• No results found

Mechanism of Synthesis of Vaccinia Virus Double-Stranded Ribonucleic Acid In Vivo and In Vitro

N/A
N/A
Protected

Academic year: 2019

Share "Mechanism of Synthesis of Vaccinia Virus Double-Stranded Ribonucleic Acid In Vivo and In Vitro"

Copied!
6
0
0

Loading.... (view fulltext now)

Full text

(1)

Copyright ( 1971 AmericanSociety for Microbiology Printedin U.S.A.

Mechanism of Synthesis of Vaccinia

Virus

Double-Stranded Ribonucleic

Acid

In Vivo

and In

Vitro

CLARENCE COLBY,1 CHRISTINE JURALE, AND JOSEPH R. KATES

Department of Biology, University of California, San Diego, La Jolla, California 92037, andDepartment of Chemistry, University of Colorado, Boulder,Colorado80302

Received forpublication2September 1970

The synthesis of vaccinia virus double-stranded ribonucleic acid (RNA) in

infected HeLa cellswassensitiveto actinomycin D, suggestingthat a

deoxyribonu-cleic acid dependent reaction is involved. Some double-stranded RNA was made

inthepresenceofcytosinearabinoside in infected cells. Double-stranded and

com-plementary RNAweresynthesized in vitro byusing vacciniacores.Thesetwo

obser-vations indicate thatsomeofthe double-strandedRNAis readfrom"early"genes.

Thedouble-stranded RNA synthesized in vitro had thesamepropertiesasthatmade

invivo. Atleast 70% of the double-stranded RNA madein vivowasin

ribonuclease-resistant form prior to sodium dodecyl sulfate-phenol extraction. In addition,

there was a complementary RNA in infected cells which could be converted to

double-stranded RNA by annealing.

Vaccinia is a large deoxyribonucleic acid

(DNA)-containing virus which replicates in the

cytoplasm of infected cells (8) and which

stimu-lates thosecellstosynthesize the antiviralprotein,

interferon (6). Field et al. (5) postulated that a

DNAribonucleic acid (RNA) hybrid might be the

inducer of interferon in cells infected withaDNA

virus; however, DNA-RNA-like synthetic

poly-nucleotideswerefoundnot toinduceinterferon in

chick embryo fibroblasts (2).

Recently, Colby and Duesberg (3) reported finding ribonuclease-resistant RNA in vaccinia

virus-infected chick cells. The RNA was

charac-terizedasdouble-strandedRNAand itwasshown

to be virus specific; i.e., it was hybridized with

vaccinia virusDNA(3, 4). ThisRNAisanactive

inducerof interferon (3).

Thepresenceof double-stranded RNA in cells

infected with a DNA virus is not restricted to

eucaryotic cells. B0vre and Szybalski (1) found

complementary RNA synthesized from the b2

region of coliphagex,and Juraleetal. (9) found

virus-specific double-stranded RNA in

T4-in-fected E.coli.Thus, themechanism of synthesis of

the vaccinia virus double-stranded RNA may

reflectagenerally interesting biological

phenome-non.

MATERIALS AND METHODS

HeLa cells were grown in monolayers in 32-oz

(ca. 900ml) prescription bottlesin 40 ml of Eagle's

S

Senior Dernham Postdoctoral Fellow of theAmerican Cancer Society. Present address: Division of Biological Sciences,

Uni-versityofConnecticut, MicrobiologySection, Storrs, Conn. 06268.

71

medium containing 5% calf serum treated for 1 hr

at 56 C. Vaccinia virus (WR strain) was grown in HeLa cells and purified as described by Joklik (7). Confluent monolayers were infected with 100 virus particles per cell for 1 hr in 1.0 ml of medium.

Radioactive RNA was prepared bydecanting the

medium and incubating the monolayers for 15 min with lOO,Ci of 3H-uridine (New England Nuclear

Corp.) in 5 mlofmedium. Theradioactive medium

was removed, and thecellswere washedwith 25 ml of ice-cold 0.15 M NaCI solution. The monolayers werethen treatedwith1.5mlofice-cold0.01 MNaCl, 0.01 M tris(hydroxymethyl)aminomethane

(Tris)-hy-drochloride(pH7.4), 0.001 MMgCl2andallowed to

stand onicefor 3 min. The cells were removed from

thesurface of theglassbyscraping and disruptedby

treatment with arotatingDounce homogenizer. The nuclei were removed by centrifugation, and sodium

dodecyl sulfate(SDS) and Pronasewereadded to the

cytoplasmic extract to final concentrations of 0.5%

(w/v) and 0.5 mg/ml, respectively. This extract was

incubatedfor 30 min at 37 C. The cytoplasmic nucleic

acids were purified by three extractions with phenol

followed by ethanol precipitation.

