Polypeptide Synthesis in Simian Virus 5-Infected Cells

JOURNAL OF VIROLOGY, July 1977,p.177-187

CopyrightC 1977 American Society for Microbiology

Vol. 23, No. 1

Printed in U.S.A.

Polypeptide Synthesis in Simian Virus 5-Infected Cells

RICHARD W. PELUSO,* ROBERT A. LAMB, AND PURNELL W. CHOPPIN

TheRockefellerUniversity, New York, New York 10021

Received for publication18February 1977

Polypeptide synthesis in threedifferent cell types infected with simian virus 5 has beenexamined using high-resolution polyacrylamide slab gel

electrophore-sis, and all of the known viral polypeptideshave been identified above the host

cellbackground.The polypeptides were synthesized in infected cells in unequal

proportions, which are approximately the same as they are found in virions, suggestingthat their relativeratesofsynthesisarecontrolled.Thenucleocapsid

polypeptide (NP) was thefirst to bedetectedininfected cells, and by 12 to 14 h

the other virion structural polypeptides were identified, except for the

polypep-tides comprising the smaller glycoprotein (F). However, aglycosylated

precur-sor

(FO)

withamolecularweightof66,000 wasfoundineach cell type, and

pulse-chase experimentssuggestedthat this precursor was cleaved to yield

polypep-tides F1 and F2. No other proteolytic processing was found. In addition to the

structural polypeptides, the synthesis of five other polypeptides, designated I

through V, has beenobservedinsimian virus5-infected cells. One of these(V),

withamolecular weight of 24,000, was found in all cells examined and may be a nonstructural viralpolypeptide. In contrast, there are polypeptides present in

uninfected cellsthatcorrespondin size topolypeptidesIthroughIV, and similar

polypeptides have also been detectedinincreasedamounts incellsinfectedwith

Sendaivirus. These findings, and the fact that the synthesis of allfour of these

polypeptidesis notincreasedineverycelltype,suggestthattheyrepresenthost

polypeptides whose synthesismaybeenhanceduponinfection. Whena

high

salt

concentrationwasused todecrease host cellproteinsynthesisininfectedcells,

polypeptidesIVand (to alesserextent) I weresynthesizedinrelatively greater

amounts than other cellularpolypeptides, as were theviral polypeptides. The

possibility that these polypeptides may play some role in virusreplication is

discussed.

Theparamyxovirussimian virus 5(SV5)has

been shown to contain five major structural

proteins, designated HN, NP, F, 5, and M (5,

23, 25). Two of theviral proteins, HNandF,are

glycosylated (15), and the larger of these (HN)

has been showntopossess both

hemagglutinat-ingandneuraminidase activities, whereas the

smaller (F)isthoughttobe involvedin

hemoly-sis and cell fusion (34-36, 38). The F

glycopro-teinof Sendai virusand Newcastle disease

vi-rus (NDV), two other paramyxoviruses, has

beenshowntobederived byspecificproteolytic

cleavage of a precursor glycoprotein,

desig-nated

FO,

whichisaccompanied by activation of

thecell-fusing and hemolyzing activities of the virion and its ability to initiate infection (12, 14, 27, 32, 35, 37). Although biologically inac-tivevirionscontaining theuncleaved

Fo

glyco-protein have beenfoundwithSendaivirusand NDV, such virions have never beenfoundwith

SV5. However, evidence for the existence of

such aprecursor has beenobtainedinthat the

SV5Fprotein, aswellasthoseof Sendaivirus

and NDV, has been found to consist of two

subunits, F1 and F2, linked by disulfide bonds

(33, 38).

Themostabundantvirionprotein, NP,

com-plexes with viral RNAtoformthenucleocapsid

(24),andthe smallestprotein, M, isthoughtto

beassociatedwiththeinnersurface of the viral

envelope (23). The remaining major structural

protein, 5, is thought to be involved in the virion RNA polymerase activity (E. Buetti and P. W. Choppin, submitted for publication),

along with NP and possibly a viral protein

present insmall amount, L. The host cell pro-tein actin (A)hasrecentlybeenfoundinsome paramyxovirions (39), but the significance of thisis notyet clear.

