Rabies mRNA translation in Xenopus laevis oocytes.

0022-538X/80/10-0133/10$02.00/0

Rabies

mRNA

Translation in Xenopus laevis Oocytes

WILLIAM H.WUNNER,* PETERJ.CURTIS, ANDTADEUSZ J. WIKTOR TheWistar Institute ofAnatomy and Biology, Philadelphia, Pennsylvania 19104

Two rabies virus-specific mRNA species were identified by analysis of their

encoded proteins after translation of the partially purified species in Xenopus

laevisoocytes.One of thesecodedforthe virion surface glycoprotein (G

protein),

and the other coded for the major structural protein of the virion nucleocapsid

(N protein). The G-mRNA sedimented in a sucrose density gradient at about

18S, and the N-mRNA had a sedimentation coefficient ofapproximately 16S.

Theirrespective translationproducts wereidentified in a radioimmunoassay with

specific

monoclonal antibody probes that recognized only G or N proteins.

Immunoprecipitates formed between the radiolabeledviral antigens synthesized

inprogrammedoocytesand their respective monoclonalantibodies were analyzed

by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.Theglycoprotein antigen translated from G-mRNA in oocytesmigrated in the gel ahead of the

virionG protein withamigration rate that was similar to that of nonglycosylated

intracellular glycoproteins from virus-infectedcells.Theresult suggested that the

branched-chain carbohydrate of G protein was notrequired for recognition by

the

particular

monoclonal antibody used. The nucleocapsid antigen translated

from N-mRNA in oocytesmigrated to the same position in the gel as marker

virion N protein. Both the electrophoretic mobility ofvirus-specific antigensin

sodium

dodecyl

sulfate-polyacrylamide gel and the antibody concentration de-pendence for

immunoprecipitations

were criteria for identifying the individual viral proteins encoded by thetworabies mRNA's.

Rabies virus, the prototype member of the

genus

Lyssavirus

withinthe

family

Rhabdovir-idae(3),replicates less efficiently than its

coun-terpartmember of genus Vesiculovirus, vesicu-lar stomatitis virus. In suitable tissue culture

systems, growth of rabies virus is

normally

slowerthan vesicular stomatitis virus at

com-parable

multiplicities

of

infection,

and virus

yield

(PFUper

milliliter)

is

usually

2 to 3 log units

lower (4). Estimates of

replication

efficiency based on the transcriptase

activity

in purified rabies virions (11, 13) and viraltranscribing

nu-cleoprotein

complexes

in vivo (22) compared with that of vesicular stomatitis virus suggest

thatthe lowerrateof

transcription

ofthe rabies viral genome RNA is

directly

related to the growthefficiency of the virus. Untilnow, detec-tionof rabies

virus-specific

mRNAin virus-in-fected cells has been hindered

by

the low level of RNA transcription, which has been aggra-vatedfurtherby the presence ofactinomycinD

required to suppress host cell RNA

synthesis

(23). Consequently, complementary

monocis-tronicmRNA molecules

corresponding

toeach of thefivestructuralgenes(L,G, N,

MI,

andM2) of the rabies genome

(10)

have not yet been

identified by

gel

electrophoresis.

However,two size classes of RNA

synthesized

in vivo in the presence ofactinomycin D,two-thirds of which

was

complementary

tothe viral genome RNA

(9),have been

described

aftervelocity

sedimen-tationanalysisin sucrosegradients. After

bind-ing this RNA to polyuridylic acid-Sepharose, 25%of thesmaller RNA (8 to25S) and 12% of the larger RNA (25to

35S)

contained

polyade-nylic acid

[poly(A)]

tractswhichwereindicative of rabies

virus-specific

mRNA(8). Inonereport

(23) five actinomycin D-resistant RNA

species

weredetected indenaturingpolyacrylamide

gel

after extraction of RNA from

persistently

rabies virus-infected BHK-21cells.

Although

molecu-larweights of the individual RNA

species

were

approximately

the size for the

expected coding

capacities

of monocistronic mRNA's of the five

structural

proteins

of the rabies

virion,

no at-temptwasmade to

identify

theseRNA

species

asviral

protein-specific

mRNA'sor

assign

gene

products

onthe basisofmolecular size.

Assignments of viral

protein

tomonocistronic

mRNA canbe made by comparing the coding capacity of eachmRNA

species

withthe size of known viral

proteins

(17, 19).The ultimate

as-signment

would be better obtained

by

transla-tion ofindividualmRNA

species

in vitro

(2, 16).

