Functional cDNA library for efficient expression of measles virus-specific gene products in primate cells.

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JOURNALOF VIROLOGY,Jan. 1986, 343-348 0022-538X/86/010343-06$02.00/0

Functional cDNA Library

For

Efficient

Expression of Measles

Virus-Specific

Gene Products in Primate Cells

TIMOTHY C. WONG*AND AKIKO HIRANO

DepartmentofMicrobiology and Immunology, Universityof Washington Schoolof Medicine, Seattle, Washington 98195

Received 1July 1985/Accepted 10 September 1985

AcDNA library designed for high-levelexpressionof measlesvirus-specificgeneproductsin mammalian cells was generated. From this library, functional clones which contained the entireprotein-codingsequences of the nucleocapsid (N) and the phosphoprotein (P) genes were isolated. By DNA-mediated gene transfer into a line of simian virus 40-transformed monkey kidney cells, the N-specific cDNA was expressed into a single

polypeptide of about 60,000Mr, which wasimmunoprecipitated by monoclonal antibodiesagainstthemeasles

virus N protein. In contrast, the P-specific cDNA could be expressed into either one or two species of polypeptides of 75,000 or 70,000

Mr,

bothof which wereimmunoprecipitated bymonoclonal antibodiesagainst

the measles virus P protein.

Measles virus possesses a negative-strand RNA genome which encodes six virion proteins: a 60,000-dalton

nucleo-capsid protein

(N), a 70,000-dalton phosphoprotein (P), a 38,000-dalton matrixprotein (M), a60,000-dalton precursor

of the fusionprotein (Fo),a80,000-daltonhemagglutinin (H), and a high-molecular-weight protein presumed to be the polymerase (L) (12).Inaddition,measlesvirus-infected cells

contain a21,000-daltonvirus-specific nonstructural protein, NS (12),which may beidenticalto theCprotein expressed fromthe P genefromanalternative reading frame (4).

Measles virus can cause acute as well as chronic infec-tions. Variousreportshavesuggestedthatchronic infections by measles virus may be caused by aberrant expression of some of these viral gene products (7, 14, 25). There is, however, little directevidencetosubstantiate these

hypoth-eses. A major obstacle in

studying

the different manifesta-tions of measles virusinfection isthe poorunderstanding of thefunctioning of itsgenes andgeneproducts. Experimen-tation is further complicated by the

complexity

of the interaction among thedifferent viral proteinsaswellashost factors.

Wewishtoapproach these problemsby studyingthe gene

products expressedfrom clonedgenes ofmeasles virus.To

facilitate these studies,a cDNAlibrarywas constructed by using a vector system which allows the expression of full-lengthcDNAclones ineucaryotic cells (20).

Africangreen monkey kidney (CV-1) cells were infected with Edmonston strain measles virus at a multiplicity of infection of 0.05. When 100% of the cells displayed

cytopathic effect,

total cellularRNA wasextractedfromthe

cultures by the addition of a solution containing 4 M

guanidine thiocyanate (Fluka),50 mMsodiumacetate, 1 mM

EDTA, 0.25% sodium sarcosyl sulfate, and 0.1 M

,B-mercaptoethanol (6). RNA waspurifiedby centrifugationat

30,000 rpm for 19 h at 20°C in a Beckman SW41 rotor

through a CsCl cushion with a specific gravity of 1.72,

dissolved in 300 ,ul ofTSE (10 mM Tris hydrochloride [pH

7.0],

0.2% sodium dodecyl sulfate, 1 mM EDTA), and

precipitated at-70°Cwith 3 volumesofabsolute ethanol in the presence of 0.3 M sodium acetate. Poly(A)+ RNA was

enriched by one passage through an oligo(dT)-cellulose column (1), precipitated with 3 volumes ofethanol in the

*Corresponding author.

presenceof0.3 Msodiumacetate,and storedprecipitatedat

-70'C until used. About 8% ofthe total RNA was

recov-ered.

For cDNA synthesis, 10 ,ug of poly(A)+ RNA from measlesvirus-infected cellswasheatedat65°C for3min and

annealed with 4 ,ug of oligo(dT)-tailed vector primer

pre-paredfrompcDV1plasmidaspreviously described(19,20).

