Evidence that vesicular stomatitis virus produces double-stranded RNA that inhibits protein synthesis in a reticulocyte lysate.

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Copyright©1982, AmericanSocietyfor Microbiology

Evidence that Vesicular


Virus Produces

Double-Stranded RNA That Inhibits Protein


in a

Reticulocyte Lysate


DepartmentofMicrobiology, The UniversityofVirginia Schoolof Medicine, Charlottesville, Virginia 22908 Received 15 April 1982/Accepted 28 June 1982

Cell-freeprotein synthesis byreticulocyte lysates was inhibited bya polyade-nylatedRNAfraction extractedfrom HeLa cells infected with vesicular stomatitis virus (VSV) but not bypolyadenylated RNAfrommock-infected HeLacells. A

similar inhibitor of cell-free protein synthesis was found in a polyadenylated fraction ofRNAtranscribed in vitro by VSV butnotinuntranscribed nucleocap-sids. Fractionation of the VSVtranscription product showed that thetranslation inhibitor segregatedwith nucleocapsids containing newly transcribed polyadenyl-ated or non-polyadenylated RNA, as determined by

oligodeoxythymidylate-cellulosechromatography. TheinhibitorspresentinVSV-infectedHeLacells and inVSV invitrotranscripts both appearedtobe double-stranded RNA,asjudged by the characteristics for inhibition of reticulocyte cell-free protein synthesis described by Hunter et al. (J. Biol. Chem. 250:409-417, 1975). The double-strandednature of the VSVRNAinhibitorwas supported by thefindingthat the translational inhibitory effect was inactivated by melting the inhibitor in the absence of salt andby micrococcal nuclease.

Infection with wild-type vesicular stomatitis virus(VSV)results inrapidinhibition of cellular protein synthesis, a genetically determined

eventthatrequires functional viral transcription

(18).Thisinhibitory effectappears tobe directed

attheinitiation of translation (23), but the under-lying mechanism isnotunderstood. It has been

clearly shown, however, that double-stranded

RNA(dsRNA), natural or synthetic, can serve

as a potent inhibitor of protein synthesis in

various in vitro translationsystemsderivedfrom mammalian cells (10, 15, 28). Protein synthesis in reticulocyte lysates is inhibited by dsRNA, which activates a protein kinase which

phos-phorylates the small subunit ofeucaryotic initia-tion factor 2 (12). Inaddition, dsRNA has been shown to be capable ofinducing interferon (7) and activating interferon-induced enzymes

which, in turn, inactivate eucaryotic initiation factor2 and activate an endonuclease and

per-haps other enzymes which inhibit protein

syn-thesis (3, 21, 27).

Viral dsRNAmayplayaroleintheinhibition

ofprotein synthesisincells infectedwithcertain viruses. The presence ofdsRNA has been

de-tected in cross-linking studies in HeLa cells infectedwithencephalomyocarditis virusorthe

tsG114 mutant of VSV (22). mRNA prepared from reovirus transcribed invitroand polyade-nylated [poly(A)+] RNA from vaccinia virus

transcribed in vitro have been shown to contain

dsRNAwhich also inhibits protein synthesis in

cell-freesystems (1,19).

We reporthere thepresenceinVSV-infected

HeLa cells but not in uninfected cells of an

inhibitor of cell-free protein synthesis which behaves like dsRNA. We also found a similar dsRNA inhibitor of protein synthesis after in

vitro VSV transcription of poly(A)+ and

poly(A)- RNA associated with viral nucleocap-sids.


Cell cultures and viruses. The wild-type San Juan strain ofthe Indiana serotype of VSV was originally obtained from the U.S.Agriculture Research Center, Beltsville, Md. (29).

BHK-21 cells were cultivated as previously de-scribed (2). VSV was grown in BHK-21 cells as outlinedpreviously(9). HeLaS3 cellsweregrownin suspension cultures in Joklik modified minimum es-sential medium (MEM) with glutamine (2 mM) and 10% calfserum.

Chemicals and radioisotopes. Rabbitglobin mRNA waspurchased from Bethesda Research Laboratories, Rockville, Md., and polyinosinate-polycytidylate


waspurchasedfrom Miles

Biochemi-cals, Elkhart, Ind. [IS]methionine(1,174.9Ci/mmol),

[3H]uridine(38.8Ci/mmol),anduniformly labeled

3H-amino acids (>75 Ci/mmol)wereobtained from New England Nuclear Corp., Boston, Mass.

Preparation ofpoly(A)+ RNA. HeLacells in 2-liter 189

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suspension cultures were grown to adensityof4 x105

cells per ml and harvested by low-speed centrifuga-tion. Cells were then suspended in 200 ml of MEM and divided into two equal portions. One portion was mockinfected with MEM, and the otherwasinfected with VSV suspended in MEM at a multiplicity of infection of 10.After adsorption of the inoculum for 30 min at room temperature, eachportion was diluted to a totalvolume of 1 liter with MEM. At 2.5 h postinfec-tion (p.i.), [3H]uridine was added to each culture to a concentration of 5 ,uCi/ml. At 4.5 h p.i., both cultures were harvested by low-speed centrifugation, washed inphosphate-buffered saline, and subjected to deter-gent-phenol-chloroform extraction as described by Palmiter (24). After ethanol precipitation at -20°C overnight, each RNA preparation was subjected to

oligodeoxythymidylate (oligo-dT)-cellulose

chroma-tography, a second ethanolprecipitation as described above, and another pass overoligo-dT-cellulose. The poly(A)+ RNA was recovered by ethanolprecipitation

and suspendedin sterile water, andabsorption read-ings were determined at wavelengths of 260 and 280 nm.

In vitrotranslation reactions.Micrococcal nuclease-treatedrabbitreticulocyte lysatewaspurchasedfrom Amersham/Searle Corp.,Arlington Heights, Ill., and translation reactions wereperformed in reaction mix-tures essentially as described by Pelham and Jackson (25). Unless otherwise noted, translation reactions were runwith K+ concentrationsof 133 mM andMg2"

concentrations of 1.83 mM.

