Isolation of a transcriptive complex from Newcastle disease virions.

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Isolation

of a

Transcriptive

Complex

from

Newcastle Disease

Virions

RICHARD J. COLONNO' AND HENRY 0. STONE*

DepartmentofMicrobiology, University of Kansas, Lawrence, Kansas66045

Received for publication 13August 1975

An activetranscriptive complex was isolatedfrompurifiedvirions of Newcas-tledisease virus. Afterdisruption withTritonX-100andhigh salt, soluble and

particulate fractions were separated by density gradient centrifugation. The transcriptive complex, recovered at a density of 1.275 g/cm:, appeared as a

nucleocapsid structure by electron microscopy. When analyzed by polyacryl-amide gel electrophoresis, the nucleocapsids consistedofthe nucleocapsid pro-tein, a minor protein of 53,000 molecular weight, and the large L protein.

Nucleocapsids possessed less than 1% ofthehemagglutinating and neuramini-dase activities originally associated with virions. The active complex synthe-sizedpredominantly11to20S RNAinvitroandapproximatelyone-fourthofthe

RNA molecules contained polyadenylic acid segments. In the presence of

S-adenosyl-L-methionine,

the RNAmolecules werecapped andmethylatedatthe 5' termini. The transcriptive complex was also capable ofmethylating exoge-nousEscherichia coli RNAinthe absence ofviral RNAsynthesis.

Newcastle disease virus (NDV) is a large, enveloped RNA virus (16) that contains a

sin-gle-stranded 50S genome (8, 16, 19) encapsi-dated bynucleocapsid protein (3, 13, 15, 21, 27, 31),designated NP protein (30). The virion

con-tains two other major proteins, a membrane protein (M) and aglycoprotein (HN), shownto

contain both neuraminidase and hemaggluti-nating activities (29). Several minor proteins havealso been detected (3, 13, 15, 27, 28).

Purifiedvirions contain a transcriptase that

transcribes the50Sgenome RNA into 18Sviral

RNA, which is complementary in base

se-quence to the genome and contains

polyade-nylic acid lpoly(A)] (18, 33). Marshall and

Gil-lespie (23) have shown that NDV genomes do

not contain stretches of polyuridylic acid

Ipoly(U)] to code for the poly(A),

suggesting

a post-transcriptional addition of poly(A) by a virion-associated enzyme.

In addition to poly(A) synthesis, we have

recently demonstrated that purified virions of NDV are capable ofcapping and methylating

the 5' terminus ofviral mRNAsynthesized in

vitro (6; R.Colonno andH. Stone, Nature [Lon-don], in press), indicating the presence of a

methyl transferase and a capping enzyme.

Thus the virion may contain as many as four

enzymes that arerelated to RNAsynthesis. Scheid and Choppin (30) have been able to

solubilize the M and the HNprotein free from

Present address: Department of Cell Biology, Roche InstituteofMolecularBiology,Nutley, NJ 07110.

the virion

by disrupting

purified

NDV with

Triton X-100

detergent

and

high

salt.

Meager

and Burke (26)

disrupted

NDV with Triton

N101

alone,

but when viewed

by

electron mi-croscopy the subviral

particles produced

ap-peared

tobe

spikeless

virions. Intheabsenceof

high

salt,

the

detergent

released

only

the HN

glycoprotein

from the virion.

Nucleocapsids

prepared

by

this and other

techniques

did not

retainRNA

polymerase

activity

(26).

Using

the

procedure

ofScheid and

Choppin (30),

Marxet

al. (24)isolateda

nucleocapsid

structure

(tran-scriptive

complex)

from Sendai virus that

re-tained the

ability

to

synthesize

RNAinvitro. We report in this communication that an

active

transcriptive complex

can also be iso-lated from NDV

by

Triton X-100and

high salt,

which

synthesizes

complementary RNA and

also retains the

ability

to

polyadenylate,

cap,

and

methylate

viral RNAin vitro.

