Acid-Soluble Material of Adenovirus

(1)

Acid-Soluble Material of Adenovirus

P. A. BOULANGER, F. JAUME, P. FLAMENCOURT, AND G. BISERTE UnitedeRecherches surla Biochimie des

ProtMines

del'INSERM, Lille,France

Receivedforpublication 22 September 1969

Two

methods

are

described

for adenovirus capsid

disruption

and extraction of acid-soluble

proteins

fromthe viral core. The acid-soluble material of adenovirus

consisted of three major

proteins, one of them being selectively extracted after mild

disruption

of the virus particle. Some chemical properties of these proteins are reported.

The presence of internal component(s) within the adenoviruscapsid, in

addition

to deoxyribo-nucleic acid (DNA), has beensuspected for a long time from

morphological considerations

(22, 24). The discrepancy observed between basic amino

acids

contents

of whole virion and

thecapsid

sub-units (2,

12-14)

suggested the existence of

an

arginine-rich

internal

protein, previously

postu-lated (18, 19). Evidence for

anew

internal

antigen

has been

given by serological

tests

(20)

and

con-firmed by

acrylamide-gel electrophoresis

of

viral

nucleoprotein

cores obtained after disruption of thevirus

capsid

(8).

Recent

findings (10, 11) have

demonstrated

the

complexity

of the adenovirus

composition

and specially of the inner core.

Acrylamide-gel

elec-trophoresis of disrupted

virions

yielded

nine

distinct

polypeptides,

three

of them

being

asso-ciated

with the

DNA-containing viral

core. More-over, two acid-extracted basic

proteins

were

re-covered after

a

mild

sequential

disintegration

of type 5

virions and characterized

immunologically

(15).

These

DNA-associated proteins

were

found

tobe

relatively

rich in

arginine (11,

15,

21). Thepresent report describes amethod for

dis-ruption of virus particles and extraction of

several

(inner?) acid-soluble proteins from

two

adenoviruses belonging

to the same

immuno-logical subgroups,

types 2

and

5. It

also

presents some

preliminary

results

on the

biochemical

properties of

these

proteins.

MATERIALS AND METHODS

Preparationof the virions.Twoserotypesof human adenovirus were studied: types 2 and 5. The virus particleswerepurifiedfrom Freonextractsofinfected KB cellskindly suppliedbyPr. J. Samaille (Depart-mentofVirology,InstitutPasteurdeLille), byuseofa procedure previously described (2). The Freon

ex-tract wasfreed of cell debris by filtrationon

hyflo-supercel under vacuum, and the virusparticlesandthe soluble antigenswereprecipitatedbyammonium sul-fateat54%saturation (pH 7.0)

overnight

at4C. The

precipitate formed was centrifuged at 2,500 X g for 30 min at 4 C. The sediment thus obtained was dis-solved in a minimum of 0.1 M tris(hydroxymethyl)-aminomethane-0.2 M NaCl buffer (pH 8.0) and chro-matographed on Sepharose 4B (Pharmacia Fine

Chemicals) equilibrated with the same buffer. The virions, excluded from the gel, eluted in the void vol-ume ofthe column. The purityofthis fraction was controlledimmunologically byusing arabbitanti-KB cell immune serum and a rabbit anti-equine serum

immuneserum, todetectpossible cellular orseric

con-taminantsfrom the culture medium.

Disruption of the virus particles. Thefractions cor-responding to virions werepooledandsubjected to a disrupting treatment. Two methods were usedfor dis-ruption of the virus capsids: amild procedure and a

drastic one.Formethod A, amilddisruption ofthe

capsids, the virus suspension was dialyzed against distilledwaterandlyophilized.Thistreatmentproved

tobe able to disrupt the virus capsids (2). For method B, a drastic disruption of thecapsids, thevirus

sus-pensionwascentrifugedat105,000X gfor 3hr, and thevirus sedimentthusobtainedwashomogenized in distilled water by means ofanUltra-Turrax

homog-enizerfor 2 min at4 C. The homogenate was then

lyophilized.

