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Replication of Sindbis Virus V. Polyribosomes and mRNA in Infected Cells

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Copyright @ 1974 American Society forMicrobiology Printed inU.S.A.

Replication

of Sindbis

Virus

V. Polyribosomes and mRNA in Infected Cells

DANIEL T. SIMMONS' AND JAMES H. STRAUSS

Division of Biology, California Institute of Technology, Pasadena, California91109

Received for publication 17 April 1974

Cells infected with wild-type Sindbis virus contain at least two forms of

mRNA, 26S and 49S RNA. Sindbis 26S RNA (molecular weight 1.6 x 106)

constitutes 90% by weight of the mRNA in infected cells, and is thought to

specify the structural proteins ofthe virus. Sindbis 49S RNA, the viral genome

(molecular weight 4.3 x 10"), constitutes approximately 10% of the mRNA in

infected cells and is thought to supply the remaining viral functions. In cells

infected withts2,atemperature-sensitive mutant of Sindbis virus, the messenger

forms also includeathird species of RNAwith a sedimentation coefficient of 33S

and an apparent molecular weight of 2.3 x 106. Hybridization-competition

experimentsshowed that90%ofthe basesequences in 33SRNA from these cells are also present in 26S RNA. Sindbis 33S RNA was also isolated from cells

infected with wild-type virus. Afterreaction with formaldehyde, this species of

33S RNA appeared to be completely converted to 26S RNA. These results

indicate that 33S RNA isolatedfrom cells infected with either wild-type Sindbis or ts2isnot auniqueand separate form ofSindbisRNA.

In cells infected with group A arboviruses

(alphaviruses), at least two species of

virus-specificsingle-strandedRNAaremanufactured

(4, 15, 16). In the case ofSindbis virus, these

twospecies ofRNA have sedimentation coeffi-cients of49Sand 26S. Sindbis 49S RNA isthe viralgenomeand has amolecularweight of 4.3

4 0.3 x 106 (3, 13). Sindbis 26S RNA has a

molecular weight of 1.6 x 106 and consists of

one-third of the viral genome (13).

Ithas been determined that 26S RNA

func-tions as a messenger in the infected cell (5, 8,

12). Since 26S RNA has only one-third of the coding capacity of the genome, it is expected

that the othertwo-thirds ofthe viralfunctions

arespecifiedbyotherspeciesof

Sindbis-specific

mRNA. Kennedy (5) and Mowshowitz (8) de-scribed a second species of Sindbis mRNA havingasedimentation coefficient of33S anda

molecularweight of 2.2to 2.4 x 10g. Rosemond

andSreevalson (12)described severalspeciesof

mRNA smallerthan 26S RNAininfected cells.

In an earlier publication (14), we obtained

indirectevidencefortheexistence ofaspeciesof

single-strandedRNAwithamolecularweightof

2.8 x 106 which contains two-thirds of the base sequence information present in 49S RNA but none of the information present in 26S RNA.

IPresent address: National Institutes ofHealth, National

Institute ofAllergy and Infectious Diseases, Bethesda,Md. 20014.

We hypothesized that this species of RNA

functions as a messenger in infected cells.

In this paper, we support the evidence that

26S RNAisthepredominantmessenger in cells infected with Sindbis virus, and we show that

49SRNA, the viralgenome, serves asthesecond

form of messenger in infected cells. We also

present evidence that Sindbis 33S RNA is a

structural variant of26S RNA and not a

sepa-rate species ofSindbis-specific RNA. No other unique speciesofRNAwasdetectedin prepara-tions ofmRNA.

MATERIALS AND METHODS

Except as described below, the materials and methods used intheseexperiments havebeen

previ-ouslydescribed (13). Theseinclude the methods for

sucrose gradient andformaldehyde sucrose gradient velocity sedimentation, analytical and preparative

acrylamide gel electrophoresis, and

hybridization-competition.