MATERIALS AND METHODS

Virus purification. NDV wasgrown in

embryo-nated hen eggs (14) andpurified as previously

de-scribed (6). Virus wasthen resuspended in 0.01 M

Tris (pH 7.3)-0.03 M NaCl and stored at -70°C. Protein concentrations were determined according totheprocedureofLowryetal.(22).Purified virions were labeled in vitro using ['4C]formaldehyde and

sodium borohydride asdescribed byMcMillen and

Consigli (25).

Isolation of transcriptive complex. Virus at a

finalproteinconcentration of 1mg/mlwasdisrupted in 5ml of1% (vol/vol) Triton X-100 (NewEngland 1080

on November 10, 2019 by guest

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Nuclear Corp.)-0.75 M KCl-0.01 M Tris-hydrochlo-ride (pH 7.3) for 20 minat20°C. The nucleocapsid

structure (transcriptive complex) waspurified free

ofsolubilizedproteinsbyisopycnicbandingina7-ml

linear gradientofD20glycerol (1.17 g/cm3)-D20

su-crose (1.37 g/cm3)buffered with 0.01 M

Tris-hydro-chloride (pH 7.3)-0.03 M NaCl. The gradientswere

centrifuged inaBeckmanSW41rotor(8°C, 90 min, 37,000rpm). Fractionationwasperformed either by an ISCO gradient fractionator (Instrumental

Spe-cialities Co.) ormanually. In the latter case,

pro-teinsthatwere solubilized by Triton and high salt wereobtained byremovingthetop5mlof the

gra-dient. The transcriptive complex, visible as a very

sharpband,waseasilyrecovered ina0.3-to0.4-ml volume with a 1-ml syringe containinga 20-gauge

needle. Solubilizedproteinswerelaterisolated free

ofthe detergent solution by butanol ether precipita-tion asdescribed byScheid and Choppin (30).

Electron microscopy. Virionsorthe transcriptive

complexataprotein concentrationof 0.1to0.5mg/ ml were appliedto a Parlodion-carbon coated grid

for 1 min andnegatively stained with 2% (wt/vol) uranyl acetatefor 30s as described by Home (17).

The negatively stained preparationswereexamined

inaPhillips 300 electron microscope.

Slab gel electrophoresis. A 10% (wt/vol) poly-acrylamide gel containing 0.13%(wt/vol) N,N'-bis-methylene acrylamide, 0.4 M Tris-hydrochloride (pH 8.7),and1%(wt/vol) SDSwaspolymerized with

0.03% (wt/vol) ammonium persulfate-0.03% (vol/ vol)tetramethylethylenediamine, and usedtopour aseparating gel (0.15 by10by16cm).Astacking gel composed of 5% (wt/vol)polyacrylamide,0.26% (wt/ vol) N,N-bis-methylene acrylamide, 0.06 M Tris-hydrochloride (pH 6.8), and 1% (wt/vol) SDS was

prepared, polymerized in the same manner, and

usedtoform 0.5-cmstacking gel.

Gelelectrophoresis wascarriedoutusing aslab gel box (Hoffer model SE 500) anda powersupply

(Buchlermodel3-1014A). Running buffer contained 0.05MTris-hydrochloride (pH 8.3), 0.38M glycine, and0.1%(wt/vol) SDS.Proteinsamples, containing

10to25,g of proteinper5to25 pl,2%(vol/vol)

f-mercaptoethanol, 1% (wt/vol) SDS, and 20% (vol/ vol)glycerol,wereplaced inaboilingwaterbathfor 1minbeforeloadingontheslabgel. Sampleswere

subjected to electrophoresis at 15 mA through the 5% stacking gel and at 30 mA through the 10%

separating gel until the bromophenol bluemarker

reached the bottom of the gel (about4h). The gels

werethenstained for 3 h with0.1%Coomassie

bril-liant blue-7% (vol/vol) acetic acid-25% (vol/vol) methanol and destained for16h withseveral

wash-ingsof 14%(vol/vol)aceticacid-50%(vol/vol) metha-nolbeforedrying.