In this report we present evidence that two rabiesmRNA'sanalyzed thus far show

individ-ual specificity for synthesis of: (i) the rabies surface glycoprotein (G protein) and (ii) the

133

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nucleocapsid (N) protein, and that each of the mRNA's is correlated with itsproduct of trans-lationonthebasis ofacorrespondence between

RNA coding capacity and polypeptide size. We describe the use ofXenopus laevis oocytes for

efficient translation of microinjected rabies mRNA's that have been partially separated by

sucrose gradient centrifugation. The assay for

mRNA is completed by the positive identifica-tion oftranslation products by selective immu-noprecipitation with monoclonal antibodies of hybridoma origin.

MATERIALS AND METHODS

Cells and virus. All mRNA preparations were

made from BHK-21/S13 cells infected with plaque-purified fixed ERA strain rabies virus. BHK-21

cell-adapted ERA rabiesvirus has been described previ-ously (5).

Infection of cells and extraction of total cell

RNA. We infected monolayer cultures of 2 x 108 BHK-21/S13 cells in roller bottles with stock virus (109PFU/ml) atamultiplicity of infection of 25 PFU percell. The viruswasadsorbedatroomtemperature

(20°C) for1hbefore infected cellswereincubatedat

37°C in thepresenceof[3H]uridine (5 ,LCi/ml; 25Ci/ mmol) in modified Eagle medium containing 0.2% bovineserumalbumin. The culture fluidwasdecanted

after 20 h, and the infected cellswere scraped into high-salt buffer containing 0.18 M NaCl in 0.01 M Tris-hydrochloride, pH 7.4, washed three times in high-salt buffer, and finally suspended in TE buffer (20 mM Tris-hydrochloride-1 mM EDTA, pH 7.5)to

which wasadded 20% sodium dodecyl sulfate (SDS)

and20mgofpronase(predigested)perml togive final concentrations of 2.5% and 200

Ag/ml,

respectively. After incubation at room temperature (20°C) for 30 minor37°Cfor 10min,totalinfected cell RNAwas

extracted twice inphenol (saturated with TE buffer), and RNAwasprecipitated from ethanol, dried under

nitrogen, and resuspended in TE buffer. The RNA

wasextractedathird time withphenol and

reprecipi-tated from ethanol. ToremoveDNA, the precipitated

nucleic acid was first dissolved in water and then adjusted to 50 mM Tris-hydrochloride (pH 7.5), 10 mM MgCl2, 2 mM CaCl2, and 50 Mug of DNase (pre-treated with sodiumiodoacetate) perml. Incubation

at 37'C for 5 to 10 min was sufficient to reduce drastically the viscosity of the solution,atwhich point the solutionwasadjustedto20 mMEDTA and 0.2% SDS followed by extraction withanequal volume of

phenol. The aqueous phase was dialyzed versus 20

mM Tris-hydrochloride (pH 7.5)-i mM EDTA and storedat-20°Cataconcentration of1mg/ml.

[35S]methionine labeling and extraction of

in-fected cell proteins. Monolayer cultures of4 x 107

to5x 107BHK-21/S13 cells in three T-75 flaskswere

infected with ERA strain rabies virus as described

above, except that regular Eagle medium with 0.2% bovineserumalbuminwasreplaced with

methionine-freeHanks medium199(GIBCO Laboratories, Grand Island, N.Y.)tolabel infected cells from 4.5to24h, 24

to 48 h, and48to 72h postinfection and uninfected

cellsfor 24 h with[35S]methionine (20 MCi/ml; >1,000 Ci/mmol). At the end of thelabeling period, cells were scraped into the culture fluid and pelleted by low-speedcentrifugation.The combined cellswerewashed three times inhigh-salt buffer andfinally suspended in 0.8mloflysisbuffercontaining0.5% NonidetP-40, 0.15 M NaCl, 5 mM EDTA, and 200 yg of phenyl-methylsulfonylfluoride per ml in 50 mM Tris-hydro-chloride, pH 7.4. The cell suspensionwas sonicated four times, 15 s each time, to break up DNA and clarified in Beckman cellulose nitrate microtubesby centrifugationinanSW 50.1rotor at12,000 xg and 4°Cfor 30min. The clarified celllysate (supernatant) wasextracted frommicrotubesbyside puncture with syringe andneedleinjected immediatelyabove the cell debrispellet. In this way, mostof thelipid that ap-pearedatthe top remained adsorbedtothewall of the tuibe. Extracted cellproteinswerestoredat-70°Cfor isolation ofviralantigen.

