Effect of Viral Double-Stranded RNA on Protein Synthesis in Intact Cells

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JOURNALOFVIROLOGY,Feb.1973,p.232-237

Vol.11, No.2 Printed inU.S.A.

Effect of Viral

Double-Stranded

RNA

on

Protein

Synthesis

in

Intact Cells

B. CORDELL-STEWART AND MILTON W. TAYLOR

Departmentof Microbiology, Indiana University, Bloomington, Indiana47401

Received for publication 17August 1972

The addition ofpurified, noninfectious, double-stranded RNA ofbovine en-terovirus, a picornavirus, to intact cells in culture results in a rapid cessation ofcellularpolypeptide synthesis. Thisinhibition isspecificforhostcell protein

synthesis since the translation of picomavirus-specific proteins isnot affected

bythe double-stranded viralRNA.

It has been recently reported that

heterolo-gous double-strandedRNAshave aninhibitory

effect on in vitroproteinsynthesis(4, 8, 9). We

report a similar inhibition ofcellular

polypep-tide synthesis when purified, noninfectious,

double-stranded RNA isolated from cells

in-fected with a picornavirus, bovine enterovirus

-1 (BEV), is added to intact cells in

suspen-sionculture. This inhibition is specific for

cell-directed protein synthesis asdemonstrated by

a lack of inhibition when the viral

double-stranded RNA is added to either BEV or

Mengo virus-infected cells at the time of

maxi-mum viral-specific protein synthesis.

MATERIALS AND METHODS

Cell, virus, and viral infections. Propagation of mouseL cells andEhrlich ascitestumor(EAT) cells, virus isolation, and infections have previously been described (5, 14, 15).

Isolation and purification of viral double-stranded RNA. After 7 to 7.5hr ofinfection, cells

were harvested by centrifugation, resuspended in

phosphate-buffered saline, and homogenized gently

in a glass Douncehomogenizer. Cell debris was

re-movedby centrifugation at 700 x g for20 min,and RNAwas extracted from the supernatant fluid at 37 C by the method of Penman(11). Theviral double-stranded RNAwas isolated by differential salt pre-cipitation (2), treatedwithribonuclease AandT,at 15

gg/ml

and 2

Ag/ml,

respectively, deoxyribonu-clease at 20 ug/ml, and Pronase at 1.0 mg/ml.The

enzymatic treatment procedure is as described by Stern and Friedman (13). The double-stranded RNA was purified by centrifugation through sucrose gra-dients as described by Bishop et al. (3) and then dialyzed against0.15MNaCl with0.001 M ethylene-diaminetetraacetic acid(EDTA)for12hr at 4 C. Radi-oactively labeled double-stranded RNA was obtained by adding3.0gCiof[5-_3H uridine perml tothe viral incubation medium1hrpostinfection in the presence

of5

Ag

ofactinomycin D per ml.

Infectivity assay of viral double-stranded RNA.Titers of infectious RNA were determined by

aplaque assay in which confluentL-cellmonolayers onFalcon plastic dishes (60 mm)wereused. Double-stranded RNA, appropriately diluted in 1 M NaCl containing 100 gg of diethylaminoethyl

(DEAE)-dextran per ml,wasaddedin avolumeof 0.05mlto

the monolayer which had previously been washed with saline and then0.6MNaCl. Aftera30-min incu-bationperiodat 37 C, themonolayerswereoverlayed

with a 1:1mixture of1.2%Noble agar and 2x Earle minimal essential media. The disheswereincubated

for 48hr, after which the agaroverlaywasremoved andthe cell sheetwasstained with0.8%crystal violet in20%ethanolforthe enumeration ofplaques.

Determination of amino acidincorporation into

polypeptides. Cellsinsuspensionwere incubatedin a 37 C shaker bath at various concentrations in

Hanks minimal essential medium and with various concentrations of radioactive protein precursors as describedfor eachexperimentinthelegends (Fig. 1, 2, 4, 5, 6). Samples werewithdrawn, and incorpora-tionwashaltedby freezingin adryCO,-ethanolbath (-70 C). Samples werethawed, bovine serum albu-minwasaddedascarrier, andsamplesweremadeto 10% trichloroacetic acid with 20% trichloroacetic acid. Next, the samples were boiledat 100C for 5 min and the resulting precipitate was washed three times with5% trichloroaceticacid, driedat37 C for 15 hr, and solubilized in 0.5 ml ofNuclear-Chicago

solvent(New England Nuclear, Boston, Mass.)for 2

hrat37 C.Samples werecounted in aBeckman liq-uid scintillation counter in a toluene-2,5-diphenyl-oxazole (NewEngland Nuclear, Boston, Mass.)

scin-tillation cocktail. Using the wide-window, fixed ISO-SET, 14C was counted at90% efficiency, while

3H was counted at 35% efficiency.

