Cloning and characterization of DNA complementary to the measles virus mRNA encoding hemagglutinin and matrix protein.

(1)

0022-538X/82/060790-08$02.00/0

Cloning and

Characterization

of DNA Complementary

to

the

Measles

Virus mRNA

Encoding Hemagglutinin and Matrix

Protein

SHMUELROZENBLATT,* CILA GESANG, VERED LAVIE, ANDFELICIA S. NEUMANN

Department of Virology, WeizmannInstitute of Science, Rehovot,Israel

Received 27 October1981/Accepted 1 February 1982

Since cloning and characterization of DNA complementary to measles virus

mRNA encoding for the nucleocapsid protein (M. Gorecki and S. Rozenblatt,

Proc. Natl. Acad. Sci. U.S.A. 77:3686-3690, 1980), two additional

measles-specific clones containing different classes ofsequenceshave beencharacterized.

Thecloned plasmids contain inserts of 480 and 530 base pairsasshown byagarose

gel electrophoresis and electron microscopy. The sizes of the mRNA species

complementary tothese insertsare 1,700 and 1,550 nucleotides, respectively,as

determinedby the Northern technique. The cloned DNA fragmentswerefurther

identified as reverse transcripts of the mRNA coding for the glycoprotein and

matrixprotein of measles virus. The major cell-free translation products of mRNA

selected byhybridizationtotheindividual cloned DNAs comigrated with the 70K

in vitro products and matrix proteins. One of the cell-free translation products

(70K) was also immunoprecipitated specifically with monoclonal antibodies

against measles virus glycoprotein.

The

measles

virus is a complex virus that

interacts

with

its

host

cell in

either an acute ora

persistent infection.

Themeasles

viroid

consists

of six structural

proteins (9,

19-21) anda

single-stranded

RNA

(molecular weight,

6 x

106)

(9,

11, 17).

Six virus-specific

mRNAs

differing

in

size have been

recognized

in

monkey

cells

in-fected with measles virus

in the presence

of

actinomycin

D (7). The

polypeptides

synthe-sized

in an

mRNA-dependent reticulocyte

cell-free

system were

subsequently identified

as

au-thentic

measles

polypeptides by electrophoretic

and

immunological

techniques

as well as

by

analysis

of

their

tryptic

peptide

patterns

(15).

Thus, it

is now

possible

to

directly

examine

measles

virus-specific

gene

expression during

the courseof infection andtodetermine whether

acute versus persistent infections canbe

corre-lated with differential patterns of gene expres-sion.

A powerful approach to

studying

differential

gene

expression

in

eucaryotic

cells and in cells

infected

with virus involves the

production

of

nucleic acid

hybridization probes

thatare

highly

radioactive and gene

specific.

In a previous

report (6), the method used to

obtain

measles

virus nucleic acid hybridization products that

are

highly

radioactive was described. A

recom-binant

plasmid

that contains most of the nucleic

acid sequences encoding measles virus

nucleo-capsid protein

was obtained. In the present

study,

we describe the isolation and

character-ization

of

two

recombinant plasmids

that

contain

partial nucleic acid

sequences encoding parts of

the measles

virus

glycoprotein (hemagglutinin)

and matrix

protein.

MATERIALSANDMETHODS

Cells and viruses. The CV-1 line of African green

monkey kidney cellswasobtained from Flow Labora-tories, Inc. (Rockville,Md.). The cellsweregrownin Eagle medium supplemented with10%calfserum.The Edmonston strain of measlesvirus, obtained from B.

Fields (Harvard Medical School),wasplaquepurified

three times in CV-1 cells. A stock was prepared by

infecting CV-1 cellsatamultiplicity of1/1,000. After thedevelopment ofa markedcytopathic effect,virus

was harvested from freeze-thaw lysates of the cells. Cell debriswasremovedbycentrifugationat1,000xg

for 10 min at 4°C, and the supernatants which

con-tained2 x 106to6x 106 PFU/ml (as titrated inVero

Africangreenmonkey cells)werestored at -90°C.

Selection of measlesvirus-specificRNA. Selectionof

RNA wascarriedoutbythemethod ofRicciardietal.

