Rabies virus protein synthesis in infected BHK-21 cells.

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Rabies Virus

Protein

Synthesis

in

Infected

BHK-21

Cells

H. PAUL MADORE* AND JAMES M. ENGLAND

The Wistar Institute of Anatomy and Biology, Philadelphia, Pennsylvania 19104 Received for publication 22 November 1976

Rabies virus-specific polypeptide synthesis was examined under hypertonic

conditions,whichselectivelyinhibitcellular proteinsynthesis. The rabies virus

proteins(L,

G,

N, M,, M2)weresynthesized throughout thecourseof infection, withlittlechangeintheir relativeratesof synthesis. The ratesofsynthesis of the G and

M,

polypeptides were more sensitive to increasingosmolarity than

those of the L,N,and M2 polypeptides. Extrapolationtoisotonicityof the results obtained under hypertonic conditions indicated that the molar ratios of the

polypeptides synthesizedundernormalconditionswere 0.4 (L), 64 (G), 100 (N),

75 (M,), and 35 (M2). A high-molecular-weight polypeptide (-190,000),

desig-nated polypeptide L, was repeatedly detected both in infected cells and in

extracellular virus. Theestimated number ofLpolypeptide moleculesper virion

was33. Thesynthesis ofa viral glycoprotein precursor, designated gp78,

pre-cededtheappearance of thematureviral glycoprotein ininfectedcells labeled

with [3H]glucosamine under isotonic conditions. In cells labeled under

hyper-tonicconditions, littleorno matureviral glycoproteinwasdetected,buta

virus-specificglycoproteinwithanelectrophoreticmobility similartothat of gp78was

observed.Thisglycoproteincould be chased intomatureviralglycoproteinwhen

the hypertonic conditions were made isotonic. These results suggest that a

reversible block of viral glycoprotein synthesisoccurs under hypertonic condi-tions.

Rabies virus, a rhabdovirus, is a negative-strand virus that containssingle-strandedRNA withamolecularweight of

approximately

4.6 x

106(40, 43).The RNAisassociatedinthevirion

with phosphorylated nucleoprotein (N),

form-ing a nucleocapsid core (40, 41). Aviral

enve-lope, which contains two non-glycosylated membrane proteins

(M,

and M2) and a glyco-protein (G), surrounds the nucleocapsid (40).

The viral glycoprotein is responsible for elicit-ing the production of neutralizing antibody against the virus (53).

Rabies virus hasaneclipse periodof6 to 12h

inBHK-21 cells andproducesvirusforseveral days withoutappreciable inhibition of cellular DNA, RNA, or protein synthesis (12, 27, 47, 52). Evidence of viral polypeptide synthesis in

rabies virus-infected cells has been limited to

the detection of virus-specific polypeptides in

the cytoplasm of infected cells by

immunoflu-orescenttechniques(47, 52)andby the isolation

ofnucleocapsidcoresafter cell fractionation (8, 39). Recently,wereported thatthesynthesis of

themajorrabies structural proteins (G, N,

M1,

M2) could be detected in infected cells labeled

withradioactiveaminoacids under hypertonic conditions (26).

The RNA of this negative-strand virus would

appear torequireavirion-associated RNA

tran-scriptase for transcription (2, 21). The L

pro-tein, which ispresent insmall amounts in

in-tactVSV virions(16, 50,51),has beenshownto

benecessaryfor RNAtranscriptaseactivity (5-7, 10, 11, 31). In contrast, the presence of L protein in rabies virions has not been clearly established (40, 42), and RNA transcriptase ac-tivityhas notbeen detected inpurifiedrabies

virions (41, 48), except possibly at extremely low levels (D. Bishop, personal communica-tion). Although virus-specific RNA

transcrip-tase activity has been detected in rabies-in-fected cells (48; H. P. Madore and F. Sokol, unpublished observations), the virus-specific polypeptide (L), which may be necessary for viral RNAtranscriptase activity, had notbeen detected in our previous analysis of infected cells labeled under hypertonic conditions (26). Inthisreport,hypertoniclabeling conditions

were employed to characterize rabies-specific polypeptide synthesis during the course of

BHK-21cell infection. Inadditiontothe major

viral structuralpolypeptides,a high-molecular-weightpolypeptide (L) has been clearly identi-fied both in infected cells and in extracellular 102

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VOL. 22, 1977

virus. The effect of increased osmolarity on

rates of synthesis ofindividual viral polypep-tides andonglycosylation of viral glycoprotein

wasalsoexamined.

MATERIALS AND METHODS

Cellsand virus. BHK-21/C13 cells (25) were seri-ally passaged in Blake bottles and maintained in minimal essential Eagle medium (MEM), supple-mented with 10% fetal calf serum(growth medium) at370C(18).Rabies virus (ERA strain) and vesicular stomatitis virus (VSV) (Indiana strain) were ob-tained from Tadeusz Wiktor of the Wistar Institute. Stocks ofrabiesvirusand VSV (108 to 109 PFU/ml) werepropagatedinBHK-21cells in a manner simi-lar to thatdescribed for the preparation of labeled virus. Both cells and virus stock were shown to be mycoplasma-free by the assay of Sedwick and Wik-tor (38).

