JOURNALOF VIROLOGY, June 1976, p.926-932 Copyright©3 1976 AmericanSociety forMicrobiology
Vol. 18,No. 3 Printed in U.SA.
Rhinovirus Multistranded RNA: Dependence of the
the Presence of
MALCOLM R. MACNAUGHTON,'* JONATHAN A. COOPER, AND NIGELJ. DIMMOCK
Department ofBiological Sciences, University of Warwick, CoventryCV4 7AL,England Receivedfor publication 15December 1975
The multistranded and double-stranded RNAs synthesized in HeLa cells infected with rhinovirus in thepresence and in the absence ofactinomycin D have been characterized by polyacrylamide gel electrophoresis and hybridiza-tion studies. The replicative form is only foundin infected cells treated with actinomycin D, whereas the replicative intermediateis found in both the
pres-enceand the absenceof thedrug.Thesignificanceof these results isdiscussed.
of picornaviruses consists ofone
single-stranded RNA that isinfec-tious
conditions (6).How-ever, RNA
complementarytothegenome is not
infectious(4, 5, 26).
ofpicornavi-ruses is present in
has been shownto
mRNA (16,24, 29).
It has been suggested that the
replicatedin a two-step proc-ess,
namely, the formation ofa
(RI) and the
replicative form (RF),
have been obtained
from human cells infected
TheRI has been shown to be
andthe RF has
double-strandedstructure (17). In our
investigated the effect of actinomycinDon
thepresence or in
actinomycin D, RFis
The inference that RF
Jfinfec-tion is discussed.
MATERIALS AND METHODS
Propagation of cells and viruses. Monolayers of HeLa cells were grown in Eagle medium supple-mented with 10% calfserum, 0.1% sodium bicarbon-ate, and antibiotics. The cells were infected with rhinovirus type2 at 0.1PFU/cell. Unlabeled infec-tious virus waspreparedfrom these cells as previ-ouslydescribed (17).
Preparation of radioactive rhinovirus-specified
'Present address: Division of Communicable Diseases, Clinical Research Centre, Harrow, Middlesex HAl 3UJ, England.
RNA. Monolayers of cells were infected with 10 PFU/cell of virus at 33 C. After 1 h the virus was
removed andreplaced with medium containing2%
calfserumand, when appropriate,1 ittgof actinomy-cin D per ml was added. After incubation for the required time, 200
,uCiof [3H]uridine (specific activ-ity, 24 Ci/mmol) (Radiochemical Centre, Amer-sham) in2mlof mediumcontaining2%calfserum
wasadded.The RNA was extracted with phenol in the presence of sodium dodecyl sulfate (17) and then precipitated with2volumes of ethanolat -20 Cfor16
Chromatography of RNAon cellulose columns. Rhinovirus RNA specieswerefractionatedby chro-matography on Whatman CF11 cellulose, which separatessingle-stranded RNA from multistranded RNA (10). Columns (11 by 1.5 cm) were packed under gravity inTNE buffer solution (0.1M
NaCl-0.05 MTris-0.001 MEDTA [disodiumsalt] adjusted
topH7.0 at 20 C withHCl) containing 0.1% sodium dodecyl sulfate plus35% (vol/vol) ethanol. The
col-umnswereloaded with RNA at concentrations not
more than 0.5 mg/ml, and during elution at room temperature the flow rate was maintained at 1 ml/
min.RNA specieswereeluted by stepwise washings ofthecolumns with0.1%sodiumdodecyl sulfatein
TNE buffer solution containing decreasing concen-trationsof ethanol (i.e., 35%ethanol, 15% ethanol, and 0% ethanol). Fractions (1 ml) were collected,
and aliquots were assayed for radioactivity. The RNA specieswereconcentrated by precipitation at
-20Cwith100,ugof tRNA as carrier and 2 volumes
Polyacrylamide gel electrophoresis of RNA.