Radioactive materials. Radioactive materials included:

[5-'Hluridine,

20 to 30 Ci/mmole;

["4C]-leucine, 58.2 Ci/mmole;

["4C]phenylalanine,

383

Ci/mmole(all threefromNewEngland Nuclear); and

[I4C]protein

hydrolysate, 54 Ci/mA (Amersham/

Searle, Arlington Heights, Ill.).

RESULTS

Infectivity of viral double-stranded

RNA. The viral double-stranded RNA used in

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the experimentsto be describedwastested for infectivity. From the same isolation, one half

ofthe total RNA initiallyisolated was prepared

as described in Materials and Methods, the

other halfwas treated similarly excepting the

ribonuclease treatment. Both 20s sedimenting

RNAs were then tested for infectivity by the

plaque assay method outlined in Materials

and Methods. It can be seen in Table 1 that

treatment of the double-stranded viral RNA

with ribonuclease drastically reduces (5 log

units) the plaque-forming ability ofthe RNA.

Therefore, the followingresults are not a

con-sequence of the replicative capacity of this

viral RNA, since ribonuclease-treated RNA

was used in subsequent experiments.

Effect of viral double-stranded RNA

on

protein

synthesis.

The effect of viral

double-stranded RNA on cellular protein

syn-thesisin intactEATcellswasexamined. A

sus-pension ofcells in culture, actively

incorpora-ting aminoacids, wassupplemented with

puri-fied BEV double-stranded RNA. As

demon-strated inFig 1, the addition ofthe viral RNA

resulted in a cessation in the incorporation of

amino acids. This probably reflects an overall

inhibition in cellular translation rather than a

reduction in the rate ofsynthesis. The

inhibi-tion can be seen to occur within 10 min after

the addition ofthe double-stranded RNA.

By

40 min aftertreatment, therate of aminoacid

incorporation has decreased by 70% as

com-pared to the control culture. Also, the

level

of

inhibition

induced by the double-stranded

RNA is identical to that

eventually

obtained

when cells are infected with complete BEV

virus (Fig. 2). The

following

experiment was

carriedoutto eliminate the

possibility

that the

inhibition by the viral double-stranded RNA

was aconsequence of analterationincell

mem-brane permeability, which would have resulted

in suppressed incorporation of protein

precur-TABLE 1. Infectivity ofdouble-stranded RNAa

RNARN

Isoltion

(PFU/,ug)

RNA/ug

Isolation (ribonuclease (uReAte) treatment) (untreated)

<1 3.2 x 105

2 <1 5.3 x 10'

aViral double-stranded RNA was isolated and

prepared as described in Materials and Methods except, whereindicated, ribonucleasetreatment was eliminated. The 20ssedimenting RNAswere plated

onconfluent L-cellmonolayersalso describedin Ma-terials and Methods. Each value presented

repre-sentstheaverageof 10platesatthreedifferent

dilu-tions ofRNA. PFU,plaque-formingunits.