The in vitro translation reaction mixtures were incubated at 31°C for the times indicated in a total reaction volume of 25 pul. In addition tothe

reticulo-cytelysate,thefollowingpreparationstobe tested for

their effect on translation were prepared by being

lyophilized undernitrogen gas: mixtures of either

3H-amino acidsat0.6,uCi/,ulor[35S]methionineat1 ,uCi/ pul and poly(A)+ RNA from either mock- or VSV-infected cells or various fractions of RNA made in vitro during aVSV transcription reaction. Also pres-ent, where indicated, were lyophilized rabbit globin

mRNAandpoly(I):poly(C). Reactionswerethen

initi-atedbyadding 25 ,ul of micrococcal nuclease-treated reticulocyte lysate which had been previously com-bined with the reaction mixbythe method described by Pelham and Jackson (25). Thelysatealso contained hemin, 0.02 mM; creatinephosphate,10mM; creatine

kinase,50,ug/ml;Ca2",1mM; EGTA

[ethyleneglycol-bis(2-aminoethyl ether)-N,N'-tetraacetic acid],2mM;

Mg2+, 1.83 mM; and K+, 133 mM (except where

indicatedotherwise, when K+ wasaddedasthe ace-tate salt). Translation reactionswere terminated and assayed foracid-precipitable material bytransferring

either 2.0- or5.0-,ul portions into 100 pl1 of Laemmli buffer andthenadding1mlof10% trichloroacetic acid (TCA).SampleswerethencollectedonWhatmanGF/ Afilters, washed with 5% TCA and 95%ethanol, air dried, and counted in BeckmanReady-SolvEP scintil-lation fluid inaBeckmanLS-230 scintillationcounter. Sodium dodecyl sulfate-polyacrylamide gel electro-phoresisand autoradiography. Portions (15 ,1l) from thein vitrotranslation reactionswerediluted with50 ,ul of sample buffer containing 2% sodium dodecyl

sulfate, 25 mM Tris(pH6.8), 100 mMdithiothreitol,

and20% glycerol, with bromophenol blue. Samples

were then kept at 100°C for3 min and loadedon a

stacking gel consisting of 4% acrylamide, 0.2% bis-acrylamide, 0.1% sodium dodecyl sulfate, and 0.125 M Tris (pH 6.8) for electrophoresis by the method of Laemmli(16) asmodified by Carroll and Wagner(4). Samples were then run for 2.5 hat 200 V on agel

containing 12.5% acrylamide, 0.1% bisacrylamide,

0.1% sodium dodecyl sulfate, and 0.375 M Tris (pH 8.7). Gels were fixed, stained with Coomassie blue, destained, and driedontoWhatman 3MM filter paper. Autoradiography was done with Kodak SB-5 X-ray film.

Invitrotranscription byVSV. TheIndianaserotype

ofVSV was cultivated and purified from BHK-21cells aspreviously described (9), and the protein concentra-tion was determined by the method of Lowry et al. (17). As previously described (4), purified VSV at a protein concentration of 2.0 mg/ml in reticulocyte standardbuffer, pH 8.0, with 15%glycerol was solubi-lizedfor 3minat4°C withanequalvolume of Triton-high salt solubilizer consisting of Triton X-100, 4%; glycerol, 15%; NaCl, 1.4 M; dithiothreitol, 7.4 mM; andreticulocyte standard buffer (pH 8.0), 50%. To this mixture was then added 5 volumes of prereaction mixture consisting of 2.0 mM each of ATP, GTP, and CTP, 0.2 mM of [3H]UTP (244 mCi/mM), and 15% reticulocyte standard buffer-30% glycerol (pH 8.0). Transcription was then initiated by the addition of 3 volumes of 0.1 mM HEPES (N-2-hydroxyethylpipera-zine-N'-2-ethanesulfonic acid) buffer (pH 8.0) contain-ing 16 mMmagnesium acetate. The reaction mixtures wereincubated for 3 h at 31°C, and the RNA was then extracted with phenol-chloroform and precipitated with ethanol. Where noted, the transcription reactions werecentrifuged for 2 h at 4°C in a Beckman SW50.1 rotor at 48,000 rpm to remove the nucleocapsids beforephenol-chloroform extraction of the RNA. The nucleocapsid pellet was recovered by suspension in 0.01M Tris(pH 7.4), and RNA from both the superna-tantandthenucleocapsid was phenol-chloroform ex-tracted andethanolprecipitated.


Comparative translational inhibitory effects of

poly(A)+ RNA extracted from VSV-infected or

uninfected HeLa cells. The initial experiments

weredesigned to determine thedegreetowhich

viral RNAnewly synthesized in infected HeLa

cells would inhibitprotein synthesis in the

retic-ulocyte lysate system. To this end, poly(A)+

RNA wasisolated 4 hp.i. from mock- or

VSV-infected suspension cultures of HeLa cells by detergent-phenol-chloroform extraction

fol-lowed by oligo-dT chromatography. Various

concentrations of poly(A)+ RNA recovered

from either VSV- or mock-infected HeLa cells

were then added tomicrococcal

nuclease-treat-edrabbit reticulocyte lysates, andcell-free

pro-tein synthesis was measured by the

incorpo-ration of[35S]methionine (orof3H-amino acids).

Theaddition of0.5to 1.0p.g ofexogenousglobin

mRNA per ml to the nuclease-treated lysates

increased the synthesis ofglobin two- to

four-fold. Three types of experiments were

per-formed: (i) relatively low concentrations of


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- -G


- -M

v= "--gb

FIG. 1. Comparative electrophoretic analysis of endogenous proteins synthesized by nuclease-treated reticulocyte lysates in the presence of: (A) no added poly(A)+ RNA (control); (B) poly(A)+ RNA from mock-infected HeLa cells (0.5


(C) poly(A)+ RNA from VSV-infected HeLacells (0.5


(D) poly(A)+ RNA from mock-infected HeLa cells (1.0


and (E) poly(A)+ RNA from VSV-infected

cells (1.0p.g/ml). Lane F,Proteins extracted from VS virions to serve as molecular weight markers: G,

-69K N, -50K; NS, -45K; and M, =29K. Also shown are markers for actin (Ac) and globin (gb). Poly(A)+ RNA was extracted from HeLa cells, mock-infected or mock-infected with VSV (multiplicity of infec-tion, -10) at 4.5 h p.i.,subjected to oligo-dT-cellulose chromatography, and added to reticulocyte lysates in a complete translation reaction mixture containing


(1,jCi/,ul)for 60 min, as described in

the text. Newly synthesized proteins were extracted and electrophoresed on 12.5% polyacrylamide slab gels and visualized byautoradiography.

poly(A)+ RNA from mock- or VSV-infected

HeLa cells were compared in the reticulocyte

lysate systemfor their effectonprotein

synthe-sisasassayed bypolyacrylamide gel

electropho-resis; (ii)increasing concentrations ofpoly(A)+ RNA from mock- or VSV-infected HeLa cells wereassayed for their effecton [35S]methionine incorporation into TCA-precipitable proteins synthesized in reticulocyte lysates in the

ab-sence orpresence ofadded globin mRNA; and

(iii)thekinetics of[35S]methionine incorporation by reticulocyte lysates was compared in the

presenceof poly(A)+RNAextracted from

VSV-and mock-infected HeLacells.

