Evidence for the existence of protomers in the assembly of encephalomyocarditis virus.

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CopyrightX 1975 American Society for Microbiology Printed inU.SA.

Evidence for the Existence of Protomers in the Assembly of

Encephalomyocarditis Virus

S. McGREGOR,* L. HALL,I AND R. R. RUECKERT

Biophysics Laboratory and Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706 Received forpublication26December1974

Two capsid precursor subunits, which sediment on glycerol gradients at 13S

and 14S, respectively, have been identified in cytoplasmic extracts of

ence-phalomyocarditis virus-infected HeLa cells. The 13S subunit, which was

detected after a 10-minpulselabel with 3H-labeled aminoacids, containedonly

capsidprecursorchainA (mol wt 100,000). Whenthe 10-minpulselabelinsuch

cells was chased for 20 min, the A-containing 13S subunit in the cytoplasmic

extractswasreplacedbya14Ssubunitcontainingequimolarproportions ofthree

chains: a, y,anda.This(e,y,a)-containing14Ssubunitcouldbe dissociatedinto

6Ssubunitswiththesamepolypeptide composition. Thesedimentation

proper-ties and thepolypeptide stoichiometry ofthesethree precursorsubunits, when

compared with those ofthe 13S,

(8,'y,a)s,

and5S,

(B,y,a),

subunits derived by

aciddissociationofpurified virions, suggest the following structural assignments:

13S, (A) ; 14S, (e,

y,a),;

6S,

(w,y,a).

The molecular weights ofthe individually

isolated capsid chains were determined by gel filtration in 6 M guanidine

hydrochloridetobe: a, 36,000; a, 32,000; ,, 29,500; y, 26,500; and6, 7,800. With

theexceptionoftheb-chain, these values areinreasonable agreement with the

values previously determined

by

electrophoresis on sodium dodecyl

sulfate-polyacrylamide gels. These data support the hypothesis that picornavirus

capsids are assembled from identical protomers

according

to the

following

scheme:

(A)-

(A)5

_(,y,a)s -

(b,f,y,a)o n(evy,a)n

where n is the numberof immatureprotomersper virion.

Picomaviruses contain four nonidentical

polypeptidechains here designateda, ,S, y, and

6 (29). All four chains are generated by

post-translationalcleavageofasingle, large(mol wt

100,000) precursor, the A chain(3, 13, 17, 22).

The virions alsofrequently contain traces ofa

fifth polypeptide,

a,

which is an uncleaved

precursorof

#

and 6chains (3, 13).The

cardiovi-ral(encephalomyocarditis [EMC]virus, mouse

Elberfeld [ME ]virus, mengovirus) chainsA,

a,

a, ,B, y, and6correspondtothepolioviral chains

NCVP-la, VP-0, VP-1,VP-2, VP-3, andVP-4,

respectively (13, 32). We have adopted the

cardiovirus nomenclature which lends itself

more readily to the description of oligomeric

structures.

Cytoplasmic extracts of poliovirus-infected

HeLa cells contain two capsid-related

struc-tures, one sedimentingat

148

and the otherat

73S (25-27). Both contain the capsid peptides 'Present address: Department ofBiology, 16-717, Mas-sachusetts Institute ofTechnology, Cambridge,Mass. 02139.

a,

'y,

aand are thought to be intermediates in

capsidassembly.Inthepresenceof

rough

mem-brane fractions from infected cells, the 14S

subunit can be assembled in vitro into 73S

protein shellswhich are indistinguishablefrom

the RNA-devoid, empty capsids found in

in-fected cells (24, 25, 27). Thekinetics of in vivo

labeling experiments are also consistent with

the postulatedprecursor role ofthese two

sub-units (14, 23).

It has been suggested that the capsid

poly-peptides are organized into two closely related

types of subunits designated "mature proto-mers" (a protomer is defined as the smallest identical subunitofan oligomeric protein [21])

(6,-,',a),

and "immature protomers,"

(w,'y,a)

(29, 30). These hypothetical subunits are

pre-sumably derived from the A chain in the

se-quence: A -

(a,y,a)

-p

(6,M,y,a).

According to the protomer hypothesis, the

immature empty capsid,

by

analogy with the

structure ofthe virion, is

comprised

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McGREGOR, HALL, AND RUECKERT mature protomers, (,

'ya!)60,

whereas the

sedimentation coefficient of the 14Ssubunit is

aboutthat expected for a pentameric structure,

(E,-y,a). (29). However,

the

stoichiometry

re-ported for the polioviral 14S precursor is E

302y5a3

(26).

Acapsid-related antigen, which sedimentsat

14S, has also been reported in extracts from

EMC virus-infected KrebsII ascites cells (15).

Inthis communicationweconfirmtheexistence

of such a 14S subunit in EMC virus-infected HeLa cells and show that it has the propertiesof a precursor, not a capsid breakdown product. We also show that this subunit can be dis-sociated intoasmaller subunit withthe

proper-ties expected of the postulated immature pro-tomer. A second subunit, which appears to be

an uncleaved precursor of the 14S subunit, is also described.

MATERIALS ANDMETHODS

Media. Medium A is Eagle minimal medium in Earle saline supplemented with 0.1 mM nonessential aminoacids and2x 10-5 Minositol (20). Medium F is Eagle minimal medium in Earle saline lacking calcium and magnesium andsupplementedwith 0.1 mM glycine and serine and 0.1% Pluronic F68. Me-dium AH is meMe-dium A containing 25 mM N-2-hydroxyethyl-piperazine-N'-2-ethanesulfonic acid buffer,pH 7.4 (3). Medium AL is medium AHlacking amino acids.

Buffers. When Dulbecco phosphate-buffered sa-line (6) is supplemented with 0.1% bovine serum albumin(BSA), it isdesignated PBSA.Buffer IV is 1 Msodium chloridein 0.02 MTris acetate (pH 7.5). TSAis 0.25Msodiumchloride,0.005M Tris acetate (pH 7.4) containing 0.01% BSA. Reticulocyte stan-dardbuffer (RSB) is 0.01 M sodium chloride, 1.5 x 10-3M magnesium chloride and 0.01 M Tris-hydro-chloride(pH 7.4).