Although much is known about the

struc-ture, composition, andfunctions of the compo-nents of SV5 and its interactions with cellsin

cytocidal and

noncytocidal

infections (6, 9, 11,

16-18), relatively little isknown aboutthe

de-177

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178 PELUSO, LAMB, AND CHOPPIN

tailsof the synthesis of its proteins. This is due largelyto the difficulties inherent in studying viral protein synthesis in a system in which host cell synthesis is not rapidly turned off (10, 13), a situation commonwith paramyxoviruses. Recently, it has been possible to study the syn-thesis of Sendai and influenza virus-induced proteins in infected cells by the use of [35S]methionine labeling, high-resolution slab gel electrophoresis, and autoradiography (20, 21). This report describes SV5-induced protein synthesis in three different cell types. The time courseofviral protein synthesis has been stud-ied by pulse-labeling, and the processing of one of theviral glycoproteins (F) has been demon-strated in pulse-chase experiments with [3H]-glucosamine.

MATERIALS AND METHODS

Cells. Monolayer cultures of a variant of the MDBK line of bovine kidney cellsand the TC7 clone of CV-1 cells obtained from James Robb were grown in reinforced Eagle medium (REM) (1) with 10% fetal calf serum, as described previously (7). Pri-mary cultures of chicken embryo fibroblasts (CEF) were grown in lactalbumin hydrolysate medium with 2% calf serum (21). Baby hamster kidney (BHK-21-F) cells for plaque assays were grown in REM with 10%calf serum and 10% tryptose phos-phate broth as described previously (13). All cells were grown onplastic petri dishes.

Virus. Stock virus was grown in MDBK cells infected with the W3 strain of SV5 (6) at a multiplic-ity of-1 PFU/cell. After a2-h adsorption period at

370C, the monolayerswerewashed with

phosphate-buffered saline (PBS), and REM withoutserum was added. After3 to 4days at 370C, the medium was collected andclarified at 3,000 rpm for 30 min in a Sorvall GSA rotor. Bovine serum albumin was added to aconcentration of 1%, and the viruswas frozen at -70'C. Plaque assayswere performed as describedpreviously (8).

Chemicals andisotopes. 3H-labeled reconstituted protein hydrolysate was purchased from Schwarz Bio-Research, Orangeburg, N.Y.; [3H]leucine and [3H]glucosamine, from New England Nuclear Corp., Boston, Mass.; [35S]methionine, from Amersham/ Searle, Arlington Heights, Ill.; sodium dodecyl sul-fate, from Gallard-Schlesinger, Carle Place, N.Y.; dithiothreitol and ammonia-free glycine, from Cal-biochem, La Jolla,Calif.;and acrylamide and bisac-rylamide, from Ames Co., Elkhart, Ind.

Growth and purification ofisotopically labeled virus. MDBK cells were infected as describedabove; afteradsorption, the monolayers were washed with PBS, and REM containing 5 ,uCi of[3H]leucine per ml was added. After 3 to 4 days, the medium was harvested and clarified, and the virus was pelleted by centrifugation at 10,000 rpm for 2 h in a GSA rotor. The pellet was suspended in 10% potassium tartrate, homogenized in a Dounce homogenizer, andlayered on a linear 15 to 35% (wt/wt) potassium tartrate gradient. After centrifugation at 22,000 rpm

J. VIROL.

in a SpincoSW27 rotor for2h, the virusband was collectedanddialyzed against 0.0625M Tris, pH 6.8. Purified virus waskept at -20°C.

Infection and labeling of cells. Confluent mono-layers on 60-mm dishes were washedthree times withPBSand inoculated with-50PFU/cell; after a 1-hadsorption, the monolayers were againwashed three times, and2mlof Eagle medium wasadded. At various times after infection, the medium was removed andreplacedwith 1mlofoneof the follow-ing: (i)REM deficientinmethionineandcontaining [35S]methionine (10 ,Ci/ml); (ii) REM deficient in aminoacidsand containinga3H-labeled aminoacid mixture (10

A.tCi/ml);

(iii) REM deficient inglucose and containing [3H]glucosamine (25 uCi/ml). After incubation at 37°C for periods as indicated in Re-sults, the mediumwasremoved, and the cellswere washed twice with PBS andlysedinasolution con-sisting of4%sodiumdodecyl sulfate,3% dithiothrei-tol, 40% glycerol,and62.5mM Tris, pH6.8. Lysates werescraped from the dish and frozenat-20°C until processed for electrophoresis. Forpulse-chase exper-iments, the radioactive medium wasremoved, the cells werewashed threetimeswithPBS, and REM wasaddedtothedishes. At the appropriate times, the cells were harvestedasdescribed above.