Oligodeoxythymidylic acid-cellulose chroma-tography.Total infectedcellRNAin TE bufferwas

heatdenatured(100°C,45s)andadjustedto 2xTNE (TNE: 0.02 M Tris-hydrochloride [pH 7.5], 0.15 M NaCl,2mMEDTA), 0.2%SDS,0.2%polyvinylsulfate andappliedto anoligodeoxythymidylicacid-cellulose column (1-cmdiameter)previously equilibratedin 2x TNE. The RNAonthecolumnwaswashedthrough with 2x TNE (20bedvolumes) at aflowrateof0.5 ml/min. Poly(A)-containing [poly(A)+] RNA that boundtothe columnwaseluted by alternatively ap-plying 1 ml of 10 mM Tris-hydrochloride (pH 7.4), 0.1%SDS, andwater. RNAfractionsweremonitored by UV absorption, and pooled RNA fractions were precipitatedtwice withethanolin 0.3 Msodium ace-tate, pH 5.5. The poly(A)+ RNA concentration was adjustedto1mg/ml foroocyteinjectionand stored at -200C.

TranslationassayinX.Iaevisoocytes.Poly(A)' RNA (1 mg/ml) wasmicroinjectedinto 10 oocytes so that eachoocyte received50ngofpoly(A)+RNA.The oocytes were incubated atroomtemperature (200C) for24h inmodified Barth medium (12). Oocyteswere labeled with[35S]methionine (>1,000 Ci/mmol),which was evaporated to dryness and dissolved in Barth medium at a concentration of 200

ACi/75

,ul per 10 oocytes.Afterincubation, oocytes werewashed three times in coldTris-glycine buffer (52 mM Tris,52mM glycine) and homogenized in 250 [L of Tris-glycine buffer per 10 oocytes. Cell debris was removed by centrifugationfor5minat12,800x g in the Eppendorf microcentrifuge, and the supernatant ofclarified cy-tosol was stored at-70°C.

RIA and immunoprecipitation with mono-clonal antibodies. A radioimmunoassay (RIA) was preparedintriplicatefor the detection of viral antigen in 10Mlof clarifiedoocyte supernatant.Selected mon-oclonal antbodies of hybridoma origin (HAb) with specificityfor virion glycoprotein (HAbG) or nucleo-capsid protein (HAbNC) were used according to de-scribed techniques (Ila). '25I-labeled rabbit anti-mouse (Fab')2 fragmentswerekindly provided by W. Gerhard of the WistarInstitute.

Viral antigens were extracted from oocytes and rabies virus-infected cells by immunoprecipitation

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withantigen-specific HAb produced inmouseascites

fluid (lla). The precipitateswerecollected by

adsorp-tiontoa10%suspension of formaldehyde-fixed Staph-ylococcusaureus (6) kindly provided by T. Dolby of

the Wistar Institute.

Viralantigenswereextracted fromoocytesafter the clarifiedcytosolwasmixed withanequalvolume of

2xlysisbuffer and further clarifiedbycentrifugation

at12,000 xg asdescribed for infected cellproteins.

Aliquots (200

p1)

of clarified lysates fromoocytesand infectedcellsweremixedwith 10

pl

of HAb in

undi-luted ascitic fluidorserial 10-fold dilutions(with lysis

buffer) for 30min in ice before25

Ad

of 10%S.aureus

suspension in lysis bufferwasaddedtoeach antigen-antibody reaction tube. Incubationwascontinued in

ice for20min with frequent (every2to3min) agitation

on aVortexmixer, and immune complexes adsorbed

toS.aureuswerepelleted by centrifugationfor 6to8

sin anEppendorf microcentrifuge. Thesupernatant

was separated from thepelletand saved for further antigen extractions; the pellet was washed several

times with 1 ml of cold wash buffer containing 5%

sucrose,1%NonidetP-40,0.5MNaCl,5mMEDTA, and 15 mMTris-hydrochloride (pH 7.4), resuspended by Vortex action,andrepelleted by centrifugation for 20 s in an Eppendorf microcentrifuge. Removal of excesscountswas monitored in50Id ofeach wash. Finally, the antigen-antibody complexes were

dis-solved in 20 p1 of modified Laemmli sample-dissociat-ing buffer (0.0625MTris-H3PO4, pH 6.8,2% SDS, 0.2% dithiothreitol, 0.1% EDTA, 10% glycerol) (14) by heat-ingat95°C for 90sfor electrophoresis in an

SDS-polyacrylamide gel. Samples were stored in 2 mM

phenylmethylsulfonylfluoride at-70°C.