Cells.HeLacells,designatedW-HeLa,were origi-nally obtainedthroughthe courtesy ofG.C.Mueller, Department of Oncology, University of Wisconsin. They were propagated in suspension in medium F supplemented with 6% bovine serum by shaking 300-mlcultures insiliconizedFlorenceflasksat37C. The cultures, which did not require trypsinization, grew with a generation time of 24 h. They were continuously maintainedatthisgrowthrateof serial dilution tokeepthecelldensitybetween 0.5 x 106to 6.0 x 101 cells per ml.

Virus. EMC virus was originally obtained from C. Fuerst,University of Toronto. It had beenadaptedby serial passagetogrowthinSarcoma 180 ascitestumor cells (12). The virus was then plaque-purifiedthree times in HeLa cells. Stock virus was prepared by growing the plaque isolate (hereafterdesignated the F2strain)inHeLa cell suspension cultures. Thestock was concentrated 50- to 100-fold by sedimentation. The pelleted virus wasresuspended and purifiedby isopycniccentrifugationincesiumchloride. Thevirus

suspension was thendialyzed against TSA buffer and stored at -70 C. Theinfectivitytiter of thestock virus was determined by plaque assay on Hela cell mono-layersand was 6.5 x 1011 PFU per ml.

Infection of suspension cultures. HeLa cells were grown in suspension culture in medium F supple-mented with 6% bovineserum. The cells were sedi-mented by low-speed centrifugation, washed once, andresuspendedinmedium AL at a concentration of 4 x 107 cells per ml. The cellsuspensionwas inocu-lated with 100 PFU per cell. After an attachment interval of 20 min at room temperature, the suspen-sion was diluted 10-fold with warm medium AL. Actinomycin D was then added to give a final concentration of 5 gg per ml. The infected cell suspension (4 x 101 cells per ml) was incubated at 37 C with gentle agitation.

Isotopic labeling and purification ofEMC virus. Isotopically labeled amino acids were added to in-fected cell suspensions at 3 h postinfection. Incuba-tion was continuedat37C. At 5 hpostinfection, the cells,containing greaterthan90% ofthe total infectiv-ity, were sedimented and washed once in cold me-dium AL. The virus was released from the cells by freeze-thawing twice. Cell debris was removed by low-speed centrifugation, and the virus waspelleted through a cushionof 30% sucrose in bufferIV. The pellet was resuspended inPBSA by flushingwith a needle and syringe and insoluble material was re-movedbycentrifugation at 12,000 x g for 5 min. The virus in the clarified supernatant fluid was further purified by isopycnic centrifugation in cesium chlo-ride, followed bygel filtration on 6% agarose equili-brated with TSA. The purified virus was stored at -70C.

Glycerol density gradient. Linear gradients of glycerol(wt/wt)containedRSB,0.1% 2-mercaptoeth-anol, and 0.1%BSA. Five-millilitergradients (Spinco SW65rotor) were generatedbythe method of Britten andRoberts (1). Twelve-milliliter gradients (Spinco SW41rotor)werepreparedby layeringfive concentra-tions ofglycerol (30, 25, 20, 15, and 10%)andstoring at6Cfor 6htoallow diffusion. Bothtypesof

gradi-entswerecentrifugedat40,000rpm and had average gravity forces of 114,000 and196,000fortheSW65and SW41rotors,respectively.Allgradientswere fraction-ated by bottom puncture. Radioactivity was deter-mined by scintillation spectrometry in scintillation solventB-10(20).

Dissociationofvirions into 13S and 5Sprotein subunits. Purified EMC virus in TSA buffer was treated with0.1%2-mercaptoethanolat room temper-ature for 10 to 15 min. The reduced virus was dissociated into 13S subunitsby dialysis against0.1 M sodium chloride in 0.05 M sodium citratebuffer, pH 5.7, at 37C for6 h. The 13S subunit was then isolated by sedimentation on a 12-ml, linear, 10 to 30%glycerolgradientat 6C for13hat40,000rpm ina Spinco SW41rotor.

Toprepare 5S subunits, reduced virions were first dissociated at pH 5.7 as described above, and the resulting 13S subunits were then dissociated into 5S subunitsby addingtothedialyzed preparation suffi-cient6Mureatobringthe finalconcentrationto1.5

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M. After 15minatroomtemperaturethe preparation

wasAppliedtoa12-ml, linear, glycerol gradient and

centrifugedat6Cfor 14 hat40,000rpminaSpinco SW41rotor.

Polyacrylamidegel electrophoresis. Electropho-resiswasby amodification (20) of the procedure of

Maizel (16). The gels contained 9.7% acrylamide, 0.3% ethylene diacrylate (vol/vol), 0.1% N,N,N',N'-tetramethylethylenediamine, 0.1%

so-diumdodecyl sulfate (SDS), 0.5 M ureaand 0.1 M

sodium phosphate at pH 7.2. The electrophoresis bufferwas0.1M sodium phosphate, pH 7.2, contain-ing 0.1% SDS and 0.01 M mercaptopropionic acid. Samples to be electrophoresed on SDS gels were

dissociatedat100C for 5 min in 1% SDS, 0.5 Murea,

and 0.1% 2-mercaptoethanol in 0.01 M sodium phos-phate buffer, pH 7.2. Electrophoresiswascarriedout at9 mApertube.

Soft gels, utilized in the purification of high-molecular-weight subunits, contained 5% polyacryl-amide (acrylpolyacryl-amide-bisacrylpolyacryl-amide ratio of 33), 0.5%

agarose, 0.1%

N,N,N',N'-tetramethylethylenedia-mine, and 0.1 M sodium phosphate buffer (pH 7.2). Thegelswerepolymerizedat37Cand then incubated atroomtemperaturefor 1 h beforeuse.The

electro-phoresis buffer contained 0.1 M sodium phosphate buffer, pH 7.2, and 0.02 M mercaptopropionic acid. Electrophoresiswascarriedout at4mApertube.

Allgelswerefractionated withaGilson automatic

gel fractionator (11). Determination of radioactivity in SDS gel fractions by liquid scintillation counting has been described (20).

Determination of polypeptide molecular weights. Theapparentmolecular weights of individ-ually isolated virus peptidesweredetermined by gel

filtration in 6 M guanidine hydrochloride accordingto the method of Fish et al. (10). The virus peptides, labeled with a 'H-labeled amino acid mixture, were

recovered from peak regions of the fractionated SDS-polyacrylamide gels by extraction with 10 gel volumes ofdistilled watercontaining 0.1% 2-mercaptoethanol and 0.1% BSA for 12 to 48 h. The proteins in this extract were carboxymethylated by the following procedure.