Polyacrylamide gel electrophoresis. Polyacryl-amide gel electrophoresis was done using a slight

modification of the procedure described previously (21);instead of anacrylamide-bisacrylamide ratio of 37.5:1, a ratio of 77:1 was used to obtain better resolution ofinfected cell lysates. Molecular weights ofpolypeptides wereestimated by their migration relativetothe following markers of known molecu-larweight: myosin, 18-galactosidase, phosphorylase a, bovine serum albumin,catalase, ovalbumin, al-coholdehydrogenase, DNase I, carbonicanhydrase,

a-chymotrypsinogen, and trypsin.

Autoradiography and fluorography. For detec-tionof 3H ingels,afluorographic procedure(2)was employed using Kodak RP-Royal X-Omat film. For [35S]methionine detection, dried gels were exposed

to Dupont Cronex 2DC X-ray film. Fluorograms wereprocessed byhand, and autoradiogramswere processed using aKodak3.5-minX-Omat.

RESULTS

StructuralproteinsofSV5virions.Figure1

shows an autoradiograph of3H-labeled, puri-fied virionsgrownin MDBKcells andsubjected to slabgelelectrophoresis. The five major

poly-peptides HN, NP, F1, 5, and M have estimated

molecularweights basedonmigration relative

to markers in this gel system of -70,000, 61,000, 52,000, 46,000, and38,000, respectively. Two minor polypeptides, L and a protein that comigrates with cellular actin, with approxi-mate molecular weights of 200,000 and43,000,

respectively, are also present. F2, the smaller

fragmentderived byproteolytic cleavageof

FO,

migrates with the buffer front in this gel and therefore is not resolved (38).

Polypeptide synthesis in SV5-infected MDBK

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POLYPEPTIDES IN SV5-INFECTED CELLS 179

L

HN

NP

F

-FIG. 1. Autoradiograph of the polypeptides of SV5 virions separated byelectrophoresis on a 10% polyacrylamide-sodium dodecyl sulfate (SDS) slab gel. Virionswerelabeledwith[3H]leucinein MDBK cells,purified,disrupted withSDSand dithiothrei-tol, andsubjectedto electrophoresisas described in thetext.Migration is fromtopto bottom.

cells. In MDBK cells, infective virions begin

toappearbetween8and9hpostinfection (p.i.),

and there is an exponential increase until 15

to 24 h p.i., followed by continuous virus

pro-duction for several days with only slight

cyto-pathic effects. Virus yields at 36 to 72 h are

[image:3.501.90.198.85.544.2]

usually in the range of 2 x 108 to 4 x 108 PFU/ mland256to 1,024hemagglutination units/ml. Figure 2 shows anautoradiograph of a lysate of infected MDBK cells labeled at various times after infection with [35S]methionine and

sub-jected to electrophoresis. By 6 h p.i., the

most prominent viral protein, NP, was

distin-guishedagainst the host cellbackground, and

by 12to 14 h all themajor virionpolypeptides, withtheexceptionof F1 and F2,weredetected. In addition to these structural proteins, three

polypeptides that are not found in virions

ap-peared to be present in increased amounts in

infected cells by 14 h. They are labeled I, II, and IV and have apparent molecular weights of -99,000, 97,000, and 78,000, respectively. As

discussed below, the available evidence

sug-geststhat thesearecellular polypeptides whose

synthesisisenhancedininfected cells.In

addi-tion tothesepolypeptides, infectedMDBKcells contain apolypeptide withamolecular weight of-24,000,designatedV(seeFig.5). This

poly-peptide ran off the gel shown in Fig. 2. The

polypeptide migrating withamolecular weight

of-66,000, labeled

FO,

willbe shown belowto

be theprecursorofpolypeptides F1 andF2. The

L protein was not detected inthese cells,

pre-sumably because it is present in a very small

amountand masked by comigrationwithhost proteins. Although the amount of each viral protein has not been quantitated precisely,

theseand similar autoradiographssuggestthat

the virion polypeptides are synthesized in

in-fected cellsinunequalamountsandarepresent

incellsinproportions similartothose foundin

virions.