Infection and mock infection of cells. The growth medium was removed from confluent monolayers of BHK-21cells in 60- or 100-mm tissue culture dishes, and the cells were infected with 50 to 100 PFU of rabies virus (ERA strain) per cell in 1 ml or

mock-infected with an equal volume of MEM supple-mented with 0.2% bovine serum albumin (0.2% BSA-MEM). Virus was adsorbed for 30 min at370C beforeunadsorbed virus and mock infection medium were removed. Both cell culture types were

incu-batedingrowth medium at370Cin a 5%C02 atmos-phere.

Labeling of virus-infected and mock-infected cells. Growth medium wasremoved, and each cul-ture wasincubatedinEarlesaltsolution plus10%

fetal calfserum at3700 for 15 min todepleteamino

acid pools. Cells labeled under isotonic conditions

were incubatedfor various times at 370C in Earle

saltsolutionplus10%fetalcalf serum with 40,uCiof 3H-labeled aminoacidmixture perml(NET-250, 15 aminoacids, New England NuclearCorp.[NEN])or 10

,ACi

of[3H]leucineper ml (TRK 170, 50Ci/mmol, Amersham/Searle). Cells labeled under hypertonic

conditions were incubated for 15 min at 370C in various hypertonic media containing different amountsof excess NaCl (toallowrunoff ofcellular

mRNA from polysomes [35]) in Earle salt solution plus 10% fetal calf serum and thenlabeled for var-ious timeswith one of the above isotopes at370Cin hypertonic media. Identical results were obtained

whenlabelingwasdone under hypertonic conditions

with [3H]leucine or 3H-amino acid mixture (after

correctingfordifferencesintherelative leucine con-tentof the individual viralpolypeptides). Cellswere

labeled with 50 ,uCi of [3H]glucosamine per ml

(NET-190, 12Ci/mmol, NEN) inisotonic or

hyper-tonic 0.2% BSA-MEM according to the procedure

previously described for amino acid labeling. The tonicity (milliosmolarity) of the various mediawas determined with an automatic osmometer (Osmette-A, PrecisionSystems, Inc., Sudbury, Mass.).

Inpulse-chase experiments, cells in 60-mm tissue culture dishes were pulse-labeled with 100 ,Ci of

[3H]leucineinhypertonic medium (550 mOsM) for 2 h and then washed twice with 5 ml of 0.2%

BSA-RABIES PROTEIN SYNTHESIS 103 MEM(290 mOsM), followed by a chase with 2 mlof

0.2% BSA-MEM (290mOsM)for 24 h at370C.

Preparation of cellular fractions. Cells labeled underhypertonic or isotonicconditionswerewashed twice withice-cold NT buffer (0.13 MNaCl, 0.05M Tris-hydrochloride, pH 7.8). Whole-cell lysates were prepared by scraping thecells from the tissue

cul-turedish with 2 ml of ice-cold NT buffer and sonicat-ing(Accousticon) for 1 min. Nuclear and cytoplas-micfractions wereprepared after Dounce homogeni-zation, as previously described (26). Total protein contentwasdeterminedbyamodified Lowry proce-dure (30), and trichloroacetic acid-precipitable

ra-dioactivitywasdeterminedby precipitation of sam-ples in ice-cold 10%trichloroacetic acid, followed by washing with ice-cold 5% trichloroacetic acid on glass-fiber filters (WhatmanGF/C).

Preparation of labeled virus. Roller bottles of con-fluent BHK-21 cells (1.5 x 108 cells/bottle) were

infectedwith 5 PFUof rabies virus (ERA strain) per

cellor 0.01 PFU of VSV (Indiana strain) per cell. Virus was adsorbed for 30 min at330Cin 10 mlof inoculum, and then 100 ml of 0.2% BSA-MEM was

addedtoeach bottle for incubation at330C. A 14C0

labeled aminoacid mixture (0.5 ,uCi/ml, NEC-445; 15amino acids, NEN) wasadded to the rabies-in-fected cells, or [14C]leucine (0.5 ACi/ml, NEC-279,

270 mCi/mmol, NEN) was added to the

VSV-in-fectedcells. Therabies-infected cellswereharvested after 4to 5 days ofincubation at 330C; the

VSV-infected cells were harvested after 12 to 16 h of

incubation at the same temperature. The culture medium containing labeled virus was clarified by centrifugationat450xg.,for 15 minat40C,and the viruswaspurified by a modification of theprocedure described by B. Dietzschold, J. H. Cox, and L. G.

Schneider(manuscript in preparation). Inbrief,the virus waspelletedfrom theclarifiedsupernatantby centrifugationin aBeckman type 19 rotorat19,000

rpmfor 120 min at40C.The viruspelletwas

resus-pendedwith a PasteurpipetteinNTE(0.13 MNaCl,

0.05 M Tris, pH 7.8, 0.001 M EDTA) buffer and layered on a 10 to 60% sucrose gradient in NTE buffer. ThegradientwascentrifugedinaBeckman

SW27 rotor at 22,000 rpm for 90 min at40C.The vi-rusband wascollected, dilutedinNTEbuffer,and

repelleted by centrifugation in an SW27 rotor at 20,500 rpmfor 90 min at40C. The virus pelletwas

resuspendedin asmallamount of NTEbuffer over-night at40C,and thenclarifiedbycentrifugationat 800 xg.,for 10 minat40C. Labeledvirusreleased

intothe culturemediumduringthe 24-h chase(Fig.