Solutions containing the isolated virion peptides

weremixed with marker proteins (never exceeding 20 mgoftotal) ina1-dram vial containing the

vacuum-dried residue from1ml of 6 Mguanidine hydrochlo-ride in 0.5 M Tris-hydrochloride, pH 8.6. After adjusting the volumeto1.0mlwithdistilled water, 4

plof2-mercaptoethanolwasadded and the solution wasincubatedfor 1 h at 45Ctocompletereduction of disulfide bonds. lodoacetamidewasthen added toa

final concentration of 0.15 M and incubation was

continuedat 45Cfor 10 min inthedark.Alkylation

wasterminatedby adding 30 &l of 2-mercaptoethanol.

Aftercooling, 300 Mgof Blue dextran2000(Pharmacia Fine Chemicals, Piscataway, N. J.) and 78 ;g of sodium 2,4-dinitrophenyl-alanine (Mann Research Laboratories, Inc., New York)wereaddedasvolume markers for the column. Gel filtration of 200-Ml sampleswascarriedouton a6%agarosecolumn (75 by 0.9 cm) equilibrated with 6 M guanidine hydro-chloride. The markerproteinswere

spectrophotomet-rically detected usingamicro-flow cell

(Instrumenta-tion Specialties Co., Lincoln,Neb.) withavolume of

50

Al.

The flowratewasmaintainedat1.5mlperh.

Determination of acid-insoluble radioactivity. Samples from glycerol gradients were placed onto Whatman 3MM filter disks and driedat37Cfor4h. The filter disks were washed three times in 5%

trichloroacetic acid at 6C for 10 min. The trichloro-acetic acid was removed by washing twice in 95%

ethanol and thediskswere driedat 100C. The filter diskswerethencounted in scintillation solvent B-10.

Material. Actinomycin D (Dactinomycin) was

ob-tained from Merck, Sharp and Dohme (West Point, Pa.). The 6% agarose (Bio-Gel A-5M) was from

BioRad Laboratories (Richmond, Calif.). The L-[4,5-'H]leucineandL-[U-14C]leucinewerepurchased from

Schwarz/Mann (Orangeburg, N.Y.); the L-[U-14C]-amino acid mixture was from New England

Nu-clear(Boston, Mass.).

RESULTS

Identification oftwocapsid-related protein precursorsubunits.There is evidence that the EMC viral A chain, which is theprecursorof all capsid protein, hasahalf-life of about 7min(4). Accordingly, we sought evidence for the exist-enceofcapsid-relatedstructureswithinthe first

10 min of synthesis. Figure lashows the sedi-mentation profile ofacytoplasmicextractfrom

EMC virus-infected HeLa cells after a 10-min pulse of

[3HRleucine.

Labeling was carried out at3.5 h postinfection when virtually all protein synthesiswasvirus coded. The bulkofthe acid-insoluble radioactivity sedimented as a broad band in the 2S to lOS region with a small shoulder cosedimenting with a 13S marker. When the label insuchpulsed cellswaschased

for 20min, the 13S shoulderwasreplaced with a prominent sharp peak which sedimented 8% faster than the 13S marker, or at about 14S (Fig. lb).

Theelectrophoretic profileofthe 13S and 14S peaks on SDS-polyacrylamide gels is shown in

Fig. 2.The 13Speak, observed after the10-min pulse, contained a dominant peak of A chain andatraceofcapsid-related B chain (3). It also

contained about 12% of the recovered label in E

chain, a noncapsid protein (3), and a trace of label in atleast one other minor, unidentified peak. The 14S peak,observed after the20-min chase, consisted predominantly of capsid-related peptides (E,a,y) and only very small

proportions of A and E chains. These results

raise the possibility that the E chain is a

component of the A-containing 13S peak, in

which case the distribution of the A and E

chains in theglycerol gradientshould coincide. To test this possibility, the distribution of

each chain in gradient la was determined by

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MCGREGOR, HALL, AND RUECKERT

j7e

L

\13S

0

U200204

0 20 4

w

a.

m

1000-0

In

z

0 20 40 0 20 40

FRACTION

FIG. 1. Sedimentation profile of cytoplasmic ex-tracts from EMC-infected cells. (a) Pulse labeled 10min;(b) pulse labeled 10 min and chased20min. Infected HeLa cells (8 x 107) were pulse labeled

with ['H]leucine (100 M&Ci per ml) at 3.5 h

postin-fection. Labelingwasallowed toproceed for 10min at 37C.Half of the cells werethen Dounce homoge-nized in cold RSB and the cytoplasmic extractwas stored in ice. The other half of the culture was sedimented and resuspended in10mlof mediumAH and the 'H label was chased by incubation for an

additional 20 min at 37C. The cells were then

homogenized. Equivalent amounts of both cytoplas-mic extracts wereplaced onto 12-ml glycerol gradients (10 to30%) and centrifuged at 6 C for 13 h at 40,000 rpm in aSpinco SW41 rotor. A [14C]13Smarker (not shown) prepared by acid dissociation of the EMC virion (seeMaterials and Methods) was included in both gradients. Thegradients werefractionated into 14-drop (270,l)fractions by bottom puncture. Sedi-mentation was fromright to left.

analyzing fractions 10 to 20 on SDS-polyacrylam-ide gels. The total radioactivity in the A and E chains in each gradient fraction was

calcu-latedandplottedagainst fraction number (Fig.

3). The small amount of label in the B chain

precluded analysis by this procedure. Also

shown in Fig. 3 are the results of a similar analysis of the 14S, E-containing peak from

gradient lb. The e, a, and y chains inthe 14S

peak allcoincided, supportingthe idea that all

are elements ofa single structure. In contrast, thelackofcoincidenceinthedistributionofthe Aand E chainsinthe 13S region ofgradientla

argues againstthehypothesis thatthe E chain is a component of the 13S peak. A more

complete analysis (not shown) indicated that

the E chain ispartof aseparate butoverlapping

component whichpeaksatabout 7S.

Thesedatasuggest that theAchain is

assem-bled into an oligomeric structure before being

cleavedintothe smallers, a, andychains. After

cleavage, the sedimentation velocity of the precursor increased from 13S to 14S. In addi-tion, it should be pointed out that while the e-containing peak was very symmetrical the A-containingpeak was not, but tended to skew toward the 14S peak (Fig. 3).