Double-stranded RNA was prepared exactly as

described previously (3). DNA and single-stranded

RNA weredigestedwith deoxyribonuclease and ribo-nuclease,repectively, and, after removing the nucleases

by three phenol extractions, the remaining nucleic acids and oligonucleotides were applied to a 6% agarosecolumn (3).

TheRNAwascharacterizedas anRNAduplex by

the following criteria: (i) nuclease sensitivity, (ii)

thermaldenaturation, (iii)Cs2SO4equilibrium density gradient centrifugation, (iv) induction of interferon in chick embryo cells, (v) self-annealing after removal

ofpossible contaminating DNA strands (3, 4). It is

important to note that only the material which is

on November 11, 2019 by guest

http://jvi.asm.org/

(2)

phenol-SDS-purified, resistant to digestion with deoxyribonuclease and ribonuclease, and excluded froma6%agarosecolumn will bereferredtobelowas vaccinia virus double-stranded RNA.

RNAwas synthesized in vitro from vaccinia virus

cores asdescribed previously (10, 11). The RNAwas synthesized in reaction mixtures which routinely contained the followingcomponents, in0.4 ml: 2 X 101" cores,0.01M2-mercaptoethanol, 0.005M

MgC92,

0.05 M Tris-hydrochloride (pH 8.4), 1 ,umole of

adenosine triphosphate, 0.5 ,umole each of cytosine triphosphate (CTP) and guanosine triphosphate (GTP), 0.02 ,umole of uridine triphoshate (UTP),

and2uCi of3H-UTP. When 3H-CTPor3H-GTP was used asthelabeled substrate, UTPwas at0.5 jAmole per 0.4 ml and the labeled nucleotide was at 0.02 ,umoleper0.4 ml and 21ACiper0.4ml.Incubationwas for 25 min at 37 C. Purification of the RNA after synthesis was by phenol extraction, and ethanol

precipitationwas aspreviously described (2).

RESULTS

Effect of inhibition of RNA synthesisonthe

syn-thesis of double-stranded RNA. Since vaccinia

virusreplicatesin thecytoplasm of infected cells,

onecanfollowviral RNAsynthesis with 15-min

pulses of 3H-uridine. Vaccinia messenger RNA

(mRNA) and a small amount of transfer RNA

arethe only RNA species in thecytoplasm that

arelabeled undertheseconditions (8).

Thedouble-stranded vaccinia virus RNA could

arise by two different mechanisms. One strand

could be transcribed from vacciniaDNA, andan

RNA-dependent RNA polymerase could catalyze

the synthesis of the complementary strand of

RNA. Actinomycin D wouldnot beexpected to

inhibitthesynthesis ofthedouble-strandedRNA

for this mechanism. Alternatively, both strands

might be copied from complementary regions of

thevaccinia DNA, in which caseactinomycin D

should inhibit thesynthesis of the double-stranded

RNA.

Actinomycin D (10 ,g/ml) was added to the

culture medium ofvaccinia virus-infected HeLa

cells 4 hr after infection. At 5 hrafterinfection,

3H-uridine-labeled cytoplasmic RNA (15-min

pulse)waspreparedasdescribed above. Controls

included 3H-RNA prepared from infected and

uninfected HeLa cells not treated with

actino-mycin D.

The results presented in Table 1 indicate that

therewas avery smallamountof 3H-ribonuclease

resistantRNAin60-minpulse-labeled uninfected

HeLa cells, confirming the results previously

re-ported by Montagnier (12). Infection with

vac-cinia causeda35-fold increasein

3H-ribonuclease-resistant RNA. Treatment of vaccinia

virus-infectedHeLa cells withactinomycin D resulted

in a 95% inhibition of RNA synthesis as

meas-ured by the incorporation of 3H-uridine. The

synthesis of vaccinia virus-directed

double-strandedRNAwas also inhibited more than 90%.