Figure 1 shows electrophoretic

autoradio-graphs of



syn-thesized for60minbyendogenousmRNAfrom nuclease-treated reticulocytelysates containing

either no exogenous RNA or exogenous

poly(A)+ RNAextracted4hp.i. from mock-or

VSV-infected HeLa cells, at concentrations of 0.5 or 1.0 ,ug/ml. Exogenous poly(A)+ RNA

from VSV-infected cells substantially reduced

the synthesis of endogenous reticulocyte

pro-teinsascompared withtheeffect ofcomparable

amounts of exogenous poly(A)+ RNA from

mock-infected cells or proteins synthesized in

the absence of any exogenous RNA. Laser

densitometry scanning and integration of the

bands shown in Fig. 1revealed -50%o reduction

in the synthesis of radioactive proteins by poly(A)+RNA from VSV-infected cells (lanes C

and E) compared with that of mock-infected

cells (lanes B and D) (data not shown). These

results indicatethatin vitrotranslation is inhibit-ed by relatively small concentrations of exoge-nouspoly(A)+ RNA extracted from VSV-infect-ed butnotfrom uninfected HeLa cells.

We next compared the effects of various

amountsof poly(A)+ RNAextracted from

VSV-and mock-infected HeLa cells by determining

the amounts ofacid-precipitable protein made

after incubation of reticulocyte lysates for 60 min. Controls consisted of determining

[35S]methionine incorporation by reticulocyte lysatesto which no poly(A)+ RNA wasadded, which provided base-line data on the residual endogenous translation activity ofthe reticulo-cyte lysates. In only one experiment,

endoge-nous mRNAwas translatedbythe reticulocyte lysates, but in the other, exogenous globin

mRNAwas added to augment the endogenous mRNA. Lowlevelsof poly(A)+ RNA (0.001 to



from either mock-orVSV-infected

cells resulted in slightinhibition of

[35S]methio-nine incorporation, averaging -15% reduction inprotein synthesis (Fig.2). Whensimilarlylow levels of exogenous globin mRNA only were

added to reticulocyte lysates, the translational


not shown). Apparently, this translational sys-temis susceptibletoinhibition bylow levels of

nonspecificexogenous poly(A)+ RNA.

Amore severereduction (upto55%)occurred

in [35S]methionineincorporation by reticulocyte lysates exposedtohigherconcentrations (0.1to

1.0,ug/ml)of poly(A)+ RNA fromVSV-infected cells (Fig. 2). By comparison, equivalent

amounts of exogenous poly(A)+ RNA from

mock-infected cells caused onlyalimited reduc-tion inprotein synthesis. Apparently,exogenous

RNA in general slightly depresses the transla-tionofendogenousreticulocytemessengers,but VSV poly(A)+ RNA has a more pronounced

effect on theprotein synthesizing machinery of

reticulocyte lysates. Larger concentrations (50

,ug/ml) of poly(A)+ RNA from VSV-infected cells resulted in greater synthesis of specific VSVproteins (datanotshown) butalsoreversed theinhibitory effectonendogenousprotein

syn-thesis, as was expected fora preparation con-taining dsRNA.

Theeffectof poly(A)+ RNAfrom mock- and

VSV-infected HeLa cells on protein synthesis by reticulocyte lysates primed with 0.5 ,ug of


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0 o 1



o 1.A

co 0 1. Cu





0 .l






withou tions c


[35S]m sample

andfilt spectrc

of addc








HeLa presen infecte




conce RNA hadn

Infac fromr

did n

mRNA poly( of .0

ne in

reticu obtair [35S]rM sure F acryla nine-l.


globin and all other endogenous proteins (data

not shown).

It was also ofinterest that less inhibition of

25 proteinsynthesis was caused by1.0thanby0.1

or0.5 ,ugofpoly(A)+ RNAfrom VSV-infected

cells per mlregardlessof whether the translation

0 - reaction was primed with exogenous globin

mRNA (Fig. 2). Protein synthesisinhibition

oc-75 - curred over a narrow concentration range(0.1to

0.5 ,ug/ml) ofVSV-infected cell poly(A)+ RNA

and was partially reversed by ahigher

concen-50- tration



A similar



been reported repeatedly with dsRNA, which

inhibits cell-free protein synthesis at low or

25 -3 10-2 intermediate concentrations, but the inhibitory

10 10-2 10-1 100 effectof which is reversed athigh concentration


pg/ml (13).

Kineticsof protein synthesis inhibition by

VSV-2. Comparative effect of poly(A)+ RNA from infected cell poly(A)+ RNA and by synthetic

or mock-infected HeLa cells on [35S]methionine dsRNA. The time course of protein synthesis

oration into proteins synthesized by reticulo- inhibition was studied in an attempt to shed light

,sates treated with micrococcal nuclease with or on thenature of the reaction. These studies were

Itexogenous globin mRNA. Various concentra- doneby adding poly(A)+ RNA (0.5 ,g/ml) from

of poly(A)+ RNA were added to 25 ,ul of a either mock- or VSV-infected HeLa cells or

ethionine (1


at 31nC. Duplicatewith synthetic poly(I):poly(C) (0.5



reticulo--swerewithdrawnat60 min, TCAprecipitated, cyte lysates and then measuring [S]methionine

tered; radioactivity was counted by scintillation incorporation into acid-precipitable material at

)metry and averaged. The final concentrations intervals after starting the reaction; no

exoge-ed HeLa cell poly(A)+ RNA in each translation nous globin mRNA was added, and therefore

nare indicated on the abscissa.[35S]methionine protein synthesis represented translation

pre-oration is expressed on the ordinate as a fraction dominantly from endogenous reticulocyte

amount incorporated by reticulocyte lysates to mRNA. Essentially parallel incorporation of

noHeLa cell poly(A)+ RNA was addedwith the [35S]methionine occurred for the first 10



iousglobin mRNA. Poly(A)+ RNA was added


lysatesin which



k)mock-infected HeLacells;(A) VSV-infected






r (0.5

cells; (0) mock-infected HeLa cells in the


was present from eithermock- or

VSV-ce of globin mRNA(0.5,ug/ml); and(0) VSV- infected cells (Fig. 3). Thereafter, there was a

d HeLa cells in the presence of exogenous marked decline in, followed by cessation of,

mRNA (0.5 ,ug/ml). proteinsynthesisinreticulocyte lysates

contain-ing VSV-infected cell RNA but not in those

containing mock-infected cell RNA.

