JOURNAL OF VIROLOGY, June 1976, p. 926-932Copyright ©3 1976 American Society for Microbiology

Vol. 18, No. 3Printed in U.SA.

Rhinovirus Multistranded RNA: Dependence of theReplicative Form on the Presence of Actinomycin D

MALCOLM R. MACNAUGHTON,'* JONATHAN A. COOPER, AND NIGEL J. DIMMOCKDepartment of Biological Sciences, University of Warwick, Coventry CV4 7AL, England

Received for publication 15 December 1975

The multistranded and double-stranded RNAs synthesized in HeLa cellsinfected with rhinovirus in the presence and in the absence of actinomycin Dhave been characterized by polyacrylamide gel electrophoresis and hybridiza-tion studies. The replicative form is only found in infected cells treated withactinomycin D, whereas the replicative intermediate is found in both the pres-ence and the absence of the drug. The significance of these results is discussed.

The genome of picornaviruses consists of onemolecule of single-stranded RNA that is infec-tious under appropriate conditions (6). How-ever, RNA complementary to the genome is notinfectious (4, 5, 26). The genome of picornavi-ruses is present in polysomes of infected cellsand has been shown to function as mRNA (16,24, 29). It has been suggested that the inputgenome RNA is replicated in a two-step proc-ess, namely, the formation of a complementarytemplate that is then preferentially copied toproduce an excess of genomic RNA (SS RNA)(6).Two replicative structures, the replicative in-

termediate (RI) and the replicative form (RF),have been obtained from human cells infectedwith rhinovirus in the presence of actinomycinD and characterized on polyacrylamide gels(17). The RI has been shown to be a multi-stranded structure, and the RF has been shownto be a double-stranded structure (17). In ourstudy we investigated the effect of actinomycinD on rhinovirus multiplication. We found thatalthough the virus multiplies with very similarkinetics in the presence or in the absence ofactinomycin D, RF is only found in cells treatedwith actinomycin D. The inference that RFdoes not exist under natural conditionb Jf infec-tion is discussed.

MATERIALS AND METHODSPropagation of cells and viruses. Monolayers of

HeLa cells were grown in Eagle medium supple-mented with 10% calf serum, 0.1% sodium bicarbon-ate, and antibiotics. The cells were infected withrhinovirus type 2 at 0.1 PFU/cell. Unlabeled infec-tious virus was prepared from these cells as previ-ously described (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 10PFU/cell of virus at 33 C. After 1 h the virus wasremoved and replaced with medium containing 2%calfserum and, when appropriate, 1 ittg of actinomy-cin D per ml was added. After incubation for therequired time, 200 ,uCi of [3H]uridine (specific activ-ity, 24 Ci/mmol) (Radiochemical Centre, Amer-sham) in 2 ml of medium containing 2% calf serumwas added. The RNA was extracted with phenol inthe presence of sodium dodecyl sulfate (17) and thenprecipitated with 2 volumes of ethanol at -20 C for 16h.Chromatography of RNA on cellulose columns.

Rhinovirus RNA species were fractionated by chro-matography on Whatman CF11 cellulose, whichseparates single-stranded RNA from multistrandedRNA (10). Columns (11 by 1.5 cm) were packedunder gravity in TNE buffer solution (0.1 M NaCl-0.05 M Tris-0.001 M EDTA [disodium salt] adjustedto pH 7.0 at 20 C with HCl) containing 0.1% sodiumdodecyl sulfate plus 35% (vol/vol) ethanol. The col-umns were loaded with RNA at concentrations notmore than 0.5 mg/ml, and during elution at roomtemperature the flow rate was maintained at 1 ml/min. RNA species were eluted by stepwise washingsof the columns with 0.1% sodium dodecyl sulfate inTNE buffer solution containing decreasing concen-trations of ethanol (i.e., 35% ethanol, 15% ethanol,and 0% ethanol). Fractions (1 ml) were collected,and aliquots were assayed for radioactivity. TheRNA species were concentrated by precipitation at-20 C with 100 ,ug oftRNA as carrier and 2 volumesof ethanol.Polyacrylamide gel electrophoresis of RNA.

