Intracellular precursors to the major glycoprotein of avian oncoviruses in chicken embryo fibroblasts.

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Copyright ©1978 AmericanSociety forMicrobiology Printed in U.S.A.

Intracellular Precursors

to

the

Major

Glycoprotein of Avian

Oncoviruses

in

Chicken Embryo Fibroblasts

DOROTHY L.BUCHHAGENt* AND HIDESABURO HANAFUSA Department of Viral Oncology,TheRockefeller University, New York,New York10021

Receivedfor publication30June1977

A 96,000-dalton glycoprotein, p(96),waspresentincellextractsobtained from gs-chf- chickenembryo fibroblasts infected with the avian RNAtumorviruses Rous-associatedvirus-2subgroupB (RAV-2) and the

Schmidt-Ruppin

strain of RoussarcomavirussubgroupA

(SR-RSV-A),

aswellasfromuninfected gsLchf+ (HE) cell extracts. It wasnot found in cellextracts from uninfected gs-chf- or

gs+chf+ (HH) cells,norfromgs-chf-cellsinfectedwithenvelope-deficient Bryan high-titer Rous sarcoma virus.

Immunoprecipitation,

kinetic, and biochemical

data indicate that this polyprotein contains information that gives rise to the major virion glycoprotein gp85.Asecondpolyproteinof80,000daltons, p(80), is also present in the RAV-2- and SR-RSV-A-infected

gs-chft

cells. This second polyprotein contains less carbohydrate than p(96), and kinetic and biochemical data indicatethatp(80)maybeanimmatureform ofp(96).

The

biosynthesis of both avian and murine

RNA

tumor

virus structural proteins in

virus-infected tissue culture cells has been

actively

studied in

recent years.

Polyprotein

precursors

to

the

internal structural virion

proteins have

been

detected in the avian

(20, 21) and murine

(11,

17,

19)

systems.

Similarly,

larger polyprotein

precursors to

the virion

glycoproteins have been

specifically

immunoprecipitated

from the

cyto-plasm of Rauscher murine

leukemia

virus-in-fected

cells

(1, 4; D. L.

Buchhagen,

Ph.D.

thesis,

Cornell

University,

Ithaca,

N.Y.,

1975), and

sev-eral

reports

have described

presumptive

precur-sors to

avian RNA tumor

virus

glycoproteins

(3,

6). This

report

describes the

finding

of

a

glyco-protein with

an apparent

molecular

weight of

96,000 daltons

that

serves as aprecursorto

the

major

avian viral

envelope

glycoprotein (gp85).

The

detailed

immunological

and biochemical

characterizations of this

precursor

protein

are

presented

here

as well as

evidence

suggesting

its

derivation from

an

80,000-dalton

glycopro-tein.

Also

presented

arekinetic data

relating

to

the

synthesis

and

disappearance

of this precur-sor

in

the

cytoplasm

of virus-infected

cells

pulsed

with radioactive amino acids and the

appearance of the

radiolabel

in the

gp85

ofintact extracellular virions.

MATERIALS

AND METHODS

Cellsand viruses. Cellsused forthepropagation ofviruses wereprepared asprimaries from embryo-nated eggssupplied bySPAFAS, Norwich, Conn., and

were grownincomplete Scherer medium containing

tPresent address: The Sidney Farber Cancer Institute, Boston, MA 02115.

5% calfserum. Only cells that typed as gs-chf by complement fixationtestsandhelper assays (8) were used for viruspropagation. Some uninfected cells that typedasgs+chfi (HH) and gsLchf+ (HE) were used in

designatedexperiments (8).

Viruses used were the Schmidt-Ruppin strain of RoussarcomavirussubgroupA (SR-RSV-A), Rous-associated virus-2subgroupB(RAV-2),and envelope-deficient Bryan high-titer Rous sarcoma virus

[BH-RSV(-)](7).Secondarycultures of cells were infected by the addition of 0.1 ml of stock virus solution to

subconfluentmonolayersof cellsin100-mm-diameter plastic petri dishes (LuxPlastics).

Antiserum. Rabbit antiserumwaspreparedby J. H.Chenusing RAV-60 gp85 that had been purified by

chromatography on Bio-Gel A-5m agarose in

guani-dine

hydrochloride-containing

buffer(5).Rabbitswere

hypenmmunizedbyoneinjectionof400,ugofpurified

RAV-60gp85 incompleteFreundadjuvant,followed

bytwobooster

injections

of 200,ugof

protein

each in

incomplete Freund adjuvant at4and 6weeks after the initial injection. A

precipitating

antiserum was

prepared byD. L. BuchhagenandG. Notaniby im-munizingagoattwice with100ugofpurifiedrabbit

immunoglobulinG incompleteFreundadjuvant.

