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0022-538X/91/073421-08$02.00/0

CopyrightC) 1991,American Society for Microbiology

Characterization of V Protein

in

Measles

Virus-Infected Cells

ELIZABETH A. WARDROPt ANDDALIUS J. BRIEDIS*

Department ofMicrobiology and Immunology, McGill University, 3775 University Street, Montreal, QuebecH3A2B4, Canada

Received 28 January1991/Accepted 22 March 1991

AneditedmRNAtranscribed fromthephosphoprotein (P)geneof measlesvirus (MV) has beenpredictedto encodeacysteine-rich proteindesignated V. ThismRNA containsasingleadditional nontemplated G residue whichpermitsaccesstoanadditional protein-coding reading frame.Suchanedited Pgene-specificmRNA has beendetectedinMV-infectedcells, butnocorresponding proteinhasyetbeenidentifiedinvivo.Wereportthe useof antiseradirected against synthetic peptides correspondingtofive different regions ofthepredictedMV

V proteinamino acidsequence toanalyse MV-specificproteins synthesized in vivoand in vitro. The MV V

protein (40 kDa) was detected in MV-infected cells in a diffuse cytoplasmic distribution, a predominant

subcellularlocalization distinct from that ofvirusnucleocapsids. The proteinwasfoundtobephosphorylated andtobemaximally synthesizedat16hpostinfection,whenMV-specific structural protein synthesiswasalso

maximal.Antiserumdirected againstapeptide(PV2) correspondingtoamino acids 65to87 ofthe Vprotein

aminoacidrecognized thePproteinbutnotthe Vprotein, indicatingthatthePand Vproteinsmaybefolded

differentlyat ornearthis region sothatthe PV2 sequence is inanexposed position at thesurface oftheP proteinbutnot atthesurface ofthe Vprotein.

Measles, ahighly contagious acute viral disease of child-hood, is caused by measles virus (MV), a member of the morbillivirus subgroup of theparamyxovirus family of neg-ative-stranded RNA viruses. Inrare cases,persistent

infec-tionwithMVresults in subacute sclerosingpanencephalitis,

a uniformly fatal central nervous systemdisease (25). For-tunately, infection with MV can be prevented by

adminis-tration ofalive attenuated virus vaccine(7, 8).

TheMV genome is a nonsegmented single-stranded neg-ative-sense RNA withanestimatedMrof4,500,000 (3, 14). The complete genomic sequence of MV has now been determined. It contains approximately 15,900 nucleotides,

the exact number depending on the virus strain (17). Six

structural proteins have their genes sequentially arranged

along the MV genome: the nucleoprotein (NP), the

phos-phoprotein (P), the matrix protein (M), the fusion protein (F), the hemagglutinin (HA), and the large or polymerase

protein (L). In addition, a small nonstructural polypeptide

(C)hasbeen identified inMV-infected cells(4).

It hasrecently been demonstrated that multiple different speciesofmRNAtranscriptscanbederived fromPgenesof a number of paramyxoviruses (6, 12, 19, 23, 24, 26, 27).

Although the process has been termed a form of RNA

editing,these different mRNAspeciesappeartoresult from

thecotranscriptional additionofnontemplatedGresiduesat atleastonespecificlocation(27).Thiscan causechangesin theprotein-coding readingframe insomemRNAs, leadingto thesynthesis ofatleasttwodifferentproteinscoterminalat theiraminotermini: the Pproteinandacysteine-rich protein termed V. Two slightly different coding strategies exist

among paramyxoviruses for the expression of P and V

proteins. TheMV and Sendai virus P proteins areencoded

by mRNAs faithful to the genomic sequence, while the

synthesisof Vprotein requiresanadditionalnontemplatedG residue within its mRNA sequence (6, 27). On the other

*Corresponding author.

tPresent address: Bio-Mega Inc., 2100, rue Cunard, Laval, QuebecH7S 2G5, Canada.

hand, during infection with simian virus 5 (SV5), mumps

virus, or parainfluenza virus type 2 or type 4, mRNAs faithful to the original genomic sequence encode V while mRNAs containing nontemplated G residues encodeP (12, 18, 19, 26). The relative frequency of mRNAs containing suchG insertions hasbeenestimatedtobe 50% forMV(6),

45% forSV5 (26), 31%for Sendai virus (27),and 40to50% formumpsvirus (19, 24).

An edited mRNAderived from the MV P gene has been identified in vivo (6) by cDNA cloning, cDNA and RNA

sequencing, and invitrotranslation. TheVprotein-specific

mRNAwasfoundtocontainanextraGnucleotideinserted at a site comprising three genomically encoded G residues

corresponding to positions 752 to 754 of the originally published P gene sequence (4). The edited mRNA was

predictedtoencodeaVprotein containing 299 amino acids, andacorresponding proteinbandcould beseenafterinvitro

transcription and translation of the corresponding cDNA

clone. The authors did not, however, provide any direct

evidence thataprotein correspondingtothe Vcoding region

was presentinMV-infected cells.