(13). ClonedDNA(10 ,ug)linearized with EcoRIwas

denatured at 100°C for 5 min and applied to

1-cm-square nitrocellulose filter pads presoaked in 0.9 M

NaCI-0.09M sodium citrate. The nitrocellulose filter

was air dried and then baked at 80°C for 2 h under

reduced pressure. Hybridization solutions (100 ,ul)

consisted of 10

p.g

of polyadenylic acid [poly

(A)]-containing RNA [poly(A)-RNA], 10 mM

Tris-hydro-chloride(pH7.4),2 mMEDTA, 0.4% sodiumdodecyl

sulfate (SDS), 10 ,ug of Escherichia coli tRNA, and

50% (vol/vol) formamide. Hybridization was carried

out at37°Cfor 16 h.poly(A)-mRNAswereeluted and

790

on November 10, 2019 by guest

http://jvi.asm.org/

(2)

plaque-purified measles virus at 0.1 to 0.5 PFU/cell. RNA was extracted with phenol and chloroform-isoamyl alcohol and then precipitated with LiCl (16). poly(A)-mRNAs were thenpurified by oligodeoxythy-midylic acid-cellulose chromatography (2).

Fractionation, extraction,and in vitro translationof

RNA obtained under denaturing conditions. poly(A)-RNA of measles virus-infected cells (20 ,ug) was denatured in the presence of 15 mMmethyl-mercuric

hydroxide and fractionated in 1.5% agarose and 7.5 mMmethyl-mercuric hydroxide as described by Bai-ley and Davidson (3). A gelfraction containing RNA of 1,000 to 2,700nucleotides wassliced into 1-mm-thick

slices, and RNA was eluted from individual gel slices

by freeze-thawing. Agarose slices were placed in Ep-pendorf tubes containing 5 ,ug of rabbit liver tRNA, 0.1 M NaCl, 10 mM Tris-hydrochloride (pH 7.4), 1 mM EDTA,0.2%SDS, and 20 mM ,-mercaptoethanol (400

jil). The agarose slices were melted(95°C for 2 min),

blended in a Vortexmixer, andfrozen immediately in liquid nitrogen. After thawing, the agarose was re-moved by centrifugation at 12,000 x g for 20 min

(4°C).The RNA waspurified from thesupernatant by

successive extractions with phenol and chloroform-isoamyl alcohol followed by ethanolprecipitationand

subsequentlytranslated invitro inreticulocyte lysates

prepared by the method of Pelham and Jackson (12).

RESULTS

Identification of

cDNA clones.

One

clone,

Cl-N,

carrying measles virus

complementary

DNA

(cDNA)

sequences

for

nucleocapsid proteins

has

previously been described (6).

Characteriza-tion of the clone revealed

an

insert

of

approxi-mately 1,420 base

pairs (Fig. 1A). The size of the

mRNA

complementary

to

the cDNA

was

1,750

bases

[including

the

poly(A)

tail];

electron

mi-croscopy

studies

using

an

R-loop technique

(Fig.

1B)

have shown that

at

least

80%

of the entire

mRNA

was present

in the clone. To

isolate

cDNA

clones with

sequences

that do

not

code

for

nucleocapsid proteins,

the measles

virus

cDNA

insert in

Cl-N

was

removed

from the

pBR322 vehicle by cleavage with PstI and

isolat-ed after fractionation

on a

1.5%

agarose

slab

gel.

The

purified

DNA

insert

was

32p

labeled

in vitro

by

nick translation and

was

used

as a

hybridiza-tion probe. Of the 39 cDNA clones

(5),

30 (containing

DNAinserts

varying

in

size from

400

to 650 base

pairs) showed

strong

hybridization

signals

to

this

probe, whereas

9

demonstrated

weak hybridization

(to the same extent as

bacte-rial

pBR322

DNA

used

as

internal

negative

control).

Upon in vitro translation of

the mRNA

selected

by hybridization

to

individual

DNA

clones

(data

not

shown),

the

nine

clones

which

did

not

hybridize with Cl-N could

be divided

into

twogroups:

eight of

themare

referred

to as

the G

group, and one is

referred

to asthe "M"

of individual clones with

PstI

(Fig.

2)

generated

two

fragments in

each clone, one

corresponding

in

size

to linear pBR322 DNA, and a second

fragment consisting of

1,420, 540, and 480

base

pairs (Cl-N, Cl-M, and Cl-G, respectively; Fig.

