Differences in the subpopulations of the structural proteins of polyoma virions and capsids: biological functions of the multiple VP1 species.

0022-538X/81/010080-12$02.00/0

Differences

in

the

Subpopulations

of the Structural Proteins

of

Polyoma

Virions and Capsids: Biological

Functions of

the

Multiple

VP1 Speciest

JOSEPH B.BOLEN, DAVID G. ANDERS, JANINETREMPY,AND RICHARD A. CONSIGLI* Section of Virology and Oncology, Division of Biology, Kansas State University, Manhattan,Kansas 66506

The structural proteins of polyoma virions and capsids were analyzed

by

isoelectricfocusingand sodium

dodecyl

sulfate-polyacrylamide

gel

electrophore-sis. Polyoma virion

VP,

wasfoundtobecomposedof six distinctspecieswhich

had pI'sbetween pH 6.75 and 5.75. Polyoma capsid

VP,

was found tocontain

fourspecieswithpI'sbetweenpH6.60and5.75.Thedifferent formsofvirionand

capsid

VP,

appeared to be generated by modifications (phosphorylation and

acetylation) oftheinitial translationproduct. The most basic of thevirion

VP,

species (pI, pH 6.75) was absent in capsids and was found to be exclusively

associated with the viral

nucleoprotein complex.

Three of the virion

VP, species

and three of the

capsid

VP, species

were found in capsomere

preparations

enriched forhexonsubunits.Two

VP, species

were

specifically

immune

precipi-tated from virionswithhemagglutination-inhibiting antibodies. These two

VP,

species were common to both virions and capsids. Polyoma virions, but not

capsids, possessed a

single

VP,

species which was immune precipitated with

neutralizing antibodies. Bothvirionand capsid VP2werefound tohave pI'sof

approximately pH5.50.Virion VP3 hadapIofapproximatelypH 7.00, whereas

capsid VP3had apIofapproximately pH6.50.

The late region ofpolyoma virus DNA

con-sistsof2,366 basepairswhich code for thevirus

structuralproteins

VP,,

VP2,and VP3 (9). The

major capsid protein

VP,

is encoded at the 3'

end of the lateregion and is translated froma

16SmRNA,whereas the minorcapsidproteins

VP2 and VP3 are encodedat the 5' end ofthe

lateregion andaretranslated from 19S and18S

mRNA's

(10).

These threeproteins comprise the

structural unitswhichareresponsiblefor

main-taining thestructuralintegrityofthevirus,but

theyarealsoessentialasreceptor sites for

infec-tionofhost cellsandashemagglutininsfor

ag-glutination of erythrocytesand areimportant in

DNApackagingand virusassembly.

In a previous report from this laboratory it

wasdemonstratedthatpolyoma capsids,which

are devoid of DNA and histones, did not com-pete with polyoma virions for specific binding sites on thesurfaceof mousekidneycells(MKC)

and were unable to inhibit virion infection of

MKC. Capsids could,however, block virion

ad-sorption toguineapig erythrocytes (GPE). Vir-ion adsorption to and infection of MKC were found to beindependentof theability of virions

toagglutinateGPE (2).Morerecently,wehave

been able to separate neutralizing antibodies

t Contribution no.81-60-j from theKansas Agricultural ExperimentStation,KansasStateUniversity.

from

hemagglutination-inhibiting

antibodies in

antisera directed

against

the

major

structural

proteinVP1 (3).

Itis clear that themajorvirusprotein

VP,

of

papovavirusesdoesnotrepresent asingle

poly-peptidebutrather is composed ofanumberof

polypeptide species

which have different pI's

(11, 16, 17). However, thefunctionsofthe var-ious

VP1 subspecies

have remained obscure.

Throughthe useof theantisera described above,

along with our understanding of the

biological

and biochemical differences between polyoma

virions andcapsids, we have nowbeen able to

assign functionstothedifferent species of

poly-oma

VP,.

MATERIALS AND METHODS

Celland viruspropagation. The preparationof

primary cultures ofMKC has been described (18).

Wild-type large-plaque polyoma virus was used to

infectcellsat amultiplicityofinfection of 10. Infected

cultures were maintained in serum-free

Dulbecco-modifiedEaglemedium(13).

Viruspurification. Virionswerepurifiedfrom the

infected-celllysateasdescribedpreviously(13). CsCl

gradients used to purify the virus were prepared as

describedby BrunckandLeick (8)and were described in greater detail previously (6, 7). Polyoma capsids

were purified from infected-cell lysatesasdescribed

previously (3).

80

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FUNCTIONS OF POLYOMA VP, SPECIES 81

Preparation of radioactively labeled virus. Purified polyoma virions orcapsids were labeled in vitrowith'25I,usingchloramine-T or the Enzymobead

method asdescribed previously (3). The preparation

of'H-amino acid-labeled polyoma virus has been de-scribedpreviously (14), as has the preparation of '2P-labeled virus (4). [14C]acetate-labeled virus was pre-pared by maintaining the infected cell cultures in serum-free culture media with 5pCi of['4C]acetate

per ml.Methyl-3H-labeledviruswas prepared by main-taining the infected cell cultures in serum-free, methi-onine-free Eagle medium with 20uCi of S-[methyl-'H]adenosyl-L-methionine.

Isolation of 48S DNA-protein complex and 18S, 12S, and 5S capsomere subunits. Radioactively labeled virions or capsids were incubated with

ethyl-ene glycol-bis(l-aminoethyl ether)-N,N'-tetraacetic acid (EGTA) and dithiothreitol (DTT), and the virus corecomplex and capsomere subunits were recovered from 5 to 20% or 10 to 30% sucrose gradients as describedby Bradyetal.(7).

GuHCI chromatography. Radioactively labeled polyoma virions were treated with 6 M guanidine-hydrochloride (GuHCl) and0.01 Mmercaptoethanol

asdescribedpreviously (4). The virion proteinswere

thenseparated bygelfiltrationthroughSephacryl

S-300 atpH5.0.

