Frog Virus 3 Replication: Analysis of Structural and Nonstructural Polypeptides in Infected BHK Cells by Acidic and Basic Two-Dimensional Gel Electrophoresis

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JOURNALOF VIROLOGY, Jan.1980,p. 18-27

0022-538X/80/01-0018/10$02.00/0 Vol. 33,No. 1

Frog Virus

3 Replication:

Analysis

of

Structural

and

Nonstructural Polypeptides

in Infected

BHK Cells by

Acidic

and Basic Two-Dimensional

Gel Electrophoresis

R. M.ELLIOTT,'t R. BRAVO,2t ANDD. C. KELLY`*

Natural Environment Research Council, Unit of InvertebrateVirology, Oxford,OXI 3UB,' and Department

of

Biochemistry,

University of Oxford,

Oxford, OXI

3QU,2

United

Kingdom

Analysis of frog virus 3-infected BHKcellsby two-dimensional,acidic and basic

gel electrophoresis showed that at least 90 infected cell-specific polypeptides

could be detected. These polypeptides represent between 70 and 85% of the

coding capacity of the viralgenome.The polypeptidesweresequentiallyinduced

in atleast three phases. The virus gradually suppressed host cell polypeptide

synthesisduring infection, although the synthesisofafew cell polypeptidesmay

be "switched off' earlyin infection.

Frog virus 3 (FV3) is alargeicosahedral

DNA-containing virus which replicates in the cyto-plasm butrequiresa functional cellnucleusfor

replicationto occur(8,9,11).Theviruspossesses a linear double-stranded DNAgenomehavinga molecular weight of about

108

(10) and so has

the potential tosynthesize about 100

polypep-tides ofaverage size. Recent studies inour

lab-oratory with sensitive high-resolution

one-di-mensional gel

electrophoresis

resolved 29

in-fectedcell-specific polypeptides(ICPs),ofwhich

22 comigrated with virus structural

polypep-tides. The ICPs appeared to be coordinately

regulatedin acascade fashion, with three

tem-poral phases (a, /3, and y) being observed (6).

Thesynthesis ofy

polypeptides

requires

synthe-sisof viralprogenyDNAto occur(5, 6).In

baby

hamsterkidney (BHK 21/13) cellstheaphase

polypeptides are detected from 2 hafter

infec-tion, the 18

polypeptides

are detected from4 h,

and the-y-phasepolypeptidesaredetected from

6h. Thepolypeptides detectedrepresent 30% of

thecodingcapacity of the viral genome.

Thedevelopmentoftwo-dimensionalgel

elec-trophoretic

techniques

to resolve

proteins by

separating proteins

inonedimension

according

to charge with isoelectric focusing (IEF) and

then separating them in the second dimension

accordingtosize with conventional

electropho-resis in the presence of sodium

dodecyl

sulfate

(SDS)andreducingagents hasproduceda

pow-erful tool to

explore

complex proteins

(13, 14).

In this research we used this

methodology

to

resolve virus-induced polypeptides in

FV3-in-t PresenFV3-in-t-'address: DeparFV3-in-tmenFV3-in-t of Microbiology, MounFV3-in-t SinaiSchool9fMedicine, CityUniversityof NewYork,New

York,NY100,9,

$ Present address: Kemisk Institut, Landgelansgade 140, DK-8000 ArhusD,Denmark.

fected cells and soexaminethenumber of

poly-peptides presentin infectedcells, to determine the effect of the virus on host cellpolypeptides,

and also to confirm the temporal induction of FV3polypeptides.

MATERIALS AND METHODS

Materials. L-[35S]methionine (840 Ci/mmol) and

125I

(100 mCi/ml) were purchased from the Radi-ochemical Centre, Amersham, England. Ampholines

werepurchased from L.K.B.Ltd.,Croydon, England.

DNase I and RNase Awereobtained from theSigma

Chemical Co.Ltd., London,England.

Cells and virus.Babyhamsterkidney (BHK21/

13)cellsand fathead minnow (FHM)cellsweregrown

and maintainedaspreviously described,and FV3was

grown and titrated in FHM cells as previously

de-scribed(4).

Radiolabeling of cellular polypeptides with

[3"S]methionine.