Inother experiments when vaccinia mRNA

syn-thesis was inhibited 98% by actinomycin D, the

synthesis ofdouble-stranded RNA was inhibited

95 %. These results aresimilar to those previously

obtained with vaccinia-infected chick embryo

cells (4) and suggest that the DNA-dependent

mechanism is the one of choice.

Effect ofinhibitionof DNAsynthesis on the

syn-thesis ofdouble-stranded RNA. At aconcentration

of 10 ,ug/ml, cytosine arabinoside inhibits host

cell and vaccinia virus DNA synthesis by 99%

(13). Only early vaccinia virus mRNA is

tran-scribed in the presence of cytosine arabinoside

(13). It was previously suggested (4) that the

double-strandedRNAis a"late" productofviral intracellular biosynthesis. Ifthis is true, onewould

expectcytosinearabinosidetoabolishthe

synthe-sis of vacciniadouble-strandedRNA.

Cytosine arabinoside (10,ug/ml) wasaddedto

the culture medium of HeLa cells immediately

after infection with vaccinia virus. At 5 hrafter

infection, the cultures were pulse-labeled with

3H-uridine, andthe total cytoplasmic RNA and

double-stranded RNA were prepared. The

amount of 3H-labeled vaccinia mRNA in the

cytosine arabinoside-treatedcells was reduced to

one-third of that in the untreated infected cells

(Table 1). However, of the RNA which was

labeled, a significant proportion was

double-stranded. Thus, it appears that some of the

TABLE 1. Effects ofinhibitors of niucleic acid

synz-thesis on double-stranded RNA

3H counts/min3H counts/min Treatmenta inphenol after nucleases extract andagarose

HeLa, 60min, labeling 4.6 X 105 195 total nucleic acids

HeLa, 15 min, labeling 2.8 X 104 35

cytoplasmic extract

HeLa + vaccinia 4.5 X 105 5,300

HeLa+ vaccinia + acti- 2 X 104 520 nomycin

(10,ug/ml),

4

to 5 hrpostinfection

HeLa+vaccinia + cyto- 1.5 X 105 820 sine arabinoside(lO,:g/

ml),0 to5postinfection

a Forthefirst treatment, uninfected HeLacells

werelabeled for 1 hrwith25

tsCi

of3H-uridine.For

the remaining treatments, cultures were labeled

for 15 min with

lOO1

uCi of 3H-uridine and cyto-plasmic RNA was prepared. All RNA prepara-tions were purified, subjected to nuclease

diges-tion,andeluted froma6%agarosecolumnas

pre-viouslv described (3).

on November 11, 2019 by guest

http://jvi.asm.org/

[image:2.491.261.455.427.586.2]
(3)

vaccinia double-stranded RNA is made from early genes.

Synthesis of double-stranded and complementary

RNA in vitro. Vaccinia virions contain an RNA

polymerase activity which directs the synthesisof

early viralmRNA (10,11).Table2showsthatthe

RNA made in vitro containsapproximatelyequal

amounts of the three nucleotides uridine

mono-phosphate, cytidine monophosphate, and

guano-sine monophosphate. 3H-ATP was not used

be-causeoftheformation ofpolyA(10).Someofthe

RNA made in vitro was resistant to a mixtureof

pancreatic and T1 ribonuclease at a high salt

concentration (Table 2). However, when we

sub-jected the RNA to annealing conditions, there

was asixfold increase in the level of

ribonuclease-resistant RNA. Finally, thermal denaturation

rendered both annealed and nonannealed RNA

susceptible toribonuclease degradation.

The level of ribonuclease-resistant 3H-RNA

afterannealing varied from3to 10%of thetotal synthesis in different experiments. The annealing

reaction isdependenton theconcentrationofthe

input RNA. Figure 1A shows that the level of

annealed ribonuclease-resistant 3H-RNA

de-creased with second-order kinetics as the

3H-RNAwas diluted. Figure 1B shows that the

an-nealingreaction was alsotime-dependent.

We carried out agarose chromatography of

annealed and nonannealed ribonuclease-treated

TABLE 2. Effect of annealinig on the amount of

ribonuclease-resistant RNAG

3H-UTPb 3H-CTP 3H-GTP Treatment (counts/ (counts/ (counts/

min) min) min)

A. Trichloroacetic acid 151,589 113 ,984 109,644

ppt

B. Ribonuclease 2,432 1,931 1,539

C. Anneal ribonuclease 14,271 11,825 8,674

D. Melt ribonuclease 206 217 208

aPurification of theRNAaftersynthesiswasby

phenol extraction and ethanol precipitation, the

RNA wasresuspended in2X SSC (SSC = 0.15 M

NaClplus0.15 Msodiumcitrate), and thesamples

were treated as follows. (A) The 5% trichloro-acetic acid-insoluble 3H-RNA was determined.