2A). The DNA was blotted onto nitrocellulose

papers,

fixed by baking in

vacuum,

and

hybrid-ized with

purified inserts of clone

N

32P-labeled

in vitro by nick translation. Hybridization

was

observed only

to

the

Cl-N insert;

no

hybridiza-tion

was

observed with Cl-G

or

Cl-M,

even

after

a

long

exposure

(Fig.

2B).

The size of the

cDNA

inserts

was

further

confirmed by electron

micro-scopic studies. Figure

3 shows electron

micro-graphs of heteroduplex

molecules

formed by

reassociation of

pBR322 and the cDNA clones,

each

one

linearized by digestion with

EcoRI,

which

does not cut the insert. The lengths of the

inserted

measles

virus

sequences appearin the

micrographs

tobe 1,420

±

20, 530 + 40, and 490 + 40

base

pairs in Cl-N, Cl-M, and Cl-G,

respec-tively, which

is inexcellent agreement with the

length determined

by agarose gel

electrophore-sis.

Heteroduplex molecules formed by

reasso-ciation of Cl-N, Cl-M,

and Cl-G

in

all three

variations

have

confirmed

that thereis no DNA

homology

among

Cl-N, Cl-M,

or

Cl-G (Fig.

3d,

e,

and

f).

FIG. 1. Electron microscopy of (A Cl-N DNA

%J W

-

IL

digested with PstI and (B) R-looping of Cl-N (1 ,ug)

linearized with EcoRI, denatured, and annealed with mRNA(5 ,ug of measles virus-infected cells (5).Insert

shows interpretive drawing.

on November 10, 2019 by guest

http://jvi.asm.org/

[image:2.487.257.450.403.625.2]

(3)

[image:3.487.130.363.77.173.2]

__

FIG. 2. Sizingof measles virus DNA clones. Plasmid DNAs digested withPstIwereelectrophoresed on a 1.5% agarose slab gel stained with ethidium bromide (A), transferred onto nitrocellulose paper (18), and hybridized with

32P-purified,

nick-translated (14) measles virus DNA insert of clone N (B). Lanes: 1, pBR322; 2,

Cl-N;3,Cl-M; 4, Cl-G. Right-hand lane:32P-labeledSimianvirus 40 DNA digested withHinfl,usedas markers.

b.p., Basepair.

Correlation of

the cloned measles DNAs with

comigrated

with

60,000-dalton nucleocapsids

[image:3.487.51.438.371.626.2]

viral gene products and mRNA species. DNA

of

prepared and

purified from cells infected with

the

individual clones

(Cl-N,

Cl-M,

and

Cl-G)

measles

virus.

Furthermore, the translation

was

separately

digested with EcoRI,

immobi-

product was

immunoprecipitated

with

anti-mea-lized onto

nitrocellulose

filters,

and

hybridized

sles

virus antibodies

(Fig. 4B, lanes 4 and 5). As

with poly(A)-RNA from measles

virus-infected

expected, no

translatable RNA was eluted from

cells. RNA was then eluted

from the

filters and

filters containing either

pBR322 DNA

(Fig.

5A,

translated in an

mRNA-dependent

rabbit reticu-

lane

4)

or

single-stranded phage

M-13 DNA

locyte cell-free translation system. As shown in

containing

cDNA

inserts of the same

polarity

as

Fig. 4A, lanes 4 and 5, the

major cell-free

measles virus

mRNA

(Fig.

4A

and

B, lanes

6).

translation

product (RNA

hybridized

to

Cl-N)

Confirmatory

evidence

was

provided

by

immu-~ ~ .

..

!~~~~~~~~~~~~~~~~~~ ~,

41 :- .

FIG. 3. Electronmicroscopy of heteroduplexDNAs.pBR322 and three clonedDNAs(Cl-M,Cl-G,

Cl-N;

1 ,ugofeach)werelinearizedwithEcoRI,denatured,andannealed(5)asfollows: pBR322and(a)CI-G,(b)Cl-M,

(c)Cl-N;(d)Cl-NandC1-M;(e)Cl-Nand

Cl-G;

(f)Cl-MandC1-G.Insetsshowinterpretive drawings. Bar,0.5

,um.

on November 10, 2019 by guest

http://jvi.asm.org/

(4)

< per, and each of the identical blots was

hybridized

to one of the three

probes.

The

nucleocapsid

and

glycoprotein

cDNA

probes

hybridized to mRNA with an approximate size

of1,750 bases (Fig. 7A and B); and the matrix

clonehybridized to mRNA of 1,550 bases

(Fig.