Extraction of acid-soluble proteins.The nucleopro-teincomplexesareinsoluble in 0.14 MNaCl,whereas the outer coatproteinsaresolubleunderthese condi-tions. Therefore, the first stepconsistedof

removing

the structural antigens released from the disrupted capsidsinto the 0.14 M NaCl supernatant fluid.

Method A. Lyophilized virus

particles

(100 mg)

weresuspended in 0.14 Msodiumchloridecontaining

0.01 M sodiumcitrate(pH 7.0) andstirredovernight at4 C. Thesuspensionwascentrifugedat 1,000X g for30min, and the supernatantfluidwasdiscarded. Thesedimentwassuccessivelywashed andcentrifuged

three timesin the sodium chloride-sodium citrate buf-fer, and the final sedimentwastwice washed withethyl

alcoholbeforebeingsubjectedtotheextraction proce-durefor histones (7).The

nucleoprotein

sedimentwas stirred with 15to20mlof 0.25NHCI for 10 min at 4C,and thesuspensionwas

centrifuged

at

1,100

X g for 30 min. The acid-soluble proteins wererecovered from the supernatant fluid by precipitation with 6

109

on November 11, 2019 by guest

http://jvi.asm.org/

(2)

BOULANGER ET AL.

volumes of acetoneat -20Covernight. Thisfraction wascalledfraction AS1. The yield of fraction AS1was approximately 2mg.

Method B.Lyophilized virusparticles (100 mg) were

homogenized in 0.14 M NaCl-0.01 M sodium citrate buffer (pH7.0) bymeansofanUltra-Turrax

homoge-nizerfor 2 min, and the suspension wascentrifugedat 1,100 X g for 30min. Thesupernatant fluid was

dis-carded, and the sediment was further washed in the same buffer by magnetic stirring at 4 C overnight. After three additional rinses and centrifugations in the sodium chloride-sodium citrate buffer, the sedi-ment was washed with ethyl alcohol. This sediment wasfirst extracted with 0.25 NHCl for 15 min at 4 C andcentrifuged at 1,100X g for 30 min. The remain-ing sediment was then subjected to a further extrac-tion with 0.25 NHClfor12hrat 4C.Theacid-soluble proteins wererecovered from the two successiveHCI extractsby precipitationwith 6 volumesof acetone at -20 C overnight, and centrifugation at 2,500 X g for 15 min. The supernatant fractions werediscarded and the two sediments werekept separately for chemi-cal analysis. The first extract was chemi-called fraction AS2 and the second one-corresponding to what was ex-tracted after AS2-was called AS3. The yield of frac-tion AS2 was 6 to 7 mg, the yield of fracfrac-tion AS3 was about 5 mg.

Disc electrophoresis. The acid-extracted proteins obtainedfrom adenoviruses 2and5werecheckedby

analytical discelectrophoresis in 15% acrylamide gels containing 6 M ureaatpH4.3, byamodification (1) of themethod of Reisfeld et al. (16) adapted to histones. Antisera. Anti-equine serum, anti-adenovirus 2 hexon antigen, whole adenovirus 2, and anti-whole adenovirus 5 rabbit immune sera were used. These two latter immune sera were prepared by

in-jectinganimals with lyophilized virus particlespurified by gel filtrationchromatography.

Amino acid analysis. Amino acid analyses were

per-formedafter24 hrofhydrolysis in 5.6NHClat110C under vacuum, by using a Technicon autoanalyzer. The tryptophan content was determined spectro-photometrically (17).

N-terminal amino acid. TheN-terminal amino acid residue was determined by the dansyl-amino acid procedure (5) adapted to proteins (23).

RESULTS

Adenovirus 5 acid-soluble components. The

acrylamide-gel

electrophoretic patterns of the

different acid extracts were

quite

different

(Fig.