Inthis articlewe report the generationand

characteriza-tionof antisera directedagainstaseriesofsynthetic peptides corresponding to different regions of the predicted amino acid sequence of the MV V protein. We have used these antiseraas probesto definitely identify and tocharacterize the in vivo pattern of V protein expression in MV-infected cells.

MATERIALS ANDMETHODS

Cells and viruses. Vero cells were grown in Dulbecco's modification of Eagle's medium supplemented with 10% fetal bovineserum.The Edmonston strain ofMV(American

Tissue Type Collection) was propagated as previously de-scribed (2).

Generationofantiseraagainst synthetic peptides. The syn-thesis of five peptides correspondingtodifferentregions of

the MV Vprotein (Table 1) wascommissionedfrom

Immu-nodynamics (La Jolla, Calif.). Peptides were coupled to

3421

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TABLE 1. Amino acid sequences of synthetic peptides usedto

generatemonospecific and polyspecific rabbit antisera against the MV P and Vproteins

Location within

Peptide Amino acid sequence Vproteina

PV1 IRALKAEPIGSLAIEEAMAA 16-35

PV2 LSAIGSTEGGAPRIRGQGPGESD 65-87

PV3 DIGEPDTEGYAITDRGSAPIS 154-174

Vi SKVTLGTIRAR 256-266

V2 RTDTGVDTRIWYHNLPEIPE 280-299

a Numberingreflectsthe sequenceofMV Vproteinassuming addition ofa single nontemplated Gresiduetothethreetemplated G residuesatpositions 252 to 254of thepublishedMV P genesequence(4)assubsequently corrected

(6).

keyhole limpet hemocyanin (KLH) or bovine serum albu-men(BSA) by addition ofan amino-terminal cysteine

resi-due before treatment with mmaleimidobenzoyl-N-hydroxy-succinimide ester. Peptide V2 (corresponding to the exact

carboxylterminusof V)was leftwithafree carboxylgroup

at its carboxyl terminus, while all other peptides were

synthesizedwithcarboxyl-terminal carbaminogroups.

DuplicateNewZealandWhite rabbitswereinitially

immu-nized with peptide-KLH conjugate in Freund's complete adjuvant. Approximately 0.2 mg ofpeptide conjugate was

injected subcutaneouslyateach of five sitesalongtheback. Rabbitsweresubsequently reimmunizedatotalof fourtimes

at 4- to6-week intervals in a similar manner with

peptide-conjugate in Freund's incomplete adjuvant. All

resulting

antisera were tested for their ability to react with the

peptides used in immunization by enzyme-linked immuno-sorbent assays (ELISAs) with peptide-BSA conjugates as

theantigen. Preimmunesera wereusedasnegativecontrols. Construction of plasmids for in vitro transcription of

mRNAsspecific tothe PandVproteins. The MV Vtranscript differs from the MV P transcript by the addition of a

nontemplated Gresidueatthe stretch of threetemplated G

residues at positions 752 to 754. We had previously

de-scribedcloning ofacDNA ofthe MV P coding region into thePstI site ofpBR322, creating pMV-P(2).We have now

usedasite-specific mutagenesis protocolto covertthis intoa

copy ofthe V protein-coding region. The P coding region consistedof1,565nucleotides bounded bya5'XhoIsite and

a3' HincII site. The plasmid containing this coding region

was digested with Sacl to release a DNA fragment 765

nucleotidesinlength thatcontainedtheGinsertion site.The

fragment was then ligated into the SacI site of M13mpl9 (Bio-Rad), and oligonucleotide-directed site-specific

muta-genesis was performed according to the instructions ofthe

supplier. The primer used for mutagenesis (Regional DNA

SynthesisLaboratory, Universityof Calgary, Calgary,

Can-ada)had the sequence

5'-dCCATTAAAAAGGG_jCACAG

AG-3',where the underlined G was the additional

nontem-plated nucleotide. Confirmation of successful mutagenesis was obtained by dideoxy chain termination sequencing of

alkali-denatured plasmid DNA performed with

deoxyade-nosine-5'-[35S]thiotriphosphate

(500 Cilmmol; Amersham

Corp.)and T7 DNA polymerase (Sequenase; United States

Biochemical Corp.) accordingtothe protocol of the

suppli-ers. The primer used for sequencing was 5'-dCTTTCCGA AGCTTGG-3' (Regional DNA Synthesis Laboratory,

Uni-versityofCalgary). The mutated Sacl fragment was inserted

into the Sacl site of pMV-P to create plasmid pMV-V,

containing the V coding sequence. Plasmids pMV-P and

pMV-V

were

digested

withXhoIandHinclltoreleasethe P and V

coding

sequences, which were then

separately

in-serted into an SP6/T7 vector

(Amersham)

to create

pAM18MV-P

and

pAM19MV-V,

respectively.