Immuneprecipitation. '25I-labeled,

GuHCl-puri-fied VP1 (100pl) wasincubated with25p1of

antihe-magglutinin,antireceptor, ornormal rabbit immuno-globulin G,allat 10pg/ml,for4hat room

tempera-ture. Afterincubation, 25p1of normal rabbit immu-noglobulin G (10 mg/ml) wasadded, followed by50

pl of goat (immunoglobulin G fraction) anti-rabbit

immunoglobulin G (20 mg/ml). The mixtures were

then incubated overnight on ice, and the immune precipitates were pelleted by centrifugation and washed three times in 10mM Trisbuffercontaining 0.25% TritonX-100and 0.15M NaCl(pH 7.8).This

protocolwasdevelopedfor immuneprecipitation re-actions in which both the antigen and antibodyare present inlowquantitiesand avoids theuseof

Staph-ylococcus aureus protein A, which leads to large

amountsofnonspecific precipitationofpolyomaVP,

(J.B.B.,unpublished data).

Two-dimensionalIEFand SDS-PAGE. Electro-phoresiswas done bythe method of O'Farrell (15). Radioactively labeled virions or capsids were

dis-rupted in2% sodium dodecyl sulfate (SDS) and 5%

mercaptoethanol for2minat90°C.Aftercoolingto roomtemperature,solidultrapureurea wasaddedto 10M, Nonidet P40wasaddedto 2%, pH 3.5 to 10

ampholines wereaddedto 2%, andmercaptoethanol

wasaddedto5%. Theampholinesusedfor the isoelec-tricfocusing(IEF;combiningpH3.5to10andpH5to 8ampholines)yieldedveryshallowpH gradients be-tweenpH5and8.pI'sweredeterminedbyslicingthe

focusing gel immediately after electrophoresis and

measuringthe pH of the individual gelsegments in distilledwater. FortheSDS-polyacrylamide slab

gel

electrophoresis (PAGE) (when second-dimensional analysiswasrequired),the

equilibrated

IEF tube

gel

wasembeddedinagarose abovethestacking gel.

Al-ternatively, the focusinggels containing

l"I-labeled

samples were first sliced into 2-mm segments, the

radioactivity of each segment was determined by counting in a gamma counter, and the peak segments were then placed in wells of a conventional SDS-polyacrylamide slab gel as described previously (7).

Quantitative assays. Cesium chloride densities were determined bythe refractive index with a

refrac-tometerand calculated by using the equation of Vin-ograd and Hearst (19). Protein concentrations were determinedby theBio-Rad protein assay, using bovine

serumalbuminas astandard. Radioactivity was quan-titated in atoluene-Triton (3:1)scintillation fluid with

aBeckmanLS-233 liquid scintillation counter. When

'Hand14C or'Hand32P isotopes were counted in the samesample, the results were corrected for the overlap of'4Cand'2Pinto the'Hchannel and for quenching. Samples labeled with fl-emitters were prepared for counting by incubating the slices with Protosol (New England Nuclear Corp.) scintillation fluid (1 liter of toluene,4g ofOmnifluor, 30 ml of Protosol; 5 ml per vial) for24 hat37°C. The vials were cooled to room temperature beforecounting.

RESULTS

IEF of

"2'I-labeled

virions and capsids.

IEF ofradioactively labeledpolyoma virions and

simianvirus40(SV40)virions has been reported

(11, 16, 17).However, in these reports the virus

proteins werelabeledby allowinginfected cells

toincubate in the presence of14C-or

:H-amino

acids,

""Pi

or [;5S]methionine. We were inter-ested in determining the pattern of IEF of in vitro

chloramine-T-'251I-labeled

polyoma virions

and comparing the polypeptide distribution of

thevirionswithsimilarly labeled polyoma

cap-sids. This type of in vitrolabeling, whichlabels

all of the virion andcapsid

polypeptides,

yielded

specific activities generally in the range of5 x

106

to 10 x 106 cpm/,ug of virus protein using

liquidscintillation spectroscopy and 1 x

108

to

5 x

10'

cpm/,ugofvirus protein using a gamma

counter to quantitate the radioactivity. Figure

1A shows the IEF distribution of

'25I-labeled

virions. Atotalofeight polypeptide specieswere

resolved by

slicing

the IEF tubegels, counting

the segments inagamma counter,andresolving

the portions corresponding to the peaks in an

SDS-polyacrylamide

slab

gel.

Virion

VP3

was

foundtohave pI ofpH 7.0, and virionVP2had

apI of5.5.Sixspecieswithmolecularweightsin the VP, range (42,000 to 45,000 [42Kto 45K])

werefound andwere

assigned

letterdesignations

A through F. The most abundant virion VP,

species (C) had a pl of pH 6.3, which is in agreementwith the results of Hewicketal. (11).

Our study revealed that the pI's of the other

VP, specieswere as follows: A,pH 6.75; B, pH

6.5; D, pH6.1; E,pH5.9; F, pH 5.75. Figure 1B

shows the IEF distribution of

'25I-labeled

cap-sids. Only five polypeptides species were

re-solved. Two-dimensionalanalysisof these

poly-peptidesrevealed thatcapsid VP2hadapI

iden-VOL. 37,1981

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82 BOLEN ET AL.

10 20 30 40 50 1

GEL SLICE

A

10 20 30 40 50 60

GEL SLICE

FIG. 1. IEFof1251-chloramine-T-labeled virions andcapsids. Equalconcentrationsof iodinated polyoma virions (A) orcapsids (B)weresubjected toIEFasdescribed in the text. Thegelsweresliced into2-mm segments,and theradioactivityin each slicewasquantitatedinagammacounter. Themolecularweights of eachof the peak fractionswereestimatedbysubsequent electrophoresisinanSDS-polyacrylamide gel, using

iodinated virionproteinsasstandards.

tical tothat ofvirion VP2, whereas capsid VP:}

hadapI ofapproximately 6.5.FourcapsidVP,

species were found (designated A' through F)

with thefollowingpI's: A', pH 6.6; B, pH6.5;D, pH 6.1; andF, pH5.75.Theletter designations

for the capsid VP1 specieswere given as those

corresponding to species found in virions. The only capsid VP, species which did not have a

correspondingvirion VP1 species was found at

pH 6.6 and was designated A'. These results

show thatpolyoma capsidslackthree oftheVP1

species found inpolyoma virions (A, C, and E)

andpossessaVP3withan alteredpI.Itshould also be noted that under thepHrangesused for these experiments, polyoma histones did not enterthe IEFgels.