6c) waspurified by layeringthe supernatant(2ml)

directlyonto a 10 to 50% sucrose gradientinNTE

buffer andcentrifugingasdescribedabove.

Polyacrylamidegel electrophoresis(PAGE). Cell

lysates and virus sampleswere prepared for PAGE after trichloroacetic acidprecipitation, as described by Sokol et al. (44), or precipitation in 5 volumes of absolute ethanol. The samples (in 8 M urea) were

subjectedtoelectrophoresis in continuous 7.5%

poly-acrylamidegelsin 0.1 Mphosphate buffer (pH 7.2) containing0.1%sodiumdodecyl sulfate (SDS) (44), or on discontinuous 10% SDS-polyacrylamide gels

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(23). The gels were fixed in 25% isopropanol-10% aceticacid,cut into 1-mmslices,and thenincubated

overnight at 330C in 5 ml of a 5% Protosol-liquid

scintillation fluid mixture.

RESULTS

Relative rate ofviral polypeptide synthesis in infected BHK-21 cells under hypertonic conditions (550 mOsM). The incorporation of 3H-labeled amino acids into viral polypeptides

wasmeasuredunderhypertonicconditions(550

mOsM) in infected cells during 1-h labeling periods at 6, 12, 24, 36, 48, and 72 h after infection. Analysis of the cytoplasmic and the nuclear fractions on PAGErevealedthatabout 85%of thelabeledrabies-specificproteinswere

in the cytoplasmic fraction, as previously de-scribed (26). From thesegels, the relativerate

of 3H-labeled amino acidincorporation into the viral polypeptides permilligram of total cellu-lar (nuclear + cytoplasmic) protein per hour wascalculated (Fig. 1).During the 1-hlabeling

period, 3H-labeled amino acid incorporation

into individual viral polypeptides was linear. Only 1.5 to 3.0% of the labeled polypeptides

were found in the medium. Analysis of these

released polypeptides by PAGE revealed that less than 10%of thesepolypeptidesco-migrated

withviral proteinsandthat therewas no

pref-erential release of any viral polypeptide into the medium. Inaddition, themedium used for

cells labeled for 2 h with [3H]leucine at 550

mOsM did not containdetectable labeledvirus

particles (detection limit,5%of290mOsM

con-trol), asdeterminedby sucrosegradient

analy-sis (10 to50% sucrose).

3H-labeled amino acid incorporation into G, N, and Ml polypeptides was detected by 12 h afterinfection, and incorporation into M2

poly-peptidewasnotedby24 hafterinfection/ (Fig.

1).The rate of3H-labeled amino acid incorpora-tion into the major viral polypeptides (G, N,

M1, M2) increased gradually for at least 72 h

afterinfection. If aminoacidsfor viral polypep-tidesynthesis areavailablefromasingleamino

acidpool andthe individualpolypeptideshave

comparable stabilities, the relative rate of

amino acid incorporation into these polypep-tidesismost likely a measure of their relative rate ofsynthesis. In Fig. 2, the relative rate of amino acid incorporation into viral polypep-tidesunder hypertonic conditions (550mOsM) obtainedfrom severalexperimentswasusedto

determine the relative number of viral protein molecules synthesized per hour at 550 mOsM. These data showthatindividual viral polypep-tides are notsynthesizedinequimolaramounts

under hypertonic conditions. The L

polypep-

100-0

a w

I. 4 0

0. a.) 0.

Q

z

a) z

w

0 0. E

80

60-40

20-I0 20 30 40 50 60 70 80 TIME AFTER INFECTION (h) FIG. 1. Rateof3H-labeled amino acid incorpora-tion intorabies viruspolypeptides under hypertonic conditions (550 mOsM). Confluent monolayers of BHK-21 cells (5 X106cells perdish) infected with 50 PFUof rabies virusper cell were labeled for 1 h at 37°C with a 3H-labeled amino acid mixture (40 puCi/ ml) in 550 mOsM medium at 6, 12, 24, 36, 48, and 72 h after infection. The cells were fractionated into nuclear and cytoplasmic fractions, and 400

pg

of proteinfrom each fraction was run on continuous 7.5%SDS-polyacrylamide gels.Radioactivity incor-porated into the viralpolypeptides in each cellular fraction was obtained by subtracting radioactivity

due to residual hostpolypeptidesynthesis from the

peaks representing the viralpolypeptides. Finally,

the sum ofradioactivity incorporated into the viral

polypeptidesinthenuclear pluscytoplasmicfractions wasdividedbythe total protein in the nuclear plus

cytoplasmicfractions to give the counts per minute incorporated per milligram of totalcellular protein.

