Newcastle Disease Virus-Specific RNA: Polyacrylamide Gel Analysis of Single-Stranded RNA and Hybrid Duplexes

Copyright0 1974 AmericanSociety for Microbiology Printedin U.S.A.

Newcastle Disease Virus-Specific

RNA: Polyacrylamide Gel

Analysis

of

Single-Stranded

RNA

and

Hybrid Duplexes

NICOLAI V. KAVERIN ANDNATALIA L. VARICH

Ivanovsky Institute of Virology,Academy of MedicalSciences, U.S.S.R.

Received for publication6July 1973

Newcastledisease virus-specific [3H

]uridine-labeled

18SRNAwasresolved by

polyacrylamide gel electrophoresis into several components with molecular weights from 450,000to 840,000.The analysis of35and 24S virus-specificRNA

alsorevealed several componentsineachsedimentationalclass. The conversion

of 18S RNA into double-stranded form by hybridization with an excess of

unlabeled virion RNA improved the resolution in polyacrylamide gels and revealedatleast six distinctcomponents.Thesamesixclasses ofhybrid duplexes wererevealed when 32P-labeled50Svirion RNAwashybridizedwithanexcessof

18S RNA. The applicability of polyacrylamide gel electrophoresis of hybrid duplexestotheanalysis of viral genome structure isdiscussed.

A large part ofvirus-specific RNA formedin

paramyxovirus-infected cells is represented by

molecules ofsubgenomic size (18 to 35S)

com-plementary to virion RNA (1, 2). Messenger

function in the synthesis ofvirus proteins was

tentatively ascribed to the complementary

RNA

by

Kingsbury asearlyas1966 (11). Since

thenanumber of datawerereported confirming

this suggestion. The most

abundant

class of

complementary

RNA,

18SRNA, possesses

sev-eral characteristics which make ita

particularly

good candidate for the role of paramyxoviral

mRNA: a "monocistronic" size

(-

7 x 105

daltons), the presence of

polyadenylic

acid-sequences (19), and

heterogeneity.

The latter

was proposed in order toexplain theability of

18S RNAto convert alargepart of virionRNA

(over 50%, i.e., >3 x 106

daltons)

into

double-stranded form

by hybridization

(2).

Polyacryl-amide gel

electrophoresis

of

paramyxovirus-specific RNA revealed a

heterogeneity

in the

region

corresponding

to 18SRNA, butno

indi-vidual specieswereresolved (12, 13).

In this paper we present a series of

experi-ments on

polyacrylamide gel

analysis

of

virus-specific RNA isolated from Newcastle disease

virus (NDV)-infected cells. Both

single-stranded RNAs andhybrid duplexes were

ana-lyzed. The latter approach was used with a

double purpose: first, toimprovetheresolution

ofRNAspeciesinthe

gel;

second,

as anattempt

to find out whether individual species of 18S

RNA

correspond

to

specific

template regions

in

the genomic RNA. For the first purpose the

hybrids oflabeled 18S RNA with an excessof

virion RNA weresubjected to gel electrophore-sis. For the second purpose the products of

hybridizationoflabeledvirion50S RNAwithan

excess of18S RNAwere analyzed.

MATERIALS AND METHODS

Egg-grown Beaudette strain of NDV (thermostable

clone C) and chicken embryo cell (CEC) monolayer

cultures were used. Theprocedures of infection and

[3H]uridine-labeling of the cells as well as labeling

and purification of the virus, RNA extraction, and

ratezonal centrifugation have been described (8).

RNA-RNA hybridization. The procedure

de-scribed by Kingsbury (11) was used with slight

modifications (8). If the product of hybridization had

tobeanalyzed further, the annealing was performed

ina2-mlvolume. Afterannealing, RNasewasadded

(final concentration 10ug/ml); thesample was

incu-bated at37C for 30 min, treated with Pronase (100

ug/ml,60minat37C) in ordertodestroyRNase(6),

and extracted twice with phenol and precipitated

with 2 volumes of ethanol and one-tenth volume of

16%sodiumacetate.

