Relationships among the polypeptides of Newcastle disease virus.

(1)

Relationships

Among the

Polypeptides

of Newcastle Disease

Virus

LAWRENCE E. HIGHTOWER,1 TRUDY G. MORRISON, AND MICHAEL A. BRATT* Department ofMicrobiology, University of Massachusetts Medical School, Worcester, Massachusetts 01605

Received for publication 18 July 1975

We have studied the relationships among the polypeptides of Newcastle

disease virus by using both kineticand tryptic peptide analyses. The results of

our tryptic peptide analyses suggest that there are at least six unique viral

polypeptides-L,HN,

Fo

(F),NP, M,anda47,000-daltonpolypeptide.The small

virion glycopolypeptide F is related to

Fo,

a glycopolypeptide found only in

infected cells. In addition, several smaller polypeptides, including a

53,000-dalton polypeptide found both in purified virions and in infected cells, are

related to the nucleocaspid protein. Kinetic analysisofeach viral polypeptide

reveals thatall of the major viralpolypeptides, with thepossibleexception of L,

arestable after an aminoacid chase. Aprecursor-product relationshipbetween

Fo

and F was notdemonstrable bypulse-chase experiments. Also, almost the

same relative amount of F, the putative product, was present in infected

cultures after either 5 or30minofradioisotopic labeling.Theseresults suggest

that

Fo

isprocessedrapidly.

Newcastle disease virus (NDV) is a

represent-ativeof the paramyxovirus group ofenveloped,

negative-stranded RNA viruses. Its genome

consists of 5.2 x 106 to 5.6 x 106 daltons of

continuous single-stranded RNA (13). A

ge-nome of this size has a maximum coding

capac-ity of about 5.4 x 105 daltons of protein.

Six to sevenpolypeptides have been detected

in purified virions by sodium dodecyl sulfate

(SDS)-polyacrylamide gel analysis (2, 7, 9, 17,

19). By the suggested nomenclature for

para-myxoviruses (24), these polypeptides include

two glycosylated membrane proteins (HN,F),

one non-glycosylated internal membrane

pro-tein(M), anucleocapsid protein (NP), and two

tothreeminorpolypeptides.The smaller virion

glycoprotein has not beendirectly implicatedin

cellular fusion induced by NDV; however, we

havetentatively adoptedthedesignationFfor

thisglycoproteinbasedonthe close similarities

between Sendai virusandNDVinprotein

com-position and biological activities. Infected

chicken embryo cell cultures contain an

addi-tionalglycopolypeptide,

Fo

(23; J. Kaplan and

M. A.Bratt,Abstr.Annu. Meet. Am. Soc.

Mi-crobiol. 1973, V291, p. 243), whichis not found

invirusparticles (1, 10, 16). The total molecular

weight of these proteinsis approximately 6 x

106,

which slightly exceeds the theoretical

cod-ing capacity of the genome. Thus, if all of these

proteins are virus coded, they may not all be

unique geneproducts.

I

Presnt

address: Microbiology Section, Univeresity of Connecticut,Storrs,Conn. 06268.

Several recent studies suggest that

relation-shipsdoexist among theproteinsof

paramyxo-viruses. Studies on the effect of proteolytic

cleavage on the size and biologicalfunction of

the virion proteins of Sendaivirussuggest that

the smallestvirion glycopolypeptide isderived

from a larger precursor (12, 24). The putative

precursormoleculehas recentlybeen detected

inSendai virus-infected cells (26; R. A. Lamb,

Abstr. Annu. Meet. Am. Soc. Microbiol. 1975,

S145, p. 237). Kinetic evidence has been

pub-lished that suggests that the NDV

nonstruc-tural glycopolypeptide

Fo

isunstable and may

be converted to one of the virion structural

polypeptides (22).

Thestrongpossibilitythat,like Sendaivirus,

at least one of the NDV virion

glycopolypep-tidesmightbe acleavage productpromptedus

to studythe relationships among the

polypep-tides of NDVbyboth trypticpeptide andkinetic

analysis. We present evidence for six unique

viral polypeptides. Our data suggest that the

nonstructural glycopolypeptide

Fo

isrelated to the small virion glycopolypeptide F. In

addi-tion, thenucleocapsid polypeptideisrelatedto

several smaller polypeptides, including a

53,-000-dalton polypeptide found in both purified

virions and infected cells.