Invitrotranscriptionandtranslation. Invitro

transcription

in the presence ofthe cap

analog

diguanosine

triphosphate

[m7G(5')ppp(5')Gm;

Pharmacia] was

performed

with SP6

polymerase

according

to the instructions of the

supplier

(Pharmacia).

The

resulting

RNA

transcripts

weretranslated in micrococcal nuclease-treated rabbit

reticulocyte

lysate

according

to the instructions of the

supplier (Promega

Corp.).

In vivo radiolabeling of infected-cell proteins. Confluent

monolayers

of Vero cells were infected with MV at a

multiplicity

of infection of10PFUpercell. Cellswere

pulse-labeledasdescribed

previously (2)

with either

Tran[35S]label

(ICN

Radiochemicals),

[35S]cysteine, [35S]methionine,

or

32p;

(Amersham

Corp.)

in minimum essential medium defi-cient in

methionine, cysteine,

or

phosphate, depending

on thelabeled substance added.

Analysis of

MV-specific proteins.

In vivo- and in

vitro-synthesized

proteins

wereincubatedin RIPAbuffer contain-ing 0.1% BSAand the

appropriate

antiserum for2 hat

4°C.

For

immunoprecipitation

of in

vivo-synthesized

proteins,

100 ,ul of cell

lysate

was mixed with 5 ,ul of antiserum. For

immunoprecipitation

ofin

vitro-synthesized

proteins,

5

,ul

of

the 50-,ul total translation reaction was mixed with 5

,ul

of antiserum. Protein

A-Sepharose

beads

(Pharmacia)

were

subsequently

addedtoeach

mixture,

andthe mixtureswere then incubated for 1 h at

4°C.

Immune

complexes

were

washed three times with RIPA buffer

containing

1% BSA and twice with RIPA buffer without BSA. Then 35

p.l

of

protein lysis

bufferwas

added,

incubatedat

37°C

for10

min,

heatedto

95°C

for3min,and

analyzed

by

10%discontinuous sodium

dodecyl

sulfate-polyacrylamide gel

electrophoresis

(SDS-PAGE)

according

to the methoddescribed

by

Laem-mli(13).

Indirect immunofluorescence. Vero cells were grown to

40%

confluence on

microscope

coverslips

before infection

with MV.UninfectedVerocellswereusedascontrols.After 16

h,

cells were washed with

phosphate-buffered

saline

(PBS)

and fixed

by

treatment with 2.5%

paraformaldehyde

for20min. Cellswere

permeabilized by

treatmentwith0.5% TritonX-100 in PBSfor10min.

Following

three washes with

PBS,

cellsweretreatedwithnormal goatserum

(Cedarlane)

for30minand thenwith

primary

antiserum for1h. Acontrol

was done without addition of

primary

antibody

for both infectedanduninfected cells. Cells werethenwashed three

times with PBS and treated with goat anti-rabbit

immuno-globulin

G

(IgG) coupled

to fluorescein

isothiocyanate

(FITC) (Cedarlane)

for30min. Cellswerewashed

again

with

PBS before inversion over a

drop

of

glycerol

containing

Citofluor

(Amersham)

on

glass

slides for

photography.

RESULTS

Preparation of antibodies directed against synthetic pep-tides. In order to study the

expression

of V

protein

in MV-infected

cells,

five

peptide

sequences werechosen from the

predicted

amino acid sequence ofthe V

protein

for the

production

of

antipeptide

antibodies

(Table 1).

Peptides

were chosen to maximize their

hydrophilicity

while also

optimizing

location and

secondary-structure

predictions.

Threeof the

peptides synthesized

(PV1, PV2,andPV3)map

totheamino-coterminal

region

ofthePandV

proteins,

and

antibodies directed

against

these

peptides

were

expected

to

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it

u I u I u I u I

200000 97 400

68000

2 0- V V > >> > > > _ - c N

a.. a.CL4 a. a. a. > > > >

M C M C M C M C M C M C MC

200000

97 400

68 000

43 000

43 000

[image:3.612.327.553.81.269.2]

29 000

FIG. 1. Analysis of [35S]methionine-labeled polypeptides from uninfected (U) Vero cells as well as from Vero cells 16 h after

infectionwith MV (I). Cell lysates were immunoprecipitated with

preimmuneserum(Pre)orpeptideantiserum PC20,PV1,PV2,PV3, Vi,orV2. Sampleswere analyzedon a10% SDS-polyacrylamide

gel. Positions ofmolecular weightmarkers (not shown)areindicated ontheright.

be able to recognize both P and V. The remaining two peptides (Viand V2)maptotheuniquecarboxyl terminus of

the Vprotein,andantibodies directed against these peptides

wereexpectedtobe able torecognize theVproteinalone. Aftersynthesis, the peptideswereconjugated with KLH, and each was used to immunize two New Zealand White rabbits. The titers of the resulting antibody response were

determinedbyELISA with preimmuneserumas anegative

control. Afteraseriesoffiveimmunizations foreachrabbit, all antisera had titers ofat least 105 except that directed against Vi. This last antiserumreached atiter of only 5 x

104. Nevertheless, the antibody titer of the antiserum

di-rectedagainst theVi peptide would normally beconsidered adequate for use in immunoprecipitation. Preimmune sera

from all 10 rabbits werepooled and then usedas anegative control. Additionally, peptide antiserum PC20 (generously provided by C. D. Richardson), directedagainsta carboxy-terminal region unique to the P protein (20) and thus not expressedinthe Vprotein, wasalso usedas acontrol.