Polyacrylamide gel electrophoresis. The

proce-duredescribedby Schincarioland Howatson(21)was

used withsomemodifications. Thegels with

acrylam-ide concentrations of2.0% or 2.4%(wt/vol)and0.5%

agarosewereused. Thepolymerizationwasperformed

at37C in tubes10 cmlongwithaninternaldiameter

of 0.8 cm. The gelswereprerun for 1h at6 mAper

tube. Tento 20

usg

ofRNAin 50Mliterswerelayered

over the gels. Theelectrophoresis was performed at

roomtemperaturefor 3 to 5 h at 6 mApertube. The

gels were either stained with methylene blueor cut

with arazor-blade device intoslices.Thelatterwere

dissolved in 0.2mlof 30%water at70C,mixedwith

15ml ofscintillation fluid (2,5-diphenyloxazole,4g;

1,4-bis-[5-phenyloxazoly]benzene, 0.1 g; ethanol,

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300 ml; toluene, 700 ml) and counted in a TriCarb scintillation counter. Ribosomal RNAs of E. coli and

ofCEC wereused as markers.

Reagents. Dactinomycin (Serva, BRD), agarose

(Koch-Light,England),acrylamide (BDH,England),

sodiumdodecylsulphate (SDS) (Matheson, Coleman

andBell,England), Pronase (Calbiochem, Los

Ange-les, Calif.), pancreatic ribonuclease (Calbiochem)

[3H]uridine (Amersham, England), carrier-free

[32P]orthophosphoric acid (Isotope, U.S.S.R.) were

used.

RESULTS

Preliminary sedimentational fractionation

and polyacrylamide

gel

analysis

of

single-stranded

virus-specific

RNA. Thedistribution

ofvirus-specific RNA in an SDS-sucrose density gradient is shown in Fig. 1. Virus-specific RNA is distributed in three broad peaks: 35, 24 and

18SRNA. Genome-size 50S RNAsediments to

the bottom of the tube under these conditions of centrifugation. Sucha pattern is characteristic

forparamyxovirus-specific RNA extractedfrom

infected cells (1, 2). Peakfractions werepooled

as shown in Fig. 1, precipitated with ethanol,

extracted withphenol forcomplete removal of

SDS,

once more

precipitated

with

ethanol,

and

applied to

polyacrylamide gel.

The

polyacryl-amide gel electrophoresisrevealed several

com-ponents in 35 and in 24S RNA (Fig. 2). Our

attention was confined mostly to 18SRNA for reasons discussed aboNe. The

analysis

of 18S RNA in 2% gel revealed a heterogeneity: two

faster moving components formed distinct

peaks; the rest were notresolved and formeda

slower moving zone (Fig. 3A). The analysis in

2.4% gel revealed six components (Fig.

3C).

Component VI in Fig. 3C is

represented by

a

shoulder, but in several experiments it was

revealed as adistinct peak. For the calculation

ofmolecular weights ofthe individual

compo-nents ofvirus-specific 18SRNA, we used

ribo-somal RNAs with known molecularweights as

standard markers. We ran 28 and 18S RNAof

CEC as well as 23 and 16S RNA ofE. coliin

parallel tubesand stainedthem with

methylene

blue. The molecular weights for 28 and 18S

RNA were 1.65 x 106 and 0.67 x

106,

respec-tively (16), whereas for 23 and 16S the values

were 1.1 x 106 and 0.53 x 106 (16). The calculated molecular weights of individual

NDV-specific 18S RNA components are

sum-marized in Table 1.

Hybridization of the labeled virus-specific

18S RNA with an excess ofvirion RNA and

the analysis of hybrids in polyacrylamide

gel. The conversion of single-stranded RNA

into double-stranded form by hybridization

3.0

C~)

x

a-1.5

bottom 5

10

15

20

top

FRACTION

NO

FIG. 1. Fractionation ofvirus-specific NDV RNA

by SDS-sucrose density gradient centrifugation.