MATERIALS AND METHODS

Viruspreparation and cell culture. Preparation and cultivation of primary and secondary chicken

embryocell cultureshave beendescribedpreviously

(3). Secondary cultures grownineither60- or

100-mmtissueculture plates for 48 h at 40 C in 5% CO2 1599

on November 10, 2019 by guest

http://jvi.asm.org/

(2)

were used in all experiments. Growth in embryo-natedeggs and purification of the AV (Australia-Victoria, 1932) strain of NDV have also been de-scribed (5, 10).

Chemicals and isotopes. Mixtures of 15 14C0 labeled amino acids (-0.1 Ci/mmol) and [35S]methi-onine (211 Ci/mmol) were purchased from New England Nuclear Corp. Trypsin (containing TPCK) was purchased from Worthington Biochemicals Corp. Acrylamide and N,N'-methylene-bis-acryla-mide were purchased from Gallard-Schlessinger Corp. Other sources of chemicals used in the prepa-ration ofpolyacrylamide gels weredescribed previ-ously (10).

General protocolfor pulse-chase experiments. Cell cultures were infected at an inputmultiplicity of 5PFU/cell. After anabsorption period of 45 min,

cultures were incubated at 40 C in 5%CO2for 6 h.

Allexperiments to be described werepetformedat 6 hafter infection. We have shown previously thatthe rate ofviral proteinaccumulationismaximaland constantbythis time (10, 11). At6hpostinfection,

theculturemedium was removed and the cells were

rinsed with Hanks balanced salt solution, pH 7.2.

Radioisotopic labelmedium (amino acid-freeEagle

minimal essential medium [MEM] supplemented

with0.01mCiof14C-labeledamino acid mixture per

ml and 2% dialyzed calf serum adjusted to pH 7.4 with NaHCO3 and prewarmed to 40 C) was then

added. The cultures were incubated at 40 C for 5

min. Next, the labeling medium was removed and

chasemedium(MEM containing five times [1 mM]

thenormalconcentrations of amino acidsplus1 mM

inalanine,asparticacid,glycine, proline, and

ser-ine, supplemented with 2% calf serum,adjusted to pH 7.4 withNaHCO3andprewarmedto40C)was

added. Under these chaseconditions, total

radioiso-topic incorporation into acid-precipitable material stops almost immediately, and incorporation into viralpolypeptides stops afterapproximately 5 min (11). Aftervarying periods ofchase, cultureswere

washed with cold Hanks salts and the cells were

solubilized directly in polyacrylamide gel sample

buffer (0.05 M Tris [pH 6.71, 1%mercaptoethanol, 1% SDS). Sampleswere stored at -20 C and were

notdialyzedbeforeelectrophoresis (10).

General protocol for accumulation experi-ments.Theproceduresused forinfectingand

label-ing cultures are the same as those described for

pulse-chase experiments, except that the

radioac-tivelabel medium contained5%ofthe normal con-centration ofMEM aminoacids. After either5or30

min of labeling, the radioactive medium was

re-moved and thecells were washed with cold Hanks salts. The cellsweresolubilized directly in polyacryl-amide gel sample buffer and stored at -20 C.

Polyacrylamidegelelectrophoresis.

SDS-discon-tinuouspolyacrylamide gelelectrophoresis was car-ried out by the method of Laemmli (14) aspreviously described(10). Gel concentrations of 8.5% were rou-tinely used, except where 6% gels wereemployedto

clearly separate the large (220,000-dalton) viral

polypeptide from thegelorigin.

Although the gel procedureused inthe present

studyissimilar tothat usedinourprevious studies

(10, 11), we haveonlyrecentlybeen abletoroutinely separate the F and NPpolypeptides. Carefulcontrol

J. VIROL.

ofseparatinggelpolymerization times (between 20

and 30min)has been a significant factor in improv-ingboth gel resolution and reproducibility.