Since the kinetics of protein synthesis

inhibi-nous globin mRNA per ml was also deter- tion bypoly(A)+ RNAfromVSV-infected cells

(Fig. 2). Under these conditions, low resembled that previously reported for protein ntrations (0.001to0.05pug/ml)ofpoly(A)+ synthesis inhibition by dsRNA (13), we tested

from either mock- or VSV-infected cells theeffect ofpoly(I):poly(C) onthe reticulocyte

o effecton reticulocyte lysate translation. lysate. Poly(I):poly(C) at the same

concentra-t, higher concentrations of poly(A)+ RNA tion (0.5 ,g/ml) which had been found to be

nock-infectedHeLa cells(0.1to1.0


optimalfor inhibiting translation had an almost ot impair in vitro translation of globin identical effect on protein synthesis as did

A. In sharp contrast, concentrations of poly(A)+ RNA from VSV-infected cells.


RNAfrom VSV-infected cells at levels If inhibition of protein synthesis caused by



markedly reduced [35S]methion- VSV-infected cellRNAisdue to thepresence of

corporation in the globin mRNA-primed dsRNA, then very high concentrations of a

ilocyte lysates. Similar results were synthetic dsRNA such aspoly(I):poly(C) should

ned when 3H-amino acid instead of reverse the inhibitory effect, as wasreported by

iethionine incorporation was used to mea- Hunter et al. (13). High concentrations (50 ,ug/

)rotein synthesis (data not shown). Poly- ml) ofpoly(I):poly(C)wereeffectiveinreversing

imide gel electrophoresis of


the inhibition of protein synthesis produced by abeled proteins synthesized under these poly(A)+ RNA fromVSV-infectedcells(Fig. 3).


revealed proportionate reductions in Moreover, poly(I):poly(C) at aconcentrationof


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10 20 30


FIG. 3. Effect of poly(A)+ RNA from VSV- or mock-infected HeLa cells and/or poly(I):poly(C) on

the kinetics of protein synthesis by nuclease-treated reticulocyte lysates primedwithglobinmRNA. Com-plete reaction mixture samples of 25 pul each were

incubatedat31°Cinthepresenceof[35S]methionine. Samplesof 2.5,uleach removedatintervals thereafter were TCA precipitated, and their radioactivity was countedby scintillation spectrometry. Added to the reticulocyte-lysate translation mixture were (0) poly(A)+ RNA(0.5 ,ug/ml) frommock-infected HeLa cells; (H) poly(A)+ RNA (0.5 p.g/ml) from VSV-infected HeLacells; (0) poly(A)+ RNA(0.5 jig/ml)

frommock-infected HeLa cells andpoly(I):poly(C) (50

p.g/ml);(O) poly(A)+ RNAfrom VSV-infected HeLa

cells plus poly(I):poly(C) (50 jig/ml); (A) poly(I): poly(C) alone (0.5



containing poly(A)+ RNA from mock-infected

HeLa cells.

These studies suggestthat the protein

synthe-sis-inhibitory factor in poly(A)+ RNA from

VSV-infected cells is dsRNA or behaves like

dsRNA, unlike the poly(A)+ RNA from

unin-fected cells.

Evidence that the protein synthesis inhibitor in

VSV-infectedcellsis dsRNA. Totestthe

hypoth-esis that the protein synthesis inhibitor is

dsRNA, we tested the susceptibility of the

in-hibitory poly(A)+ RNA from VSV-infected

HeLa cells to melting, micrococcal nuclease,

and the K+ optima. Heating the RNA to100°C

and cooling it quickly in HEPES-KOH buffer

abolished the capacity of infected-cell poly(A)+

RNA to inhibit protein synthesis in a

reticulo-cytelysate,whereasheating and cooling it under

reannealing conditions in 0.3 M NaCl had no

effect on its inhibitory properties (Table 1).

Similar treatment did not affect the inactive

poly(A)+ RNA from mock-infected cells, but the

capacity of poly(I):poly(C) to inhibit protein

synthesiswassimilarly abolished by melting itat

100°C and cooling it quickly in HEPES buffer. It seems likely, therefore, that double-stranded-ness or significant secondary structure are as

essentialfor theinhibitory effect of

VSV-infect-edcellpoly(A)+ RNAoninvitro protein

synthe-sisas theyare for the effect ofpoly(I):poly(C).

Micrococcal nuclease is capable of digesting

double-stranded nucleic acids (10). Therefore,

we tested this enzyme by the procedure of

Pelham and Jackson (25) for nucleasetreatment

TABLE 1. Effect ofmelting and micrococcal nuclease onthe residual fractionalcapacityofpoly(A)+ RNA fromVSV-infected HeLa cells andpoly(I):poly(C)toinhibitproteinsynthesisbyreticulocyte lysatesa

Fractional[35SJmethionine incorporationaftertreatmentinindicated medium

RNA(source) Melting(100°Cfor 1min)b Micrococcalnuclease'

Control HEPES 0.3 MNaCI Control Treated

Poly(A)+ (mockinfected) 1.01 1.04 1.06 0.84 0.96

Poly(A)+ (VSVinfected) 0.65 1.02 0.58 0.57 1.03

Poly(L):poly(C) 0.68 1.09 0.57 0.76 1.08

a Parallel translation reactions of micrococcal nuclease-treated reticulocyte


containing exogenous

globinmRNAwerecarriedoutin the presenceorabsenceof RNA inhibitors. Reaction mixtureswereincubated

at31°Cinthe presenceof[35S]methioninefor 60 min, and thenduplicate


sampleswereTCAprecipitatedand filtered,andtheradioactivitywascounted byscintillationspectrometry.The data shownrepresent [35S]methio-nine incorporation expressed as afractionofthatincorporated in an equivalent reaction mixture containing globinmRNA (0.5,ug/ml)but no otherexogenous RNA.