NDV-infected CEC were labeled with [3H]uridine

from 7 to10hpostinfection after 2 h of pretreatment

with dactinomycin (2

Ag/ml).

SDS-phenol-extracted

RNA was layered on 50-ml 15 to 30% SDS-sucrose

density gradient and centrifuged inanS W-25.2 rotorof

aSpinco L2ultracentrifuge at 20,000 rpm for 16 h at

25 C. Fractionswerecollected and radioactivity was

determinedinsamples (one-twentiethof the fraction

volume). Chosen fractions werepooled as shown in

the figure (a, b, c) for further analysis. Arrows

indicate theposition of CEC 28 and 18S ribosomal

RNA(28.8 and17.5Svedberg units as determined by

analytical centrifugation in a Spinco E

ultracentri-fuge).

withcomplementarystrands has been shown to

facilitatethe resolution of RNA in

polyacrylam-ide gels (7). Annealing of [3H

]uridine-labeled

NDV-specific 18SRNA with an excess of

unla-beled virion RNA rendered 95 to 100% of the

labelribonuclease resistant. When the product

of hybridization after ribonuclease-Pronase

treatment (see Materials and Methods) was

analyzed in2% gel, six peaks were consistently

resolved (Fig. 4). The components may be

tentatively identified with the peaks of

single-stranded RNA(Fig.3C). If the doubled

molecu-lar weight of the respective single-stranded

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[image:2.493.263.448.63.360.2]

VIRUS-SPECIFIC

N

0

C.L

7t

B

6

C 5

x

t0 4

28

3

2

18S

0

20

30 40 50

60 70 80

10 20 30 40 50 60 70 80

[image:3.493.49.438.62.333.2]

FRACTION NO

FRACTION

NO

FIG. 2. Electrophoresis of 35 and 24SNDV-specific RNA in polyacrylamide gel. RNA from pooled sucrose

gradientfractions (a and bregions, Fig. 1) was precipitated with ethanol and re-extracted with phenol, and a

partofit was analyzed in2%oacrylamide-0.5% agarose gel. A, 35S RNA (region a) electrophoresis during 5h;

B, 24S RNA(region b)electrophoresisduring4h. Arrowsindicate the positions of marker RNA run in parallel

gels.Migration here and in all electrophoregrams is toward the anode on the right.

component is ascribed to each class of the

hybrids, the dependenceoftheir relative

mobil-ity on the log of molecular weightseems to be

fairly linear (Fig. 5). This confirms toacertain

extentthe identification ofhybrid classes with

individual single-stranded components. This

argument, however, is based on anassumption

that the dependenceofrelativemobility onlog molecular weight described forsingle-stranded RNA (14) holds also forhybrid duplexes.

Hybridization of labeled 50S virion RNA

withanexcessofvirus-specific 18SRNAand

the analysis of hybrids in polyacrylamide

gel. Virion RNA ofegg-grown Beaudette strain

of NDV is represented by molecules with a single polarityand does notproduceany

signifi-cant amount of double-stranded structures

when self-annealed (9, 18), differing in this

respect from Sendai virus (18, 20). For this

reason labeled virion NDV RNA may be used

forannealingwithanexcessofcomplementary virus-specific RNAforasubsequent analysisof hybridization product.

Labeled virion50SRNA wasobtained either

from [32P]labeled NDV purified by centrifuga-tion through potassiumtartrate (8, 10) orfrom

the virus partially purified by differential

cen-trifugation. In the latter case thefractionation

of RNA in SDS-sucrose gradient provides a

sufficiently pure preparation of50SRNA (Fig.

6), whereas the lossofvirusduring purification

isnegligible.

The

hybridization

withanexcess ofunlabeled

18S RNA should convert 50 to 60% of virion

RNA into

double-stranded

form (2).To besure

that 18S RNA was really present in excess, a

series ofdilutionsof18S RNApreparation was

used in every experiment. The amount of 18S

RNAinthesample usedforfurtheranalysis, as

shown in Table 2, may be considered

saturat-ing. Theproduct ofhybridization after

ribonu-clease treatment had a sedimentation coeffi-cient closetotheoneexpectedforthe duplexof

18SRNAmolecule (Fig. 7). The analysisofthe

product of annealing inpolyacrylamidegel(Fig.