Sample preparationfor electrophoresis has been

described previously(10). Samples were boiled for2

minbefore theywere layered on gels. After

electro-phoresis, the gels were fixed in 10% acetic acid,

sliced longitudinally,and dried, and the

radioactiv-ity was analyzed by autoradiography with Kodak

Royal X-omat film (12-h to 1-week exposure). The

resulting autoradiograms were scanned with an

Or-tecmicrodensitometer.

Tryptic peptide analysis. (i) Sample

prepara-tion. Cellcultures wereinfected and incubated as

described above. At 6 h postinfection, infected

cul-tures werelabeled with 0.5 mCi of[35S]methionine perml in methionine-free Eagle MEMfor 30 min.

Cultures were solubilized directly in gel sample

buffer. Extracts derived from 6.7 x 105 cells were

subjectedtoelectrophoresison asinglegel.

Radioactive virus particles were prepared by in-fecting approximately 108 cells as described above. At 2 to 3 h postinfection, the cells were washed with Hanks salts, and Eagle MEM containing 1% the normal concentration of methionine and supple-mented with 0.125 mCi of[35S]methionine per ml and 2% dialyzed calf serum was added. After an incubationperiod of approximately 15 h at 40 C in 5%CO2,theculture medium was collected and the viruswaspurified as follows. After centrifugation at 27,000 x g for 10 min to remove cell debris, the supernatant was collected andsedimentedat82,500 xg (25,000 rpm) for 1 h in a BeckmanSW27 rotor at 4Cthrough a 20% sucrose layer onto a 65% sucrose-deuterium oxide pad. The virus band at the inter-face of the sucroselayers was thencollected,diluted threefold with cold standard buffer (10), and layered on a 20 to 65%sucrose-deuterium oxide linear gra-dient. Centrifugation was carried out in a Beckman SW27 rotor at 82,500 x g for 16 h. The peak of

radioactivitywaspooled and stored at -20 C.

Ap-proximately one-halfof the entire preparation was

subjectedtoelectrophoresison asinglegel.

(ii)Analysis.Radioactiveviralpolypeptidesfrom virions and infected cultures were separated on

SDS-polyacrylamide gels. Thegelswerethen

proc-essed forautoradiographyasdescribed above. After the viral bands were located by autoradiography, discretebandswereexcisedfromthegelsand incu-bated overnight at 37 C in 1 ml of 1% ammonium bicarbonate containing 100,ugof trypsin. The tryp-sin-bicarbonate solution was removed and the gel slice was reincubated for4 h at 37 Cin fresh 1% ammonium bicarbonate-trypsin solution. The two solutions werecombined andlyophilized.The result-ing peptides were washed twice in water and re-solved by paper ionophoresis at pH 3.5 as described previously (8, 15). After electrophoresis, the paper was either cut into 1 cm strips and analyzed by liquid scintillationcountingorexposed to X-ray film for 2 weeks. In the lattercase, theresulting

autora-diogramswere then scanned withan Ortec

micro-densitometer.

RESULTS

Stability of viral polypeptides.

Precursor-productrelationshipsaresometimes

http://jvi.asm.org/

(3)

NDV PROTEIN SYNTHESIS 1601

ble by kineticanalysis of proteinaccumulation. Wehaveusedtwodifferentexperimental

proto-cols to study the kinetics of protein

accumula-tion. In each experiment, the proteins from

infected cultures were separated on

polyacryl-amide gels to monitorthe incorporationof

radio-active aminoacids into viralpolypeptides. We

have shownpreviously that these gels also

con-tainradioactive cellular proteins,thesynthesis

of whichisonly partiallyinhibitedduring

infec-tion (10, 11). Therefore, electropherograms of

the proteinsofuninfected cultures were

super-imposed on the remainingcellularbackground

in the gel patterns of proteins from infected

cultures to estimate thehost-cellcontribution.

The fraction of the total radioactivity in agelin

each viralpolypeptide wasestimatedby

meas-uring the peak area that remained after

sub-tracting out the host-cell contribution. This

method of quantification, which utilized

densi-tometer tracingsof autoradiograms,isless

accu-rate than the double-label difference analysis

(10) ofpolyacrylamide gels; however, it is

ade-quatewhen theproportionofhost-cell proteins

issmall.