(B) Ribonuclease was treated with 10mg of

pan-creatic ribonuclease plus 3pg of T1 ribonuclease

per mlat 37 Cfor 15 min in2X SSC, and the 5% trichloroacetic precipitable counts per minute

weredetermined. (C) The RNA wasannealed for

6 hr at 65 Cin2X SSC andtreated as in B. (D)

Samplesdiluted to O.1X SSC beforeor after

an-nealing (shown here for after annealing) were

heatedto 100C, cooled, andtreatedasinB.

bAbbreviations: UTP, uridine triphosphate;

CTP, cytidine triphosphate; GTP, guanosine

triphosphate.

z

-._

0

cs

.U)

40

cc I 0 z

cr

E 0. 0 t)

z

C

l

I

0 0

0

0

800

6001

4001

800

600

400

200

2.5 5.0 7.5 10 15 25

Dilution

B

IS

1 2 3 4 5 6 7 8 9

Time of Annealing (Hr.) FIG. 1. Concentration and time dependentce of the annealingreaction. (A)RNA waspreparedanidpurified

in the standardmannerby using vaccinia virus cores (10). The RNA was diluted as indicated and

an-nealedfor 6 hrat 65 Cin 2X SSC (0.3 M NaCI,

0.03M trisodium, citrate). After annealing, the RNA

was treatedwith ribonucleaseasdescribed in Table 2, and the 5% trichloroacelic acid insoluble 3Hcounts perminuteweredeterminied. (B) Samples of RNAwere

annealedas in (A) for different periods oftime, and treated with ribonuclease; the 5% trichloroacetic

acid-precipitable 3H counts per minuite were determinied

3H-RNAaspreviouslydescribed(3).Aportion of

the 3H-RNA was excluded from the agarose

column in both cases, indicating the presence

of high-molecular-weight ribonuclease-resistant

RNA with and without annealing. Only 0.074%

of the3H-RNA which hadnoprior annealingwas

excluded from the column, whereas the amount

excluded increased to 0.34% for 3H-RNA which

wasannealedbeforeribonucleasetreatment.

Table 3 indicatesthat the excluded RNA was

resistant to both ribonuclease and ribonuclease

plusdeoxyribonuclease, butitbecamecompletely

sensitive to ribonuclease afterthermal

denatura-tion.Themelting temperatureofthe

ribonuclease-resistant RNA excluded by the agarose column

A

0

.

Is

-1

on November 11, 2019 by guest

http://jvi.asm.org/

[image:3.491.251.444.80.384.2] [image:3.491.49.239.393.503.2]
(4)
[image:4.491.61.254.76.184.2]

TABLE 3. Nuclease sensitivity ofpurified ribonuclease-resistant RNAa

Treatment 3H-counts/min Per centoftotal

A. Trichloroacetic acidppt.. 1,604

B. Ribonuclease... 1,542 93.7

C. Ribonuclease +

deoxy-ribonuclease... 1,437 82.5

D. Melt and ribonuclease... 74 4.6

aAnnealedRNA waspreparedandsubjectedto

ribonuclease digestion and agarose

chromatog-raphy. The excluded RNA was dialyzed versus 0.01 MTris (pH 7.4),MNaCI.Samplesweretreated

asfollows. (A) Five percent trichloroacetic

acid-precipitablecounts per minute were determined.

(B) Ribonucleasewas treated with 20,gof

pan-creatic ribonucleaseplus2pgofT,RNase per ml

in 0.1 M Tris (pH7.4), 0.2 M NaCl for 20 minat37 C. Then treatmentproceededasin A. (C) AsinB,

but the solutionwas made 0.004 MMgCl2and 50

,pg

of DNase per ml(ribonuclease-free,

Worthing-ton) wasadded for 20 minat37C. (D)Samplewas

heatedto100 C for 15 min,cooled, and treatedas

in B.

was determined as previously described (3). A

sharptransition withaTm of 76 Cwas observed

(Fig. 2).