Polyacrylamide gels (2.2%) supported by 0.5%
aga-rose were made by the procedure of Loening (18) with certainmodifications (20). Thegelswere poly-merized in 10-cm tubeswith internal diameters of
0.7cm.Afterapre-electrophoresis of30min at 50V, up to 60,mg ofRNA wasloadedonto eachgeland
subjectedtoelectrophoresis for3 to5hat 50V.After electrophoresis the gelswereextruded andanalyzed
on a Gilfordgel scanner for materialabsorbing at 260 nm. The gels were frozen, sliced into 1-mm
disks, and solubilized, and the radioactivity was
measuredaspreviously described (17).
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Hybridization. The hybridization method used
wasessentially that of Avery (1). Radioactively
la-beled RNA preparations were dissolved in 0.02x
SSC (0.15M NaClplus0.015Msodium citrate, pH
7.0)and denaturedby heatingat98Cfor4minand then rapidly brought to 6x SSC and 65C. When appropriate,excessunlabeled virus RNAwasadded
at this stage. Samples were diluted after 4 h of
incubation inwaterat37Ctobring thesalt
concen-trationto1x SSC. Pancreatic andT, ribonucleases
(Sigma Chemical Co. Ltd., London)wereaddedto50
,ug/ml and170units/ml,respectively, and digestion
was allowed to proceed for 20 min at 37C. The reaction was stopped by the addition of250 ,mg of
yeast RNA and an equal volume of ice-cold 10%
trichloroacetic acid. Other samples were
precipi-tated without priorenzymedigestion to determine the total acid-precipitable radioactivity.
Precipi-tateswerecollectedoncelluloseacetatefilters (Mil-lipore Ltd., London) andwashed twice with cold5%
trichloroacetic acid and ether. The filtersweredried
underaninfraredlamp andassayed for
Infectivity titrations. The infectivity of virus
of HeLacellsseeded into petri dishes (5cmin
diam-eter)(31). The disheswereinoculatedwith 0.2mlof
virusdiluted in Eagle medium containing4%calf serum,which wasallowed toadsorb for 90min at
33C. The inoculum was removed, and the dishes were incubated with 5 ml ofan overlay medium
containing0.5% (wt/vol) agarose (30) at33C for4
days. The cells were fixed with formal saline, the agar wasremoved, andthe cellswerestainedwith 1%gentian violetin 20%ethanol.
Inhibition of protein synthesis in rabbit
reticulo-cytelysatesby double-strandedRNA. Protein syn-thesisin rabbitreticulocyte lysateswas measured
according to established procedures (9).
Double-stranded and multiDouble-stranded RNAs from8x 106 cells
wereseparated by cellulose CF11 chromatography. RNAfrom thepeak fractionwasseriallydiluted in
ofreticulocyte lysate containing 25 JLMhemin.
As-says were preincubated at 30 C for 30 min before adding 10 ,IA of a solution containing 2.5 ,iCi of
[3H]leucine (specific activity, 2.0 Ci/mmol) (Radi-ochemical Centre, Amersham) and other
compo-nentsneeded forprotein synthesis (9). Aftera
fur-ther 30min ofincubation,
re-moved, hydrolyzed with alkali, precipitated with
trichloroacetic acid, and assayed for radioactivity. Incubationsinthe absenceofaddedRNA incorpo-rated 39,000 counts/min for a 10-1A sample. The
dilutionof RNAinhibitingprotein synthesis by50%
The kinetics of inhibition were characteristic of
double-stranded RNA. Under thesame conditions a preparation of polyriboinosinic-polyribocytidylic
acidhada50%inhibitoryconcentration of3 x 10-10
Effect ofactinomycin Donrhinovirus
mul-tiplication.Certain strains of
poliovirushave been shown to be sensitive to
(7, 13, 28).
cells were infected with
productionof virions. Table 1 shows
production of infectious
expo-nential increase (from5 to 8 h
,ug/ml) hasonly a
inhibitory effect during this time on the
of infectious virus. However, its rate of
production or final yield after 12 h of infection
the production of infectious virus, butit
negligible effect on the total amount of
viral RNA species synthesized during infection.
