Isolation and Properties of Poliovirus Minus Strand Ribonucleic Acid

Copyright ( 1970 American Society for Microbiology

Isolation

and

Properties

of

Poliovirus Minus

Strand Ribonucleic Acid

POLLY ROY AND D. H. L. BISHOP

InistitutteofCanlcerResearch, Columbia Unziversity, New York, New York 10032

Received for publication 23June 1970

Poliovirus

minus

strands

were

purified from double-stranded polio ribonucleic

acid.

The minus strands haveabase

ratio

complementary

to

that of the viral

ribo-nucleic acid and

arenot

infectious.

The complementary (minus) strand

to the

Q3

bacteriophage

genome

(18)

is not

only

an

excel-lent template for

the

Qf

replicase,

butalso is an

intermediate

in the in vitro

replication

of

Q3

ribonucleic

acid

(RNA; 11, 21, 22).

Without host

factors

(12, 21), Q3 replicase

cannotbe

template-duplicated by

the viral

RNA, although

it willuse

minus strands very

efficiently (1).

This suggests

that

depletion

of host factors in an infected cell

could

be a

regulatory

mechanism

allowing only

production of plus

strands late in infection. Free

minus

strands are

produced

in vitro before free

plus strands, although

later

predominantly plus

strands

are

synthesized

(21, 22).

It isnotknown whetherfreeminus strandscan

template-duplicate

the enzymes

responsible

for

replicating

thegenomes ofRNAvirusesof

higher

organisms.

With this in

mind,

we describe here

the isolation and characterization of

poliovirus

minus strands.

MATERIALS AND METHODS

Themethodsemployedtoobtain poliovirusminus

strandsweresimilartothosedescribedby Polletetal.

(18) for the isolation of bacteriophage Qfl minus strand RNA. Modifications of their procedure were developed to accommodate problems specific to

handling poliovirus RNA. The basic procedure

in-volved melting double-stranded poliovirus RNA and annealing theseparatedstrands toan excessof polio-virus RNAfragments.Thehybridsoformedwas iso-lated by agarose chromatography andthe denatura-tion-annealing process wasrepeated. Afterafinal de-naturation, full-length minus strands were separated from plus strand fragments by centrifugation. The minus strands so obtained were contaminated, as judged by annealing studies, with less than 5%', plus strand RNA, and, from infectivity assays, with less than 0.01% full-length plus strands.

Preparation of poliovirusand poliovirus RNA

frag-ments. Poliovirus was obtained from infected HeLa S3 cells by employing the observations ofEhrenfeld etal.(10) that detergent lysisof HeLa cellssolubilizes

the cellular membranes, liberating the intracellular

virus but leaving the nucleic intact. Log-phase cells wereinfected with wild-type (ts+) Sabin poliovirusat amultiplicity of infection of 10to 30plaque-forming units (PFU) per cell. The cells were grown under standardconditions (5, 14) and wereharvestedafter 8 hr. Infected cells, 108 cells per ml of TSM buffer [0.01 M tris(hydroxymethyl)aminomethane (Tris),

pH 7, 0.05 M NaCl, 0.005 M MgCI2], were lysed by freeze-thawing thrice in detergents (10; 1% NP-40,

0.5sc deoxycholate), and nuclei were removed by centrifugation at 800 X gfor 5 min.The lysate was adjusted to contain 0.5%c, sodium dodecyl sulfate (SDS; 15) andwascentrifugedat40,000rev/minfor 2.5 hrat 10C inaSpincoSW41 rotorover abottom 1-mlpad of CsCl (p 1.40) tocollect the virus and a 1.5-ml pad of 47C%/G sucrosetoretain membrane resi-dues. The virus bandwasremoved, the CsCl concen-tration was adjusted togive a density of 1.34 g/ml, and the preparation was centrifuged for 36 hr at

40,000rev/minand5C inaSpincoSW41 rotor.The virus bandfrom this equilibrium gradient centrifuga-tion wascollected and usually yielded virus havinga 200:1 particletoPFUratio.

