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0022-538X/82/030792-09$02.00/0

Poliovirus Empty Capsid Morphogenesis: Evidence for

Conformational

Differences

Between

Self-

and

Extract-Assembled Empty Capsids

J. ROBERT PUTNAK AND BRUCE A. PHILLIPS*

Departmentof Microbiology, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15261

Received 14 September1981/Accepted5November 1981

In this paper we describe the use of specific proteinases, surface-specific radioiodination, and antigenic reactivity in conjunction with isoelectric focusing for probing the conformations of different polioviral empty capsid species. Naturally occurring empty capsids (called procapsids) with an isoelectric point of 6.8 were resistant to proteolytic digestion by trypsin or chymotrypsin, as were empty capsids assembled in vitro in the presence of a cytoplasmic extract prepared from poliovirus-infected HeLa cells. In contrast, self-assembled empty

capsids (isoelectric point,5.0) weresensitivetobothproteinases. Capsid proteins

VPO and VP1 were attacked predominantly, whereas VP3 was resistant to

cleavage. Unpolymerized14Sparticles possessedatrypsin sensitivitywhich was

qualitatively similar to that of self-assembled empty shells. Surface-specific

iodination of virions and procapsids labeled VP1 exclusively. In contrast,

radioiodination of self-assembled empty capsids labeled predominantly VPO.

After radioiodinationthe sedimentationcoefficientcorrectedto water at20°C, the

isoelectric point, and the trypsin resistance of the procapsids remained

un-changed. Procapsids and extract-assembled empty capsids were N antigenic,

whereas self-assembled empty capsids were H antigenic. Self-assembled empty

capsids were notconverted to pH6.8 trypsin-resistant structuresby incubation

with avirus-infected cytoplasmic extract. However, 14S particles assembled in the presence ofamock-infected extract formed empty capsids, 20% of which resembled extract-assembled emptyshells as determinedbythe above-described criteria. These and related findings are discussed in terms of empty capsid structure andmorphogenesis.

The assembly of poliovirus empty capsids from precursor 14S particles was first demon-strated to occur in vitro in the presence of

cytoplasmic extracts prepared from

poliovirus-infectedHeLacells(13, 16). Later,itwasshown

that 14S particles can self-assemble, forming emptycapsid-like structures visiblebyelectron

microscopy (14). Unlike extract-mediated

as-sembly, this latter reaction demonstrated a marked dependence upon the concentration of 14Sparticles. The nature of the assembly-facili-tating activity in extracts is not known, but this

activitydoes notappear to be due to free

endog-enous14Sparticles (12).

We recently reported that self- and extract-assembledemptycapsids could bedistinguished from one another by (i) isoelectric focusing under nondenaturing conditions and (ii) rate-zonal centrifugation on sucrose density

gradi-ents (17). However, the polypeptide composi-tions of thesecapsids, as well as the apparent sizes and isoelectricpoints (pI)of their individ-ual polypeptides, appeared to be identical.

Therefore, we concluded that the two species of invitro empty capsids differed only in conforma-tion.

The topography of virus particles, including thetopography ofpicornaviruses,hasbeen stud-ied extensively by usingsurface-specific labeling

techniques, suchasradioiodination(5, 8, 9, 19).

When these techniques were performed with chloramine T, H202, or other oxidizing agents, tyrosine residues were labeled almost exclusive-ly. In an effort to obtain surface specificity, a glucose oxidase-lactoperoxidase enzyme system has been used with carrier-free 1251 (5). More recently, a solid-state oxidizing reagent,

chlor-oglycoluril, wasdeveloped (6). Based upon the

criterion ofpreservationof virusinfectivity,this reagent appeared to be more gentle than either chloramine Torlactoperoxidase. Studies have alsoindicated thatchloroglycolurilisasefficient aschloramineTandmore surface-specific than lactoperoxidase (10).

Another technique for studying topography hasbeenthe useofdegradative enzymes. With

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POLIOVIRUS EMPTY CAPSID MORPHOGENESIS

the exception of foot-and-mouth disease virus, picornavirusesareveryresistanttothe actionof proteolytic enzymes. Treatment of foot-and-mouth disease virus with trypsin caused a de-creasein its infectivity and cell attachment and altered its antigenicity (3). An analysis of tryp-sin-treated virusby polyacrylamide gel electro-phoresis (PAGE) in the presence of sodium dodecyl sulfate (SDS) showed that VP1 was cleaved, suggestingthat thispolypeptidewas at the surface of the virus particle. Both VP1 and VP2 were cleaved when the 12S particles pro-ducedasby-products of virus degradationwere exposedtotrypsin (7).

Inthisworkwestudied the surface configura-tions of the various polioviral capsid speciesby using proteolytic enzymes, surface-specific ra-dioiodination, and specific immune antisera. We presentevidence concerning the conformational differences between self- and extract-assembled empty capsids, as well as heat-denatured pro-capsids. We found thatvirus-infected cytoplas-mic extracts possess a morphopoietic factor(s) which directs empty capsid assembly and deter-minescapsid conformation.

MATERIALSANDMETHODS

Virus.Type 1poliovirus (Mahoney strain)wasused

for this work. The purification methods used have

been describedpreviously (16).

Cells.HeLacells(S3)werecultivated insuspension

culturebyusingEagle minimal essential medium

sup-plemented with Spinner salts, 2 mM glutamine, and

5%calfserum.Noantibioticswereused in routine cell

propagationcultures.