BHK cells were grown in small plastic petri dishes (30 mm; 3 x 105 cells) at 37°C

overnight. Cellswereinfectedat25to30PFU of FV3

percellbyadsorbingat roomtemperaturefor1hand thenwashingwith theGlasgowmodification ofEagle

minimal essential medium. Theinfectionwasallowed

toproceedat28°C.Cellswerepretreatedwith radio-labeling medium (2% dialyzed calf serum in Earle balanced saltsolution)for15minbeforebeing pulsed

with 100,uCiofL-[35S]methionine (in1ml)for1hat

28°C. The isotope was removed, and the cellswere

washed three times in ice-cold phosphate-buffered

saline and thenpelleted.The cellswereresuspended

in 100

p1

of 10 mM Tris-hydrochloride (pH 7.4)-50

mMNaCl-5mMMgCl2 containing25,ug ofDNase I

perml and25,ugofRNase A perml. The cellswere

sonicated andthenincubatedonice for 30 min. The cellswereimmediatelyfreeze dried.

Two-dimensionalgel electrophoresis of poly-peptides. The procedures followed wereessentially

thosederivedbyO'Farrelland co-workers(13, 14).

Freeze-driedcellswereresuspendedin 100,lIoflysis

buffer(9.4Murea,2% NonidetP-40,1.6%ampholines

18

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ANALYSIS OF FV3 POLYPEPTIDES 19

[pH 5 to 7], 0.4% ampholines [pH3.5 to 10], 5%

/-mercaptoethanol), and 250,000cpmof material(about

20

Ad)

wasappliedtoeachelectrofocusing gel. The first-dimension separations were performed

eitheras(i)acidic 4% polyacrylamide gels (130 by2.5

mm) containing2%ampholines (1.6% pH5to7,0.4% pH3.5to10)or(ii) basicnonequilibrium pH gradient

electrophoresis (NEPHGE) 4% polyacrylamide gels (130 by2.5mm) containing2%ampholines (1% pH7

to9, 1%pH8to9.5) exactlyasdescribedbyO'Farrell

and co-workers(13, 14).Both IEFandNEPHGEgels

werethen immersed in equilibrium buffer(0.06Tris,

pH 6.8,4% SDS, 10%,8-mercaptoethanol,20% glycerol)

and incubatedatroomtemperaturefor30min before

being applied tothesecondSDS-polyacrylamide di-mension. Gelstobe stored frozenwereincubated for

15min only,andthe incubationwascompleted after

defrosting.

The second-dimension separations by SDS-poly-acrylamide gel electrophoresis of the proteins

con-tained in theIEFgelswereaccomplished by layering

theequilibrated gelsontoastacking gel (4.75%

acryl-amide,0.024%bisacrylamide, 0.1% SDS,0.0625M Tris-hydrochloride, pH 6.8) which overlaid a 15%

acryl-amidegel (0.1% SDS,0.375 MTris-hydrochloride, pH

8.8). A 1% agarose gel made up in electrophoresis

buffer andcontaining0.01%bromophenol bluesecured

thegelinplace.Theelectrophoresis buffer contained

0.025 MTris,0.192 Mglycine,and0.1% SDS, pH8.8.

Electrophoresiswasperformedat6to10mA for14to 18h. The slab measured19by16cm.The gelswere

processedforfluorography (2) by being fixed in 45% methanol-7.5%acetic acid-47.5%waterfor1h,

incor-poratingPPO(2,5-diphenyloxazole), drying,and then

being exposedto Fuji X-ray filmasdescribed inan

accompanyingpaper(6).

The isoelectricpoints of individual polypeptidesare

givento thenearest0.01 pH unit for convenience of

identification. The valueswere obtained by

extrapo-lation and do notrepresent measurements made to

thenearest0.01pHunit.

One-dimensional SDS-polyacrylamide gel

electrophoresis.This wasbased onthe second

di-mensiondescribed above and followedprocedures

de-scribed inanaccompanyingpaper(6),exceptthat the

reservoir bufferwastwice concentratedand the

acryl-amide-bisacrylamideratiowasincreasedto200:1from

75:1; 15%acrylamide gelswere run.The samples

con-tained ampholines as described for the

two-dimen-sional work andweremixed withanequal volume of

10%SDS-10% /3-mercaptoethanol-15%glycerol-0.02%

bromophenolbluein 1 MTris-hydrochloride,pH6.8.

lodination of FV3 particles with 125I using

chloramine T. This was performed exactly as

de-scribedbyMooreetal.(12).

RESULTS

Incorporation of [35S]methionine into

tri-chloroacetic acid-insoluble material. It has

been claimed that FV3 is apotent inhibitor of

protein synthesis in cells infected with live or

inactivated virus(3). Wewereunabletoconfirm these observations since the incorporation of [35S]methionine into trichloroacetic

acid-insolu-ble materialwasatanequivalentorhigherlevel

than control cells formost of the infection(Table 1). The data reported indicate the results

ob-tained with materialrunonboth the

one-dimen-sional and the two-dimenone-dimen-sional gels. Similar

re-sults have been obtained in numerous other

experiments.