Co-electrophoresis with "4C-amino acid-labeled ra-bies virusproteinsidentifiedtheviral-specificpeaks.

tide,especially,issynthesizedat a rate approx-imately170-foldlessthan thatof the N polypep-tide. The presence of this polypeptide in in-fected cells is attimesobscuredby other high-molecular-weight polypeptides at the top of SDS-polyacrylamide gels.

Effect ofincreasing tonicity on viral poly-peptide synthesis. The rates ofNandM2 poly-peptide synthesis were enhanced relative to the ratios ofL, G, and

M,

polypeptide synthesis withincreasing tonicity (Fig. 3). The incorpora-tion ofradioactive leucine into all viral polypep-tides was inhibited astonicity increased (e.g., incorporation into viral polypeptides at 550 mOsM is 25% of incorporation at 290 mOsM [26]). Therefore, the enhancement in rate of syn-thesis ofNandM2(as compared with the other

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VOL.22,1977~~~~~RABIESPROTEIN SYNTHESIS 105

U.-i

100-0 5 ~at

'a -i

z 25

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10 2I0 3I0 40 50 60 70 80

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TIME AFTER INFECTION (h)

FIG. 2. Relative numberofviralpolypeptide mole-cules synthesized under hypertonic conditions (550

m~sM)atdifferenttimesafter infection.

Radioactiv-ity (31-labeled amino acid or[3H]leucine)

incorpo-rated into individual viralpolypeptidesperhourwas determined in several experiments by 7.5%

SDS-polyacrylamide gel analysis ofcell extracts as de-scribed in Methods. Correctionsweremadefor

differ-ences in the relative leucinecontentof

steady-state-labeled viral polypeptides (the ratio of 3H-labeled aminoacidmixturef[3H]leucineis:L, 0.78;G,1.03; N, 1.21;

Ml,

0.80;M,,0.72).Thepercentageof

radio-activity incorporated into individual viral

polypep-tidepeaks(countsperminuteincorporatedinto indi-vidual viralpolypeptidepeaks/totalcountsper min-ute incorporated into viralpolypeptides x 100) was convertedtomoleculesofprotein fromtheirrespective

molecular weightsandnormalized to100molecules

ofNpolypeptide.The molecularweightsusedforthe calculationwere:L, 190,000(seetext);G, 80,000; N, 62,000; M,, 40,000; M2, 25,000 (40). Except forthe 12-h andthe 36-h timepoints,thevalues obtainedare derivedfrom two tofourseparateexperiments. viral

polypeptides)

reflects a relative, rather

thananabsolute,increaseintherateof

synthe-sis of these

proteins.

Over therangeof tonicities examined (450to

650mOsM), the

plots

of the relativerateof

syn-thesis versus

tonicity

of the individual viral

polypeptides

were linear. If we assume that thislinear

relationship

extendstoisotonic

con-ditions, ashasbeen shown for VSV

polypeptide

synthesis

(32),the datain

Fig.

3,when extrapo-latedto

isotonicity,

give

anestimateof the

rela-tive ratesof

synthesis

ofindividual rabiesvirus

polypeptides

under isotonic conditions

(Fig.

3, dotted lines). The percentage of total

virus-specific radioactivity

incorporated

into indi-vidual viral

polypeptides

at

isotonicity

(290 mOsM) was estimated to be: L = 0.5%, N =

34%,G = 33%,

Ml

=25%, and

M,

= 8%. These

valueswereusedtocalculate the relative num-ber of viral

protein

molecules

synthesized

un-derisotonicconditions (Table1) and showthat, underisotonicconditions,theL, G, N,

M1,

and

M,

polypeptides

were not

synthesized

in equi-molar amounts. Furthermore, the relative

number of

polypeptide

molecules

synthesized

wasnot

comparable

to therelative number of

polypeptide

molecules present in extracellular virus (Table 1).

Virus-specific high-molecular-weight

poiy-peptide (L) in infected cells and in purified

virus.

Synthesis

of a

high-molecular-weight

polypeptide

was

repeatedly

detected

by

PAGE

in infected

(Fig.

4a) but notin uninfected cells

(Fig.

4b). This

polypeptide, designated

the L

polypeptide,

wasalsopresentin

purified

rabies virions

(Fig.

5a) and

co-migrated

with L

poly-peptide

of VSV.

Analysis

of

purified

rabies yiin-ons labeled with [3

Hlglucosamine

and 14 C-la-beled amino acids indicated that theL

polypep-tide of rabies virus was not

glycosylated

and

co

Z

50-0 C.

C.)

-.

40-a. Cl)

CC

-J o

20-0

I-0

z

W.)

a.

250 350 450 550 650

mOSM

FIG. 3. Effect of increasing tonicity on relative

ratesofrabies viruspolypeptide synthesis. Confluent monolayers (5 X 106 cells/60-mm dish) of rabies-infected (50 PFU/cell) and mock-infected BHK-21 cellswerelabeledat24hafter infection for1 h with

[3H]leucine (10 Ci/ml) under isotonic(290 mOsM)

orhypertonic(450, 500,550, 600,and 650 mOsM)

conditions. Whole-cell lysates (400 Mgof protein) of infected andmock-infected cells were run with '4C-amino acid-labeledprotein markers on continuous 7.5%SDS-polyacrylamide gels. The[3H]leucine in-corporationinto the viralpolypeptide peaks

attnibut-able to residual cellularprotein synthesis was sub-tractedafter normalizing thepolypeptidepattern of themock-infectedtotherabies-infectedcells. Thenet

[3H]leucineincorporationintovirus-specific polypep-tides (aftercorrection fordifferencesin relative

leu-cinecontentofthe viralpolypeptides) wasconverted

tothepercentage oftotalvirus-specificcounts inthe individualviralpolypeptides.