RNA was extracted from virus by procedures de-scribed previously (6) and was dissolved in 0.005 M

ethylenediaminetetraacetate (EDTA), pH 7,ata con-centration of1mg/ml. Viral RNA fragmentswere ob-tainedby adding

20pliters

of1 MNaOHpermlof RNA, incubating atroom temperature for5 min, and neu-tralizing with I M HCI. The fragments so obtained werecentrifuged in a glycerol gradient (10 to 30% glycerol, 0.1MLiCl, 0.01 MTris buffer, 0.005MEDTA,

pH 7) for 6 hrat40,000rev/minand 5 C, byuseofa Spinco SW41 rotor (see Fig. 1). The low molecular weight viral RNA fragments (average, 12S) were col-lectedby alcohol precipitation and dissolved in 0.005

M EDTA, pH 7, togiveaconcentration of1

mg/ml.

Preparation of labeled double-stranded (DS) and multistranded (MS) poliovirus RNA. Poliovirus-in-fectedHeLa cellswerelabeled from 50min postinfec-tion with 3H-uridine (10to

50,uCi/ml)

or 32P-ortho-phosphate (50

uCi,ml)

andweregrownfor6hr;the cellswerethenlysed by detergentsasdescribed above. After removal of the nuclei, the total RNAwas ex-tracted, andthebulk ofthesingle-strandedRNAwas precipitated by I NINaCl (3, 4, 8). BoththeDS-and

604

Prinztedin U.S.A.

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I5 .30

0

20 -2 (L

3I

05 10

A

10 20 30

FRACTION

FIG. 1. Poliovirus fragments. Fragments of

poliovi-rus RNAwerepreparedasdescribedin Materials and

Methods. Thefragmenits werecentrifuged for 6hr on

a 10 to 30% glycerol gradienit with 3H-labeled 28S

HeLa ribosomal RNA. The indicatedfractions were

pooledand usedfor annealingstudies.

MS-RNAas wellastransfer RNA(tRNA) were

alco-hol-precipitated fromthesupernatantand loadedon a

2% agarose column (3, 17). Agarose columns were runin 0.1MNaCl,0.01 MTrisbuffer,0.005MEDTA,

and0.1% SDS, pH 7; severaldays priorto use,they

werewashed in thesamebuffer saturated with

diethyl-pyrocarbonate (DEPC) to remove alltraces of

nu-clease (20). The DEPC was washed out with fresh bufferinview oftheobservation by Oberg (16) that

DEPC destroys the infectivity of poliovirus RNA.

Agarose chromatography of the total infected-cell

RNA is shown in Fig. 2. The predominantly

ribo-nuclease-resistantDS-RNA and MS-RNA were re-coveredin the void volume (Fig. 2) and were

sub-sequently further purified by glycerol gradient

cen-trifugation (see Fig. 4).Nodeoxyribonucleicacidwas

detectablein thepostglycerolRNApreparations. Denaturation of DS and MS poliovirus RNA. No

attemptwasmade to separatetheDS- from the

MS-RNA in the 18S postglycerol fractions.The ribonu-clease resistance of these RNA preparations was usually between 80 and90%. Several conditions for

denaturation of the RNAwereexamined.Incomplete

melting was thegreatest difficulty encountered. If

la-beledRNAwasmelted at 10or100ug permlof0.001

MEDTA, pH 7, by heatingat100 Cfor 90sec,then,

no matter what concentration it was diluted to, it

reannealed to recover 70 to 80% of the label in a ribonuclease-resistant form. This

concentration-inde-pendent reannealingindicates thatthestrandsduring

this (melting) procedure were not completely

sepa-rated and consequently reassociated with each other

when incubatedat70C in0.4 MNaCl. Thisoccurred

evenwitha100-foldexcessof unlabeledfragmentsin

theannealingcocktail (Fig. 3,lineA). Heatingat100

C forlonger periods gave similar results. Heating at

OD,2 5

VOID

50 100 150 200 250

FRACTION

FIG. 2. Separation ofRNAfrom infected cells on

2%agarose.Acolumn of2% Agarose(approximately

2liters in volume, 140cm inheight) was prepared as

describedinMaterials andMethods. Infected-cellRNA

(100 mg) in 40 ml of column buffer was loadedand

7.5-ml fractionswerecollected.

z

A

4-'

01 05 10 5 IC 50 100

RNA /ig/ ml

FIG. 3. Anntealing poliovirus double-strantded RNA

after melting at different concentrations. Poliovirus

double-stranded RNA washeatedin0.001MiEDTAat

100 C for 90secat100

,ug/ml

(curve A), dilutedtothe

anniealing concentration indicated, and incubated at

70 Cin0.4MNaCl for 15min priortodeterminationof

the ribonucleaseresistance. Alterniatively (curveB),thle

RNAwasheatedat100 Cat theannzealinzg concentra-tion, cooled, brought to 70 C and 0.4 m NaCl, and

annealed for15 min beforedetermintationt ofthe

ribo-nucleaseresistance.

these concentrations under pressure at above 100 C

gavegood melting, asjudged bylow ribonuclease

re-sistance,but resulted inarecoveryofonly 10%ofthe

RNAasfull-length singlestrands(compare Fig. 4B).

