Separation and Characterization of Soluble Adenovirus Type 9 Components

(1)

Separation

and

Characterization

of

Soluble

Adenovirus Type 9

Components

E. NORRBY, B. NYBERG, P. SKAARET, AmN A. LENGYEL1

DepartmentofVirology,School of Medicine, KarolinskaInstitutet,Stockholm, Sweden

Receivedforpublication2August 1967

Fourdifferentsoluble components ofadenovirus type 9 (Rosen's group II) were

identified. These were a complete hemagglutinin (HA), an incomplete HA,

com-ponentscarrying group-specific comple'ment-fixing (CF) antigen, and components

identified only by their hemagglutination-inhibition (HI) antibody consuming

capacity andantigenactivity inCFtestswithan antiserum againstcomplete HA.

Thecomplete HA sedimented relatively rapidly. It was composed of 12 pentons

(vertexcapsomersplus

projections)

aggregatedintotheformof apentagonal

dodec-ahedron. Thelength

of

the projectionswas about 12 to 13 m,. Thus they appeared

longer thanthecorresponding structures oftypes 3 and 11, but shorter than those

oftypes 4 and 5. The rate of sedimentation of completeHA oftype 9 was

inter-mediatetothose

of

thecomplete HA oftypes 3 and 11. TheincompleteHA

sedi-mentedtogetherwith componentscarrying group-specificCFantigen,butcould be

separated from those by

anion-exchange

chromatography. Two different antigens

were present in

incomplete

HA. One couldabsorb a group-specific

hemagglutina-tion-enhancing antibody, and was sensitive to treatment with trypsin. The other

antigen could absorbthetype-specificHIantibody andwas notdestroyedby trypsin.

Inadditiontothe

incomplete

HA,aseparatepopulationofmoreslowly sedimenting

components showed a capacity to absorb HI antibody. These components could

also beidentified inCF tests when an antiserum against completeHAwas applied.

Theincomplete HA,group-specificCFantigen,andslowly sedimentingHIantibody

absorbing componentsaresuggestedtorepresent

isolated

penton, hexon, andfiber

components,

respectively.

Adenoviruses are

separated

into three sub.

groups on the basis of their

hemagglutinating

activity

(14).

Studies on soluble components have been

mainly

concerned with members of

subgroup III,

e.g., types 2 and 5

(8,

13,

17, 20),

and, rrore

recently,

with members of

subgroup

I, e.g., types 3 and 11

(9-11).

Soluble nonvertex

capsorr

ers, vertex capsomers

plus projections,

and isolatedvertex

projections

were

distinguished

in

preparations

of alltheseserotypes. These

com-ponents have been named

hexon,

penton, and

fiber

antigens

(5),

and this

terminology

will

be

used in the

following

presentation. However,

for

reasons

given

ina recentreview

(Current Topics

of

Microbiology

and

Immunology,

in

press),

the term

"antigen"

will be

replaced

by

"component."

The

complete

hemagglutinin

(HA)

ofmembers

of

subgroup

Iwasidentifiedas onemorekind of

soluble component, and was found to be

com-IOn leave from the Institute of

Microbiology,

University MedicalSchool,Budapest, Hungary,on a

World HealthOrganizationresearch

fellowship.

posed of12 pentonsaggregated intheform ofa

pentagonal dodecahedron. No similar structure

has been identified in preparations of members

of subgroup III, with the exception of type 4

(12, 18). Members ofsubgroups I and m also

appear to differ with regardto the

immunologi-cal complexity of their fiber components (11,

17).

Against the background of this diversity in

structural and functional characteristics of

solu-ble components of members of

subgroups

Iand

III, it was considered of interest also to study

the

corresponding

components ofa member of

subgroup II. Adenovirus type 9 was chosen as

an object for this study. Available information

oncharacteristicsofmembers of

subgroup

II is

meager, andmainly derivesfrom studies carried

out by

Wigand's

group

(1, 2,

19). They

have

demonstratedthat some of the

hemagglutinating

activity

of "whole" virus material is associated

with

virions,

but that the

major

part of it is carried by a soluble HA. This soluble

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nent is distinct from the soluble group-specific complerrent-fixing (CF) antigen, as

demron-strated by anion-exchange chromatography (4) and columnelectrophoresis experiments (15). In

the following, results are presented ofattempts to identify additional soluble components and to analyze the relationship between different virusproducts with regardtotheir structure and function.