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VOL.37,1981~~FUNCTIONSOF POLYOMA

VP,

SPECIES 83

Two-dimensional

analysis

of

"'I1-labeled

virions and

capsids.

Conventional O'Farrell

(15)

two-dimensional

electrophoresis

of

"'M5-a-beled virion and

capsid

preparations

is shownin

Fig.

2.Whenthe

autoradiographs

were

exposed

forshort

periods (Fig.

2Aand

C),

the

VP,

sub-species

could be observed with some success.

However,

it is clear that

during equilibration

of

the IEF

gels,

diff-usion of the focused

species

occurred which

prevented

the observationof the

distinct

patterns

observed whenthe

single

IEF

dimension was

analyzed.

This

problem

was

un-doubtedly

complicated by

the

1"5I labeling

of the

proteins,

whichtends to

fog

the film

surrounding

theactual

polypeptide

spot.

L-onger

exposureof

the

autoradiographs clearly

showed the

migra-tionofthe minor

proteins

VP2

andVP3

(Fig.

2B

and

D).

Both virion and

capsid

VP2 and VP:3

migrated

as doublets in the

autoradiographs

shown.

However,

the

degree

of

separation

of the

VP2

and

VP3

doublets varied

considerably

be-tween

experiments. Again,

thealteration in the

migration

of

capsid

VP3 in IEF dimension can

beseen.

Differential

labeling

ofvirion and

capsid

proteins

with

"'I1. Experiments

were

per-formedto compare theIEF

patterns

of virions

and

capsids

labeled in vitro

by

the chloramine-T and

Enzymobead

techniques.

In vitro iodina-tion of virions and

capsids

with chloramine-T labels both internal and extemnal

proteins.

In

vitro iodination of these virus

particles

with

Enzymobeads,

however,should

only

label

exter-nal

proteins

with

exposed tyrosine

residues.

In-deed,

preliminary experiments

showed that

io-dinationof virions withchloramine-T

efficiently

labeled virioncapsomeresand the

DNA-protein

core, whereasiodination with

Enzymnobeads

la-beled

only

the virion capsomeres (data not

shown). We were therefore interested to see

whateffect thesetwotypesofiodinations would haveonthe IEF distribution of virion and

capsid

proteins.

Comparing Fig.

3A and B, it can be

seen that virion

VP,

species

Awasnot labeled with

Enzymobeads.

Notethat in this

figure only

the distribution of VP1

species

is shown.

Fur-thermore,theratio of

species

Ctothe

remaining

species

was

drastically

decreased after

Enzymo-bead iodination, whereas the relative levels of

species

B, D, E, and F were not

substantially

altered. When thesame

experimnents

were

con-ducted with

capsids, only

minor alterations in the ratios between the VP1

species

were ob-served

(Fig.

30 andD).Theseresults

suggested

ISOELECTRIC

fOCUSING

*lo

A a

0

vo VP1 C

0

yp3 VP3~*VP3

FI..IEadSD-PE

f

hormie T idnaedvrin n

cpsdprten.

ocsngwan0h

horiontldmenionwit acdit

inreaing romlef torigt.

SS-PGE as nteveticl dmenion

Aferth

irt-ienioa

fcuin,th gl

wreeuiibatdan

eetrphrse

i teseon imnso

asdscriedyO'arrll

(5).(A)

utordioraphof olyoa cpsid aftr

a4-h

xpoure;(B)autoadi

ogrphofolom casdSfe

4hepsr;()atrdorp

foym

iin

fe

xoue

(D)autradogaphofplyma

irinsaftr

a24heposre VOL. 37,1981

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84 BOLEN ET AL.

i

a

!r4

IL

*1

L

S

45

.4

SOI

ao 30

9-r as d%

GEL SLICE GL SLIU

FIG. 3. IEFof chloramine-T- andEnzymobead-iodinated virionsandcapsids. Virionsand capsids were

labeled in vitro with either the chloramine-TortheEnzymobead technique. Thesamples were then analyzed

byIEF and SDS-PAGE as described in the text. (A) Chloramine-T-iodinated virions; (B)

Enzymobead-iodinatedvirions;(C)chloramine-T-iodinatedcapsids; (D) Enzymobead-iodinated capsids.Only the positions

of virion and capsidVP,speciesareshown.

that virionVP, subspeciesAandpossiblysome

ofspeciesCwereinternal in the virionstructure.

Since suchdiscrepancieswerenotobservedwith

capsids,itwasthoughtlikelythatpolyoma

cap-sids harbornointernal proteins. Capsid VP2 and

VP:i,

likewise, exhibitedno changes in their de-gree of iodination. (The extent of capsid VP:3

labeledwithEnzymobeadswascalculated from

single-dimension SDS-polyacrylamide gels, and

all structural proteinratios in virion and capsid

experiments were calculated in an analogous

fashion[data notshown].) Invirions, however,

we were able to calculate that approximately

20% of the virion VP3 and 2 to5% of the virion VP2 was internal. These calculations were in agreement with the results ofBrady et al. (7),

who observed small amounts of VP2 and VP:3

associated with thepolyoma DNA-protein

com-plex after virion dissociation with EGTA and DTT.

Isolationof virionDNA-proteincomplex;

virion and capsid capsomere subunits. Since thepriorexperiments suggested that

vir-ionVP, speciesA andperhapssome ofspecies

Cwere

internal,

we wereinterested in determin-ing which of the virionspecieswere associated

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FUNCTIONS OF POLYOMA VP, SPECIES 85

with the DNA-protein complex. T

was obtained by dissociation of

(chloramine-T) virions with EGT. followedby velocity sedimentation ciated materialon5to20%sucrose

separatethecorecomplex from the

(7). The iodinatedcores werethen

SDS-PAGE and by IEF. The resu

the core are shown in Fig. 4. TI

species associated with the DNA-I plex was species A. Small amount:

VP3werefound when theSDS-pol

gels were autoradiographed for pi

riods, butwerenotobserved in the

to their low concentration. These gested that a single species of p

(species A)wasassociatedwith th(

Furthermore, these experimentsle'

ulation that perhaps the tertiary

5-A

4-3

2.