Reversetranscriptionwascarriedout at42°C for30min in 30 ,ul of reaction mixturecontaining50 mMTrishydrochloride (pH 8.3), 8 mMMgCl2, 50 mM KCl,2 mM dithiothreitol, 2 mMeach dATP,dGTP, dCTP, and TTP, 100 U ofreverse

transcriptase from avian myeloblastosis virus (Bethesda

Research Laboratories), and 20 Uof RNasinRNase

inhibi-tor (Promega Biotech). The addition ofoligo(dC) residues [oligo(dC) tails]tothe 3' endof the cDNAwascarriedout at

37°C for5minbypublished proceduresin areaction volume of 30 ,ul (19). After

digestion

with enzyme

(HindIII),

the

cDNA vector molecules were

cyclized by ligation

with an oligo(dG)-tailedlinkerfragmentderived from the PL1 plas-mid, which contains the simian virus 40 (SV40) promoter

region (20).The RNAmoietyin the recombinant constructs was removed by RNase H and replaced with DNA by a DNA polymerase I reaction as previously described (20).

TheresultingcDNAlibrarywasstored inaliquotsat

-20°C.

Asmallportion of thecDNAlibrary (ca.

5%)

wasused to

transform Escherichia coli HB101(17) and screenedforthe presence ofmeasles virus-specific sequences by hybridiza-tion (13). 32P-labeled

gel-purified

DNA fragments derived from cDNA clones representing portions of the measles virusNand Pgenes (kind gifts from S. Rozenblatt [3, 10,

22]) were used as probes under hybridization conditions previously described (27). Three clones containing

N-specific

sequences andsix clones withP-specific sequences were identified. Together, these clones represented 12% of thetotalcDNA clonesscreened,indicatingthat themeasles virus-specific RNA represented a sizeable portion of the

intracellular poly(A)+ RNA in the measles virus-infected cells.

Restriction enzyme analysis showed that all three

N-specificclones and one of theP-specificclonesmight contain full-lengthcDNAinserts. Digestion ofoneoftheN-specific

clones (pcD-N7) with

BamHI,

which released the cDNA insert from the vectorDNA,producedalongfragmentwith

electrophoretic mobility corresponding to ca. 1.8 kilobases

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FIG. 1. Structuresof thefull-lengthcDNA clonesrepresentingthe Nand thePgenesof measles virus.pcD-N7andpcD-P8 DNAs (0.5 p.g) digested with restriction enzyme BamHI (lanes B and C, respectively) or XhoI (lanes I and J, respectively) were separated by

electrophoresisin 0.7%agarosegel and transferredontonitrocellulose filterpaper(23). Lanes:DandE,Southernblotofthematerials inlanes B andC,hybridizedwithanN-specificDNAprobe;Fand G, thesameblothybridizedwithaP-specificDNAprobe;Kand L,Southernblot

of theDNA in lanes Iand J,hybridizedwithaP-specificDNAprobe;N, pcD-N7 DNAdigested with EcoRV;0,P,andQ, products ofpcD-P8

DNAdigestedwithBalI,PstI,andSstI,respectively; A, H, and M,DNAsize markers.Aschematic representation ofthe pcD-N7and pcD-P8 clonesisdepicted, showing the locationsof theSV40promoterandpolyadenylation signals (hatched boxes) and theextentof thegel-purified DNAprobesused. Directionoftranscriptionisindicated byarrows.Otherarrows areexplainedinthetext.

(kb) (Fig. 1, lane B). Hybridization withanN-specific DNA probe confirmed that the 1.8-kb fragments in this clone contained N-related sequences (Fig. 1, lane D). Further restriction enzyme analysis (not shown) suggested that the 1.8-kbfragment spanned from the 3' terminus to aBamHI siteclosetothe5' terminusof theNgene(withrespect tothe [+]-strand mRNA [Fig. 1]).

With theP-specific clone(pcD-P8), similar digestion with BamHI produced a 1.4-kb fragment and a0.5-kb fragment

(Fig. 1, lane C, arrows). Only the 0.5-kb fragmenthybridized withtheP-specific DNA probe (Fig. 1, lane G). These results suggested the presence ofan internal BamHI site in theP

gene,andthe1.4-kbBamHIfragment inpcD-P8 represented sequences in the 5' region ofthe P gene which were not

recognized by the P-specific probe representing the 3'

se-quences (Fig. 1).

This interpretation proved tobecorrect. Digestion of the

cDNAcloneswith XhoIreleasedthecomplete inserts (1.95 kb) from both pcD-N7 and pcD-P8 DNAs(Fig. 1, lanesI and

J). The 1.95-kb fragment released from pcD-P8 contained P-related sequences which hybridized to the P-specific probe (Fig. 1, lane L).

