Relationship between the methionine tryptic peptides of simian virus 40 and BK virus tumor antigens.

(1)

Relationship Between

the Methionine

Tryptic Peptides

of

Simian Virus

40 and BK Virus Tumor

Antigens

DANIEL T. SIMMONS,l* KENNETH K. TAKEMOTO,2 ANDMALCOLM A. MARTIN'

Laboratory of Biology of Viruses' and Laboratoryof ViralDiseases,2 National Institute of Allergy and

InfectiousDiseases, Bethesda,Maryland20014

Received for publication31May1977

The monomer

form of

BK virus

(BKV)

tumor

antigen (T Ag)

was

immunopre-cipitated from

extractsof BKV-transformed cells and had a molecularweight of

approximately

113,000.

This

compared

with 97,000 for the molecular

weight

of

either

BKV or simian virus 40

(SV40)

T

Ag

from

lyrically

infected cells. The

SV40

and

BKV T

Ag's from productively

infected cells were

compared

by

examining their methionine-labeled tryptic peptides.

Out of a total of 20

SV40-and

21

BKV-specific peptides,

there were seven

pairs

of similar

peptides

onthe

basis of

ion-exchange chromatography.

These

coeluting peptides

contained

ap-proximately

25 to 30%

of the total methionine

radioactivity. Similar results

were

obtained when the

tryptic

peptides of

SV40

T

Ag from

lyrically infected

cells were

compared with those of

BKV T

Ag from virally

transformed cells.

Since the

initial isolation of

BK virus

(BKV)

from the

urine

of

a

renal transplant

recipient on

immunosuppressive therapy (6),

a number

of

reports

have appeared which have shown

a

relationship between the

nucleic acid and

pro-tein components

of

this human papovavirus

with

those of simian

virus 40

(SV40). For

exam-ple, Mullarkey

et

al.

(12)

and Wright and

Di-Mayorca

(20)

demonstrated that the

sizes

and

relative

proportionof the

structural

proteins

of

BKVwere

only

slightly different from those of

SV40.

Although the

peptide compositions of the

capsid

proteins

of

these

twopapovaviruses

dif-fer

significantly

(20),

the structural

proteins of

these

twoviruses cross-react

weekly,

as

demon-strated

by immunofluorescence

(13, 15),

anti-body neutralization

(15), or

immunoelectron

microscopy (6). However, the tumor antigen

(T

Ag), induced by SV40

or BKV in

virus-infected

or

-transformed

cells,

reacts

quite

strongly

with

serum

directed against the heterologous

T

Ag

(15).

Howley

et

al.

(7)

showed that the molecular

weight of

BKVDNA was 3.45 x

10"

compared

with 3.6 x 106

for

SV40 DNA

and that

the two primate papovavirus

DNAs shared

approxi-mately

20 to 25%

of their

base

sequences. Khoury et al. (10) demonstrated that this base sequence

homology

was

localized

tothe regions

of

the virus

DNAs transcribed late

in

infection

(late

regions). Recent experiments by P.

How-ley and

M. Martin (manuscript in

preparation)

indicated

a small degree of sequencehomology

involving

5 to

6%

of the early gene regions of SV40 and BKV DNA. Furthermore, T.

Kelley

and collaborators (personal communications)

have

detected extensive

homology

throughout

the early regions of these DNAs, using

less stringent

conditions for

hybridization.

Because

the SV40 and

BKV T

Ag's

cross-react

strongly

in

immunological

assays

(15)

and

because

some

base

sequence

homology

exists

between the regions of the

virus

DNAs that

are

believed

to

code for

T

Ag (the early regions),

we

have

examined the

sizes

and

peptide

composi-tions

of the

twopapovavirus T

Ag's.

In a

subse-quent manuscript

(submitted for

publication),

we

will

report

that the

molecular weights of

the T

Ag's isolated by immunoprecipitation from

extracts

of

infected monkey cells,

SV40-transformed cells,

or

BKV-infected human cells

are

indistinguishable

(97,000)

by acrylamide

gel electrophoresis.

In this

study,

the size of BKV T

Ag

isolated from

virally transformed

hamster

cells

was

examined and shown

to

be

significantly larger (113,000

daltons)

than the

corresponding

protein

from

lyrically

infected

cells.