Radiolabeling

ofviru8esand cells.Whole

radio-labeledviruswaspreparedby

labeling

infectedcells

with

L-'4C-amino

acids(NewEngland Nuclear)at25

,uCi in 5 ml of Scherer medium containing5% calf

serum andlackingtryptosephosphate broth fortwo 16-hperiods,followed bya 16-hchaseusingScherer

mediumcontainingboth 5% calfserumandtryptase phosphatebroth(complete Scherer medium).

Cultureswerepulsedfor thetimes indicated bythe addition of25uCiof

'4C-labeled

L-amino acids in 1.0 ml of Earle balanced saltsolutiontotheinfectedcells

in100-mm-diameterplates;chaseswere performed by replacing thelabelingmedium with 10mlofcomplete

Scherer medium. Pulses and chaseswere terminated bywashing the monolayers twicewith 10 ml ofcold

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846 BUCHHAGEN AND HANAFUSA

phosphate-buffered saline. Labeled sugarswereused

inthesame manner at thefollowing concentrations: D-['4C]mannose (10uCi/ml); D-['4C]glucosamine (10

,uCi/ml);

D-[3H]galactose

(200

uCi/ml);

and

L-[3H]fu-cose(200uCi/ml).

Inhibitors.D-Glucosaminehydrochloride (Mathe-son,Coleman, and Bell) or2-deoxy-D-glucose (Calbi-ochem)wasaddedtocomplete Scherer medium at a concentration of10mM.

Virus purification. Radiolabeled virus was puri-fiedasoutlinedpreviously(18) by pelleting thevirus fromculturesupernatantsthrough 20% sucrosein0.01 M Tris (pH 7.4)-0.1 M NaCl-0.001 M NaEDTA (TNE) onto a cushion of 60% sucrose-TNE in an SW27.1 rotor at 119,000 x g for 3 h at 4°C. The cushioned viruswasthen banded in15 to50%

sucrose-TNEgradients inanSW27.1rotor at 119,000xgfor

16hat 4°C. Banded viruswaspelletedinatype40 rotor at111,000xgfor90minat4°C,andviruspellets

werekept frozenat-70°Cuntil needed.

Immunoprecipitation. Confluent monolayers of chickenembryo fibroblastswerelabeled with the ra-dioactive precursoraccordingtothe particular

exper-iment,andcytoplasmicextracts werepreparedas de-scribedby Vogtand Eisenman(20) bytheaddition of

abuffercontaining0.5% Nonidet P-40 and 0.5% sodium

deoxycholate.To200 ug of totalcellularprotein per reactionwasadded themonospecific antiserumata final dilution of1:40. This mixturewasincubatedat

37°C for1handat4°C for16h.Precipitating

antise-rum wasthenaddedat a5:1ratiotothe first antise-rum;the reaction mixtureswereincubated at 37°C for

15min and thenat4°Cfor4h. Immuneprecipitates

were collectedby centrifugationina Sorvall RC2-B

centrifuge usinganSE-12 rotorat 10,000 xgfor 15

minat40C.

SDS-PAGE. Sodium dodecyl sulfate-polyacryl-amide gel electrophoresis (SDS-PAGE) was per-formedasfollows. Gradientpolyacrylamide slab gels (7.5 to 15%) were prepared using modifications of Laemmli (14).Separating gelswere13.0 cmlongand

1.25 mm thick; 4.5% stacking gelswere 1.5 cmlong.

Samplesweresolubilized in solutionshaving the fol-lowing final concentrations: 0.0625 M

Tris-hydrochlo-ride(pH 6.8),2%SDS(Matheson,

Coleman,

andBell),

0.025 M

dithiothreitol,

10%

glycerol,

and 0.01%

brom-ophenolblue. Gelswere runat20mAconstant current

and were fixed and stained with 0.25% Coomassie

brilliant blue in 45% methanol-10% acetic acid.

Pro-gressive destaining was first in 45% methanol-10% acetic acid and then in 7.5% methanol-5% acetic acid. Gelswere impregnated with PPO

(2,5-diphenyloxa-zole)accordingtotheprocedureof Bonner andLaskey

(2) and dried.Autoradiographywascarriedoutwith Kodak RP Royal X-Omat medical X-ray film at

-70°C;filmsweredevelopedin aKodak

Rapid-Proc-essautomaticdevelopingunit.