Preparation of virus-related particles, purification,

radio-labeling,andassembly reactions. Welabeled14S

particles,emptycapsids,andpoliovirionswith14C-or

3H-aminoacid mixtures andpurifiedthemas

previous-ly described (17). Likewise, assembly reactions were

carriedout asdescribedpreviously (13, 14).

Protein determinations.Proteinwasmeasuredbythe

Bradfordmethod(2).

Agarosegelisoelectricfocusing. Isoelectricfocusing

ofpoliovirus-related particles and polypeptides was

carried out in gels at pH 3.5 to 9.5, as described

previously (17).

Acrylamide gel isoelectric focusing. Focusing was

carriedoutingels containing4.8%acrylamide

mono-mer,0.27%bisacrylamide,8 M urea, and2%Nonidet

P-40in double-deionized water.These reagentswere

passedover amixed-bed ion-exchangeresin(Bio-Rex

RG501-X8) before use. Then2%ampholine (pH 3.5 to

9.5;LKB) wasadded, and the gel was polymerized in

the presenceofasolution containing 0.01%

ammoni-umpersulfate, 0.05% TEMED

(N,N,N'N'-tetrameth-ylethylenediamine), and 0.005% riboflavin phosphate

for1hunderafluorescentlight. Samples were

solubi-lized in1%SDS-5% 3-mercaptoethanolat 100°C for 3

min andcooled, and then 9 volumes of 10 M urea-4%

Nonidet P-40 was added. The gels were prefocused at

200 Vfor1h,and then the samples were focused from

thecathodeat200Vfor1h, at 400Vfor1 h, at 600V

for5h, andat800Vfor 30 min. The catholyte was 0.5

N NaOH, and the anolyte was 0.5 N H3PO4. After

focusing, the gels were fixed and fluorographed as

previously described (17).

Radioiodinations. Radioiodination of the purified

particles was carried out in the presence of

1,3,4,6-tetrachloro-3a,6a-diphenylglycoluril

(chloroglyco-luril). The choroglycoluril (dissolved in 0.1 ml of

chloroform) was added to a borosilicate glass test tube

(12 by 75 mm), and the solvent wasevaporated under

an N2 gas stream. The protein to be iodinated was

added together with 500 ,uCi of carrier-free Na

"25I

buffered with 20 mM Na2HPO4 (pH 7.4) in a total

volumeof 0.1 ml. The reaction wascarried outfor 10

min at0°C, and then the mixture was transferred to a

polypropylene tube containing 10 ,ulof 250 pM

KI-0.01% 3-mercaptoethanol. The iodinated proteins

wereseparated from thefree "25Ibycentrifugationon

15 to30%o sucrosegradients inphosphate buffer (pH

7.2) containing 25p.MKI and0.001%

P-mercaptoeth-anol. Centrifugationwas in an SW41 rotor at35,000

rpmfor 4 h at18°C.Fractionswere collected from the

bottom of the tube and assayed for radioactivity.

Immunoprecipitation of virus-related particles.

Spe-cific anti-N and anti-Hsera weregenerouslyprovided

byR.Rueckert and A. Mosser(Universityof

Wiscon-sin).Anti-N serumwaspreparedagainst purified po-liovirus in rabbits and was adsorbed with heated virus. Anti-H serum to heated virus was made and adsorbed with native virions. Dilutions of antisera were

pre-paredin NET (0.1 MNaCl,1mMEDTA, 0.01 MTris,

pH 7.2) or, more satisfactorily, in IPbuffer (20mM

P04, pH 7, 0.1 M NaCl, 1 mMEDTA, 0.1% bovine

serum albumin, 0.01% Triton X-100, 0.01% SB-14

[Zittergent; Calbiochem]) in 1.5-ml polypropylene

tubes. Thevolumes used were 15 to 30

RIp.

Aknown

amountof labeled antigen (1,000 to 5,000 dpm) in 5

RI

orlesswasadded to eachdilution tube. After1 hof

incubation atroomtemperature (20°C)with continu-ousvigorousshaking,sufficientIPbufferwasaddedto

bringthevolume to 85p.1,and then 15,u1ofprotein

A-Sepharosebeads(50% suspensioninNET)wasadded.

Incubation was continued for 60 to 90 min, as

de-scribedabove. The tubes werecentrifugedat15,600x

g in an Eppendorf model 5412 centrifuge, and the

resulting supernatant fluids were carefully collected

and counted. Thebeads were washed twice with 100

RI of cold IP buffer and then suspended in 50p.lof

buffer and counted. Preimmune antisera at several dilutions (usually 1:10 and 1:100) were run as

con-trols.Specificimmune precipitation was calculated by

thefollowingtwomethods: (i) percentageof

disinte-grations immunoprecipitated = [(disintegrations per

minute in immunepellet- disintegrations per minute

incontrolpellet)/(disintegrationsper minute recovered

-disintegrationsperminute in control pellet)] x 100;

and(ii) percentage ofdisintegrations

immunoprecipi-tated= 100-[(disintegrations per minute in immune

supermatant)/(disintegrations perminuteincontrol

su-pernatant)]. Recoveries were 80 to 100%o, and the

values obtained by the two methods were nearly

identical. Controlpelletscontained approximately5%

orlessof the recoveredradioactivity.

Treatment of particles with proteolytic enzymes.

Preparations of particles or proteins were treated with trypsin (tolylsulfonyl phenylalanyl chloromethyl

ke-tone treated) or chymotrypsin in sodium phosphate

buffer(pH 7.4)at20°C. A ratio of enzyme to protein of

793

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794 PUTNAK AND PHILLIPS

1:10(wt/wt) was used. The reaction was stopped by

adding a 2- to 10-fold excess of soybean trypsin

inhibitor or 0.1 mMphenylmethylsulfonylfluoride (for

chymotrypsin).