Isolation ofSindbis-specificpolysomes. Chicken embryo fibroblastswereinfected withSindbisvirusas previously described(13). The cellswerelabeled with

[5-_3Hluridine

(specific activity 22.4 Ci/mmol) for

variousperiods oftimeand/orwith ["4C]aminoacids (Schwarzhydrolysate,specific activity50mCi/mmol) for 2 min. Insomecases, thecellsweretreated with50

tig

ofcycloheximideperml(NutritionalBiochemicals Corp.) for 7minbeforeharvestingtheinfectedcells. The flask ofcellswasrapidly cooled to0 C inan ice-waterslurry,and thecellswerewashed three times

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in ice-cold 0.01 M Tris, 0.01 M KCI, and 0.0015 M MgCl, (pH 7.4). The cell monolayerwasscrapedoff thesurface of theflaskwitharubberpolicemaninto

0.5ml of thesamebuffer. After 10 minat0Ctoallow the cellstoswell,thecellswerehomogerizedwith10 to 15 strokes ofaglass Douncehomogenizer. Nuclei and unbroken cellswereremovedby centrifugationat 1,000 x gfor 8 minat0C.The supernatantwasmade 0.5%with respect to deoxycholate (Matheson, Cole-man & Bell), andparticulate materialwas removed by sedimentation at 10,000 x g for 10 min. The polysomes and othercytoplasmicmaterial present in theclarifiedsupernatantweresedimented inalinear 15to40%sucrosegradientintheabovebufferfor2.25 h(unless otherwisestated)at40,000rpm and1.5C in theSpinco SW40orSW41rotor.

If thegradientwas tobeanalyzedforradioactivity, it was firstpumped through aflow cell ina Gilford recording spectrophotometer to determine the posi-tion of the monosome band. The material in each fraction was precipitated with trichloroacetic acid andcounted in toluene-fluor scintillation fluid.

If the labeled RNA in the gradient containing

Sindbis-specific polysomes was to be analyzed by velocity sedimentation, the gradient was first frac-tionated directly into 2-ml beakers containing 20 lAiters of 10% sodium dodecyl sulfate (SDS). The RNA from selected fractionswassubjectedtosucrose

gradient velocity sedimentation (14) for 4.5 h at

40,000 rpm in theSpincoSW41 rotor.

Preparation of mRNA from infected cells. To prepare mRNA, polysomesweresedimentedthrough

asucrosegradient such that thoseconsistingofmore

than 10 ribosomes were pelleted in the centrifuge tube. The gradient above the pellet was carefully removed by aspiration, and the pellet was resus-pended in 0.2 ml of0.01 M Tris, 0.01 M KCl, and 0.0015MMgCl2(pH 7.4). The solutionwasmade0.01

MinEDTAtorelease the mRNA fromribosomesand 100Ag/mlindextran sulfate(Sigma ChemicalCo.)to

inhibitRNase. To separate the released mRNA from nucleocapsids present in the polysomal pellet, the material was sedimented in a sucrose gradient, as

described above, for thesedimentation ofpolysomes withtheexception that the time ofcentrifugationwas increasedto 3h. Thegradientwascollected inbeakers containing 20 sliters of 10% SDS, and a volume of eachfraction was counted forradioactivity.TheRNA inthepooledfractionswas made 0.2 M inpotassium acetate(pH 6.0) andprecipitated with 2.5volumes of ethanol overnight at -20 C. The RNA was resus-pended in 0.01 M Tris, 0.06 M NaCl, 0.001 M EDTA, and 1% SDS (pH 7.2) and extracted twice with phenol-chloroform (9) at 0C and twice more at room temperature. The RNA was then subjected to electro-phoresisinanacrylamide gel as previously described

(13).

Preparation of Sindbis 33S RNA. Sindbis 33S RNA was prepared from chick cells infected with eitherwild-typeSindbis virusof ts2.

When thesourceofthe33SRNAwascellsinfected withwild-type Sindbis (HR), total cellular RNA was prepared aspreviously described (13, method A, but without diethyl oxydiformate). The RNA was

sub-jected to sedimentation in a 15 to 30% sucrose

gradientin0.01MTris,0.06MNaCl,0.001MEDTA,

0.2%SDS(pH7.2)for4.5hat40,000rpm and 23 C in

theSpincoSW41 rotor,andthefractions

correspond-ing to Sindbis 26S RNA and 33S RNA were pooled

together. The RNA was precipitated with ethanol,

andtheresuspendedRNAwas subjectedto

prepara-tivegelelectrophoresis (13).Thefractionscontaining

33S RNA were combined andthe RNA was

concen-tratedbyethanolprecipitation.

When thesourceofthe33SRNAwascellsinfected with ts2 at 39C, polysomes were prepared and

subjectedtosedimentation, asdescribed above.The

fractionscontainingpolysomeswith4to10ribosomes

weremade 1%with respecttoSDS, and thesamples were extracted withphenol-chloroform (9). The spe-cies of33SRNAwaspurified byelectrophoresisina

preparative acrylamidegel (13).