SDS-polyacrylamide gel electrophoresis. Dis-continuous Laemmligelsof 10%SDS-polyacrylamide

wereprepared essentiallyasdescribedelsewhere (14),

exceptslabgelswerecast1.5mmthick and13cmlong

betweenglass plates (20).The 0.8-cmstacking gelof 4%acrylamidecontained 14sampleslots.Thestacking gel buffer system was modified from the Laemmli

systemby substitutingTris-H3PO4for Tris-hydrochlo-ride(21). Gelswereloadedwith 50

pl

ofsample (1to

5,tg ofprotein; 20,000to 150,000 cpm), and electro-phoresiswasperformedatroomtemperaturefor4to

5 h at 20mA until the migration front followedby bromophenol blue that was added to each sample

before electrophoresiswas 1 cmfrom thebottom of thegel.Afterelectrophoresis,thegelswerefixed and

stained in 50% methanol-7% acetic acid containing 0.25% Coomassie brilliant blue. Gelsweredestained by gentle rockingin50% methanol-7% acetic acid and photographedbeforedryingundervacuum.Driedgels wereautoradiographed byexposuretoRP Royal X-Omat(Kodak) film at-70°Cwith Cronex lightning-plusintensifying screens. Exposedfilms were

devel-opedinKodakliquid X-ray developerfor5min,put in 25%acetic acid for 10 s,fixed for 5minin Kodak rapid fixer,washed for 30min,and dried.

RESULTS

Translation of rabies mRNA in X. laevis oocytes. Our initial assay of virus-specific mRNA translation inprogrammedoocyteswas

perforned with total poly(A)+ RNA (ERA-RNA)extracted from ERA rabies virus-infected cells. Similarly preparedRNA from uninfected

cells served as the control. Viral antigens

syn-thesized in oocytes injected with ERA-RNA

were detected by RIA with HAb specific for

virion glycoprotein and nucleocapsid antigens. Table1 shows thatapositiveresponsewas

ob-tained, which suggested that theoocyte system

was capable of faithfully translating rabies

mRNA. Theresponsewith HAbGwas

approxi-matelyeightfold, and HAbNcwasapproximately

twice thebackground levels observed with

oo-cytes that were programmed with poly(A)+

RNA from uninfected cells (S13-RNA) or

con-trol uninjected (-RNA) oocytes. Whole virus antigenadjustedtoaprotein concentration of 2

jug/ml

atthesametimegavea typicalresponse

with the HAbG and HAbNc in RIA (lla, llb). Since the detection of viralantigenwasatalow

level, the RIAwas repeated onundiluted and

one-half and one-fifth dilutions ofa secondset of oocyte lysates containing viral antigens to demonstrate that theresponse,although slight,

wasreal and concentration dependent. The

re-sults clearly showed that the detection of viral antigen in oocytesprogrammed with ERA-RNA

wasconcentrationdependent; the values for

un-dilutedextract(190 and 266cpmwithHAbG and HAbNc, respectively) werereduced to approxi-mately 50% (56 and 120 cpm) and 20% (23 and 57cpm) in the one-half andone-fifth dilutions of theextract.

Toincrease theefficiency of translating rabies mRNA inoocytes,wefractionated thepoly(A)+

RNA from virus-infected cellsona sucrose

den-sity gradient and collected different sizeRNAs (based onsedimentation coefficients) fromthe

gradient in pooled pairs of adjacent gradient fractions. The RNAfromeachpoolwas

concen-tratedto1mg/mlandmicroinjectedintooocytes fortranslation.InFig. 1,theresponseinseparate RIAs withHAbG andHAbNC to translation prod-ucts in oocytes programmed with individual pooled mRNA fractions is shown across the

TABLE 1. RIAofviralantigenproducedin X. laevisoocytesprogrammedwith total

poly(A)

+RNA

"Icpmbound in the pres-enceandabsenceof:

Antigen source

HS-PBSa HAbG HAbNC

Virus 292 5,910 5,250

Oocyte-RNA 12 39 27

Oocyte+S13-RNA 22 25 24

Oocyte+ERA-RNA 19 207 50

aTen percent

-y-globulin-free

horse serum (HS-PBS)wasusedasdiluent formonoclonal antibodies. VOL. 36,1980

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136 WUNNER, CURTIS, AND WIKTOR

1.0

0.D.

0.5-fI

nn

i

1liI

0 10 20

Fracti o no.