One-dimensional SDS-polyacrylamide gel electrophoresis of polypeptidesin

FV3-infected BHKcells. Thesequential induction

of FV3 ICPs on a gel system identical to the

two-dimensional gel electrophoretic second

di-mension is showninFig. 1;apolypeptideswere

detected from 2 h after infection, polypeptides

were detected from 4 h, and the onset of y

synthesis was at 6 h. In thisparticular

experi-mentthe and y phases were slightly late, so

the

,8

polypeptides predominated at6 h and the

-ypolypeptides, whichwerebarelydetected at 6

h, predominated later. Basically the same

pat-ternofpolypeptideswasobservedonthosegels

as on ourstandardSDS-polyacrylamide gel

elec-trophoresis gels (6), although two additional bands are detected in purified virus

(polypep-tides X and88) whicharenotnormallydetected

and X isnotpresentas anICP.There arealso

"anomalies" in therunning of certain polypep-tides inthe molecularweightrangeof3x 104to

6 x 104. In these experiments the acrylamide-bisacrylamide ratiowasdifferent than that used

forourstandardconditions (6), and the samples

contained 4 M urea and 0.5% ampholines, so

some anomalies may have been due to these differences. The ICPsare labeled according to

the nomenclatureused inan accompanying pa-per(6).

Nomenclaturefortwo-dimensionalICPs.

The ICPs detected by IEF and

SDS-TABLE 1. IncorporationofL

-[35S]methionine

into trichloroacetic acid-insoluble material in FV3-infectedBHK cellsatvarioustimesafter infection

Trichloroacetic

acid-msolublecounts'

Control 45,529

FV3 2 55,018

FV3 4 41,647

FV3 6 54,543

FV3 9 44,879

FV3 12 42,748

FV3 24 46,997

aCountsperminuteobtainedafterprecipitationof

material in 6 x 104cells infectedat25 to30PFUof

virusper cell andpulse-radiolabeled with 5MCiof

L-[35S]methioninefor1h.Precipitationwasachievedby addinganequalvolume of ice-cold20%trichloroacetic acidandincubatingonicefor60min.Thecontrolwas

uninfected cells held at the infection temperature

(280C).

VOL. 33, 1980

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20 ELLIOTT, BRAVO, AND KELLY

tCP

U

2

4

6

9 12 24

FV3

VPiNS

_-'' '

''--14 98

88 ---78

r

SESfe

_-

---69 i ---4

-4 4

[image:3.504.68.257.69.368.2] [image:3.504.270.462.285.610.2]