Protein synthesisin primary CEF cells. In

primary CEF cells, few SV5 virions are

pro-duced,i.e., <105PFU/ml, thereis nodetectable

hemagglutinin (<2 hemagglutination units),

andnonon-hemagglutinatingparticles are

re-leased. Therefore, thepossibility ofadefectin

viralproteinsynthesisinCEFcellswas

investi-gated.Eventhoughlittleor novirus isreleased

fromthecells,all the known viralproteins are

synthesized

(Fig. 3). The most abundant viral

protein,NP,wasdetected before the other viral

proteins, as early as4hp.i., and by14 hall the known virion polypeptides except

F1

and F2 were seen, includinga small amount ofthe L protein which wasjust detectableat 14 to 18h. Inaddition to the virion proteins, the glycopro-tein precursor,

FO,

was clearly seen in these

autoradiographs. As shown above in MDBK

cells, therewerealso large

polypeptides

whose

synthesis was increased in these cells and

which were not present in purified virions.

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180 PELUSO, LAMB, AND CHOPPIN

4U

4I 6U 6I 8U 8I

1OU

10I 12U 12I

A*. 4.si, I_ ato 66._11- _m*

*-Wk.

LI-.tAh i 4 i; ; X 4 ;

;iB| \ F j; rmeI.- *I

Si'

.*sea*

.w

r.-.u.Alw'

J. VIROL.

14U 14I 16U 16I 18U

1E8

4~~~~~~~~~~~~~~~~~~~~~~~~~4

4 ^ J. X

FIG. 2. Time courseofsynthesis of SV5polypeptidesinMDBK cells. Cellswereinfectedandlabeled with

[35S]methionine for60min at varioustimesafterinfectionasindicated.Inthis andsubsequentfiguresthe numbersrefertothe timeafterinfectionatwhich thelabelwasadded.I,Infectedcelllysates;U,

uninfected

celllysates.Whole-celllysateswereprepared forelectrophoresisandautoradiographyasdescribedinthetext.

Thepolypeptidesofunlabeled virions wereincludedoneachgelas markers.

Thesearedesignated I,II, IV,andV,and their

apparentmolecularweightsweresimilar in the two cell types. However, there was one

poly-peptide, III (molecular weight, -86,000), that

wasdetectedin CEF but not in MDBK cells.

Protein synthesisin theCV-1 line of

mon-key kidneycells.Virus-specific protein

synthe-siswasexaminedinthe TC7clone ofCV-1cells,

whichexhibitcontact inhibition ofgrowth (30),

todetermine ifsynthesisof minor viralproteins would be more easily detected in these cells.

Figure4showstheresultsobtained with CV-1 cells pulse-labeledatvarious times after infec-tion. Inaddition to themajorvirionstructural

polypeptides and Fo, polypeptides IV and V

wereclearlyseeninthesecells, and theL pro-tein, though still present in a small amount,

was detected at 12 to 18 h in these cells. No otherprecursorsorminorproteinswereseen.

Attempts to diminish host protein

synthe-sis. Toobviate furtherthe inherentdifficulties

in studying viral protein synthesis in cells in

which thereis no inhibitionof hostcell protein

synthesis, attempts were made to

selectively

reduce therateofsynthesis of cellular

proteins.

Neither growing cells at

330C

nor the use of

actinomycinD (0.05to5

Ag/ml)

was

successful

inthisregard;however,the exposure of MDBK

cellstohypertonic conditions priorto and

dur-ingthelabelingperiod,aprocedure thatcauses

adecrease in the initiation ofcellular

protein

synthesis (28, 31), resultedin some

degree

ofa

selectiveinhibition of host

synthesis.

Theviral

polypeptides HN,

FO,

NP, 5, and M, although

synthesized

at decreasedrates as

compared

to

cells notexposed tohigh salt, were

prominent

againstthe inhibited host cell

background (Fig.