FIG. 1. Detectionof translation products in X. Iae-vis oocytesprogrammed with poly(A)+ RNA from rabies virus-infected cells. Total infected cell RNA was separated on oligodeoxythymidylic acid-cellu-loseasdescribed inthetext.Thepoly(A)+RNAwas

thenfractionatedina5 to23%(wt/vol)sucrose den-sitygradient bycentrifugationat76,400x gand10°C

for17hinanSW41rotor.The RNA inpooledpairs of adjacentfractions was concentrated to 1 mg/ml andmicroinjectedintooocytesfortranslations. The oocytelysatesweretestedbyseparate RIAsfor

trans-lationproducts with monoclonal antibodiesagainst

the glycoprotein or nucleocapsid protein, and the counts above background (38 cpm) areplotted for

eachpairofgradientRNAfractions.rRNA(28Sand 18S) and 4S RNA markers were sedimented in a

parallelsucrosegradientand detectedbyUV absorb-ance at 260nm.

RNAgradient. Sincethe virion Gprotein hasa

molecular weight of approximately 70,000 (mi-nuscarbohydrate),the sizeof mRNA coding for thisprotein(G-mRNA)would beexpectedtobe 1.8 kilobases long and sediment at about 18S.

Similarly,the Nproteinwithamolecularweight

of 60,000 would be coded for byanmRNA (N-mRNA) of 1.6 kilobases sedimenting at about 16S. The response with HAbG was greatest in oocytesthatwereprogrammedwith mRNA that sedimented further into the sucrose gradient, whereasthe responsetoHAbNC was greatestin oocytesthatwereprogrammed with mRNA that

sedimented moreslowly. The results suggested that the rabiesG-mRNAwaslarger thanrabies N-mRNA, whichisconsistent withthe hypoth-esis that the size of rabies monocistronic mRNA'sand thesize of the encodedpolypeptide aredirectly related.

Extraction of viral antigen from oocytes programmed with rabies mRNA. Xenopus oocytes were injected with unfractionated

poly(A)' ERA-RNA or withpooled, fraction 7 mRNA (gradient fractions 13 and 14, Fig. 1); the latter showed a strong response in RIA to both HAb(; and HAbNC. The injected oocytes were incubatedfor 24 hinthepresence of

[:35S]methi-oninetolabelthe translation

products

of exog-enousmRNA. After

translation,

aliquots

ofthe oocytelysateweremixed with undilutedorserial

10-fold dilutions of ascitic fluid

containing

HAbG, and

antigen-antibody complexes

were

precipitated with

formaldehyde-fixed

S.aureus.

Dissociated protein in the

immunoprecipitate

wasanalyzed in

SDS-polyacrylamide gel.

After

electrophoresis,

the stained

gel

was

compared

withtheautoradiogramof thesame

gel.

Inboth thestainedgel andits autoradiogram, the

back-ground protein was minimal, thus making it

possible to identify the virus-specific antigens.

Ascites fluid protein, extracted with each

im-munoprecipitate in the stained gel (Fig. 2),

de-creasedproportionallywith eachdilutionof the

monoclonalantibodyfluid used until none was detected in thefinal(10-4) dilution.Viral antigen labeled with [35S]methionine was identified in

Fig. 2 (autoradiogram) by the same dilution criterion, assuming that the amount of antigen

interacting with antibody was proportional to the concentration of antibody present in the

reaction mixture. A labeled polypeptide

(Go)

correspondingtothenonglycosylatedform of G

protein (15) was detectable in diminishing amountswithantibodydilutions of

10-1

to10-4, suggesting specificity for HAbG. Moreover, the

specificity of the reaction was reflected in the increased amount of radiolabeled

Go

going from

undiluted to a

10-1

dilution, which suggested that a 1:10 dilution of antibody-containing fluid wasoptimalforantigen-antibodyinteraction. All otherlabeled polypeptidesappearing in the gel weredetected in near equal quantities regardless ofantibody dilutions, suggesting they were

non-specifically

adsorbed and co-precipitated.

Thelysatesupernatantsthat were saved after each reactiontoextractantigen with HAbG were

subsequently mixed with HAbNc in undiluted and serial 10-fold dilutions of ascitic fluid,

re-spectively. The stained gel and its autoradi-ograminFig.3showed that ascites fluid proteins and labeled antigen, respectively, diminished with each dilution of antibody. The major

[35S]methionine-labeled

polypeptide precipi-tated from oocytes also migrated coincidently withvirionNprotein marker. Both observations provide -evidence that N-mRNA was translated faithfullyinoocytes. The apparenthomogeneity ofNantigen in the SDS-polyacrylamide gel was illustrated by the lack of shoulder peaks associ-ated with theN protein peak in tracings of the autoradiogram (data not shown).