dlii Sb

~~~~~~~~~~~~~~~~~~~.

-x .

...-3--5 4

---34

}; -29

-27 -23

22

18

15 16

2

lb

a-FIG. 1. Sequential induction of ICPs in BHK cells

infected with FV3. Cells were pulse-labeled with

[35S/methioninefor1hatthetimes indicated across the top of thefigure (in hours). Purified FV3 was

co-run to indicate structural polypeptides. ICPs are

identified by estimates of their molecular weights

(xl103) (6)and are assignedasvirus particle (VP) or nonstructural(NS). U, Uninfected.

NEPHGEare shown in

Fig.

2and

cataloged

in

Table 2. The

polypeptides

are identified

by

a

combination of molecular

weight (to

thenearest

1,000) andisoelectric point (to thenearest 0.01

pH unit), so that the

major

structural

poly-peptide

ofFV3 is58,5.58 and the

major

nonstruc-tural basic

polypeptide

is35,7-10. The

pH

range

is indicated for basic polypeptides because

nonequilibrium

electrophoresis

was

performed

(14).

The various polypeptides were designated a,

fi,

and y for

comparison

with one-dimensional

gel polypeptides (6). The

assignment

was not

rigorous because the

requisite

"function-block-ing" experiments had not been

performed

yet

(6). For the purpose of thispaper, the timing

andapparentamountof

synthesis

wereused for

assignment;

a

polypeptides

were synthesized

from 2 h

(although

apparent isomers of

poly-peptide 16,5.48weredetectedfrom4

h),

,8

poly-peptides were detected at 4 or 6 h and subse-quently synthesized in similar amounts, and y polypeptides were synthesized at 6 or 9 h and predominated late in infection.

Acid IEF and SDS-acrylamide gel

elec-trophoresis of FV3 ICPs. Uninfected BHK

cells contained well in excess of 200polypeptides

resolvedby SDS-IEF (Fig. 3,gel i). By 2 h after

infection three ICPs were detected, all of which

were nonstructuralpolypeptides (Fig. 3, gel ii).

These three polypeptides (ICP 46,5.55, ICP

45,5.47, ICP 16,5.48) are by definition a

polypep-tides. Most cell polypeptides continued to be

synthesized,

albeit at a reduced rate, and some

major cell

polypeptides were markedly reduced.

At 4 h after infectionthese three a polypeptides

were synthesized in large amounts, and an

ad-ditional fourpresumptive isomers of polypeptide

16,5.48 were detected (Fig. 3, gel iii and Table

2). Seven additional polypeptides,

all

presump-pH

0

I-S 2

C:

4c

-J

Lu

-5

0

I

9

K114

T

100 0

_

-76

2

0

.

Lu

m 4 38 D

35 a

0

2

-18 3

FIG. 2. Diagrammatic representation ofICPs de-tectedon(A) SDS-IEF and(B) SDS-NEPHGE

two-dimensionalgels. ICPs tentativelyassignedas

struc-turalpolypeptides are shown asclosed areas, and nonstructuralpolypeptidesareshownasopenareas

or areindicatedNS.

J. VIROL.

;,.h:-::%:- MOO*

:.4--s'. -Zx

32 .-!:;:,!!:.- .-

'--No

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VOL. 33, 1980 ANALYSIS OF FV3 POLYPEPTIDES

21

TABLE 2. Catalog ofICPsdetected inFV3-infectedcellsbytwo-dimensionalgelelectrophoresis

Structural Period Equivalent Structural Period Equivalent

Polypeptide or nonstruc- detected a,13'

one-dimen-

| Polypeptide ornonstruc- detected a,or

one-dimen-turala (h) or-y sionalgel tural'~ (h) or-y sionalgel

I

I~__

Polypeptideit

J j polypeptide

NS NS S NS S NS S NS NS NS S NS S S S S NS NS NS NS NS NS S NS S S NS NS NS NS NS NS NS S NS NS NS S NS NS NS NS NS NS 9-24 9-24 9-24 9-24 6-24 9-24 4-24 9-24 9-24 9-24 6-24 9-24 6-24 9-24 6-24 9-24 9-24 6-24 4-24 9-24 9-24 9-24 6-24 6-9 12-24 12-24 12-24 4-12 2-24 4-? 2-24 12-24 12-24 9-24 12-24 6-24 12-24 6-24 6-9 12-24 12-24 12-24 9-24 9-24 Y y Y y y y 0 y y y y y y y y y y y /3 y y y .7 y y Y a a y y y y p y y y y y y .7 ICP 76yVP orICP 78yVP ICP69,BVP ICP 55-yVP ICP52NVP ICP46NVP ICP40-yNS ICP 37yVP 29,4.14 29,4.18 29,4.46 27/28,4.50 24,4.76 24,4.88 24,6.37 23,6.46 22,4.76 22,4.88 22,5.23 22,6.53 18,5.91 17,7.00 16,4.931 16,5.22 16,5.30 16,5.48 16,5.70 16,7.00 15,4.86 15,6.52 15,6.80 14,4.14 14,4.57 13,6.25 Basic 115,7-10' 114,7-10 100,7-10 100,7-lObi 100,7-lObii 100,7-10biii 76,7-10 67,7-10 55,7-10 47,7-10 38,7-10 38,7-lOb 35,7-10 24,7-10 24,7-lOb 18,7-10 15,7-10 14,7-10 13,7-10 12,7-10 NS S? S NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS S NS NS NS NS S S S NS S S S S NS NS NS NS S NS NS NS S S S NS S S 9-24 9-24 6-24 4-9 6-24 6-24 6-24 6-24 6-24 6-24 6-24 6-24 6-24 6-24 4-24 4-24 4-24 2-24 4-24 6-24 4-9 4-24 6-24 9-24 6-24 12-24 9-24 12-24 6-24 6-24 6-24 6-24 9-24 4-24 9-24 9-24 6-24 4-24 4-24 9-24 6-24 9-24 9-24 12-24 9-24 6-24 Y Y /3 1 /3 / /3 / 13 1 Y / 1 1 a a a a a / 13 / Y Y Y Y Y Y .7 0 0 y /3 y Y /3 p Y Y 0 y y Y y Y I Y3 Y3 Y7 ICP27yVP ICP29,BVP ICP23,8NS ICP23,BNS ICP23,BNS ICP2313NS ICP22yNS ICP 16aNS ICP 12yVP ICP 114yVP ICP98,BVP ICP66,8NS ICP44,/VP? ICP35flNSd ICP12yVP Acidic 86,5.76b 82,5.76 76,6.85 72,5.53 72,5.63 72,5.73 69,6.93 59,5.93j 59,6.11 59,6.22 59,6.35 59,6.5U 58,5.411 58,5.48 58,5.58 58,5.69J 58,6.99 55,5.39 55,6.93 54,5.82 54,5.94 54,6.12 52,5.39 49,4.76 49,5.13 49,5.23 49,4.35 47,5.45 46,5.55 46,5.63 45,5.47 43,5.60 41,5.57 38,5.30 38,5.56 35,4.57 34,5.56 34,6.56 34,6.70 33,5.35 33,5.65 32,5.53 31,4.64 31,5.35

S,Structural; NS,nonstructural.The brackets onthetableindicateapparentcharge isomers.

b For the acidicpolypeptidesthefirst number isthe molecularweight

(x103),

andthe second number is the isoelectric point

(pH).

Forthebasicpolypeptidesthe firstnumber is themolecularweight(XI03),and the second numberis thepHrangeresolved.

dSeetext.

tive

f polypeptides,

were

observed, including

the anumber of

additional

ypolypeptideswere de-structural polypeptide 69,6.93. Little further tected (Table 2). Minor yICPsweredetected at

suppression of cellular synthesisoccurred.By6 12and 24 h(Fig.3,gels vi and vii). At 24 h after

h ,Bpolypeptide synthesis waswell under way, infection,anumberof /3and y polypeptides had

and an additional 15

,B

ICPsweredetected(Fig.

declined

in synthesis, although the main virus

3,gelivand Table 2).At 6 height-ypolypeptides structural polypeptides werestillsynthesized in

were detected, includingthe major virusstruc- quantity (Fig. 3, gel vii). About 70 ICPs were

tural

polypeptide

58,5.58.By 9h (Fig. 3, gelv) resolved during infection.

these

polypeptides

became the prominent -y The structural polypeptides contained in FV3

polypeptides synthesized, althoughatthis time and resolvedby SDS-IEF are shown in Fig. 3,

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(5)

4

MW

pH 5

tni7- T'',

76-58- 2

34-

24-

16-

76-4w._

58-

34-"':

24-J. VIROL.

pH

*-_tM #* -16

16-

_