H

10 20 30

GEL SLICE FIG. 4. IEF of polyoma virion 48S complex. Chloramine-T-iodinated vir sociated in vitrobytheEGTA-DTTme et al. (7), and the 48S DNA-protein

isolatedafter velocitysedimentationi,

crosegradients. Thecomplexwasthe,

IEF and SDS-PAGEasdescribedint,

'his complex virion

VP,

species C prevented theexposureof

'25I-labeled some of tyrosine residues of this polypeptide,

A and DTT therebyresulting inalowerdegree of

Enzymo-of the disso- bead iodination sincethis specieswasclearlynot

gradientsto associated with thecorecomplex.

acapsomeres Previous results from this laboratory have

analyzed by shown that, after EGTA-DTT dissociation of

dts of IEF of virions, three groups of capsomere subunits

he only VP, could be resolved by velocitysedimentation of

protein com- the dissociatedvirus(7).Thesethreegroupshad

s of VP2 and sedimentationvalues of18S, 12S,and5S.

Simi-lyacrylamide lartreatmentof capsids also yieldssimilar

cap-rolonged pe- somere sedimentationpatterns(J.B.B.,

unpub-IEFgel due lished data). The interest in these virion and

results sug- capsid capsomeresubunitsstemsfrom the fact

)olyoma VP1 that the 18S and 12S capsomeres contain

pri-avirus DNA. marily VP,. Electron microscopic analysis re-Itothespec- vealed distinct hexon arrangements (12S) and

structure of hexon aggregates (18S). No distinct structures

could be observed in the virion or capsid 5S

material, which contains primarily structural

[8 proteins VP2 and VP3 (7). The distribution of

-7 the various virion and capsid VP1species in the

-6

18S,

12S,

and 5Scapsomeres is shown in Table

5 1.Note that inthese

experiments only

the virion

andcapsid preparations labeled with125Iin vitro 4 thatyielded protein distributions closely

resem-bling in vivo 3H-aminoacid-labeled virions and capsidswere used (see below). Virion 12S cap-somerescontainedprincipally VP1 species B,

C,

andD, and capsid 12S material was composed

mostly of species A', B, and D. The same VP,

species also madeupthemajorityof virionand

capsid 18S capsomeres, but were enriched in

40 50

species

B. Virion5Scapsomereswere

composed

principally

of VP,

species

E and

F,

whereas

DNA -protein

capsid

5S capsomerescontained

mostly

species

ions

DNAproem

d D and F. These resultssuggestthatthe hexon

ions were dis-*

ethod

of Brady capsomeres of polyoma virions are made up of

complex was

VP1

and

species

B, C,

and D and those of

capsids

n 5 to 20% su- aremade up of VP1 species A', B,and D. These

-nanalyzed by experiments lead us tospeculate thatpolyoma

hetext. capsid speciesA'

(pl,

pH 6.6) playsarole

equiv-TABLE 1. DistributionofVP,species incapsomeresubunits"

Virion-s()" Capsids(si.`

Sample

Total A B C D E F Total A' B D E F

18S 21.3 48.2 30.1 20.0 0.8 0.9 20.5 35.2 56.3 18.5

12S 66.3 30.4 40.0 27.6 0.5 1.5 65.3 48.3 26.1 22.3 3.3

5S 12.4 4.5 1.2 0.6 42.3 51.4 14.2 9.3 41.3 49.4

"Capsomeresubunitswereisolated fromsucrosegradientsafterEGTA-DTTdissociation of chloramine-T-iodinatedvirions andcapsids.

"Theareaof thepeakfor eachVP, speciesisexpressedasthepercentageof the totalareaoccupiedbythe speciespresent in thesample. Each value is the average ofanumber ofseparate determinations ofpooled samples.

'The amount ofradioactivityin each capsomere speciesis expressed asthe percentage of the total VP, radioactivityrecovered fromthegradients.

VOL. 37,1981

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86 BOLEN ET AL.

alent to that of virion

species

C

(pl, pH

6.3)since

theirrelative distributionsinthecapsomere

sub-units were similar. The results may also shed

somelightonthenatureof thepolyomapenton.

Since distinctpenton capsomeres couldnotbe

observed in the 18S and 12S

subunits,

it has

beensuggested that the

building

blocks of the

pentonsreside in the5S

region

(7). Thus,it is

possible that virion

VP,

species E and F and

capsid

VP,

speciesDandFareinvolvedinthe

penton structure.

Post-translational modifications of the

virion and

capsid

VP1

species.

We next

soughtto determine the nature ofsomeofthe

modifications of thevirion andcapsidVP, spe-ciesby labeling polyoma-infected cellswith

dif-ferentradioactiveprecursors. The infectedcells

were labeled with

:2pO4,

['4C]acetate,

or

H-amino acids, and the virions and

capsids

were

purifiedthereafter. Equalamountsof32P-or

'4C-labeled virions and

capsids

and 3H-labeled

viri-ons andcapsids were

mixed,

and the mixtures

wereexamined by IEF,

SDS-PAGE,

and

IEF-SDS-PAGE. The results of IEF of 3H-amino

acid-labeled virions and

capsids

are shown in

Fig.5. Itisimportantto notethat the number of

polypeptide speciesobserved and theirpI'swere

the same as the results obtained when

chlora-mine-T-'251-labeled

virionsand

capsids

were

ex-aminedinthismanner

(see

Fig. 1).

These results

suggested that in vitro iodination of

polyoma

virionsandcapsids didnot

appreciably

alter the

pl's of the virus

proteins. However,

these results

also indicated thattherelative amounts ofthe

individualspecies couldbe altered to some

ex-tentduring theinvitro

labeling

procedure. Thus,

to quantitate the protein

distribution,

only in

vivo 3H-amino acid-labeled virion and capsid

proteinswereused.

The results of the mixing experiments are

shown inTable 2 andagain pointout the

dra-matic differences between polyoma virion and

capsid proteins.Fourof the

polyoma

virionVP,

species (A, D,E, and F) and two of the capsid

VP1

species (D and F) were foundto be

phos-phorylated. OftheVP, speciescommonto both

virions andcapsids which were also

phosphoryl-ated,virionspeciesD and Fexhibitedtwice the

level of

:p2p

incorporationasdid capsid species D and F. Two of the virion

VP,

species (C andD) and three of the

capsid VP, species

(A', D,and

F)werefoundtobe

acetylated.