Materials. 14C-amino acids (100 .Ci/ml) and

3H-amino acids (1 mCi/ml) were obtained from New

England Nuclear Corp., Boston, Mass. Na125I (100

mCiIml;carrier-free; in NaOH, pH 8 to 10) was also

obtained from New England Nuclear Corp.

Chlorogly-coluril(IODOGEN)wasobtained from Pierce

Chemi-calCo., Rockford, Ill. Bio-Rex mixed-bed resin was

obtained fromBio-Rad Laboratories, Richmond,

Cal-if.Trypsin (209 U/mg;N-tosyl-L-argininemethylester)

and soybean trypsin inhibitor were obtained from

Worthington Diagnostics, Freehold, N.J.

Chymotryp-sin A4 was obtained from Boehringer Mannheim,

Indianapolis, Ind. Phenylmethylsulfonyl fluoride was

obtained fromCalbiochem,LaJolla, Calif. Protein

A-Sepharosebeads werepurchased from Pharmacia Fine Chemicals, Inc., Piscataway, N.J.

RESULTS

Todescribe moreprecisely the kinds of empty

capsids used in these experiments, we used a

previously definedterminology, in which the pl and density (in grams per cubic centimeter) in CsCl are designated by a superscript and a subscript, respectively(17). Inthis terminology

procapsids derivedfrom infected cellsare

abbre-viated PRO, and empty capsids assembled in vitro are abbreviated E.C.

Trypsin sensitivity of poliovirus-related parti-cles. Polioviral procapsids

(PROT:83),

extract-assembled empty capsids(E.C. 9), self-assem-bled emptycapsids

(E.C.5:

%),and 14Sparticles weretested forsusceptibilitytotrypsin. Purified

or partially purified particles were treated at

20°Cwithtrypsin at a ratio of enzyme to protein of 1:10 (wt/wt). The reaction mixtures were sampled at 0, 10, 20, 40, and 60 min, and the samples wereanalyzedby SDS-PAGE. Figure 1 shows ananalysis ofthe'4C-aminoacid-labeled

particles after trypsin treatment. As this figure

shows,

PRO6

81 and

E.C.6-9

were very resistant totryptic cleavage, as judged by the intactness of their polypeptides. However,

E.C.5:

%, the product of the self-assembly of 14S particles, and the14S precursor particles themselves were sensitive to trypsin;VP0andVP1 were attacked selectively.

Densitometric scanning of a fluorogram such asthatshown in Fig. 1permitted kinetic analyses

ofthe reactions(Fig. 2). The reactions of PRO

1.31 (Fig. 2A)and

E.C1

19

(Fig. 2B)

with

trypsin

showed that both of these empty capsids were relatively insensitive. A limited amount of VPO appeared to be vulnerable. From these data, it wasdifficult to assess whether VP1 or VP3 was attacked.After60 min,75 to80%of the

structur-alpolypeptidesremained intact. Whether a

siz-R6.8

E.C.

PRO

6 o E 6.8

0 0 0 0 0 0 0

y- N1 v co 0w- N V (

14

S

03 %- CM qt (O

E o o o 0

o cs- q XD

a b C d e f g h i k I m n 0 p q r s t

FIG. 1. Effect oftrypsinonpoliovirus-relatedparticles. 14C-labeled procapsids

(PRO`

1;lanesathrough e),

extract-assembled empty capsids (E.C.8 ; lanes fthrough j), 14S particles (lanes k through o), and

self-assembledemptycapsids

(E.C.1-09;

lanespthrough t)werepurified aspreviouslydescribed(17). They were

reacted withtrypsin(tolylsulfonyl phenylalanylchloromethylketonetreated)ataratiooftrypsintoproteinof

1:10(wt/wt) for 0, 10, 20, 40,or60minat20°C.Afterthisincubation,a2-to10-foldexcessofsoybeantrypsin

in-hibitorwasaddedtothemixtures,whichwerethensolubilizedby adding2%SDSand100 mMdithiothreitolat

100°C for 3 min. Electrophoresis was on 15% polyacrylamide gels as previously described (17). After

electrophoresisthegelswerefixed,andVPO, VP1,andVP3 werevisualizedbyfluorography.

0

Vp~~~~~~~~~~~~~~~~~~

w-X4..i 4&

V. ;: ijjjSoZ!S:d}X^.

VP3 S. ..

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POLIOVIRUS EMPTY CAPSID MORPHOGENESIS 795

FIG. 2. Kineticsofcleavageofpolioviralstructural

proteins by trypsin. (A) Purified "4C-labeled

procap-sidsweretreated withtrypsin(tolylsulfonyl

phenyla-lanyl chloromethyl ketone treated) at20°Cfor0, 10,

20, 40, or 60 min and then subjectedtoPAGE in the

presenceofSDS,asdescribed in thelegendtoFig.1.

The percentagesofVP0(0), VP1 (A),and VP3 (0)

that remained uncleaved were determined from a

densitometric scan ofthe developed fluorogram and areexpressed aspercentages of the radioactivity

re-maining.(B) Purified extract-assembled empty capsids (E.C.68). (C) Purified self-assembled empty capsids (E.C.5). (D)Purified 14S particles.

able fraction of these particles were completely trypsin resistant or whether approximately 20%

ofthe polypeptides in any given particle were

accessible to attack was not determined. In contrast,

E.C.1%29

(Fig. 2C) and 14S particles

(Fig. 2D) were attacked at much higher initial

rates, and a substantial percentage ofVPO and VP1 molecules were cleaved to small peptides after 10 minof incubation. Cleavage of theVPO in both types ofparticles was essentially com-plete by40 min. There appeared to be a small fraction of VP1 molecules associated with

E.C.5

29which

resisted attack. However, VP3 wasunique in its trypsin resistance in both types

ofparticles. These results also suggested that

theVPO in

E.C.5:0

particles was more accessi-ble to trypsin initially than the VPO in 14S particles.