In the firstprotocol, infected and uninfected

culturesweregivena5-minpulseof radioactive

aminoacidsfollowed by eithera2-min(Fig. 1A)

or a 60-min (Fig. 1B) chase with excess

unla-beled amino acids.

Fo,

the putative precursorof

F, the small virion glycopolypeptide, did not

decrease significantly during the chaseperiod.

Infact, all of the majorviralpolypeptides(HN,

Fo,

F,NP,M),withthe possible exceptionofthe

L(large) polypeptide, were stable. The L region

has been more clearlyresolved on 6%gels(not

shown). Under these conditions, wehave con-sistently found an approximately 50% decrease

in the relative amount of L after a 30-min

chase, with little further reduction after a

60-minchase. Therefore, it is possible that at least

part oftheLregion is unstable.

Using thesecond protocol, we determined the

effect of varying the length of the radioisotopic labeling period on the accumulation of viral

polypeptides. Infected and uninfected cultures

were labeled with radioactive amino acids for

either 5 min (Fig. 2A) or 30 min (Fig. 2B). After

thelonger labeling period, the relative amount

of

Fo

decreasedby approximately 30%, whereas

I.

CL

0)

[image:3.502.107.393.328.614.2]

Migration

FIG. 1. Gel electropherograms of polypeptides from infected and uninfected cultures after amino acid chases. Infected (solid line) and uninfected (broken line) cultures were labeled for 5 min with radioactive amino acids andthen chased for either 2 (A) or 0 (B)min with unlabeled amino acids. Samples of the solubilized culturesweresubjectedtoelectrophoresison8.5%SDS-disc gels. The densitometer tracings of the autoradiograms of gels run inparallelhave been superimposed and the cellular protein backgrounds have been normalized.

16, 1975

http://jvi.asm.org/

(4)