RESULTS

Immunoprecipitation

of proteins from

the

cytoplasm

of

infected

cells. Rabbit

anti-RAV-60 gp85 serum was used to

precipitate

pulse-labeled proteins

from the

cytoplasm

of

chicken

embryo

fibroblasts that had been

in-fectedwith RAV-2 and SR-RSV-A viruses(Fig. 1, lanes b and c). The major protein that was

precipitated migrated in this SDS-PAGE slab gelsystem with anapparentmolecularweight of 96,000 andisdesignated p(96).Asecond

protein,

p(80), of

approximately

80,000

daltons,

wasoften precipitated by the anti-gp85 serum; however, the quantityof thisproteinintheimmune pre-cipitates was highly variable from one experi-ment to the next,

although

it was present in

a b C d

*% w-.

w44

p

(96)

-p

(80)

--iel I

_ #1.

_

r0

*l

FIG. 1. SDS-PAGEanalysisofimmunoprecipitates ofpulse-labeled chicken embryo fibroblast cytoplasm. Cytoplasmicextractsofgs-chf cells wereprepared aftera20-minpulsewithL-"4C-aminoacids. Portions of theextractswerereacted withrabbit anti-RAV-60 gp85serum, and theresulting precipitateswere sub-jectedtoelectrophoresisin 7.5 to 15%gradientslab

gels. Electrophoresis wascarried out at20mAper gel (constantcurrent). Thefollowinggs-chf cell ex-tracts wereused: lanea,uninfected; laneb, RAV-2-infected; lane c,SR-RSV-A-infected;andlaned,

BH-RSV(-)-infected.

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PRECURSORS TO GLYCOPROTEIN OF AVIAN ONCOVIRUSES

largest

quantities in lysates from cells infected

with RAV-2 virus. p(96)

was not

detected

in

immune

precipitates obtained by using either

uninfected

gs-chf

cells

(lane

a)

or

BH-RSV(-)-infected

gs-chf

cells

(land d). Further,

neither

p(96) nor

p(80)

was

precipitated by

ratantisera

prepared against

purified proteins

p27, p19,

and

p15

derived

from

RAV-2

(D.

L.

Buchhagen,

unpublished data). Conversely,

the

specificity

of

the

rabbit

anti-gp85

serum was

further

estab-lished

by its failure

to

precipitate

the

major

virion

proteins p27, p19,

and

p15. Many

protein

bands,

including

those

of

approximately 96,000

and

80,000

daltons,

were

detectable

in all

im-mune

precipitates including those

obtained from

uninfected gs-chf-

cells

and

were

assumed

to

be

comigrating host

proteins.

The dark band

at

approximately

40,000

to

50,000 daltons

is an

artifact

produced

by the

presence

of

large

amounts

of

immunoglobulins

in

the

precipitates.

Presence

of

p(96) in cells having

dif-ferent levels

of

helper activity. Uninfected

chicken embryo fibroblasts that

were

either

gs+chf-, gs+chf+

(HH),

or

gsLchf'

(HE)

were

pulse-labeled

with radioactive amino

acids

for

1E*

.->i

20

min, and immune

precipitates

were

prepared

as

described

previously,

by using equal

quanti-ties

of

total

protein

from each

cell lysate. p(96)

was

detectable

at

low

levels in

extracts obtained

from

pulsed

gsLchf+

(HE)

cells

(Fig.

2, lanes e

and

f),

but not in those from

gs-chf

(lane b)

or from

gs+chf+

(HH)

cells

(lanes

c and d). The

p(96)

from

the

gsLchf+ (HE) cells

comigrated

with

that obtained

from

RAV-2-infected

gs-chf

cells

(lane

a).

Only

immune precipitates from

RAV-2-infected

cultures.

(lane

a) had

demon-strable p(80). The failure

todetect

either

p(80)

in any

uninfected

cell

lysates

or

p(96)

in

gs+chf

(HH)

cells may

be

dueto

the

insensitivity

of the

detection method

chosenormay

be due

to a

real

absence of the

proteins

studied.

Evidence

that both

p(96) and

p(80)

are

glycosylated.

To

determine whether

the

pro-teins

p(96) and p(80) that

are

specifically

rec-ognized

by

an

antiserum

to a

virion

structural

glycoprotein

are

themselves

glycosylated,

cells

infected with RAV-2

were

pulsed for

20 min

with either

D-['4C]mannose,

D-["4C]glucosa-mine,

D-[3H]galactose,

or

L-[3H]fucose

(Fig.