Polypeptides in the 2S to lOS region. The monomeric form of the A chain, with a mass of 100,000daltons, would be expected to sediment

atabout6S(29).. A search for such a monomeric

structure inthe 2S to10S region of the gradient (Fig. la) revealed less than 2% of the total radioactivity in this region in A chains. How-ever, after the 20-min chase (Fig. lb), approxi-mately 10% of the total label in the 2S to 10S region was found in e chains. A trace of label was also found in capsid-related peptides in a narrow band at the top of the gradient. The

bulk of the label in the 2S to 10S region was

a 400r

z u 3001-0

LU

Zj E

5o2002

0 50 100 150 200

FRACTION

[image:4.505.62.256.60.261.2]

FRACTION

FIG. 2. Electrophoretic profile of peak fractions from densitygradients inFig. 1. Samples of 200MI were electrophoresed on 20-cm, SDS-polyacrylamide

gelsat 9 mAper tube for approximately18 h.(a)13S subunit(fraction 17) from gradienta inFig.1.(b)14S subunit(fraction 14) from gradient b inFig.1.

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14S

13S

I

1'\

I I'

F

-I

I

0

15 FRACTION

FIG. 3. Distributionof virus-coded peptides inthe 13S to 14S region of sedimentation gradients of cytoplasmic extracts. Gradientfractions from Fig. 1

(a and b) were analyzed by SDS-polyacrylamide gel

electrophoresis. Theamountof each virus peptideper

fraction was calculated and their distributions were

plotted. The values plotted for the A chain and E chainwerefrom (a); the value for theechainwasfrom

(b) (Fig. 1). The distribution ofaand-ychains (not

shown) coincided with that of the e chain. The

maximum values for each peptide were: A, 1,163

counts/min; E, 544counts/min; and e,2,576counts/

min. A chain, *--*; e chain, 0--- 0; and E chain, O--O.

associated with the noncapsid polypeptides E

(molwt56,000),H (12,000),and I(11,000).The Echain sedimentedatabout7S and theHand

Ichainsatabout5S.Theamountof these three

noncapsid proteins inthe2Sto10Sregionwas

increased at least fivefold when the extracts

were treated with 0.5% Nonidet P-40 (NP-40)

beforecentrifugation.

Distribution of A chains in the pellet. In the course of these experiments, 40 to 60% of the total A-chain label in the cytoplasmic extracts

was found in the membrane-rich pellet at the bottom of the glycerol gradients. When cyto-plasmic extracts of pulse-labeled, infected cells were centrifuged on discontinuous sucrose gra-dients (5), 30 to 35% of the A chain was found in the rough endoplasmic reticulum band; an

additional 5% was found in the smooth endo-plasmic reticulum band. In an attempt to detectamonomeric (6S) form of the A chainon

membranes, pellets from glycerol gradients were solubilized with 0.5% NP-40 and sedi-mented on glycerol gradients. A chains were found in the 148 but not in the 6S region. This result suggests that the hypothetical A chain, monomericsubunits are rapidly assembled into the 14S form. Another possibility is that A chains werepresent, but, because of a tendency to precipitate or attach to surfaces, were not solubilizedby NP-40 treatment.

Electrophoretic purification ofcapsid pre-cursors on nondissociating polyacrylamide

gels. When material from the 14S region of a

density gradient (Fig. lb) waselectrophoresed

on nondissociating (no SDS) polyacrylamide

gelsatpH7.2,twocomponentswereobserved, a

largepeak I andaslowerpeakII(Fig. 4). Peak II

[image:5.505.56.251.60.448.2]

wastypically lessabundant than that shown in Fig. 4. To examine the possibility that these peaks might correspond to the A-containing,

13S precursor and the cleaved, 14S precursor

subunits, each component was eluted from the

gel and its polypeptide composition was deter-mined by electrophoresis on

SDS-polyacryla-mide gels.

The electrophoretic profile of peak I (Fig. 5)

revealed three major polypeptides; these comi-grated with the c, a, and y chains of the EMC virion. Sedimentation analysis of peak I on glycerol gradients further showed a single peak

sedimenting at 14S (not shown); hence the

majority ofthe applied 14S material survived

electrophoresis and elution without

degrada-tion. The total lack of E chains in this profile further supports our earlier conclusion that the E chain is not an integral element of the 14S

subunit. In addition, the absence of f chains indicatesthat the 14Ssubunit is a precursor of the virion and not a degradative product.

A similar electrophoretic analysis of peak II revealed93% of the recovered radioactivity in E, a, and ychains (not shown); these chainswere

present in the same relative proportions as in peakI.Tracesoflabel were also foundincapsid precursorchains A (0.7%) and B (0.7%) and in a

100-%

i

lIo

0 LA-z

LJ

0

Lii

a--L50

0

10

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McGREGOR, HALL, AND RUECKERT

1500_

0

1000_

cc I Io

w~~~~~~~~~~~~~

500

10

30

50

[image:6.505.57.252.62.362.2]

FRACTION

FIG. 4. Electrophoretic profile of the intact 14S

precursor subunit on a 5% polyacrylamide gel (no SDS). The 14S subunitwasisolatedfrom cytoplasmic extractsof[3H]leucine-labeledEMC-infected cellsas

described inFig. lb. Afterdialysis against RSB, the subunit was further purified by sedimentation in a

secondglycerol density gradient. The 14Speak frac-tions werepooled andelectrophoresedona5% poly-acrylamidegel containing 0.1 M sodiumphosphate, pH 7.2, and 0.02Mmercaptopropionate but lacking SDS (see Materials and Methods). Electrophoresis

was carried out at 4mA per tubefor 32 h. The gel

wasfractionated and the protein subunitwaseluted into distilled water containing 0.1% 2-mercapto-ethanol. Fractionswereincubatedforatleast 12 hat 6Cbefore sampleswereremovedfordeterminationof radioactivity.

noncapsidchain G (4%).Hence, peakIIcannot contain more than a trace amountofsubunits

composedof Achain. It consists insteadmainly

ofpeakI-related material, possiblyinthe form ofanaggregate, aconformeroreven a

dissocia-tion product. Because of its smaller size, the

mobility ofsuch a dissociation product would

normally be expected toexceed that ofpeakI.

However, similar electrophoretic studies of the

virion-derived 13S, (,y,a)5, and 5S,

(0,,y,a),

subunits(see below) yieldedtheopposite result;

i.e., the 5S subunit migrated slower than the

13S. Such a result could be explained by a

decrease in the isoelectric point of the protein accompanying the dissociation of the 13S sub-unit.