bPoly(A)+RNA(0.1pug/ml)isolated fromeither mock- or VSV-infected HeLa cellsorpoly(I):poly(C)(0.5p.g/

ml)inHEPES-KOH (pH 7.2)wasincubated at 100°C for 1 min andthenimmediately quenchedonice.Duplicate poly(A)+ RNA or poly(I):poly(C) samples were similarly treated in the presence of0.3 M NaCl or carried throughthe sameprocedure without heating(controls).

cPoly(A)+RNA(0.1 pug/ml)isolated from mock- or VSV-infected HeLa cells orpoly(I):poly(C)(0.5,ug/ml)in 20 mM Tris(pH 8.2)and0.01MCaCl2weretreated with 7.5 U of micrococcalnucleasepermlbythe methodof PelhamandJackson(25). After treatment for 30 min at 20°C, the nucleasewasinactivatedbyadding EGTAto the reaction mixture toaconcentration of 0.02 M. Each control was treated identically but the micrococcal nuclease wasomitted.

VOL. 44,1982 193

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O0 -- 1 I I


E 300


_ 200

133 157 181 205 229

K+ Concentration


Fig. 4. K+ optima for in vitro trar mRNA-dependentreticulocytelysatesint of (A) poly(A)+ RNAfrom mock-infected

(0.5 ,ug/ml);(0)rabbitglobinmRNA(0.5

poly(A)+RNAfromVSV-infected HeLac

ml);(l)poly(A)+ RNA from VSV-infected

(0.5 ,ug/ml)pluspoly(I):poly(C)(50,ug/ml);

poly(C) alone (0.5 ,ug/ml). All reactions

translation mixtureswereincubatedat3104

in the presence of[35S]methionine (1 p.C

catesamples of 5 ,ul eachwerethenwithc

precipitated, filtered, and counted by liqu

tion spectrometry.

centration for

optimal [35S]methionine

incorpo-ration to about 205 mM, as compared withthe

100 to 150 mM K+ concentration which was

optimalfor translation in the presence of

equiva-lentconcentrations of exogenous globin mRNA

or poly(A)+ RNA from mock-infected HeLa

cells (Fig. 4). Moreover, whereas inhibitionby

VSV-infectedcellpoly(A)+ RNA wasabolished

by the presence of 50


ofpoly(I):poly(C) per

ml,the K+concentration for optimaltranslation

reverted to -133 mM (Fig. 4). These results are

in close agreementwith the K+ optima obtained

267 by Baglioni et al. (1) for reticulocyte lysate

4) translation under conditions of noinhibitionor

inhibition by dsRNA. Electrophoretic analysis

nslation by of the protein products synthesized in the

pres-:he presence ence ofVSV-infectedcell poly(A)+ RNA

inhibi-HeLacells tors indicated that both cell- and virus-specific


(0) proteins were inhibited at the lower (100 to 150

ells (0.5 pg/

mM) concentration of K+ (Fig. 5). By

compar-I HeLacells ing mock-infected with VSV-infected cells, it



can be seen that


RNA from VSV-C for 60min infected cells


,ug/ml) substantially


i/pA); dupli- globinsynthesis (-50% reductionasdetermined

irawn, TCA by densitometry scanning). This inhibition of

iid scintilla- globin synthesis was reversed byincreasing the

K+ concentration (compare lanes C and D

[mock] with lanes C and D[VSV]). When inhibi-tionofproteinsynthesiswasreversed byhigher

ofreticulocyte lysatestodetermine its effecton

the capacity ofpoly(A)+ RNAfrom uninfected andVSV-infected cells and ofpoly(I):poly(C)to

inhibit protein synthesis. Micrococcal nuclease

treatmentof 0.5,ugofpoly(A)+RNAfrom

VSV-infected cells per ml or of 0.5 ,ug of poly(I): poly(C) per ml abolished theirability to inhibit

protein synthesis byreticulocyte lysates

recon-stituted with exogenous globin mRNA. Micro-coccal nuclease had little or no effect on

poly(A)+ RNA controls from mock-infected HeLa cells(Table 1).

The effects ofmelting andmicrococcal

nucle-ase treatment indicate that double-strandedness

may be the critical property of infected-cell poly(A)+ RNA thatenables ittoinhibit protein synthesis. Another property of dsRNA is its

ability to shift the optimum concentrations of potassium required for translation of vaccinia virus transcripts in thereticulocyte lysate

trans-lationsystem(1).Baglionietal.(1)reportedthat the optimum concentration ofsaltforthe

trans-lation of vacciniavirus mRNA is influenced by

thepresenceofadsRNAinhibitor which

copuri-fies with vaccinia virus poly(A)+ RNA. There-fore,wechosetoexamine theK+ concentration required for optimal reticulocytelysate transla-tion under conditransla-tions ofdsRNAinhibitionor no

inhibition. Poly(I): poly(C)orVSV-infected cell poly(A)+ RNA (0.5 ,ug/ml) shiftedthe K+


AB ,C 0 E



_avb _uumom*mm_A_W d --Ac


FIG. 5. Comparative electrophoretic analysis of endogenous proteins synthesized by nuclease-treated reticulocyte lysates in the presence of various K+ concentrations and poly(A)+ RNA (0.5 ,ug/ml) from mock-infected (lanes A-E, Mock) or VSV-infected (lanes A-E, VSV) HeLa cells. In vitro translation reactions wererun in the presence of increasing K+ concentrations(mM) of (A) 133; (B) 157; (C) 205; (D) 229; (E) 267. Reactions were incubated at 31°C for 60 minin the presence of[35S]methionine and then sub-jected to electrophoresis on 12.5% polyacrylamide slab gels and visualized by autoradiography as de-scribed in the text and in the legend to Fig. 1. Migration of actin (Ac) and globin (gb) and of VS virion protein NS and Mare shown; VSV protein N wasobscured byactin, and proteins G and L were not detected.