1). The results depended on the extraction pro-cedure employed. The mild disruption of virus capsidsfollowed byashortHCIextractionyielded

a major protein band (fraction

AS1,

Fig. la),

slightly

contaminatedby threeorfourminor

com-ponents. The drastic

disruption

of virus

capsids

followed

by

a shortHCI extraction

yielded

three major components

(fraction

AS2, Fig. lb); the slowest

migrating

one seemingly corresponds to fraction AS1 obtained after mild

disruption.

An

extended HCI treatment of the

remaining

sedi-+

b

_.

.

.';:;

oxR

E>q

|'%

P_

...; -'9.S.-.' ....

.E.

[image:2.496.268.462.64.245.2]

b

j.

...'.,.,

.>::.::.:....-::.:z..'.:....f

,,,,,,O,.n

;n.o...

Z

,*

C

U

d

FIG. 1. Acrylamide-gel electrophoretic patternz of the

differenzt

acid-soluble fractions obtainied from adenovirus 5. The gel was formed of15% acrylamide in 0.5Mpotassiumi acetate

(pH

4.3) containing 6 M urea. (a) Fractioni AS1 extractedby 0.25 N HC after mild disruption of the virus capsid; (b) fraction AS2 extracted by draistic disrutptioni of the virus capsid, followed by a short HCI treatment; (c) fractionz AS3 obtained from the remaininlg sediment of the AS2 extraction, by ani extendedstirrinigin 0.25NHCI; (d)

purified hexoii antigeni riunl iunzder the same electro-phoretic coniditionis; (e) hexonz, pentton, anid fiber anti-genis mixture. AS1 conitainis onie major proteiln; AS2 con1sists of three major proteinis, anid the slowvest one corresponids to the

mjcjor

componenit of AS1; AS3 con-tainis several slow-migrating componients. Adenovirus 5 structural anttigents were Iot detected in the acid-solublceextracts.

mentafterextraction ofAS2yieldedseveral

slow-migrating components (fraction AS3, Fig. lc) which were also present in the other extracts as

minor contaminants.

These three extracts were tested against anti-equine serum,anti-hexon antigen,andanti-whole adenovirus 5 immune sera. Anti-horse, rabbit-immune serum was used to detect possible

con-tamination from cell culture medium. No reac-tionwasobservedwiththese antiserafor fractions

AS1 and AS2. However, fraction AS3 gave a faintprecipitation line with the anti-whole

adeno-virus 5 immune serum,

showing

apatternof

par-partial identity with hexon

antigen (Fig.

2).

The amino acidcompositions ofthe three

dif-ferent acidextracts fromadenovirus 5

(AS1,

one major protein; AS2, three major proteins; and AS3, several

slow-migrating

components)

are presented inTable 1. The general characteristics are ahigh contentof acidic amino

acids;

a high

contentofglycine, alanine,and

leucine;

lowvalues

110

J. VIROL.

on November 11, 2019 by guest

http://jvi.asm.org/

(3)

ACID-SOLUBLE MATERIAL OF ADENOVIRUS

[image:3.496.102.377.67.193.2]

FIG. 2.

Gel-diffusion

precipitation tests. Left:

immunodiffusion

withthe AS3 fraction in thecenitral well; (a) anti-Ad5lyophilized virions; (b) anti-hexon antigeni; (c) anti-horse serum. Right: IS, anti-Ad5 virionis in the cen-tralwell;Iand 4, AS3; 2, AS1; 3 and 6;htexon antigent; 5, AS2.Areactionz of partialidentity forms between AS3 andhexon.

TABLE 1. Aminioacidcompositionisof threedifferenzt

HCI extractsfrom adenioviruses 2and 5

Aminoacida ASP THR SER GLU PRO GLY ALA CYS VAL MET ILE LEU TYR PHE LYS HIS ARG TRP A B B/A LYS/ARG Adenovirus5 ASi 8.1 5.9 11.3 10.6 5.6 14.5 12.1 0.0 5.6 0.3 2.9 4.6 2.3 1.6 3.7 1.8 9.1 NDc 18.7 14.6 0.78 0.40 AS2 9.7 5.5 6.4 11.6 5.2 8.6 7.8 1.0 6.3 1.8 4.8 7.9 3.2 3.4 6.9 2.5 6.3 1.1 21.3 15.7 0.74 l 1.10