Polyacrylamide gels (2.2%) supported by 0.5% aga-rose were made by the procedure of Loening (18)with certain modifications (20). The gels were poly-merized in 10-cm tubes with internal diameters of0.7 cm. After a pre-electrophoresis of 30 min at 50 V,up to 60 ,mg of RNA was loaded on to each gel andsubjected to electrophoresis for 3 to 5 h at 50 V. Afterelectrophoresis the gels were extruded and analyzedon a Gilford gel scanner for material absorbing at260 nm. The gels were frozen, sliced into 1-mmdisks, and solubilized, and the radioactivity wasmeasured as previously described (17).

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RHINOVIRUS MULTISTRANDED RNA 927

Hybridization. The hybridization method usedwas essentially that of Avery (1). Radioactively la-beled RNA preparations were dissolved in 0.02xSSC (0.15 M NaCl plus 0.015 M sodium citrate, pH7.0) and denatured by heating at 98 C for 4 min andthen rapidly brought to 6x SSC and 65 C. Whenappropriate, excess unlabeled virus RNA was addedat this stage. Samples were diluted after 4 h ofincubation in water at 37 C to bring the salt concen-tration to 1 x SSC. Pancreatic and T, ribonucleases(Sigma Chemical Co. Ltd., London) were added to 50,ug/ml and 170 units/ml, respectively, and digestionwas allowed to proceed for 20 min at 37 C. Thereaction was stopped by the addition of 250 ,mg ofyeast RNA and an equal volume of ice-cold 10%trichloroacetic acid. Other samples were precipi-tated without prior enzyme digestion to determinethe total acid-precipitable radioactivity. Precipi-tates were collected on cellulose acetate filters (Mil-lipore Ltd., London) and washed twice with cold 5%trichloroacetic acid and ether. The filters were driedunder an infrared lamp and assayed for radioactiv-ity.

Infectivity titrations. The infectivity of virussamples was titrated by plaque assay on monolayersof HeLa cells seeded into petri dishes (5 cm in diam-eter) (31). The dishes were inoculated with 0.2 ml ofvirus diluted in Eagle medium containing 4% calfserum, which was allowed to adsorb for 90 min at33 C. The inoculum was removed, and the disheswere incubated with 5 ml of an overlay mediumcontaining 0.5% (wt/vol) agarose (30) at 33 C for 4days. The cells were fixed with formal saline, theagar was removed, and the cells were stained with1% gentian violet in 20% ethanol.

Inhibition of protein synthesis in rabbit reticulo-cyte lysates by double-stranded RNA. Protein syn-thesis in rabbit reticulocyte lysates was measuredaccording to established procedures (9). Double-stranded and multistranded RNAs from 8 x 106 cellswere separated by cellulose CF11 chromatography.RNA from the peak fraction was serially diluted in100 mM KCI, and 5-j.l aliquots were added to 35 ,ulof reticulocyte lysate containing 25 JLM hemin. As-says were preincubated at 30 C for 30 min beforeadding 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-nents needed for protein synthesis (9). After a fur-ther 30 min of incubation, 10-IAI samples were re-

moved, hydrolyzed with alkali, precipitated withtrichloroacetic acid, and assayed for radioactivity.Incubations in the absence of added RNA incorpo-rated 39,000 counts/min for a 10-1A sample. Thedilution ofRNA inhibiting protein synthesis by 50%was calculated by interpolation on a standard curve.

The kinetics of inhibition were characteristic ofdouble-stranded RNA. Under the same conditionsa preparation of polyriboinosinic-polyribocytidylicacid had a 50% inhibitory concentration of 3 x 10-10g/ml.