7C). The difference in hybridization of the

ma-trix clone and clone G is further demonstrated

in

Fig.

7C' where the blot

hybridized

with Cl-M

2 3 4 5 _A

*i

FIG. 4. Products ofthe in vitrotranslation ofRNA

selectedby hybridizationtoM-13phageDNA witha

plus polarity (N) insert (complementary to mRNA)

and minus polarity insert. [35S]methionine-labeled

polypeptides synthesized in a reticulocyte cell-free

system (11) werefractionated on an SDS-10o poly-acrylamide gel. The dried gel was fluorographed at

-90°C (4). (A) Translation products. (B) Immunopre-cipitates. Translationproducts under direction of: no

added RNA(lanes 1), total cytoplasmicpoly(A)-RNA

of uninfectedcells(lanes 2),ormeaslesvirus-infected

cells(lanes 3);RNAselectedby hybridizationof total

cytoplasmicpoly(A)-RNAtoimmobilized Cl-N DNA

(lanes 4); phageDNAwithplus polarityN insert(lanes

5); or minus polarity insert (lanes 6). Molecular

weights (M.W.) shownwerethefollowingmethylated (methyl-14C) proteins of known molecular weight,

fromtop tobottom:myosin phosphorylase B, bovine

serumalbumin, ovalbumin, and carbonic anhydrase.

noprecipitation techniques with monoclonal

antibodies (obtained from B. Bloom and V. ter

Meulen) raised against nucleocapsids and glyco-proteins (Fig. 6). We concluded from these

results(Fig. 4, 5, and 6) that the cDNAsinserted

in the recombinant clones are specific for the

measlesvirus nucleocapsid, the

non-glycosylat-ed precursor of the glycoprotein (hemaggluti-nin), and the matrix protein.

Cl-N, Cl-M, and Cl-G DNAswere32p labeled

in vitro by nick translation and were used as

hybridization probestothree identical

diazoben-B

.- .>

...k

FIG. 5. Productsof theinvitrotranslationoiKINA

selectedbyhybridizationtopBR Cl-M, Cl-G,and

Cl-N DCl-NAclones. [35S]methionine-labeled polypeptides

synthesizedinareticulocyte cell-freesystem(11)were fractionatedon anSDS-10% polyacrylamide gel. The

driedgelwasfluorographedat-90°C (4). (A)

Transla-tionproducts. (B) Translation productsafter

immuno-precipitation with anti-measles guinea pig serum as

describedpreviously (10).Translationproductsunder

direction of: noadded RNA(lanes 1), total

cytoplas-mic poly(A)-RNAofuninfectedcells(lanes 2), RNA

from measles virus-infected cells isolated 24 h after

infection(lanes 3), RNA selectedby hybridizationof

total cytoplasmic poly(A)-RNA to immobilized

pBR322 DNA (lanes 4), Cl-N DNA (lanes 5), Cl-M DNA (lanes 6), and Cl-G DNA(lanes 7). Molecular

weight (M.W.)markersasinFig.4.

B

40.

S"

"1"-NW _;_...._

low.,Oa

4"mwxmw

on November 10, 2019 by guest

http://jvi.asm.org/

[image:4.487.52.240.76.364.2] [image:4.487.256.447.226.508.2]

(5)

794 ROZENBLATT ET AL.

FIG. 6. Products of in vivoand in vitro translation of RNA selected hybridization topBRCl-N andCl-G immunoprecipitated with monoclonal measles virus-specific antibodies. [35S]methionine-labeled proteins of measlesvirus-infected cells and polypeptides synthesized in a reticulocyte cell-free system (11) were fractionated

on an SDS-10% polyacrylamide gel. The dried gel was fluorographed at -90°C (4). Gels show translation products of poly(A)-RNA of measles virus-infected cellsimmunoprecipitated with guinea pig anti-measles virus

sera(A, F). Immunoprecipitationwith rabbit monoclonal antibodiesagainst nucleocapsid protein: (B) in vivo-labeledproteins of measles virus-infected cells; (C) in vitro products ofRNAselected by hybridization of total cytoplasmic poly(A)-RNAtoimmobilized Cl-N DNA immunoprecipitatedasin B;(D) in vivo-labeled proteinsof measles virusimmunoprecipitated with rabbit monoclonal antibodies against glycoprotein; (E) in vitro products ofRNAselected byhybridization of total cytoplasmic poly(A)-RNAtoimmobilizedCl-G DNA immunoprecipi-tated as in D; (G) products as in C immunoprecipitated with glycoprotein antibodies; (H) products as in E

immunoprecipitated with nucleocapsid antibodies.

was

hybridized later

with

Cl-G.