Detection ofVproteininMV-infectedcells. To determine

whether theMVV protein was indeed synthesized invivo,

[35S]methionine-labeled proteins from MV-infected Vero

cells were immunoprecipitated with our different antisera

and analyzed by SDS-PAGE. The MV P protein has a

predicted size ofapproximately 72 kDa (17), while the V

protein has beenpredicted tobeapproximately 40 kDa (6).

Noproteinsweredetectedwhenuninfected-celllysateswere

immunoprecipitated with any of our peptide antisera or when infected-cell lysates were immunoprecipitated with

preimmune serum(Fig. 1).However,twopolypeptideswith

migrations correspondingto70and40 kDaweredetected in MV-infected cell lysates in patterns corresponding to the

predicted specificity of the different peptide antisera and wereconsideredlikelytobethe PandVproteins. Evidence in support of such identification is that (i) the 70-kDa

polypeptide migratedatthesamepositionasthe P

immuno-precipitated by thePC20 antiserum (Fig. 1,lane 2), (ii) the

29 000

FIG. 2. Analysis of polypeptides from Vero cells 16 h after infection withMVfollowingdifferentialradiolabeling with

[355]me-thionine (M) or [35S]cysteine (C) and immunoprecipitation with preimmune serum (Pre) or peptide antiserum PC20, PV1,PV2,PV3,

Vi,

orV2. Samples were analyzed on a10%SDS-polyacrylamide gel. Positions of molecularweight markers (not shown)areindicated ontheright.

70-kDapolypeptidewasrecognized by antisera predictedto

bespecifictoboth the PandVproteins (PV1,PV2,andPV3) butnotby antiserapredictedtobespecifictothe Vprotein

alone

(Vi

and V2), and (iii) the 40-kDa polypeptide was

recognizedby theV-only specific antiserumV2 aswellasby

antiseraspecifictoboth Pand V (PV1 and PV3) but notby

the P-onlyspecific antiserum PC20.

Despite multiple attempts at immunoprecipitation and

autoradiographs ofmuch longer exposure and greater

inten-sitythan those shown, neitherthe

Vi

northe PV2 antisera could detecta band correspondingto the V protein. How-ever, the

Vi

peptide was predicted tobe the most

hydro-phobic ofoursynthetic peptides. The region of amino acid sequence unique to V encoded after the G insertion site contains only 62 amino acids. The

Vi

peptide sequence

seemed, after that of V2, to be the most favorable for antibody recognition based on secondary-structure

predic-tions. Suchpredictionsdo not, however, guaranteeantibody recognition ofthe native protein. The PV2 antiserum was able to immunoprecipitate the 70-kDa polypeptide but not

the 40-kDa

polypeptide,

although the sequence of the pep-tidewasderivedfromaportion oftheP/V amino-coterminal region. This may indicate that the P and V proteins are

folded differently at or near this region so that the PV2 sequence is in an exposed position at the surface of theP

proteinbutnot atthe surface ofthe Vprotein.

Immunoprecipitation of differentially radiolabeledproteins

from MV-infected cells. In order toprovide more evidence that the 70- and 40-kDa polypeptides indeed

represented,

respectively, the P and V proteins,MV-infected cellswere

separately and differentially radiolabeled with either

[35S]methionine

or

[35S]cysteine,

immunoprecipitated

with

our different

antisera,

and then

analyzed by

SDS-PAGE (Fig. 2). The MV P protein contains 11 methionine and 6

cysteine residues, whereas the V

protein

is

predicted

to

contain5methionine and 11

cysteine

residues.Itcanbeseen in thecontrol lanes, where the PC20antiserum was

used,

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PV1 V2

UN 12 14 16 18 20 22 UN 12 14 16 18 20 22

200000

InVitro InVivo

NORNA P V

PV1V2 PV1V2 PV1 V2 PV1V2 200 000

97400

97400

68000

43000

*_ 68000

43000

4,. 29000

FIG. 3. P and V protein synthesis late in infection. Vero cells were labeled with [35S]methionine at the times noted (in hours) following infection with MVandimmunoprecipitated with peptide antiserum PV1 or V2. Samples were analyzed on a 10% SDS-polyacrylamide gel. Positions of molecular weight markers (not shown)areindicated on theright. UninfectedVerocells(UN)were used as acontrol.

that the efficiency ofcysteine labeling was notas great as

thatof methioninelabeling. WhileabandrepresentingtheP

protein labeled with methionine was clearly visible, no

equivalentbandrepresentingPproteinlabeled withcysteine

could beclearly identified.Thiscould be duetoanumber of

factors, includingdifferentialstabilityof the radiochemicals

or their differential transport into cells, as well asdifferent

cellularamino acid pool sizes. The Pproteinwas alsomore

readily labeled with methionine than with cysteine when

immunoprecipitated with the PV1, PV2, or PV3 antiserum

(Fig. 2). Radiolabeling ofthe 40-kDapolypeptide with

cys-teine, onthe otherhand,wasrelativelyeasiertoaccomplish (Fig. 2, lanes immunoprecipitated with PV1, PV3, and V2

antisera). The data therefore support the identification of these twopolypeptides asthe MV P andVproteins.