However, meltingatRNAconcentrations below 1

,ug/

mlresultedinstrand separation, asjudged bythe ob-servation that reannealing was

concentration-de-pendent (Fig. 3,lineB).Thisconcentration-dependent

reannealing indicates that the strands during the

melting procedure were completely separated. The

reasonthatmeltingis betterat1,ug/mlthan 10

,g/ml

could bethatdilution ofsomecontaminating divalent cationoccurs.Alternatively,itcouldbeanexpression

of some primary sequences reannealing at a

faster-than-normal rate, even at 100 C. As annealing is a

concentration-,temperature-, andtime-dependent

proc-ess,dilution ofthe RNA for meltingtherefore favors

strandseparation.

The procedure adopted was, therefore, as follows.

RNAwasaddedto0.001 MEDTA, pH 7, previously

equilibratedat100 C inaboiling-water bath,togivea

605

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concentration of 1 ,ug/ml or less. After 90 sec, the

solution was rapidly cooled in iced water. The

ribo-nucleaseresistance was then determined byincubating

asample for20min at 37 C in 0.4 M NaClwith (per

ml) 10 ,ug each ofribonuclease Aand T1. The

resist-anceof melted RNA was5%0. Single-stranded

polio-virus RNA has a ribonuclease-resistant core of

4%

under these

conditions.

Glycerol gradient separation of melted and

un-melted native DS-RNA is shown in

Fig.

4.

Melting

RNA in dimethylsulfoxide (13) gave essentially the

sameresults and asimilar recovery of

full-length

RNA

fromthe DS- and MS-RNA preparations.

Annealing with poliovirus RNA fragments. To

melted DS-RNA (at 1.0 to 0.1

pg/ml),

poliovirus

A

RNAfragmentswereadded togiveaconcentration of

5 ,ug/ml; the mixture was adjustedto70C and was

made 0.4 M NaCl. After 15 min, the solution was

cooled and theRNA wascollectedby alcohol

precipi-tation. The time course ofannealing with or without

fragments is shown in Fig. 5. Usually 40 to 50% of

the label became ribonuclease resistant as compared

to anincreased resistance of 4% without fragments.

Isolation of the annealed RNA. The RNA was

chromatographed through 4% agarose, and the

an-nealed RNA was recovered from the void volume.

Under thse conditions, single-stranded poliovirus

RNA and poliovirus fragments arerecovered in the

included volumeof the gel (seeFig. 6). The annealed

RNA soobtained was 96% ribonuclease-resistant.

Subsequent minus strandpurification. The hybrid of

10 20 30

FRACT ION

FIG. 4. Gradient centrifuigation ofnative (A) and

melted (B) poliovirus double-stranded RNA. (A) 32p_ labeledpoliovirus double-stranded RNA and 3H-labeled poliovirus RNA were prepared and centrifuged on a glycerol gradient, as described in Materials and

Methods. (B) Double-stranded 32P-labeled poliovirus

RNA was heated together with 3H-labeledpoliovirus

RNA in 0.001MEDTA atI Mg permland100C for 90 sec and was similarly centrifuiged. Ceentrifugation throughl 10 to 30% glycerol in 0.1 M NaCl, 0.01 m Tris, 0.005 vr EDTA was for 6 hrat5 CinaSpinco SW41 rotor.

WITHFRAGMENTS

/

WITHOUT FRAGMENTS

--- ---* -3-0-20_

10 20 30

T M E (MIN)

FIG.

5.

Annealinig

melted

poliovirus

double-stranded RNA to

poliovirus

fragments.

Poliovirus 3H-labeled double-stranded RNAwasmeltedata

concentration

of

0.1ug permlandannealedwithor without 5 ug ofviral 30

fragments

per mlfor the timesindicated at 70 Cand 0.4.A NaCIasdescribed in MaterialsandMethods.The

ribonuclease resistance was determined as describedin

Materials

and

Methods.