IN

-- G

IA

r - -- - - A -6 N1

CT----I- I I

VOL. 22, 1977

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TABLE 1. Relative number of viral polypeptide molecules synthesized

Conditions L G N MI M2

Hypertonic (550 mOsM)a 0.60 32 100 31 79 Isotonic(290mOsM)b 0.40 64 100 75 35 Relative no. of viral poly- 1.9 101 100 52 86

peptide molecules per virionc

aPercentageof3H-labeledaminoacidincorporatedinto virus-specificpolypeptides at 550 mOsM (Fig. 3)converted torelative number of viral polypeptidemolecules synthe-sized per hour, asdescribed in Fig. 2. Thenumber of N polypeptideswas setequalto 100.

bExtrapolatedpercentage of3H-labeledamino acid in-corporated into virus-specific polypeptides at 290 mOsM (Fig. 3), converted to relativenumberof viral polypeptide molecules synthesized per hour. The number of N polypep-tide molecules was set equal to 100.

cDerived from gel analyses of three separate

prepara-tions ofpurified rabiesvirus (ERA strain), uniformly la-beled with 14C-amino acids (see text). The value for N polypeptidewassetequal to 100.

7

0-0~x

I

thus did not appear to be a dimer of the viral

glycoprotein (Fig. 5b).

Number ofL protein molecules per virion.

An estimate of the number of L protein mole-cules per virion was made from data derived from PAGEanalysisofthreedifferent

prepara-tions ofpurified rabiesvirusuniformly labeled

with"4C-labeledamino acids(Table 2). Approx-imately33 Lproteinmolecules wereestimated

tobe present perrabies virion. The numberof each ofthe othermajor structural protein mole-culespervirion estimated from these data

cor-respondedtothe valuesreportedbySokol et al.

(40).

Analysisof viralglycoprotein biosynthesis.

Avirus-specifiedglycopolypeptide labeled for2 h with[3H]leucineinrabies-infected cellsunder

hypertonic conditions consistently migrated in

PAGE faster than themarker virion

glycopro-tein (Fig. 6a). Since this component was not detected inuninfectedcellslabeled under

simi-40 50 60 70 80 90 100,

50

25

0c o

5 0

2 5

FRACTION NUMBER

START

FIG. 4. Virus-specific high-molecular-weight polypeptide (L) in infected cells. (a) Rabies-infected (50

PFU/cell) or (b) mock-infected BHK-21 cell monolayerswere labeledfor1 h with [3H]leucine(10 uCi/ml)

underhypertonic conditions (550 mOsM)24 hafterinfectionat379C, and400-pgportionsofproteinofthe whole-celllysates wereanalyzedondiscontinuous10%SDS-polyacrylamide gels.['4C]leucine-labeledVSV wassubjectedtoco-electrophoresis with the celllysates. Thearrowsrefertothe VSV markerproteins.

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RABIES PROTEIN SYNTHESIS

5 0

X

0 4

4 M12 3 Q

A

2J

i~%~ ~

I~~~~~~

3 1S 2

4

I-_ 10 20 30 40 50 60 70 80 90 100 11o +

STiRT FRACTION NUMBER

FIG. 5. Virus-specific high-molecular-weight polypeptide (L) inpurified rabies virus. Roller bottles of BHK-21 cells (1.5 x 108cellslbottle) were infected with rabies virusat5PFU/cell; the infectedcellswere

grown in 0.2% BSA-MEM for 4 days at 33°C in the presence of[3H]leucine (2 pmCi/ml) alone, or [3H]glucosamine (10 pXilml) and ["4C]leucine (2.5 pCi/ml). The virus was harvested and purified as

described inMaterials and Methods.(a) Analysis of[3H]leucine-labeledrabies virus with[14C]leucine-labeled

[image:6.508.141.375.64.344.2]