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K+ concentrations, specific VSV I

and M became more visible (Fig. 5 tein Nwasobscured by the actin ban proteins G and L couldnot be dete data also demonstrate thepresence RNA from infected cells of intact N

notgreatly degraded in these prepa:

These data on the K+ optima of

areconsistent with thehypothesis th lation inhibitor in VSV-infected ce RNAis dsRNA.

Translation inhibitors produced

transcription of VS virions. The foi denceforadsRNAinhibitor ofprote

present in VSV-infected but not

HeLa cells implicateseitheraviralI

virus-induced cell product as the inhibitor. Direct evidence foraviralI

soughtby testingRNAsynthesized

transcription of VS virions for it!










Jl ax






10-3 10-2 10-,

[RNA], pg/ml

FIG. 6. Comparative effect of RNA tracted from in vitro transcripts of V

protein synthesis bymicrococcalnuclea

ticulocytelysatesprimedwithglobin ml ml). Transcriptionreactions of VS virio: and extraction of RNA transcripts fron reaction mixtureorfrom pelleted nucle

supernatant aftercentrifugation are des text. The conditions used for translatio:

cytelysates and60-min incorporation of nine induplicate5-,ul samples (shownon

ateach concentration ofexogenousRN

the abscissa) are similar to those desc legend to Fig. 2. The type of RNA ac translation reaction is asfollows: A, pc

isolated fromcomplete reaction mixture fuged) after in vitro VSV transcription; RNAisolated from thesupernatant ofa transcription reaction after nucleocaps movedbycentrifugation;0,RNA isolatei

ednucleocapsidafter invitro VSVtrans

0,RNAisolated from control VSVnucle

mock transcription in the absence ofni phosphates.

proteins NS inhibitprotein synthesis



). VSV pro- Standardprocedures for in vitrotranscriptionby

id, and VSV purifiedVS virionswere used to produce RNA

cted. These transcripts (4), which were then separated

inpoly(A)+ by oligo-dT-cellulose chromatography into

viral mRNA poly(A)+ and poly(A)- RNA as described

rations. above. These invitro-producedVSVtranscripts Ftranslation contained viral mRNA that could be translated

ratthetrans- in reticulocyte lysates; as expected, the major

11 poly(A)+ VSV proteins synthesizedwere N, NS, and M


by in vitro Figure 6 shows the effects ofincreasing

con-regoing evi- centrations of various RNA fractions isolated insynthesis from the VS virion transcription reaction on

uninfected proteinsynthesis by micrococcal


or a edreticulocyte lysates supplemented with

exog-translation enousglobin mRNA. Asnoted, [35S]methionine productwas incorporation was inhibited 40 to 45% by by cell-free poly(A)+ RNA (0.5 ,ug/mlorgreater concentra-s ability to tions) isolated by nhenol-chloroform extraction of complete VS virion transcription reactions from which nucleocapsids had not been

re-moved. In contrast, when transcribed nucleo-capsidswereremovedbycentrifugation, equiva-lent concentrations of supernatant poly(A)+


RNA had no effect on



The RNA pelleting with virion nucleocapsids aftera 60-min transcription, ormock transcrip-tion in the absence of nucleoside triphosphates, wasextractedwithphenol-chloroform and

test-ed for its effect on reticulocyte lysate transla-tion. Protein synthesis was inhibited by RNA extractedfromnewly transcribed nucleocapsids

at concentrations of0.05 ,ug/ml orgreater but

10° 1< 1 notby untranscribedtemplateRNA atany con-centration (Fig. 6). These data indicate that RNAnewly transcribed from virion

nucleocap-sidsinhibits protein synthesis only in association

fractions ex- with virion template RNA, which itself is not

S virions on inhibitory.

se-treated re- RNAs extracted from pelleted nucleocapsids

RNA(1.0F±g/ that had been transcribed in vitro were also

ns for 60 min fractionated by oligo-dT chromatography into

n a complete poly(A)+ and poly(A) RNA and


individ-ocapsids and ually for theircapacityto



synthe-n of










RNA extracted from


tran-Xtheordinate) scribednucleocapsid both inhibited reticulocyte

'A(shown on


whereas the RNA extracted from

cribed in the untranscribed VS virion nucleocapsids had no

dded to each effect (Fig. 7). In fact, it appears that the





nucleocap-:s (notcentri- sidswas moreinhibitoryatlower concentrations

1, poly(A)+ than was thepoly(A)+ RNA.


VSV These data support the


that the


from pellet-


RNA is of viral



scription; and like the VSV-infected cell inhibitor, is dsRNA,





by newly

transcribed virion

ucleoside tri- RNA complexed with the nucleocapsid

tem-plate. 44,


on November 10, 2019 by guest




gen beb

syn tru]

sim fou this inh bili


exp the


the the ext un Coi mR idei run per tho



m-0 c




F" coni ed I



nuc: thei sep; olig oft

fron effe


witt conr isol,


afte isol, scri

Evidence that the protein synthesis inhibitor [35S]methionine was not significantly different

erated by in vitro VS virion transcription for the first 10 min in reactions containing

iaves like dsRNA. If the inhibitor of protein poly(A)+ or poly(A)- RNA extracted from

tran-ithesismadeduring VS virion transcription is scribed nucleocapsid or controls without

inhibi-ly dsRNA, it should behave in a manner tor. Thereafter, the rates of protein synthesis

iilar to that of the poly(A)+ RNA inhibitor diminished greatly in reactions containing either indinVSV-infectedcells. Tofurther examine poly(A)+ (0.5 ,ugIml) or poly(A)- (0.05


srelationship,wetested the protein synthesis RNA extracted from transcribed nucleocapsids, iibitorin VS viriontranscripts for its suscepti- to levels of -40 to 50% inhibition of translation ity to poly(I):poly(C), melting, micrococcal by 40 min. In each case, including the control, ,lease, and shifts in K+ concentration in poly(I):poly(C) reversed the inhibitory effect of

)eriments similar to those reported above for thepoly(A)+ and poly(A)- RNA extracted from VSV-infected cellinhibitor. transcribed nucleocapsids (data not shown). The Phe kinetics of protein synthesis inhibition in kinetics of the translation-inhibitory effects of reticulocytelysate system wereexamined in poly(A)+ and poly(A) RNA from VS virion presence of poly(A)+ or poly(A) RNA nucleocapsid transcripts and the reversal by