B.

~~~~~~~~~NP

o7 L HN FQF M DYE

[image:4.502.118.403.71.440.2]

Migration

FIG. 2. Gelelectropherogramsofpolypeptides from infectedanduninfected cultures after different labeling

periods.Infected (solidline)anduninfected (broken line)cultureswerelabeledforeither 5 (A) or 30 (B)min

with radioactive aminoacids. Samples ofthe solubilized cultures were subjected to electrophoresis and

processedasdescribedinthelegendtoFig.1.

that of F increased by the same amount. The

relative proportions of HN, NP, and M

re-mained constant during this period, while L

increased two- to threefold inrelative amount.

Because of its size (approximately four times

larger than the other viral polypeptides), L

shouldachieve maximum specific activity more

slowly after addition of radioisotopes. This lag

probably accounts for its apparent increase in

relative amount.

Tryptic peptide analysis. No evidence for a

precursor-product relationship between

Fo

and

F wasfoundin pulse-chase experiments;

how-ever, studies in which thelabeling period was

varied revealeda weak reciprocal kinetic

rela-tionship between these glycopolypeptides. It

was particularly important to determine the

relationship of L to other viral polypeptides

since a 220,000-dalton polypeptide could be a

possibleprecursorfor,or aggregateof, several

of the smaller polypeptides. To obtain more

conclusive evidence for unique and related

pro-teins, we carriedouttryptic peptide analyses.

Radioactively labeled polypeptides from both

purified virusparticles (Fig. 3A) and infected

cultures(Fig. 3B)wereseparatedon

SDS-poly-acrylamide gels. Discrete bands were excised

from the dried gels anddigested withtrypsin.

The resulting tryptic peptides were separated

by paperionophoresis atpH 3.5.

Evidence for six unique gene products.

The tryptic peptidepatternsofpolypeptides

iso-lated from virions and infected cultures are

comparedin Fig. 4 and 5. Figure4 shows the

http://jvi.asm.org/

(5)

trypticpeptide patternsofpolypeptidespresent

in large enough amounts to be conveniently

analyzed by autoradiography. The tryptic pep-tide patternsof the 47,000-dalton polypeptides

that were present in small amounts and

re-quired analysis byliquid scintillation counting

are shown in Fig. 5. Six distinctly different

tryptic fingerprints were obtained from the

viral proteins L, HN, F, NP, M, and a

47,000-daltonpolypeptide. The trypticpeptidepattern

of this 47,000-dalton polypeptide is different

from thepatternobtained for the46,000-dalton

actin-like polypeptide, which is a major

poly-peptideinuninfected cells (notshown).

There-fore, this polypeptide does not correspond to

anydetectablepolypeptideofsimilarsize inthe

uninfected cell andisprobablyof viral origin.

Pairs ofpolypeptides of similar

electropho-reticmobilityisolatedfrom virions andinfected

cellshavealso beencomparedin Fig. 4 and5.

Polypeptides of similar size fromeither source

havesimilar tryptic fingerprints, thus

confirm-ingtheir identity.

Related viral polypeptides. Several

rela-tionships among the viral polypeptides have

been revealedby tryptic peptide analysis.

Fo,

thenonstructural viral glycopolypeptide, is

re-lated to F, the smaller virionglycopolypeptide

(Fig. 6A). The six majorpeptides of F all have

counterparts amongthe peptides of

Fo.

In

addi-tion,

Fo

contains two to three methionyl

pep-tides notpresent in F. There is some ambiguity

in the correspondence between minor tryptic

peptidesofhigher mobilityinthe pattern ofF

and those derived from

Fo.

These species may

arise by contamination of the F preparation

with NP, which migrates very close to F on

gels. Alternatively, there may be changes in the mobility of some of the F peptides as a

result ofprocessing by either glycosylation or

cleavage.

The 53,000-dalton polypeptide (designated

NP1 in Fig. 3) isolated from both purified

vi-rions and infected cultures is related to the

nucleocapsidprotein NP (Fig. 6B). Inaddition,

several minorpolypeptidesofmolecularweight

45,000(NP2) and 43,000(NP3) isolated from

in-fected cultures have tryptic peptide patterns

that are very similar to NP (Fig. 7).

DISCUSSION

Trypticpeptideanalysis suggests that

Fo

and