The apparent sizes ofthe inserts in pcD-N7 and pcD-P8

correspondedwell with the apparentsizes of the correspond-ing intracellular RNAasdeterminedby gelelectrophoresis.

Totestif thesecDNA clonescontained thecomplete coding sequences of the N and Pgenes, we analyzed them further by restriction enzyme mapping. Digestion of pcD-N7 with

EcoRVproduced a0.74-kbfragment (Fig. 1, lane N),

signi-fying that thetwoEcoRVsitesinthe Ngene werepresentin

thisclone. Previous studies showed that the firstEcoRVsite waslocated in the 5'-untranslatedregion and the secondata

position 715 nucleotides from the AUG start site in the

coding region (21). These results indicated that pcD-N7 contained thecomplete codingsequences of the Ngene.

Similaranalysis showed that pcD-P8 containedtwoBalI

sites 0.88 kb apart (Fig. 1, lane 0). Since the 5'-proximal BalI siteis81 nucleotidesfromthestartofthecoding region (4), clone pcD-P8 contained at least most ofthe protein-codingsequences of the P gene up tothe 5'-proximalBalI

site. Based onthesedata and datanotshown,the organiza-tion of pcD-N7 and pcD-P8 is depicted in Fig. 1. The

restriction enzyme maps ofthese N-and P-specific cDNA

clones corresponded well with those of the previously

de-scribedcDNA clones ofthe measles virus (4, 21).

The pcD expression vector contains the SV40 early

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FIG. 2. Expression ofN-andP-specificRNAin COS cellstransfectedwithcloned measlesgenes.COScellsweretransfected with 30,ug

of pcD-N7orpcD-P8 withoutcarrierDNAasdescribedin thetext.At 1day (31h)or2days (56 h)posttransfection,totalcellularRNAwas

extractedwith 4 Mguanidine thiocyanate and purified by centrifugation throughaCsClcushion(6). PurifiedRNA(20 ,ug)waselectrophoresed

in1%agarosegels in thepresenceof methyl mercurichydroxide (2). The resolvedRNAwastransferredontonitrocellulosefilterpaper(24)

andhybridized withanN-specific DNA probe (panelA)or aP-specificDNAprobe (panel B) under conditions previously described (27).As

control, total cellularRNAwaspreparedfrom COS cells infected with measles virusatatime when 10to 30% of the cultures showed cytopathic effect. Lanes:AandB, mock-transfectedCOScellsat1 and 2days,respectively;C andD,cellstransfected withpcD-N7at1 and 2 days, respectively; E and F, cells transfected withpcD-P8 at 1 and 2 days, respectively; G, measles virus (MV)-infected COS cells. Arrowheadsareexplained in thetext.

moterjuxtaposed with the inserted cDNA (Fig. 1). Splicing and polyadenylation signals are provided in the 5' and 3'

sequences flanking the cDNA insert. To testthe biological activity of the full-length cDNA constructions, pcD-N7 and pcD-P8wereintroduced intoSV40-transformed CV-1 (COS) cells (9) by DNA-mediated transfection.

Briefly, 1.8 x 106 COS cells were seeded onto 60-mm culture dishes and transfected with 10 or 30 ,ug of cloned

pcD-N7 and pcD-P8 DNA withorwithout carrier DNA(30 ,ug total), using the calcium phosphate coprecipitation method(11)asmodifiedbyWigleretal.(26) and Wongetal. (27). At 7 h posttransfection, cultures were shocked with growth medium containing 10% dimethyl sulfoxide for 15

min, replenished with fresh growth medium containing 10% fetalcalfserum,andmaintainedat37°C. Total cellular RNA was extracted at 1 day and 2 days posttransfection and analyzed by electrophoresis in denaturingagarosegels.

Cultureswhich received the N- and theP-specific cDNA clones expressed high levels of N- and P-specific RNA, respectively (Fig. 2A, lanes C and D; B, lanes E and F). In cellstransfected with clonepcD-P8, amajor RNAspecies of

ca. 2.5 kb was detected by hybridization with aP-specific DNAprobe butnotwithanN-specific probe (Fig. 2B, lanes E andF). This RNAspecieswasroughly 0.8 kb longer than theP-specific RNA in measles virus-infected COS cells (Fig. 2B, lane G). The longer length of the RNA in the transfected cells corresponded tothe additional 5' and 3' sequences in the cDNA construction, assuming that RNA transcription

wasinitiatedattheSV40promoterandterminated nearthe

SV40polyadenylation signal.