10

5L

:

5

*e

30 60

FRACTIOr

FIG. 6. Agarose

(4%1)

chromatography of polio-hybridRNA. Thesynthetic hybrid of 32P-labeledminus strands and unlabeled polio fragments (see Materials

and Methods) was chromatographed with 3H-labeled viral RNA ona 4% agarosecolumn (90 by 0.9 cm).

The indicated fractions were pooled for subsequent

minulsstrandpurification. 15

10

0

5

10*

50 z

cr

N

Ox

a.

3J 0: LY

10 20

FRACTION

I in 5

2

0

a. 0

POOL

6

Io

,0

a.3 0

N:

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labeled minus strands and unlabeled viral fragments was

melted

andreannealed again to a 20-fold excess of

viral

fragments as described above. This second

hy-brid,

after agarose chromatography, was denatured

and centrifuged on a glycerol gradient (Fig. 7); the

indicated fractions were pooled and used to determine

theproperties of poliovirus minus strands.

Base-ratio determination, infectivity assays. The

base ratios of 32P-labeled RNA were determined as

described previously

(7).Infectivity ofsingle-stranded

RNA and DS-and MS-RNA, with the use of

DEAE-dextran, wasdetermined by the agar suspension

tech-nique

described

byBishop and Koch (8).

Stocksandmaterials. HeLa S3 and theSabin

polio-virus strains were kindly given to us by D. Summers

and E. Ehrenfeld. Agarose was

obtained

from

Bio-Rad Laboratories, Richmond, Calif., and DEPC,

from Naftone Inc., New York, N.Y.

RESULTS

Purity of the poliovirus minus strands. Four

methods were used to determine the

purity

of

poliovirus minus strands prepared

as

detailed in

Materials and

Methods.

(i)

Contamination of minus strands by original

plus strand RNA. It has

been shown in

Materials

and Methods

(Fig.

5)

that

added

plus strand

frag-ments anneal to minus strands in the

annealing

cocktail. To demonstrate the loss of original

plus

strand RNA

from

the

DS-RNA preparation,

32P-labeled

viral

plus strands

were added

to

3H-labeled DS-RNA, and minus strands were

pre-4

3

I0

,0~

I2

In

10

FRACTION

Io

_x0

4 E

0

CY)

20

FIG. 7. Glycerol gradient centrifugation ofmelted

32P-labeledpoliovirus hybrid RNA. Poliovirus hybrid

RNA (containing 32P-labeledpolio minus st?anzds and

unlabeled poliovirus fragments) was melted and cen-trifuged with3H-labeled 28S HeLa ribosomalRNAona 10 to30%X0 glycerol gradient for 6 hr. The indicated 35Sfractions werepooled for further analysis.

pared as described in Materials and Methods.

The purified minus strands contained less than

0.08% of the original 32P-labeled viral plus

strands-equivalent to 1% plus strand RNA in

the final minus strand preparation (Table 1).

(ii)

Contamination of minus strands with plus

strand RNA. The extent of fragmented and

com-plete

plus strand

contamination

of the minus

strand

preparation was determined by

self-annealing

at

a

concentration of 5 ,ug

of

RNA per

ml

(see

Materials and Methods). The ribonuclease

resistance

before

and

after annealing was 4 and

5%,

respectively.

Furthermore,

annealing with

added, unlabeled, plus strand fragments gave

95%

resistance of the label.

Consequently,

there

was

less

than 5%

plus

strand

contamination of

the minus

strand

preparation.

(iii)

Infectivity of

the minus strand

preparation.

The

infectivity of purified viral plus

strand RNA,

the initial

DS-RNA

(containing

DS-

and

MS-RNA), and the minus strand RNA

preparation

was

determined

by use

of

DEAE-dextran as

described

by

Bishop and Koch (8). The

results,

recorded

in Table 2, indicated that minus strands

are

not

infectious

and

that

there was

less

than

0.01%

contamination

of

the

preparation

with

infectious

plus strands. The infectivity of the

DS-RNA, which is slightly

greater than

would

be

expected

from its plus strand content,

probably

reflects

the better

survival of

DS-RNA during

the

plating

procedure (8).

(iv)

Base ratios of

purified

plus and minus

strands

as well as DS-RNA. The base

ratios

of 32p_

labeled

plus

and minus strand

RNA,

as

well

as

that of

the DS 18S

RNA, are

given in Table

3.