3)

as

described in Materials

and Methods.

Under

d

e

g

5

..

lI

p

(96

i-p

(

80

-I.X

g

:.

sbFF

e-:

.

*-: :s

..

Or.

.OF

?-

If

ow1

FIG. 2. SDS-PAGEanalysisofimmuneprecipitates preparedasdescribedin thelegendtoFig. 1.Lane a,

RAV-2-infected gs-chf;laneb, uninfectedgs-chf;lanec,

gs+chf

(Hit),

embryo 3006; laned,gs+chf

(Hid,

embryo 3010; lane e,

gsLchf (HE),

embryo3007; lanef,

gsLchf

(HEJ,embryo 9838; laneg,purified

L-'4C-amino

acid-labeled RAV-2 virus.

847

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848

BUCHHAGEN AND HANAFUSA

L

1'

'K,

f

l)

dUb

F. .¢ O}

3 "

viruses (4, 9, 12, 13). It

should

be noted that cell

morphology

was

normal

after the 8-h

pretreat-ment,

and

the patterns of

labeled

cellular

pro-teins

nonspecifically precipitated

in theimmune

complexes

were

similar for both treated

and

untreated cultures.

Kinetics of synthesis of p(80),

p(96),

and

gp85. A

series of

immune

precipitates obtained

from

RAV-2-infected cultures that had been

pulsed

with

L-'4C-amino

acids

forperiods

rang-ing from 2 to 20 min, or

pulsed

for 20 min and

then

chased

with

complete

mediumlacking the

label for

1 to 4

h,

are

depicted

in Fig. 5. Only

ad

b

P

(96'\I

p

(SO.)

(-.

W.F.,

.3r1 >

W. Mp

FIG. 3. Immune precipitates of extractsfromcells labeledfor20mineach asfollows: lane a,

D-["Cl-mannose; lane b, D-/'4C]glucosamine; lane c,

D-3H]galactose;lane d,

L-[H]fucose.

these

conditions,

both

p(96) and

p(80)

could

be

labeled with

mannose

(lane

a) and

glucosamine

(lane b);

galactose

(lane c)

could be detected

only in p(96), and fucose (lane d) could not be detected in either

protein.

Sensitivity

of

the

synthesis of p(96) and

p(80)

toknown

inhibitors of

glycosylation.

Pretreatment of

virus-infected

cells

for8 hwith

either

2-deoxy-D-glucose

or

D-glucosamine

hy-drochloride

atconcentrations of10mM

substan-tially

decreased the

subsequent

20-min

incorpo-ration

of

L-14C-amino

acids intop(96) and p(80) as shownin

Fig.

4

(lanes

b andc). This finding is consistent with the known effects of these drugs on the maturation of various enveloped

FIG.

4. Immuneprecipitates ofextractsfromRA V-2-infected

gs-chf

cells that had beenpretreated for 8h with10mM concentrations ofinhibitorsbefore

receiving 20-minpulses of L-

'4C-amino

acids. Cell

extracts:lane a,untreated;laneb, 2-deoxy-

D-glucose-treated;lanec,D-glucosamine hydrochloride-treated. J. VIROL.

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PRECURSORS TO GLYCOPROTEIN OF AVIAN ONCOVIRUSES

a b c e f

...

g . h. ........

x

-y

_-P(96)- ,

p

(80)-4 3 t

FIG. 5. Immuneprecipitatesfrom RAV-2-infectedgs-chf cells thatwerepulsed with L-"C-amiroacids.

Pulsetimes: lanea,2min; laneb,5min;lanec,10min; laned, 15min;lanee,20min;lanef, 20-minpulse, 1-hchase; laneg,20-minpulse,2-hchase;laneh, 20-minpulse,3-hchase; lanei, 20-minpulse, 4-h chase; lanej,L-14C-aminoacid-labeled RAV-2 virus.