5, cf. Fig. 2). Figure 5 also shows that the

synthesisof

polypeptides

I andIV

appeared

to

be lessinhibitedininfectedcells thanin

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POLYPEPTIDES IN SV5-INFECTED CELLS 181

46

ma

66

6I 86

8I

1Ju

l1

126 121

146 14I 166 16i 18U 18I

I i *i ;

i .-LA

X

n~~~

i iI

E i~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

W ~~~~~~~~~~~~

km 1

l".

r

1

I

IT

* 4

i

U

i:

I

#W W

FA-

m

I

I.

Ft {sr

=a

-,

-~-a;

*v

_

|6i

r

* 4

VW-_ o suW

* _S *ASr an

FIG. 3. Timecourseof SV5polypeptide synthesisin primaryCEFcells. Cellswerelabeled for 60 min with

[35S]methionineatthe times indicatedandprocessed for electrophoresis and autoradiographyasdescribedfor

Fig.2.

fectedcells. This becomesapparentifone

com-paresthe synthesis of thesepolypeptides,

par-ticularly IV, with those of other cellular pro-teinswhoseratesofsynthesis appeared similar

ininfectedand uninfected cells. Thus, high salt appeared to cause less inhibition not only of

viral polypeptides, but also oftwo presumed cellular polypeptides whose synthesis was

en-hanced in infectedcells. This finding raises the possibility that these proteins mayplay arole

in virusreplication.

Figure5alsoemphasizes the value of methio-nineasalabelinstudies of SV5 proteins, since

the labeling patterns ofviral polypeptides ob-tained with thetritiated amino acidmixture in

theseexperimentswassimilartothat obtained

withmethionine (cf. Fig. 2).

Comparisonofpolypeptide synthesisinSV5 andSendaivirus-infected cells. Toinvestigate

furtherthe originofpolypeptides Ithrough V, whose rates ofsynthesis in infected cells ap-peared to be greater than that in uninfected cells, thepatternsobtaineduponinfection with

SV5andSendaiviruswerecompared.

Polypep-tides thatcorrespondtopolypeptidesIthrough

IVinSV5-infected cellswerealso foundin Sen-daivirus-infected cells (Fig. 6).Thisfinding,in addition to the factthat proteins with similar

electrophoretic mobilitieswere found in

unin-fected cells, providessuggestive evidence that these are host polypeptides whose synthesis

is enhanced after viral infection rather than

virus-coded polypeptides. However,

polypep-tide V (molecular weight, -24,000) of SV5-in-fected cells does notcorrespond to a proteinin

Sendai virus-infected cells, nor does poly-peptide C (molecular weight, -22,000) in

Sen-dai virus-infected cells have a counterpart in

II

I

L

I'

i

p I

4

4

,-d*

L

I

zHN

---

Fo

-NP

5

A

M

6

VOL. 23, 1977

i i

. . 0

L ..

i

A _4...

,4w

4094

'w'..:,...,

;,_

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182 PELUSO, LAMB, AND CHOPPIN

shows a representative experiment in which CEFcells werepulsed for 60 min at 17 h p.i. and then chased. By 30 min into the chase period, the amount of

Fo

wasgreatly decreased, andby 45 min it was not detectable, but F. had ap-peared, suggesting a precursor-product rela-tionship. The other cleavage product, F2, was

present in the dye front on this gel but is re-solved in gels shown below (Fig. 8 and 9). In contrast to the rapid chasing of

Fo

into F1 and F2, the M protein did not decrease until 3 h, and

nootherproteins werechasedout in up to 5 h.

The decrease in M is consistent with the

hy-pothesis that thesynthesis of this polypeptide

represents arate-limiting step in the

matura-FIG. 4. SV5 polypeptide synthesis in CV-1 cells. Atthe times indicated, the cells were labeled with

[P5S]methionine for60min andprocessed for electro-phoresisandautoradiography.

SV5-infected cells. This suggests that poly-peptide V may be a virus-specific,

nonstruc-turalpolypeptide, as waspreviously suggested

forpolypeptide CinSendai virus-infected cells (21).