RNA synthesis and purification. Standard

tran-scriptaseassays (0.1 ml) contained50 mM Tris-hy-drochloride (pH7.3), 120 mMNaCl,0.4mMMnCl,,

0.015% (vol/vol) Triton N101, 3 mM dithiothreitol,

0.7mMCTP,0.7 mMGTP,0.7mMUTP(reducedto 0.09 mM when [3H]UTP [21 Ci/mM] was used as

label), 0.7 mM ATP (reduced to 0.9 mM when [a-32P]ATP [15.3 Ci/mM] was used as label), and 1.5

ACi of[3H]UTPor 1 ,Ci of [a-32P]ATP. When the

transcriptive complex was assayed, the NaCl was reducedto 30 mM and Triton N101waseliminated. Methylation assay conditions were identical except that the 50 mMTris-hydrochloride (pH 7 .v-asused in place ofTris-hydrochloride (pH7.2') X - al pH in

each case was the same), and 0.78 A ,Ci) S-adenosyl-L-[methyl-3H]methionine (1 -C. Ci/mmol) was added. Allreaction mixtures contained 25 to 45

,Agofprotein (purified virions ortranscriptive com-plex) and wereincubated at 32°C for 6 h. Labeled product RNA was phenol-SDS extracted and puri-fied as previously described (6).

Analysis of in vitroproduct RNA. Procedures for rate zonal centrifugation, RNase digestions, near-est-neighbor analysis, DEAE chromatography, poly(U)-cellulose chromatography, and hybridiza-tions have been described previously (6, 7).

Hemagglutination titration. Tritrations, using 2.5 ,ug of protein (intact virions,solubilized protein, ortranscriptive complex), were performed at4°Cfor 2 h asdescribed by Choppin (5).

Neuraminidase assay. Samples containing 2.5 ,ug of protein (solubilized protein, intact virions, or transcriptive complex) were assayed, using neura-minlactose (Sigma) as a substrate, for 50min at 37°C asdescribed by Scheid and Choppin (30) and Ami-noff (2). Oneneuraminidaseunit was defined as the amount ofenzyme that releases 1 nmol of N-acetyl-neuraminic acid in 1 min at 37°C.

RESULTS

Isolation of transcriptive complex.

Experi-ments were performed to identify the virion

proteins and the structural components of the virionthatare involved in RNA synthesis. To

provide a sensitive and convenient assay for

viral protein,purified NDVwaslabeledin vitro

with

['4C]formaldehyde

(25). Purified virions

werethendisrupted with Triton and high salt, and the solubilized and particulate fractions

wereseparated by centrifugationon aD.O glyc-erol-sucrose gradient (Fig. 1). After fractiona-tion,all of the '4Cradioactivelabelwaslocated either in the solubilized fraction at the top of the gradient or ina sharpband having a den-sity of1.275 g/cm:1. To determineifthe

virion-associated transcriptase resided in either of

these peaks, portions of each gradient fraction

were assayedfor [H]UITPincorporation in an invitroRNA polymeraseassay.Asseen inFig.

1, all of therecovered polymerase activity was

associated with the structure havingadensity of1.275g/cm3,which will bereferred toasthe transcriptive complex. The specific activity of

thetranscriptivecomplexvaried between prep-arations, but always exhibited a 2- to 20-fold

increase in specific activity as compared with virions(datanotshown).

Since the transcriptive complex appeared to have the same densityaspurified nucleocapsid

(24, 29, 33), it was of interest to confirm this

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a-1

9

U z w

x

F-0 :

a-z

10LA

z

I m

(0

I C

K

u 50

i

xA-2 4 6 8 10 12 14 16 18 20 22 FRACTON NUMBER

FIG. 1. Isolation ofa transcriptivecomplex fromNDV.Purifiedvirions,disruptedwith1%(vollvol)Triton X-100and 0.75 MKC1, wereisopycnically banded in aD/Oglycerol-sucrose gradient. After fractionation, samples (20pi)of eachfraction (0.4 ml) were assayed for [3H]UTP incorporation (*) in an RNA polymerase assay. Aftertrichloroacetic acid precipitation, the samples were counted under double label conditions to simultaneously determine the amountof RNAsynthesizedand the amountof14C-labeled proteins (0) in the sample.

observation using electron microscopy.