!2~0

10

z<

~~~~~~~0/

/

OO /o i O o

10 20 30 40 S0 *0

FIG.1. Effect of viraldouble-stranded RNA on cel-lularprotein synthesis. "4C-protein hydrolysate was added to a culture of EAT cells (2.5 x 0l cells per ml) to a final concentration of 2.0

;Ci/ml.

7he cul-ture wasimmediately divided. After 10 min of incu-bation at 37 C with slight agitation, BEV double-strandedRNA, in a smallvolume of2x SSC(SSC=

0.15M NaCl, 0.015 M sodium citrate, pH 7.0), was added to one culture to a concentration of 20ug/ml,

and anequal volumeof2 x SSCwasadded to the control culture. Samples were prepared for analysis ofamino acidincorporationasdescribed in Materials and Methods. Symbols: 0, untreated control cells; 0, double-stranded RNA-treated cells. Arrow indi-catestimeofRNAaddition.

sors. Viral double-stranded RNA-treated cells

and untreated cells were monitored for their

respective ability to incorporate amino acids.

The rate and extent of uptake ofradioactive

amino acids in Fig. 3 indicatesnodifference in

membrane

permeability

between cells cultured

withorwithout the viral RNA.

To test the specificityofthis

inhibition,

the

effect ofBEV double-stranded RNA on

viral-specific protein synthesis was investigated. At

145 min postinfection with BEV virus, a time of linear viral-specific protein synthesis (Fig.

4), BEV double-stranded RNA was added to

the culture. Noinhibition in the accumulation

ofviral-specificproteinsbythe viral RNA can

be detected, even 60 min aftertreatment(Fig.

5). To show that this was viral-specific protein

synthesis, the culture wasassayedat6 hr after

virus infection for virus production. A normal

burst size for BEV-infected EAT cells (25

plaque-formingunits per cell) was obtained by

plaqueassay determination.

To further test the specificity of the

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CORDELL-STEWARTAND TAYLOR

wasobtained for

virally

infected cells (datanot

D

shown).

. Nature of the cell-associated RNA. Cells

were incubated with labeled double-stranded

RNA for 30minatwhich timetheywereplaced

in RNA-free medium and incubated further for various intervals. The cell-associated RNA

D * was then analyzed as to its double- or

single-stranded nature on the basis of ribonuclease resistance. After a total of 30 min of incuba-tion, 98.8% ofthe RNA was foundto have

re-tained its double-strandedness. However, at

60 min incubation, only 65% was resistant to A

.,,

ribonuclease,

and

by

120 min

only

25.5% was

double-stranded RNA (Table 2). Although

the double-stranded RNA appears to become

QC5 i.'o

2.

. single-stranded (ribonuclease sensitive) with

t

0.5

1.0 1.S

2.0

time, it would seem from Fig. 2 that the RNA

HOURS

is

not capable of translation, precluding the

FIG. 2. Inhibition of cellular protein synthesis by viral double-stranded RNA. BEV double-stranded RNAataconcentration of25gg/mlin 2x SSC (see Fig. 1 legend) was added toasuspension culture of EATcells(107cellsperml). After the addition ofthe RNA, samples of 101 total cells were removed and pulse labeled for10minutes with5,uCiof l 4C-phenyl-alanine. The pulsewashalted byfreezing the sample at-70Cinadry ice-ethanol bath. Sampleswerelater

assayed forhot trichloroacetic acid-precipitable ma-terial. The data are expressed aspercentages of an

identically labeled control towhichanequal volume of2 x SSCwasadded.Also shown isthe inhibitory effect by BEVvirus in a culturehandled similarly. (Infection by the virus is described in reference 5.) Each point was determined from theaverage of duplicate samples. The arrow indicates the initial

time of treatment with the double-stranded RNA

and virus. Symbols: A, double-stranded RNA

treated; A, BEVinfected.

tor,the effect of BEVdouble-stranded RNAon the protein synthesis of another picornavirus, Mengo virus, wasexamined. Adding BEV RNA at a time of increasing accumulation of viral proteins in thereplication cycle of Mengo virus (15),noalterationintranslationwasnoted(Fig. 6).

Extent of incorporation of viral double-stranded RNA into whole cells. Using radi-oactively labeled BEV double-stranded RNA, the rate and amount of RNA associated with whole cells was determined. Cells were

incu-bated in the presence of the double-stranded RNAat20,ug/ml. Thoroughlywashed cell