Preparation ofviral nucleocapsids.

Nucleocap-sids were isolated from detergent-treated Sindbis viruswhich hadbeenpurifiedaspreviouslydescribed

(10).The virussolution wasdilutedthreefold in 0.01

MTris,0.01MKCl,and 0.005 MEDTA(pH7.4), and

Triton X-100(Sigma Chemical Co.) was added to a final concentration of0.5%. The nucleocapsids were sedimented in a 15 to 40% sucrose gradient in the

same buffer for 3 h at 40,000 rpm and 1.5 C in the

Spinco SW40 rotor. The fractions containing the

nucleocapsidswerepooledand storedfrozen.

RESULTS

Polysomes

ofinfected cells. Polysomes

pre-pared

from cells infected with Sindbis virus

served as the source of the mRNA in these

experiments.

Itwastherefore

important

to

ana-lyze

the size ofthese polysomes and to deter-mineif

they

wereinvolvedinprotein

synthesis.

When cells infected with wild-type (HR

strain) Sindbis virus were briefly labeled for 2

min with

"4C-labeled

amino

acids,

the sedi-mentable

radioactivity

was associated

primar-ily

with monosomes,

disomes,

and trisomes

(Fig.

1),

indicating

that these structures were

involved in

protein

synthesis. Smalleramounts of amino acid label were incorporated into

larger

polysomes.

The polysomes were also la-beled with

[3HJuridine

which was added at 30 min after infection in the presence of

ac-tinomycin

D

(in

order to label

only

virus-specific

RNA).

In

addition,

the uridine label

was

incorporated

intoviralnucleocapsidswhich

sediment at 140S. In these gradients, most of

the

nucleocapsids

sedimented slightly faster

thantrisomes andattheposition shown bybar

bin

Fig.

1.

Relatively

littleaminoacidlabelwas

associated with

nucleocapsids

afteronly 2 min of

labeling.

In cells infected with certain mutants of

Sindbis virus (ts2, ts5, tsl3, and tslO6), the

synthesis

ofviral nucleocapsids istemperature

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sensitive (1,2,E. Strauss, personal

communica-tion).When polysomeswereprepared from cells

infected withts2atthe restrictivetemperature, uridine label was found in polysomes as

ex-pected, but relatively little uridine had been incorporated into nucleocapsids (Fig. 2) (nu-cleocapsids, if present, would be found near

fraction 27 in this gradient). These polysomes

were also labeled with amino acids in a 2-min

pulse showing that all classes of ts2polysomes wereinvolved in proteinsynthesis.

Because polysome preparations from cells infected with ts2 werefree from large amounts ofnucleocapsids, we were able to calculatethe relative amountsof[14C]aminoacidsto [3H ]uri-dine incorporated into each class of polysomes. Thus, in Fig. 2, the ratio of [14C ]amino acidto [3H]uridine label increasedasthe polysomesgot larger. This was expected on the basis that

larger polysomes contain a greater total length ofpolypeptide chains perunitlength of mRNA

thando smaller polysomes. The "4C-to-3H ratio in the polysomes of Fig. 1 could not be calcu-lated due to the presence of large amounts of nucleocapsids in thegradient (see below).

How-ever, these resultssuggest that polysomes from cells infected with wild-type Sindbis virus are

smallerthan polysomes from cells infected with

ts2.

Furthermore, to show that polysomes were

12

-t

-

4-FRACTION NUMBER

FIG. 1. Polysomes from cells infected with

wild-type Sindbis virus. 1.5 x 107 chickenembryo cells

wereinfectedwith wild-type (HR)Sindbis virus and

labeled with 20uCi of [5-3H]uridinepermlfrom0.5

to4.5 hafterinfectionandwith 100 MCiof[14Clamino

acidsperml(Schwarz hydrolysate) for2min before

cell harvesting. The polysomes were prepared and

sedimented. The bars labeleda, b, c, and dreferto

certainfractionswhichwerepooled for analysis ofthe

labeled RNA (see Fig. 4). Thearrow on theright in this and all other figures represents the top of the

gradient. Symbols: -, [3H]uridine label;

---[14C]aminoacid label.