RESULTSEffect of actinomycin D on rhinovirus mul-

tiplication. Certain strains of poliovirus havebeen shown to be sensitive to actinomycin D

during their multiplication cycle in humancells (7, 13, 28). HeLa cells were infected withrhinovirus to see if actinomycin D affected theproduction of virions. Table 1 shows the effect ofactinomycin D on the production of infectiousvirus in HeLa cells during the period of expo-nential increase (from 5 to 8 h postinfection).Actinomycin D (1 ,ug/ml) has only a slightlyinhibitory effect during this time on the appear-ance of infectious virus. However, its rate ofproduction or final yield after 12 h of infectionwas not affected.Not only does actinomycin D have little effect

on the production of infectious virus, but it alsohas a negligible effect on the total amount ofviral RNA species synthesized during infection.However, the relative amounts of RI and RFproduced under these conditions were difficultto calculate, as these species were obscured bycellular RNAs, presumably heterogeneous nu-clear RNAs and rRNA precursors, when noactinomycin D was present during infection. Toanalyze RI and RF further, double-strandedand multistranded RNAs were separated fromsingle-stranded RNAs by cellulose CF1l chro-matography (17).

Double-stranded and multistranded RNAssynthesized in the presence and in the absenceof actinomycin D. Figure 1 shows polyacryl-amide gel profiles of [3H]uridine-labeled dou-ble-stranded and multistranded RNAs obtainedfrom infected or noninfected HeLa cells incu-bated in the presence or absence of actinomycinD. The cells were labeled from 6 to 8 h during

TABLE 1. Production of infectious rhinovirus inHeLa cells in the presence and absence of

actinomycin Da

IncubationTreatment time (hours PFU/ml (x 102)1

postinfection)No actino- 5 9.8mycin D 6 29

7 3408 3,100

Actinomy- 5 6.7cin D (1 6 20,Lg/ml) 7 250

8 1,400a Monolayers were infected at a multiplicity of 10

PFU/cell for 1 h at 33C. The inoculum was removedby washing the cell sheet three times, and the incu-bation was continued in Eagle medium containing2% calf serum and appropriate amounts of actino-mycin D. At the times indicated cells were scrapedinto the medium and disrupted before the total in-fectivity was assayed.

b These results are averages of duplicate samples.A similar experiment produced similar results.

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928 MACNAUGHTON, COOPER, AND DIMMOCK

the exponential period of increase of infectiousvirus (17). All the manipulations were done atthe same time under identical conditions, andeach profile is derived from the same number ofHeLa cells. RNA was extracted, and then dou-ble-stranded and multistranded RNAs wereseparated from single-stranded RNAs on cellu-

DNA

oA-*A

4- ~ RF

3

RI2

'C

c DNAo C5

lose CF11 columns by elution with differentconcentrations of ethanol (19). Single-strandedRNAs were eluted with 15% ethanol, and mul-tistranded RNAs were eluted with 0% ethanol.The elution patterns ofRNAs from cells treatedwith and without actinomycin D were thesame. Treatment of the fractions of Fig. 1 with

FractionsFIG. 1. Polyacrylamide gel electrophoresis of double-stranded and multistranded RNAs extracted from

rhinovirus-infected and uninfected HeLa cells. These RNAs were separated from single-stranded RNAs oncellulose CFI1 columns. (A) RNA from rhinovirus-infected cells labeled with [3H]uridine from 6 to 8 hpostinfection in the presence of actinomycin D; (B) RNA from rhinovirus-infected cells labeled with 13H]-uridine from 6 to 8 h postinfection in the absence of actinomycin D; (C) RNA from uninfected cells labeledwith [3H]uridine for 2 h in the presence ofactinomycin D; (D) RNA from uninfected cells labeled with PH]-uridine for 2 h in the absence of actinomycin D. The arrows are taken from the absorbance at 260 nm traceindicating the position ofadded "marker"' HeLa DNA.

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RHINOVIRUS MULTISTRANDED RNA 929

10 ,ug of DNase per ml for 1 h at room tempera-ture did not alter the profiles on polyacrylamidegels.Both RI and RF were found in infected cells

treated with actinomycin D (Fig. 1A), but noRF was found in cells infected in the absence ofactinomycin D, and the only multistranded spe-cies had a mobility similar to RI (Fig. 1B). Wehave called the latter species putative RI (pRI).It is interesting to note that this pRI appears tobe larger in size than the RI obtained frominfected cells treated with actinomycin D.These profiles from infected cells were com-

pared with parallel profiles of multistrandedRNAs obtained from noninfected cells incu-bated with (Fig. 1C) or without (Fig. 1D) actino-mycin D. Some multistranded RNAs were ob-tained under both conditions, and a greateramount was obtained from the culture that didnot receive the drug. This RNA appeared to bevery large, as it only just entered the gels, andit may be heterogeneous nuclear RNA, which isknown to be partly double stranded (15, 27). Inboth cases the profiles of the peaks were differ-ent from those from infected cells, but the back-ground radioactivity was identical in infectedand noninfected cultures.