sam-pleswere then assayed for uptake of the RNA. A linear increase in cell-associated label could be observed over a2-hr incubationperiod (Fig. 7). At this time, this represented approxi-mately 1.8 pg per cell. Similar incorporation

possibility that a newly

synthesized

viral

pro-tein is the mediatorofthe inhibition observed.

DISCUSSION

The data presented here further suggest

that the inhibition of cellular protein synthesis

\ 3

70

, 2

0

o 1. v

10 20

MINUTES

FIG. 3. Effect of viral double-stranded RNA on

cellpermeability. EATcellswereincubatedat37 C in phosphate-buffered saline at a concentration of

4 x 106cellspermlfor1hr. Thecellswere then pel-leted andresuspended toa concentration of2 x 106

cellsper mlinphosphate-buffered saline containing

2

gCi

of "4C-protein hydrolysateper ml plus25 Ag ofviraldouble-strandedRNAperml in2x SSC(see

Fig. 1 legend) oranequivalent volumeof2 x SSC.

Samples were extractedanddiluted1:100 insaline,

washedthreetimes insaline,andthefinal cell pellet

wassolubilized inNCSat37Covernight. Symbols:

A, untreated control; A, double-stranded RNA

treated. lOC

wso

c

6C

0

z 0

I

4C

20

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--1EAT -0-L-CELLS

hoursafter infection

FIG.4. BEV-induced inhibitionofhostprotein syn-thesis. Cells were infected as previously described (5).Atvarious time intervals after infection, samples of2x 101cellswerepulse labeled for 10 min with 10

ACi

oft4C-phenylalanine. Sampleswereanalyzed for hot trichloroacetic acid-precipitable material. Values are reportedaspercentages ofanuninfected control culturepulse labeled in thesamefashion.

by picornavirus double-stranded RNA seen

in vitro reflects the inhibition occurring in

in-fected cells. This is demonstrated by an

inhi-bition oftranslation in intact cellswhen

nonin-fectious viral double-stranded RNA is added

tothe culture medium. The inhibitionisrapid,

observable as early as 10 min after treatment

with the viral RNA. Allowing 5 min for the in-corporation of the double-stranded RNA (2),

this inhibition may actually be commencing

within 5 min ofaddition ofthe inhibitor. The

inhibition inthe rate of amino acid

incorpora-tion was morethan 70%, 40 min after thewhole

cellscome in contact withthedouble-stranded

RNA. Thisinhibitionintranslationby the viral

double-stranded RNA is not a consequence of

decreased membrane permeability since the cells show no alteration in theuptake of amino acids after treatment.

Reisolation ofthe viral double-stranded RNA fromtreated cells at a time of maximum inhibi-tion indicated that the RNA had retained nearly 99% of itsdouble-stranded nature deter-mined by its resistance to ribonuclease. This suggests that single-stranded viral RNA by

it-self is not the inhibitor. That viral

double-stranded RNA is responsible for inhibition of

host cell translation in virally infected cells is

also evidenced, though only indirectly, by the

necessity of a functional, single-stranded ge-nome; ultraviolet-inactivated virus (12) or hy-droxylamine-treated virus (10) has no effect on

cell-specific protein synthesis. Also, Hunt and

Ehrenfeld (9) demonstrated that

single-stranded viral RNA did not inhibit in vitro syn-thesis of hemoglobin at concentrations at which the double-stranded RNA is inhibitory. This inhibition of translationby picornavirus

double-stranded RNA appears specific. There

is nodetectable inhibition of protein synthesis inpicornavirus-infected cellsbythe viral inter-mediate. This is seen for both homologous

(BEV) and heterologous (Mengo virus)

poly-peptide synthesis employing BEV double-stranded RNA. This is consistent since BEV

and Mengovirus can replicate productively in

simultaneously infected cells. Therate of

incor-poration ofthe double-stranded RNA is

identi-cal for both picornavirus-infected and

unin-fected cells. Approximately 0.8 pg per cell can

be foundafter a 1-hrincubation period

reflect-180 190 200 210

.1.UT 5,

"I",r[ Po"t S C.6 V0",I,,, -" T

'-FIG. 5. Effect of BEV double-stranded RNA on protein synthesis in BEV-infected cells. EAT cells

wereinfected with BEVat amultiplicity of infection of50 aspreviously described (5).At 135min

postin-fection, 2.0MCiof "4C-leucineper ml was addedto the culture. The culture was then divided. After10 min of incubation at 37 C, BEV double-stranded