Treatment ofall four types ofparticles with chymotrypsin yielded similar results in terms of relative susceptibilities (data not shown).

Sensitivity

of heatedprocapsidstotrypsin treat-ment. Previously, we reported that when

PRO6

81particles were heated at 40 to 46°C, they assumed apI of 5.0 and a density of 1.29g/cm3 (17). Wealso reportedpreliminary results which indicatedthat heatedprocapsids lost a substan-tial amount of their VP0 molecules, yet sedi-mented at 70S orgreater.

The change in the pI values of theprocapsids caused by heating is shown in Fig. 3. The reaction was complete within 20 min, and the heated particles focused at pH 5.0. A similar reaction was demonstrable at 37°C (data not shown). The sedimentation coefficient (correct-ed to water at 20°C) of heated procapsids was

determinedby sucroserate-zonalsedimentation

together with differentially labeled, unheated procapsids. Figure 4 shows thatheated procap-sids had asedimentation coefficient whichwas5 to10%greater than that of unheatedprocapsids.

Purified procapsids labeled with 14C were

heated at 43°C for up to 20 min and then ana-lyzed for trypsin sensitivity. The trypsin

sensi-tivity of these procapsids, which was

deter-mined as described in the legend to Fig. 1, was increased by heating. After heating, predomi-nantly VPO was cleaved to smallpeptides,which migrated at the gel front (data not shown; see Fig.2C). VP1 was alsoattackedbut wascleaved only incompletely, generating a highly stable fragment that was only slightly smaller than

CO

O

*-~

£

E

0 C 0E

Nv

anode

pH 5.0_

pH

6.8-,

r

IFt 4

I

cathode

a

b

c

d

FIG. 3. Effect of heating on the pI of purified

poliovirus procapsids.Procapsids were heated at43°C

for 2, 5, 10, or 20 min. Isoelectric focusing was

performed inaprefocused (200 V, 1 h) agarose gel.

The pH was determined by using an Ingold

micro-probe surface electrode. VOL. 41,1982

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796 PUTNAK AND PHILLIPS

70a3I

.2-I

S10 1 ba 1o 155 30

fractions

FIG. 4. Effect of heat on the sedimentation

coeffi-cientofpurifiedpoliovirus procapsids. (A) Unheated

14C-labeledprocapsids (0)weremixed with unheated

3H-labeled procapsids (A)andcentrifuged on a 15 to

30%osucrosegradient in an SW41 rotor at35,000rpm

for 4h at 18°C. (B) 14C-labeledprocapsids (0)were

heated at 43°C for 20 min before being mixed with

unheated3H-labeledprocapsids(A).Themixture was

centrifuged as described above. The sedimentation

coefficients wereestimated by integration.

VP1. The identification of this fragment as a

cleavageproduct of VP1 is described below.

Heated procapsids treated with trypsin were analyzed by rate-zonal centrifugation. Most of the radioactivity sedimented at 80 to 85S, with only a small amount at the top of the gradient

(datanotshown). Therefore, most of the

cleav-age products produced by trypsin treatment must have remained associated with the parti-cles under theseconditions.

Surface-specific radioiodination as aprobe of particle conformation. Since

PRO1.81

and

129 were resistant to trypsin, we had no

information aboutthe surfaceconfigurations of

theseparticles. To study the procapsids further, we used a recently introduced surface-specific radioiodination technique (10).

Virus particles,

PRO681,

and

E.C.59

were purified, and 1 ,ug of purified material was reactedwith0.1, 1.0, or 10.0 ,g of chloroglyco-luril in the presence of 500 ,uCi of carrier-free

Na125I, as described above. lodinated particles

were separated from free 125I by sucrose rate-zonalcentrifugation. Wefoundthatprocapsids wereiodinated to specific radioactivities of2 x

106,

4 x 106 and5 x 106dpm/,ug with 0.1, 1.0, and 10.0 ,ug of chloroglycoluril, respectively. When theiodinated procapsidpreparationswere

subjectedtoSDS-PAGE andautoradiographed,

labelwasfound almost exclusively inVP1(Fig. 5, lane e).

Wedetermined thataratio of chloroglycoluril to protein of 1:1 was optimal for radioiodina-tion, and this ratiowas used in theiodination of poliovirus and

E.C.':

%. A specific radioactivity of 5.5 x 106dpm/,Lgwasobtained for virus,and a value of 8 x

105

dpm/nLg

was obtained for

E.C.5:

%.Like

PRO681,

poliovirusparticles were also iodinated primarilyin VP1 (Fig. 5, lane d). In contrast,

E.C.129

particles were iodinated primarily in VPO, although some impuritieswere also labeled (Fig. 5, lane f).

0

0.o

'ttt Ns N N

_

cc

_.-VP0

-VP1

._ _ _ 0-VP3

[image:5.491.50.243.58.276.2]

a

b c

d

e f

FIG. 5. Analysisof thepolypeptidesof

poliovirus-relatedparticles aftersurface-specificradioiodination.