Attempts to electrophoretically purify the A-containing 13S peak from pulse-labeled, cy-toplasmic extracts were frustrated by thesmall amount ofradioactivity available and by seri-ous losses encountered during storage and ma-nipulation of this material.

Dissociation of the 14S capsid precursor

with urea. Figure 6 shows the sedimentation profile of the 14S subunit after a30-min treat-ment with 1.5 M urea at 35 C. Approximately 55% ofthe 14S subunitswasdissociated by this treatment into material which sedimented just ahead of a 5Smarker; this smaller component wasassigned a nominal sedimentation value of 6S. An additional 30% of the radioactivity sedimen,ted as undissociated 14S subunits. The remaining 15% of the material was adsorbed to the centrifuge tube. To minimize the loss of material due to adsorption, the 6S region was collected into siliconized glass vials. When ex-aminedby electrophoresis on SDS-polyacrylam-ide gels, the 6S material exhibited a polypep-tide composition very similar to that of the parent, 14S subunit (Fig. 7; see also Table 4,

below).

13S and 5S subunits from pH-dissociated

EMC virions. ME virus (7) and mengovirus

(18), which are serologically related to EMC

virus, can be dissociated through a 13S-14S intermediate, (,y,a)8, intoprotomeric 5S sub-units,

(B,y,a).

In this section we describe the properties of the analogous subunits derived from pH-dissociated EMC virions (Fig. 8). The

sedimentation profile of the dissociation

prod-uct of

[ICJleucine-labeled

EMC virions (Fig.

8a) was indistinguishable from that of the

(#,,y,a),

subunits from ME virus (data not

shown). Weassumetherefore that the 13S-14S

subunitsderived from EMC viruscorrespondin

structure tothose from ME virus.

The

(#,-y,a),

subunit of ME virus has

previ-ously been assigned a sedimentation value of 14S (7); on this basis, the (e,y,a)-containing

precursorsubunitofEMC virus wasassigned a

valueof15S(29).The sedimentation coefficient

ofthemengovirus

(#,,y,a)5

subunit, determined

by analytical ultracentrifugation, has been reported to be 13.4S (19). In this paper, we

have attempted to simplify the nomenclature

by adopting a nominal value of 13S for the

(#,,y,a),

subunit ofMEvirus, EMC virus, and

mengovirus. This leadsto acalculatedvalueof 14S for the EMC virus capsid precursor (Fig. lb), avaluewhich has beenextensivelyusedin

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500

0 50 100 150 200

FRACTION

FIG. 5. SDS-polyacrylamide gelpattern ofthepurified 14Sprecursorsubunit. The subunit elutedfrom fraction 14 to 15ofFig. 4 was coelectrophoresed with ["4CJEMC virus on a20-cmpolyacrylamide gelat 9 mA pertube forapproximately15h. Arrowsindicate thepositions oftheEMCvirionpolypeptides.

5S E

14S

20 1500X

13S 1200

z

4 ~~~~~~

~9006-L1000

300

0 30

w

O.. 20 50 100 150 200

2 ~~~~~~~~~~~~~~~~~~~FRACTION

a. FIG. 7. SDS-polyacrylamide gelpattern of the6S

~500-

peak fr-om Fig. 6. Electrophoresed on a22-cm poly.

acrylamide gel at 9 mA per tubeforapproximately18 h.

describing the poliovirus capsid precursor,

whichpresumably hasa similarstructure.

The 13S subunit was

completely

dissociated

) 10 20 30 40 into smaller 5Ssubunits by treatment with 1.5

FRACTION M urea at room temperature (Fig. 8b). Its

FIG. 6. Sedimentation profile of the 14S subunit stability differed significantly in this respect after treatment withurea. Thepurified14Ssubunit from that of the 14S precursor subunit which

from Fig. 4was heated at 35Cfor30 minafterthe was only partially dissociated even when ex-additionof 6 M ureatogiveafinalconcentrationof posed to anequivalenturea concentrationfor a 1.5M urea. It wassedimentedon a12-mllinear15 to

35%glycerol gradient at 6 C for14 h at40,000rpmin a longer

time

at a higher temperature (35 C Spinco SW41 rotor. A

[14CJ5S

subunit was included rather than 21 C).

as a sedimentation marker (not shown). A parallel SDS-polyacrylamide gel analysis of the 13S gradient contained a ["C ]13Smarkerpreparedfrom and 5S subunits demonstrated that both sub-EMC virus(notshown). units were

composed

ofa, ,B,and ychains

(Fig.

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a 9). The distribution of

[H

H]leucine

in the

pro-14S

files of the twosubunits was very similar

(see

Table 3).

Stoichiometry of polypeptides in the EMC virion. The number of polypeptide chains of species i (nf),

and

molecular weight (mi), in a

5000_

capsid

of known mass (M) can be calculated

using equation (1) if the relativedistribution of radioactivity

(r,)

and the relative specific

activ-ity

(a1)

are known:

X ni

=

(ai)(ri)

(M)/(m)

(1)

2500 Purified EMC virions, labeled with amixture of

2500 15radioactive amino acids, weredisrupted with

SDS and electrophoresed on SDS-polyacrylam-ide gels. The distribution of radioactivity in each polypeptide peak was determined (Table 1, column 2). Assumingthat all five chains were

labeled to a constant specific activity by the radioactive amino acid mixture, the average

0

10

20 30 40 chain stoichiometry was calculated using

pep-FRACTION tide molecular weights determined by two

dif-ferent methods: (i) electrophoresis on

SDS-polyacrylamide gels, and (ii) gelfiltration chro-matography in 6 M guanidine hydrochloride.

b4S

The

stoichiometry

obtained usingthe

electro-14S

l phoretically determined molecular weights was

f2a60#57769b

45

(Table 1), comparedtoaformula of

,f2a0:5'ys06,s

predicted for a

60-protomer

capsid containing two immature protomers.

500_

Agreement

with the theoretical

stoichiometry

fell well within the 6 to 10% reliability limit attributed to theelectrophoretic method(8, 33), except for the two smallest chains, y(15% high) and6(25% low). Theapparentmolecularweight

0 of the y chain of ME virus, which is

electro-phoretically indistinguishable from that of

250 EMC virus, is known to be sensitive to small

changes in electrophoretic conditions, varying

from 23,000 to 26,500 (8, 29). Anapparently low delta chain content of the virion has also been observed with ME virus and was traced to an

anomalous molecular weight value (30, 31).