Extraction of viral antigen synthesized inrabies virus-infected cells. The compari-sonofputative viral antigens derived from rabies mRNAtranslation inoocytes with viral antigens

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RABIES VIRUS-SPECIFIC mRNA's 137

TRANSLATION

IN OGCYTES TOTAL mRNA FRACTION 7rRNA

I- -- r

HAbG OIL (LOGIO): ND -I -2 -3 -4 ND -I -2 -3 -4

AFP- -

-AFP - .1

-NN

M2

-FIG. 2. SDS-polyacrylamide gelelectrophoresisof immunoprecipitatesformedbetween oocyte translation products andglycoprotein-specific monoclonal antibody (HAbo). X. laevis oocytes wereprogrammed to

translate total(unfractionated) poly(A)+RNAfromrabiesvirus-infectedcellsorpooled fraction 7(gradient fractions 13 and 14) ofsucrose gradient-fractionated RNA and labeled with

[uS]nmethionine

(Fig. 1). Translationproductsweremixed withHAbGin undiluted ascitesfluid (ND)orserial10-folddilutionsofthe asciticfluidcontaining antibodyandimmunoprecipitatedwithS.aureus asdescribed in thetext.Protein in theimmunoprecipitateswasvisualizedafterSDS-polyacrylamide gel electrophoresisinthestainedgel (top) and the autoradiogram ofthesamegel (bottom).Ascitesfluid proteins (AFP) and rabies virus structural proteins(G, N,Ml,andM2)in lane1 werevisible in the stainedgel.Presumptivenonglycosylated glycoprotein

(Ga)migrated aheadofglycosylatedvirionprotein (G).

synthesized in vivo was a necessary adjunct in

the characterization ofoocyte translation prod-ucts. Welabeledcellswith

[35S]methionine

be-tween 4.5and24hand from24to 48hand 48 to 72 hafterinfection with ERA rabies virus. Un-infected cells werelabeled with[35S]methionine for24h ascontrols and processed inparallelto the infected cells forthe extraction of viral

an-tigens. Cell

lysates

weremixedfirstwithHAbG to extract theintracellular viral G protein in a

procedure similartothat described foroocytes.

Neither ascitic fluid nor S. aureus was

preab-sorbed with unlabeled uninfectedBHK-21/S13

cell proteins in these experiments. Figure 4

shows the result ofSDS-polyacrylamide gel elec-trophoresis of HAbG-specific antigen extracted

from infectedcellsby immunoprecipitation with

S. aureus. A

single polypeptide, designated

Go,

wasextracted in

decreasing

amounts asantibody dilutions decreased from

10'

to

10-4,

suggesting specificity between

Go

polypeptide and HAbG. Another

polypeptide

that co-extracted with the

Go

polypeptide

wasvisible between

Go

andNin

theautoradiogram. The

electrophoretic

mobility

VOL. 36,1980

MI

--G

Go

--o--r.

'2

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[image:5.510.91.385.71.428.2]

138 WUNNER, CURTIS, AND WIKTOR

FIG. 3. SDS-polyacrylamidegelelectrophoresis ofimmunoprecipitates formedbetween oocyte translation products andnucleocapsidprotein-specific monoclonal antibody(HAbNc).X. laevis oocytelysatesthatwere usedto extractrabiesvirus-specificantigens with HAbG(Fig. 2)weresimilarly mixed with undiluted(ND)or serial10-fold dilutions of ascitic fluid-containing HAbNC. Immunoprecipitates were collected andproteins werevisualizedafter SDS-polyacrylamide gel electrophoresis in the stained gel (top) and the autoradiogram (bottom).Protein markersareasdescribed inFig.2.

of this polypeptide corresponded to that of a

cellularpolypeptide from S.aureusin the sub-sequent immunoprecipitation with HAbNC (see

Fig. 7) and inimmunoprecipitationswithHAbG

from uninfectedcells (Fig. 5). Thesenonspecific

polypeptides were extracted in quantities that wereindependentofantibody concentration.

Thedetection ofaHAbG-specificintracellular

antigen,whichmigratedinSDS-polyacrylamide gelfaster thanpurifiedvirionGproteinmarker,

suggested that the intracellular polypeptidewas the immature (not fully glycosylated) form of the virion surface Gprotein (15). We repeated the extraction with HAbG and two extended growth cultures of rabies virus-infected cells

(multiplicity of infection = 25) which were la-beled with[35S]methioninefrom24 to 48hand

48to72hpostinfectioninanattemptto extract thefully glycosylated (mature) G protein from

these cells.The electrophoretic mobility of the

HAbG-specific antigen (Go) extracted aslateas 48 to 72h (Fig.6)wasidenticaltothat of theGo polypeptideobtained 24hpostinfection.