8) revealedthe samecomponentsasthe analysis

ofhybrids oflabeled 18SRNA (Fig. 4).

Itshould bekeptinmindthatRNAduplexes

inFig.8arerepresentedbyfragments oflabeled

viralgenome RNAhybridizedwith correspond-ing unlabeled virus-specific RNA. Such frag-mentsmay beexpectedtobepresent in

equimo-255

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9

A

8

7

N O6

5

C-,

4

3

2

10 20 30 40 50 60 70 80

FRACTION NO

B

28

S

18S

10 20 30 40 50 60 70 80

FRACTION NO

C

28S 23S

20 i

N~ ~ ~ FATO NO

C-)

10

10 20 30 40 50 60 70

[image:4.493.61.455.60.544.2]

FRACTION NO

FIG. 3. Electrophoresis of NDV-specific 18S RNA inpolyacrylamidegel. A, Total RNA extracted from

un-infected CEC labeled for 3 h with [3H]uridinewassubjectedtoelectrophoresis in 2%o gel for3.25h.B, Virus-specific 18S RNAwasisolatedfromsucrosedensitygradient (Fig.1, region c) andapartof itwassubjectedto

electrophoresis in 2%gel for 3.25 h. C, 18S RNAwasanalyzed in 2.4% gel for4h.

lar amounts. In an attempt to calculate the relations obtained in three experiments are

molarrelations,weascribedthedoubled molec- shown inTable 3. The componentsIII, IV, and

ularweight of the correspondingsingle-stranded V were consistently present in approximately

component to each class ofthe duplexes and equimolar amounts, whereas the molar

rela-dividedthesumofradioactivityineach peak by tions for components I, II, and VI were lower

the respective molecular weight. The molar and morevariable. 12

cN .

0-C.,)

6-I

1*

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[image:5.493.44.234.73.187.2]

TABLE 1. Molecularweights of the componentsof

NDV-specific 18SRNAa

Component Mol wtb No. of

no. (x 10-') determinations

I 446 1

II 521 9.9 4

III 608 7.8 6

IV 696 4.9 6

V 760 7.2 6

VI 840 9.7 6

aThree different preparations of

virus-specific

3H-labeled 18S RNA were used. Peak I either migrated

out ofthegel (if an attempt was made to resolve the

heavier components) or was poorly resolved. One of

the experiments is shown in Fig. 3C. 'Mean ± standard error.

4.

N

O 3

x

a.

C-,

2

10 20 30 40 50 60 70 80

FRACTION NO

FIG. 4. Polyacrylamide gel electrophoresis of

la-beled 18S RNA converted into double-strandedform

by hybridization with an excess of virion RNA.

3H-labeled virus-specific RNA was isolated as

de-scribed in the legend to Fig. 1 and annealed with

unlabeled virion RNA.A 300-agportionoftotal 18S

RNA containing200,000counts/minoflabeled

virus-specificRNAwasmixed with290ugofunlabeled 50S

RNA,annealed,andtreatedasdescribed in Materials

and Methods. The annealingrendered

ribonuclease-resistant91%of3H-18S RNA. Theresulting

prepara-tionwasdissolved in 0.2 mlof electrophoresis buffer.

A0.05-mlportionwasappliedto2%gelandrunfor4

h.

DISCUSSION

The heterogeneity of

paramyxovirus-specific

18S RNA had been shown with the use of

polyacrylamide gel

electrophoresis

for Sendai

virus (12) and for NDV (13),

although

the

number of size classes hadnotbeendetermined.