Sucrose gradient sedimentation in 15 to 30%

sucrose was carried out on the

ribonuclease-resistantRNA excluded byagarose, indicating a

rangefrom7.5 to 10.5Sforcoredouble-stranded

RNA (Fig. 3). This value is in good agreement

with the sedimentationcoefficient ofthevaccinia

double-strandedRNAsynthesizedin vivo

(3).

Effect ofannealing on the amount ofvaccinia

virus 3H-double-stranded RNA made in vivo. Our

results with thevaccinia RNA

synthesized

invitro

prompted us to reinvestigate the kinetics of

appearanceof thevaccinia virus double-stranded

RNA intheinfected cell. Confluent

monolayers

of

HeLacells were infected with vaccinia virus. At

1,3, and5hrafter

infection,

cultureswere

pulse-labeled with100

,uCi

of

3H-uridine,

and thetotal

cytoplasmic nucleic acids were

prepared.

After

removing the

contaminating

DNA

by digestion

with

deoxyribonuclease (100

,ug/ml,

60 min,

37C),each3H-cytoplasmicRNA

preparation

was

purified by three phenol extractions and three

ethanol precipitations. The

purified

RNA was

divided into two

equal

samples.

From one,

double-stranded RNA was

prepared

as above.

The other wasallowedtoself-annealfor 6 hr and

then

double-stranded

RNA was

prepared.

The

results are

presented

in Table 4. It is clear that

complementary RNA is made

throughout

the

infection

cycle.

Is there double-stranded RNA inside the cell?

It maybeargued that only complementaryRNA

exists inside the cells and that the SDS-phenol

extraction catalyzes theannealingof theRNA to

give helical structures. This argument was tested

by the following experiment. 3H-cytoplasmic

RNA was prepared as above. Before extraction

with SDS and phenol, NaCl and ribonuclease

wereadded to final concentrationsof 0.25 M and

100

,g/ml,

respectively. After incubating the

mixture at 37 C for 60

min,

double-stranded

RNA wasprepared by the standard SDS-phenol,

nucleasedigestion, agarosechromatography tech-nique. As a control, duplicate cultures were

infected at the same time and double-stranded

RNAwasprepared as usual. At least 70% of the

double-stranded RNA purified by this technique

was in a form that is resistant to ribonuclease

100

.

z

co75

w

50

c)

z

-

40

60

80

100

TEMPERATURE

OC

FIG. 2. Effect of temperature on

ribonuclease-re-resistantRNA. RNA excludedby agaroseas inTable2

was dialyzed versus 0.02 M Tris (pH 7.4), 0.01 M

NaCl. Samples were made 0.001 M

ethylenediamine-tetraaceticacid and heatedtotheindicatedtemperature

for10min,andrapidlycooledonice.Eachsamplewas

made 0.2M NaCI and treated with ribonucleaseasin

Table 2. The trichloroacetic acid-insoluble RNA was determined.

CVi

on November 11, 2019 by guest

http://jvi.asm.org/

[image:4.491.264.457.276.573.2]
(5)

16

0

12

k.)

4

23S 16S 4S

4 8 12 16 20

FRACTION NO.

FIG. 3. Sucrose gradient analvsis of ribonuclease-resistant RNA excludedfrom 6% agarose. The RNA was run ona 15 to30% (w/v) sucrose gradient

con-taining

0.1%70

sodiumdodecylsulfate, 0.1 MNaCI, and 0.01 M ethylenediaminetetraacetic acidfor 16 hr at

27,000 rev/min at 25 C in a Spinco SW27 rotor. Bacillus subtilis RNA wasusedas a marker.

TABLE4. Effect ofanniealing on the amount of

WH-double-stranded RNA made in vivo

3Hcounts/min Hcounts/min Hcounts/min Timeafter afterphenoland afterribonu- after annealing,

infection(hr) deoxyribo- cleaseand ribonuclease,

nuclease agarose and agarose

1 1.0 X 105 560 800

3 2.0 X 105 2,400 3,600

5 1.8 X 105 2,440 4,600

before extraction with

phenol (Table 5).

Con-sideringtheseverityoftheribonucleasedigestion, it is

likely

that most, ifnot

all,

of the

double-stranded RNA isolated by this technique is

helical RNA inside the cell.

DISCUSSION

We find thatthe synthesis ofvaccinia

double-stranded RNA is inhibited

by

actinomycin D.