5b_

AS3 10.3 5.6 6.6 11.3 5.0 9.8 8.2 0.4 5.7 1.1 4.9 7.9 3.0 4.0 7.6 2.3 5.1 1.2 21.6 15.0 0.69 1.49 Adenovirus2b AS1 7.8 7.2 13.7 6.5 4.7 11.7 12.4 0.0 5.8 0.3 2.1 3.6 1.6 3.2 4.4 3.2 11.7 ND 14.3 19.3 1.35 0.38 AS2 10.0 5.9 6.1 10.0 6.0 8.2 8.8 1.1 6.1 1.9 4.6 7.9 3.1 3.5 5.9 2.2 7.5 1.2 20.0 15.6 0.78 0.79 AS3 12.0 5.2 6.4 10.9 5.0 10.2 9.3 0.0 6.3 0.0 4.8 8.1 2.0 3.7 8.1 2.0 4.5 1.5 22.9 14.6 0.64 1.80

a ASP, aspartic acid; THR, threonine; SER,

serine; GLU, glutamic acid; PRO, proline; GLY, glycine; ALA, alanine; CYS, cysteine; VAL, valine; MET, methionine; ILE, isoleucine; LEU, leucine; TYR, tyrosine, PHE, phenylalanine; LYS, lysine; HIS, histidine; ARG, arginine; TRP,tryptophan;A, acidic;B, basic.

bAS1 correspondsto onemajorprotein, AS2to

three major proteins, and AS3 to several slow-migrating components. The amino acid contents

are expressed as moles/100 moles of all amino acids found.

cND, not determined.

for

sulfur-containing aniino acids, and

the

pres-ence

of

tryptophan. The mostremarkable

point is

the

composition

of the

fraction

AS1

containing

one

major protein;

this protein hasahighglycine,

alanine, serine,

arginine, and

glutamic acid

con-tent

and

is

relatively

leucine-

and

lysine-poor.

Three

N-terminal amino

acids were found

in

fraction

AS2: glycine, alanine, and

threonine;

traces of

dansyl-proline

were also found, but probablycorrespond to some contaminant.

Frac-tion

AS1 possesses

glycine

astheN-terminal

resi-due.

Adenovirus2acid-soluble components.The

acid

extracts

from

adenovirus2

virions

are

quite

simi-lar to

adenovirus

5 extracts

(Fig.

3). After

mild

disruption of

the

adenovirus

2

capsids,

HC1

ex-traction

yielded

one

major

band

(fraction AS1,

Fig. 3a), which corresponds to the slowest

mi-grating

band

of

the three components

extracted

after

drastic disruption

and short HC1 treatment

(fraction

AS2,

Fig. 3b).

Extended HCI

extraction

yielded

slow-migrating

components, asfor

adeno-virus5

(fraction

AS3,

Fig. 3c).

Thislatterextract

faintly

reacts with anti-adenovirus 2 immune

serum,

with

a

pattern

of

partial

antigenic

identity

with

hexon

antigen.

The

other

acid extracts

do

not reactwith the different antisera tested. The

amino

acid

compositions

of the

adenovirus

2 acid-extracted

proteins

obtained

with

the

dif-ferent

procedures

are

presented

in

Table

1. The general chemical characteristics are the same as

for the adenovirus 5 acid-extracted

proteins.

A

high

glycine, alanine, serine,

and

arginine

con-tent wasalso found in AS1 fraction from adeno-virus 2, but some differences were

observed.

Adenovirus2AS1 hasahigher basic amino acids

contentand a lower

glutamic

acid content. The high

glutamic

acid content of

adenovirus

5

AS1

VOL.