Time course of V protein synthesisduringMVinfection. To determine the time course ofV protein synthesis in vivo,

cells were labeled with

[35S]methionine

at different times

followingMVinfection. Labeledproteins were immunopre-cipitated with PV1 or V2 antiserum and analyzed by SDS-PAGE. Neither the P nor the V protein was detectable before 8 to 10 h postinfection (data not shown). Figure 3

shows levels of synthesis ofthe P and V proteins at later times after infection. Maximal synthesis occurred at 16 h afterinfection,atwhichtimeapproximately80to90% of the cells exhibited cell fusion. The time of maximal in vivo synthesis of MV structural proteins for the virus stock used had also previously been determined to be 14 to 18 h after

infection (data not shown).

Analysis of polypeptides synthesized in vitro. To confirm the specificity of the peptide antisera for the P and V

proteins, theantisera weretested for their ability to

immu-noprecipitate in vitro-synthesized P and V. Two plasmids, pAM18MV-P and pAM19MV-V, containing, respectively, the P and Vcoding regions,wereconstructed so as tobe able

todirectthe invitrotranscriptionof RNAs specific to the P

and V

proteins.

Rabbitreticulocyte

lysate

wassubsequently

29000

FIG. 4. Comparison ofPand Vproteins synthesized in vivoand

in vitro. [35S]methionine-labeled polypeptides from Vero cells 16 h after infection with MV and [35S]methionine-labeled polypeptides resulting from in vitro translation in rabbit reticulocyte lysate of RNA transcribed in vitro from linearized DNA from plasmids pAM18MV-P (P) and pAM19MV-V (V) wereimmunoprecipitated withpeptide antiserum PV1 orV2 and analyzed on a 10% SDS-polyacrylamide gel. Positions of molecular weight markers (not shown) are indicated ontheright.

used for in vitro translation reactions with the in

vitro-transcribedRNAs.

Polypeptides of 70 and 40 kDawere immunoprecipitated from in vitro translation reactions by peptide antisera and

were not presentin control invitro translationreactions to

which RNAwas not added (Fig. 4). In vitro-synthesized P protein was consistently found tobe less abundant than V protein. This is probablythe result of the inherently lower

efficiencyof thereticulocyte lysate systemin thetranslation

of higher-molecular-weight species but may also reflect differential in vitro stability of the proteins. Proteins were also immunoprecipitated from MV-infected cell lysates to compare the migration of in vivo- and invitro-synthesized

proteins.Polypeptides synthesizedinvitrocorrespondingto

both Pand Vmigrated slightlyfaster thanthecorresponding proteins synthesized in vivo. This probably indicates that these proteins exhibit different degrees of posttranslational modificationin vivo and in vitro.

Proteins synthesized in vitro were differentially labeled with either [35S]methionine or [35S]cysteine of equivalent total andspecificactivityandanalyzedby SDS-PAGE (Fig. 5). Under these conditions, the intensity of labeling ofthe 70-kDapolypeptidewas atleastasgreatwithmethionineas

withcysteine. The intensity of labeling of the 40-kDa

poly-peptide was, however, significantly greater with- cysteine thanwith methionine. This pattern of labelingis consistent with the different expected ratios of methionine to cysteine in the P and V proteins. Since stability, cell transport, cellularamino acidpool size, etc., are not factorsin vitro,

these results stronglyconfirm theidentityof theseproteins.

Analysis of 32P-labeled P and V proteins. In order to

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canclearlybe seen that the V protein isalsophosphorylated

in vivo.

Subcellular localization of the MV P and V proteins. In ordertodetermine the exact cellular localization of the P and V proteins in MV-infected cells, indirect

immunofluores-cenceanalysiswasperformed.Uninfected cells exhibitedno

fluorescence with any of theantisera (Fig. 7). Similarly, no

fluorescence was observed in uninfected or MV-infected cells that were not treated with primary antibody (data not

shown).The PV2antiserum(specificonlytoPproteinwhen used for immunoprecipitation) resulted in specific fluores-cencein small discrete cytoplasmic inclusions. This result is similar to that described previously (4) for a peptide antise-rum against a carboxy-terminal region of P which is not presentin V. Hence, thespecificity of immunofluorescence resulting from use of the PV2 antiserum does seem to represent P alone. The V2 antiserum(specificonly to the V protein when used for immunoprecipitation) resulted in a different pattern, a diffuse cytoplasmic fluorescence. The PV1 antiserum (specific to both the P and V proteins when used for immunoprecipitation) gave a result appearing to combine thepatterns obtained with the PV2 and V2 antisera.