Similar results showing that RF is undetect-able in the absence of actinomycin D (M. R.Macnaughton, unpublished data) have been ob-tained with rhinovirus-infected human embryolung cells.

Both RI and RF species are infectious (6), andwe have shown that RI + RF and pRI frominfected HeLa cells are infectious to similardegrees when titrated by plaque assay on mon-olayers of HeLa cells. This result shows thatpRI is at least partially virus specified. Thefollowing experiments were performed to showthat RI and pRI are essentially the same spe-cies.Treatment of pRI with pancreatic RNase (50

,ug/ml) and T, RNase (170 units/ml), which di-gest single-stranded but not double-strandedRNAs (1), converted pRI into a double-strandedRNA species with a mobility similar to RF (Fig.2). A similar result is obtained on treating RIwith RNase (17). This result is consistent withthe view that pRI is like RI, which comprises atemplate molecule of the same size as the ge-nome and is partly hydrogen bonded to nascentmolecules that have single-stranded "tails."These tails are removed by RNase treatment,leaving a double-stranded structure ofthe samesize as RF consisting of one discrete and onesegmented molecule (6).Characterization of pRI by hybridization.

Multistranded and double-stranded RNAs pre-pared by CF11 cellulose chromatography fromHeLa cells infected in the presence or absenceof actinomycin D were used in these studies.We know by polyacrylamide gel electrophoresisthat the preparations from actinomycin D-treated cultures contain only RI + RF' and1ERI

2

20 40 20 40Fractions

FIG. 2. Polyacrylamide gel electrophoresis of double-stranded and multistranded RNAs extracted fromrhinovirus-infected HeLa cells without actinomycin D treatment. These RNAs were separated on celluloseCF11 columns from single-stranded RNAs. The RNAs were labeled with [3H]uridine from 6 to 8 h postinfec-tion in the absence ofactinomycin D. (A) UntreatedRNA; (B) RNA incubated with 50 pg ofpancreatic RNaseper ml and 170 units ofT, RNase per ml in I x SSC at 37 C for20 min. The arrows are taken from the absorb-ance at 260 nm trace indicating the position ofadded marker HeLa DNA.

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930 MACNAUGHTON, COOPER, AND DIMMOCK

those from the untreated cultures contain onlypRI. Table 2 shows the percentages of 3H-la-beled RI + RF and pRI that self anneal oranneal with a large (50-fold) excess of unlabeledvirion RNA. The data show that about 37% ofRI + RF and about 21% of pRI hydridized toexcess virion RNA and hence consist of viralcomplementary RNA. Thus, at least this pro-portion of the multistranded pRI consists ofvirus-specified RNA.Assuming that the amount of labeled virion-

type RNA present in the multistranded speciesexceeds the complementary RNA (see above),then, by definition, the amount of complemen-tary RNA present is half the value that resultsfrom self annealing (RNase resistance countsper minute). One can thus derive the ratio ofself-annealed RNA to the independent meas-urement of complementary RNA obtained byannealing with cold virion RNA. This valuecomes in all experiments close to the expectedvalue of 2. This result shows that essentially allRI + RF and pRI are viral RNA species, sinceany contamination with cellular RNAs wouldhave resulted in less annealing with unlabeledvirion RNA and hence a ratio of self annealingto annealing with unlabeled virion RNA of lessthan 2.The proportion of RNA complementary to

virion RNA in RF is 50% (Macnaughton, un-published data). Our preparation of multi- anddouble-stranded RNAs from actinomycin D-treated cells contain about equal proportions ofRI and RF. By annealing the combined RI + RFwith virion RNA, we find that the mixture con-

tains 37% complementary RNA. Since this fig-ure is less than 50% we see that the majority ofRNA in the RI synthesized in the presence ofactinomycin D is of the same polarity as virionRNA. This result also argues that the propor-tion of viral complementary RNA in pure RI

must be less than 37%. Hence our figure of 21%viral complementary RNA in pRI is consistentwith it being a structure consisting largely ofRNA of the same polarity as virion RNA. Ourfigure is close to the value of 19% complemen-tary RNA reported for poliovirus RI (21).