5,

1970

III

on November 11, 2019 by guest

http://jvi.asm.org/

[image:3.496.43.233.277.537.2]

(4)

a

b

c

FIG. 3. Acrylamide-gel electrophoresis of the acid-extractedproteins from adenovirus2. (a) AS1; (b) AS2; (c) AS3. The HCIextractions werecarriedout as

de-scribedinthe legend of Fig.1.Theelectrophoretic pat-terns are quite similar to those of adenovirus S acid

extracts.

could be due to some acidic contaminating

ma-terial.

The same three N-terminal amino acids, gly-cine,

alanine,

andthreonine, werefound in

frac-tion AS2; fraction AS1 has glycine asthe N-ter-minalresidue.

DISCUSSION

Two simple procedures-mild and drastic-havebeendescribed fordisruption of the adeno-virus capsid and extraction of acid-soluble

pro-teins

from

theviralcore,after

removing

the solu-bleantigensinthe 0.14MNaClsupernatant

frac-tion. Thenucleoprotein complexesareinsoluble in

0.14 M NaCl (unlike the

adenovirus

structural

antigens), and the acid-soluble proteinsare

there-fore extracted from theviralnucleoprotein core.

Our mild disruption procedure followed by a

shortHCI extraction yieldsonemajorcomponent (AS1); a drastic

disruption

with a short HCI

treatment yields three major proteins (AS2), among which the slowest migrating one

corre-spondsto componentAS1.No structural antigens

werefound in theAS1 andAS2extracts,asshown

by

immunological

control and analytical acryl-amide-gel electrophoresis. However, it is likely that the 0.14 MNaCl sediment contains, besides

nucleoprotein,

some

morphological

components

belonging

to

incompletely disrupted capsids;

an

extended HC1 treatment

is able

to partially

hydro-lyze these capsid morphological subunits. This

can

explain

the

presence,

in

AS3

fraction

(ob-tained

by

a

prolonged stirring

in 0.25 N

HCI

after drastic

capsid disintegration),

of a

component

sharing antigenic determinant

with hexon

antigen.

The

acid-extracted proteins

are

quite different

from adenovirus structural

antigens

in

physical

and

chemical

properties. Our

analytical

disc

electrophoreses

were run

by

the

procedure

usually

employed for

histones,

with

15% acrylamide

gel

containing

6

M

ureaat

low pH

value.

Under

these

conditions,

the

adenovirus structural

antigens-hexon,

penton,

and

fiber-of

high molecular

weights (300,000

to

60,000) hardly

enter the

gel and remain

near

the

origin

(Fig. ld and le),

whereas the

migration of the

acid-soluble

pro-teins

corresponds

to

molecules of

approximately

10,000

to

50,000.

This

range

of

molecular sizes

is in

agreement

with the

results of Maizel

et

al.

(10)

for the

internal components

V, VI,

and VII

(44,000, 24,000, and

24,000,

respectively).

The

investigators who studied the adenovirus

core

(8, 15, 21) employed

5%

acrylamide gels

for

analytical electrophoresis, but this acrylamide

concentration is

not

suitable for

a

good

fractiona-tion

of

low-molecular-weight

proteins

as

histones.

It is

therefore impossible

to assert

whether

the

fractions

we

have

obtained correspond

to

pre-viously found proteins.

The

amino acid

composition

of

the

whole

acid-soluble material

(fraction AS,) differs from that

of

structural antigens (2),

especially in

basic

amino acids

content

(Table 1). More significant

is the

analysis of

the

AS1 fraction from

adeno-viruses

2

and

5,

because of

its

high degree of purity

(Fig.

la

and

3a). This chemical

composition is

characterized

by

a

high

glycine,

alanine,

serine,

and

arginine content, which has

never

been

de-scribed

for

any

adenovirus

protein. Such

a

high

alanine and serine

content

is

found in F2b histone

from calf

thymus (4), but this histone has

a

higher

lysine

contentand is

relatively

arginine-

and

gly-cine-poor in

comparison with AS1. An important

result is the

presence

of tryptophan

in

fractions

AS2 and AS3,

which

confirms the

findings by

Maizel

etal.

(10,

11) of

a

fair

amount ot

trypto-phan in

the

core

proteins.