Such anomalies are now known to occur

com-0)

10

20 30 40 monly inSDS-gelelectrophoresis with

polypep-0FRACTION

tides smaller than 15,000 daltons (8, 28). To examine this issue with EMC virus, the FIG. 8. Sedimentation profile of the 13S (a) and 5S molecular weight ofeach

polypeptide

was deter-(b) subunits derived

from

the EMC virion by acid minedby gel'filtration chromatography in 6 M dissociation. The 13S subunitwasprepared by

dialyz-ing purified,thiol-reduced,

['4C]leucine-labeled

EMC guanidine

hydrochloride

using

isolated,

virusagainst 0.1M sodium chloride, 0.05 Msodcbm [H

Jleucine-labeled

chainsextractedfrom SDS-citrate at pH 5.7 (see Materials and Methods). The polyacrylamide gels (Table 1). The stoichiome-dissociated virus was sedimented on a 12-mi, linear,

10 to 30% glycerol gradient at 6 C for 12 h at 40,000 temperature. The dissociated virus was then sedi-rpm in a Spinco SW41 rotor. The 5S subunit was also mented on a12-ml, linear, 15to35%glycerolgradient

prepared

from the

purified

virion. After acid dissocia- at 6 C for 14 h at 40,000 rpm in an SW41 rotor.A

tion of the virus at pH 5.7 in0.1M sodium chloride, it ['H114S marker was included inboth gradients (not was further treated with 1.5 M urea for 15min at room shown). Sedimentation isfromrightto left.

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Lu z

I

U"

0)

(-FRCTvv

FRACTION

FiG. 9. SDS-polyacrylamide gelpattern of the 13S and 5S subunits derivedfrom the virion. The peak fractions from Fig. 8 (a and b) were electrophoresed on separate 30-cm polyacrylamidegelsat 9 mA per tube for approximately20h.

[("C

EMCviruswascoelectrophoresedwith eachsubunit(notshown).Arrowsindicate the positions of the EMC virionpolypeptides.

[H'H]13S

subunit(---);

['H]5S

subunit (--).

TAmLE 1. Molarproportionsofpolypeptide chains in EMC virions

SDS-gelelectrophoresis 6MGUHCLgelfiltrationb Peptide radioactivity(%)a Apparent No. of chains Apparent No. of chains

molwtC perviriond molwt perviriond

1.1±0.1 40,000 1.6 36,500 200 1.7

a 35.0 ± 0.1 34,000 59.7 32,000±100 63.4

0 29.3 ± 0.2 30,000 56.6 29,500±200 57.6

ly 27.5 ± 0.3 23,000 69.3 26,500± 150 60.2

a 7.0 0.1 9,000 45.1 7,800 ± 150 52.1

aThe averagevalues (4 averageerror)of three determinationsutilizing purifiedEMCviruslabeledwitha mixtureof 15"'C-labeledaminoacids.Thestandarderror ofcountingwas3% for echain.

bTheapparentmolecularweightsweredeterminedbygelfiltrationchromatographyofisolatedpeptidesin 6 Mguanidinehydrochloride(GUHCL).Theaverageerrorsgiven indicatetheprecisionof two orthree separate determinations.

cFromButterworth et al. (3).

dThe number of chains per virion was calculated using equation(1) (see text).

trycalculated with thesemolecularweight

val-ues was

(*63058n7062.

The molecular weight

value required to obtain a value of 58 delta chains per virion from the radioactivity

distri-bution data is 7,000 rather than the value of

7,800actually found. These values can be

com-pared with similarly determined values for the

delta chain: EMC virus, 6,800 (2); ME virus and mengovirus, 7,200; and poliovirus, 7,600 (31). Using the average molecular weight value

of all these determinations, 7,300, yielded a

calculated delta chaincontentof56per virion.

There is good agreement between the chain

stoichiometry calculated bythesemethods and

that expected for a 60-protomer virion. This is

possibly fortuitous considering the 7 to 10%

reliability limit attributed to the gel filtration

method ofdetermining molecularweights, and theuncertainty ofthe errorinvolved in

assum-ing the polypeptides are labeled to a constant

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specificactivity by the amino acid mixture (10). Inaddition, there is also goodagreementonthe mass of the capsid protomer calculated by different methods: 100,000 daltons for the A

chain (3); 97,000 and 96,000 daltons for the immature (e + 'y +a) andmature(6 +,B+ y+

a) protomersusing themassvaluesdetermined by SDS-gel electrophoresis; and 95,000 and 96,000 daltons for the immature and mature protomersusing themassvalues determined by gel filtration chromatography.

The nonintegral amounts ofe chain (1.6 and

1.7)observedintheseexperimentsdonot

neces-sarily reflect a 15 to 20% error in a true

stoichiometry value of 2. A nonintegral value could be attributedtoaheterogeneous

popula-tion of virions containing 0, 1, 2, 3 or more

immature protomers (7). For example, a value of 1.6 might represent a mixture of virions,

40% containing one immature protomer and

60% containing two. In this case, the average stoichiometrywouldappeartobe

E,.,a60#68.d860-Distribution of leucine residues in capsid polypeptides. [3

H]leucine

is incorporated into

EMC viral protein without significant conver-sion to other amino acids (D. Omilianowski, personalcommunication). Therefore,the

distri-bution of label intheelectrophoretic profilesof

eachof thecapsid-precursorandcapsid-product subunits could be used to measure their chain

stoichiometry if the leucine content of each

capsid chainwere known. These valuescan be

computedfrom theradioactivity profileof puri-fied 3H-labeled leucine virions by assuming thepolypeptide stoichiometry predicted bythe

60-protomer model.

The capsid protein of EMC virus is reported

to contain 8.3 mol% leucine (9). Assuming a

mass of 111 daltons per average amino acid

residue (29), the leucine content ofEMC virus protein iscalculated to be (0.083/111 =) 7.5 x

10-4mol/g ofprotein.Hence, aprotomerwitha massof96,000daltons would contain about (7.5

x 10-4x 96,000 =) 72leucine residues anda

60-protomercapsidabout (60x 72=)4,300leucine

residues.