:racted from VS virionnucleocapsidsthathad poly(I):poly(C) were similar to the data for

VSV-lergone transcription at 31°C for 60 min. infected cell RNA (Fig. 3) and provide further ntrols contained only exogenous globin evidence for the dsRNA nature of these

transla-LNA and, asdescribed inthelegend to Fig. 3, tion inhibitors present in association with

tran-ntical duplicate translation reactions were scribed nucleocapsids.

in the presence of 50 ,ug of poly(I):poly(C) In experiments similar to those for

VSV-ml. Theresults wereessentially thesame as infected cell RNA (Table 1), we found that the

)se reported in Fig. 3; incorporation of translation inhibitory effects of RNA from whole

VS virion transcripts and nucleocapsids or

poly(A)+ or poly(A)- RNA from transcribed

nucleocapsidscould beinactivatedbymelting or bymicrococcalnuclease treatment. Inhibition of

1.25- protein synthesis by poly(A)+ and

poly(A)-RNAextracted from pelleted nucleocapsids

af-1.0 - ter in vitro VS viriontranscriptionreactions was

reversed by exposing the RNA to


for 1

0.75 min andquick cooling in 20 mM HEPES-KOH

buffer (pH 8.2) before adding it to micrococcal

nuclease-treated reticulocyte lysates

supple-0.50- mented with exogenous globin mRNA. Melting

either poly(A)+ or poly(A)- RNA in the

pres-0.25 ence of 0.3 M NaCl preserved their ability to

10- 10-2 10-1 100 10 inhibit protein synthesis. Similarly, treatment


with micrococcal nuclease also reversed the

protein synthesis-inhibitoryactivity ofRNA

ex-tracted from whole VSV transcription reactions,

IG. 7. Effect on protein synthesis of increasing unfractionated transcribed nucleocapsids, or

Icentrationsofpoly(A)+ andpoly(A)- RNAisolat- poly(A)+ and poly(A)- RNA extracted from

from VSV nucleocapsids after in vitro transcrip- transcribednucleocapsids (datanotshown).

Mi-i. Asdescribed in thelegend to Fig. 5 and in the crococcal nucleasetreatmentandmelting

mark-t,after60-min transcriptionof purified VSvirions,




leocapsids were separated by centrifugation, and edly reducedal o


t nhesis-inhibitory

ir RNA wasextracted withphenol-chloroformand activity of all of the nucleocapsid-RNA

tran-arated into poly(A)+ and poly(A)- fractions by scripts,as wasthecaseforpoly(A)+ RNA from

,o-dT chromatography. Increasing concentrations VSV-infected cells(Table 1), thus strongly

sug-:hese RNA extracts and a control RNA extracted gesting that this inhibitory effect is due to

-nuntranscribednucleocapsidsweretested for their dsRNA.

:ct on[35S]methionine incorporation bymicrococ- Finally, we tested theK+ optima for cell-free

nuclease-treatedreticulocyte lysates supplemented protein synthesis under conditions of inhibition h exogenousglobin mRNA (1.0pLg/ml). The data +

npare the translation-inhibitory effects of (S) RNA by


and poly(A) RNA extractedfrom

ated from mock-transcribed VSV nucleocapsids; VSV nucleocapsids after


for 60

poly(A)+ RNAisolated fromVSVnucleocapsids


Theresults were similar to those shown in

-r a 60-min transcription; and (A) poly(A)- RNA Fig. 4 for K+ optima for translation in the

atedfrom VSVnucleocapsidsafter a60-mintran- presence ofpoly(A)+ RNA from VSV-infected

iption. cells as the inhibitor. The presence ofpoly(A)+

on November 10, 2019 by guest



(0.5 ,ig/ml) or





ex-tracted from transcribed nucleocapsids shifted the K+ optima for protein synthesis to -200

mM, as compared with 140 to 150 mM for

uninhibited translation. Moreover, whenpoly(I): poly(C)wasalsopresent atconcentrations of 50

,ug/ml with the poly(A)+ and poly(A)- RNA

inhibitors, the K+optimafor reticulocyte lysate translation reverted to 140 to 150 mM. Once again, these dataare consistent with previously reported studies (1) on K+ optima for globin mRNA translation and reinforce the postulate thattheinhibitoryfactor(s)behaves like dsRNA probably derived from VSV transcripts base-paired with nucleocapsid RNA.


In vitro translation by rabbit reticulocyte

ly-sates is sensitive to inhibition by dsRNA (10,

13). Typically, inhibition by dsRNA occurs abruptly but only afteralag period of10 to 15

min, before which time protein synthesisoccurs atcontrol rates. This inhibition of protein

syn-thesis is associated with the disappearance of detectable


ribosomal subunit complexes (8). The addition of eucaryotic initia-tion factor 2, which under control conditions

promotes the binding of met-tRNAfto the 40S

ribosomal subunit, can reverse dsRNA inhibi-tion ofprotein synthesis (6, 14, 15). An inhibitor

activated by dsRNA has been shown to be

presentinreticulocyte lysatesand tohave

pro-tein kinaseactivitythatishighly selective for the small subunit of eucaryotic initiation factor 2

(11). In additionto its biphasic kinetics, inhibi-tionofproteinsynthesis in thesesystems occurs

over alimited dsRNA concentration range and

can readily be reversedbythe addition ofhigh

concentrations (>10 ,ug/ml) of either naturally occurring orsynthetic dsRNA (13). Other cell-free protein synthesizing systems have been showntobesusceptibletoinhibitionbydsRNA,

butthese systems donotappearto beas sensi-tivetodsRNAasthe rabbitreticulocyte system

is (12, 28).

Several groups have also reported inhibition of cell-free translation by viral dsRNA. The replicative intermediate of poliovirus has been showntoinhibit translation in cell-free systems

derivedfrom HeLa cells (5) and rabbit

reticulo-cytes (10). In addition, there are reports of

dsRNA inhibitory activity associated with

mRNApreparations. dsRNAinhibitory activity hasbeen reported tobepresent inpreparations ofpoly(A)+ RNAtranscribed in vitroby vaccin-iavirus and purifiedby oligo-dT-cellulose chro-matography (1). Such activity has also been observed in reovirus mRNA preparations

tran-scribed in vitro (1, 19). It should be noted that poly(A)+ RNA isolated from the cytoplasm of

normal uninfected BSC-1 cells also induces a

dsRNA-activated protein kinase in a cell-free


interferon-treat-ed BSC-1 cells, butwith anefficiency about 1%

that ofpuredsRNA such aspoly(I):poly(C)(26).