The minus strands were

essentially

comple-mentary in base

composition

to

the

plus strand

preparation,

and the

base

ratio

of

the

DS-RNA

is as

expected (2,

8, 9, 19,

23).

DISCUSSION

Two

forms

of experiment

are

possible

with

purified

minus strands.

Although

minus

strands

are

not

infectious,

it is

possible, if

the

minus

strand

genome was

replicated by

the

replicase

coded

for

in the

plus

strand

genome, that in the

presence

of

plus strands the minus strand genome

could

be

expressed.

DS

poliovirus

RNA

is

infec-tious

(8);

consequently,

it

should be

possible

to

hybridize

plus

and minus strands

of different

genotypes,

purify the

DS-RNA,

and test its

infectivity

with

regard to the

expression

of the

two

genotypes.

Advantage

can

also be taken of

the

observation that DEPC

destroys

the

infec-tivity of

single-stranded

RNA but

not

DS-RNA

(16).

The

second

form

of

experiment

in

which minus

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TABLE 1. Loss

of

original plus stranidsfrom original double-strandedpoliovirus RNAa

Double-stranded Single-stranded

3H-RNA 32P-RNA Calculated Calculated labeled

Prepn minusstrand (9Hand32p)

content plusstrand content Counts/min Amt Counts/min Amt

Ag lg pg pg

Original

2 X 105 100 6 X 104 10 45 65

First

Agarose

4 X 104 20 5 X 103 0.8 15 5

Second

Agarose

... 2 X 104 10 60 0.01 10 0.07

Postglycerol gradient. 1.2 X 104 6 <40 <0.008 6 <0.04

a3H-labeled double-stranded RNAwas mixed with 32P-labeled single-stranded

35S

poliovirus RNA

and minus strands wereisolated as described inMaterials and Methods. For the first and second

an-nealing, 500 and400,Agof viralfragmentswereused,respectively. The minus strand content of

double-strandedRNA (column 5) wascalculated from thelabel rendered ribonuclease-resistant after annealing

asmallportion of melted double-stranded RNAto a100-fold excess of unlabeled fragments. This figure

isonlyaroughestimate. It wasassumed that the unannealable portion of the label was poliovirus plus

[image:5.489.44.237.255.340.2]

strand RNA (column 6).

TABLE 2.

Intfectivity

ofdouble-strantded and purified

poliovirus single-stranded minus and viral

(plus) RNAa

Prepn PFU per pg_RNA of

Native double-stranded RNA

18,000

Poliovirus minus strand RNA... 0

Poliovirus RNA...

24,000

aRNAsamples (1.0,0.1, and

0.01

,ug) in

DEAE-dextranwereincubated withHeLacells

according

to the procedure described by Bishop and Koch

(8). RNA

samples

were prepared as described in

Materialsand Methods.

TABLE 3. Base ratios ofdouble-stranded (DS)

anzd

purified poliovirus

single-stranided minius anid

viral (plus) RNAa

Component Poliovirus Poliovirus Poliovirus

ComponentDS RNA minus RNA RNA

Cytosine ... 23.2 23.3 24.1

Adenine ... 26.8 23.2 30.3

Guanine ... 23.1 23.3 22.4

Uridine... 26.9 30.0 23.3

a32P-labeled RNA samples were prepared and

purified as described in Materials and Methods.

Base ratios (mole

%7)

after alkali hydrolysis were

obtained as described by Bishop, Mills, and

Spiegelman (7).

strands can be used involves the

purification of

polio replicase.

A

polio

replicase preparation

which,

without

template addition, synthesizes

single-stranded

35S and DS 18S poliovirus

RNA

has been

recently

isolated

from infected

HeLa

cells by

Ehrenfeld

et

al.

(10).

This

enzyme, in

our

hands, apparently

synthesizes only plus strand

RNA, even in the 18S DS product. This suggests,

therefore, that the enzyme has bound to it minus

strand templates. If so, then it is logical to look

for

dissociated enzyme with minus strands as

templates, especially in view of the observation

that

Q3

replicase purified from host factors

can

be

template-duplicated by

minus,

but not by

plus, strands (1).

ACKNOWLEDGMENTS

Weappreciatethe advice and encouragement of Sol Spiegelman and theexperttechnical assistance ofUlrikeStadler.

This investigation was supported by Public Health Service researchgrantCA-02332from the National Cancer Institute.

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