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) forvariousperiods 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 times552
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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. Polysomespre-pared
from cells infected with Sindbis virusserved as the source of the mRNA in these
experiments.
Itwasthereforeimportant
toana-lyze
the size ofthese polysomes and to deter-mineifthey
wereinvolvedinproteinsynthesis.
When cells infected with wild-type (HR
strain) Sindbis virus were briefly labeled for 2
min with
"4C-labeled
aminoacids,
the sedi-mentableradioactivity
was associatedprimar-ily
with monosomes,disomes,
and trisomes(Fig.
1),
indicating
that these structures wereinvolved in
protein
synthesis. Smalleramounts of amino acid label were incorporated intolarger
polysomes.
The polysomes were also la-beled with[3HJuridine
which was added at 30 min after infection in the presence ofac-tinomycin
D(in
order to labelonly
virus-specific
RNA).
Inaddition,
the uridine labelwas
incorporated
intoviralnucleocapsidswhichsediment at 140S. In these gradients, most of
the
nucleocapsids
sedimented slightly fasterthantrisomes andattheposition shown bybar
bin
Fig.
1.Relatively
littleaminoacidlabelwasassociated with
nucleocapsids
afteronly 2 min oflabeling.
In cells infected with certain mutants of
Sindbis virus (ts2, ts5, tsl3, and tslO6), the
synthesis
ofviral nucleocapsids istemperature1974 553
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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
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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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[image:3.493.266.457.70.225.2] [image:3.493.62.255.330.531.2]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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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 theaveragesize 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 26SRNAasmarker
(Fig.
7).They
consisted primar-ilyof26S RNA and of smallamountsof anotherspecies 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 RNAwas 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._EU)
-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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[image:5.493.67.257.58.346.2] [image:5.493.267.464.62.324.2] [image:5.493.270.461.419.589.2]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.
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