Afterpurification,theparticles were radioiodinated in

the presence ofchloroglycoluril (1 ,ug/,ug ofprotein)

and 500 ,Ci ofcarrier-free 125I. The reaction was

carried out at 0°C for 10 min. The particles were

reisolated by sucrose rate-zonal centrifugation and

thensubjectedtoPAGEasdescribed in thelegendto

Fig.1.Lanesa,b, andccontained"C-labeledextract

(ext.), poliovirus particles (P.V.), and procapsids

(PRO) forcomparison. Lane d contained 125I-labeled

poliovirus particles (1251P.V.), laneecontained

125i-labeled procapsids (1251 PRO), and lane f contained

125I-labeled self-assembled empty capsids (125I

E.C.5-°). The structural polypeptides VPO, VP1, and

VP3 areindicated byarrows.

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POLIOVIRUS EMPTY CAPSID MORPHOGENESIS 797

Theradioiodinatedprocapsidsweretested for susceptibilitytotrypsin,aswellaspl.Wefound thatthese procapsidsweretrypsinresistantand focusedatpH6.8(datanotshown). Thus,based on these criteria, the particles had not been denatured. The iodinated procapsidswerethen heated at 43°C for 15 min, after which they focusedatpH 5.0 (datanotshown). The heated procapsids were also treated with trypsin for 20 min at 20°C and then analyzed by SDS-PAGE (Fig. 6). A 32-kilodalton cleavage product of VP1, designated VPla, was observed. In addi-tion, the smaller cleavage products VPlb (17 kilodaltons) and

VPlc

(11 kilodaltons) werealso detected.

Antigenic determinants of E.C.1.9 and

E.C.I50

. Based onprevious reportby Mosseret

al. (A. S. Mosser,S. W. Hong, J. Icenogle, and R. R. Rueckert, Abstr. Annu. Meet. Am. Soc. Microbiol.1981, T125,p.258),weexaminedthe

antigenic determinants ofprocapsids, aswellas

extract-mediated and self-assembled empty cap-sids. Poliovirions (datanotshown)and procap-sids isolatedfrom virus-infectedHeLacells and

purified in CsCl gradients were N antigenic.

E.C.1 29

formedby incubating14Sparticleswith

w 0L 10X 0.