Elec-tron micrographsofintact virions (Fig. 2A, B)

andisolated transcriptive complex (Fig. 2C, D) indicated that theactivetranscriptive complex

was indeed a nucleocapsid structure.

Treat-mentwithTriton-high salt completelyremoved theoutermembrane of the virion, and the

nu-cleocapsid was no longer confined within a tightlypacked corestructure.

Polypeptides in the transcriptive complex. Virions, transcriptive complex,and solubilized

proteinswerenextanalyzedby gel electropho-resis (Fig. 3). Slab gel electrophoresis was

foundtogive better resolution of proteins than

standard disc gels, revealingasmany aseight bandsinintact virions.Besidesthe threemajor proteins, HN, NP, and M, several minor pro-teinswerefoundincludingthelarge L protein.

After Triton-high salt disruption, most ofthe

HNandMproteins,alongwithpartofthelarge

L protein and a minor protein having an

ap-proximate molecular weight of 50,000, were

found in the solubilized fraction. The isolated

transcriptive complex, which retained the abilty to synthesize RNA, contained

predomi-nantlythe NP, a minor protein having an

ap-proximate molecular weight of 53,000, and a

reduced amount of the large L protein. Trace

amounts of HN andM proteinwere also pres-ent.

Enzymatic activities of solubilized

pro-teins. Bothneuraminidaseand

hemagglutinat-ing activitieshave been showntobeassociated

with theglycoprotein HN (30).Todemonstrate that the transcriptive complexwas free of the HNprotein,solubilizedproteinsand transcrip-tivecomplexwereextracted withbutanol-ether

andresuspendedatidenticalprotein

concentra-tions.Equalamountsofeach,along with intact

virions, were assayed for both neuraminidase and hemagglutinating activities. Results (Ta-ble1)indicate that thesolubilized fraction

con-tained96Uofneuraminidaseper mgofprotein

compared with 0.1 for the transcriptive

com-plex, and1.6 x 106hemagglutinatingunitsper mg ofprotein compared with 2.4 x 103 forthe

isolated complex. Thus,treatmentwith Triton-high salt left the transcriptive complex

vir-tually devoidof any enzymatic activity

associ-ated with the HNprotein.

Size of RNA product synthesized by

tran-scriptive complex. RNAwassynthesizedinthe presence of [a-32P]ATP to determine the

sedi-mentation properties of RNA synthesized in

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FIG. 2. Electronmicrographs of (A,B) NDV virions and(C, D)thetranscriptivecomplex.Allspecimens

werenegatively stained with uranyl acetate. (A, x42,000; B, x132,000; C, x73,000; D, x127,000)

vitroby the transcriptivecomplex. The result-ingproduct RNAwaspurified byphenol

extrac-tionand Sephadex chromatography before

sedi-mentation on a 15 to 30% (wt/wt) sucrose

gra-dient (Fig. 4). Results indicate that the RNA synthesized by the transcriptive complexis

het-erogenous insize, rangingfrom2to28S,with a majority ofthe RNA sedimenting in the 11 to 20S region of sucrose gradients. The smaller

peakofRNAsedimenting at6S may be due to

fragmentation of the nucleocapsid structure

(Fig. 2C and D), resultinginapretermination of nascent RNA chains duringRNA synthesis.

Thus, the majority of RNA synthesized by the transcriptive complex is slightly smaller than

the predominant 188 mRNA species synthe-sized by purified virions (6) or isolated from NDV-infected cells (18).

Presence of poly(A) on viral mRNA. Since virions of NDV synthesize complementary

RNA-containing poly(A) segments (33), we nextdetermined whether theabilityto

synthe-size poly(A) was retained in the transcriptive complex. RNA wassynthesizedinvitroby viri-ons and by the transcriptive complex in the presence of either [3H]UTP or [a-32P]ATP.