It

would be

of great

interest

to carry out

this tryptophan

determina-tion

on each

acid-soluble

protein,

after

further

fractionation and purification.

The

N-terminal

amino acid analyses confirm

the

findings by

Laver et

al.

(9) for

the

adenovirus

core,

by

another

technique.

These

authors found

glycine

and

alanine

as

major

N-terminal

residues,

and in some

preparations,

VIROL.

Im

on November 11, 2019 by guest

http://jvi.asm.org/

[image:4.496.67.260.71.312.2]

(5)

ACID-SOLUBLE MATERIAL OF ADENOVIRUS

threonine,

but this

latter amino

acid

was

con-sidered

as a

contaminant.

Our results

imply

that one

of

the two most

rapid migrating

components

of

AS2

possesses threonineas N-terminal amino

acid, since

AS1,

which

corresponds

to the

slowest

one, has

glycine

asN-terminal.

Are these

proteins histones? They

cannot be properly

called histones since

they contain

trypto-phan and

a

high

amount

of acidic amino acids

(Table 1).

However,

these

proteins

were

extracted

by

the

procedure for

the

isolation

of

histones or

histone-like

proteins (or

both).

As

histones,

they

can

be

easily dissolved

in

distilled

water, they are

deeply stained

by amido black, and

they

have an

electrophoretic

migration

similar to

basic

proteins. Their acidic character

can

be

tentatively

explained

in

several

ways.

(i)

As

already

suggested

(21), it

may

be that

an

acidic internal

component

is rendered

acid-soluble by its close

association

with

amore

basic

component.

(ii)

"High

aspartic-glutamic histones" have been

previously

reported

(6), and

especially

in

metabolically

active tissues

(3). (iii) Regarding

the HCI

solubility of these

adenovirus internal

pioteins

and their

cathodic

migration when

run in

acrylamide

gel

at

low

pH

values, it

can

be

postulated

that

they

have

an

excess

of basic

charges,

owing

to

amidification of

their acidic

groups.

Only

determination of

the

amide

groups can

elucidate

this

point.

Without

being "histones,"

as

chemically defined,

the

acid-extracted

proteins

of

adenovirus have

perhaps

an

histone-like role,

as

DNA-associated

proteins.

If

this

were

the

case,

these internal

proteins

would

have

a

major

function in adenovirus

replication.

ACKNOWLEDGMENT

This investigation was supported by a Convention de

Re-cherchen°67-00-537 from theDelegation Generaleala Recherche ScientifiqueetTechnique.

ADDENDUM IN PROOF

After submission of this report, wedetermined the amide content of the AS2 fraction extracted from

adenovirus 2 by amino acid

analysis

after total

en-zymatic hydrolysis (by using Pronase and

leucine-aminopeptidase,

successively); 60%

of the free

car-boxylic groups of

aspartic

and

glutamic

acids were

foundto beamidified. This result thus confirms that

AS2 contains an

important

excess of basic

charges,

whichexplainsitsphysicalandchemicalproperties.

LITERATURE ClIED

1. Bonner, J.,G. R.Chalkley,M.Dahmus,D.Fambrough, F.

Fujimura, R. C. Huang, J. Huberman, R. Jensen, K.

Marushige, H. Ohlenbusch, B. M. Olivera, and J.

Wid-holm. 1968. Isolation and characterization of

chromo-somal nucleoproteins, p. 3-65. In L. Grossman and K.

Moldave (ed.),Methods inenzymology,vol. XIIB. Aca-demic PressInc.,NewYork.

2. Boulanger, P. A., P. Flamencourt, and G. Biserte. 1969. Isolation and comparative chemical study of structural proteinsof theadenoviruses 2 and 5: hexon and fiber

anti-gens. Eur. J. Biochem. 10:116-131.

3.Boulanger, P. A., F.Jaume,Y. Moschetto, andG. Biserte. 1969. Isolation of histones from virus-induced tumors.

Fed. Eur. Biochem. Soc. Letters 4: 291-294.