Fare related; however, kinetic studies did not

(n,

c]0

B.B

Fo

NP 47K

NP3

O|L HN F [ NP NP2 M DYE

0.

[image:5.502.106.389.358.623.2]

Migration

FIG. 3. Gelelectropherograms ofpolypeptides from purifiedvirionsand infectedculturesusedfor tryptic peptide analysis. Polypeptides from purified virions (A) and infected (B) cultures labeled with [3S]methionine

weresubjectedtoelectrophoresis on8.5%SDS-discgels. Densitometer tracings weremade from

autoradi-ograms in which the major viralpolypeptides were overexposed to detectpolypeptides present in small

amounts. Thisaccountsfor the distorted proportions of themajorviral peaks.

A.

L HN F NP NP 47K M DYE

AIAAA

I

l

nA~~~~~A

A

VOL. 16, 1975

http://jvi.asm.org/

(6)

HNC

HNV v

Fc

F

NPC

NPv

47KV

Mc it

mtit5

_J _{_} _{_} _w

0

[image:6.502.72.458.62.497.2]

Migration

FIG. 4. Trypticpeptidepatterns of the major unique viral polypeptides. The majorpolypeptides were

excisedfromthe driedgelsshown inFig.3 anddigested with trypsin, and the resultingmethionyltryptic

peptides wereresolved bypaperionophoresis. The densitometer tracings of the autoradiograms oftryptic

peptides derivedfrompolypeptides ofsimilar sizeisolated frompurifiedvirions (subscript v) and infected

cultures(subscript c) are compared. The polypeptide47K,was notpresent in large enough amounts to allow

analysisofitstrypticpeptidepatterns byautoradiography.Theanalysis of this minor polypeptide by liquid

scintillationcounting is shown in Fig. 5. All of the peptidepatterns shown in each figure were analyzed in parallel.

reveal a clear product-precursor relationship.

The fact that almostthesamerelativeamount

ofFispresent after either5or30min of

label-ing may indicate that the processing occurs

quickly.Iftheprocessingisfast, it couldescape

detection in pulse-chase experiments since

ra-dioactive viralproteinscontinuetoaccumulate

for at least 5 min after addition of chase

me-dium (11). Pulse-chase experiments do show

that arelatively stable population ofFoexists

that is either not processed or processed very

slowly. Cultures maycontain twopopulations

ofcells, one capable of processing Fo and the

othernot. Alternatively, onlyaportion of the

Fo glycopolypeptides may be processed in the

infected cell. W

G)

.2_

m

http://jvi.asm.org/

(7)

Our findingofarelatively stablepopulation

of

Fo

isdifferent froma

report

(22)

that

showed adramatic chase of

Fo

intoeither NP or

F (these polypeptides were not resolved). The

difference is apparently not due to virus strains

since we obtained similar results with either

strain AV or strain L-Kansas, the virus used in the previous study. This discrepancy may be

due tophysiological differences in the cell

cul-tures used in the two studies, particularly if

processing ismediated by cellular enzymes.

20

0

xX

E

15-,, 10 C

0

c0

I 0 20 40 60

[image:7.502.51.243.202.313.2]

Fraction

FIG. 5. Trypticpeptide patterns of minor unique viralpolypeptides. The47,000-daltonregion was

ex-cised from thegels shown in Fig. 3 and processed for

trypticpeptide analysis as described in the legend to Fig. 4, except thatthe amount of radioactivity was

determinedby liquid scintillation counting instead of

by autoradiography. Thetrypticpeptide patterns of

the47fKK)-daltonpolypeptide isolated from both

vi-rions (0) and infected cultures (0) are compared.

A, Fo

B, NPc

'C'

NP,c

0 NP,v

O

The molecular mechanism fortheprocessing

of

Fo

and F in NDV infection is not known.

There is no directevidence for proteolytic

cleav-age, such as exists for Sendaivirus. It islikely

that processing occurs by analogous

mecha-nisms of proteolytic cleavage in both viruses;

however, there are several otherpossible

mech-anismsfor generating relatedpolypeptidesthat

migratedifferentlyonpolyacrylamidegels.For

example, thesamepolypeptide mightbe glyco-sylated to varying extents in the cell.

Differ-encesin glycosylation could alterthemobility

of apolypeptideinagel. Alternatively,asingle

mRNA could have twoinitiation or termination

sites for protein synthesis. The translation of

such an mRNA wouldyieldtwopolypeptides. It

is also possiblethat viral transcriptionresults

intwo mRNAswith overlapping sequences.