After purification, the labeled product RNA

wasassayedfor the presence ofpoly(A) by enzy-maticdigestionwithRNaseA + T1, using 14C-labeled synthetic poly(A) and 28S ribosomal

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WHOLE

SOLUBLE

VIRON

PROTEINS

ACTIVE COMPLEX

FIG. 3. Polyacrylamide slabgelelectrophoresisofNDV polypeptides. Virionsweredisruptedwith

Triton-high salt, andthe resultingtranscriptive complex was isolatedby isopycnic banding.Solubilizedproteins wereisolated free ofTritondetergent bybutanol-etherprecipitation beforegelelectrophoresis.Protein samples

(10to25ugin5to20pi)wereheatdenaturedin1%(wtlvol)SDS,2%(vollvol),3-mercaptoethanol, and20%

(vollvol) glycerol,and werethensubjectedtoelectrophoresis on a10%SDS-polyacrylamideslab gel.

TABLE 1. Assay forneuraminidase and hemagglutinating activities

Samplea Neuraminidase

(UlXa10t2]/mg

(U/mgpotein) protein)

Intact virions 52 324

Solubilized pro- 96 16,000

teins

Transcriptive <0.1 24

complex

" Proteinsamples contained 2.5ggof protein in25

,A of 10 mM Tris-hydrochloride (pH 7.3)-30 mM NaCl.

RNA as controls. Results (Table 2) show that the AMP-labeled product RNA of both intact

virions and the transcriptive complex contain

8% and 11% RNase resistance, respectively, compared with the 100% RNase resistance of

synthetic poly(A). Both UMP-labeled product

RNAand28SribosomalRNA showedlessthan

1% RNase resistance. All the RNA

samples

were found to be totally sensitive to digestion withRNaseT2, indicatingthat eachradioactive label residedin RNA.

A further indication of the presence of poly(A)onviral RNAtranscriptswasobserved upon passage of the labeled RNA through a

poly(U)-cellulose column (Table 2). Controls showedthat 100% of the synthetic poly(A)and 0.5% of the 28S ribosomal RNAwereretained by the poly(U)-cellulose column. When [32P]AMP-labeled product RNAwasdenatured

andchromatographed, 17% of the RNA synthe-sized by virions and 24% of the RNA

synthe-sized by the transcriptive complex were

re-tained by the column. Proofthat the poly(U)

column was separating moleculeson thebasis

of the presence of poly(A) was obtained after

digestion of bound and unbound RNA with

RNase A + T,. More than 96% of the RNase

resistance resided in RNA that bound to the

NP-53K

50K

J. VIROL.

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column (data not shown). These results indi-catethat thetranscriptive complex retains the ability to synthesize viral RNA-containing poly(A).

Size of in vitro poly(A). The mechanisms involved in controlling the size of viral poly(A) segmentshave yetto be determined. Itwas of

interest to determine what effect, ifany,

Tri-ton-high salt disruption haduponthese

control-ling mechanisms. Product RNA labeled with [32P]AMP was digested with RNase A + T1,

chromatographed free ofmononucleotides, and

18S 28S

6

0

x

E4

C.

Ca4

0 10 20 30

FRACTION NUMBER

FIG. 4. Size determination ofRNAsynthesized by transcriptive complex. RNA synthesized in vitro in thepresenceof [a-32P]ATP by the isolated

transcrip-tive complex wasphenol-SDS extracted,

chromato-graphed on a Sephadex G-50 column, and

sedi-mentedon a15to30%linearsucrosegradient. After

fractionation, each gradient fraction (0.4 ml) was

assayed for[32P]AMP-labeled RNA (0) using PCS (AmershamlSearle). Thepositionsofunlabeled18S

and28Smarkersareindicated byarrows.

sedimentedonsucrosegradients. Unlabeled

N-formylmethionyl tRNA from Escherichia coli

andlabeled chickenembryofibroblasts4SRNA

were sedimented in a parallel gradient. After

fractionation, each fractionwasassayedfor

ra-dioactivity (Fig. 5). Nearly identical poly(A)

segmentsweresynthesized by both virions and

the transcriptive complex. The poly(A) seg-mentswereveryheterogeneous, ranging in size

from 4to 11S,with amajorpeakof

radioactiv-ity sedimenting at 4.4S. No poly(A) segments smaller than 4S weredetected.