Inhibition of protein synthesis in reticulo-cyte lysates by double-stranded RNA. Wehave estimated the amount of double stranded-ness in rhinovirus RI and RI + RF by measur-ing the inhibition of protein synthesis in reticu-locyte lysates (9). Table 3 shows that there isconsiderably more inhibition of protein synthe-sis by RI compared to double-stranded RNAfrom uninfected HeLa cells, although this inhi-bition is less than for a mixture of RI + RF.Assuming that this inhibition is a measure ofdouble strandedness (9, 14), then there is con-siderable double strandedness in RI.

DISCUSSIONDuring infection with picornaviruses, RI has

been shown to be the intermediate precursor ofprogeny virus SS RNA. In vivo, it is the firstRNA species to be labeled by brief exposure toradioactive precursors (3, 17), implying that itis the site of nascent RNA. Similarly, in vitro,using virus polymerase preparations, RI hasbeen identified as the precursor of progeny SSRNA (12, 25). However, the role of RF in thesynthesis of picornavirus RNA has never beenreadily understood, although it is now gener-ally regarded as an end product of replication(2, 3, 12, 17), and evidence has accumulatedshowing that RF is a consequence of replicationrather than an intermediate thereof (3, 22, 23).Nevertheless, RF has been shown to be infec-tious (6).Our results show that in rhinovirus-infected

cells not treated with actinomycin D, the only

TABLE 2. Annealing of3H-labeled double-stranded and multistranded RNAs from rhinovirus-infected HeLacellsa

Annealingwith excess RNA com- Self-anneal- Self-an-

Total counts/ unlabeled vir- plementary ing RNase re- nealingTreatment min in viral ion RNA: to virion sistance RNase re- Ratio (b/c)

RNA RNase resist- RNA (%) (counts/min) sistance(a) ance (counts/ (c/a) (b) (%)

min) (c)

Incubation with ac- 14,621 5,760 39.4 10,311 70.5 1.8tinomycin 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 1.9actinomycin D 10,772 1,991 18.5 3,943 36.6 2.0(pRI) 52,386 11,762 20.5 20,861 39.8 1.9a The RI + RF and pRI were obtained from infected cells labeled 6 to 8 h postinfection with [3H]uridine

and separated from single-stranded RNAs by chromatography on CF11 cellulose.

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RHINOVIRUS MULTISTRANDED RNA 931

TABLz 3. Inhibition ofprotein synthesis inreticulocyte lysates by double-stranded and

multistranded RNAs from rhinovirus-infectedand noninfected HeLa cells

Reciprocal of RNATotal dilution producingcounts/ 50% inhibition of

RNA speciea *n in un- protein synthesisdilutedRNAb Uncor- Normal-

rected izedcRI + RF from infected

cells treated with ac-112 70 128tinomycin D (1 Zgg 1,126 720 1,280ml)

RI from infected cellsnot treated with acti- 3,916 820 420nomycin D

Double-stranded RNAfrom uninfected cells 168 NDd NDtreated with actino-mycin D (1 jig/ml)

Double-stranded RNAfrom uninfected cells 8,414 280 70not treated with acti-nomycin Da RNA in each case was extracted from 8 x 106

cells.b All double-stranded and multistranded RNAs

were separated from single-stranded RNAs by chro-matography on cellulose CF11 columns. Only thepeak fraction from 0% ethanol eluate was used inthe assay.

c Normalization to arbitrary units assuming thesame specific activity of uridine in each sample.

d ND, Not done. Too little double-stranded RNAwas available for an accurate determination of theRNA dilution producing 50% inhibition of proteinsynthesis.

virus replication structure found is RI. Al-though the action of actinomycin D on the repli-cation of the viral RNA is not yet understood,we suggest that in rhinovirus replication RI isthe only true replication structure and that RFis an artifactual structure derived from RI as aresult of actinomycin D treatment.