Simultaneous

hybridization

with Cl-N and Cl-G could not

resolve the

two mRNA species (data not shown).

_a

s

-

_

imp

Correlation

of measles virus mRNA

species

and

their

corresponding

gene

products.

poly(A)-RNA

(20

,ug)

was

fractionated

under

denaturing

condi-tions from

measles

virus-infected

cells

(methyl-.1Mmm ,, .*f-_.m-:i-.

won_

FIG. 7. Sizing of measlesvirus mRNAbyhybridizationofCl-N, Cl-M and Cl-Gtototalpoly(A)-RNA from

cells acutely infected with measles virus. Poly(A)-RNA from measles virus-infected cells (lanes 1) and from

uninfected cells(lanes 2)wasfractionatedunderdenaturing conditions (1)in1.5% agarose gel. The RNA was

transferredtodiazobenzyloxymethyl paper and hybridized with nick-translated (14) plasmid [32P]DNA; Cl-N (A), Cl-G (B), and Cl-M (C)werehybridizedlater withCl-G(C').32P-labeled18S and 28SrRNA wereusedas markers.

AN-,

owxmw

.P%.., 1:

...-

...::.

21 1.

O"

ot-.I

....

..:,.

,t,

C '", .:..

on November 10, 2019 by guest

http://jvi.asm.org/

[image:5.487.131.359.72.240.2] [image:5.487.109.391.441.603.2]

(6)

MEASLES VIRUS cDNA CLONES 795

mercuric hydroxide

agarose gel). Gel segments

(3

cmlong) containing fractionated mRNA in a

size range of 1,000 to 2,700

nucleotides

were

sliced, and mRNA waseluted

from each gel slice

as

described

above. RNA recovery in this

elu-tion

procedure was 60%, as determined by

32p-labeled

rRNA markers. Eluted mRNA was

translated in

an

mRNA-dependent

rabbit

reticu-locyte cell-free

translation system. Measuring

[35S]methionine incorporation

induced by each mRNA

fraction,

twomajor peaks were obtained

corresponding

in size to 16S and 14S (data not

shown). The

[35S]methionine-labeled

polypep-tides immunoprecipitated

with guinea pig

anti-measles virus

sera were

characterized

by

elec-trophoresis

on a

10% SDS-polyacrylamide gel

(Fig.

8).

Under

the

fractionation conditions

used,

migration of

rRNA markers was found to

be

linear with

respect to the

logarithm

of

their

molecular weight. The peak fraction for each

translation

product could easily be determined.

Fractions for polypeptides

were as

follows:

frac-tion 9 (2,115

nucleotides) for

a 65K

protein,

fraction

14(1,735

nucleotides)

for a 60K

protein,

fraction

15 (1,677

nucleotides) for

a 70K

and

a 40K

protein, and

fraction 18

(1,515

nucleotides)

for

a

35K

protein (Fig.

9

and

Table

1).

DISCUSSION

A

clone

containing measles virus

DNA

se-quences

which

specify

most

of the nucleotide

sequences

coding for measles virus

nucleocap-N~ ~~~ .._~ ~ ~ ~~~ ~~~~~~~... Ci~~~~~~~~~~~~~~~~..

,N,

M.. _0

N- _~mmqAm_&_

_ _ W

e..

...sls

.."...mi. i_No ?--.X

do

am.

FIG. 8. Measles virus polypeptides obtained by in vitro translation of fractionated mRNA. Equal reaction volumes of

[35S]methionine-labeled

in vitrotranslation products, obtained with mRNA eluted from each gel slice,wereimmunoprecipitated with anti-measles guinea pig serum. (a) no exogenous mRNA; (b) mRNA from uninfected CV-1 cells; (c) mRNA from measles virus infected cells; (1 through 30) mRNA eluted from successive slices ofagel segment containing fractionated mRNA of 2,700 (fraction 1) to 1,000 nucleotides (fraction 30). VOL.42, 1982

I-, 1,

j A

on November 10, 2019 by guest

http://jvi.asm.org/

[image:6.487.91.409.253.613.2]

(7)

C. 'i "I I-1- (i _' _?_C

S-.Iii

.. a.