p(80) could be detected during a 2-min pulse (lane a), whereas both p(80) and p(96) were detected after a 5-min or longer pulse (lanes b-e). Likewise, p(80) almost entirely disap-pearedfrom thecytoplasm within1haftera 20-min pulse (lane f), but the level of p(96) di20-min- dimin-ished more

gradually

beginning 2 h into the chase period. The protein bands designated y andzrepresentstableproteinsthatarepresent in all immune precipitates and most probably are hostproteins. The assignment ofproteinx

to either cell or virus was more difficult since thisband seemed to diminish

rapidly

aftera 20-min pulse, althougharesidual bandwas detect-ableafterlongchaseperiods.These datacanbe comparedwith densitometertracingsofagelon whichvirusesharvested from thesupernatants ofthe chased culturesweresubjectedto electro-phoresis (Fig. 6, a-e). Itisreadilyapparent that

themajor internal virion proteinsarefully

rep-resented in the harvested viruses asearly as 1

h after thepulse; however, gp85 didnotbecome detectablein maturevirions until 3 h after the

pulse (Fig. 6c). These kinetic studies are sum-marizedin Fig. 7. The 2-hlagbetween the

dis-appearanceof50%of the

cytoplasmic

p(96) label

present in the 20-min

pulsed-and-chased

sam-ples and the appearance of 50% of the final incorporation leveloflabelinthegp85 ofmature

virions should be noted.

DISCUSSION

Thepresencein

virus-infected

cells ofa poly-proteinprecursorto the avian virion

glycopro-teingp85 representstheidentification ofa sec-ondmajor class ofprecursorstothe virion

struc-tural proteins. The precursor to the internal virion

proteins, pr76,

containsinformation which eventually gives rise to

p27,

p19, p15, and p12 proteins (21). The

immunoprecipitation,

kinetic, and biochemical data

presented

here confirm

thata

glycosylated polyprotein

of96,000 daltons

eventuallymatures toa smaller

glycoprotein

of 85,000 daltons. This p(96) most likely corre-spondstothep90

polyprotein

reported by Eng-land et al. (3) and the gp92 of Moelling and Hayami (15).

Asecondimmune

precipitated

protein, p(80),

appears

in,

and chases from, immune

precipi-tates prior to p(96); thus, p(80) mayrepresent animmature formof the

glycoprotein

precursor

p(96) and may be analogous to the putative d

-gp85

-p27

_pl9s

p1 2 -p1

5,

plO

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850

BUCHHAGEN AND HANAFUSA

d

b e

gp85 qp85

C f

gp85~~~~~~~g8

|bll^ #tyl&L~~~~~~~~~~~~~~~~

85WVi-lE}

FIG. 6. Scanningdensitometer tracings ofan

au-toradiogram obtained

by subjecting

to

electrophore-sispurifiedRAV-2 virus that had been obtained

from

thepulsed-and-chased culturesdescribed in the

leg-endtoFig. 5. The cultureswere

pulsed for

20min withL-14C-aminoacids and then chased

for

the

fol-lowingtimes:(a)1h; (b)2

h; (c)

3

h; (d)

4

h;

and

(e)

6h.

(fl

Purified L-'4C-aminoacid-labeled RA V-2 virus that had been labeled

for

16h.

glycoprotein precursor

p70

described

by

Halpern

et

al.

(6). Further,

both

p(80)

and

p(96) contain

glucosamine

and

mannose,

two

sugars that are

often

present in

core

carbohydrate

structures

attached to

protein backbones through

glucosa-mine

linkages

to

asparagine

residues.

The

detec-tion of

galactose

only

in

p(96) may indicate that

p(96)

is more mature

than

p(80)

with

regard

to

glycosylation,

although

the

presence of

this

sugar

in a

much

lower

quantity

in

p(80)

cannot

be

ruled

out. The

inhibition of the

synthesis

of

p(96) and p(80)

by

D-glucosamine,

in contrast

to an

accumulation

ofa

smaller, nonglycosylated

protein that is not cleaved, suggests that the

addition

of the carbohydrate residues occurs

during translation and may be requisite for its

continuation. Hayman et al. (9) had determined

that pr76

synthesis occurred, but that cleavage

was

greatly reduced under

a

similar glucosamine

block.

No intracellular gp85 was detected in these

investigations,

although England

et

al. (3) could

demonstrate that their

presumptive precursor

chased to intracellular gp85.

Our

failure to detect

the mature viral

glycoprotein

in

the

cytoplasmic

extracts

can

be

explained

in any of

several ways.

One

possibility is that the embryos used to

pre-pare the

monolayers for our experiments came

from a

different flock of

chickens than those

used

by the other

investigators

and may have a

different

or

altered

pathway for glycoprotein

precursor

processing.

Another

possibility is that

gp85 may

be very

firmly

associated with the

cellular

plasma

membrane and is not released

by the addition of

detergents;

it would then be

lost in

the material

pelleted during

the

prepa-ration of the

cytoplasmic

extracts. The 2-h lag

that

was

observed between the

disappearance

of

50%

of the labeled

cytoplasmic

p(96) and the

appearance

of labeled gp85 in virions at 50%

maximal level suggests that there may be

se-questration

of the

p(96) in

a

membrane fraction.