FRACTION NUMBER

FIG. 2. Polysomesfromcellsinfected withts2.The

experimental procedures were as described in the legendtoFig. 1withthe exception that the cellswere infected with ts2 at39C. Symbols: -, [8Hjuridine

label;---, ['4Claminoacid label.

notdegraded during the isolationprocedure, we

prepared polysomesfrom uninfected cells after

a brieflabeling period with amino acids. Most

of the sedimentable radioactivity was

associ-ated with large polysomes consisting of five or more ribosomes, whereas relatively little label was in monosomes or disomes (Fig. 3). More-over, as shown below, the RNA isolated from

polysomes of infected cells was usually unde-graded. These results indicate that, under the conditions of infection, polysomes from cells infected with either wild-type Sindbis or ts2 weresmaller than those from uninfected cells.

Characterization of the RNA from gra-dients of Sindbis-specific polysomes.

Poly-somes labeled with [3Hluridine were prepared

fromcellsinfected withwild-type(HR) Sindbis virusandsubjectedtovelocity sedimentationin

a sucrose gradient. In this gradient, theprofile of3Hradioactivitywassimilartotheoneshown inFig.1. Fourdifferentsamplesof thisgradient

werepooledasshownbythe bars labeleda,b,c,

or d in Fig. 1, and the structures in these samplesweredisrupted withSDS. The RNA in

eachsamplewassedimentedinalinearsucrose

gradient in the presence of E. coli rRNA (Fig. 4). Sample (a), which apparently consisted of

large polysomes, and sample (b), which

con-sisted of trisomes, tetrasomes, and viral

nu-cleocapsids, contained both Sindbis 26S RNA

and49SRNA (Fig. 4[a] and [bI). On the other

hand, samples (c) and (d), which consisted of

monosomes andslowersedimenting structures, respectively, contained primarily 26S RNA

(Fig. 4[c] and [d]). The interpretation of these

results is complicated by the presence oflarge amounts of nucleocapsids which sedimented

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fasterthan 140S throughout the bottom half of the gradient containing the polysomes. For

example, it was shown that after EDTA

treat-ment and resedimentation of the material in

Fig. la, 35% of the 3H label was in viral nucleocapsids. This observation and the data

FRACTION NUMBER

FIG. 3. Polysomesfrom uninfected cells. A culture

ofuninfected chickenembryocells was labeled with

100ACiof[3H]aminoacidsperml(Schwarz hydroly-sate) for2min beforecellharvesting. Thepolysomes

weresubjectedtovelocity sedimentation,asdescribed

inthelegendtoFig.1with theexceptionthat the time

of centrifugationwasreduced to2 h.

FRACTION NUMBER

FIG. 4. Characterization of the RNA from gradi-ents of Sindbis-specific polysomes. Chick cells

in-fectedwithwild-typeSindbisviruswerelabeledwith

20 MCi of [5-3H]uridine per ml from 0.5 to 4.5 h

postinfection. Thepolysomeswerepreparedand

sedi-mented,and thegradientwascollected for analysis of

the RNA. The RNA in each of the four samples, correspondingtotheregions ofthegradient shown by

bars a, b, c and d in Fig. 1, was subjected to

sedimentation in a 15% to 30% sucrose gradient at 40,000rpm and 23Cfor 4.5h in the Spinco SW41 rotor using '4C-labeled E. coli rRNA as markers. Symbols: (a-d) -, 'H-labeled Sindbis-specific RNA inbars a-dof Fig. 1,respectively;---, "4C-labeled E. coli rRNA.

shown in Fig.4probably indicate that thelarge majority ofthe 49S RNA in these gradients of

Sindbis-specific polysomes was in the form of

nucleocapsids, whereas most of the 26S RNA was associated with ribosomes.

TheRNA in each of thesamples (a)through (d) was treated with RNase to detect the presenceoflabeleddouble-stranded RNA. Only sample (d) contained RNase-resistant RNA, and thiswasshownto consistof the threeforms

ofSindbis replicative forms (RF) (13) (datanot

shown). The conclusion was that only sample (d)contained theSindbisreplicative

intermedi-ates.

mRNA in cells infected with wild-type

Sindbis virus. To isolate mRNA, polysomes

fromcellsinfected withwild-type Sindbis virus

were sedimented such that those consisting of

more than 10 ribosomes were pelleted in the centrifuge tube. For this experiment, the cells weretreated withcycloheximidetoincreasethe

average sizeof thepolysomes (7) andtoreduce the relative amounts of nucleocapsids in the pelleted material. The polysomes in the pellet

were dissociated with EDTA, and the compo-nents weresedimented in asucrose gradient to separate released RNA (i.e., mRNA) from

nu-cleocapsids (Fig. 5). The polysomal pellet

con-tained relatively little uridine label in

nu-cleocapsids as shown by the small amounts of 3H radioactivity sedimenting with the

nu-cleocapsid marker. Figure5 shows as well that the RNAreleased by EDTAwasassociatedwith

structures (presumably RNA-protein

com-plexes) sedimenting at approximately 35S to

40S (sample I), and at60Sto70S (sample II).