Nearest-neighbor analysis. As afinal proof

ofthe presence ofpoly(A), the nearest neigh-bors of the[32P]AMP residuesweredetermined.

The RNase-resistant RNA synthesized by the transcriptive complexwaspooled from the4 to

11S regions ofsucrose gradients (Fig. 5) and

hydrolyzed with KOH. Results (Table 3) show

that 95.5% of the RNase-resistant RNA

con-tainsAMPas anearestneighbor, proving that it is indeed poly(A). Approximately 2% ofthe

radioactivity migrated asGMP, perhaps

repre-sentingthe baseadjacenttopoly(A).

Assay for methyl transferase activity. We previously (6)demonstratedthatpurified NDV contains anenzyme that transfers the methyl group from S-adenosyl-L-methionine to viral RNAinvitro. To assayfor methyl transferase

activity,theisolatedtranscriptivecomplexwas

incubated in an in vitro transcriptase reaction

containing S-adenosyl-L-[methyl-3H]methio-nine and examined for 3H incorporation into viral RNA. Results (Table4) indicate that the transcriptive complex retained the ability to

methylate the RNA synthesized in vitro.

Addi-tionofS-adenosyl-L-homocysteine inhibited 3H incorporation by 89%, indicating that only the methyl group of

S-adenosyl-L-[methyl-3H]methionine was incorporated into viral

TABLE 2. RNasedigestionandpoly(U)-cellulosechromatography ofin vitroproductRNA

RNA sample % Resistance

%

Bound to

poly

(U)-cel-RNaseA+T,a RNaseT2b lulos

Controls

'4C-labeled poly(A) 100 0.5 100

'4C-labeledchickenembryofibroblast 0 0.8 0.5

2&S rRNA Virion

[32P]AMP productRNA 8.0 0.1 17.3

Transcriptive complex

[32P]AMP productRNA 11.3 0.1 24.5

[3H]UMPproduct RNA 0.7 0.3

aRNAsampleswereincubated with50,ug of RNase A permland2,ug of RNaseT,per ml for30minat

37°C.

bRNAsamplesweredigestedwith 20 UofRNaseT2perml for 5 hat

37aC.

' More than97%of thelabeled RNAloaded onthecolumnwasrecovered.

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1086 COLONNO AND STONE

1-1

3

x

1

0\j

b 2

(-A

3

x

0

10 20

FRACTION NUMBER

FIG. 5. Sizedeterminationofin vitropoly(A). RNA synthesized invitroin thepresenceof[a-32P]ATP by

virionsorby the isolated transcriptive complexwaspurified and treated withacombinationofRNase A and

T,. The RNase digest was phenol-SDS extracted, chromatographed on a Sephadex G-50 column, and

sedimentedon a15to30%linearsucrosegradient. Samples (100pi1) ofeachgradient fraction (0.4 ml)were

assayed for [:'2P]AMP-labeled RNase-resistant RNA synthesized byvirions(O)orthetranscriptivecomplex

(0).As4S markers, unlabeledN-formylmethionyl tRNA from E.coli(arrow)and 14C-labeled 4StRNA from

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chickenembryo fibroblasts (---)weresedimentedinaparallel gradient.

TABLE 3. Nearest-neighbor analysisof Poly(A) synthesizedinvitroby NDV transcriptivecomplex

Migration cpm recovereda %Totalcpm

re-covered

AMP 674 95.9

CMP 6 0.8

GMP 16 2.3

UMP 7 0.9

"In each experiment,morethan93% of the radio-activity loaded on the thin-layer plate was

re-covered.