Therefore, we conclude thatthis RNA arises via

a DNA-dependent reactionmechanism.

Ribonuclease resistant RNA synthesized in

vitro by vaccinia cores possesses properties

similar to those of ribonuclease-resistant RNA

isolated from vaccinia virus-infected cells and

T4phage-infectedbacteria (3, 4, 9). It is

interest-ing to note that the increase in

ribonuclease-resistant RNA after annealing corresponds to a

similar phenomenon observed for the in vivo

vaccinia RNA isolated throughout infection.

Since cores synthesize only "early" RNA (11), we may regard these results as proof that vaccinia virus does produce complementary RNA early

ininfection. Ourresults with cytosine

arabinoside-treated cells offer further confirmation of this

idea.

Itisimportant to note that the annealing of the

ribonuclease-resistant RNA made in vitro is both concentration- and time-dependent. These results

support the modelinvolving the transcription of

complementary regions of vaccinia DNA and rule

out thepossibilitythat theribonucleaseresistance

could be theresultof a folding back of a

single-stranded molecule to give a lengthy hairpin

structure.

TABLE5. Effect ofpretreatment with ribonuclease before phenol extraction ont double-stranded

RNA

'Hcounts/min 3H counts/min Per-Treatment inphenol after nucleases cent-extract andagarose aeo

Standard

proce-dure... 3.2 X 1063.7 X 103 1.16 Pretreatment with

ribonuclease 2 X 104 2.7 X

103

0.84

CONVERGENT TRANSCRIPTION

- ~ ~ ~~~~~~~VACCINIA

C

COMPLEMENTARY

1 I RNA

1 INTRACELLULAR

111~ ds-RNA

1RNase

PURIFIED ds-RNA

DIVERGENT TRANSCRIPTION

5'1 ,^5'

3 < - L --~~~~~~ 31 1

3R

RNase

FIG. 4. Models ofconvergent and divergenit trani-scription. RNA is transcribedfromboth strandsof the vacciniaDNAsuch that molecules with complementary regionsaresynthesized. Someofthese molecules form the appropriate base pairs andare converted to intra-cellular double-stranded RNA with single-stranded

regions (Sw20 = 9 to22S). Duringpurification of the

double-stranded RNA, thesingle-stranded portions are removedby ribonuclease digestion. Inthecase of con-vergent transcription, the terminal sequences of the complementary RNA are found in double-stranded RNA, whereas the initial sequences ofcomplementary

RNA become double-stranded RNA in the case of divergent transcription. ds-RNA, double-stranded

RNA. IC',

on November 11, 2019 by guest

http://jvi.asm.org/

[image:5.491.42.237.67.280.2] [image:5.491.248.443.267.519.2] [image:5.491.45.240.362.462.2]
(6)

Duesberg and Colby

(4) reported

that the

intracellular

form of the double-stranded RNA is

a heterogeneous

population

of molecules with

sedimentation coefficients

ranging

from9to22S.

Treatment with ribonuclease converts these

molecules into a homogeneous population of

double-stranded

molecules of 9 to lOS.

Thus,

the

intracellular

formappearstobea

population

of

molecules

sharing

common size

double-stranded cores and

having single-stranded

por-tionsof various lengths.Thesedata

coupled

with

the above arguments that the double-stranded

RNA is

synthesized

via a

DNA-dependent

reac-tionmechanismareconsistent withthepattern of

convergent transcription

recently suggested by

B0vre and

Szybalski (1)

for the b2

region

of

coliphage x.

Alternatively,

the vaccinia

double-stranded RNA could arise from a pattern of

divergent

transcription (Fig.

4).

Both models

involve the

transcription

of

complementary

regions of theDNA;

they

differfrom each other

with respect to the promoter sites or

regions

of

initiation of

transcription.

It should be

em-phasized that neithermodel demands the

simul-taneous transcription of the

complementary

sequences. Indeed, the

finding

of

complementary

RNA in the

cytoplasm

argues against

simul-taneoustranscription.

It was

previously

shownthat the

purified

vaccinia

double-strandedRNAisavery potentinducerof

interferon in chick cells

(3).

The demonstration

thatatleast 70%ofthepurified double-stranded

RNA is in a ribonuclease-resistant form in the

cytoplasm

of infected cellssuggestsmore

strongly

that this

population

of molecules is

responsible

for the induction of interferon in cells infected with

vaccinia virus.