The minor

struc-tural proteinsof both virions and capsids (VP2

and

VP:j)

werefound to bephosphorylated but

notacetylated. Ourattempts to label virions and

capsids with

S-[methyl-:H]adenosyl-L-methio-nine to determine whetherany of the virus

pro-teinswere

methylated

have been unsuccessful.

Although asmallamountof label has beenfound

associated with purified virions and slightly

more has been found associated with capsids,

thedegree ofincorporationwassufficientlylow

thatanalysis by SDS-PAGEorIEFyielded

in-conclusive results.

Immune

precipitation

and IEF ofGuHCI

column-purified VP1. In a previous report

from this laboratory, polyoma virion proteins

VPI,

VP2,and

VP:3

and histones were separated

by using GuHCl gel

filtration

(4). We

utilized

this technique toisolate

'25I-labeled

virion

VP1

todetermine whether theGuHCl

column-puri-fiedVP, showedanIEFpattern similartothat

foundpreviouslywithpolyoma virions. The

re-sults shown inFig. 6clearly demonstrate that,

indeed,allofthe

VP,

species foundduring IEF

ofvirions were found after IEF of the

GuHCl

column-purified

virion

VP,.

Samples ofthe same

pooled VP, preparation were then used in

im-mune precipitation experiments with rabbit

anti-polyoma hemagglutinin and anti-polyoma

receptor. In anearlierreport (3), it was

demon-stratedthat these two antibody activities could

beseparated by polyomacapsid affinity column

chromatography from antiserum prepared

againstproteolytic cleavageproducts of the

ma-jor structural protein

VP,

in

SDS-polyacryl-amide gels. In immune precipitation experi-ments,itwasfoundthat, after EGTA-DTT dis-sociation ofpolyoma virions (conditions known

to lead to the breakdown of

VP,),

the

anti-polyoma

hemagglutinin specifically precipitated

a16K-daltonVP1

cleavage product,

whereasthe

anti-polyoma receptor

specifically

precipitated

a14K-dalton

VP,

breakdownproduct. Whenthe

same virion

preparation

wasdissociated under

conditions which minimized VP1

degradation,

both

anti-polyoma

hemagglutinin and anti-re-ceptorantibodiesimmuneprecipitated 44K-dal-ton VP1. The

goal

of the experiments with

GuHCl

column-purified

VP1wasthus to

deter-mine whether any one or a group of the

VP,

species would berecognized by oneor both of

these

antibody preparations.

The results of these immuneprecipitationexperiments are shown in

Fig. 7.Twoof the VP1specieswere

specifically

immuneprecipitatedwithanti-polyoma

hemag-glutinin (Fig. 7A). These two species

corre-sponded to

VP1-D

and

VP,-F.

Onlyone ofthe

VP1

species(species E)wasimmuneprecipitated

withanti-polyoma receptor immunoglobulin G

(Fig. 7B). These results suggest that the

VP1

speciesassociated with thehemagglutinating

ac-tivityofpolyomavirionsconsist of

VP1

species

Dand F. It isinterestingthatthese two species

are also foundon

polyoma

capsids which have been shown to compete for the same

cellular

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FUNCTIONS OF POLYOMA VP, SPECIES 87

0 lo.

A, 8. o8

0

O 6-o6

,C E

a

.X

4-GEL SLICE

8

I-6

XC

5 4

60

a -8

-6:

-5 -4

10 20 30 40 50 60

GEL SLICE

FIG. 5. IEFof3H-aminoacid-labeled virions andcapsids.Polyoma virionsandcapsidswerepurified from

thelysates of infectedcells grown in the presenceof3H-aminoacidsduringtheperiodofvirusinfection.

3H-aminoacid-labeled virions(A)andcapsids(B)werethenanalyzed byIEFandSDS-PAGEasdescribed in thetext.

receptors asvirions on thesurface of GPE (2). Theseresults furthersuggestthatVP, speciesE

isresponsible for the virion-associatedreceptor

activityofpolyomavirions which isrequiredfor

specific adsorption and infection of primary

MKC since the anti-polyoma receptor anti-bodiesspecifically inhibitedpolyomavirion

ad-sorptionto MKC butnot toGPE (3).

Further-more, VP,-E is found only on polyoma virions and is absentin polyoma capsidswhich fail to

compete with virions for receptor sites on the surface ofMKC (2).

DISCUSSION

The central reason for comparing the IEF

patternsofpolyomavirion and capsidproteins

stems from previous studies conducted in our

laboratory which demonstrated distinct

biologi-cal,biophysical,andbiochemicaldifferences

be-tween these two populations of virusparticles

(2, 3, 12,20).MackayandConsigli (12) reported

thatthe fate ofpolyomacapsidswithin cellswas

distinct from the small

population

ofpolyoma

virionswhichwereresponsibleforcellular infec-VOL. 37,1981

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88 BOLEN ET AL.

TABLE 2. IEFofpolyomavirion andcapsid structuralproteins

Protein Species pI totalo P/'H" '"'C/'H' Virions

VP, A 6.75 8.6 0.2

B 6.50 18.0

C 6.30 37.8 0.05

D 6.10 13.2 0.40 0.25

E 5.90 6.6 0.60

F 5.75 5.8 0.80

VP2 5.50 4.8 1.00

VP:, 7.00 5.2 1.10

Capsids

VP, A 6.60 34.6 0.20

B 6.50 18.5

D 6.10 20.9 0.20 0.40 F 5.75 16.0 0.40 0.30

VP2 5.50 5.4 0.80

VP:, 6.50 4.6 1.00

"Theareaof thepeak foreach of theproteinsand

proteinspecies isexpressed asthepercentage of the

totalareaoccupied by the protein or protein species present inthesample.Eachvalue isthe average of a number of separate determinations of pooled

'H-aminoacid-labeledsamples.

'Ratio of "Pi-labeled proteins to :'H-amino

acid-labeled proteins.

'Ratio of["4C]acetate-labeledproteinsto 'H-amino acid-labeledproteins.

c

p

8

7

613

2-2 5

0

-U~~~~~~~~~~

10 20 30 40 50

[image:9.496.61.252.77.283.2]

GEL SLICE

FIG. 6. IEF of GuHCI-chromatographed virion VP,. Chloramine-T-iodinated virion proteins were

chromatographedon aGuHClcolumn asdescribed

inthetext.TheVP, fractions from the column effluent

werepooled, dialyzed againstTrisbuffer (pH7.4)to

removeexcessguanidine, and analyzed by IEF and

SDS-PAGEasdescribed in thetext.