i ^

~~~~~Pro6-1. pr6.

01

a T- EC6.

10,

ANTI-H s

0

5

0 c;

satedPro

1 2 3 4 5 -O

AbConc.(-LogDil'n.)

FIG. 7. Reactivities of procapsids (Pro681),

ex-tract-assembled emptycapsids(E.C.t ),

self-assem-bledempty capsids

(E.C."

°:),and heatedprocapsids

(broken line) to specific anti-N and anti-H antisera.

Notethat heatedprocapsids didnot reactwithanti-N

serum at a10-2 dilution (A). Particleswere-obtained

as described in the text. Heated procapsids were

obtained by treatment ofpurifiedprocapsids at43°C

for15min.AbConc.,Antibody concentration;Dil'n, dilution; IMMUNE-PPTE,immuneprecipitate.

ib4_

p.-VP1 *--VP1a

-.00h omn

WYalioqgl

.4

laop

4-VPlb

-VPlc

a

b

c

d

e

f

FIG. 6. Effect of trypsin on 1"MI-lat

procapsids. Poliovirus procapsids were

radioiodinated as described inthe legei

Lane a, "M5I-labeled procapsids; lane b

procapsids treated with trypsin; lanec

procapsids heatedat43°C for 20 min and

withtrypsinat20°Cfor 20 min; lane d,r

labeled procapsids; lane e, 14C-labele

treatedwithtrypsin for 20 minat20°Cas

the legend to Fig. 1; lane f, 14C-labele

heated at 430C for 20 min and then

trypsin.Electrophoresiswascarriedout

acrylamide gels asdescribedinthelege

The major trypsin cleavage products

VPla, VPlb,andVPlc.

infected cell extracts were also N antigenic. In contrast, self-assembled empty capsids i

h

-n

VP 0

(E.C.1

%) and heated

(43°C,

15

min)

procapsids

-.-vYP were H

antigenic

(Fig.

7). These and other r-VPla? findings are summarizedinTable1.

P+ VP3 Evidence for a

conformational

difference

be-tween

self-assembled

empty

capsids

(E.C.50

°)and

-VP1b mock-infected extract-assembledPreviously(17), wereported that a small number

empty

capsids.

of procapsid-like, pl 6.8 empty shells were

VP-

c formed when concentrated 14S

particles

were

allowed to assemble in the presence of cytoplas-micextract frommock-infectedcells. To charac-terize these empty capsids, we studied their

susceptibility totrypsin.

Self-assembled empty capsids

(E.C.51%29)

and

beled

VP

iin

empty

capsids

assembled in the presence ofa

purified and mock-infectedextractwereisolated

by

sucrose

nd to Fig. 5. rate-zonal

centrifugation.

In this

experiment,

2,521I-labeled

E.C.1os

sedimented at 71S, whereas

mock-in-125I-labeled fected extract-assembled empty capsids

sedi-Ithen treated mented at 75S (data not shown). Both types of

*eference

14C- empty capsids were treated with trypsin as

de-d

procapsids

scribed in the legend to Fig. 1 (Fig. 8). The sdescribed in resulting gel

patterns

were distinctly different,

treated

with

indicating

that a different setof

cleavage

sites

in 20%poly- was

accessible

ineachtypeof

particle.

Notable :ndto Fig. 1. were the 60%o conservation of VPO and the of VP1 were almost totalconservation of VP1 in

mock-infect-edextract-assembled empty capsids.

VOL.41, 1982

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TABLE 1. Characteristics of empty capsids formed in vitrobyassembly of14Sparticles in the absence (i.e.,

self-assembly) or presence of poliovirus-infected cell extracts

Type ofempty I Density inCsCl a

Ant.g.i.t

Surface Trysin

capsids (g/cm3) s ntigemcity polypeptide susceptibility

Self-assembled 5.0 1.29 71 Hb VPO Yes

Extract-assembled 6.8 1.29c 75 N VP1 No

a

s20,W,

Sedimentation coefficient corrected to water at

200C.

bSome N determinants may exist.

cProcapsids differed fromextract-assembledempty capsids only in density in

CsCl

(i.e., 1.31

g/cm3).

The trypsin-treated particles were also ana-lyzed by sucrose rate-zonal centrifugation. The sedimentation coefficient (corrected to water at

20°C) of trypsin-treated

E.C.5:0

was shifted

from 71S to 60S, whereas the sedimentation

coefficient of the mock-infected

extract-assem-bled empty capsid species remained unchanged

aftertrypsinization (data not shown). The ability

of mock-infected or virus-infected extracts to convert E.C.1 particles into trypsin-resistant

structures could not be demonstrated, which was consistent with the inability of extracts to convert E.C. ° into E.C. 9 or PRO 81, as determined by pl analysis (17). These findings strongly suggested that mock-infected extracts contain afactor thatdirectsalimitednumberof

14S particlesinto a similar kind of empty shell

(E.C689) as the putative morphopoietic factor in poliovirus-infected cells.

DISCUSSION

Previously, we reported that the

self-assem-bled emptycapsidsof poliovirustype 1 hada I of5.0 andadensityof 1.29 g/cm3(i.e.,E.C.129) and that the empty capsids assembled in the presenceof infected-cell extracthadapIof6.8

(i.e.

E.C.619)

(17).Procapsids,whichapparently

exist in virus-infected cells, alsofocused atpH

A

E

c 4

vpo - . _ _

vp1 -- -: -.oI

VP3 -is_ ma_ _ _

6.8, but they had a density of 1.31 g/cm3 (i.e., PRO

13).

At that time we ascribed the differ-ences in pl values todifferences in capsid

con-formation because the two species possessed

polypeptides which were identical in apparent

molecular weight and pI. In thispaper we pre-sent more direct evidence for differences in capsid conformation.

Wefoundthat

PRO68

1 and

E.C.69

were both

very resistant to the proteinases trypsin and

chymotrypsin. Incontrast,

E.C.1:29

wasreadily

attacked by both enzymes, as were the precur-sor 14S particles. The polypeptides of E.C.l12

and 14S particles that were most readily at-tacked were VPO and VP1. We conclude from these results that the configurations of VPO and VP1 are such that extensiveregionsof these

molecules lie exposed at the surfaces ofthese

particles. The configurations of these

polypep-tides must bedifferent in PRO6

31

and

E.C.1

29,

so astobe resistant toproteolysis.This does not eliminate the possibility thatsignificant regions of thesepolypeptidesare onthe exteriorsofthese

shells, but only means that their secondary,

tertiary, and quaternary conformations make

them inaccessible to the active site of the

en-zyme. VP3 was the only polypeptide that was resistant toproteolyticcleavage in all four

parti-c

0

B

0 0 0 0

v N t CD

-woom

FIG. 8. Comparison of the trypsin sensitivity of self-assembled empty capsids (E.C.1: (B) with the

sensitivity ofcapsidsassembled in the presence ofcytoplasmic extractfrom mock-infected cells (A). After assemblythe emptycapsidswerepurified bysucroserate-zonalcentrifugation,treatedwithtrypsin,and then analyzed byPAGEasdescribed in thelegendtoFig.1.