It is clear that two RIs must be involved inthe production of large quantities of genomicRNA from a single molecule of genome RNA(6). In the first RI, the input virus genome RNAis transcribed to form complementary RNA,and this RI would contain an excess of comple-mentary or negative RNA strands. This speciesis called negative RI. The complementary viralRNAs must then be replicated to form a largenumber of genomic or positive RNAs. Thisprocess presumably occurs in a positive RI con-taining an excess of genomic RNA strands. Ourhybridization data suggest that under our con-ditions of infection and labeling the majority of

RI consists of genomic strands, and thus thegreater part ofthis RI consists of positive RI. Atpresent, we are trying to identify negative RI,which we would expect to be found during ear-lier stages of rhinovirus replication.On infecting HeLa cells with poliovirus type

1 (strain LSc 2ab) in the presence and absenceof actinomycin D (1 Ag/ml), we have observedthat poliovirus RF exists in the infected cellswhether or not the cells had been treated withactinomycin D. Thus, in this case RF is not anartifactual structure caused by actinomycin D.However, the effect of actinomycin D on

poliovirus multiplication is known to vary withthe strain of poliovirus used (7, 13, 28). Thus,although we are suggesting that during natu-ral infections with rhinovirus type 2 no detecta-ble RF is produced, this hypothesis appears notto hold for all picornaviruses.Our results may have implications for cer-

tain aspects concerning the multiplication ofother viruses, since double-stranded RNA mol-ecules are suggested to have specific effects,such as the induction of interferon (32), cyto-pathic changes (11, 30), and inhibition of pro-tein synthesis (8, 9, 14). We have shown byinhibition of protein synthesis in reticulocytelysates with double-stranded RNA that there isconsiderable double strandedness in RI, andthis may be enough to induce the effects cur-rently ascribed to RF.However, before considering the implications

of these results further, we need to know howgeneral are these phenomena and in particularto what extent the RFs of other picornavirusesdepend on actinomycin D for their existence.

ACKNOWLEDGMENTSThanks are due Cheryl Penny for valuable technical

assistance during part of this work and to S. I. Koliais foruseful discussions. Actinomycin D was a gift from Merck,Sharp and Dohme Research Laboratories.

This work was supported by a grant from the MedicalResearch Council, London, England.

LITERATURE CITED1. Avery, R. J. 1974. The subcellular localization of virus-

specific RNA in influenza virus-infected cells. J. Gen.Virol. 24:77-88.

2. Baltimore, D. 1968. Structure of the poliovirus replica-tive intermediate RNA. J. Mol. Biol. 10:359-368.

3. Baltimore, D., and M. Girard. 1966. An intermediate inthe synthesis of poliovirus RNA. Proc. Natl. Acad.Sci. U.S.A. 56:741-748.

4. Bechet, J. 1972. Isolation ofthe minus strand ofenceph-alomyocarditis virus RNA. Virology 48:855-857.

5. Best, M., B. Evans, and J. M. Bishop. 1972. Double-stranded replicative form of poliovirus RNA: pheno-type of heterozygous molecules. Virology 47:592-603.

6. Bishop, J. M., and L. Levintow. 1971. Replicative formsof viral RNA. Structure and function. Prog. Med.Virol. 13:1-82.

7. Cooper, P. D. 1966. The inhibition of poliovirus growthby actinomycin D and the prevention ofthe inhibition

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by pretreatment of the cells with serum or insulin.Virology 28:663-678.

8. Cordell-Stewart, P., and M. W. Taylor. 1973. Effect ofviral double-stranded RNA on protein synthesis inintact cells. J. Virol. 11:232-237.

9. Ehrenfeld, E., and T. Hunt. 1971. Double-strandedpoliovirus RNA inhibits initiation of protein synthe-sis by reticulocyte lysates. Proc. Natl. Acad. Sci.U.S.A. 68:1075-1078.

10. Franklin, R. M. 1966. Purification and properties of thereplicative intermediate of the RNA bacteriophage R17. Proc. Natl. Acad. Sci. U.S.A. 55:1504-1511.