Asimilarpatternof RNAexpressionwasobserved in cells

transfected with pcD-N7 (Fig. 2A). The major species of N-specific RNA was 2.7 kblong (Fig. 2A, lanes C and D), alsoabout0.8 kblonger than the 1.85-kb N-specific RNA in

measles virus-infected cells (Fig. 2A, lane G, arrow). The size of the major species of N-specific RNA in the transfected cells was also consistent with the assumption

that it was derived from utilizing the SV40 promoter and

polyadenylation signals. Additional species of RNA both

longer and shorter than themajor specieswerepresentin all transformants. Since each transformed culturewasderived

from an independent event of transfection, it seemed

un-likely that the consistent appearance of discrete species of N- and P-specific RNA in the transformants was due to

randomaggregationordegradation. More likely, premature

termination and readthrough transcription might have pro-duced the aberrant RNAspecies. In this connection, previ-ous workers using this vector system have reported the presence ofseveral RNA species derived from differential utilization ofthetwosplicing signalsintheupstream vector sequences (20). We have not pursued these possibilities further.Inany case,theamountsofN- andP-specificRNA

in the transfected cells could beas highorhigher than that presentin thesamecellsacutely infected with measles virus. Totestif theobservedintracellularmeasles-specific RNA wastranslated intomeasles virus proteins, parallel cultures transfected with pcD-N7 and pcD-P8 were labeled with [35S]methionine for 23 h starting from 8 h or 1 day posttransfection.Labeledproteinswereimmunoprecipitated withantiseraagainst theNorPprotein and analyzed by gel

electrophoresis.

A single measles virus-specific protein ofca. 60,000 Mr

was detectedon day 2 in cultures transfected with pcD-N7

(Fig. 3, lane D, asterisk). Thisprotein comigrated with the N protein in measles virus-infected COS cells (Fig. 3, lane G). Moreover, a protein withidenticalelectrophoretic mobility

was immunoprecipitated from pcD-N7-transfected cultures

by monoclonal antiserum against the measles virus N protein '2.5Kb

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FIG. 3. Expression of N- and P-specific proteins in COS cells transfected with cloned measles genes. Parallel cultures of COS cells transfected with pcD-N7 and pcD-P8 as described in the legend to Fig. 2 were labeled with 50 ,uCi of[35S]methioninefor23 h starting at 8 h(lanes C, E, L, and N) or 33 h (lanes D, F, M, and0)posttransfection, such that the time of harvest corresponded to the time points used inthe RNA experiment shown in Fig. 2. Labeled proteins were immunoprecipitated and analyzed by electrophoresis in 6 to 18% gradient sodium dodecyl sulfate-polyacrylamide gels (15, 16). As a control, measles virus-infected COS cells were similarly labeled when 30% of the cellsdisplayed cytopathiceffect. Lanes: A andI,'4C-labeledprotein molecular weight markers (Bethesda Research Laboratories); B, J, and K,mock-transfected COS cells; C, D, E, and F, pcD-N7-transfected cells; L, M, N, and0,pcD-P8-transfected cells; G, H, and P, measles

virus-infectedCOS cells. Antisera used were against the total viral proteins (lanes C, D, G, J, L, and M) or were monoclonal antisera against the Nprotein (lanes B, E, F, and H) or against the P protein (lanes K, N,0,and P) (5,18). Due to the difference in avidity of the antisera, exposure timefortheproteins immunoprecipitated by the monoclonal antisera was longer. Asterisks are explained in the text.

(Fig. 3, lane F). This 60,000-Mr protein was notpresent in

themock-transfected cultures (Fig. 3, lanesB and J) nor in

the cultures transfected with the P-specific cDNA clone

(Fig.

3,lanes Land M).

Similaranalysis of the cultures transfected with pcD-P8

revealed a measles virus-specific protein of ca. 70,000 to

75,000Mr

(Fig.

3, lane M).This proteinwas recognized by monoclonal antiserum against thePprotein (Fig. 3, lane

0)

and

comigrated

with thePprotein in measles virus-infected COS cells

(Fig.

3, laneP).

These results provided direct evidence forthe biological

activity

of the full-length cDNA clones and confirmed the

identities ofthe clonedNand Psequences.