After deproteinization, the RNA in each of

samplesIand IIwassubjectedtoacrylamidegel

electrophoresis (Fig. 6). Sample I consisted

primarily of 26S RNA (Fig. 6[a]), whereas 49S RNA was the predominant species of RNA in

sample II (Fig. 6[b]). Other species ofSindbis mRNA could possibly be present in sample II

since another band(or bands) could be hidden under the leading sideofthe49S RNApeak in

Fig.6(b). Approximately 10% ofthelabelin the

mRNA fraction (samples I and II) was in 49S RNA. These results indicate that 26S RNA is

the predominant species of mRNA in infected

cells, butthat49SRNAhas messengerfunction aswell.

mRNAin cells infected withts2. Since the

virus-specific polypeptides synthesized in cells infected with ts2 are different in size from the

polypeptides synthesized in cells infected with

wild-type Sindbis virus (17), we characterized

thespecies of mRNA in ts2-infected cells. The mRNA forms weresubjected to electrophoresis

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I

7

x X

C

E E

t4-4

2-2'

FRACTION NUMBER

FIG. 5. Disruption of polysomeswith EDTA.1.5x

107chickenembryocellswereinfectedwithwild-type (HR) Sindbis virus and labeled with20ACiof

[5_3H]-uridineper mlfrom 3 to 5 hafter infection. Seven minutesbeforecellharvesting,themediumwasmade

50

Aglml

withrespecttocycloheximidetoincrease the

averagesize of the polysomes. Thepolysomes were sedimentedat40,000rpmfor2h15min and1.5C ina

Spinco SW40rotor, and thepolysomes in thepellet

weretreated with EDTA. The materialwassubjected

tovelocitysedimentation. 14C_labeledpurified Sind-bis nucleocapsids were added as a marker. Bars labeled I and II refer to certain fractions of the

gradientwhichwerepooledforanalysisofthe RNAby acrylamide gel electrophoresis (seeFig.6). Symbols:

-, [3H]uridine label,; ---, l4C_labeled Sindbis

nu-cleocapsids.

inan

acrylamide

gel using purifiedSindbis 26S

RNAasmarker

(Fig.

7).

They

consisted primar-ilyof26S RNA and of smallamountsof another

species of RNA with an apparent molecular

weightof 2.3 x 10".This secondspeciesofRNA

has been detected

previously

in cells infected with SemlikiForest virus orwithSindbis virus (6) and in mRNA preparations from these infected cells (4, 8). Although little 49S RNA

was detected in this preparationofmRNA, we

havefound49SmRNA in otherpreparationsof

polysomesfromcells infected with ts2ateither

the restrictiveorpermissive temperature. Characterization of Sindbis 33S RNA. The

speciesofRNA withamolecularweightof 2.3 x

10" (33S RNA) was isolated from polysomes of

I

L;

N~ ~ ~ FAT ON NUMBE

rih.SmosSapl (b)a n Sindbis0SHlbe

25-6~~~~~~~~~6'

x~~~~~~~l61

_EN

in)sampleII3ndinsmlI,repcily 2---,154C-labele E. coli rRNA._E

U)

-5-5

20 40 60 8

FRACTION NUMBER

FIG. 6. mRNA from cells infected with wild-type

Sindbis virus. The RNA in each ofsamplesIand II

(Fig. 5)wassubjectedtoacrylamide gel

electrophore-sis. "4C-labeled E. coli rRNA wereaddedasmarkers and the direction ofelectrophoresis wasfrom leftto right. Symbols: (a) and (b) -, 'H-labeled Sindbis mRNA in sample I and in sample II, respectively;

-,--1C-aee

EE. coli rRNA.