~~~~~~~~~~~~~~~...

'-,,.

FIG. 3. Distribution of[35S]methionine-labeled polypeptides in FV3-infected BHK cells analyzed by SDS-IEF.Gel i, Uninfected cells radiolabeled for I hat28°C; gel ii, infected cells radiolabeledat2to3h; gel iii, infected cells radiolabeled at4to 5h; gel iv, infectedcellsradiolabeled at6to 7h; gelv, infected cells

radiolabeledat9to10h; gel vi, infected cells radiolabeledat12to13h; gel vii, infected cells radiolabeledat 24 to25h; gelviii, purified[35S]methionine-labeled FV3 structural polypeptides. The ordinaterepresents

molecularweight (MW)(xl3).

gel viii. Mostpolypeptides detected in virus

par-ticles possessed ICP counterparts, although a

few hadnoobviousICPorcellularpolypeptide

counterpart.Virusparticles presented consistent

profiles, and so this discrepancy may be due

eithertoenrichment ofminorinfectedcell

pro-teins in the virus particles or to processing on

encapsidation and release.

Basic NEPHGE and SDS-acrylamide gel electrophoresis ofFV3 ICPs. In contrast to

the acidicsystem,fewICPsweredetected in the

basicpH7 to pH 10 analysis. The 20 polypep-tides detected are shown diagrammatically in

Fig.2 andsequentially inFig.4. Asummaryof

theirproperties isgiveninTable 2.

NoICPsweredetected2 hafterinfection(Fig.

4, gel ii), and therewasnotamarked alteration

in the synthesis of cell polypeptides, although the synthesis of certain cell polypeptides was

obviously becoming suppressed. At4h the

syn-thesis of three nonstructural ,B polypeptides (ICP 67,7-10; ICP 38,7-lOb; ICP 35,7-10) was

detected,and by 6 hanadditional four structural

B8polypeptidesweredetected (Fig. 4, gels iii and

iv), which increased in relative amountof

syn-thesisat9 h(Fig. 4, gel v). At 6 honestructural

ypolypeptide (ICP 12,7-10) wasdetected.

Sub-sequentlyat12and 24 hlittlealteration in ICP synthesis occurred, although cell polypeptide

synthesiswasfurther suppressed.

Identification of structuraland nonstructural polypeptides by SDS-NEPHGEwas aproblem.

6 7 4

iii

6

14 im

iv IAf j~ 'a'!

VW 4

0-MW

-76.

-58

-34

-24

-16

-76

-58

-34

..-24

I

:

;..4

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pH

5

"Isan if,.