The supernatants of the infected and unin-fected cell lysates once extracted with HAbG werere-extracted,respectivelywithHAbNC, ND, and

10-1

to 10-4 dilutions. An analysis of the

immunoprecipitates from 24-h-infected cells is shown in Fig. 7. In the autoradiogram of the

SDS-polyacrylamide gel,a 35S-labeled polypep-tidewasdetected withanelectrophoretic

mobil-itywhichcorrespondedtothat of virion N-pro-tein marker, and it diminished in quantity as

antibody concentration was decreased. An

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[image:6.510.130.417.71.415.2]

RABIES

VIRUS-SPECIFIC

mRNA's

139

ERA/BHK 0-24H

HAbGDIL(LOGIr):'ND

-I -2 -3 4

AFP - -.

-

N-M

-stainedgel andautoradiogram.Thattheseother

polypeptides were of cellularoriginappearingin near constant amounts regardless of

antibody

(HAbNc)concentration was corroboratedbythe uniform recovery of cellular proteins from

un-infected cells (datanot

shown).

The co-extrac-tion of N and M1 antigens from infected cells with HAbNC suggested that HAbNC either had * dualspecificity for the solubleantigens or was

monospecificfor Nprotein and

co-precipitated

BHK

HAbG

DIL(LOGIO):

ND -I -2 -3 -4

AFP-

-G

MI

AFP

G0

G

N-AFP

-

T

1

#

.v

M2

-FIG. 4. Immunoprecipitation of 3S-labeled BHK-21 cell-derived viral antigens with HAbG. Rabies virus-infectedcell lysates wereprepared24h after infectionandmixed withHAbGin undiluted ascites fluid (ND)orinserial10-folddilutionsofthe ascitic fluid. Theantigen-antibodycomplexeswere precipi-tated withprefixedS.aureusandanalyzedby SDS-polyacrylamide gel electrophoresis as described in thetext.Ascitesfluid proteins (AFP)and rabies virus proteins(G, N,M,,andM2)were seenclearlyin the stainedgel(top). Theautoradiogram (bottom)showed nonglycosylated virus-specific glycoprotein

(Ga)

whichmigratedaheadofthe virion structural glyco-protein(G).

ditional labeled

polypeptide

corresponding

to virionM1

protein

alsodecreasedwith serial di-lutions of

HAbNC.

Detection of both N and

M1

antigensbythis criterionwasinmarkedcontrast totheotherdetectable

polypeptides

inboth the

N-

Mt-

M2-FIG. 5. Immunoprecipitation of35S-labeled unin-fected BHK-21 cellproteins with HAbG. Uninfected cells were labeled with

[3Slmethionine,

and cell lysates were mixed with HAbG in ascites fluid as prepared in Fig. 4. The immunoprecipitates were resolved on SDS-polyacrylamide gelandvisualized in the stainedgel (top) and in the autoradiogram (bottom) alongwith "4C-labeledproteinsofpurified rabiesvirions.

36,1980

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[image:7.510.44.236.74.471.2] [image:7.510.250.440.183.582.2]

G

ERA/BHK 48-72 H P.l.

Go

0 0 0

-iND

z

0

a-

-1

F

<,

0 4c

-2

-3

N

Ml M2

d:I

/~~~~~~~

products inXenopusoocytes andby

correlating

the size ofmRNA and that of its encoded

poly-peptide.

The resolution of rabies virus-specific G-mRNA andN-mRNA

by

sucrose gradient

cen-trifugation

coincided with thesedimentation of

purified

vesicular stomatitis

virus-specific

G-mRNA andN-mRNA ofcomparablemolecular

weights,

respectively, thatwerepreviously

sep--4

FIG. 6. Microdensitometer tracings of autoradi-ogramsshowingvirus-specific antigen (Go)from ra-biesvirus-infected BHK-21cellsimmunoprecipitated withHAbG. Top tracing is of 4C-labeledvirion (G, N, M,,andM2)protein markers.Eachtracing belowthe top is an immunoprecipitate in which virus-specific antigen synthesized and labeled from 48 to 72 h postinfection interacted withHAbG inundiluted(ND) orserial10-fold dilutionsof ascites fluid.

with itthe

Ml

proteinas anintegral partof the nucleocapsid structure. Experiments arein prog-ressto rule out theunlikely possibility of dual

specificity by reacting HAbNC with antigen translated from purified M,-mRNA

microin-jectedinto oocytes.

