyl-3H label had incorporatedintothe

mu-tantssRNAsofthethreesizeclasses.Asimilar sedimentation profile was also observedin an

analysis of the wild-type viral ss RNAs synthe-sizedinvivoat37°C underthe samecondition, except thatthelabelingof infected cellswasfor

5 h (6 to 11 h). Inboth analyses, the relative

amounts of the methyl-3H label incorporated

into the s-size RNA was slightly less than the expectedproportions onthe basis ofaninverse relationship to the molecular size of the respec-tive

1,

m, andsclasses.

(ii) In vitro methylation. To determine whether the mutant subviral particles derived from infected cells at

370C

are capable of

syn-J.

thesizing the methylated ss RNAs in vitro, a cytoplasmic pelletwas obtained from mutant-infected cells at370Cfor 9h,and it was used as a source ofthe mutantsubviralparticles. The material inthe pellet was treated with chymo-trypsinandincubatedat370C for 90 min in the presence of the optimal concentrations of all components describedbyShatkin (40). The

re-action mixture was thencentrifuged, and the sedimentable material wasseparatedfrom the supernatant. Under this assaycondition,most of themutant ssRNAs

synthesized

were associ-ated with the sedimentablematerial, and little was present in the supernatant fraction. The centerpanel shows thesedimentationprofileof the in vitro transcriptsisolated from the sedi-mentable material. The methyl-3H and 14C la-belswereincorporatedintothenascent mutant

ss RNAsof thethreesizeclasses (1, m, ands), and the relative proportionsof both labelswere

inversely related to the molecular size of the respective sizeclass. Thischaracteristic pattern of methyl-3H incorporation indicates that the mutant subviral particles present in cells in-fected at 370C are capable ofmethylatingthe mutant ssRNAsintheprocessofinvitro

tran-scription.

In vitro transcription by the chymotrypsin-treated wild-type virion was carried out for comparison. The reaction mixture was incu-bated for 60 min at

450C,

the optimal assay temperaturefor thewild-type viral cores. Un-der thiscondition, the wild-typessRNAs

tran-scribed were released from the core

particles

during the period of theassay.The sedimenta-tionprofile shownintheright panelwas simi-lartothatreported by Shatkin (40). These data inFig. 6 supportthe conclusion that methyla-tionof themutant ssRNAssynthesized in vivo

at370C is not restricted.

Effect of temperature shift-down of mu-tant-infected cells. The inhibition of produc-tionof the infectious mutant virus can be re-versed by shifting the temperature of mutant-infectedcells from 37 to300C,and the maximal yield of infectiousvirus can be obtained. It is of interest to ascertain whether the protein-syn-thesizing ability of the mutant virus-specific polysomes has been restored in these shift-down cells. Two sets of an equal number of mutant-infected cells were maintained at

370C

for 11 h. One set was harvested at 11 h. The

other set was shifted down to 30°C and was maintained for an additional4h. TheP-150 was prepared from each set of the mutant-infected cells,and theprotein-synthesizing ability ofthe P-1i0 was assayed in vitro at

300C

in the

ab-sence of the S-150. The results are shown in

Fig. 7. Theprotein-synthesizing ability of the

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REOVIRUS

ts 261-b mutant virusspecificssRNA's Invivo at370 In vitroat 370

Wild-typevirusspecificssRNA's invitro at450

25r- _I _m _s

H |

20H

5'

4

1

'CQJ

E

1:0

100RNose

0 10 20 30

I m s

I-I MZ

rII

11

'C.

Ec

-

>t

w

t

Il

0 10 20 30

Fraction number

0 10 20 30

FIG. 6. Velocitysedimentation analysis ofts261-bmutantvirus-specificsingle-strandedRNAssynthesized invivo and in vitroat37`C. (Left) ts261-bmutantvirus-specificssRNAssynthesized andmethylated invivo

at 370C. The mutant-infected cells (108) were maintained at 370C in methionine-free spinner medium

supplemented with 20 mM sodium formate, 20 puMguanosine, 20 PM adenosine, and 5%fetal bovine

serum.Infectedcells were labeled with ['4C]uridine (50 PCi) andL-[methyl-3H]methionine (2.2 mCi) for