VSV(12.5-cm gel). (b)Analysis of [3H]glucosamine-and[14C]leucine-labeled rabies virus(11-cm gel). TABLE 2. Number ofpolypeptide molecules per

rabies viriona

Poly- ~~~~~~~Daltons

Mole-peptid

%Total radio- Ml (oyp- cules species activity tide/virion)

i/vir

ion) L 1.85 + 0.94 190,000 6 x 106 33 G 42.67 + 1.30 80,000 145 x 106 1,815 N 32.94 ± 0.43 62,000 112 x 106 1,806 M, 11.17± 0.19 40,000 38 x 106 951 M2 11.36 ±0.73 25,000 39 x 106 1,547

aPercentage of totalvirion-associated 14C-labeled amino

acidincorporatedintotheindividual viral polypeptides was determined. Thenumber ofdaltonsof each viral polypep-tideper virion wasdetermined by multiplying the percent-ageof eachviralpolypeptide in the virion times the molecu-lar weightof the total polypeptide (340.4 x 106 daltons) in thevirion(calculated from the ratio of polypeptide to RNA [74:1]inthe virion, assumingthe RNA molecular weight to be 4.6 x106daltons[40]).The number of each polypeptide molecule per virion wascalculated by dividing the number ofdaltonsof eachpolypeptide per virion by the molecular weight of that polypeptide. The molecular-weight values for the G, N, M,, and M2 are from Sokol et al. (40). The molecular weight of the L protein was estimated at 190,000 onthe basis ofco-electrophoresis with the VSV L protein (Fig. 5a) (49).

lar conditions (notshown) and could be quanti-tatively precipitated with monospecific antise-rum directed against purified virion glycopro-tein (B. Dietzschold, H. P. Madore, and J. M.

England, unpublished observations), it was

considered to be virus specific. When rabies-infected cells were labeled for 2 h with

[3H]leucine under hypertonic conditions and chased for 24 h in isotonic medium, a large

proportion of the radioactivity that was

previ-ously associated with the faster-migrating in-tracellular glycopolypeptide shifted to the slowermigrationrateofthe virionglycoprotein

(Fig. 6b). A small proportion of the

radioactiv-ity associatedwith the intracellular glycopoly-peptide still migrated more rapidly than the virionglycoprotein (Fig. 6b). The [3H]leucine-labeled glycopolypeptide that was synthesized under hypertonic conditions was incorporated into virusreleased into the mediumduringthe 24-hchase in isotonicmedium, asshownby co-migration with the rabies 14C-labeled virion

glycoprotein that was labeled in isotonic

me-VOL. 22, 1977 107

I

0

2

I

0

a.)

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z

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N0201 A000

u

IS

25M

-c. I c-)