Rhabdovirus 13S RNA, which is similar to

paramyxoviral 18S RNA in many respects,had

been shown to consist of several (up to eight) components (21, 22). In the experiments pre-sented in this paper, the resolution power of the

gel wasincreased (7) by conversion of 18S RNA

into hybrid duplexes, and the population of18S

RNA was resolved into sixdistinct components (Fig. 4). The range of the sizes of differenit

classes of

RNA,

asdetermined by the analysis of single-stranded 18S RNA preparation (Table 1) roughly corresponds to the expected size of mRNAs for viral proteins (14). The sum of molecular weights of allthe components is -3.4 x 106,

i.e.,

-50 to 60% ofthe

molecular

weight

of virion RNA (4, 5). This value is in good agreement with the size of the part of viral genome RNA which may be converted into

double-stranded formby hybridization withan

excess of18SRNA (2, 8; see alsoTable 2) and

probably serves as a template for 18S RNA

synthesis.

Itshould be takeninmind,however, that the

analysisofsizedistribution of18S RNAcannot

give an answer to the question whether a

particular RNA component has an individual

base sequence,i.e., whether itis atranscript of

3t

6.3

6.2

6.1

6.0

+

5.9

-5.8

S 0

0.4

0.5 0.6 0.7

Rf

FIG. 5. A dependence between relative mobility

and assumed molecular mass of hybrid duplexes.

Relative mobility ofhybrid duplexes was calculated

from theexperimentrepresentedinFig.4.Sixpoints

correspond to the sixpeaksresolved in thegel. The

individual value ofmolecular mass foreach hybrid

component is taken to be equal to the doubled

molecularweightof thecorrespondingsingle-stranded

component(Table 1).

257

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[image:5.493.43.236.258.468.2] [image:5.493.246.434.371.580.2]

2C-0

C-)

iS

l+

Bottom 5 10 15 20 Top

FRACTION NO

FIG. 6. Isolation of 50S RNAfrom partially

puri-fied NDV. RNA was extracted from 32P-NDV

par-tiallypurified by differential centrifugation, layered

on a 39-ml 15 to 30% SDS-sucrose gradient,

cen-trifugedfor15hat 18,000 rpm and25C inanSW27

rotorofaSpinco L 65ultracentrifuge. Radioactivity

wasdetermined insamples,andfractionswerepooled

asshown in thefigure.

TABLE 2. Demonstrationofsaturatingamountof

NDV-specific 18S RNAatannealingwith32P-labeled

virion50S RNAfor furtheranalysisof hybridization

producta

18S RNAfromNo.RNase RNase

Sample NDV-infected (counts treat- Annealed

no. cellsa g

mmin)

ment

sample)'

1 70 640C 410 64

2 53 636 424 66.5

3 35 610 381 62.5

4 1.3 716 37 5.2

5 0 690 23 3.3

a32P-labeled 505virion RNA (0.1Ag, 1,300counts

per min per sample) was annealed with different

amountsof 185 RNA.Inthereactionmixture usedfor

furtheranalysis (Fig. 7, 8), theexcess of18 over505

RNAwasthesame asinsample 1 inthetable.

ITotalamountofRNAinthesample, represented

mostly by cellular RNAfrom the188 zone.

cMean oftwoparallelsamples.

an individual gene, a fragment of a larger

transcript, or a transcript of two

neighboring

individual genes. The latter possibility cannot

be

disregarded

as it had been

reported

that

nucleotidesequences identical to 18S RNAare

present in35S RNA (2).

To study the regions of 50S virion RNA

serving astemplates forvirus-specific 18S RNA,

we attempted to cutthe labeled 50SRNA into

fragments complementary to individual size

classes of 18S RNA. The possibility of this

approachwas notevidentapriori: it wasbased

on a presumption of the existence of "spacer"

sequences among the template regions in 50S RNA. Such spacers would remain

single-stranded and ribonuclease-sensitive after

an-nealing with an excess of 18S RNA. If such

spacers were absent, the annealing would

pro-duce a population of hybrids containing a 50S

RNA molecule as one strand and several 18S

RNA molecules stuck end-to-end as the other

strand. In this caseribonuclease might be not

quite efficient in "cutting" such structures

between twoadjacent 18S RNA molecules.