9 h (2to11 hafterinfection). ActinomycinD(15pg) waspresentfor2 hpriortoinfectionandthroughout the labelingperiod. Theprocedures for extraction andpurification ofssRNAsand foranalysis of velocity

gradientcentrifugation were as described in Materials and Methods and in Table 2. Hydrolysis ofRNAs

ineachfraction bypancreatic RNase (3 pglml) was carried outas described by Gomatos (18). (Center) ts261-b mutant viral ss RNAs synthesized and methylated in vitro at370C. The cytoplasmic pellet was

obtainedfrom cellsinfectedat370C for9 h with the ts261-b mutant in thepresenceofactinomycin D. The pelletwasresuspendedin0.01 MTris (pH 8.1)andwasusedasasourceofmutantsubviralparticles. The detailed procedures for in vitro synthesis of RNAsandforextraction andpurification ofssRNAsfrom the

assaymixturewere asdescribedinMaterials and Methods. In vitroassaywasat370C for90min,and the reaction mixturewascentrifugedat17,000rpmfor30min.ThessRNAswereextracted andpurified from

pelletedmaterials andanalyzed by velocity gradient centrifugation. (Right) Wild-typeviralssRNAs synthe-sizedinvitroat450C byviralcoresderivedfromthepurified wild-typevirus. Theproceduresusedforinvitro transcription and methylation by wild-typeviralcores werethesame asthosedescribedbyShatkin(40).The ssRNAs releasedasfree fromviralcoreswerepurifiedasdescribed inMaterialsand Methods.

ts261-b P-150 from the shift-down cells

in-creased almost 10-fold, and the ability to

syn-thesize thepolypeptides ofo- sizeand those of

140,000 to 120,000 daltons was markedly

re-stored (Fig. 7, center). These data imply that

the mutantvirus-specific polysomes have been

modifiedinafunctionally activestructure

dur-ing theperiod of shift-down incubation atthe

permissive temperature. When thesameP-150

fromtheshift-down cellswasassayedat300Cin

thepresenceofthets261-bS-150 (370C) (Fig. 7,

lowerpanel), theprotein-synthesizingability of

theP-150decreased by 70to80%suggesting the

presenceofaninhibitor(s) in themutantS-150

(370C).

Analysis ofpostribosomalsupernatant.The

S-150 from cells infected with the ts261-b

mu-tant at 370C did not manifest a stimulatory

effect on the viralprotein synthesis catalyzed

by the P-150 (Fig. 5, 7). Its effect was rather

inhibitory insomecases. Asfor the nature ofa

components)responsible for this inhibitory

ef-fect, thets261-b S-150 (370C)might contain an

increasedamountofanenzyme,likeanuclease

that hydrolyzed the viral mRNA's or cleaved

the virus-specific endogenous polysomes. For

elucidation of thisproblem,anexperimentwas

carriedout todetermine theextentof

hydroly-sisofthe free virus-specific ssRNAsduring in

vitroincubation withanS-150 sample. Three

S-I

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42 IKEGAMI

[IiProteinsynthesisin vitro at30° byts 261-b P-150 obtained from cells infected for11 hrs at370

Q) 5_- 10 H H

a 6

u 0 20 40 60 80 100 0 20 40 60 80 100 120 Time (minutes) Mobility (mm)

[21 Protein synthesis in vitro at 30° by ts 261-b P-150 obtained from the infectedcells incubated for 4 hrs at300 after temperature shift down from 371 at11 hrs.

AdditionofS-150 fromncellsinfectedwith ts261-bat37°to the261-b P-150frominfected cells after shiftdown.

10 20 h4

5 010 2

0 20 40 60 80 100 0 20 40 60 80 100 120 Time(minutes) Mobility (mm)

FIG. 7. Restoration ofprotein-synthesizingability of ts261-b P-150 from mutant-infected cells after shifting down to 300C from 370C. Two sets ofan

equal number of cellswereinfected with ts261-b

mu-tantat370C. At 11 hafter infection, the ts261-b P-150 (370C) wasprepared from one setof infected cells. Anothersetof infected cellswas transferredto30'C and incubated for an additional 4 hat300C. The ts261-bP-150(370C/300C)wasprepared from it. The

assay and the subsequent analysis of the products

werecarriedoutinthesame manner asdescribed in

thelegendtoFig.5.(Top)Protein-synthesizing abil-ity invitroat30°C ofts261-b P-150 (37°C) (1.3mgof