MATERIALS AND METHODS

Virus and cell cultures. The prototype strain

("Hicks") of adenovirus type 9 was used. Before

arrival in this laboratory, it had been passed3 times onKBand 10 timesonHeLacells.Materialtobeused in thepresent experiments was preparedinahuman bonemarrowcellline,denoted Masa cells (7),orina

human embryonic lung cell line, Lu 106 cells. The

techniques for handling these cells were previously

described (9, 10).

Medium and cells harvested atanadvanced stage

of virus-induceddegenerationwereconcentrated 10to

20times by forced dialysisagainstpolyethylene glycol

(Carbowax 6000, Union Carbide Chemicals Co.,

New York, N.Y.). Theconcentrate was frozen and

thawedthreetofivetimes,afterwhichcelldebriswas

eliminated. Threeconsecutiveultracentrifugations for

1 hr at 20,000 rev/min (rotor 40, Spinco Division,

BeckmanInstruments Inc., LosAngeles, Calif.) were

thenperformedtoremovevirionsand emptycapsids

from the material. Infectivitytiterswerereducedbya factor of105or morebythis treatment.

Preparation ofimmunesera.Antiserawereprepared

byhyperimmunizationof rabbits withconcentratesof

different prototype adenovirus strains. Amounts of 4 mlof material mixed with Freund'scomplete

adju-vant were given intramuscularly as a primary

immunization. Thiswas followed 4 to 5 weeks later

byanintravenous booster of 1 to2ml. Theanimals

were thenexsanguinated after another week.

Determination of complete and incomplete HA. Serialtwofold dilutions (0.4 ml) of material in

phos-phate-buffered0.067 M,(pH 7.2) physiological saline

(PBS), or the same buffer containing heterotypic

adenovirusantiserum,wereusedfordetermination of

complete and incomplete HA, respectively. The

hemagglutination-enhancement (HE) antibody titer of serawas determined in chessboardtitrations against a reference preparation of incomplete HA prepared

by erythrocyte absorption of"whole" virus material

(10, andseebelow).TwotofourHEserumunitswere

employedper dilution intests for incomplete

adeno-virustype9 HA, andthe testswere incubated for 1

houratroomtemperaturebefore addition of

erythro-cytes. Either fresh rat or human 0 erythrocytes

(0.2 ml)wereaddedintheform ofa0.5%suspension.

Readings were taken by bottom patterns. The final

tubeexhibitingacompleteorclearcut partial

aggluti-nation was considered to contain one unit of

HA (HAU).

Hemagglutination-enhancement and hemagglutina-tion-inhibition antibody consumption (HEC andHIC) tests.Thetheoreticalbackgroundtothesetests,which

were used foridentification ofvertexcapsomers and

projections, respectively, present either inanisolated

orcombined form, wasalready described (11). Both testscomprisedthemixing of serial twofold dilutions

(0.2ml) ofmaterial with 0.1 ml ofadilution of

homo-typic antiserum. After incubation ofthese mixtures

for 1 hr at roomtemperature,0.1 ml ofanindicator

antigen was added. This antigen was representedby

incomplete adenovirus type I I HA (10; J. Gen.

Virol., in press) and "whole" virus materialof type9

in the HEC and HIC tests, respectively. The

homo-typicantiserumwasdilutedtocontaintwo hemagglu-tination-inhibition (HI) or HE units per 0.1 ml,

referringtoeach oneof thesetwoantigens.Thehighest dilutionofmaterial whichcould absorb all HE

(ab-senceofhemagglutination) orHIantibodies (presence

ofhemagglutination) was consideredto containone

HEC or HIC test unit (HECU and HICU),

respec-tively.

Determinationof CF antigen activity.Themodified

drop technique described by Svedmyretal. (16) was

used. Twounitsofserumandtwounitsofcomplement

wereapplied perantigen dilution.Group-specificCF

antigen was determined with antivirion sera against

type 5. Antigens, capable offormingapart of

com-plete HA, were assayed by use of antisera obtained

after immunization withpurifiedpreparations of this

component(seebelow).Noneof theserausedreacted

withnonviral components.

Zonal centrifugation. Thetechniqueforpreparation,

handling, and harvesting of linear 5 to 20%sucrose

gradients was already described (9).