The result shown inFig. 8indicates that the

"cutting" is performed at appropriate sites,

because thepopulation oftheduplexes (Fig. 8)

issimilarinthe number and size of the

compo-nents totheoneobtained afterhybridization of

6

28 S

18S

4S

C-,x

X

bottom 10 20 30 40 50 top

FRACTION NO

FIG. 7. Sucrose density centrifugation of hybrid

duplexes obtained by annealing of 32P-virion RNA

with an excess of18S RNA. 32P_virion50S RNA (3

,og,27,000counts/min)isolatedasshown inFig.6was

annealed with 2 mg of unlabeled 18S RNA from

infected cells. These conditions were shown to be saturating (Table 1). The product of hybridization

wastreated asdescribed in the text and divided into

two equal parts. One part was analyzed in sucrose

density gradient. The speed, 23,000 rpm, and the

otherconditions of centrifugation were as described in

thelegend toFig. 1. Theotherpart was analyzed in

polyacrylamide gel (Fig. 8).

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20

X 16

C.,

10 20 30 40 50 60 70 8o

FRACTION NO

FIG. 8. Polyacrylamide gel analysis of hybrid

du-plexes obtained by annealing of 32P-virion RNA with

an excess of 18S RNA. The product of the

hybridiza-tion experiment described in Fig. 7was analyzed in

polyacrylamidegel.Electrophoresis in2%ogel for4.25

h.

labeled 18S RNA withanexcessof virionRNA (Fig. 4).

Itshould be noted that if eachcomponent of

18S RNA isa transcript ofan individual tem-plate region, the amount of label in hybrid peaks (Fig. 8) should be distributed in equimo-lar ratios. As one can see from Table 3, this is

notgenerally thecase: only thecomponentsIII,

IV, and V are present in equimolar amounts, whereas there is a deficit of the label in the otherpeaks. The values of molar ratios for the components I, II, and VI were variable, but always less than 1.0, especially for the

compo-nentVI. Thesumofradioactivity in peaks I, II,

and VIslightly exceeds the amount needed for

the component VI to be present in equimolar

relation with components III, IV, and V. One

cannot exclude the following possibility: the

component VI is a composite of components I

and II, i.e., an mRNA-transcript oftwo neigh-boringgenes.Suchasituationcould introducea

biasinto the equimolardistribution of the label

in the population of hybrid duplexes. These considerations indicatethat, although the

popu-lation of 18S RNA isrepresented byatleast six

components (Fig. 3, 4), one cannot conclude

with certainty whether theyarethetranscripts ofsix or five template regions of the genomic

RNA.

Thedatapresentedin thispaperindicate the

applicability ofpolyacrylamide gel electropho-resis ofhybrid duplexes for the analysis of the

viralgenomeswhicharenotfragmentedinsitu.

Further elaboration of the method and its

[image:7.493.45.228.59.235.2]

application to DNA-containing viruses might facilitate the study of viral genome structure and function.

TABLE 3. Molar relations of 32P-hybrid duplexesa

Componentno.

Exptno.

I II III IV V VI

1 0.85 0.85 1.07 0.95 1.0 0.22

2 0.52 0.67 1.09 1.03 1.0 0.74

3 0.69 0.79 0.93 1.03 1.0 0.43

aThe sum ofradioactivityineachpeak was divided

bythe assumedmolecularweightof thecorresponding

component. The value for component V is taken as

1.0.

b The conditions of annealing and the reaction

mixture weresimilar to those described in the legend

toFig. 7. The fractionationprocedure in the gelwas as

inFig. 8.

After this paper had been submitted for

publication, we became acquainted with a

re-cent work on this subject (3) where

virus-specific 18S RNA is resolved into several species

inpolyacrylamide gel. Our results(Fig.3C) are

in good agreement with the data published by

Collins andBratt.

ACKNOWLEDGMENTS

We wishtothankG. Petrovskyforthe analytical determi-nation ofsedimentation coefficient of ribosomal RNA and T. Logutenkovaforexcellent technical assistance.

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