protein) inthe absence of S-150 (370C) and electro-phoretic analysis of the in vitro product. (Center) Protein-synthesizing ability in vitroat30°C of ts261-bP-150 (37°C/30°C) in the absence of ts261-b S-150 (370C) andelectropherogram of thein vitroproduct. 3Hlabeldenotespolypeptides from the wild-type vir-ionsubjectedtocoelectrophoresis. (Bottom) Protein-synthesizing ability of ts261-b P-150 (370C/30°C) in thepresenceof ts261-b S-150(37°C) and electrophe-rogram of the in vitro product. The ts261-b S-150 (37°C) wasobtainedfromcells infected for 1I hat 37°C, and the protein concentration added to the reactionmixture was0.8mg.

150 samples (ts261-b 150 [370C], wild-type S-150 [370C], and mock-infected S-150 [370C]) were prepared from cells infected or mock-in-fected at 370C for 11 h. Two kinds of virus-specific ss RNAs (ts261-b ss RNAs and wild-type ss RNAs) were isolated from infected cells that had been exposed to [14C]uridine and

L-[methyl-3H]methioninefor9h at370C.Thesess

RNAs were used asthesubstratefordetection of nuclease activity. Each methylated viral ss RNA wasincubated in vitro at300C with each of theS-150 (370C)samples, and the radioactiv-ity in theacid-precipitable material was deter-mined at the timesindicated. Enzymatic activ-ity that hydrolyzed the virus-specific ss RNAs was present in all three S-150 (370C) samples (Fig. 8). Both ss RNAs were hydrolyzed to a similar extent. Judged by 14C counts (upper panels), 20 to 50% ofthe original radioactive countsremained in theacid-precipitable mole-cules. The observeddifferences in the extent of hydrolysis byeachS-150 appear to result from the differences in the protein concentration of each S-150 tested (see legend to Fig. 8). The methyl-3H-labeled portion of the RNA mole-cules was relatively insensitive to an enzyme presentin theS-150 (lower panels). About80% of themethylatedRNAs,either the intact mol-ecule or the oligonucleotides derived from it, appear tobeprotectedfrom the cleavageof the methyl-3H-containing 5'-terminal nucleotides. From these data, it is reasonable to conclude that the ts261-bS-150(370C)does not contain an increased amount of nuclease, and the inhibi-tory effect of this S-150 (370C) onprotein syn-thesis appears to be attributed to a compo-nent(s)otherthananuclease.Thenatureofthe inhibitory component(s) remains to be eluci-dated.

DISCUSSION

The ts261-b mutant isolated in this labora-torysynthesizesin vivoreduced amounts of the virus-specific ds RNAs and ss RNAs

through-out the infective cycle at the nonpermissive temperature,370C. Despitethe reductioninthe total amounts synthesized, the 10 segments of the mutant ds RNA andthreesizeclasses of the

ss RNAswere synthesized in the proper rela-tiveproportions.The methylation of the newly synthesized ss RNAs was not blocked in the mutant-infected cells at370C.Thecapabilityof the ts261-b mutant subviral particles to tran-scribe the virus-specific ss RNAs in vitro at 370C was comparable to that of the wild-type subviralparticles. As judged from the capabil-ity tosynthesize reovirus-specific ds RNAs and

ssRNAs,the ts261-bmutantcanbeplacedinto the category of dsRNA-positivemutants, like

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ts261-b SS RNAs

% [iJ 14C-Uridinelabel

WT SS RNAs

ol [2] C-Uridinelabel

100

t 80

0 z

a 60

x, 40

!0 O

4-0 20

0.§

.q 100'