Pulse-chase experiments in SV5-infected cells. Inthe aboveexperimentsinthree differ-ent cell types, aglycoprotein corresponding to

F1 was not detected. However, another poly-peptide that migrated slightly faster than HN

was consistently found. By analogy to Sendai

virus (21, 35, 37, 38), thiswasthoughttobe

FO.

This precursor has never been found on SV5

virions;therefore, it is presumably processed by proteolyticcleavage before release of the virus inall cells thusexamined, anassumption

sup-ported by therecentfindings that the F protein ofSV5, as well as those of Sendai virus and

NDV, consists oftwodisulfide-linked polypep-tides, F1 andF2(38).To investigate whether the polypeptide observed in SV5-infected cells is

suchaprecursor,pulse-chaseexperimentswere

performed in all three cell types. Figure 7

an

mmA*

-11 b

!r--\4

--lt

p:

FIG. 5. Effect ofhighsalt concentration on

poly-peptide synthesis in SV5-infected MDBK cells. (A)

Polypeptides ofSV5 virions grown in MDBK cells and labeled with [PH~leucine. (B) Polypeptides of infectedcells labeledfor60minat14hp.i. with 3H-amino acids in the presence of0.1 M NaCl. (C)

Polypeptides of infected cells labeledfor 60 min at

14 hp.i. with 3H-amino acids in isotonic medium. All samplesweresubjectedtoelectrophoresisonthe

same slabgel. Fluorography wasperformedas de-scribed in thetext.

H

of,-momm

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POLYPEPTIDES IN SV5-INFECTED CELLS 183

L

HHN

ILI

HN-NP

5

A

M

20% gel, it is evident that F. and F2 are both appearing in the chase. Thus, the experiments shown in Fig. 8 and 9 strongly suggest that

F0

isprocessed to yieldF1 andF2. These results, together with the previous findings thatF. and F2 are disulfide linked on SV5 virions (38), es-tablish that SV5-infected cells contain an

F0

protein that isproteolytically cleaved to yield twopolypeptide chains in a manner analogous to Sendai virus and NDV.

DISCUSSION

_____F

The use of

high-resolution polyacrylamide

____N

gel electrophoresis, autoradiography, and fluo-rography (20, 21) hasmade it possible to study SV5protein synthesis in the presence of ongo-ingcellular synthesis. The use of several differ-entcelltypes hasmade it possible to identify all the known viral proteins and has also revealed

severalproteins that are notfoundin the virion

--B but whose

synthesis

is enhanced in infected

--M cells.

By

4 to6 h

p.i.

the most abundant viral

polypeptide, NP, was seen, and by 12 to 14 h all

FIG. 6. Comparison ofpolypeptidessynthesizedin SV5- and Sendai virus-infected primary CEF. At 18hp.i. thecells were labeled with[35S]methionine for 30 min, andcell lysates were then prepared for electrophoresis and autoradiograhy. Left lane, Unin-fected cells; middle lane, SV5-inUnin-fected cells; right lane, Sendai virus-infected cells.

tion ofenveloped viruses (21, 22). There is a

slight alteration in the mobility ofHN as

Fo

disappears, which could represent processing,

suchas changesinthe

carbohydrate

portionof

theglycoprotein. Although proteolytic cleavage

involvinga significantportion ofHN doesnot

appear to occur, removal of a small peptide cannotbeexcluded.

Glycoprotein

synthesis.

To obtain further

evidence that the transient

polypeptide,

FO,

is

the precursor to F1 and F2, infected cellswere

labeledwith

[3H]glucosamine.

Proteins

HN, F1,

and F2 were

labeled,

aswell as

Fo

(Fig.

8). To establish the precursor-product relationship,

pulse-chaseexperimentswere

performed

using

[3H]glucosamine. When

Fo

diminished, F. and

F2 appeared (Fig. 9, left). This is shown more

clearlyintheright panelofFig. 9,inwhich the

samples were subjectedto electrophoresis in a

20% gel; both F1 and F2 are clearly resolved.

Although it was not possible to detect a

de-crease in

FO

since it migratedwith HN in this _fls

^.

s-w F1 w

e .J

^ it fl

A;;E

| |

| ;

-9

-*

W 4 *at4 to f

An..