RNA wasaddedto oneculturetoaconcentrationof 20 ug/ml. The control was treated with an equal volumeof RNA suspension buffer. Symbols: 0, un-treated BEV-infected cells; 0, double-stranded RNA-treated BEV-infected cells. Arrow indicates timeofRNAaddition.

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CORDELL-STEWART AND TAYLOR

1S

v s

10~~~~~~

6/0

10 20 30 40 SO 60 70

'145 1SS 165t '175 185 195 20S

II-INUI1S POST St"V INICtTION

FIG. 6. Effect of BEV double-stranded RNA on protein synthesisinMengo virus-infected cells. EAT

cellswereinfected with Mengovirusatamultiplicity of infection of80aspreviouslydescribed(13).At 180 min postinfection, 1.0 pCi of "4C-protein hydrolysate permlwasaddedtotheculture and then the culture

was equally divided. The procedure follows thatas described in the legend of Fig. 4. Symbols: 0,

un-treated Mengo virus-infected cells; 0,

double-stranded RNA-treated Mengo virus-treated cells.

TimeofRNA addition markedbyarrow.

ing 20% adsorption. Weigers and Koch report

that, in the absence of polycations,

approxi-mately20% ofpoliovirus double-stranded RNA

is adsorbed to HeLa cells after a 15-min incu-bation period (16). These authors also state

that the degree of double-stranded RNA ad-sorbed is not dependent on RNA

concentra-tion and has only a slight dependence on cell concentration. In oursystem, a 15-min incuba-tion results in about 7 to 9% RNA adsorbed,

and this amount increases as the incubation

period is lengthened. It should be noted that

the poliovirus double-stranded RNA used by

Weigers and Koch was not treated with ribo-nuclease which may increase its adsorption to cells. Whether the BEV double-stranded RNA

taken up by cells is intracellular oradheres to the cell surface iscurrently being investigated.

Preliminary evidence suggests it is

intracellu-lar.

The mechanism of inhibition of host cell translation by the viral intermediate is un-known. Theinhibition observed mayrepresent

an interaction between the double-stranded RNA and ribosomes, transfer RNA (tRNA), essential factors, or messenger RNA (mRNA).

Ribosomes and tRNA canbe eliminated since

such interactions should produce nonspecific

inhibition. Thus the possible siteofspecificity

is the mRNA, the essential factor(s), or both.

From in vitro studies (1, 9), there is indirect evidence that the initiation processof thehost

is inhibited. It is also suggested by Ehrenfeld

and Hunt (9) thatan initiation factorrequired for polypeptide synthesis is depleted during

incubationwiththe viral double-stranded RNA

and that its formation or regeneration is

pre-2.0 10

0 20 40 O 100

FIG. 7. Rate and extent of incorporation ofBEV double-stranded RNA into whole cells. EATcells, at a concentration of5 x 106 cellsperml, were incu-batedin thepresenceof20pgof 3kH-double-stranded

RNAperml(510countsperminper,g).Cellsamples were removed and washed three times with saline. Samples were thenanalyzed for cold trichloroacetic acid-insoluble material by precipitation onto

mem-branefilters (Millipore Corp.).

TABLE2. Secondary structure of cell-associatedRNAa Total Ribonuclease- % Ribo-time of Untreated treated nuclease-resist-incubation (counts/min)

(min) (counts/mm) ant RNA

30 987 975 98.9

60 1026 667 65.0

120 1002 256 25.5

aEhrlichascites, at aconcentration of 4 x 106 cells per mlin Hanks minimal essential medium were treated with 3H-double-stranded viral RNA at a concentration of 20

ug/ml. The suspension culture was kept at 37 C under slight agitationfor 30min,afterwhich thecells were washed with 37 C minimal essential medium, resuspended to the samecell concentrationinRNA-free medium, and incubated further. Samples of8 x 101total cells were removed, pel-leted, resuspendedin 2 x SSC, and then phenol extracted at 37C. Theresulting aqueous phase was divided equally; one-halfwas treatedwith 10pgofpancreatic ribonuclease per ml and2 pgofribonucleaseT, perml at 37 C for 20 min, the other half was incubated similarly without ribo-nuclease. Thesampleswerethen analyzedforcold trichloro-aceticacid-insoluble material.

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ventedby this RNA. Further evidence from in vitrostudieswithahemoglobinprotein

synthe-sizing system suggest that, in the absence of

mRNA,complexes areformed between the

ini-atorformylmethionyl tRNA (met-tRNAF) and

40sribosomal subunits,areversalofthe normal

function ofcodon recognition. In the presence

of poliovirus double-stranded RNA this