't 80

%

60

40

A

I

~~~~~B

-A.:Buffer

B:261-bS-150(37 \ C lC: WT S-150 (37) D D:Mock-infectedS-150 (37 D

[3]

3H-methyl

label

h

KAA

BCD

'I ~~~~B

100

80 60

40

A

B

c

_ -uD

201 0

100

80

[4]

3H-methyl

label

A

B 60_

401-201_

I

0 30 60 90 0 30I 60 90

Minutes

FIG. 8. Detectionof nuclease activityinS-150thathydrolyzes free virus-specificssRNAs. Theanalysisat each timepoint for detection ofnucleaseactivityinaparticularS-150(37°C)wascarried out in the presence

ofprotein-synthesizingmixture inafinalvolumeoflOOg0 inindividual tubes that containedaknownamount

of labeledts261-bssRNA orwild-type ssRNA. ProteinconcentrationsoftheS-150tested were210 pg of ts261-bS-150 (37°C),300pgof wild-type S-150(37°C),and 370pgofthemock-infectedS-150(37C).Inthe controlassay, bufferA was included inplace oftheS-150. The in vitro incubation was at30°C. Samples takenat0, 30, and 90 min weremixed With 2 mlof5% trichloroaceticacid andkeptat0°Cfor30min. Theremainingacid-precipitable,labeled RNAswerecollectedonmembranefilters(Millipore Corp.).Thess

RNAs usedwereisolatedfrom cells infectedand labeled(2 to11 hpostinfection) at37°Candpurifiedas

describedinthelegendtoFig. 6 and in Materials and Methods.

those ofclasses A, B, F, and G, which have been isolated and characterized byFields and

Joklik andtheircollaborators (7, 12, 23, 25).

A major temperature-sensitive restricted

function of the ts261-bmutantwasexpressedat

the translational leveland resulted in the

inhi-bition of theviralproteinsynthesis.

The studies using thein vitro protein-synthe-sizing systemreconstituted withanendogenous

polysomalfraction, P-150, andapostribosomal

supernatant,S-150,from reovirus-infectedcells

demonstratedthat restriction of the viral

pro-tein synthesis in the mutant-infected cells at

3700 wasattributedtothe defectivefunctions of

bothmutantvirus-specific polysomesand

com-ponents present in thepostribosomal

superna-tant.Thesynthesisofthemutantviralproteins

wasrestrictedatsomestep(s)ofelongationand

completion of the peptide chains. Under the

experimental conditions in this study, it was

not clear whether the initiation of the viral

peptidechainswasblocked.

When P-150 and S-150 were obtained from

reovirus-infected cells at the permissive

tem-perature and in vitro proteinsynthesis by the

P-150was assayed intheabsence of the S-150,

the P-150 synthesized mostly polypeptides (43,000to33,000 daltons) of thea size and

poly-peptides of 140,000to120,000 daltons and those

of69,000to55,000daltons. The polypeptides of thelattertwosizeranges arevirusspecific, as

theycanbeprecipitated by thereovirus-specific

immune serum, but their apparentmolecular

weights, determined by theirmobilities in SDS-phosphate gel, donot correspondtoanyof the

virus-specific polypeptides reported previously (31, 32, 45,46). Wheninvitro protein synthesis bytheP-150wasassayedinthepresenceofthe

S-150, theelectrophoreticpatternofthe result-ing product showed polypeptides of88,000 to

70,000daltons and thedisappearance of poly-peptides of69,000to 55,000daltons. Ithas not yetbeen determined whether thesetwoevents

occurred by a direct conversion of the latter

)I--v_

20_

43

I

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44 IKEGAMI

polypeptides (69,000 to 55,000 daltons) to the polypeptidesof the Asize (88,000to70,000 dal-tons),or whether they are unrelated. The effect of the added S-150 onthe in vitroprotein syn-thesis directed by the active P-150 probably results fromthe supplement ofthe active fac-tors, such as elongation factorsoramino acyl-tRNA synthetases, which are necessary for elongation and completionof thepeptidechain andsome of which mayberesponsible for the regulation oftranslational frequency.It is also conceivablethatapossible effect of the added S-150maybe partly relatedtophosphorylationor glycosylation. As aresult, the mobility ofthe modified viral polypeptides

(/i

size) maybe al-tered, since it has been found that the ,u-size polypeptides derived from thewild-type vision arephosphorylated (27) and glycosylated (26). Cross and Fields have reported that one or two species ofthe

ak-size

polypeptides synthe-sizedinvivo atthe nonpermissive temperature

(390C)

by thets mutantsofclassesA, D,F, and

G manifestan aberrent electrophoretic mobil-ity in ahigh-resolution PAGE system. The mo-bilityisslightly fasterthan thatof the control, but the altered mobility remains within the range of the migration distances of the

4u-size