The leucine contentof each chaincannowbe

calculated fora capsid of knownstoichiometry (Table 2). For example, the number of leucine residues in a virion complement ofa chains is

(0.330 x 4320 =) 1,430, or (1,430/60 =) 24

leucine residues per a chain. The results of

similarcalculations for each chain in the virion are summarized in Table 2 for a 60-protomer

capsid containing (n) immature protomers,

wheren = 2 andn = 3. The calculatedleucine

contentofa, 13, y, and6chains isnotaffectedby

the variation in (n).However, in thecaseofthe

echain,the number ofleucineresiduesis45ifn

= 2, but30ifn = 3.The latter valueagreeswith

theleucinecontentof plus6chains, whichare

the cleavage products of the e chain. This

suggests that n = 3, i.e., the average virion in

thispreparation contained three echains.

The alternative interpretation, that n = 2,

would require the loss of 15 leucine residues during the cleavage of theechain.This hypoth-esisrequiresthat themassoftheechain exceed thatofitscleavage products (13 + 6)by atleast 1,700daltons (the mass of15leucineresidues).

Adiscrepancyofthismagnitudemight conceiv-ably go undetected since the estimated uncer-taintyofthe molecularweight determinationis

on the orderof 10%, or 43,600daltons, for the

epsilon chain.However, such asequencewould

TABLE 2. Distributionofleucineresidues in thepolypeptides ofEMCvirus

Two e chains per virion Threeechains per virion

Peptide Recovered No. ofleucine No. ofleucine

radioactivity (%)a No.of chains residuesper No. of chains residuesper pervirionb chainc pervirion" chainc

2.12+0.04 2 45.5 3 30.0

a 33.01+0.23 60 23.8 60 23.8

ft 30.7240.37 58 22.9 57 23.3

y 25.18 0.18 60 18.2 60 18.2

a6 I 8.98+0.12 58 6.7 57 6.8

aThe average values(4+ averageerror) oftwodeterminations utilizing

[(H

lleucine-labeled

EMC virus. The

chainswereseparatedon SDS-polyacrylamide gels.

bAssuming60protomerspervirion,including(n)immatureprotomers (e,-y,a)and(60-n)matureprotomers Ml,$,y,a).

cThe number of leucine residues per chain was calculated by dividing the product of the total leucine residuesinthe virion and the fraction of['H

#leucine

in aparticularpeptidebythenumber of chains per virion. Itwasassumed thatthe virion contained 4,320 leucine residues.

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havetobeveryleucine rich(>50%) in orderto

have such a small mass. Further evidence against this hypothesis isdiscussed below.

Peptide stoichiometry in capsid-related subunits. Electrophoretic profiles of [3H Jeu-cine-labeled 13S,

(f,,y,a),

, and 5S,

(M,y,a),

subunits(Fig. 9), derived by acid dissociation of EMCvirions, providedveryreproducible distri-butions of radioactivity. These aresummarized in Table 3 (columns 3 and 5, respectively).

These data were used to calculate the molar

stoichiometry of the polypeptide chains assum-ing that the a chain contained 24 leucine

residues; the ,B chain, 23; and the y chain, 18

(Table 2, column 6). The chainstoichiometry of the 13S and 5S subunits fell within a few

percent of the theoretical equimolar ratios (Table 3, columns 4 and 6). These results strongly reinforcedourconfidence in the values determined for the leucine content of each

virion chainandin theability of this methodto determine chain stoichiometry in capsid sub-units withconsiderable precision.

It should be noted here that the calculated chainstoichiometry dependsonthedistribution

ofleucine in the subunit and inthe virion, but

not onthe gross leucine contentor massofthe

average amino acid residue in the capsid

pro-tein. Thus an error inthe value of0.083 mol% used for the leucinecontentofthevirioncapsid

orinthe value of111forthemassoftheaverage

amino acid residue would change the apparent

number of leucine residuesperchainbutnotthe

relative chain stoichiometry.

Thechain stoichiometry of the14Sprecursor subunit and the 6S subunit was determined assuming the e chain contained 30 leucine residues (Table 2, column 6). The ratio ofall threechainsinthe14Ssubunit fell within 2% of

theequimolar values predicted by theprotomer TABLE3 3. Distributionof['H]leucineinthe13S and 5S subunits derivedfromthe virion

13Ssubunit 5S subunit

Peptide Fraction of leucine Recovered Recovered

residues' radioactivity Molarratioc radioactivity Molarratioc

(%)b (%)d

a 24/65 36.3 L0.7 0.98+ 0.02 36.3 + 0.8 0.98 + 0.02

0 23/65 36.1+0.5 1.02+ 0.02 35.9+ 0.8 1.01 + 0.02

ly 18/65 27.6+1.0 1.00+0.04 27.8+0.3 1.00+0.01

aFrom Table 2,

last

column, assuminga(#,-y,a)protomercontains 65 leucine residues.

bThe average values (+ average

error)

of four determinations utilizingthepurifiedsubunit labeled with

['H leucine.The 13S subunitwaspreparedby dissociating thepurifiedvirionasdescribedinFig.8.Thechains wereseparatedonSDS-polyacrylamide gels (seeFig.9).

cThe relative molaramountofeach chain (n) wascalculatedfrom the relationn = r/m, where(r) isthe fractionofradioactivity and (m) isthe fraction ofleucineresidues in thechain (column 2).Forexample, the numberof achains wascalculatedtobe0.363 . 24/65 = 0.98.

dThe average values (+ averageerror) of five determinationsutilizing thepurified subunit labeled with

['H leucine.The5Ssubunitwasprepared by dissociatingthe13S subunitasdescribedinFig.8.Thepeptides

[image:11.505.52.448.504.604.2]

wereseparatedonSDS-polyacrylamide gels (see Fig. 9).

TABLE 4. Distribution of['H]leucineinthe14Sprecursorand its 6S subunit

14Ssubunit 6S subunit

Peptide Fraction of leucine Recovered Recovered

residues' radioactivity Molarratioc radioactivity Molarratioc

(%)b (%)d

30/72 42.4 0.6 1.02+ 0.02 44.1+1.0 1.06 0.02

a 24/72 32.6+ 0.6 0.98+ 0.02 33.1 + 0.5 0.99+ 0.01

y 18/72 25.0+ 0.3 1.00+0.01 22.8 ± 0.6 0.91±0.03

aFromTable 2.

bThe average values (i average error) of three determinationsutilizingthepurifiedsubunit labeled with

['Hleucine.