5

4

3

3

5-

2-0

a.

C. 5.-H

4.-

3.-2

--8

-6

1L

5

-4

-8

-6X 5 4

10 20 30 40 50

GEL SLICE

FIG. 7. IEF of GuHCl column-purified VP,

im-muneprecipitation. Samples ofthe Chloramine-T-iodinated VP, isolatedasdescribed in thelegendto Fig.6wereused in immuneprecipitationexperiments withantihemagglutinin andantireceptorantibodies

as described in the text. The immuneprecipitates

wereanalyzed byIEF. Thefigureshows thefocusing patternofGuHCIVP,plusantihemagglutinin (A)or antireceptor(B). Forclarity,thebackgroundcounts obtained by incubatingnormal rabbit

immunoglob-ulin Gwith theGuHCIVP,have beensubtracted.

tion. After virion adsorption to cells, a small percentage ofthe original inoculum was

trans-ported to the nucleus, where the virions were

uncoated (12, 20), and early transcriptional

ac-tivitieswereinitiated (20).Themajority of the

originalvirionswerefound in cellularlysosomes,

wheretheyweredegraded. Aftercapsid

absorp-tiontocells, nosignificant nuclear transport of

these"empty" virusparticleswas detected.

In-stead, almost the entire capsid population was

sequestered into cellular lysosomes and

de-graded(12). Subsequently, itwasreported that

thereasonfor this differential fate of virions and

capsidswithin cells resulted fromthemannerby

which virions and capsids adsorbed to the cell surface (2). Although capsids efficiently

com-petedwith virions for receptorsitesonthe

sur-face of GPE, capsids could not compete with virions for receptor siteson MKC, which were

requiredforsuccessful infection. This study also

demonstrated that thereason only 7 to 10% of

theinput virions ever entered thenucleus was

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FUNCTIONS OF POLYOMA VP, SPECIES 89

because the number of specificsurface receptors to which virions, but not capsids, could bind was

limited.Recently, the maximum number of

spe-cific virion receptor sites on the surface of quies-cent primary MKC has been estimated to be

approximately 10,000 per cell (J.B.B.,

unpub-lished data). In the same report, it was reported that anadsorption mutant of polyoma (Py 235),

originallydescribed by Basilico and DiMayorca

(1), which lacked the ability to adsorb to and

agglutinate GPE, nevertheless possessed an

equalabilitytospecificallyadsorb to and infect

MKC at 32°C as did wild-type virions. These

results demonstrated that specific adsorption to

MKC and infection of these mouse cells was independent of the ability ofpolyoma virions to

adsorb to and agglutinate GPE. In a recent

report (3), we were able to demonstrate the

separation of neutralizing antibodies from

he-magglutination-inhibitingantibodies in antisera

directedagainst cleavage productsof virionVP1

which migrated in the histone region of

SDS-polyacrylamide gels. Itwasfound that the

anti-genic determinantsresponsible for

hemaggluti-nation-inhibiting activity of the serum were

present on a 16K-dalton polypeptide, whereas those antigenic determinents responsible for

neutralizationactivity of theserum were present

on a 14K-dalton polypeptide. These two

poly-peptideswerebothderivedfrom 44K-dalton

vi-rion

VP,.

Theseresults strongly suggested that

themajority of the diverse biological functions

observed were dependent on the major

struc-turalprotein VP1.

In this report we have shown distinct differ-encesbetween thesubpopulations of the

struc-tural proteins ofpolyoma virions and capsids.

The results presented here demonstrate that

polyoma virionspossess sixspeciesofthe major

structural protein

VP,

in addition to the two

minorstructuralproteins VP2and

VP:). Polyoma

capsids were found to contain only four VP,

species in addition to VP2 and VP:3. Multiple

species of

polyoma

virion

VP, differing

in their

pl'shave beenreported previously (11, 17). How-ever, the number ofspeciesand thefunctionsof these

multiple species

were not clear. It is of

considerable interest that O'Farrell and

Good-man(16)

reported

thatSV40virionsalso possess six distinct VP1 species. Such results suggest thatthispattern ofsix

VP,

species(eventhough the pl's of these species vary considerably be-tween polyoma andSV40) may be commonto

allpapovaviruses.Our

experiments

haveallowed

ustotentatively assignfunctionstothe various

speciesofVP,and have aidedin the

comprehen-sion of howVP1possesses thepotentialfor such

biological diversity.

The major structural protein (VP,) of

poly-oma virions consists of six species which have pI's between pH 6.75 and 5.75. On

two-dimen-sional analysis these species all had molecular

weights in the 42K to 45K range. The molecular-weight diversity observed was not due to con-tamination of our standard wild-type virions with defective virus or deletion mutants, as freshly plaque-purified virus samples yielded

identical electrophoretic patterns. The

differ-ences in pI's of the VP1 species conceivably

result from the different degrees and types of modifications observed (Table 2). Four of the

VP,

species were differentially phosphorylated (A, D, E, and F) and two of the species were

acetylated (C and D). In our analysis to date,

only speciesBappearstobeunmodified. Similar

experimentswithcapsids revealed only four

VP,

species withpI's betweenpH 6.60 and 5.75. Two

of the capsid

VP,

specieswere phosphorylated

(D and F). It is interesting that the degree of

phosphorylation as determined by the ratio of

32P/3H

ofthesetwocapsid VP1 specieswasonly

halfthatobservedwith virion

species

D and

F,

although the significance of this remains

ob-scure.

Similarly,

theimportance ofthree ofthe

capsid

VP, species

A',C, andDbeing

acetylated

isalsounclear. Aswithvirions, capsidVP1

spe-ciesB does notappeartobemodified via

ace-tylationor

phosphorylation.

Analysis

ofthe

mi-nor virion and

capsid

structural

proteins VP2

and VP3 also revealed that differences exist.

VirionVP3had apl of pH 7.00,whereas

capsid

VP3 had anapparent pl of pH 6.50.

Again,

the

significance of this observationisnotunderstood

atthepresent time.