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POLIOVIRUS EMPTY CAPSID MORPHOGENESIS 799

cles. This finding appears to be moot because

under nocircumstances was VP3 degraded

ex-tensively by trypsin or chymotrypsin (see be-low).

Each structural polypeptide appeared to have

a different reactivity with trypsin, as

demon-strated by the differences in the initialcleavage rates (Fig. 2). Cleavage at one site may make previously hidden sites accessible. Forexample, VP3 in 14Sparticles wascompletely resistantto

cleavage until most of the VP0 and VP1

mole-cules had been digested (Fig. 2D); however, even after 60 min most of this polypeptide remained intact.

Itis not evident whether the limiteddegrees of trypsin sensitivity demonstrated by

PRO68

1and E.C

.1.29were

due to asmall number of sensitive sites in every particle or to alarge number of sites in a smallpopulationofparticles. Previous-ly, we reported that

PRO6:81

underwent a con-version to pI 5.0 particles when it was heated (17)(Fig. 3). The heatedprocapsidsalsobecame

trypsinsensitive. Although it isconceivablethat

asmall percentage ofprocapsidsare denatured during purification, much of which is carried out at 20°C, 20 to 25% appears to be too large an amountand is inconsistent with thehighenergy

ofactivationobserved (about 450kJ/mol;

previ-ously, weerroneously reported thatthis energy

of activation was 119 kJ/mol) (17). Also, we

never found more than a few percent of the

particles in procapsid preparations with a pI of

5.0.Therefore,wetentatively favor the idea that

there are afewtrypsin-sensitive sites on every

particle and that these sites reside on certain

VPO molecules or VP1 molecules or both. We have reported the results ofpreliminary

experimentswhich indicated that heated

procap-sids lose most of their VPO molecules (17). It now appears that contaminating proteinases could be responsible for this observation.

In-deed, VPOwaspredominatelycleavedin heated

procapsids by trypsin. Heated procapsids also showed an increased sedimentation rate of ap-proximately 80S (Fig. 4). The exact nature of

this heat-induced change is unclear, although

again it most likely reflects a change in capsid

conformation. It is noteworthy that although

E.C.5%0

and heated procapsids were both H antigenic (Fig. 7), they were not identical in susceptibility to trypsin (Fig. 1 and 6). Procap-sids in extracts appeared to be more resistant to

heating than purified

PRO6.81

(unpublished

data), but this may have been due to a protective effect exerted by other proteins or differences in

theionicenvironment.

Aftertrypsin treatment the sedimentation co-efficient of heated procapsids was conserved, indicating that most of the cleavage products remained particle associated, probably held in

place by electrostatic or hydrophobic interac-tions. These bonds should be susceptible tohigh concentrations of salts or to urea and detergents. On theother hand, trypsin treatment of

E.C.29

yielded particles with a sedimentation coeffi-cient of about 60S and a largeamountof radioac-tivity at the top of thegradient.

Since virus particles,

PRO61

, and

E.C.69

were resistant to proteolytic cleavage,we were unable to obtain topographical information by this method. Therefore, chloroglycoluril-cata-lyzed radioiodination of exposed tyrosine resi-dues was used to probe the surface configura-tionsof these particles. Both virus particles and

PRO68

1 were labeled primarily in VP1. The

hypothesis that VP1 is the predominant surface

polypeptide in most picomavirions has been

supported by thefollowing lines of evidence: (i) the radioiodination of VP1 by the lactoperoxi-dase and chloramine-T method (4, 5, 9), (ii) surfacelabeling ofVP1withaceticanhydride (8) andN-succinimyl proprionate (19), (iii) the tryp-sinsensitivity of VP1 in foot-and-mouth disease virus (3), and (iv) virion neutralization by anti-body to VP1 (9). Coxsackievirus appears to be the only exception to date (1). On the other hand, published data dealing with the topogra-phyofprocapsids is scanty (5). Itwascommonly

believedthat theconformation of polioviral

pro-capsids was different from that of virions since

procapsids reportedly did not attach to

virus-susceptible cells or cross-reactimmunologically with virions (18). Recently however, data

con-flicting with this notion have been obtained

(Mosser et al., Abstr. Annu. Meet. Am. Soc. Microbiol. 1981). Also, procapsids and virus

particleshavealmostidenticalisoelectricpoints

(17), and both types of particles were

radioiodin-ated primarily in VP1 (Fig. 5). Evidently, the

cleavageofVP0 that occurs upon RNA

encapsi-dation may not have the profound effect upon

capsidconformation thatwas oncebelieved.

In contrast tothe otherpolioviral capsid

spe-cies,

E.C.5l0

shells wereradioiodinated

primari-ly in VPO, a finding thatcomplements the

sus-ceptibility ofthispolypeptidetotrypsin (Fig.1).

The factthat VP1 was also cleaved by trypsin butwas notiodinated may indicate that cleavage of VPO isaprerequisiteto VP1 susceptibilityto trypsin (Fig. 2) or that the exposed portions of VP1 lack tyrosine residues. In any case, the exposed regions of VPO andVP1 aredifferent in self-assembledemptycapsids than in procapsids or

E.C.61

9 particles, and this difference may accountfor their different pI values. Finally, our

experiments showed that

PRO6-8

and

extract-assembledemptycapsids(E.C.1:29)were N anti-genic, whereas self-assembled empty capsids

(E.C.5

:) and heated PRO6

81

were Hantigenic

(Fig.7). Thus, there appears to be a correlation

VOL. 41,1982

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between the pl and the predominant antigenic determinantsof poliovirus empty shells;i.e.,pl 5.0 empty shells are H antigenic, and pl 6.8 emptycapsids areNantigenic. Note that polio-virions have a pI of 6.7 to 6.8 and are N antigenic.

How is the conformation of empty capsids determined? The conformation of E.C.l% is determined by information contained within its constitutent14S particles. These capsids appear to require no accessory factors for assembly. However,

E.C.5:0

particles are not found in vivo, and those formed in vitro by the self-assemblyreaction could not be converted into pl 6.8 procapsid-like structures by incubation in