Anion-exchange chromatography. Columns

meas-uring 1.5 X 40cm were packed with diethylamino-ethyl (DEAE)-Sephadex A25 (Pharmacia Fine Chemicals, Uppsala, Sweden). Prior to packing, the

gel was allowed to swell in an 0.04 M

tris(hydroxy-methyl)aminomethane (Tris) chloride buffer of pH

8.4 and washed carefully in the same buffer. The

temperature of fractionation was about 17 C. The

flow rate was maintained at approximately 5 to 10 ml cm-2 hr-1. Linear NaCl gradients were used for

elution of material retained by the column. A 4- to 8-mlamount ofmaterial, dialyzedagainstthe

above-mentioned Tris chloride buffer, was fractionated in each experiment. Equal fractions (volume of 3.0 to

3.5 ml) werecollected by use of an automatic drop

counter.

Electron microscopy. The negative-contrast

tech-niquewas used. Onedrop ofa 2%sodium

tungsto-silicate (STS) solution wasmixed on dentalwaxwith

onedropof thematerialtobeexamined.Priortothis,

the latterhad beendialyzed againsta1%ammonium

acetatesolution.Bovine albumin inafinal

concentra-tion of 0.01% was added if needed to improve the

spreadingofmaterialdroppedontothecarbon-coated

grids. Examinations weremade by use ofaJEM-SY

electron microscope at primary magnifications of

40,000to50,000.

Treatment with trypsin. Preparations to be treated

were dialyzed against a solution containing 0.15 M

NaCl,0.05 M Trischloride buffer (pH 7.2) and 0.001 M

CaCl2. Stock solutions oftrypsin (twicecrystallized,

Fluka AG, Buchs SG, Switzerland) and soybean

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SOLUBLE

5 10 15 20

Fraction number

FiG. 1. Separation ofsoluble adenovirus type 9

com-ponents by zonalcentrifugationi in a linear 5 to 20%

sucrose gradient at 25,000 rev/min (SW25, Spinco)

for 5.5 hr. Fractions were collected dropwise through

the bottom of the tube (left). The following activities

were recorded: O, complete HA; *, incomplete HA

determined inthe presence of an antiadenovirus type 15

serum; 0, group-specific CF antigen assayed in tests

includinganantiadenovirus type 5 serum.

trypsin inhibitor (five times crystallized, Nutritional

BiochemicalsCorp., Cleveland, Ohio) were prepared

in the samebuffer.

RESULTS

Zonalcentrifugation ofa mixture of all soluble components. As in previous experiments (9, 10, 12), the zonal centrifugation technique was used

as a means to trace the possible occurrence of a

rapidly

sedimenting complete HA. The results of

one such experiment are presented in Fig. 1.

Under the conditions of centrifugation, the

complete HAwas recovered close to the bottom

of the tube. In the low-density region of the

gradient, group-specific

CF and an incomplete

HA were recovered. Tests with rat and human 0 erythrocytes gave a similar distribution of both

complete and incomplete HA. The incomplete

HA was demonstrable in the presence of an

antiserum against type 15, another

mrember

of

Rosen's subgroup II, but also in the presence of other heterotypic antisera, e.g., against types 3

(subgroup I) and5 (subgroupIII).

Comparison of the rate of sedimentation of

complete adenovirus types3,9, and11 HA. Since

previous findingshaverevealed thatthecomplete

HA of adenovirus type 11 sediments markedly

morerapidlythan the corresponding component

ofadenovirus type 3 (Norrby, J. Gen. Virol., in

press), it was considered of interest to relate the

sedimentation characteristics of type 9 complete

HA to that of the other two complete HA. It

was found (Fig. 2) that the completetype 9 HA

sedirrented irore slowly than the complete type

11 HA, but still significantly more rapidly than

thecorrespondingtype3 HA.

Ultrastruclural characteristics of complete adenovirus type 9 HA.Complete HA, purified by zonal centrifugation as shown in Fig. 1, was

used for electron

nf

icroscopy. As

might

have

been anticipated, this HAwas found tohave, in

principle, the

sanme

composition as the complete HA oftypes 3 (9) and 4

(18),

i.e.,

a

symmetrical

aggregate of 12

capsorrerlike

structures carrying

projections, each located at the facets ofa

pen-tagonal dodecahedron

(Fig. 3).