Capsid

and virion

VP2

had a commonpI of

pH

5.50.Further

analysis

ofall ofthe virion and

capsid

VP, species, VP2,

and

VP3 using other

analytical techniques,

suchas

tryptic peptide

mapping,

anda wide

variety

of

temperature-sensitive

and deletion mutants is

currently

under way in this

laboratory.

It is

anticipated

thatsuchan

approach

will be

helpful

in

answering

manyofthe

questions

raised inthis

investigation.

The variety of

presumably

host-contributed

post-translational

modifications of the virus

structural

proteins,

particularly VP1,

suggests

that suchmodifications couldallowanumber of different functions to be expressed by a

single

protein.Atthe present

time,

this appearstobe

thecase withpolyomavirus andrepresents

an-otherexampleof the virus'abilitytoefficiently

utilize and expand its limited genetic coding

capacity.Our results suggest thatpolyomavirion

VP, may serve atleast four distinct functional roles. The first is a structural function. The

experiments in which the various VP1

species

weredifferentiallyiodinated with

Enzymobeads

orchloramine-T

(Fig.

3)demonstrated that

VP,

VOL. 37,1981

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90 BOLEN ET AL.

species A was an internal protein whereas the

remainingVP, specieswere available for

exter-nal labeling. Analysis of the EGTA-DTT-de-rived capsomere subunits (Table 1)

demon-strated that virionVP,species B, C, andDwere

presentincapsomerepreparations enriched with

hexon subunits, whereas species A, E, and F

wereabsent. These resultssuggestthatpolyoma virion hexonsarecomposed ofVP,species B, C,

and D.They alsosuggestthatVP,species E and F may be involved in the penton capsomere

subunitsof the virion. Thus, potentiallyfive of

the six VP1species performastructural function

for the virus. Similar analysis of capsids

sug-gested thatVP, speciesA' incapsids likely

per-forms thesamefunctionasvirion VP1species C

(Tables 1and2). In capsids, therefore, the

hex-onsareapparently composedofVP, speciesA',

B, and D. Since capsids lackaVP,species which

physically corresponds to virion species E, it

appears that capsid species D, along with F, mightcompensatestructurally for the lack of E. Minorproteins VP2 and VP3werefoundboth in

capsomerefractionsbeingaccessible forsurface iodination withEnzymobeads andinassociation with the DNA-protein core (7; J.B.B.,

unpub-lisheddata).At thistime, the roles ofVP2 and VP;3asstructural entitiesorregulatory proteins, orboth, unfortunately remain unresolved.

The second role thatpolyoma VP, possesses

is exemplified by virion VP, species A. This species,asnotedabove,couldnotbelabeled in vitro by the Enzymobead technique (Fig. 3B) andwasfoundtobe theonlyVP,species firmly associated with the virion DNA-protein core

(Fig. 4). Furthermore, VP, species A was not

found in capsids (Fig. 1A, 3C and D, and 5; Tables 1 and 2) or in significant amounts in

capsomere subunits (Table 1). The function whichthis DNA core-associatedVP,species

ful-fillsisnotclear at this time. ThatVP,species A, themostbasic (pl,pH 6.75) oftheVP,species, is apparently exclusively associated with the DNA-protein complex of the virus implies that its role may not be structural in nature. One function VP, species A may serveissuggested

by the recent experiments by Brady et al. (5; personal communication). They demonstrated that 110S SV40 nucleoprotein cores derived

from intact240S SV40 virions by EGTA-DTT dissociationcontained VP,and asmallamount ofVP2inadditiontotheproteinsnormallyfound

associated withtheSV40 55Sminichromosome (i.e., VP.,and histones). In additiontokeeping the 110Scorecompact,theassociatedVP1-VP2

significantly modified the transcriptional activ-ity of the core in the presence of Escherichia

coliRNApolymerase. The presenceofthelate

virusproteinsresulted intranscription of 16to

18SRNAwhichhybridizedtothe"early" SV40

DNA region. In the absence of VP1-VP2, the

RNA transcription product was only 4 to 6S.

These results lead to the speculation that the same events may take placewith polyoma

nu-cleoproteincomplexes. Experimentstotestthis

hypothesisarecurrentlybeingconducted in our

laboratory. Nevertheless, the experiments of

Brady et al. clearly point out the probability

that VP1 andperhaps VP2,

normally

considered

only structural entities, alsopossess regulatory

functions.

The remaining two functions which can be

attributed topolyoma

VP,

deal with the virus'

ability to adsorb to mammalian cells such as

GPE andprimary MKCor mouse

embryo

cells.

The datapresentedinthisreportindicate that

threeofthe virion VP1

species (D, E,

and

F)

are

involvedinsuchactivities.

VP,

speciesDand F

appear to be common to both virions and cap-sids, although their distribution among

capso-mere subunits, degree ofmodification, and

ab-solute concentrationdifferto some extent

(Ta-bles 1 and 2). Our data suggest, however, that

whereasthecapsidD and Fspeciesappeartobe

slightly different from theirvirion counterparts,

theymayfulfillsimilarfunctions. These results

alsopoint out the possibility thatsome ofthe

modifications observedmay notnecessarily

dic-tatefunctionalchangesotherthan

allowing

con-formational diversity of the

VP,

molecules

re-quired for assembly of virusparticles. The

ad-ditional biological functions attributedtosome

of thesespecies could thus be dependentonthe

presence of other unidentifiedmodifications or in many instancesthey couldbeindependent of

suchalterations. The functionalidentity of

vir-ion species D and F wassuggested by the

im-mune precipitation experiments (Fig. 7).

Anti-bodieswhoseonlyknownfunctionwastoinhibit virion orcapsid absorption to and agglutination

of GPE could specifically recognize only

VP1

species D and F. Thus, it appears that virion

VP1

speciesDandF areinvolved withthe virus'

abilitytoadsorbtoandagglutinateGPE.These

results further suggest thatcapsid VP, species Dand Fprobably

fulfill

the samefunction,since virions andcapsids have an equal ability to cause

hemagglutinationwhich canbe equally inhibited

bythehemagglutination-inhibitingantisera (3).