6 7

MW

-76

-58

-34

-24

-16

-76

-58

2-34

-24 4

uWO

4

[image:6.504.53.451.64.429.2]

* -16

...

--[35S]methionine-labeledFV3particlesdisrupted with lysis bufferor lysis buffer plus0.1% SDS

failedtoproduce reproducible patterns.Purified

FV3 subsequently radiolabeled with

"25I

using chloramine T demonstrated that three major polypeptides detected late in infection were

basic structural polypeptides. Considerably more '25I-labeled polypeptides were present in

virus particles than were observed as ICPs in

infected cells late in infection (and in

SDS-NEPHGEresolving purified

[35S]methionine-la-beledvirus), and thisweascribein part to

chlor-amineT-inducedchargealterations in the poly-peptidesand in part todifferences inthe methi-onineandtyrosinecontentsof thepolypeptides. There is also the possibility ofunlabeled

con-taminating host proteins becoming iodinated by chloramineT.

Comparison of SDS-IEF and SDS-NEPHGE. The ICPs detected over the pH range4 to10areshowndiagrammaticallyinFig.

2 and in Fig. 5. A total of 70 acidic and 20basic

polypeptidesweredetected.Thetotalmolecular

weight of these polypeptides was 3.89 x

106,

whichrepresents77% of the coding capacityof theFV3genome (Table 3).

A number of ICPs detected by

one-dimen-sional SDS-PAGE analysis appearednot to be

detected by SDS-IEF and

SDS-NEPHGE.

ThesefrogFV3 ICPsincludedICPs104,93,88, 61, 58, 18, and 9 (whichare all minora, fi, and

ypolypeptides whichco-electrophoresewith

cel-lularpolypeptides) and ICP 34, amajor

struc-tural

ft

polypeptide. With the exceptionofICP

34, thesepolypeptides eithermayhavean

isoe-lectric point of pH 7.00 andso arenotdetected

atthe first-dimension gel origin (pH 7.0) above material which hasnotentered the gel ormay

haveisoelectric pointsoutside the pHrange4 to

10. The total molecular weightofthe ICPs

de-tected bytwo-dimensional gelanalysis andthe unresolved polypeptides (with the exception of

pH

5 6 7 4

4 MW

v

76-

58-

34-vii

....

IL

*

0%~ ~ ~ ~ ,

..k

4'

24-

16-vi

76-

58--._ ,@ - .

* II

viii

34-

24-16-

_Ab4

.

-FIG. 3. v-Vini VOL. 33, 1980

- %4

...

--A-.

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(7)

pH

10 7

i I4mor"

am 4W

iii MW

--loc' -76

-38

-35

-15 _---12

ii _iv

-100

-76

...

W..-.N

l_..4.

_ .,A W

.

....

o 1. - .,.

I' B. M ~

:

. _ -38

-35

-15

-12

FIG. 4. Distributionof[35S]methionine-labeledpolypeptidesinFV3-infected BHK cells analyzed by SDS-NEPHGE. Geli, Uninfectedcellsradiolabeledfor1hat28°C; gel ii,infected cells radiolabeledat2to3h;

gel iii, infectedcells radiolabeledat4to5h;gel iv,infectedcellsradiolabeledat6to 7h; gelv,infected cells

radiolabeledat9to10h; gel vi,infectedcellsradiolabeledat12to13h;gel vii, infected cells radiolabeledat 24to 25h; gel viii,purified FV3 structuralpolypeptides 125I iodinatedusingchloramine T. The ordinate representsmolecularweight (MW) (x103).

ICP 34 [see below]) was 4.325 x 106, which is

equivalentto85.6% of the virusgenome (Table

3).

Effect of FV3oncellularpolypeptide

syn-thesis. Over400polypeptidesweredetected in

uninfected BHK cellsandaretoonumerousto

realistically andprofitably catalog. At 24 h after infection 50 cell-specific polypeptides were

de-tected. Since FV3hasnomarked effectontotal

protein synthesis in BHK cells (Table 1), it is obviousthatthe virussubverts thecapacity of

thecellprotein-synthesizingsystems to

synthe-size itsownvirus-specific proteins. Thisappears

to occur by suppressing cell protein synthesis

ratherthanby actively switching offcell

synthe-sis. Most cell polypeptides appear to become gradually undetectable with respect to time,

which isindicative ofsuccessfulcompetition of virussynthesis with cell synthesis. Certain poly-peptides do, however, declinerapidly in synthe-sis(e.g.,polypeptidesatposition 60,5.01 and four polypeptides withintherange80 to 100,4to5).