com-plex formation is prevented (6). This

met-t-RNAF subunit complex seems to require an

initiation factor(s) (7). Ascione and Vande

Woude (1) report the existence of a

virus-spe-cific initiation factor isolated only from cells infected with foot and mouth disease virus,

another picornavirus. This factor stimulates

the binding of N-acetyl-aminoacyl-tRNA to

viralmRNA in a foot-and-mouth disease virus

RNA-dependent system in vitro. Host

initia-tion factors isolated from uninfected cells do

not permit translation of the viral message.

This factor may be insensitive to the

transla-tional inhibitory action of the viral

double-stranded RNA, whereas a corresponding

cellu-larfactor may not.

However, the workof

Speyer

etal. (4) would

suggest that the inhibition seen by

double-stranded RNA is specific for the message. In

an E. coliin vitroprotein synthesizing system

directed by f2 and

T4

mRNAs, the

synthetic

double-stranded RNA,

polyinosinic-polycyti-dylic acid inhibitsonly theformersystem.

Also,

it was shown that the conformation of the f2

RNA

plays

aroleinthe inhibition.

A well defined in vitro

study

employing

cellular,

viral,

and

synthetic

messages and

cell and viral initiation factors may

help

to

elucidate the mechanism ofinhibition

by

picor-navirus double-stranded RNAs.

ACKNOWLEDGMENTS

The authors wish to acknowledge the finetechnical as-sistance of Elodee A. Hopper andMengovirus donatedby

Suzanne Prather.Thiswork wassupported bygrantsfrom thePublic HealthService,no. CA10417fromthe National Cancer Institute, and from the DamonRunyon Memorial fund(DRG1018). B. C.S.is apredoctoraltraineesupported byPublic Health ServicegeneticstraininggrantGM12from theNationalInstituteforGeneral Medical Sciences.

LITERATURE CITED

1. Ascione, R., and G. F. Vande Woude. 1971. Ribosomal factors effecting the stimulation of cell-free protein synthesis in the presence of foot-and-mouth disease virus ribonucleic acid. Biochem. Biophys. Res. Com-mun.45:14-21.

2. Bishop, J.M., and G. Koch. 1967. Purification and char-acterization of poliovirus-induced infectious double-strandedRNA. J. Biol. Chem. 242:1736-1743. 3. Bishop, J. M., D. F. Summers, and L. Levintow. 1965.

Characterization of ribonuclease-resistant RNA from poliovirus-infected HeLa cells. Biochemistry 54:1273-1281.

4. Chao, J., L.Chao,and J.Speyer. 1971.The inhibitionof f2 RNA directed E. coli protein synthesis by Poly rI rC in vitro. Biochem. Biophys. Res. Commun. 45: 1096-1102.

5. Cordell-Stewart, B., and M. W. Taylor. 1971. Effect of double-stranded viral RNA on mammalian cells in cul-ture. Proc. Nat. Acad. Sci. U.S.A. 68:1326-1330. 6. Darnbrough, C., T. Hunt, and R. J. Jackson. 1972. A

complex betweenmet-t-RNAFand native 40s subunits in reticulocyte lysates and its disappearance during incubation with double-stranded RNA. Biochem. Bio-phys. Res. Commun. 48:1556-1564.

7. Editorial. 1972. Protein synthesis at Cold Spring Harbor. Nature N. Biol. 239:223-224.

8. Ehrenfeld, E., and T. Hunt. 1971. Double-stranded poli-ovirusRNA inhibits initiation of protein synthesis by reticulocyte lysates. Proc. Nat. Acad. Sci. U.S.A. 68: 1075-1078.

9. Hunt, T., and E. Ehrenfeld. 1971. Cytoplasm from polio-virus-infected HeLa cells inhibits cell-free haemoglo-binsynthesis. Nature (London) 230:91-94.

10. Koschel, K., H. Tauber, and E. Wecker. 1971. The inhi-bitionofcellular protein synthesis in HeLa cells after infection with active and inactivepoliovirus. Z. Natpr-forsch 26:798-803.

11. Penman, S. 1966. RNA metabolism in the HeLa cell nu-cleus. J. Mol. Biol. 17:117-130.

12. Penman, S., and D.Summers. 1965. Effects on host cell metabolism following synchronous infection with polio-virus.Virology 27:614-620.

13. Stern,R., and R. M. Friedman. 1971. Ribonucleic acid synthesis in animal cells in the presence of actinomycin D.Biochemistry. 10:3635-2645.

14. Taylor, M. W., B. Cordell, M. Souhrada, and S. Prather. 1971.Viruses as an aid to cancertherapy: regression of solid and ascites tumors in rodents after treatment with bovineenterovirus. Proc. Nat.Acad. Sci. U.S.A.

68:836-840.

15. Wall, R., and M.W.Taylor. 1970. Mengovirus RNA syn-thesis inproductive and restrictive cell lines. Virology 42:78-85.

16. Weigers, K. J., and G. Koch. 1972. Interaction of polio-virus-induced double-stranded RNA with HeLa cells. Arch. Biochem.Biophys. 148:89-96.