11. Gawes, D. J., P. J. Wright, and P. D. Cooper. 1975.Poliovirus temperature-sensitive mutants defectivein cytopathic effects are also defective in synthesis ofdouble-stranded RNA. J. Gen. Virol. 27:45-59.

12. Girard, M. 1969. In vitro synthesis of poliovirus ribonu-cleic acid: role of the replicative intermediate. J. Vi-rol. 3:376-384.

13. Grado, C., S. Fisher, and G. Contreras. 1965. The inhi-bition by actinomycin D of poliovirus multiplicationin HEp 2 cells. Virology 27:623-625.

14. Hunter, T., T. Hunt, R. J. Jackson, and H. D. Robert-son. 1975. The characteristics of inhibition of proteinsynthesis by double-stranded ribonucleic acid in re-ticulocyte lysates. J. Biol. Chem. 250:409-417.

15. Jelinck, W., and J. E. Darnell. 1972. Double-strandedregions in heterogeneous nuclear RNA from HeLacells. Proc. Natl. Acad. Sci. U.S.A. 69:2537-2541.

16. Kerr, I. M., R. E. Brown, and D. R. Tovell. 1972.Characterization of the polypeptides formed in re-sponse to encephalomyocarditis virus ribonucleic acidin a cell-free system from mouse ascites tumor cells.J. Virol. 10:73-81.

17. Koliais, S. I., and N. J. Dimmock. 1973. Replication ofrhinovirus RNA. J. Gen. Virol. 20:1-15.

18. Loening, U. E. 1967. The fractionation of high-molecu-lar-weight ribonucleic acid by polyacrylamide-gelelectrophoresis. Biochem. J. 102:251-257.

19. Macnaughton, M. R., and N. J. Dimmock. 1975. Poly-adenylic acid sequences in rhinovirus RNA speciesfrom infected human diploid cells. J. Virol. 16:745-748.

20. Martin, B. A. B., and D. C. Burke. 1974. The replica-tion of semliki forest virus. J. Gen. Virol. 24:45-66.

21. Mitchell, W. R., and D. R. Tershak. 1973. The synthe-sis of complementary ribonucleic acid during infec-tion with [Sc poliovirus. Virology 54:290-293.

22. Noble, J., and L. Levintow. 1970. Dynamics of polio-virus-specific RNA synthesis and the effects of inhibi-tors of virus replication. Virology 40:634-642.

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24. Oberg, B. F., and A. J. Shatkin. 1972. Isolation ofpicornavirus protein synthesis in ascites cell extracts.Proc. Natl. Acad. Sci. U.S.A. 69:3589-3593.

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26. Roy, P., and D. H. L. Bishop. 1970. Isolation and prop-erties of poliovirus minus strand ribonucleic acid. J.Virol. 6:604-609.

27. Ryskov, A. P., V. R. Farashyan, and G. P. Georgiev.1972. Ribonuclease-stable base sequences specific ex-clusively for giant dRNA. Biochim. Biophys. Acta262:568-572.

28. Schaffer, F. L., and M. Gordon. 1966. Differential in-hibitory effects of actinomycin D among strains ofpoliovirus. J. Bacteriol. 91:2309-2316.

29. Smith, A. E. 1973. The initiation of protein synthesisdirected by the RNA from encephalomyocarditis vi-rus. Eur. J. Biochem. 33:301-313.

30. Stewart, W. E., and E. de Clercq. 1974. Relationship ofcytotoxicity and interferon-inducing activity of poly-riboinosinic-polyribocytidylic acid to the molecularweights ofthe homopolymers. J. Gen. Virol. 23:83-89.

31. Stott, E. J., and G. F. Heath. 1970. Factors affectingthe growth of rhinovirus 2 in suspension cultures of L132 cells. J. Gen. Virol. 6:15-24.

32. Tytell, A. A., G. P. Lampson, A. K. Field, and M. R.Hilleman. 1967. Inducers of interferon and host re-sistance. III. Double-stranded RNA from reovirustype 3 virions (Reo 3-RNA). Proc. Natl. Acad. Sci.U.S.A. 58:1719-1722.

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