Previous studiesshowed that under certainconditions,the

measles virus P protein could exist as two electrophoreti-cally distinct species: P(75,000Mr) and P2 (70,000 Mr) (12).

Weobservedthatcloned pcD-P8DNAcouldoccasionallybe

expressed into two forms ofP-related proteinsin

transfec-tion experiments. COS cells infected with measles virus

producedtwospecies ofP-relatedproteins,PandP2,which

were immunoprecipitated by anti-measles virus serum as well asbymonoclonal antiserum againstthe Pprotein(Fig. 4, lanes A and F, respectively). In the same experiment,

COS cells transfected with pcD-P8 likewise produced two

P-related proteins which comigrated withP and P2. These proteinswere also recognized by the samepolyclonal and monoclonal antisera (Fig. 4, lanes B and G, respectively,

asterisks)and were notpresent inmock-transfectedcultures

(Fig. 4,lanesD,E, I,andJ).Theseresultsshowedthat the cloned P gene could be expressed into eitherone or both

speciesofP-relatedproteins (PandP2)intheabsence ofany other measles virusgene function. In contrast, the N-specific clonepcD-N7neverproducedmorethan asingle speciesof

N-specific proteinintransfectionexperiments.

Tryptic peptide analysis showed that P and P2 shared extensive homology (12). However, no precursor-product relationship was demonstrable, nor was significant differ-ence in theirextent ofphosphorylation detected (12). This

situation is reminiscent of that of the C and C' proteins of

Sendaivirus, which arethoughttobegenerated by alterna-tive translationinitiation from different AUG start sites on thesame readingframe ofa single speciesof mRNA (8).

Recently,athirdproteinof21,000daltons(protein C)was

found to be encoded by the measles virus P gene in an

alternativereading frame(4). Protein Cwas notrecognized bythe antisera used in this study. However, invitro

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FIG. 4. Expression oftwospeciesofP-relatedproteins by pcD-P8inCOS cells. COS cellsweretransfected with 10,ugofpcD-P8in the

presenceof 20 ,ugofsalmon spermDNAas acarrier. Transfected cellswere labeled with 50 ,uCiof[35S]methioninefor 24 h starting at

26 h (lanesB, D, G, and I)or 51 h(lanes C, E,H, andJ)posttransfection. Labeledproteins wereimmunoprecipitated andanalyzedby

electrophoresisin 10% sodiumdodecyl sulfate-polyacrylamide gels.Lanes:Aand F,measles virus(MV)-infectedCOScells; B,C, G,and H,COS cells transfected withpcD-P8; D,E,I,andJ, COS cells transfectedwith30 jig ofsalmonspermDNA. Lanes AthroughErepresent proteins immunoprecipitatedwithpolyclonalantiserumagainstmeasles virusproteins.LanesFthroughJrepresentproteins immunoprecipit-ated withmonoclonal antiserumagainstthePprotein.Molecularweightmarkers(x103)areshownonthe sideof eachpanel. Asterisksare

explained in thetext.

lationexperiments indicated thatprotein Cwasalsoencoded

by pcD-P8(unpublisheddata).

Theexperiments described here directly verified the iden-titiesof the N and P genesmapped nearthe 3' terminus of the measles virus genome. Aspointed outpreviously (20), the vector system used in this study is highly efficient in

obtaining full-length cDNAclones. We have sinceanalyzed over70additionalP-specific cDNA clonesfromthislibrary. Atotal of70% of the analyzed clones contained full-length cDNA inserts(unpublished data). Thus, this cDNA library couldprovide valuable information about theexact5' struc-tures of the measles virus mRNAs. Furthermore, statisti-cally this cDNA library likely contains additional functional viralgenesusefulforunderstanding the othergenefunctions

ofmeaslesvirus.Experiments toidentify these clonesarein progress.

WeespeciallythankCarol Miller,University of Southern Califor-nia School of Medicine, for providing the measles virus, her expertise,andherlaboratoryfacilities for theinitiationof thisstudy. We thankS.Rozenblattforprovidingthemeasles cDNAprobesand E.NorrbyandW.Bohn forprovidingthe monoclonal antisera. We also thank Tulan Do, Greg Wipf, and Kent Beech for technical assistance.

This work was supported by grant MV-238fromthe American Cancer Society, aBiomedical ResearchSupportgrant, andby the

Graduate School Research FundfromtheUniversityofWashington School of Medicine.

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