The

capsorrer-like structures hadan outer diameter of 70 to60

A,and they formeda coreofcompleteHAwith

a diameter

varying

between 240 and 290 A

de-pendingupontheorientation ofthe

particle.

The

projectionsextendingfrom thecorehada length

of 110 to 140 A, thus

giving

the "star"-shaped

HAanoverall diarreter of500to560 A.

Interaction between different soluble

compo-nents and erythrocytes. Preparations of soluble

adenovirus type 9componentswere

subjected

to

fourconsecutive

absorptions

withrat or, in most

experirrents,

human 0 erythrocytes. The latter

type ofcell was

preferred

because

they

were less

prone to undergo spontaneous

lysis.

The

con-centrations of

complete HA,

incomplete

HA,

and

group-specific

CF

antigen

were determined

in the different samples. The results from one

experiment are shown in Table 1. The complete

HA was rapidly eliminated by the treatment,

HAU per

0.4ml 3

[image:3.471.35.223.52.221.2]

Fraction number

FIG. 2. Zonalcentrifugationofa mixtureof

adeno-virus type3, 9, and 11 materialsin a linear5 to

20%lo

sucrosegradient (left = bottom oftube) for5.5 hrat

25,000 rev/min (SW 25, Spinco). Symbols: 0, HA

activitydetermined withmonkey erythrocytesat37 C.

After incubation of thetests at4 C,onl.y theleftpeak

of activity remained, revealing that this represents

complete adenovirus type 11 HA; *, HA activity

determinedwith rat erythrocytes.

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a

b

C'.:f ...

FIG. 3. Ultrastructuralappearanceofparticles

pres-ent in apreparation of complete HA purifiedby rate

zonalcentrifugation (see Fig. 1). The threeparticles

have been interpretedasbeing viewedalonga (a)

two-fold, (b) threetwo-fold,and (c) fivefoldaxis ofsymmetry.

X400,000.

whereas the incomplete HA was not so well

absorbed. Thetiter ofincomplete HA, asshown

in Table 1, wasnot significantly reduced.

How-ever, the outcome ofthe different experiments

waspartly dependent upon the titers of the

ma-terialpriortoabsorption. Ifthe ratioof

erythro-cyte concentration to quantities of biological activity washigher than in the experiment

illus-trated in Table 1,acomplete elimination of both

complete and incomplete HA could be obtained. In contradistinction, the titer of group-specific CF antigen always remained unchanged by the absorptions.

It has been reported that receptor-destroying

enzyme (RDE) caneliminate the agglutinability

of human 0 erythrocytes by some members of

Rosen's subgroup II (6). We therefore investi-gated the possibility that treatment with RDE could be used as arreans ofbreaking the

asso-ciation between complete HA and erythrocytes, which normally is irreversible. Packed

erythro-cytes from the first cycle ofabsorption ofvirus

material were washed three times in PBS to remove components which had not attached to the cells. Theerythrocyteswerethenresuspended in a standard reagent cholera filtrate product (N.V. Philips-Roxane, The Netherlands) diluted

1:8inPBSandincubated for 1 hr at room

tem-perature. The red cells were then removed by

low-speed centrifugation. As can be seen from

Table 1, this treatment of the cells with RDE ledto an elution of allcomplete HA. The eluate

did not contain any group-specific CF antigen.

In a similar way, an elution of complete HA

from agglutinated rat erythrocytes could be

ob-tained by treatment with RDE. However, in

connection with elution ofHA, therewas a

con-siderable lysis of these latter cells, because of

theirgeneralfragility.

Zonal centrifugation was used as a means to

characterize the

product

eluted from red cells.

Figure 4 illustrates the quantitative distribution

in sucrosegradients of complete andincomplete

HA in unabsorbed material and an eluate from redcells.Whereas theformer materialcontained

both complete and

incomplete

HA, only the

[image:4.471.52.242.72.361.2]

former HA was demonstrable intheeluate.