4. Butler,J. A. V., E. W.Johns,and D. M. P.Phillips. 1968.

p.221. In J. A.V. Butler and D. Noble (ed.),Progressin biophysics and molecular biology, vol. 7. Pergamon

Press,Oxford.

5. Gray,W.R.,andB.S.Hartley.1963.Afluorescentend-group

reagent forproteinsandpeptides.Biochem. J.89:59 P. 6. Johns,E.W.1964.p.52.InJ.Bonner and P.0.P.Ts'O(ed.),

The nucleohistones.HoldenDay,Inc.,SanFrancisco. 7. Johns,E.W.,D.M.P.Phillips,P.Simson,andJ. A.V.

But-ler. 1961. Theelectrophoresisofhistones and histones frac-tionsonstarchgel.Biochem. J.80:189-193.

8. Laver,W.G.,H. G.Pereira,W.C.Russel,andR.C. Valen-tine.1968.Isolationofaninternal component from aden-ovirustype5. J. Mol. B;ol. .37:379-386.

9. Laver,W.G.,J. R.Suriano,and M.Green. 1967. Adenovius

proteins. II. N-terminal amino acid analysis. J. Virol. 1:723-728.

10.Maizel,J.V., Jr.,D.0.White,andM. D.Scharff.1968. The

polypeptidesof adenovirus. I. Evidence formultiple protein

components in the virion andacomparisonof types2, 7A,

and 12. Virology 36:115-125.

11. Maizel,J.V., Jr.,D.0.White,andM. D.Scharff. 1968. The

polypeptidesofadenovirus.II. Solubleproteins,cores,top

components andthestructure ofthe virion. Virology 36: 126-136.

12. Pettersson,U.,L.Philipson,and S.Hoglund.1967. Structural proteinsofadenoviruses. I. Purification and characterization of the adenovirustype2 hexonantigen. Virology 33:575-590.

13.Pettersson,U.,L.Philipson,andS.Hoglund.1968.Structural

proteinsofadenoviruses. II. Purification and characteriza-tion of theadenovirus type2fiberantigen.Virology 35:204-215.

14.Polasa, H., and M. Green. 1967. Adenovirus proteins. I. Aminoacidcomposition ofoncogenicandnononcogenic

humanadenovirus.Virology31:565-567.

15.Prage,L.,U.Pettersson,andL.Philipson.1968. Internalbasic

proteinsinadenovirus.Virology 36:508-511.

16. Reisfeld,R.A.,V.J.Lewis,andD. E.Williams. 1962.Disk

electrophoresis of basic proteins and peptides on

poly-acrylamide gels. Nature 195:281-283.

17. Spies,J.R.,and D.C. Chambers.1949.Chemical determina-tionoftryptophaneinproteins.Anal. Chem.21:1249-1266. 18. Rouse, H. C., and R. W. Schlesinger. 1967. An

arginine-dependent step in thematuration of type 2 adenovirus. Virology 33:513-522.

19. Russel,W.C.,andY. Becker. 1968.Amaturation factorfor adenovirus. Virology 35:18-27.

20. Russel,W. C.,and B. E. Knight. 1967. Evidence foranew

antigen within the adenovirus capsid. J. Gen. Vircl. 1:

523-528.

21. Russel, W. C., W. G. Laver, and P. J. Sanderson. 1968. Internalcomponents of adenovirus. Nature219:1127-1130. 22. Russel,W.C.,R.C.Valentine,and H. G. Pereira. 1967. The effect of heaton the anatomy ofthe adenovirus. J. Gen. Virol. 1:509-522.

23. Schmer,G. 1967. Quantitative Bestimmungvon10-4

Atmole

N-terminalen Aminosauiren beiImmunoglobulinen durch Markierung mit 1-Dimethyl-amino-naphthalene-5-sul-fonyl-chlorid. Z.Physiol. Chem. 348:199-204.

24.Valentine,R.C.,andH.G.Pereira.1965.Antigensand struc-tureofthe adenovirus.J. Mol. Biol.13:13-20.

113

VOL.5, 1970