However, the relative amounts of RI andRF
produced under these conditions were difficultto
calculate, as these species were obscured by
cellular RNAs, presumably heterogeneous
RNAs and rRNA precursors, when no
during infection. To
analyze RI and RF further, double-stranded
single-stranded RNAs by cellulose
Double-stranded and multistranded RNAs
the presence and in the absence
dou-ble-stranded and multistranded RNAs obtained
infected or noninfected HeLa cells
the presence or absence of actinomycinD.
labeled from6 to 8
TABLE 1. Productionof infectiousrhinovirus in HeLacells in the presence and absenceof
Treatment time(hours PFU/ml(x 102)1
No actino- 5 9.8
mycin D 6 29
Actinomy- 5 6.7
cin D (1 6 20
a Monolayers wereinfectedat amultiplicity of10
PFU/cell for 1 h at 33C. The inoculumwasremoved bywashingthe cell sheet threetimes,andthe incu-bation was continued inEaglemedium containing 2% calf serum and appropriate amountsof actino-mycin D. At the times indicatedcellswerescraped
intothe medium anddisruptedbefore thetotal
bThese results areaverages ofduplicatesamples.
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928MACNAUGHTON, COOPER, AND DIMMOCK
exponential period of increase of infectious
the same time under identical
derived from thesame
HeLa cells. RNA was
dou-ble-stranded and multistranded RNAs were
separated from single-stranded RNAs on
CF11 columns by elution with different
of ethanol (19).
eluted with 15%
RNAs were eluted with 0% ethanol.
The elution patterns
of RNAs from cells treated
without actinomycin D were
Treatment of the fractions of
FIG. 1.Polyacrylamidegelelectrophoresis ofdouble-stranded and multistranded RNAs extractedfrom rhinovirus-infected anduninfected HeLa cells. These RNAswereseparatedfrom single-strandedRNAson
cellulose CFI1 columns. (A) RNAfrom rhinovirus-infected cells labeled with[3H]uridinefrom 6 to8 h postinfection in the presence of actinomycin D; (B) RNA fromrhinovirus-infected cells labeled with 13H]-uridinefrom6 to 8h postinfection in the absence of actinomycin D; (C) RNAfromuninfected cells labeled
[3H]uridinefor2h in the presence of actinomycin D; (D) RNAfromuninfected cells labeled with
PH]-uridinefor2h in the absence ofactinomycinD. Thearrows are taken from the absorbance at 260nmtrace indicatingthe position of added"marker"'HeLa DNA.
on November 10, 2019 by guest
of DNase per ml for1
Both RIand RF
treated with actinomycinD
(Fig. 1B). We
have called the latter species putative RI (pRI).
It is interesting to note
that this pRI appears to
than the RI obtained from
infected cells treated with actinomycinD.
profiles from infected
obtained from noninfected
incu-bated with (Fig. 1C) or without (Fig. 1D)
multistranded RNAs were
ob-tained under both conditions, anda
obtained from the culture that didnot
This RNA appearedto
heterogeneous nuclear RNA, which is
double stranded (15, 27). In
that RF is
undetect-able in the
with rhinovirus-infected human
Both RI and RF species are infectious (6), and
have shown that RI+
and pRI from
infectedHeLa cells are infectious to similar
degrees when titrated by plaque assay on
cells.This result shows that
is at least partially virus specified. The
following experiments were performed to show
and pRI are essentially the samespe-cies.
Treatmentof pRI with pancreatic RNase (50
RNase (170 units/ml), which
single-stranded but not double-stranded
RNAs (1), converted pRI into a double-stranded
RNA species with a mobility similar to RF (Fig.
2). A similar result is obtained on treating RI
with RNase (17). This result is consistent with
the view that
is like RI,
which comprises a
template molecule of thesame size as
ge-nomeand is partly
hydrogenbonded to nascent
moleculesthat have single-stranded "tails."
These tails are removed by RNase treatment,
leaving a double-stranded structure of the same
consisting of one
discrete and one
pRI by hybridization.