FIG. 5. Analysis of invitro-synthesized P and V proteins differ-entially radiolabeled with either[35S]methionine(M)or[5S]cysteine (C)atequivalent total and specific activities. Linearized DNAfrom plasmids pAM18MV-P (P) and pAM19MV-V (V)wastranscribed in vitro, and the resulting mRNAsweretranslated in vitro inarabbit

reticulocyte lysate. Polypeptides were analyzed on a 10%

SDS-polyacrylamide gel. Positions of molecular weight markers (not shown)areindicatedonthe right.

determine whether the V proteinwas phosphorylated,

MV-infected cells were pulse-labeled with

32pi

at 16 h after infection,immunoprecipitated, and analyzed bySDS-PAGE (Fig. 6). The P protein has previously been shown to be highly phosphorylated (10), and ourresults confirmthis. It

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FIG. 6. Analysis of polypeptides from uninfected Vero cells (U)

andfrom Vero cells 16 hafterinfection with MV (I). Cellswere pulse-labeled for 1 h with32Pi.Celllysateswereimmunoprecipitated withpreimmuneserum(Pre)orpeptide antiserum PC20, PV1,PV2, PV3,Vl,orV2.Sampleswereanalyzedon a10%o SDS-polyacryl-amidegel. Positions of molecular weight markers (not shown)are indicatedontheright.

DISCUSSION

Since Thomasetal. (26) reported that the SV5 P protein

wasexpressed fromanedited mRNA transcript,anumber of groups have reported similar RNA editing in transcripts arising from the Pgenes ofanumber of other paramyxovi-ruses. The discovery of this type of RNA editing came

together with the discovery of a previously unknown

paramyxovirus-encoded protein which has cometo be des-ignated the V protein. Analogous V proteins havenowbeen

detected in cells infected withanumberof paramyxoviruses.

Pprotein-specific monoclonal antibodies were used for the

detection of V protein in SV5- and parainfluenza virus type 2-infected cells (23, 26). Antisera directed against synthetic peptides were similarly used to detect V in mumps virus-infected cells(19, 24). An edited P gene-specific mRNA has been detected in MV- and Sendai virus-infected cells, but therehas beennodirectevidence for the in vivo existence of

either a Sendai virus- or MV-specific V protein. Synthetic

peptide-specific antisera directed against the MV Vprotein

wereproduced in the present study for this purpose. With

these antisera, acysteine-rich polypeptide was detected in

MV-infectedcellsbyimmunoprecipitation and

immunofluo-rescence analysis. The V proteinwasfound tomigrate as a

40-kDapolypeptide. Rimaet al. (21) reported the detection ofan MV-specific 40-kDa protein possibly related tothe P protein that comigrated with the MV M protein and used partialproteasedigestion tosuggestthat itwasnotsimplya

breakdownproduct ofP. Wenowreportthe definite identi-fication of the MV V protein, andwe have also found itto comigrate withthe Mprotein (datanotshown). This proba-bly explains previous difficulties in recognizingand identify-ing the MV V protein. No strong signal at 40 kDa had

previously been detected in MV-infected cells after

immu-noprecipitation with antibodies directed against the exact amino terminus of the Pprotein(4).Thismaybe becausethe

carboxyl terminus ofparamyxovirus P proteins is, in

gen-eral, moreimmunogenic than the restof the molecule(28).

Thegelmigration ofP and Vproteinsimmunoprecipitated

from MV-infected cells was compared with that of those

immunoprecipitatedfrom in vitro translation reactions. The

invitro-synthesized P and Vproteinswerefoundtomigrate slightly faster than those immunoprecipitated from

MV-P v

M C M C

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FIG. 7. Indirectimmunofluorescenceanalysis of theMV PandVproteinsinuninfected Verocells and Vero cells 16 h afterinfection with MV. Cellswerefixed in2.5% paraformaldehyde in PBS,permeabilized with0.5% Triton X-100 inPBS,and then incubatedsequentiallywith normal goat serum,peptide antiserum, and FITC-conjugated goat anti-rabbit IgG. (A)Uninfected, PV2antiserum;(B) MVinfected, PV2 antiserum; (C) uninfected, V2antiserum; (D)MVinfected,V2antiserum; (E) uninfected,PV1antiserum;(F) MVinfected,PV1antiserum. UVphotomicrographs takenat x400magnification. Photomicrograph exposure times forpanels A,C,and Ewereadjustedtobe thesame asthosefor panels B, D, and F.

infected cells (Fig. 4). This probably indicates that different posttranslational modifications occur in vivo and in vitro.