We foundatleastthree smallerpolypeptides

invirions andinfected cultures that arerelated

tothenucleocapsid protein. It is known that the

nucleocapsid proteins of paramyxoviruses are

susceptibletospecific cleavages atlimited sites

(18, 20). The size of the cleavage product

de-pends upon the proteolytic enzyme used, and

the cleavage can apparently be accomplished

by cellular proteases in the absence of

exoge-nousenzymes. It islikely that at least some of

the nucleocapsid fragments NP1_3 are

gener-ated bycellular proteases withdifferent

specific-ities, althoughother mechanisms such as

pre-mature termination during transcription or

Migration

FIG. 6. Trypticpeptidepatternsof themajorrelatedviral polypeptides.Polypeptides wereprocessed for

trypticpeptide analysis and analyzed by autoradiographyasdescribedinthe legendtoFig.4. (A)Foand F from infected cultures arecompared; (B) NP isolatedfrom infectedcultures is compared with the53/X

dalton region (NP1)isolated from both virions and infected cultures.

1'.

II - '

l _

19

i

http://jvi.asm.org/

[image:7.502.56.450.409.620.2]

(8)

c c

.r_

z

0 20 40 60

Fraction

FIG. 7. Trypticpeptidepatternsof minor related viralpolypeptides. Polypeptides wereprocessed for

trypticpeptide analysis and analyzed by liquid scin-tillation countingasdescribed in thelegendtoFig.5. The NP polypeptide is compared with the 45,000-dalton (NP2) and43,000-dalton (NP3) polypeptides isolatedfrom infected cultures.

translation havenotbeen ruledout.

The tryptic peptide analysis of the L

polypep-tide suggests that this polypeptide is unique

andnotanaggregateof smaller viral

polypep-tides. Infected cells contain a plus-stranded

viral RNAlarge enough (35S) tocode forL (4;

B. Spanier-Collins and M. A. Bratt,

manu-scriptinpreparation). RecentlyLhas been

syn-thesized in a cell-free systemprogramed with

the 35S RNA from infected cells (B.

Spanier-Collins, C. W. Clinkscales,M. A. Bratt, andT.

G.Morrison,manuscriptinpreparation).These

data support the hypothesis that L isa

virus-codedpolypeptide.

The possibility that at least part of the L

regionmay beunstable is interesting because

atleastoneviral activity, replication (butnot

transcription), inparamyxovirus-infected cells

requires continuous protein synthesis (21, 25).

A polypeptide ofapproximately the same size

as L is also found associated with

nucleocap-sids isolated from NDV virions (R. J. Colonno

and H. 0.Stone,Abstr. Annu. Meet. Am. Soc.

Microbiol. 1975,S223,p. 250).Recently,alarge

polypeptide has also been detected in

ribonu-TABLE 1. Unique virala polypeptides

Polypeptide Mol wt

L 220,000b

HN 67,000'

Fo 60,000c

NP 56,000

47K 47,000

M 41,000

Total 491,000d

aPolypeptides found in virions and infected cells

but not inuninfected cells are considered virus spe-cific. The possibility that some of these proteins are induced cellular polypeptides has not been rigor-ously eliminated.

bComigrates with rabbit myosin on6% polyacryl-amide gels.

cEstimates for non-glycosylated form based on

size of in vitro translation product on gels (T.G. Morrison, S. R. Weiss, B. Spanier, L. E. Hightower,

and M. A. Bratt, unpublished data).

dApproximate coding capacity is 540,000+54,000 daltons.

cleoprotein complexes isolatedfrom Sendai

vi-rus-infectedcells (26).

We have presented evidence for six unique

viral polypeptides. These polypeptides have a

total molecular weight of 491,000 according to

our best size estimates (Table 1). This total

approaches the maximum coding capacity of

the genome. The number of unique

polypep-tides found here isingood agreementwith the

number predicted from both studies on

tempera-ture-sensitivemutants (five to six

complementa-tion groups; J. Ebel-Tsipis and M. A. Bratt,

manuscriptinpreparation) and the analysis of

viral mRNA on gels (five to six different size classes; 6).

ACKNOWLEDGMENTS

We gratefully acknowledge the technicalassistanceof Eivor Houriand thehelp ofGayle Hightowerinthe prepara-tionof themanuscript.

This work was supported by research grant BMS 75-05024 to M. A. BrattfromtheNational ScienceFoundation, Public Health ServicegrantAl12467-01 to M. A. Brattfrom the National InstituteofAllergy andInfectious Disease, and grant VC-167toTrudyG. Morrisonfrom the American CancerSociety.