49z;.^

*

to. 9,.< g '.4 } I

Air i I Et ^ ... * t i

|+wAa_at_

I C. .4._Ww

r-.-a-W_

e____a*.___...W

.- drage -a > or

_

b_-_-*._1*_

And___

_a,,

5 3,7 Ad 6C 3rr Bhr

L

-IIIC

___NP

_-

FAN

_Fo

-""NP

5

-

A

M

- -S|~_ _-Y--A

F

FIG. 7. Pulse-chase experiment in SV5-infected

primaryCEFcells.Cellswerepulsed for60 minwith

[f5S]methionineat17 hp.i. After the pulse, the radio-active medium was removed, thecellswerewashed three times withPBS, and REMwasadded. Atthe timesindicated, the cells wereprepared for electro-phoresis andautoradiography.

.. 26h dw

A"

a*

AM

am

Joe&

I

.-AV joinVWIM."s"O

m

-mmmmr-.. No

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184 PELUSO, LAMB, AND CHOPPIN

t_

PR,

;"

W

FIG. 8. Synthesis ofglycoproteinsinSV5-infected MDBKcells.Cellswerelabeled for2h with [3H]glu-cosamine (25 ,uCi/ml) at 17 h p.i. and processed forelectrophoresis andfluorography asdescribedin

the text. Marker virions (right lane) were labeled with[3H]leucine.

ofthe viral structural polypeptides except F1 andF2werepresentin amountssufficienttobe

detected. Although the amount ofeach viral polypeptide synthesized ininfected cells could not be determined precisely by this method, examination of many autoradiographs

indi-cates that they are synthesized in

approxi-mately the same unequal proportions as they arefoundinvirions, whichsuggeststhat there

is control ofviral polypeptide synthesis.

Simi-lar findings have been reported for NDV (12)

andSendai virus (21, 29).

Inpulse-chase experiments a precursor

gly-coprotein, F0, has been identified in SV5-in-fectedcells; thisappearstobe convertedto two

smaller

polypeptides,

F1

and

F2.

Unlike Sendai virus and

NDV,

the

Fo

protein

ofSV5 hasnever beenseen onmature

virions, indicating

that it is

susceptible

to

cleavage by

proteases

present

inawide

variety

ofcells. Thismaybe duetoan

extreme

sensitivity

tothesameprotease found in many

cells,

or to

susceptibility

to a wide

variety

of proteases, unlike

wild-type

Sendai

virus,

or

NDV,

which are

susceptible

to

tryp-sin,

or to the Sendai

mutants,

which

require

other

specific proteases (27, 35,

37).The demon-stration of

cleavage

of

Fo

in infected cells to

yield F1

and

F2

andthe

finding

ofthelattertwo

disulfide-linked

polypeptides

on SV5 virions

(33,

38)

have established the

generality

of the

activation of

paramyxovirus

virions

by

cleav-ageofthe

F0

glycoprotein,

eveninthecaseofa virus in which the uncleaved precursor is not foundonmaturevirions.

The

finding

of

cleavage

of the

Fo

protein of

SV5 inthe nonpermissive CEF cells has ruled

out the

possibility

that lack of

cleavage

is

re-sponsible

forthefailure of these cellsto

produce

virus.

Similarly,

thelack ofsynthesisof any of

theknown virion

proteins

has also beenshown

not to be the

explanation.

Although the

possi-bility

that there is ablock in theproductionof progeny genome strandsofviral RNA hasnot

been

excluded,

the

high

levels ofsynthesis of virion

polypeptides

in CEP cells suggest that thedefect lies atsomelaterstepinthe

assem-bly

process, e.g., in the proper sequence of

events at the cell membrane. Further studies

are

required

toexplainthefailure of

production

of enveloped viruses from cells in which the

known viral proteinsare

synthesized.

Intwo avirulent strains ofNDV, but not in other

strains,

Nagai

andco-workers (27) dem-onstrated theexistence ofaprecursor

(HNO)

to

the viral

hemagglutinin-neuraminidase

glyco-protein

(HN).

We have attempted to detect such aprecursor inSV5-infected cells, usinga

variety

ofexperimental conditions,

including

short pulses with [35S]methionine, high salt concentrations to reduce host cellprotein

syn-thesis,

and zincions,which have been shown in other systemsto inhibit cleavage ofviral pro-teins

(3, 4,

19).