L

W 10- 50

Z

~~~~~~~~0

1020~ ~3 4 0 60 7

---r

---F

35- 30-

25-20 6 P10u0

ti5 (05

i0 (0PK

-

2.5I

10 20 30 40 50 60 70°

SAT FRACTION NUMBER

FIG. 6. Pulse-chaseanalysis ofintracellular viral

glycoprotein synthesized under hypertonic

condi-tions. Cultures(5 X106cells/60-mmdish)of rabies-infected (50PFU/cell) BHK-21 cells grown at370C for24 h were eitherpulse-labeled with [3H]leucine (100 gCi/ml) for2 h at 550 mOsMat 370C (a) or

pulse-labeledfor2hat550mOsMandthenchased for24hat37°Cin290mOsM medium (b). Whole-cell lysates containing 50,000 trichloroacetic acid-precipitablecpm (a) and(b) andextracellular virus from (b) above containing 30,000 trichloroacetic acid-precipitablecpm(c)wereanalyzedon discontin-uous10%SDS-polyacrylamidegels. Thearrows

in-dicate 14C-amino acid-labeled rabies virus marker proteinssubjectedtoco-electrophoresiswiththe

sam-ples.

dium (Fig. 6c). These results indicatethat hy-pertonicconditions cause the accumulation of a morerapidly migratingform of the viral

glyco-protein. During an isotonic chase, however,

this more rapidly migrating form of

glycopro-tein is converted to a form that co-migrates

with virion glycoprotein and becomes associ-ated withextracellularvirus.

In effortsto detectthe synthesis ofa faster-migrating formof viral glycoprotein under iso-tonicconditions(290 mOsM),infected and unin-fectedcellswerelabeled with [3H]glucosamine. As showninFig.7aandb, aprominent hetero-geneouspeakof[3H]glucosaminelabel was de-tected intheinfected butnotin the uninfected

celllysates.Thisviral-specificglycopolypeptide showed two components on the gel, a major component, whichco-migratedwith thevirion

glycoprotein, and a minor component, which

migrated morerapidlyas ashoulder. Both

com-ponents could be quantitatively precipitated

withantibody directed against virion

glycopro-tein (B. Dietzschold, H. P. Madore, and J. M.

England, unpublished observations). We have

designatedthe morerapidly migrating

compo-nent gp78 to distinguish itfrom the

glycopro-tein present inextracellular virus (gp8O). The faster-migrating component exhibited approxi-mately thesameelectrophoreticmobilityasthe virus-specific peak labeled with [3H]glucosa-mineunderhypertonic conditions (Fig. 7cand d).

To examine thepossibilitythat gp78 was an

intermediateinthebiosynthesis of rabies virus glycoprotein (gp8O), infected cells were pulse-labeled with [3H]glucosamine under isotonic conditions forintervals from 30min to4 h (Fig. 8). After 30 min oflabeling, the majority of

[3H]glucosamine, whichwasassociated with

vi-rus-specificglycopolypeptide, was inthe faster-migrating component, gp78 (Fig. 8a).After1h

of labeling, a portion of the [3H]glucosamine

label was in a shoulder of the gp78 peak that migrated with the gp8O marker (Fig. 8b). After

2h (Fig. 8c) and 4 h (Fig. 8d) oflabeling, the majorityofthe[3H]glucosaminelabelwas

asso-ciatedwithgp8O. This shift of[3H]glucosamine label fromgp78 togp80 withincreasedlabeling

time suggests that gp78 is a precursor ofthe

mature viralglycoprotein (gp8O).

DISCUSSION

Using hypertonic conditions to selectively

suppress cellular protein synthesis, we have

characterized several aspects of rabies virus protein synthesis in BHK-21 cells. The rabies virus structural polypeptides L, G, N,

M,,

M2

are synthesized continually throughout the

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RABIES PROTEIN SYNTHESIS 109

1Y

0

N

0.

w

z

4 0

C-)

IS

en

I20

I.0

-2

0

4 0

-30 -15

10 20 30 40 50 60 70 80 90 160 10 20 30 40 50 60 70 80 90 100

[image:8.508.56.455.59.382.2]

FRACTION NUMBER

FIG. 7. Labelingofrabies-infectedoruninfected cells with[3H]glucosamineunder isotonic (290mOsM)or

hypertonic(550mOsM)conditions. Confluent monolayers(5 x 106cells/60-mm dish)ofBHK-21 cellswere eitherinfectedwithrabies virus (100PFU/cell) (a and c)ormock-infected(bandd)andincubatedat37'C

for40 h.Cells were then labeled under isotonic conditions(290mOsM) for2hwith[3H]glucosamine(50 XciI

ml) (a and b). After pretreatment for 15 min withhypertonicmedium (550mOsM),other cells werelabeled

for2h inthe same hypertonic medium containing[3H]glucosamine (50 puCi/ml)(c andd). The whole-cell

lysates containing27,000 trichloroaceticacid-precipitable cpm wereanalyzedon discontinuous10%

SDS-polyacrylamidegels. Arrows indicate 14C-amino acid-labeled rabies virus proteins.

courseof infection. Incontrasttothesynthesis of the NS polypeptide ofVSV, which has been showntobepreferentially

synthesized

early

in infection (17), none of the rabies-specific

poly-peptides appearedtobesynthesizedat agreater

relativerateearlyininfection.

The relative rates ofsynthesis of the rabies

virus polypeptides do not correspond to their relative proportions in the intact virion. This observation indicates that theprotein

composi-tion ofvirions is not solely determined by the relativerate atwhich the viralpolypeptidesare

synthesized andsuggests thatadditional mech-anismsunderlieviral assembly.In the absence ofdata that compare the absolute rate of

syn-thesis of a given polypeptide to the rate at

which thatpolypeptide isincorporated into

vi-rus, noconclusioncanbe made concerningthe

rate of synthesis of a given viral polypeptide and arate-limitingstep inviral assembly.

Lodish(24)hasproposedamodel for the regu-lation ofa- and f3-globin synthesis which

pre-dicts that a reduction in the overall rate of

peptide chaininitiation willcausethe preferen-tial translation of mRNA species with higher

rate constants of peptide initiation. Since hy-pertonicconditions have been shownto specifi-cally suppress peptide chain initiation, this model can account forthe differential inhibi-tioncausedby hypertonicconditionsof the

syn-thesis ofvariouscellular (33)andviral (rabies, VSV [32], and MMTV [36]) polypeptides.

Ifthis modelis indeed correct, the different

sensitivities amongthesynthesesof L, N, and M2, aswellasG andMl,polypeptidesto

hyper-tonic conditions suggests that at least three

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I

0

a-0)

30min

8.0-5

1.0-

ft7

II

II I I II

z

f 200

<n 16.0

0

°

12.0-80

ID

6.0-4.0

2.0

-5.0

TI

-2.5 N,

o

X X

(

E

2

J

0

9 A,

-50

-25

I-0

z

0 20 30 40 50 60 70 80 10 20 30 40 50 60 70 80