LITERATURE CITED

1. Alexander, D.J., andP. Reeve. 1972.Theproteinsof Newcastle disease virus.2. Virus-inducedproteins. Microbios 5:247-257.

2. Bikel, I., and P.H.Duesberg. 1969. Proteins of

Newcas-tle disease virus and of the viral nucleocapsid. J. Virol. 4:388-393.

3. Bratt, M.A., and W. R. Gallaher. 1969. Preliminary analysisof therequirementsfor fusionfrom within and fusionfrom withoutby Newcastlediseasevirus.

Proc.Natl.Acad.Sci. U.S.A. 64:536-543.

http://jvi.asm.org/

[image:8.502.65.254.73.331.2] [image:8.502.264.458.82.191.2]

(9)

4. Bratt, M. A., andW. S. Robinson. 1967. Ribonucleic acid synthesisincells infected with Newcastle dis-easevirus. J.Mol. Biol.23:1-23.

5. Clavell,L.A., and M. A. Bratt. 1972.Hemolytic interac-tionofNewcastle disease virus and chicken erythro-cytes. II. Determining factors. Appl. Microbiol. 23:461-470.

6. Collins, B.S., andM. A. Bratt. 1973.Separationof the messengerRNAs ofNewcastle diseasevirusbygel electrophoresis. Proc. Natl. Acad. Sci. U.S.A. 70:2544-2548.

7. Evans, M. J., and D. W.Kingsbury.1969.Separationof Newcastledisease virus proteins ofpolyacrylamide gelelectrophoresis. Virology37:597-604.

8. Goldman, E., and H. F. Lodish. 1971. Inhibition of replication ofribonucleic acid bacteriophage f2 by superinfection with bacteriophage T4. J. Virol. 8:417-429.

9. Haslam,E.A., I. M.Cheyne, andD.0. White. 1969. Thestructural proteinsof Newcastle disease virus. Virology39:118-129.

10. Hightower,L. E., and M. A. Bratt. 1974.Protein synthe-sis in Newcastle diseasevirus-infected chicken

em-bryocells. J.Virol. 13:788-800.

11. Hightower,L.E.,andM. A.Bratt.1975.Protein metab-olism during thesteadystate of Newcastledisease virusinfection.I.Kinetics ofaminoacid and protein accumulation.J. Virol. 15:696-706.

12. Homma,M., and S.Tamagawa.1973.Restoration ofthe fusion activity ofLcell-borneSendaivirusbytrypsin. J.Gen. Virol. 19:423-426.

13. Kolakofsky,D., E. Boy de la Tour, and H. Delius. 1974. Molecularweight determinationofSendaiand New-castle diseasevirus RNA. J.Virol. 13:261-268. 14. Laemmli,U. K. 1970. Cleavage of structural proteins

during theassembly of the head ofbacteriophageT4. Nature(London)227:680-685.

15. Lodish,H. 1968.Bacteriophage f2 RNA: control of

trans-lation andgeneorder. Nature(London) 220:345-349. 16. Lomniczi, B., A.Meager,and D. C.Burke.1971. Virus

RNA andproteinsynthesisincellsinfectedwith dif-ferent strains ofNewcastle disease virus. J. Gen. Virol. 13:111-120.

17. Meager, A., and D. C. Burke. 1973. Studiesonthe structural basis of the RNApolymerase activityof Newcastle disease virus particles. J. Gen. Virol. 18:305-317.

18. Mountcastle,W.E.,R. W. Compans, L.A.Caliguiri, and P. W.Choppin. 1970.Nucleocapsidprotein sub-units of simian virus5,Newcastle disease virus,and Sendai virus. J. Virol.6:677-684.

19. Mountcastle,W.E., R.W.Compans, andP. W. Chop-pin.1971.Proteins andglycoproteinsof paramyxovi-ruses: a comparison ofsimian virus 5, Newcastle diseasevirus,andSendai virus. J. Virol. 7:47-52. 20. Mountcastle,W.E.,R. W.Compans,H.Lackland,and

P.W.Choppin.1974.Proteolyticcleavageofsubunits ofthenucleocapsidoftheparamyxovirus simian

vi-rus5.J.Virol.14:1253-1261.

21. Robinson,W.S.1971.SendaivirusRNAsynthesisand nucleocapsid formation in the presence of cyclohexi-mide.Virology44:494-502.

22. Samson,A.C.R.,and C. F. Fox. 1973. Precursor pro-teinforNewcastle diseasevirus. J. Virol.12:579-587. 23. Samson,A. C.R., andC. F. Fox. 1974. Selective inhibi-tion ofNewcastledisease virus-inducedglycoprotein synthesisby D-glucosamine hydrochloride. J. Virol. 13:775-779.

24. Scheid, A.,and P. W.Choppin. 1974. Identification of biological activities ofparamyxovirusglycoproteins. Activation of cell fusion, hemolysis, and infectivity byproteolytic cleavage of an inactive precursor pro-teinofSendaivirus.Virology 57:475-490.

25. Weiss, S. R., and M. A. Bratt. 1975. Effect of cordycepin (3-deoxyadenosine) on virus-specific RNA species synthesized in Newcastle diseasevirus-infectedcells. J. Virol. 16:1575-1583.

26. Zaides,V.M.,L.M.Selimova, 0. P. Zhirnov, and A.G. Bukrinskaya. 1975. Proteinsynthesisin Sendai vi-rus-infectedcells. J. Gen. Virol. 27:319-327. VOL. 16,

http://jvi.asm.org/