Whether the inhibitory activitypresentin these

mRNA preparations is due to the presence of

contaminating non-mRNA dsRNA or the

pres-ence of mRNAs with sufficient dsRNA

charac-ter toactivate theproteinkinase is not clear.

Wereporthere thatVSV-infected HeLa cells,

when subjected todetergent-phenol-chloroform extraction, yieldaninhibitor of cell-freeprotein synthesis thatcopurifies withpoly(A)+ RNA. A

similar RNA inhibitor was found after in vitro

transcription of purified virions. The VSV RNA inhibitors described here display the following characteristics previously described by Hunter

et al. (13) for dsRNA inhibitors ofprotein

syn-thesis: (i) the inhibitor is effective only over a

limited concentration range; (ii) inhibition

oc-cursonlyafter alag periodof 10 to 15 min; (iii)

theinhibitioncanbe reversedbyhigh

concentra-tions of a synthetic dsRNA such as poly(I): poly(C).

In addition, we have demonstrated that the VSV inhibitors ofprotein synthesis are inacti-vatedbymeltingat100°C and by the additionof

micrococcal nuclease. Baglioni et al. (1) were

able to show that the K+ optimum for protein synthesis in reticulocyte lysates is shifted

up-ward by thepresenceofadsRNAinhibitor. We also observed similar K+optimawith the

inhibi-tors isolated from VSV-infected HeLa cells and VSVtranscription reactions.Moreover,we

not-ed that the shift to ahigher K+ optimum in the

presenceof either inhibitor could be reversedby

highconcentrations ofpoly(I):poly(C).

Moyerand Banejee (20)previouslyreported the formation of double-stranded transcriptive

intermediates after in vitro VSV transcription reactions. They estimated that41% of the

prod-ucttranscribed in vitro remained in the formof suchtranscriptive intermediates after transcrip-tion in vitrofor2h. Weestimate thatasmuchas

25 to30%of the transcribed product pellets with

thenucleocapsid template after VS virion

tran-scription; analysis of this pelleted RNA before phenol extractionby electrophoresisunder

non-denaturingconditions in 0.7% agarose indicates

that a majority of the 32P-labeled product runs

with the unlabeled template. These results are

further evidence for the presence of dsRNA transcriptive intermediates in VSVtranscription reactions. Basedontheseobservations,we

pro-pose that the inhibitors that copurify with

poly(A)+ RNA from VSV-infected HeLa cells

andVSV transcription reactions aredsRNAs.

The presenceof dsRNAinhibitorsinpoly(A)+

RNApreparations has been reported for other 197

on November 10, 2019 by guest



systems (1). Atpresent we are unable to state unequivocally that the in vivo-produced

inhibi-torreported here isaviralproductrather thana

cellular product induced by viral infection.

However, the presence of a similar inhibitor

aftertranscription in vitro of VS virions free of

cellular materialsupportsthe evidence that viral

RNA is the inhibitor of protein synthesis.


This researchwassupported byPublic Health Servicegrant

no. AI-11112from the National Institutes ofHealth,grantno. PCM77-00494 from the National Science Foundation, and

grantno.MV-9E from the American CancerSociety.J.R.T. is apostdoctoraltrainee oftraininggrantno.CA-09109 from the

National CancerInstitute.


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on November 10, 2019 by guest



FIG. 1.gelsinfectedcompletetheandchromatography,tion,RNApoly(A)+cellsvirionsPoly(A)+[35S]methioninepoly(A)+mock-infectedreticulocyteshownendogenousjig/ml);-69K Comparative electrophoretic analysis of proteins synthesized by nuclease-treated lysates in the
FIG. 1.gelsinfectedcompletetheandchromatography,tion,RNApoly(A)+cellsvirionsPoly(A)+[35S]methioninepoly(A)+mock-infectedreticulocyteshownendogenousjig/ml);-69K Comparative electrophoretic analysis of proteins synthesized by nuclease-treated lysates in the p.3
FIG.2510-3 p.4
TABLE 1. Effect of melting and micrococcal nuclease on the residual fractional capacity of poly(A)+ RNAfrom VSV-infected HeLa cells and poly(I):poly(C) to inhibit protein synthesis by reticulocyte lysatesa


Effect of melting and micrococcal nuclease on the residual fractional capacity of poly(A)+ RNAfrom VSV-infected HeLa cells and poly(I):poly(C) to inhibit protein synthesis by reticulocyte lysatesa p.5
FIG. 3.poly(C)fromreticulocyte-lysatepoly(A)+cells;cellswerecountedinfectedthereticulocytepleteincubatedmock-infectedSamplesp.g/ml); Effect of poly(A)+ RNA from VSV- or HeLa cells and/or poly(I):poly(C) on kinetics of protein synthesis by nuclease-treated
FIG. 3.poly(C)fromreticulocyte-lysatepoly(A)+cells;cellswerecountedinfectedthereticulocytepleteincubatedmock-infectedSamplesp.g/ml); Effect of poly(A)+ RNA from VSV- or HeLa cells and/or poly(I):poly(C) on kinetics of protein synthesis by nuclease-treated p.5
Fig. 4.mRNA-dependent K+ optima for in vitro reticulocyte lysates
Fig. 4.mRNA-dependent K+ optima for in vitro reticulocyte lysates p.6
FIG. 5.jected229;concentrationsreactionswasconcentrationsendogenousreticulocyteminscribedvirionMigrationslabmock-infected(lanes Comparativeelectrophoreticanalysis of proteins synthesized by nuclease-treated lysates in the presence of various K+ and poly(A)
FIG. 5.jected229;concentrationsreactionswasconcentrationsendogenousreticulocyteminscribedvirionMigrationslabmock-infected(lanes Comparativeelectrophoreticanalysis of proteins synthesized by nuclease-treated lysates in the presence of various K+ and poly(A) p.6
FIG. 6.tracted Comparative effect of RNA from in vitro transcripts of V
FIG. 6.tracted Comparative effect of RNA from in vitro transcripts of V p.7