RNA. Todetermine if the methyl transferase

wasabletomethylate other RNAtemplatesin

the absenceofviralRNAsynthesis,the ribonu-cleosidetriphosphateswerereplacedbyvarious RNA templates. Results (Table 4) show that

there was little or no 3H incorporation with RNA templates such as poly(A), poly(G), and yeast carrier RNA, but incorporation was

ob-served usingstripped tRNA fromE. coli W. Location of methyl groups in RNA

prod-ucts. The methyl-1H-labeled RNAs were next examined for the presence ofmethyl label ina

TABLE 4. Assayfor methyl transferase activityin

transcriptivecomplex

cpm[3H]SAM

Additionw incorporated/mg

protein

None 250

0.7mMATP,CTP, GTP, UTP 13,450

0.7 mMATP, CTP, GTP, UTP+ 1 1,550

mMSAM

0.1mgPoly(A) (MILES) 350

0.1mgPoly(G) (MILES) 950

0.04 mg Yeast carrier RNA 1,400

(MILES)

0.1mgStripped tRNA fromE.coli 1]8,650

W (GIBCO)

't Beforeadditions, reaction mixtures contained 50

mM Tris-hydrochloride (pH 7.8), 30 mM NaCl, 0.4

mM MnCl2, 3 mM dithiothreitol, and 0.78 ,uM

[3H]SAM (S-adenosyl-L-[methyl-3H]methionine).

5'-terminal "cap" structure. Methyl-labeled RNA was phenol extracted, hydrolyzed with

KOH, andchromatographedon a DEAE-cellu-lose column. Results demonstrated that all of themethyl-3Hlabel in RNAsynthesized by

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15

10

5

15

CY

20 40 60 duO

FRACTON &JM8ER

FIG. 6. DEAE-cellulosechromatography of kaline digest of RNA synthesized in vitro. RP beledinvitrointhepresenceofS-adenosyl-L-[m

3H]methionine by either virions orthetransci

complex was hydrolyzed with KOH and chrc

graphedon aDEAE-cellulose column (0.6by1

aspreviously described (6). The elutionpattern

oligonucleotide markers resulting from RNase tionof wheatgermrRNA isindicated by thea andthesodium chloride concentration(---) w

tained fromastandardcurve.Column fraction

assayed for radioactivity (0)aspreviously desi

(6). (A)Methyl-3H-labeled viral RNA synthesi,

virions; (B) methyl-3H-labeled viral RNA s*

sized by the transcriptive complex; (C) meth labeled RNA from E. coli methylated by the scriptivecomplex.

ons (Fig. 6A), and the transcriptive con

(Fig. 6B) elutedas asingle peak havinga

charge, and has been previously identifi

7MeGpppGp (Colonno and Stone, in p

DEAE chromatography of the methyl-3H-la-beledtRNA (Fig. 6C), methylated by the

tran-scriptive complex, demonstrated that the

methyl label was not incorporated into a

5'-terminal structure, but instead eluted as an

internal methylated base (-2 charge), which

02 lateridentifiedas 7MeG (datanotshown).

Hybridization of in vitro-labeled RNA to

50S genomes. To demonstrate that the RNA

QI productssynthesized byvirionsandby the

iso-latedtranscriptivecomplexwere faithful

tran-scripts of the genome, in vitro RNA products were hybridized to purified NDV 50S genome

RNA. These hybridization experiments (Table

5) show that 97% of the radioactive label was

x incorporated intovirus-specificRNAexceptfor

methyl-3H-labeledtRNA. In thecaseof

methyl-Q2}3H-labeled

tRNA,

more than

90%

of the zH

label was not incorporated into virus-specific

RNA, proving that the virion-associated

methyltransferasewasabletomethylate

exog-01 enousRNA.

DISCUSSION

Purifiedvirionsof NDV contain atleastfour

enzymatic activities relatedto RNA synthesis:

RNA transcriptase (18), methyltransferase (6),

capping (Colonno and Stone, in press), and

02 poly(A) polymerizing activities (33). To date,

the specific polypeptides involved in each of these enzymatic activities are not known. The ol membrane proteins HN and M, along with the

lipidenvelope of thevirion, canberemovedby

disrupting purified virions with Triton X-100 detergentand high salt(30).Afterdisruption,a

nucleocapsid structure remains (Fig. 2) which retainstheabilitytosynthesize complementary

RNA in vitro(Table5)and is therefore calleda anal- transcriptive complex. In addition to genome

VA la- RNA,theisolatedcomplexwascomposedofthe

wethyl- nucleocapsid protein N, a minor protein with

rmpato

an

approximate

molecular

weight

of

53,000,

t7cm)

Iofthe TABLE 5. Hybridization of invitrolabeled RNA to

diges- 50S Genome RNA

irrows

was

ob-swere

,cribed 'zedby ynthe- lyl-3H-

tran-nplex ,-4.5 ed as

ress).