FRACTION NUMBER FRACTION NUMBER

START lSTART

FIG. 8. Kinetic analysisof intracellularviral glycoproteinsynthesis.Confluent monolayers(5 x106 cells!

60-mmdish) wereinfected with rabies virus (100PFUlcell),and thenincubated for40hat37TC. Cellswere

exposedto[3H]glucosamine (50

gCi/ml)

inisotonicmediumfor 0.5 h (a),1h(b), 2 h (c), and4h (d). Whole-cell lysates (450 pgofprotein) were analyzed ondiscontinuous 10%SDS-polyacrylamidegels. 14C-amino acid-labeled rabies virus proteinsweresubjectedtoco-electrophoresis with the cell lysates. Totalincorporation of[3H]glucosamineintoacid-precipitablematerialas afunction of time is shown in the insert of (c).

ribosome-binding sites (with different initia-tion efficiencies) exist in rabies virus mRNA. Since eukaryotic mRNA apparently contains only one functional initiation site (9, 13, 22), thesedata suggest that there isaminimum of threediscrete rabies virus mRNA species. Con-sistent with this argument are observations that the synthesis ofindividual poliovirus pro-teins, whichare translated fromasingle

poly-cistronic mRNA molecule with one initiation site (13, 35), does not show differential inhibi-tionbyhypertonic conditions (34), whereas the synthesis of individual VSV proteins, which are translated from several monocistronic mRNAmolecules (4, 20, 28, 29, 46), is differen-tially inhibited by hypertonic conditions (32,

34).

A majordifferencebetween rabies virus and VSVhas been the inabilitytodetect RNA

tran-scriptase activity in purified rabies virions (41, 48). RNA transcriptase activity could not be

detected in purified rabies virions by in vitro

assays that were sensitive enough to detect RNAtranscriptasewithaspecific activity 4,000 times less than that present in VSVvirions (H. P. Madore and F. Sokol, unpublished observa-tions).

In VSV, the high-molecular-weight L poly-peptide has been shown to be necessary for RNA transcriptase activity (5-7, 10, 11, 31). Since a high-molecular-weight polypeptide analogous to the VSV L polypeptide had not

beenclearly demonstratedinrabiesvirions(40, 42), itwaspossibletoattribute thedeficiencyin RNAtranscriptase activity inrabies virionsto

the absenceorextremely lownumber of L poly-peptide molecules per virion. In the present work, however, wehave detecteda high-molec-ular-weight polypeptide (with an electropho-retic mobility similar to that of the VSV L

polypeptide) in rabies virions and in infected

cells, and we have estimated that each rabies b Ih

I

1

I

I I

III

I

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RABIES PROTEIN SYNTHESIS 111 virion containsapproximately 33 Lpolypeptide

molecules (Table 2). In comparison, aVSV vir-ion contains about 60 L protein molecules (3). Therefore, if we assume that the Lpolypeptides of rabies virus and VSV arefunctionally simi-lar, the extremely low level of RNA transcrip-tase activity in rabies virions does not seem to result from an absence or extremely low num-ber of L polypeptide molecules in the rabies virion.

In rabies-infected cells labeled for 2 h with [3H]glucosamine under isotonic conditions, two forms of virus-specific glycoprotein are ob-served: the major form, designated gp80,

co-migrates with the viral glycoprotein; a minor

form, designated gp78, is seen as the

faster-migrating shoulder of the major viral glycopro-teinpeak (Fig. 7a). Kineticanalysis of the

bio-synthesis of these two forms has revealed that

the initial appearance of the faster-migrating gp78 and the subsequent accumulation of the

gp8Oiscoupled withthe slowdisappearance of

the gp78(Fig.8a-d).These resultssuggest that the faster-migrating gp78 isanintermediatein

the synthesis of gp8O. Furthermore, gp78 may represent anincompletelyglycosylatedform of the viral glycoprotein, since neuraminidase-treated viralglycoproteinmigratesonPAGE at

a rate that approximates that of intracellular

gp78 (B. Dietzschold, unpublished

observa-tions). Glycoproteinsynthesis of another rhab-dovirus, VSV, also appears to involve incom-pletelyglycosylatedintermediates that migrate faster than the mature glycoprotein in

SDS-polyacrylamide gels (1,4, 17, 20, 28, 29,45, 46).

The rabies-specific glycoprotein in

infected

cellslabeledwith aminoacids (Fig. 6a) or with

glucosamine (Fig. 7c) underhypertonic

condi-tions migrates in SDS gels with a mobility similar to that of gp78. The observation that this faster-migrating glycoprotein can be chased under isotonicconditions into an

intra-cellular

glycopeptide

that co-migrates with

gp8O and is subsequently released with

extra-cellular virions (Fig. 6b and c) suggests that hypertonic conditionsmay reversiblyinterfere withthe biosynthesis of rabiesvirus glycopro-tein. The faster migration rate of the product

synthesized under hypertonic conditions

sug-gests that interference occurs at an

intermedi-ate that requires supplementaryglycosylation

to formthematureviralglycoprotein. Whether this component is (i) identical to gp78, (ii) an-other intermediate not resolved from gp78, or

(iii)anaberrantstepinviralglycoprotein

mat-uration remains to bedetermined. A

compari-sonof the sizeandionexchange distribution of

the oligosaccharides of the viral glycoproteins

isbeing carriedout to answerthisquestion.

The reversible block of rabies glycoprotein

biosynthesis caused by hypertonic conditions

appears tobeanalogoustothe reversible block in Semliki forest virus glycoprotein synthesis, which occurs wheninfected cells are grown in

sugar-free medium (15). In contrast to hyper-tonic and sugar-free conditions, the impaired glycosylation of influenza (19, 37) and Semliki forest virus (14)glycoprotein intermediates

in-duced by 2-deoxyglucose or excess glucosamine cannotbereversedafterremovalof these inhib-itors. Thisirreversibleblockageinglycoprotein biosynthesis isattributedto incorrect glycosyl-ationinduced bythe inhibitors (15).

ACKNOWLEDGMENTS

The skilled assistance of Len Milewski and Len Kaplan isgratefullyacknowledged.

This work wassupported by Public Health Service grant AI-09706 from theNational Institute for Allergy and Infec-tiousDiseases, a fellowship (CA-01463) from the National CancerInstitute,andgrant RR-05540 from the Division of Research Resources.

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