Such differences would not be surprising since the rabbit reticulocyte lysate system has been shown to contain a

variety of posttranslational processing activities whichmay leadtogel migration differences. These include proteolysis (15), phosphorylation (9), acetylation (22), and isoprenyla-tion (29). The Pprotein is knowntobe phosphorylated, and sincethe Vproteinshares theamino-terminal 231 of its 299 amino acids with the amino terminus of the P protein, the possibility ofphosphorylation of the V protein was

exam-ined. Immunoprecipitation of32P-labeledproteinsfrom MV-infected cells indicated that the Vproteinwasindeedhighly phosphorylated(Fig. 6). Differential phosphorylationinvitro

and in vivo of the Vprotein may explain differences ingel migration. This does not exclude the possibility that other

modifications may distinguishthe V protein synthesized in

vivo fromthat synthesizedinvitro.

Patersonand Lamb (19)have reportedthat anadditional

mumpsvirus-specific protein,designatedI,of

approximately

19kDa is encodedbyanadditional

species

ofeditedmRNA

transcribed from the virus P gene. In this transcript, four

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nontemplated G residues are added instead of thetwowhich give rise to the P mRNA. This results in a shift into yet another reading frame distal to the G insertion point which is distinct from that of either P or V. Takeuchi etal. (24) have reported immunoprecipitation of a mumps virus-specific

protein of approximately 24 kDa with antiserum directed against a synthetic peptide corresponding to a part of the

amino-coterminal region of P and V. This protein may well be the product of an mRNA to which four nontemplated G residues are added. An analogous protein could potentially beencoded by MV if G residues were added to the insertion

point in the P gene transcript to cause a shift into the +1

reading frame relative to that of P (i.e. 2, 5, 8, etc.). Translation of such a message would terminate only five codons afterthe insertion site. Cattaneo et al. (6) reported that 2 of 19 cDNA clones specific to MV P gene-encoded mRNA species had insertions of three G's, but no clones wereobtained which could encode a putative MV I protein. However,thismayonlyreflect examinationof aninsufficient number orunrepresentative sampling of cDNA clones. We wereunabletodetect any suchputativeMV-specificprotein with our antisera forimmunoprecipitation analysis of

MV-infected cell lysates. We were, in some instances, able to detect proteins of low molecular weight, but these did not appear to be additional species of amino-coterminal yet

carboxy-dissimilar proteins since they were also

immuno-precipitated with the PC20 antiserum, which is directed

against acarboxyl-terminal region of the P protein (Fig. 2). The exact nature ofthese proteins has not yet been fully determined.

The MV Vprotein isdiffuselypresentin the cytoplasmof MV-infected cells (Fig. 7). The P protein localizes in a

distinctly differentpattern, in small brightcytoplasmic inclu-sions. Earlier studies have shown that such cytoplasmic inclusionsalsocontainviralnucleocapsids(11, 16). Since the P protein thus appears to be a marker for nucleocapsids,

most MV V proteindoes not therefore appear to be associ-atedwith nucleocapsids duringthe period of maximal virus

protein synthesis. In fact, when MV-infected cells were

analyzedwith an antiserum able torecognize both P and V, amixture of both of thesedistinctfluorescencepatterns was seen.Thisdoesnot,however, exclude the possibilitythat a very small proportion of V protein may nonetheless be

associated with nucleocapsids. A number ofinvestigators

have reported that anti-P antibodies are also able to

immu-noprecipitate the V, NP, and possibly also L proteins of some paramyxoviruses and have taken this to imply an

association ofVwithtranscriptional complexes(24, 26). We

also found that asmall amount oftheNP protein could be

immunoprecipitated when either P- orV-specific antiserum

was used. However, we have previously found that small amounts ofthe NPcould be immunoprecipitated with

anti-serum directed against the MV hemagglutinin (1). The

sig-nificance ofthis phenomenon remains unclear.

The function of the paramyxovirus V protein remains

obscure. An open reading frame for V protein is highly

conservedamongparamyxoviruses, and itthus seemslikely that it has an important biologicalrole. Since the cysteine-rich domain in theunique region ofparamyxovirus V

pro-teins resembleszincfinger motifsthat have beenidentifiedas

participatinginbinding withnucleicacidsorproteins,it has beensuggested that the Vproteinmay be aregulatory factor involved in virus RNA orproteinsynthesis(6, 26). Delinea-tion of the exact nature of the funcDelinea-tion of the Vproteinwill

involve additional research, likely requiring the ability to

produce large amounts of purified protein for functional

assays or elsethe developmentof virusmutantsdeficient in

theexpression ofVprotein.

ACKNOWLEDGMENTS

Wethank Talia Pakos for excellent technicalassistance andPeter Liston and Francoise Blain forhelpful discussion.Wethank Chris-topher Richardson for the generousgift ofpeptide antiserum PC20 andforstimulating scientific discussion.

This workwassupported byaresearchgrant from the Multiple SclerosisSociety of Canada.E.A.W. was supportedbyfellowships

from theRoyal VictoriaHospital Research Institute and theRoyal Victoria Hospital Department of Medicine. D.J.B. is a Chercheur-Boursier-Clinicien ofleFonds de laRechercheenSanteduQuebec.