Virion VP, species E appears to represent the virion protein which is required for successful virus adsorption to specific cellular receptors, which results in infection of MKC and mouse

embryo cells. Thus,

VP,

species E apparently

fulfillsthe role of thepolyomavirion-associated

receptor. Two lines of evidence support this

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FUNCTIONS OF POLYOMA VP, SPECIES 91

contention. Primarily, VP1 species E is found

onlyonvirions,never on capsids.Since, as

dis-cussed above,capsids donot compete with

viri-ons forspecific cellular receptors (2), it seems

logical that capsids would lack the correspond-ing protein present on virions. Similarly, since

capsids andvirions share the same

cell-associ-ated receptor on GPE (2), one would expect these two typesofvirusparticlestoshare

com-mon hemagglutinins. As discussed above, this

appears tobethecase.Second, antibodieswhose

onlyknownactivitywas toinhibit virion

adsorp-tionto mousecells,but not toGPE,specifically

recognizeddistinct antigenicdeterminantsonly

on

VP,

species Einimmuneprecipitation

exper-iments (Fig.7B).

An additional point of interest concerns the

precursor-product relationshipbetween the

VP,

species. Thepulse-chase experiments performed

byO'Farrell andGoodman (16) suggestedthat

themostacidic of theSV40

VP,

species actedas

aprecursor to twoadditional

VP,

species which

focused

immediately adjacent

totheprecursor.

If the same relationship is also present with

polyoma, then species F would represent the

precursorforspeciesDandE.Sucha

relation-ship would indicate that the

VP1

species

in-volved in cellular adsorption activities are

de-rivedfromacommonprecursor which is distinct

from other

VP1

species, possibly through

de-phosphorylationof theprecursor(Table2).

Thisstudyhasallowedus toassign functions

tothe VP1species ofpolyoma virus. In the future

we anticipate being able to better understand

the molecular interactions which result in the

modified forms of

VP1

and to determine what

other functions

VP,,

VP2,and VP3mightexpress

during thecourseof

polyoma

infection.

ACKNOWLEDGMENIS

Thisinvestigationwassupportedby Public Health Service grant CA-07139fromtheNational Cancer Institute. R.A.C.is aSenior Scientist with the Mid-America Cancer Center Pro-gram.

We expressourappreciationtoDiane Potts and Viola Hill forexcellent technical assistance andtoSueStefanskiforhelp inpreparationof thismanuscript.We also thank M. S. Center forhelpful discussions.

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1. Basilico, C.,andG.DiMayorca. 1974.Mutant of poly-omavirus withimpaired adsorption toBHK cells. J. Virol. 13:931-934.

2. Bolen, J. B., and R. A. Consigli. 1979. Differential adsorption ofpolyoma virions andcapsidsto mouse kidney cells andguineapig erythrocytes.J. Virol.32: 679-683.

3. Bolen, J. B., and R. A.Consigli.1980. Separationof neutralizingandhemagglutination-inhibiting antibody activitiesandspecificityofantiseratosodiumdodecyl sulfate-derived polypeptides ofpolyomavirions. J. Vi-rol. 34:119-129.

4. Brady, J.N., andR.A.Consigli.1978.Chromatographic separation ofthepolyoma virus proteins and renatur-ation ofthe isolatedVP,majorcapsidprotein. J. Virol. 27:436-442.

5. Brady, J.N., C. Lavialle, andN. P.Salzman. 1980. Efficienttranscription of a compact nucleoprotein com-plex isolated from purified simian virus40virions. J. Virol. 35:371-381.

6. Brady, J. N.,V. D.Winston, and R. A.Consigli.1977. Dissociation ofpolyomavirusbythe chelation of cal-cium ions found associated with purified virions. J. Virol.23:717-724.

7. Brady, J.N., V. D. Winston, and R. A.Consigli.1978. Characterization ofaDNA-protein complexand cap-someresubunits derivedfrompolyoma virusby treat-mentwithethyleneglycol-bis-N-N'-tetraacetic acid and dithiothreitol. J. Virol. 27:193-204.

8. Brunck,C. F., and V. Leick.1969.Rapidequilibrium isopycnicCsCl gradients. Biochim. Biophys. Acta 179: 136-144.

9. Deininger, P., A.Esty, P. LaPorte, and T. Friedmann. 1979.Nucleotide sequence andgenetic organization of thepolyoma lateregion:features common tothe poly-omaearly region and SV40. Cell 18:771-779. 10. Fried, M., and B. Griffin. 1977. Organization of the

genomes ofpolyoma virus and SV40. Adv. Cancer Res. 24:67-114.

11. Hewick,R.M., M.D.Waterfield,L K.Miller,and M. Fried. 1977.Correlationbetweengenetic loci and struc-tural differences in thecapsid proteinsofpolyomavirus plaquemorphologymutants.Cell11:331-338. 12. Mackay, R.L.,and R. A.Consigli.1976.Earlyevents

in polyoma virus infection: attachment, penetration, and nuclearentry.J.Virol.19:620-636.

13. McMillen, J., M. S.Center,andR.A.Consigli.1976. Origin ofpolyoma virus-associated endonuclease. J. Vi-rol. 17:127-131.

14. McMillen,J., and R. A.Consigli. 1974.In vitro radio-isotopic labeling of proteins associated with purified polyomavirions. J. Virol.14:1627-1629.

15. O'Farrell,P.H. 1975.Highresolutiontwo-dimensional electrophoresis of proteins. J. Biol. Chem. 250:4007-4021.

16.O'Farrell, P. Z., and H. M. Goodman. 1976. Resolution ofsimian virus 40 proteins in whole cell extracts by two-dimensionalelectrophoresis: heterogeneity of the major capsid protein. Cell9:289-298.

17.Ponder, B. A. J., A. K. Robbins, and L. V. Crawford. 1977.Phosphorylation of polyoma and SV40 virus pro-teins.J. Gen. Virol. 37:75-83.

18. Smith, G. L, and R. A. Consigli. 1972. Transient inhi-bition of polyoma virus synthesis by Sendai virus (parainfluenzaI). I.Demonstration and nature of the inhibition by inactivated virus. J. Virol. 10:1091-1097. 19. Vinograd, J., andJ.E.Hearst. 1962. Equilibrium

sed-imentation ofmacromolecules and viruses in a density gradient. Prog. Chem. Org. Nat. Prod. 20:372-422. 20. Winston, V.D., J. B.Bolen, and R. A.Consigli.1980.

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VOL. 37,1981

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