FIG. 9. Summary of the peak fraction foreachpolypeptide.

sid

protein

was

reported elsewhere

(6). The

isolation and

characterization of cDNA clones

that

appear to

specify the nucleotide

sequences

coding

for measles virus matrix

protein

and the

non-glycosylated

precursor

for

hemagglutinin

protein

are

reported

here.

A

series of 39 clones

containing measles virus

sequences were

identi-fied

by colony

hybridization, using

a

measles

virus-enriched

[32P]cDNA

probe (S.

Rozenblatt,

F.

S.

Neumann,

and C.

Gesang, submitted for

publication). The advantage of this

probe

is that

it

recognizes viral

nucleic acid

sequences

specif-ically and thus directly

discriminates

among

clones

containing plasmids

with virus DNA

in-serts.

Translation in vitro of the measles virus

RNA

purified by

hybridization selection

has

shown that the

cloned

double-stranded

cDNA,

contained within

Cl-M, encodes

a

portion

of the

measles

virus matrix

protein.

The

major

radioac-tive

polypeptide product

synthesized

in

a

cell-free

reticulocyte

translation

system

comigrated

with

authentic

matrix

protein and

was

the

only

polypeptide

immunoprecipitated by

antisera

against

measles virus. The double-stranded

TABLE 1. Correlation between measles virus mRNAsandpolypeptides

Molwt

Frac- Molwt No. of coding Molwt

tion of baesi capacity of

tion_fl* mRNA₍₁₀

₅₎

bA_mNA

_mRNAa

Ofa

protein

_(10-s)

(1O-3)

9 7.2 2,115 84.0 65

14 5.9 1,735 68.1 60

15 5.7 1,677 65.7 70

40

18 5.15 1,515 58.9 35

aCalculating coding capacity: RNA length was cor-rectedfor the poly(A) tail considered to be 100 nucleo-tides; the average molecular weight of an amino acid

was 125.

cDNA contained within the clone G encodes a

portion of the measles virus non-glycosylated precursorforhemagglutinin protein. Two major

radioactive polypeptide products were

synthe-sized inacell-free reticulocyte translation

sys-temusing the RNAs selected by hybridization;

thelarger product migrated atabout 70K, and

the second migrated at 40K. This result was

obtained with seven independently isolated

cDNAclones ofgroupG.The only polypeptide

immunoprecipitated with anti-measles virussera

was the 70K polypeptide. Since polypeptides

synthesized in a cell-free reticulocyte are not

glycosylated, it is not surprising that the

non-glycosylated precursor for the hemagglutinin

migrated faster than theauthentic protein during

electrophoresis. The nature of this polypeptide

was determined by theuse ofmonoclonal

anti-measles virus glycoprotein sera. The nature of

the40Kpolypeptide is notknown.

A previous attempt was made to correlate

mRNA species with virus polypeptides on the

basis of molecular weight (8). In the present

study, the sizes ofindividual mRNA species of

measles virus were determined by two

tech-niques. In one approach, poly(A)-RNA from

cells acutely infected with measles virus was

fractionated on agarose gels under denaturing

conditions

(Glyoxal)

and transferred to

diazo-benzyloxymethyl paper. The viral sequences

were then detected by hybridization with

32p-labeled individual cDNA clones (Cl-G, Cl-N,

and Cl-M). This study has shown that the sizes

of the mRNAs encoding the non-glycosylated

precursorforhemagglutinin and the major viral

nucleocapsid protein are similar (1,700 bases),

although the molecular masses of the two

pro-teins,asdeterminedbyelectrophoresis in

SDS-polyacrylamide gel electrophoresisare70K and

60K, respectively. The mRNA coding for the

matrixprotein is 1,550 bases long. The second

more general approach involved the separation

of different mRNA species into homogenous

w

..M.