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1088 COLONNO AND STONE

andthe large 150,000-molecular-weight L pro-tein, with traces of HN and M proteins (Fig. 3).

Investigations by Bishop and Roy (4) with vesicular stomatitis virus (VSV), a

rhabdovi-rus, havealsoshown that neither the G nor M proteins are required for RNA transcription.

Emerson and Wagner (10, 11)demonstratedan L protein requirement for RNA synthesis by VSV, and Emerson and Yu (12)have recently shown a requirement for the NS protein.

Per-haps the L, N, and 53,000-molecular-weight

proteinsof NDVareanalogoustotheL,N,and NSproteinsof VSV.

The transcriptive complex isolated from

NDV synthesized an RNA in vitro that was

heterogenous andslightly smallerinsize (Fig. 4)thanthat synthesized by purifiedvirions (6).

This may be due to the isolation procedures,

sincethe transcriptivecomplex didnot appear as an intact nucleocapsid structure (Fig. 2). However, the isolated transcriptive complex didretaintheabilitytosynthesizepoly(A) seg-ments on viral RNA in vitro (Tables 2 and 3; Fig. 5). Attempts to synthesize poly(A) using

several knownprimers inthe absence ofRNA

synthesis have been unsuccessful. Thisresult wouldseem to indicate that the RNA transcip-taseand poly(A) polymerase activities are

cou-pled. Removal of the outer membrane of NDV

had no apparent effect on the size of poly(A)

segments attachedtomRNA (Fig. 5), suggest-ing that the secondary structure of

nucleocap-sids doesnotplayarole indeterminingthesize

ofpoly(A) segments.

Wehave recently identified the 5' terminus

of NDVviral RNA synthesized invitro in the presence ofamethyl donoras 7MeGpppGpPyp

... (Colonno and Stone,inpress).Similar

stud-ies using the isolated transcriptive complex demonstrated that the methyl transferase and

capping activities (Table 4, Fig. 5) are also retained. Both the virions and thetranscriptive complex were able to synthesize methylated viralRNA in vitro, whichcontains a

5'-termi-nal structure, 7MeGpppGp (data not shown).

Unlike the poly(A) polymerase, the methyl

transferase may be a separate enzyme activity

fromthe RNAtranscriptase. Thisis supported

by the findingthatthevirion-associatedmethyl

transferase can methylate exogenous RNA such as E. coli tRNA. Analysisofthe

methyl-labeled E. coli RNA showed that it was not virus specific (Table 5) and that the methyl

label did not reside in a capped 5'-terminal

structure. Instead, the methyl label appeared

to exist as a single base methylation (Fig. 6), which has been further identified as 7MeG

(datanotshown).

Similar experiments by Abraham and Baner-jee (1) have demonstrated that nucleocapsid structuresisolated from VSVby high salt and

Triton disruption alsoretainthe abilityto syn-thesize viral mRNA that is capped, methyl-ated, and contains poly(A) segments identical

tothosesynthesizedbywhole virions. Analysis

of the active nucleocapsid structure by poly-acrylamide gels demonstrated the presence of L, N, and NS with a traceofM protein. Thus, VSV, arhabdovirus, andNDV, a paramyxovi-rus, mayutilizesimilarenzymaticmechanisms totranscribe andmodifyviral mRNA.

ACKNOWLEDGMENTS

Wewish to express our gratitude to David Wright and Jeanette Robbins who provided skilled technical assistance. Theexcellenttechnical assistance of Lorraine Hammer and the use ofMcCollumLaboratory's electron microscope facil-ity is gratefully acknowledged. We wish to thank Janis McMillen and Richard Consigli for the gift of ['4C]formaldehyde-labeledNDV virions.

This research wassupportedby Public Health Service grantAI-11127 from the National Institute ofAllergy and Infectious Diseases.

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