However,

wehave been unable

todetect suchaprecursororto detect the syn-thesis of any other large unstable proteins in

infected cells. Similarly, attempts to find an

HNo

precursor in Sendai virus-infected cells have notbeen successful (21; Lamband

Chop-pin, unpublished data). Thus, the question

re-mains open of whether synthesis of an

HNo

protein islimitedto afew strains, such as the

two avirulent strains ofNDV, or is a

general

feature of paramyxoviruses;however,if the

lat-teris the case, it hasescapeddetectionin exten-J. VIROL.

'.1

".K;p

4 -"

-, .4

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[image:8.501.105.219.72.430.2]

POLYPEPTIDES IN SV5-INFECTED CELLS 185

0 .iI I

I * ii_I

.4~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~..

a

MO

-HN.IF

0'* --F

2

2

FIG. 9. Pulse-chaseofglycoproteinsinSV5-infectedMDBK cells. Cellswerepulsed for60minat17 hp.i. with[3H]glucosamine (25 pXi/ml)and then washed threetimes withPBS,and REMwasadded. At thetimes

indicatedthecellswereprocessedasdescribedforelectrophoresison a10%gel (left) with[3H]leucine-labeled marker virus in theright lane,or on a20%gel (right)with[3H]glucosamine-labeledmarker virus in theright lane.

siveexperiments with SV5 and Sendai virus. Aninterestingaspectof thepresentstudies is the observations relating to the polypeptides whose synthesis is stimulated in infectedcells but which are not foundin the virion. One of these, V (molecular weight -24,000), doesnot

appeartocorrespond to apolypeptidefound in

uninfected cells, and thusmayrepresenta

non-structural protein analogous to the

22,000-dal-ton,presumed nonstructural polypeptide C

syn-thesizedinSendai virus-infected cells(21). The

function of these proteins is at present

un-known. In additiontoprotein V, thereareother

polypeptides in both SV5- and Sendai virus-infected cells (polypeptides I through IV) whose origins and functionsarenotcertain. However,

the available evidence suggests that these are

host polypeptides whose synthesis may be

en-hanceduponinfection. This conclusion is based onthefindingthat therearepolypeptideswith similarelectrophoretic mobilities in uninfected

cells, thatthe number detecteddependsonthe

cell type,e.g., three in MDBK cells and four in

CEFcells, and thatthesynthesisof four similar

polypeptides appears to be enhanced in cells infected with Sendai virus. When a high salt

concentration was used toselectively decrease thesynthesis of host cellproteins,the synthesis

of most cellular polypeptides was greatly

di-minished relative to SV5 proteins; however, synthesis of polypeptide IV, and, to a lesser

extent, I, was not inhibited as much as other

hostcell polypeptides. Thissuggeststhat these polypeptides are translated more efficientlyin

infectedcells than in uninfected cells and raises thepossibility that theymayplaysome rolein

the replication of paramyxoviruses, a concept

supported bythefindingofenhancedsynthesis

ofapparently host polypeptides in Sendai virus-infected cells (Fig. 6). Whether these proteins

are significant in virus infection, or,

alterna-tively, their apparentincreased synthesis is a

nonspecific effect following infection, remains

tobe determined. Therearepossibleroles that

could be envisaged for host proteins in para-myxovirusinfection, e.g., as acomponentofan

enzyme involved in the replication ofgenome

RNA, but there is no evidence at present on

E,

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[image:9.501.54.443.69.354.2]

186 PELUSO, LAMB, AND CHOPPIN

which to base any assignment of function to

these polypeptides. However, their detection

serves to stimulate investigation of possible roles for host proteins whose synthesis is en-hanced in infection by these nontransforming RNAviruses.

ACKNOWLEDGMENTS

We thank Ann Duncan for excellent technical assist-ance,E. Gershey for CV-1 cells, A. Scheid for helpful dis-cussions.

This research wassupported by Public Health Service research grantAI-05600 from theNational Institute of Al-lergyand Infectious Diseases and research grant PCM76-09993from theNational ScienceFoundation,andby Insti-tutional National Research Service Award TE-CA09256 from the NationalCancerInstitute, under which R.W.P. is apredoctoral trainee.

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