^S

on November 10, 2019 by guest

http://jvi.asm.org/

[image:7.487.130.367.70.209.2] [image:7.487.50.242.515.633.2]

(8)

density gradients and sedimented

together

with-in the 16S to 18S region. Electrophoresis on

agarose-methyl mercuric hydroxide

gels proved

to

be a suitable fractionation method; the

mRNAs could be resolved and

recovered from

the

gel while retaining their

biologically active

form. The present study indicated the presence

of

four mature functional mRNA species: 2,115,

1,735, 1,680, and 1,515 nucleotides in size, in

direct correlation with the size of their encoded

proteins. A 65K protein encoded

by a

2,115-nucleotide mRNA is of an unknown nature and

could be a

candidate for the Fo fusion protein.

The 60K protein, corresponding to

an mRNA of

1,735 nucleotides, and the 35K

protein,

corre-sponding to an mRNA of 1,515 nucleotides, are

most

likely the nucleocapsid (N) and the matrix

(M)

proteins,

respectively. The 70K to 72K

protein

encoded by a fraction

containing mRNA

of 1,680 nucleotides is the non-glycosylated

pre-cursor

for the G protein hemagglutinin. This

same mRNA fraction was also found to encode a

40K

polypeptide; the nature of the 40K protein

is not yet clear.

ACKNOWLEDGMENTS

S.R. thanks E. Winocour for his support and encourage-mentand B. Bloom and V. ter Meulen forprovidingmeasles virus monoclonal antibodies.

This work wassupportedin partbygrants(toS.R.)from the VolkswagenwerkStiftung,theU.S.-Israel Binational

Founda-tion,andthe IsraelMultipleSclerosisSociety. S.R.holds the

CharlesH. RevsonCareerDevelopmentChair.

LITERATURE CITED

1. Alwine,S.C., D. J. Kemp, and G. R. Stark. 1977. Method

for detection ofspecificRNAsin agarosegels bytransfer todiazobenzoyloxymethyl-paper and hybridization with DNAprobe. Proc. Natl. Acad.Sci. U.S.A.74:5350-5354.

2. Aviv,H., and P. Leder. 1972.Purification ofbiologically

active globin messenger RNA by chromatography on

oligothymidylic acid-cellulose. Proc. Natl. Acad. Sci.

U.S.A.65:1408-1412.

3. Bailey,J. M.,and N.Davidson.1976.Methylmercuryas a reversibledenaturingagent for agarosegel

electrophore-Electronmicroscopicevidence forsplicing of SV40 late mRNA.Cell 13:783-790.

6. Gorecki, M., and S. Rozenblatt. 1980. Cloning of DNA

complementary to the measles virus mRNA encoding

nucleocapsid protein. Proc. Natl. Acad. Sci. U.S.A.

77:3686-3690.

7. HaUl, W. W., W. R. Kiesling, and V. ter Meulen. 1978. Membraneproteinsof subacutesclerosing

panencephali-tis and measlesviruses. Nature (London) 272:460-462. 8. HaUl, W. W., W. R. Kiesling, and V. ter Meulen. 1978.

Biochemical comparison of measles and subacute

scleros-ingpanencephalitis viruses, p. 143-156. In R. D. Barry and B. W. J. Mahy(ed.),Negativestrand viruses and the host cell. Academic Press, Inc., London.

9. Hall, W. W., and S. J. Martin. 1973. Purification and characterization of measles virus. J. Gen. Virol. 19:175-188.

10. Lamb, R. A., P. R. Etkind, and P. W. Choppin. 1978.

Evidence forninthinfluenza viral polypeptide.Virology

91:60-78.

11. Morgan, E. M., and F. Rapp. 1977.Measles virus and its associateddiseases. Bacteriol.Rev.41:636-666. 12. Pelham, H. R. B., and R. J. Jackson. 1976. An efficient

mRNA-dependent translation system from reticulocyte

lysates. Eur. J. Biochem. 67:247-256.

13. Riccardi, R. P., J. S. Miller, and B. E. Roberts. 1979.

Purification andmapping of specific mRNAs by

hybrid-ization selection and cell-free translation. Proc. Natl.

Acad.Sci.U.S.A. 76:4927-4931.

14. Rigby, P. W. J., M. Dieckmann, C. Rhodes, and P. Berg. 1977. Labelling deoxyribonucleic acid to high specific

activityin vitroby nick-translation with DNApolymerase

I.J. Mol. Biol. 113:237-251.

15. Rozenblatt, S., M. Gorecki, H. Shure, and C. Prives. 1979.

Characterizationof measlesspecific proteins synthesized

in vivo and in vitro fromacutelyandpersistentlyinfected cells. J.Virol. 29:1099-1106.