ThechainswereseparatedonSDS-polyacrylamide gels (see Fig. 5).

cDescribed in footnote c, Table 3. For example, the number of e-chains was calculated to be 0.424 30/72 =

1.02.

dThe average value (i average error) of four determinations utilizing the purifiedsubunit labeled with ['H eucine. Thechains wereseparated on SDS-polyacrylamide gels (see Fig. 7).

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hypothesis (Table 4, column 4).

However, thepeptideratio of the 6Ssubunit,

which had been generated by dissociating the 14S subunit with urea, deviated significantly from the equimolar values expected for an

immature protomer (Table 4, column 6). Nor-malizing to the e chain yielded a ratio of 1.00:0.93:0.86 for the e, a,and ychains, respec-tively. Thisdiscrepancycould be accounted for by partial degradation of about 15% of the immature protomers. Such degradation would notbe surprisinginview oftherelativelyharsh urea treatment required to dissociate the 14S precursor intothe 6Ssubunit (see above).

DISCUSSION

The results reported here reveal a satisfying internal consistency between the chain ratios predictedbythe protomerhypothesisand those actuallyfound for the virion and its four related subunits. Moreover, the finding of exact (i.e., within a few percent) equimolar stoichiometry

inthe 14S precursorsubunit, using the leucine compositions calculated for each chain in the virion, justifies a previously unstated, but fun-damental, assumption.Thatis, the 14S

precur-sor subunit loses little, if any, mass (leucine

13S

14S

(A)~~~~~~~~~~o (A5 -*

Or')5

((,

CAPSID ASSEMBLY

VIRION

CAPSID DISSOCIATION

S9

r.

a)S

': r.

a)

<

5S

13S

residues) during or after the maturation cleav-age to form the virion.

In fact, leucine might have been lost during maturation in at least two different ways. One, suggested by the result in Table 2, is by elimination of a small, undetected, leucine-rich fragment from the e chain when it is cleaved to formthef, and 6 chains. Another way is by loss of chains, e.g., delta chains, from the capsid during maturation or purification of the virions.

In either case, the apparentleucine content of

theechain,calculatedbysumming thenumber

ofleucine residuesinthe , and 6 chains, would have been low. This would have resulted in a high estimate for the relative number ofechains in the immature protomer. The fact that this did not occur allows us to make two conclusions:

(i)the leucine content of the , and 6 chains is a good measure of the leucine content of the e chain; and (ii) the purified EMC virion prepara-tion did contain the assumed full complement ofeach chain. The latter is reinforced by a sim-ilarconclusion drawn previously in studies with ME virus (31).

Our current view of the early stages of capsid assembly, and the structural relationship of the subunits involved, are summarized in Fig. 10.

6S

_-- r

a)

RNA

,y,

a

)2

-3

RNA

's+ '

.a)(:

a)4

PRECI

PI

TATE

FIG. 10. Model for theassembly and dissociation of the EMC virion. The capsid precursor protein, A, is assembled into 13Ssubunits, whicharethoughttobepentameric oligomers. Cleavageofeach A chain at two specific processingsites then follows. The8%increase insedimentationvelocity attending this processing step suggests aconformational change preceding assembly of14Ssubunits into the 60-protomer capsid. The 14S precursorcould be dissociated with 1.5 urea into a 6S subunitcontaining approximatelyequimolar proportions ofe, y,andachains andfitsthedescriptionofanimmature protomer. EMCvirionscanbe dissociated into 13S and 5S subunitsindistinguishablein sedimentationvelocityandchainstoichiometryfromthose of ME virus. Hence, the dissociation pathway appearstofollow that proposed previously for the serologically related ME virus(7).

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Also shown are thesubunits which are derived

by dissociation ofthevirion.The assignmentof

a pentameric structure to the 13S and 14S

precursorsubunitsisstilltentativeand isbased

on measurements of sedimentation position. Theseindicate that the14Sprecursorsediments

8% faster (Fig. lb) than the

(B,y,a),

subunit,

which has a known mass of about 425,000 daltons (7). The molecular weightsofparticles ofsimilarshapeanddensityarerelated to their sedimentation velocities by the relationship

S1/S

2 =

(MJ/M2)

213or, inrearrangedform,M2 =

Mi(Si/S2)- /2. Assuming thegradient is

approxi-mately isokinetic for the two particles, the mass ofthe14S precursor subunit is calculated to be 480,000 daltons. This is exactly the mass ex-pected for a pentamer of 96,000-dalton proto-mers.

However, a similar calculationyields amass ofonly 425,000 daltons for the 13S,

A-contain-ing precursor. Since cleavage processing is

un-likelytoincreasethemassordensityofthe13S

subunit, the 8% increase in the sedimentation

velocity accompanying its maturationevidently

reflects adecrease in the hydrodynamic radius. This in tum implies a substantial conforma-tional change. In view of the surface changes

implied by marked differences between the antigenic determinants of the 14S, 73S, and virion ofpoliovirus (reviewed in reference 29),

the occurrence of such conformational changes

is not unprecedented. Indeed, it now seems likely that substantial rearrangements of sur-face structure may accompany each step in picornavirus assembly.

A monomeric form of the capsid precursor,

polypeptide A, which is knowntobeaprimary

gene product in infected cells, would be

ex-pected to sedimentat5Sto6S(30).However,a

search for such a form, or for its cleaved

derivative,

(,,y,a),

was negative. It is possible

that the monomeric A chain, which is thought

to be synthesized on the rough endoplasmic

reticulum, is assembled on the membrane

be-fore its release in thesoluble 13S form orhasa

rate of assembly which is rapid enough to

precludeappreciableaccumulation of the

mono-meric form. Absence of the

(e,-y,a)

monomer suggests that polymerization of the A chains may be prerequisite for chaincleavage.

Ithas been suggestedthateachpicornavirus

protomer requires two different types of inter-subunit bonding regions to accomplish capsid

assembly (30). Ordering of atwo-step bonding

sequence might be controlled by adelay

infor-mation of the stereospecific, complementary surface configuration required for the second

bindingstep until the productofthefirststep is

complete. We suggest that the conformational

shift accompanying the cleavage of the 13S subunit may representjust such an unmasking of a second bonding site, and that the double cleavage of the A chain may berequiredtoallow sufficientconfigurational change to accomplish

thisunmasking.

ACKNOWLEDGMENTS

This research wassupported bygrantVC 26C fromthe American CancerSociety.R.R.R. holds aFacultyResearch award(PRa-106)from the American CancerSociety.

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