ACKNOWLEDGMENTS

This investigation was supported by Public Health Service grants CA-10802 from the National Cancer Institute and IROlA-10841-02 from the National Institute for Allergy and InfectiousDiseases.

LITERATURECITED

1. Bovre,K. E., andW. Szybalski. 1969. Patterns of convergent andoverlappingtranscription within theb2 region of coli-phageX.Virology 38:614-626.

2. Colby, C., andM.J.Chamberlin. 1969. Thespecificity of inter-feroninduction in chickembryo cells by helical RNA. Proc. Nat. Acad.Sci.U.S.A.63:160-167.

3. Colby, C., andP. H.Duesberg. 1969.Double-stranded RNA invaccinia virus infectedcells. Nature (London) 222:940-944.

4. Duesberg, P. H., and C.Colby. 1969. Onthe biosynthesis and structure of double-stranded RNA in vaccinia virus-in-fected cells.Proc.Nat. Acad. Sci. U.S.A.64:396-403. 5. Field, A. K., G. P. Lampson, A. A., Tytell, M. M. Nemes, and

M.R.Hilleman. 1967. Inducers of interferon and host re-sistance.IV.Double-stranded replicative formRNA

(MS2-RF-RNA) fromE.coliinfectedwith MS2-coliphage. Proc. Nat.Acad. Sci. U.S.A. 58:2102-2108.

6. Glasgow, L.A., andK.Habel.1962. Theroleof interferonin vaccinia virus infection ofmouse embryo tissue culture. J. Expt.Med. 115:503-512.

7.Joklik,W.K. 1962. Thepreparation and characterizationof highly purified radioactively labelled poxvirus. Biochem. Biophys. Acta 61:290-301.

8.Joklik,W. K. 1968.In H.Frankel-Conrat (ed.), Molecular basis of virology, p. 576. Reinhold PublishingCo., New York.

9.Jurale, C., J. R. Kates, and C. Colby. 1970. Isolation of double-strandedRNAfromT4phage infected cells. Na-ture (London) 226:1027-1;29.

10.Kates, J. R., and J.Beeson. 1970. Ribonucleic acid synthesis in vaccinia virus. I.Themechanism of synthesis and re-leaseofRNAinvacciniacores.J. Mol. Biol. 50:1-18. 11.Kates, J. R.,andB.McAuslan. 1967.Messenger RNA

syn-thesis by a"coated" viralgenome. Proc. Nat.Acad. Sci. U.S.A. 57:314-320.

12. Montagnier, L. 1968.Presenced'un acide ribonucleique en doublechaine dans des cellulesanimales. C.R. H.Acad. Sci. Ser.D267:1417-1420.

13. Oda,K.S., and W. K.Joklik. 1967. Hybridizationand sedi-mentation studieson"early" and "late" vaccinia messen-gerRNA.J. Mol.Biol.27:395-419.

on November 11, 2019 by guest

http://jvi.asm.org/

Figure

TABLE 1. Effects of inhibitors of niucleic acid synz-thesisondouble-strandedRNA
TABLE 2. Effect of annealinig onribonuclease-resistant RNAG
TABLE 3. Nuclease sensitivity of purifiedribonuclease-resistant RNAa
TABLE 5. Effect of pretreatment with ribonucleasebefore phenol extractionont double-stranded

References

Related documents

studies indicate that, when cells are pretreated with IUdR prior to infection, greater amounts of infectious virus per cell are produced. Even though the final virus titer obtained

control condition were exercising compared to 90% of participants in the IE (i.e. 23 out of 24) in the IE and BTN conditions indicated that participating in the study. encouraged

In other words, if a NO is characterised by the lowest reputation rating, we quantify the mean relative error in ex ante expected utility the NO is going to incur by bidding

In Section III, the spectral flux and group delay function are derived for the auditory spectrum, a fundamental frequency estimation algorithm using the auditory model is presented

Table 8 - Quantitative results from pre-search questionnaire (Dyslexia Information) – Note: * signifies problem only in real world. In summary there is no strong evidence of

We also know that notionally some of the tax relief given to employees on pension contributions goes to those who appear to contribute to state ‘pay as you go’ pensions schemes –

Using data from previous studies, we estimated that with 400 swabs per year, an effectiveness of 20% would be detected with 79% power for our primary outcome of PCR-confirmed

Figure 6 shows the maximum likelihood pedigree that results without using the.