8-N 6-0 x

E 4- '-I

2-10 20 30 40 50 60

FRACTION NUMBER

FIG. 7. mRNA from cells infected with ts2. Chicken cellsinfected withts2 at39Cwere labeled with20,Ciof[5-3H]uridine per ml from0.5 to 3.25h postinfection. The mRNAfraction was isolated and the RNAwassubjectedtoacrylamide gel electropho-resisusing purified 32P-labeled Sindbis 26S RNA as marker. Symbols: -, 3H-labeled ts2 mRNA;

---32P-labeledpurifiedSindbis 26S RNA.

-8

-6N

x t

C III

-4 E I'I

N4 II

In~~~~~~I

-2

0E 2.3x106 E

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POLYRIBOSOMES

cellsinfected with ts2atthe restrictive

temper-ature and purified by preparative gel electro-phoresis. A hybridization-competition

experi-ment using 33S RNA was performed to

deter-mine whether or not this form of Sindbis-specific RNA was separate and distinct from Sindbis26SRNA. When 33S RNAwas hybrid-ized to Sindbis double-stranded RNA, unla-beled 26S RNAwas abletoeffectivelycompete

with it for the sites on the complementary

strand of the double-stranded RNA (Fig. 8). Only about 10% of the base sequences in 33S RNAwere not incompetitionwithexcess unla-beled 26S RNA. The curve for the control experiment where 26S RNA was hybridized to

Sindbis double-stranded RNA in the presence

ofincreasingamountsofunlabeled 26S RNA is also showninFig.8.These resultsindicate that

atleast 90% of the base sequences in33S RNA from cells infected with ts2 are alsopresent in 26S RNA. It is unclear whether the 10%

non-homologybetween26S and 33S RNArepresents a real difference between these moleculesor is

due tocontamination ofthe 33S RNA

prepara-tion with fragments derived from 49S RNA.

If, as the hybridization-competition results

indicate, 33SRNA isastructural variantof26S

RNA, denaturation of this form of RNA should

convertitto26S RNA. For thefollowing

experi-ment, 33S RNAwasisolatedfromtotalcellular RNA after infection with wild-type Sindbis virus and was purified by electrophoresis in a

preparative acrylamide gel. Thenative form of

the RNA was subjected to sucrose

gradient-velocity sedimentation with E. coli rRNA as

W

D 100

' 80

E 60

E 40

020

l00 200 300 400 500

[image:6.493.36.230.442.558.2]

COMPETING RNA ng/tube

FIG. 8. Hybridization-competition experiments with33SRNA.108 chicken embryocellswereinfected with ts2 at 39 C and labeledwith 250 MCiof H 32PO4

permlfrom0.5to4.5hpostinfection. The33S RNA

wasisolated from polysomes. The annealing mixtures

contained20ngofdouble-strandedRNA (13),2ngof

"2P-labeled26Sor33S RNA, and variousamountsof unlabeledpurified 26SRNA. TheRNA was

hybrid-ized aspreviously described (13). Symbols: 0,

Hy-bridizationof32P-labeled 33S RNA; 0,hybridization

of 32P-labeled 26S RNA.

markers (Fig. 9). In agreement with its size estimatedby gel electrophoresis, the RNA had a

sedimentation coefficient of 33.5S. However, when the purified RNA was denatured by heatinginformaldehyde, it sedimented with an

apparent molecular weight of only 1.4 x 106 (Fig. 10). After reaction with formaldehyde,

Sindbis 26S RNA sedimented with this same apparent molecular weight (13). It appears, therefore, that when 33S RNA from cells in-fected with wild-type Sindbis virus was dena-tured with formaldehyde, it was converted to

26SRNA.

DISCUSSION

Polysomes isolated from cells infected with

either wild-type Sindbis virus or ts2appear to be smaller, under the conditions of infection, than polysomes of uninfected cells. This is in contrast with theresults obtainedonpolysomes

ofcellsinfected with poliovirus (11).

Sindbis26S RNA is thepredominant

messen-ger in cells infected with eitherwild-type

Sind-bis or ts2. Kennedy (5), Rosemond and Sree-valsan (12), and Mowshowitz(8) havereported similar conclusions. Wefound that Sindbis 49S RNA serves as messenger as well, but to a

lesserextentthan26S RNA. The majorityofthe 49S RNA in our gradients containing Sindbis-specific polysomeswas foundin nucleocapsids, whereas the majority of the 26S RNA was

probably associated withribosomes.