Such a

phenomenon has been reported for the

murine

glycoprotein

precursor (22), and it is

possible

that the

subsequent maturation steps

include

specific proteolytic cleavages. These

later steps

in

virus maturation are the subject of

our current

investigations. It also should be

pointed

out

that,

contrary to

the findings of

100 so

E

E 60 2 40 20

1 2 3 i + i

Choselength (hours)

FIG. 7. Plots of the amounts of label present in p(96) (0) in the immuneprecipitates and in virion

gp85(0) atdifferent timesaftera 20-minpulse of

RAV-2-infected

culturesusingL-

-4C-aminoacids. The datawereobtainedby integrating theareas under peaks in densitometer tracings (not shown) derived

fromthe datapresented in Fig.5and 6.Allvalues have been normalized to those obtained for the

amountof p(96)immunoprecipitatedfrom cells that had beenpulsedfor20min andfor the amountof

gp85present invirions harvestedafter an 8-h cold chasefollowinga20-minpulse.

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(7)

others

(3),

no

gp37

was detected in

immune

precipitates

obtained from

cytoplasmic

extracts

by using

the

anti-gp85

serum. This failure

may

be explained by

the same

possibilities raised

with

regard

to the absence of

gp85

from the

cytoplasm

of the infected cells or

may

be due

to

alack of immunoreactive

sequences

between

gp85 and gp37, as has been

suggested by Mosser

et al.

(16).

ACKNOWLEDGMENTS

We thank M. S.Halpernof the Wistar Institute for helpful discussionsandforcommunicating results prior topublication. Thiswork was supported by Public Health Service grants CA-18213-01and CA-14935-03 from the National Cancer In-stituteandby DamonRunyon-WalterWinchellFellowship GrantDRG-54F awardedtoD.L.B.

LITERATURE CITED

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Figure

FIG.ofpulse-labeled
FIG.ofpulse-labeled p.2
FIG. 3.labeledmannose;3H]galactose; Immune precipitates of extracts from cells for 20 min each as follows: lane a, D-["Cl- lane b, D-/'4C]glucosamine; lane c, D- lane d, L-[H]fucose.
FIG. 3.labeledmannose;3H]galactose; Immune precipitates of extracts from cells for 20 min each as follows: lane a, D-["Cl- lane b, D-/'4C]glucosamine; lane c, D- lane d, L-[H]fucose. p.4
FIG. 4.2-infected8receivingextracts:treated; h Immuneprecipitates ofextracts from RA V- gs-chf cells that had been pretreated for with 10 mM concentrations of inhibitors before 20-min pulses of L- '4C-amino acids
FIG. 4.2-infected8receivingextracts:treated; h Immuneprecipitates ofextracts from RA V- gs-chf cells that had been pretreated for with 10 mM concentrations of inhibitors before 20-min pulses of L- '4C-amino acids p.4
FIG. 5.Pulselanej,1-h Immune precipitates from RAV-2-infected gs-chf cells that were pulsed with L-"C-amiro acids
FIG. 5.Pulselanej,1-h Immune precipitates from RAV-2-infected gs-chf cells that were pulsed with L-"C-amiro acids p.5
FIG.85WVi-lE}toradiogramsistheendwithlowing6h.that 6. Scanning densitometer tracings of an au- obtained by subjecting to electrophore-purified RAV-2 virus that had been obtainedfrom pulsed-and-chased cultures described in the leg- to Fig
FIG.85WVi-lE}toradiogramsistheendwithlowing6h.that 6. Scanning densitometer tracings of an au- obtained by subjecting to electrophore-purified RAV-2 virus that had been obtainedfrom pulsed-and-chased cultures described in the leg- to Fig p.6
FIG. 7.gp85gp85dataamountchasep(96)RAV-2-infectedpeaksfromhavehad Plots of the amounts of label present in (0) in the immune precipitates and in virion (0) at different times after a 20-min pulse ofculturesusingL--4C-amino acids
FIG. 7.gp85gp85dataamountchasep(96)RAV-2-infectedpeaksfromhavehad Plots of the amounts of label present in (0) in the immune precipitates and in virion (0) at different times after a 20-min pulse ofculturesusingL--4C-amino acids p.6

References

Related subjects : chicken embryo fibroblasts