the presenceofvirus-infected cellextracts (17).

Evidently, additional information in the formof an assembly factor or factors is required for the assembly of PRO681 in vivo and

E.C.689

in vitro.

Wefoundthatincubation ofpartially purified 14S particles with mock-infected cell extracts resulted in the assembly of empty capsids, some (about 20%) of which focused at pH 6.8 (17) and were more resistant to trypsin than self-assem-bled empty capsids (Fig. 8). The significance of an activity in uninfected cells which affects empty capsid conformation, at least in vitro, remains obscure. Perhaps this activity fortu-itously mimics avirus-coded morphogenetic fac-tor. Another possibility is that it interacts with the viral proteins which function in capsid as-sembly, analogous to the interaction between the bacterial protein groE and phage p31 in T4 headmorphogenesis (11). However, the fact that this activity was not detectable in HeLa cells infected with poliovirus defective-interfering particles (15) or a temperature-sensitive mutant (C. K. Drescher and B. A. Phillips, Abstr. Annu. Meet. Am. Soc. Microbiol. 1979, S89, p. 254; C. K. Drescher, J. R. Putnak, and B. A. Phillips, submitted for publication) strongly sug-gests that the putative morphopoietic factor is not aderepressed or induced host protein.

ACKNOWLEDGMENTS

Wegratefully acknowledge the excellent technical assist-ance of Mary-LouWong-Chong.

This work was supported by Public Health Service grant Al-08368 to B.A.P. from the National Institutes of Health.

ADDENDUM IN PROOF

Recently (Marongiu et al., J. Virol. 39:341-347,

1981), it has been reported that emptycapsids isolated

from poliovirus-infected cells were dissociated into

14S subunits under mild alkalineconditions (pH 8.5)at

4°C. Empty capsids made in vitro by self-assembly of

isolated 14S particles could not be similarly

dissociat-ed. Performing the same experiment on our own types

of empty shells, we found that PRO`1 and E.C.49

were alkali labile, whereas E.C.51% was stable under

the same conditions. The14S particles produced from

the pH 8.5 dissociation of PRO0 31 at 4°C did

self-assemble when the pH was lowered to 7.4 and the

temperaturewasraisedto37°C;atypical self-assem-bled shell, E.C.5%, wasthe only product of such a reaction.

LITERATURECITED

1. Beatrice, S. T., M. G. Katze, B. A. Zajac, and R. L. Crowell. 1980. Induction ofneutralizing antibodies by the coxsackievirus B3 virion polypeptide, VP2. Virology 104:426-438.

2. Bradford, M. 1976. A rapid and sensitive method for the quantitation ofmicrogramquantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248-254.

3. Brown, F. 1978. Structure-functionrelationships in the picornaviruses, p. 49-72. In R. Perez-Bercoff (ed.), The molecular biologyofpicornaviruses. NATO Advanced StudyInstituteSeries,vol. 23. Plenum Press, New York. 4. Carthew, P. 1976. The surface nature ofproteins ofa bovine enterovirus, before and after neutralization. J. Gen. Virol. 32:17-23.

5. Carthew,P., and S. J. Martin. 1974. The iodination of bovineenterovirus particles.J.Gen. Virol. 24:525-534. 6. Fraker,P.J., and J. C.Speck, Jr. 1978.Protein and cell

membrane iodination with a sparingly soluble chloroa-mide, 1,3,4,6-tetrachloro-3a,6a-diphenylglycoluril. Bio-chem.Biophys. Res. Commun. 80:849-857.

7. King, A. M. Q., and J. W. F. Newman. 1980. Tempera-ture-sensitive mutants of foot-and-mouth disease virus withaltered structuralpolypeptides. I. Identification by electrofocusing. J. Virol. 34:59-66.

8. Lonberg-Holm,K., and B. E. Butterworth. 1976. Investi-gationof the structureof polio-andhuman-rhinovirions throughtheuseofselective chemicalreactivity.Virology 71:207-216.

9. Lund, G.A., B. R. Ziola, A. Salmi,andD. G. Scraba. 1977. Structure of themengovirion.V.Distribution of the capsidpolypeptides with respect tothe surface ofthe virusparticle.Virology78:35-44.

10. Markwell, M. K., and C. F. Fox. 1978. Surface-specific iodination of viruses and eucaryoticcellsusing 1,3,4,6-tetrachloro-3a,6a-diphenyl-glycoluril. Biochemistry 17:4807-4817.

11. Murialdo,H., andA.Becker.1978. Headmorphogenesis ofcomplexdouble-stranded deoxyribonucleic acid bac-teriophages. Microbiol.Rev.42:529-576.

12. Perlin,M., and B.A.Phillips. 1975. In vitroassemblyof polioviruses. IV. Evidence for the existence oftwo as-semblysteps in theformation of emptycapsidsfrom 14S particles.Virology 63:505-511.

13. Phillips,B.A.1969. In vitroassembly of polioviruses.I. Kinetics of theassemblyofemptycapsidsand the role of extractsfrom infectedcells.Virology39:811-821. 14. PhUUlips,B. A.1971. In vitroassemblyofpolioviruses.II.

Evidencefor the selfassemblyof 14Sparticlesinto empty capsids.Virology44:307-316.

15. Phillips,B.A., R. E. Lundquist,and J. V. Maizel, Jr. 1980.Absenceof subviralparticlesandassembly activity in HeLacells infected withdefective-interfering particles ofpoliovirus. Virology100:116-124.

16. Phillips,B.A.,D. F.Summers,andJ.V.Maizel,Jr. 1968. Invitroassemblyofpoliovirus-relatedparticles.Virology 35:216-226.

17. Putnak, J. R., and B. A. Phillips. 1981. Differences between poliovirusempty capsids formed in vivo and thoseformed in vitro:arole for themorphopoieticfactor. J.Virol. 40:173-183.

18. Rueckert,R. R.1976.On thestructureandmorphogenesis ofpicornaviruses,p. 131-200. In H. Fraenkel-Conrat and R. R. Wagner (ed.), Comprehensive virology, vol. 6. PlenumPress,New York.

19. Wetz, K., and K. 0. Habermeli. 1979. Topographical studiesonpolioviruscapsidproteins bychemical modifi-cation andcross-linking with bi-functional reagents. J. Gen.Virol. 44:525-534.

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Figure

FIG. 1.extract-assembledassembledreactedhibitorelectrophoresis1:10100°C Effect of trypsin on poliovirus-related particles
Fig. 2C).fragmentonly VP1 was also attacked but was cleaved incompletely, generating a highly stable that was only slightly smaller than
FIG. 4.forcientheatedunheated3H-labeled30%ocentrifugedcoefficients14C-labeled Effect of heat on the sedimentation coeffi- of purified poliovirus procapsids
FIG. 7.asforobtaineddilution;tract-assembledbled(brokenNoteserum described Reactivities of procapsids (Pro681), ex- empty capsids (E.C.t), self-assem- empty capsids (E.C."°:), and heated procapsids line) to specific anti-N and anti-H antisera
+2

References

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