Multistrandedand double-stranded RNAs
by CF11 cellulose chromatographyfrom
cells infectedin the presence or absence
actinomycin D were usedin
know by polyacrylamide gel electrophoresis
that the preparations from actinomycin
D-treatedcultures contain only RI +
20 40 20 40
FIG. 2. Polyacrylamidegelelectrophoresis
ofdouble-stranded and multistranded RNAs extracted
fromrhinovirus-infectedHeLa cells withoutactinomycinDtreatment. These RNAswere
separatedoncellulose CF11 columnsfromsingle-strandedRNAs. The RNAswerelabeled with[3H]uridine
postinfec-tion intheabsenceofactinomycin D.(A)UntreatedRNA;(B)RNAincubated with 50
pgofpancreaticRNase per ml and170 unitsof T,RNase per mlinI xSSCat37Cfor20min.Thearrows aretakenfromthe
absorb-ance at260 nm traceindicatingthepositionofadded marker HeLa DNA. VOL. 18, 1976
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930 MACNAUGHTON, COOPER, AND DIMMOCK
those from the untreated culturescontainonly pRI. Table 2 shows the percentages of 3H-la-beled RI + RF and pRI that self anneal or anneal withalarge (50-fold)excessofunlabeled virion RNA. The data show that about37% of RI + RF and about 21% ofpRI hydridized to excess virion RNA and hence consist ofviral complementary RNA. Thus, at least this pro-portion of the multistranded pRI consists of virus-specified RNA.
Assuming that the amountoflabeled virion-typeRNApresentin themultistranded species exceeds the complementary RNA (see above), then, bydefinition, theamount of complemen-taryRNApresentis half the value that results from self annealing (RNase resistance counts per minute). One can thus derive the ratio of self-annealed RNA to the independent meas-urement ofcomplementary RNA obtained by annealing with cold virion RNA. This value comesin allexperiments close tothe expected value of 2. This result shows that essentially all RI + RFand pRIare viral RNAspecies, since
any contamination with cellular RNAs would haveresulted in less annealing with unlabeled virion RNA and hencearatio of selfannealing toannealing withunlabeled virion RNA of less than 2.
The proportion of RNA complementary to virion RNA in RF is 50% (Macnaughton,
un-published data). Ourpreparation ofmulti-and double-stranded RNAs from actinomycin D-treatedcells containabout equalproportions of RIand RF.Byannealingthecombined RI+ RF
withvirionRNA,wefind that themixture
con-tains 37% complementary RNA. Since this fig-ureis less than50%weseethatthe majority of
RNA inthe RI synthesized inthe presence of actinomycin Dis of thesamepolarity asvirion RNA. Thisresult also argues that the propor-tion ofviral complementary RNA in pure RI
mustbe less than 37%. Henceour
viralcomplementary RNA in pRI is consistent with it
beinga structure consisting largely of RNA ofthe same
polarityas virion RNA. Our figure is closeto the value of 19% complemen-tary RNA
proteinsynthesis in reticulo-cyte lysates by double-stranded RNA. We have estimated theamountofdouble stranded-nessinrhinovirus RI and RI + RFby
measur-ingtheinhibition ofprotein synthesis in
reticu-locyte lysates (9).Table 3 shows that there is considerablymoreinhibition ofprotein synthe-sis
byRI compared to double-stranded RNA from uninfected HeLa cells, although this inhi-bition is less than for a mixture of RI + RF. Assuming that this inhibition is a measure of double strandedness (9, 14), then there is con-siderable double strandedness in RI.
During infection with picornaviruses, RI has beenshowntobe the intermediateprecursorof
progeny virus SS RNA. In vivo, it is the first
RNAspecies tobe labeled by briefexposure to
radioactive precursors (3, 17), implyingthat it is the site ofnascent RNA. Similarly, in
vitro,using virus polymerase preparations, RI has beenidentified as theprecursorofprogenySS RNA (12, 25). However, the role of RF in the synthesis of picornavirus RNA has neverbeen
readily understood, although it is now gener-ally regarded as an end product ofreplication (2, 3, 12, 17), and evidence has accumulated showing that RF isaconsequenceofreplication ratherthananintermediate thereof(3,22, 23). Nevertheless, RF has been shownto be infec-tious (6).