DISCUSSION

Ourresults demonstrate thata high

propor-tion of the FV3 genome appears to be

tran-scribed and translated into presumptive virus-specific proteins during a productive infection.

Theresults also showthattwo-dimensionalgel electrophoretic analysis ofFV3 ICPs is vastly superiortoconventional one-dimensional anal-ysis and that it could be the method of choice

for the futureanalysis ofFV3polypeptide

me-tabolism.

7

MWr

100-

76- 38-

35-zF. ...

15- W

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35-

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ANALYSIS OF FV3 POLYPEPTIDES

7 MW

100-76-;

38-35- t

*

12.

12* ...

100- .

76-.e

38-

-

35- 15-12-

O-ab

10

7

V EaS* ,

pH

10 ..

MW

-100 -76

-38 -35

Theresultsobtainedontwo-dimensional gels

confirm that there are atleast three temporal

phases ofpolypeptide synthesis in FV3-infected BHKcells, substantiatingouraccompanying

ob-servations(6).This is atvariance with thereport by Granoff (9) that all FV3ICPsaresynthesized

from the initiation of infection

(although

there

aresubsets ofICPsdistinguished by theirrate

ofsynthesis [15]). Autoradiographic

overexpo-sure of the gels shown in this paper failed to

demonstrate any of the /8 or y ICPs at times

earlierthanthosereported (unpublished data),

and so we are confident that there was not a

detectionproblem.

Theseparation of FV3ICPsaccordingtotheir isoelectric pointsintheIEF and NEPHGE di-mensions allowedacatalog of isoelectricpoints

tobecompiled (Table 2), and this is particularly useful in the IEF separation. Our information showsthatthebulk of theFV3ICPshave isoe-lectric points in the pH 4to 7range, including

the major structuralpolypeptide (ICP 58,5.48). Observations on the major structural

poly-peptide showthatthere is apparent isomerism ofthispolypeptide withpolypeptidesofdifferent charges but similar molecularweights appearing in the cell. Thispolypeptide is phosphorylated butisnotdetectably sulfatedorglycosylated (6),

and so some isomerism may be attributed to

phosphate-created charge differences. Not all

phosphorylated ICPsshowisomerism (e.g.,ICP

69,6.93) andso someisomerism maybe caused

byas-yet-unidentified posttranslational

modifi-cation. Anumber of other polypeptidesappear

to show charge isomerism and are shown in

Table 2.

Onone-dimensionalgelsICP34(a majorICP)

appears to be a structural polypeptide,

al-thoughontwo-dimensionalgels nocounterpart

is found. Acomparison of IEF and SDS-NEPHGEindicatesthatamajorftnonstructural

polypeptide (ICP 34,7-10) is found on

SDS-pH

_ Isas

e

.a

Vi .1/6/f ; J.

-15 -12

_K:

E...

Viii

-100

-76

-38 -35

a4

[image:8.504.50.450.76.427.2]

i

FIG. 4. v-vuii

-15

-12

25

VOL. 33, 1980

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... .w

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(9)

4 MW 1

ab .

76-58- - ._

34- r

24-

16-Iv

-100

-76

-38 -35

-15

-12

S.A. . ....-.

FIG. 5. Distribution oftotal[35S]methionine-labeledpolypeptidesinFV3-infectedBHK cells;acomparison

of SDS-IEF and SDS-NEPHGEat4and12hafter infection.Gels i and iii, SDS-IEF and SDS-NEPHGE,

respectively, 4h; gel ii and iv, SDS-IEF and SDS-NEPHGE, respectively, 12 h. The ordinate represents

molecularweight (MW) (x103).

NEPHGEgels from 4to24hand thataminor

y structural polypeptide (ICP 34,6.56) occurs

from 6 to 24 h on SDS-IEF gels; therefore, it

appears that ICP 34 resolved on

one-dimen-sionalgelsrepresents twopolypeptides and that the structuralcomponentof the virus particle is

anacidicypolypeptide.

Early in infectionnobasicapolypeptidesare

detected, which may be considered surprising

since thevirusappearstohaveaneffectonthe

expression ofthe cell genome,and the interac-tion of basicproteins with DNAorRNAorboth

may provide a mechanism of inhibition. The

absence ofproteins with basic isoelectric points doesnotpreclude the possibility thatsomeother

ICPs have basic regions capable of such inter-action.

Ourdata indicate that reasonable quantities of FV3 (about 30 PFU/cell, 20 virus particles

perPFU) failtocausecellpolypeptide synthesis

to be switched offrapidly, although there is a

gradual suppression of cell polypeptide synthesis

asthe infectionprogresses.Thiscontradicts

ear-lierpublishedwork(8),but is inagreementwith

observations ofJ.C. S. Clegg and S. I. T. Ken-nedy (personal communication) and R. W. Schlesinger (personal communication). FV3, like herpesvirus (7), undoubtedly possesses

struc-turalpolypeptides which can inhibit cell RNA

and protein synthesis, as elegantly shown by

pH

7

J. VIROL.

_pu, w0

10

...