Mowshowitz (8) also presented evidence that 49S RNA possesses messenger activity. In her experiments, treatment ofthe polysomes with

EDTA or puromycin caused a decrease in the amounts of49SRNA associated with

fast-sedi-menting structures and a corresponding in-creaseof49S RNAinstructuressedimentingat

140S withviral nucleocapsids. Although it was

assumed that the 49SRNA was released from

polysomes, it ispossible that some or all of the

newlyappearing49SRNA in the140Smaterial wasdueto analteredsedimentation behaviorof

nucleocapsids in the presence of EDTA. We

have found that fast-sedimentingstructures do contain significant amounts of nucleocapsids which can be released by EDTA treatment. In ourgradients, released49SRNAsedimentsas a

ribonucleoprotein complex at 60 to 70Sversus

140Sfornucleocapsids(or forreleased49SRNA intheexperiments of Mowshowitz [8]),and can

be distinguished from nucleocapsids. It is also noteworthy that released26SRNA sedimented as a ribonucleoprotein complex of 65S in the

experiments of Mowshowitz (8), versus 35 to

40S in our experiments. The reason for these

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x

_E

In

FRACTION NUMBER

FIG. 9. Sedimentationof33SRNA insucrose

gra-dients. Chicken cells were infected with wild-type

(HR) Sindbis virus and labeled with [5-3H]uridine from 0.5 to 4.5 h postinfection. The 33SRNA was isolated from total cellular RNA and subjected to velocitysedimentationasdescribed in the legend to Fig. 4. Symbols: -, 3H-labeled 33S RNA;

---"4C-labeled E. coli rRNA.

20 40 60 80

FRACTION NUMBER

FIG. 10. Sedimentationof33SRNAafterreaction with formaldehyde. 33S RNA was prepared, dena-turedbyheatinginthepresenceofformaldehyde (12),

and sedimented inaformaldehyde-sucrose gradient

for10.5 hat40,000rpmand 23C in theSpincoSW40 rotor.Formaldehyde-treatedE. coli rRNAwereused as markers. Symbols: -, 3H-labeled formaldehyde-treated 33S RNA; ---, "4C-labeled

formaldehyde-treated E. coli rRNA.

differences in sedimentation coefficients is not

clear. Itmaybe duetothe different

sedimenta-tion conditions or to the use of different host

cells.

In vitro and, by inference, in infected cells, 26S RNA codesfor the structuralproteinsof the

virus (D. T. Simmons and J. H. Strauss, J.

Mol. Biol., in press; R. Cancedda and M. J. Schlesinger, Proc. Nat. Acad. Sci. U.S.A., in

press). In infected cells, the remaining viral functions seem to be coded for by 49S RNA.

Theproteins specified by the 49S RNA genome

arenot known but they presumably include one

or two RNA replicases and regulatory proteins neededin small quantities. In lysates of rabbit reticulocytes, 49S RNA specifies eight or nine polypeptides ranging in molecular weight from

60,000 to 180,000 (D. T. Simmons and J. S.

Strauss, inpress). It isnotknown if these poly-peptidesaresynthesizedininfected cells.

A 33S RNA species from arbovirus-infected

cells was firstdetected by Levin and Friedman (6) and shown to have messenger activity by Kennedy (5) and by Mowshowitz (8). We have

confirmed the existence of a 33S RNA species

and its apparent messengerfunction. However,

our results indicate that 33S RNA is not a unique species ofSindbis RNA.When 33SRNA

isolated from cellsinfectedwithwild-type Sind-bis virus was denatured with formaldehyde, it

appeared to be completely converted to 26S

RNA. Hybridization-competition experiments indicatethat 33S RNA from cells infectedwith ts2shares at least90%ofits base sequences with 26S RNA.

In apreviouspublication(14), wepresented a

model for the replication of Sindbis-specific

RNA whichshowedthesynthesisof a

hypothet-ical unique species of single-stranded RNA

(molecular weight 2.8 x 106) equivalent to two-thirdsof the genome. Arigorous searchfor this species ofRNA has failed. We have aban-doned the possibility that 33S RNA is the

molecule in question. Ifthis RNA molecule is ever released from the template, it must be rapidly degraded.

ACKNOWLEDGMENTS

We wishtoacknowledgethe expert technical assistance of Edith M. Lenches and Sharman E. S.Christophinpreparing the cultures of chickenembryofibroblasts. We also wish to thank Ellen G. Strauss forhelpful suggestionsinwritingthis manuscript.

Thisinvestigationwassupportedin part bythe National Science Foundation grantno.GB 31763X andbythe Public Health Service grant no. GM 06965 from the National Institute of General Medical Sciences.D.T.S.wassupported

by Public Health Service Training grant no. 5-T1-GM-86 from theNational Institute of GeneralMedical Sciences.