Our results show that in rhinovirus-infected cellsnottreated with actinomycin D, the only
TABLE 2. Annealing of 3H-labeled double-stranded and multistranded RNAs fromrhinovirus-infected HeLa cellsa
withexcess RNAcom- Self-anneal- Self-an-Total counts/ unlabeledvir- plementary ingRNasere- nealing
Treatment mininviral ion RNA: tovirion sistance RNase re- Ratio(b/c) RNA RNaseresist- RNA(%) (counts/min) sistance
(a) ance(counts/ (c/a) (b) (%)
Incubation withac- 14,621 5,760 39.4 10,311 70.5 1.8
tinomycin D (RI 34,079 11,849 34.8 22,579 66.3 1.9
+ RF) 28,089 10,725 38.2 18,960 67.5 1.8
Incubation without 17,414 4,009 23.0 7,791 44.7 1.9
actinomycin D 10,772 1,991 18.5 3,943 36.6 2.0
(pRI) 52,386 11,762 20.5 20,861 39.8 1.9
aThe RI + RF andpRIwereobtained from infected cells labeled6 to8 hpostinfection
andseparatedfrom single-stranded RNAsbychromatographyonCF11cellulose.
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TABLz 3. Inhibition ofproteinsynthesis in reticulocyte lysates by double-stranded and multistrandedRNAsfrom rhinovirus-infected
and noninfected HeLa cells
ReciprocalofRNA Total dilutionproducing counts/ 50%inhibitionof RNAspeciea *nin un- proteinsynthesis
RNAb Uncor- Normal-rected izedc RI + RF from infected
cells treated with
tinomycin D (1
Zgg1,126 720 1,280 ml)
RI from infected cells
not treated with acti- 3,916 820 420 nomycin D
fromuninfected cells 168 NDd ND
treated with actino-mycin D (1
from uninfected cells
8,414280 70 nottreatedwith
a RNA ineach case was extracted from 8 x
bAll double-stranded and multistranded RNAs wereseparated fromsingle-strandedRNAsby chro-matography on cellulose CF11 columns. Only the peak fractionfrom0% ethanol eluatewasused in theassay.
dND, Not done. Toolittle double-stranded RNA wasavailable for an accurate determination of the RNA dilution producing 50% inhibition of protein synthesis.
Al-though theactionof actinomycin D on the
of the viral RNAis not yet
rhinovirus replication RIis
and that RF
derived from RIas a
result of actinomycin Dtreatment.
large quantities of genomic
single molecule ofgenome
RI, the input virusgenome
wouldcontain an excess
ofcomple-mentary or negative RNA
replicatedto form a
positive RNAs.This process
presumablyoccurs in a
RI consists of genomic strands, and thus the greater part of this RI consists of positive RI. At present, we are trying toidentify negative RI, which we would expect to be found during ear-lier stages of rhinovirus replication.
Oninfecting HeLa cells with poliovirus type 1 (strain LSc 2ab) in the presence and absence of actinomycin D (1
Ag/ml),we have observed
that poliovirusRF exists in the
infected cellswhether or not the cells had been treated with
RFis not an
caused byactinomycin D. However,
the effectof actinomycin D on
poliovirus multiplication is knownto
the strain of poliovirus used(7, 13, 28). Thus,
althoughwe are suggesting
natu-ral infections with rhinovirustype 2 no
produced, this hypothesisappears not to
hold for allpicornaviruses.
have implications forcer-tain aspects
have specific effects,
induction of interferon(32),
cyto-pathic changes(11, 30),
and inhibition ofpro-tein
synthesis(8, 9, 14). We have shown
lysates with double-stranded RNA that thereis
considerable double strandednessin
induce the effects
considering the implications
of these results
for their existence.
Thanks are dueCheryl Penny for valuable technical assistanceduringpartof this workandtoS. I. Koliais for usefuldiscussions. ActinomycinDwas agiftfromMerck,
Sharpand Dohme ResearchLaboratories.
This workwassupported bya grantfrom the Medical ResearchCouncil, London,England.
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