TABLE 1. Effect of four consecutive erythrocyte

absorptions ondifferentbiological

activities ofapreparation of

solubleadenovirus type 9

components

CompleteHA Incom- Group-No.ofabsorptionsa (units/0ml pleteHA specitic

M) (unfits! CFunits/

m) 0.4

ml)b

0.62mlc

0 12,800 256

1 128 1,024 256

2 <4 1,024 256

3 <4 1,024 256

4 <4 512 256

Eluate from cells of 12,800 -'d <4

absorptionI

aEachindividualabsorption wasperformed by

addition ofpackederythrocytesto afinal

concen-tration of10%.Afterincubationfor1 hr at room

temperature, thecellswereremovedby low-speed

centrifugation.

bDetermined in the presence ofan

antiadeno-virus type 15serum.

cAssayed in CF tests by use ofan

antiadeno-virustype5serum.

dNot measurable due to the presence ofhigh

concentrations ofcompleteHA.

eWashed and packed erythrocytes were

resus-pendedin standard reagent cholerafiltrate,diluted

1:8in PBS, to theoriginalvolume of the absorbed

material.

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TABLE 2. Effect of trypsin treatmentofdifferent soluble components ofadenovirus type 9

Treatment Complete HA Incomplete HECU HICU Grou Typeofprepna withb units per HAunits 0p4ler 0IC mer specificSFU

trypsinb 0.4ml per 0.4mlc . m . m per 0.02mId

Complete HA _ 3,200 NTe NT NT <2

+ <8 <8 NT NT NT

Incomplete HAplus group-specific _ <4 256 8 2 128

CFantigen + NT 16 <2 2 128

SlowlysedimentingHIC test <<4 <4 <2 4 <2

positivematerial + NT NT NT 4 NT

aComponentspreparedbyzonal

centrifugation

(Fig.

1 and

5)

wereused.

bIncubation withtrypsin inafinalconcentration of

0.2%

for 3 hrat37 C inawater bath. Controls

-receivedbuffersolutionwithout trypsin.

cAssayedin the presenceofanantiadenovirustype15serum.

dDetermined byuseofanantiadenovirustype 5serum.

eNot tested.

HAUper

0.4ml

128r

5 10 5

Fraction nurnmer

FIG. 4. Distribution of hemagglutinat tested in thepresenceofanantiadenovirustj

ofpreparations ofsolubleadenovirustype9

priortoabsorptionwith (0) andafterelut

erythrocytes (X). Separation of comp

achievedbyzonalcentrifugationundersimile

asinFig.1.

Zonal centrifugation of soluble

excluding complete HA. Materials fr thecomplete HA had been removed cyte absorption (see Table 1) were st

zonal centrifugations at 23,000 rev/m

rotorSW 25) for 45 hr. Under these

of fractionation, incomplete HA a

specificCFantigenwererecoveredas

peak of activities in the high-density

the gradient (Fig. 5). HECtest, andso

HIC test,positivematerial and CFan

onstrated withananti-HA serum alsc

similar distribution. However, a co

fraction of the latter two activities v

by more slowly sedimenting compon

tions 12 to 14, Fig. 5). No clearcut

ltination was seen in the absence of I

antiserum in the same region of the gradient. However, in the presence of such a serum, the

bottom patterns at low dilutions offractions 12

to14werenotidentical withthat of the

erythro-cyte control containing PBS and heterotypic

antiserum. There was someform ofaggregation

of cells, whichwas most clearly revealed by the

relatively more sluggish slipping of sedimented

cells in these

fractions,

as

compared

with other

negative fractions, whenthe rack was held in an

inclinedposition.

Further separation of soluble components by anion-exchange chromatography. Fractionation

on the anion-exchanger DEAE-Sephadex A25

20 25 was tested as an additional

technique

for

sepa-rationof soluble type 9components. Linear0.05

ring activity to 0.3M NaCl

gradients

were found to

give

a

ype15serum good resolution of components in the elution

components diagram. As can be seen from Fig. 6, the se-Fion from rat quence of elution was incomplete HA,

group-7onents was specific CF antigen, and complete HA.

Appar-arconditions ently the former two activities were carried by

different solublecomponents.

Effect of trypsin treatment on the activity of

components different soluble components. Preparations of

rom which complete HA,

incomplete

HAmixed with

group-by

erythro-

specificCFantigen, andslowlysedimentingHIC

abjected

to test positive material, which had been separated

in

(Spinco

by zonal centrifugations as described in Fig. 1

conditions and 5, were used in these

experiments.

The

nd group- preparations weretreated with trypsin in afinal

acommon concentration of

0.2%

for 3 hr in a water bath

region

of

(37 C). The activity ofthe enzyme was stopped omeof the by addition ofequal amounts of soybean

tryp-sthgen

dem-

sin inhibitor. The results of one

experiment

are

) showed a illustrated in Table 2.