LITERATURE CITED

1. Burge, B.W.,andE. R. Pfefferkorn. 1966. Isolation and characterizationof conditional-lethalmutantsof Sind-bis virus.Virology 30:204-213.

2. Burge, B.W., and E. R. Pfefferkorn. 1968. Functional defects oftemperature-sensitive mutants of Sindbis virus.J. Mol. Biol. 35:193-205.

3. Dobos, P.,and P. Faulkner. 1970. Molecularweightof Sindbis virus ribonucleic acid as measured by poly-acrylamidegelelectrophoresis. J. Virol. 6:145-147.

J.-VIROL.

on November 10, 2019 by guest

http://jvi.asm.org/

[image:7.493.67.259.54.228.2] [image:7.493.65.259.307.478.2]
(8)

4. Friedman, R. M., B. H. Levy, and W. B. Carter. 1966.

Replication of Semliki Forest virus: three forms ofviral

RNAproduced during infection. Proc.Nat.Acad. Sci. U.S.A.56:440-446.

5. Kennedy, S. I. T.1972.Isolation and characterization of thevirus-specified RNA species foundon

membrane-bound polyribosomes of chick embryo cells infected with Semliki Forest virus. Biochem. Biophys. Res. Commun.48:1254-1258.

6. Levin, J. G., and R. M. Friedman. 1971. Analysis of arbovirus ribonucleic acid forms by polyacrylamide gel electrophoresis. J. Virol. 7:504-514.

7. Lodish, H. F., D. Housman, and M. Jacobsen. 1971.

Initiationof hemoglobin synthesis. Specific inhibition by antibiotics and bacteriophage ribonucleic acid. Biochemistry 10:2348-2356.

8. Mowshowitz, D.1973.Identificationofpolysomal RNAin

BHK cells infected by Sindbis virus. J. Virol.

11:535-543.

9. Penman, S. 1966. RNA metabolism in the HeLa cell nucleus. J. Mol. Biol. 17:117-130.

10. Pierce, J. P., E. G. Strauss, and J. H. Strauss. 1974.

Effectof ionicstrengthonthebindingofSindbisvirus

tochickcells. J. Virol. 13:1030-1036.

11. Rich, A., S. Penman, Y. Becker, J. E. Darnell, and C.

Hall. 1963. Polyribosomes: size innormal and

polio-infected HeLa cells. Science 142:1658-1663. 12. Rosemond, H., and T. Sreevalsan. 1973. Viral RNA's

associated with ribosomes in Sindbis virus-infected HeLa cells. J. Virol. 11:399-415.

13. Simmons,D. T.,andJ. H. Strauss.1972.Replication of

Sindbisvirus. I. Relativesizeand genetic contentof

26S and 49S RNA. J. Mol. Biol.71:599-613. 14. Simmons, D. T., and J. H.Strauss.1972.Replication of

Sindbis virus. II. Multiple forms of double-stranded RNA isolated from infected cells. J. Mol. Biol.

71:615-631.

15. Sonnabend, J., L. Dalgarno, R. M. Friedman, and E. M. Martin. 1964. A possible replicative form of Semliki Forest virusRNA. Biochem. Biophys. Res. Commun.

17:455-460.

16. Sreevalsan, T., and R. Z. Lockart, Jr.1966.

Heterogene-ous RNA's occurringduring the replication ofwestern

equine encephalomeylitis virus. Proc. Nat. Acad. Sci. U.S.A.55:974-981.

17. Strauss, J. H., B. W. Burge, andJ. E. Darnell. 1969.

Sindbis virus infection of chick and hamster cells: synthesis of virus-specific proteins. Virology 37:367-376.

on November 10, 2019 by guest

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Figure

FIG. 2.experimentalinfectedlegendlabel; Polysomes from cells infected with ts2. The procedureswereas described in the to Fig
Fig.la, 35%of the 3Hlabel wasin viralnucleocapsids. This observation and the data
FIG. 5.gradient50averageuridinesedimented(HR)Spincominutes107wereacrylamidetolabeledcleocapsids.bis-, velocity Aglml [3H]uridine Disruption of polysomes with EDTA
FIG. 8.izedperofunlabeledwascontainedbridizationwithwith"2P-labeled 32P-labeled Hybridization-competition experiments 33S RNA
+2

References

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