polypeptides (8). Thesetsmutants arecapable of synthesizing at 390C most of the reovirus polypeptides intheproperrelative amounts (8, 25). In contrast tothe ts mutants of classes A, F, and G, the ts261-b mutant was unable to synthesize the polypeptides of the sizes corre-sponding to the reovirus-specific capsid and noncapsid polypeptides in the mutant-infected cellsat

370C.

Thiswasdue to the impairment of the mutant virus-specific polysomes, which wereunabletorespondtothe additionof active soluble factors that stimulate the synthesis of the completed lengthof the viralpolypeptides.

The S-150obtained from the ts261-b mutant-infected cells at

370C

(see Fig. 5 and 7) had no stimulatory effectonthe synthesis of theviral polypeptides of allsizeclasses. The ineffective-nessof this S-150 appears to result from some defective functions of soluble proteins rather than an action of a nuclease, since no increased nuclease activity is found in thisS-150 obtained frommutant-infected cells at

370C.

Alternatively, the ineffectiveness of the

mu-tantS-150

(370C)

may be due to a limited avail-ability of soluble components, which are re-quired for an efficient translation. Since high-speed supernatants from wild-type

virus-in-fected cells contain a quantity of virus-specific polypeptides (20;unpublishedobservation),

fur-ther work will be necessary to elucidate a

role(s)inprotein synthesis for reovirus-specific soluble proteins synthesized at

370C

by the ts261-b mutant.

In the high-speed supernatantfrom frog vi-rus-infected cells, a newly synthesized frog vi-rus-specific protein kinase has been identified which isultimately assembledintovirion parti-cles. Ininfectedcells,certaints mutantsof this virus fail to synthesize the virus-specific pro-tein kinase at the nonpermissive temperature (41). This evidence from another system sug-gests a possibility of the presence of reovirus-specific protein kinase. A possible role of the proteinkinase inthe regulationof protein syn-thesis has been indirectly demonstratedinthe reticulocyte lysate (29). Underconditions such asthe incubationofthelysateabove30°Cinthe absenceof heminorthe addition of ds RNAs or oxidizedglutathione tolysate, the initiation of the peptide chainis inhibited(2, 6, 9, 10). This inhibition is due tothe formation ofa transla-tional inhibitor, which is formed in the high-speed supernatant (10, 29). The inhibition of peptide chain synthesis by the translational inhibitor can be reversed by the addition of cyclicAMP (10,29)ortheadditionof the active initiationfactors that areinvolvedinbinding of the initiator

Met-tRNAf

to the 40S ribosomal subunits (2, 6, 9), suggesting that the transla-tionalinhibitormayresembleaninactiveform of cyclic AMP-dependent protein kinase and that the phosphorylation of a particular pro-tein(s)mayplayanimportant role inthe regu-lation of proteinsynthesis.

The ds RNAs are inducers ofproduction of interferon as well as inhibitors of protein syn-thesis. In reovirus-infected cells, the parental ds RNAs are conserved within the parental subviral particles (42), the progeny ds RNAs

arereplicated withinanewlysynthesized parti-cle structure (39, 44), and free ds RNAs have never been detected inanalysis of cellextract (17, 39). However, it is conceivable that an undetectable amount of free ds RNAs, if they

are present in mutant-infected cells at

370C,

may engage information of a translational in-hibitor similartothat foundinthereticulocyte lysates.

Another consideration is that the inhibition of the synthesis of the ts261-b mutant viral proteinsat

370C

may bedue toanactionof the induced interferon. That this would -not be the

caseissupported by(i) the evidencereported by Lai and Joklik (28) that during the infective cycleat

390C

withanyof thets mutantsof the sixclasses (A, B, C, D, E, andG), the level of interferon production is approximately

one-sixth of thatproduced bythewild-typevirusat

thesametemperature, and that theamountof interferon producedcorrelates with the yields of the infectiousvirus;and(ii)myfindingsthat during the infective cycle at

370C

with the ts261-b mutant, the synthesis of the cellular

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and viral proteins is inhibited and the inhibi-tion of the viralprotein synthesis is reversed by shifting the mutant-infected cells from 37 to 300C.

ACKNOWLEDGMENTS

I thankShirley Keelfor technical assistance. I am in-debtedtoPeterJ.Gomatos andthemembers of this labora-toryformanyhelpful discussions.

Thisinvestigation wassupported by Public Health Ser-vice grantCA-08748 fromthe National Cancer Institute.

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