)nsiderable This treatment destroyed all complete HA. It

vas carried also eliminated

all

HEC test

positive

activity,

ents

(frac-

but did notreduce the HICtest

positive activity

herragglu- detectable in mixtures of incomplete HA and

heterotypic group-specific CFantigen. Similarly, there was a

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NORRBY ET AL.

CFU 0.02r

64

32

16 8 4

per ml

I-0- 0 -*-

\

0

_-3 6 9 2

Fractiori

numbe-FIG. 5. Distribuition of biological activities of a

preparation ofsoluble adenovirus type 9 components,

from which the complete HA had been removed by

erythrocyte absorptions, after zonal centrifugation in a

linear 5 to 20% sucrose gradient at 23,000 rev/min

(SW25, Spinco) for 45 hr. Fractions were collected

through the bottom of the tube (left). The following

activities were recorded: (a) incomplete HA

deter-mined in the presence ofan antiadenovirus type 15

serum; (AL)HECand(A) HIC test positive material;

(0)

group-specific and (a) HA-specific CFantigens

determined by titrations with antisera against

adeno-virus type 5 and complete HAof type 9, respectively.

HAUper CFU -r

0.4n albuminpeak C02.

2048r 0 1 p<5 132

I

8I

q 'o-o-o

35 45 55 65

Fraction number

FIG. 6. Recovery ofcomplete HA (0), incomplete HA (U), andgroup-specificCFantigendetermined with antiadenovirus type5serum (0) after fractionationon

the anion exchanger DEAE-Sephadex A25. Different components were eluted by introduction ofa linear

0.05 to 0.3 M NaCI gradient in 0.04 M Tris chloride

(pH8.4).

marked reduction of incomplete HA

activity

of

the latter. However, the titer of

group-specific

CF antigen rerrained unchanged by the

treat-mrent. Slowly sedimenting HIC test positive

mraterial, finally, also was unaffected by

incuba-tion withtrypsin.

DISCUSSION

The complete HA of adenovirus type 9 was

found toappear in the form of aggregates of 12

capsomer-like structures each carrying a

projec-tion. Structures with

principally

the same

sym-metrical organization were already identified in

preparations of purified comrplete HA of

adeno-virus type 3 (9), 4 (18), 11 (Norrby, J. Gen.

Virol., in press) 13, 15, and 19 (Gelderblom et

al.,J.Gen.Virol.,inpress). Ithas beensuggested

that a completeHA of this type is formfed by 12

pentons, i.e., vertex capsomers plus projections,

aggregated aroundan internalcomponent

(9).

Different complete HAexhibit some variation

in structural details. As an example, it can be

mentioned that the

complete

HA of adenovirus

type 11 sedimented rruch faster than the

corre-sponding component of type 3. This was

inter-pretedasbeing dueto adifference in mass of the

postulated internal component of thesetwoHA

(Norrby, J. Gen. Virol., inpress). In the present

study,thesedimentationrateof thecompletetype

9 HAwasfoundtobeintermediate betweenthose

ofthe types 3and 11 completeHA.The

explana-tion for this behaviorintermsof structural

differ-ences mustremain

partly

amatterofspeculation.

The relatively longer

projection

of type 9 as

compared withtypes 3 and 11

might

bea factor

of

imrportance.

However, an increase in relative

length of

projections

can influence the

sedimen-tationrateintwodifferent ways. Itcancause an

increase in mass, but at the same time it can

increase the frictional ratio of comnponents. It

can be mentioned that the complete adenovirus

type 4HAsedimented with about thesame rate

astype 3 HA,

although

their

projections,

which arecarried bycoresof similar

dimensions,

differ

in length, about 17 and 10 mg, respectively (9,

18). Thus, it appears unlikely that the variation betweensedimentation ratesof the complete HA of types 3 and 9 could be explained bya differ-encein this respect only. The dimensions of the

complete HA of type 9, excluding projections,

are similar to, or possibly slightly larger than,

those ofthe complete HA of type 3. The srrall

difference, iffurther confirmed, could be caused

by structural dissimilarities in either the capso-merlike orthesuspectedinternal component.

