Copyright©1982, AmericanSocietyfor Microbiology
Evidence that Vesicular
Double-Stranded RNA That Inhibits Protein
Reticulocyte LysateJAMES R.THOMASANDROBERT R. WAGNER*
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).
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
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
Biochemi-cals, Elkhart, Ind. [IS]methionine(1,174.9Ci/mmol),
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
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
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
[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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A B C D E F
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
jig/ml);(C) poly(A)+ RNA from VSV-infected HeLacells (0.5
jig/ml);(D) poly(A)+ RNA from mock-infected HeLa cells (1.0
jig/ml);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
[35S]methionine(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
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
p,g/ml)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
co 0 1. Cu
withou tions c
HeLa presen infecte
conce RNA hadn
mRNA poly( of .0
reticu obtair [35S]rM sure F acryla nine-l.
globin and all other endogenous proteins (data
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
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
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
uCiturl)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
k)mock-infected HeLacells;(A) VSV-infected
cells; (0) mock-infected HeLa cells in the
icg/ml)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
jig/ml)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.
WRNAfrom VSV-infected cells at levels If inhibition of protein synthesis caused by
jig/mlmarkedly 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
[35S]methio-the inhibition of protein synthesis produced by abeled proteins synthesized under these poly(A)+ RNA fromVSV-infectedcells(Fig. 3).
:ionsrevealed proportionate reductions in Moreover, poly(I):poly(C) at aconcentrationof
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TRANSLATION INHIBITED BY VSV dsRNA
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
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
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
globinmRNAwerecarriedoutin the presenceorabsenceof RNA inhibitors. Reaction mixtureswereincubated
at31°Cinthe presenceof[35S]methioninefor 60 min, and thenduplicate
5-l.dsampleswereTCAprecipitatedand 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
133 157 181 205 229
Fig. 4. K+ optima for in vitro trar mRNA-dependentreticulocytelysatesint of (A) poly(A)+ RNAfrom mock-infected
ml);(l)poly(A)+ RNA from VSV-infected
poly(C) alone (0.5 ,ug/ml). All reactions
in the presence of[35S]methionine (1 p.C
catesamples of 5 ,ul eachwerethenwithc
precipitated, filtered, and counted by liqu
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
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
l&g/ml);(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
completecan be seen that
poly(A)+RNA from VSV-C for 60min infected cells
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
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
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
A B C D 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
transcription of VS virions for it!
10-3 10-2 10-,
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
nuclease-treat-productor 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
poly(A)-RNA extracted from
tran-Xtheordinate) scribednucleocapsid both inhibited reticulocyte
translation,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.
60-mimVSV These data support the
translation-inhibitoryRNA is of viral
scription; and like the VSV-infected cell inhibitor, is dsRNA,
by newlytranscribed virion
ucleoside tri- RNA complexed with the nucleocapsid
on November 10, 2019 by guest
sim fou this inh bili
the the ext un Coi mR idei run per tho
F" coni ed I
nuc: thei sep; olig oft
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
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
ERNA)4jg/mIwith 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
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
poly(A)eand poly(A) RNA extractedfrom
ated from mock-transcribed VSV nucleocapsids; VSV nucleocapsids after
poly(A)+ RNAisolated fromVSVnucleocapsids
mi.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
met-tRNAf-40Sribosomal 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)
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
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
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on November 10, 2019 by guest