REFERENCES

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3. Baczko, K., M. Billeter, and V. ter Meulen. 1983. Purification and molecular weight determination of measles virusgenomic RNA. J.Gen. Virol. 64:1409-1413.

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6. Cattaneo, R., K. Kaelin, K. Baczko, and M. A. Billeter. 1989. Measles virus editingprovides anadditionalcysteine-rich pro-tein. Cell 56:759-764.

7. Enders, J. F. 1962. Measles virus: historicalreview, isolation and behaviorin various systems. Am. J. Dis. Child. 103:282-287.

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genes ofhumanparainfluenzatype4Aand4Bviruses and RNA editing at transcript of the Pgenes: thenumberof G residues added isimprecise. Virology 178:321-326.

13. Laemmli,U. K.1970.Cleavageof structuralproteinsduringthe assembly of the head ofbacteriophage T4. Nature (London)

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14. Lund,G.A.,D. L.J. Tyrell,R. D.Bradley,andD.G. Scrabba. 1984. Themolecularlengthof the measles virus RNA and the structuralorganizationof measlesnucleocapsids.J.Gen. Virol. 65:1535-1542.

15. Mumford, R. A., C. B. Pickett, M. Zimmerman, and A. W. Strauss. 1981. Protease activities present in wheat germ and rabbitreticulocyte lysates. Biochem. Biophys. Res. Commun. 103:565-572.

16. Nakai, T.,F.L.Shand,and A. F. Howatson.1969.Development

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17. Norrby, E., and M. M. Oxman. 1990. Measlesvirus, p.

1013-1044. In B. N. Fields andD. M. Knipe (ed.), Fields virology, 2nded. Raven Press,Ltd.,NewYork.

18. Ohgimoto,S., H. Bando, M. Kawano, K. Okamoto, K. Kondo,

M. Tsurudome,M. Nishio, andY.Ito. 1990.Sequenceanalysis of Pgeneof humanpara-influenzatype2virus: P and cysteine-richproteins are translatedby two mRNAs thatdiffer by two

nontemplated G residues.Virology177:116-123.

19. Paterson, R. G., and R. A. Lamb. 1990. RNA editing by G nucleotide insertioninmumpsvirusPgenemRNAtranscripts. J. Virol.64:4137-4145.

20. Richardson, C. D., A. Berkovitch, S. Rozenblatt, and W. J. Bellini.1985. Use ofantibodies directed againstsynthetic pep-tidesforidentifyingcDNAclones,establishing readingframes, anddeducingthegeneorderofmeasles virus. J. Virol. 54:186-193.

21. Rima, B. K., S. A. Lappin, M. W.Roberts, andS. J. Martin. 1981. A study of phosphorylation ofthe measles membrane protein. J.Gen.Virol. 56:447-450.

22. Rubinstein, P., and J. Deuchler. 1979.Acetylatedand

nonacety-lated actins inDictyostelium discoideum. J. Biol. Chem. 254: 11142-11147.

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nontemplated nucleotide additionsarerequiredtogeneratethe PmRNA ofparainfluenzavirus type2since the RNAgenome encodesproteinV.Virology 177:338-390.

24. Takeuchi,K., K.Tanabayashi,M.Hishiyama,Y. K.Yamada, A.

Yamada, and A. Sugiura. 1990. Detectionandcharacterization

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25. ter Meulen, V., J. R. Stephenson, and H. W. Kreth. 1983.

Subacute sclerosingpanencephalitis.Compr. Virol.18:105-185.

26. Thomas, S. M., R. A. Lamb, and R. G. Paterson. 1988. Two

mRNAsthatdifferbytwonontemplatednucleotides encode the aminocoterminal proteinsPand Voftheparamyxovirus SV5. Cell54:891-902.

27. Vidal, S., J. Curran, and D. Kolakofsky. 1990. Editingof the SendaivirusP/C mRNAbyG insertionsoccursduring mRNA synthesisviaavirus-encoded activity. J. Virol.64:239-246. 28. Vidal, S., J. Curran, C. Orvell, and D. Kolakofsky. 1988.

Mappingofmonoclonal antibodiestotheSendaivirus Pprotein and the location of itsphosphates. J. Virol.62:2200-2203. 29. Vorburger, K., G. T. Kitten, and E. A. Nigg.1989.Modification

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Figure

TABLE 1.generate Amino acid sequences of synthetic peptides used to monospecific and polyspecific rabbit antisera against theMV P and V proteins
FIG. 1.uninfectedgel.preimmuneinfectiononVi, Analysis of [35S]methionine-labeled polypeptides from (U) Vero cells as well as from Vero cells 16 h after with MV (I)
FIG. 3.followingpolyacrylamideantiserumwereusedshown) P and V protein synthesis late in infection
FIG. 6.andpulse-labeledindicatedwithPV3,amide Analysis of polypeptides from uninfected Vero cells (U) from Vero cells 16 h after infection with MV (I)
+2

References

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