DISCUSSION

Two rabiesvirus-specific mRNA species have been identified as monocistronic RNA

[image:8.510.82.268.76.417.2]

tran-scripts, each coding for distinct viral polypep-tides. Thiswasdone by using monoclonal anti-bodies specific for rabies mRNA translation

FIG. 7. Immunoprecipitation of35S-labeled BHK-21 cell-derived viral antigens with HAbNc. Rabies virus-infected cell lysates (24 hpostinfection) were

mixed withHAbNc after the same lysates were

ex-tracted with HAbG (Fig. 4). Antigen-antibody

com-plexes formed with HAbNC in undiluted (ND) and serial10-fold dilutions ofthe ascitic fluid were pre-cipitated and analyzed by SDS-polyacrylamidegel electrophoresis as described in Fig. 4. Theprotein designations are described also in Fig. 4 for the stainedgel(top)and autoradiogram (bottom).

J. VIROL.

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[image:8.510.286.469.176.550.2]

RABIES VIRUS-SPECIFIC mRNA's 141

arated by polyacrylamide gel electrophoresis (18). Separation of poly(A)+ RNA into

G-mRNA- andN-mRNA-rich fractionsresulted in a 5-to 10-fold greater response in the RIA com-pared withunfractionated poly(A)+ RNA. The

synthesisof viralantigen (1 to 4 ng) in

individ-ually programmed oocytes possibly varied due to the actual amounts ofmRNA injected into

each oocyte and the"recruitment" rateof

dif-ferent mRNA's fortranslation(1).These factors

arereflectedintheincreased RIAresponsewhen oocytes wereprogrammed withsucrosegradient fractionatedmRNA's.

Theisolation of immunochemicallypureviral

antigens fromprogrammed oocytes by

sequen-tialimmunoprecipitation with monoclonal anti-bodies demonstrated that singlepreparationsof mixedmRNAspecies could be analyzedin this system. We noticed that monoclonal antibody specific for virion G protein interacted with a

polypeptide,

Go,

which is synthesized in both Xenopusoocytes and BHK-21 cells. This poly-peptide migrated in SDS-polyacrylamide gel electrophoresis ahead of the G protein marker, suggestingthat

Go

representstheimmature(not

fully

glycosylated)

form of rabies virionG

pro-tein (15). Since the

Go

polypeptide reactedwith themonoclonalantibody,weconcluded that the carbohydrate moiety of the mature G protein

was not part of the critical antigenic

determi-nant. The absence ofan oocyte HAbG-specific polypeptide with the same

electrophoretic

mo-bilityas mature Gprotein indicated eitherthat

Go polypeptide

was not

synthesized

on

mem-brane-boundpolyribosomeswhere it

might

have

been

glycosylated

orthat

glycosylation

wasnot

sufficient to produce a molecule

equal

to the molecular sizeof the virion G

protein.

Theextent

of

glycosylation

of

foreign proteins

in oocytes

hasnot beendetermined.

Nevertheless,

the oo-cytetranslationsystemwas not

expected

to

per-form the same

post-translational

modifications

on the rabies-specific

glycoprotein

that take place in virus-infected mammalian cells (7). If glycosylationdid occur in theoocytetranslation

system, itwould

presumably

modify

transmem-brane molecules destined for export from the

oocyte,and suchproteins would

probably

notbe

found in clarified oocyte

lysates.

Weattribute absence of rabies

virus-specific

glycopolypeptide

with electrophoretic mobility of G protein in virus-infected cellstofractionation; i.e., such a

glycopolypeptide has been detected

previously

when whole cells were

analyzed (15).

We con-clude that themature

glycopolypeptide

is mem-brane associatedand therefore isnotpresent in ourclarifiedcell

lysates.

Xenopus oocytes

provide

a suitable in vivo

translation system for the identification of rabies

virus-specificmonocistronic mRNA's. The assay systemcoupled with the RIA with monoclonal antibodies has beenanintegralpart ofour pro-gram to clone the rabies genes in bacterial

plas-mids.

ACKNOWLEDGMENTS

Wegratefully acknowledge the excellent technical assist-ance ofSally Shane, Erik Whitehorn, and Lynn Clompus.

This work was supported by Public Health Service research grantsAI-09706 from the NationalInstitute of Allergyand

Infectious Diseases and RR-05540 from the Division of Re-searchResources.

ADDENDUM IN PROOF

Pennica et al. (Virology 103:517-521, 1980) reported theidentificationof five rabies virus-specifiedmRNA species inacid-urea-agarose gels. Four of the mRNA species were translated in vitro into products that wereidentical to authentic virus proteins. The poly-peptidecoding assignments of the individual mRNA species showed a relationship between sizes ofmRNA andtranslation product which corresponds to the re-sults that we report inthis paper.

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