The complete HA of type 9 was effectively elirr inated byerythrocyte absorptions.The

inter-1106

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8

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1 <1

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action between HA and cells appeared to be

irreversible, but an elution of HA could be

ob-tained by treatment with RDE. Treatmrent with

K104 was less effective in bringing about an

elution of complete HA (Norrby and Lengyel, unpublished data). The relationship between

adenovirus type 9 and myxovirus receptors on

red cells is a subject to be studied further. The

effect of treatment of cells with Formalin

indi-cateda possible difference in nature ofreceptors

fortheseviruses(3).

Onthe conditionthat theratios ofthevarious

biological activities were favorable and proper

quantities of erythrocytes were used in

absorp-tion experiments, a product containing

incom-plete HA, but no complete HA, could be

ob-tained. With relatively

higher

concentrations of

erythrocytes, both types of components were

eliminated. This behavior is similar to that of

type 11 components (Norrby, J. Gen. Virol., in

press), but different from that of type 3

compo-nents. T'he incomplete type 3 HA could not,

under the different conditons tested, be

elim-inated by absorption with red cells alone (10).

The incomplete type 9 HA sedimented together

with group-specific CF antigenin sucrose

gradi-ents, but could be

separated

from the latter

component

by

erythrocyte

absorptions,

as was

discussed

above,

anion-exchange

chromatogra-phy and also gel filtration on Sephadex G200

(Norrby, unpublished data). It should be

men-tioned that the sequence of elution of various

adenovirus type 9 components from anion

ex-changers is different from that of the correspond-ing components of other serotypes studied

(8,

10). Therelative

positions

for

group-specific

CF

antigen and

complete

HA described above

cor-respondtothose

previously

described

by

Gelder-blometal.

(4)

for different members of Rosen's

subgroupII.

The

incomplete

HA appears to be

composed

of two different parts. One part was

group-specific,

could absorb HE

antibody,

and was

trypsin-sensitive.

It should be noted that the

immunological

specificity

of this

group-specific

antigen

is different from that of

group-specific

CF

antigen,

determined

by

an adenovirus type

5 antivirion serum

(see below).

The other part

of the

incomplete

HA was

type-specific,

could absorb HI

antibody

and was not sensitive to

treatment with

trypsin.

A

comparison

with

experiences

gained

from studies of type 3

(11)

indicates that the

incomplete

type 9 HA

repre-sents pentons.

Accordingly,

thetwo parts of this component most

likely

represent vertex capso-mers and

projections,

respectively.

The

relation-ship between the occurrence of

incomplete

HA andtoxin

activity

of virus materialcouldnot be

includedin the present study since no activity of

the latterkind was demonstrable even in highly

concentrated virus preparations of type 9. In further analogy with results obtained in studies of type 3 (11), it appears likely that the slowly sedirrenting HIC test positive type 9 component, also identified in CF tests with an

anti-HA serum, represents fiber components.

The slight aggregation of erythrocytes incubated

with the presumed isolated fiber components in the presence of heterotypic antiserum is a

phe-nonrenon whichrequires further analysis.

Fiber components, when forming a part of the complete HA, exhibited a length of about 12 to 13 m,u. It is ofconsiderable interest to note that

this value suggests the occurrence of one more

dirrensional class of fiber components in

addi-tion tothose hitherto identified. Previous studies

have demonstrated projections with a length of

about 10 m,u in adenovirus types 3 (9) and 11

(Norrby, J. Gen. Virol., in press), about 17 m,u

intype 4 (18), and about 25

m,i

in types 2 (Pet-tersson et al., personal communication) and 5 (17, 21). Theseclasses with different fiber lengths

conform with adenovirus subgroups proposed by

Rosen (14), the only exception being the anoma-lous memberofsubgroup III, type 4.

The group-specific CF antigen of type 9 was

carried by soluble components different from

thosediscussedabove, asinferred fromresults of

fractionation by anion-exchange

chromatography

or gel filtration on Sephadex G200 (Norrby,

unpublished data). It appears likely that this

group-specific CF antigen, like the

correspond-ing antigen oftypes 5 (17) and 3 (9), is carried

bynonvertex capsorrers, i.e., hexoncomponents.

ACKNOWLEDGMENTS

Theexcellent assistance of UlfSchonningin

connec-tion with electron microscopy studies and of Ulla

Carlsson and Birgitta Lindstrom in performance of

biologicaltestisgratefully acknowledged.

Thisinvestigationwassupportedbygrantsfrom the

SwedishMedicalResearchCouncil (project noK

67-16x-548-03 andno. B67-16x-74443C).

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