MW

III

-100 -76

40-, .:...

t

ii

-38 -35

-ao

*w .

-15 -12

76-

58-34-. w

.m

24--

16-I

wgl.

jr..%-,

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TABLE 3. Total molecularweights ofFV3-induced classesofICPs detectedbyvariousgel analytical

methods and the percentagesofthe FV3 genomerequiredfortheirsyntheses

Summedmol wt of thefollowingICPclasses:

Analytical method

,a a ya Sb NSb

SDS-IEF 1.71 x105(3.4)c 6.85x105 (13.6) 1.933x106(38.3) 8.31 x105(16.5) 1.958 x10W (38.8) SDS-IEF corrected 1.07x105(2.1) 6.85x105 (13.6) 1.523x10W (30.1) 6.57x105(13.0) 1.658x10W (32.8)

for isomerismd

SDS-NEPHGE 6.02 x105 (11.9) 5.03 x105(10.0) 6.35 x105(12.6) 4.70x 105(9.3) SDS-NEPHGE 3.02 x105(6.0) 5.03 x105(10.0) 3.35 x105(6.6) 4.70 x105(9.3)

corrected for isomerism

Unresolved' 6.1x104(1.2) 2.48x105(14.9) 1.22 x105(2.4) 3.25x 105(6.4) 1.06 x105(2.1)

Totalf 2.32 xi05(4.6) 1.535 x10"(30.4) 2.558x106(50.6) 1.791 x10W (35.5) 2.534x106(50.2)

Total corrected for 1.68 x105 (3.3) 1.235x106(24.5) 2.148x106 (42.5) 1.317 x106 (26.1) 2.234x106(44.2) isomerism

aa,

fi,

andy refertothetemporalclasses ofICPs resolved (6,7).

bSStructuralpolypeptides; NS,nonstructuralpolypeptides.

'Thenumbers in parentheses are the percentages of the FV3 genome(molecular weight, 1x 108[12]) responsiblefor the synthesis ofindividual classes ofICPs, assuming that the whole genome is available for translation and transcription and that no overlapping genes occur.

d Forcorrections forpossible isomerism,see textand Table 2.

'Unresolvedpolypeptides are ICPs which are resolved on one-dimensional gels but havenotwo-dimensional counterparts (see text).

fThe total molecularweight for a plus f plus y polypeptides was 4.325x 106or85.6% of the FV3 genome(3.551x106and 70.3%correctedforisomerism). Forstructuralplusnonstructuralpolypeptidesthemolecularweight totalswerethesame as

those for aplus fiplusypolypeptides.

Aubertinetal. (1), but it is doubtful that

suffi-cientquantitiesof theseproteinsareintroduced

into cells undernormalphysiologicalconditions tosuppresshostpolypeptide synthesis

dramati-cally.

ACKNOWLEDGMENTS

Wethank J. S.Knowland for his interest in thesestudies.

R.M.E. received a Science Research Council postgraduate studentship. R.B. thanks the Comision Nacional de Investi-gacionCientificayTechnologica deChile, the Royal Society

of London for an Exchange Fellowship, and the Medical ResearchCouncil forfinancial assistance.

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2. Bonner, W.M., and R. A. Laskey.1974. Afilmdetection

method fortritium labeled proteins andnucleic acids in

polyacrylamide gels. Eur. J. Biochem.46:83-88. 3. Drillien, R.,D.Sephner, and A.Ki-n.1977.Cell killing

by frog virus3:evidence forcellkillingby single viral

particlesorsingle viral units. Biochem. Biophys. Res. Commun.79:105-111.

4. Elliott,R.M., M. K. Arnold, and D. C.Kelly. 1979. The

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5. Elliott, R. M., A. Bateson, and D. C. Kelly. 1980.

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6. Elliott, R. M., and D. C. Kelly. 1980. Frog virus 3

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10. Kelly, D.C.,and R. J.Avery.1974.Frogvirus3 deoxy-ribonucleicacid. J. Gen. Virol. 24:339-348.

11.Kelly, D. C.,and J. S. Robertson. 1973. Icosahedral

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Envelopeproteinsof vesicular stomatitis virions:

acces-sibilitytoiodination.Virology61:292-296.

13.O'Farrell, P. H. 1975.Highresolutiontwo-dimensional

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250:4007-4021.

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