Furthermore, the

methionine-labeled

tryptic

peptides of

SV40 T

Ag from

productively

infected cells

were

compared with those of

BKV T

Ag from

lyrically

infected and

virus-trans-formed cells. The results indicate

that out of a total of 20SV40- and 21 BKV-specific peptides,

there

were six or seven pairs of similar peptides on

the basis

of coelution fromChromobead

ion-exchange columns.

MATERIALS AND METHODS

Labeling and cellextraction conditions. Primary

cultures of African green monkey kidney cells

in-319

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fected with SV40 (500 PFU/cell) were labeled with

100

/LCi

ofL-[methyl-3H]methionine per ml (specific

activity, 7Ci/mmol) between 22 and 23.5 h

postinfec-tion in methionine-free minimal essential

me-diumcontaining 2%dialyzed calf serum and 10-4 M

L-1-tosylamide-2-phenyl-ethylchloromethyl ketone

(TPCK) added to prevent proteolytic degradation of

T Ag. Secondary cultures of humanembryonic

kid-neycells infected with BKV (2 to 5 PFU/cell) were

labeled with 50jiCiofL-[35S]methionineper ml

(spe-cific activity 327 Ci/mmol) between 85 and 86.5 h

postinfection. Ten minutes before cell harvest,

TPCK was added to afinal concentration of 10-4 M.

BKV-transformed hamster cells (14) growing in roller bottles and at 80% of confluency were labeled with100,.tCiofL-[35S]methionineper mlfor1.5hin

minimal essential medium lacking unlabeled

me-thionine and supplemented with 10% fetal calf

se-rum. TPCK (10-4 M)wasadded 30 min before cell

harvest.

Labeled cells were washed twice with ice-cold 0.02

M Tris (pH 7.4)-0.001 M Na2HPO4-0.137 M NaCl

(Tris-buffered saline) and collected into the same

buffer. The cells were pelleted at 1,000xg for10min

at 0C and resuspended in a small volume of

Tris-buffered saline at pH 8.0 containing 15% glycerol,

0.001M dithiothreitol, and 250 ug of

phenylmethyl-sulfonylfluoride per ml. The material was sonically

treated for twoperiods of30 seachat 30%maximum

output, using a Branson Sonifier. Nonidet P-40 and

sodium deoxycholate were added to the sonic

ex-tracts to a final concentration of 0.5%.

Preparation of Staphylococcus aureus.

Heat-killed suspensions of protein A-bearingS. aureus

(Cowan I strain, NCTC 8530)wereprepared

accord-ingto themethod of Jonsson and Kronval (9). The

suspensions werestoredinsmallsamplesat-70°C.

Before use, the bacteria were washedonce in 0.02 M

Tris (pH 8.0), 0.001MNa2HPO4, 0.137MNaCl,15%

glycerol, 0.001 M dithiothreitol, 0.5% Nonidet P-40,

and 0.5% sodium deoxycholate andresuspended in

the samebufferto afinal concentration of10% (wt/

vol).

Conditions forimmunoprecipitationandgel

elec-trophoresis.Cellextracts wereclarifiedby spinning

at40,000 rpm for40minat2°CintheSpincoSW 50.1

rotor. Supernatants werecarefullyremoved and

in-cubated for 1 hat 0°C in the presence of20 1.l of

hamster normal serum or anti-T serum (1:320 or

1:640asassayed byimmunofluorescence)per ml of

extract. Washed protein A-bearing S. aureus was

then added(0.2mlof a 10%suspension perml ofcell

extract)tobindimmunecomplexestothe surface of

the bacteria (9), and the materialwasincubated for

anadditional30 min at0°C.Thebacteriawere

cen-trifuged at 2,000 xg for 10min at 2°C and washed

twicein ice-cold Tris-buffered saline, pH 8.0,

con-taining 15% glycerol, 0.001 M dithiothreitol, 0.5%

Nonidet-40, and 0.5% sodium deoxycholate and

twiceinphosphate-buffered saline. The final

bacte-rialpellets were resuspended in asmallvolume of

electrophoresissample buffer (0.075MTris-PO4, pH

8.6, 2%sodiumdodecylsulfate, 2%

2-mercaptoetha-nol, 0.002% bromophenol blue, and 15% glycerol)

and incubatedat60°C for5min.Afterpelletingthe

J. VIROL.

bacteria,the supernatants were carefullyremoved,

heated to 100°C for 7min, and subjected to

electro-phoresis through polyacrylamide slab gels (20%

acrylamide-0.1% bisacrylamide in the separating

gel and5% acrylamide-0.12% bisacrylamide in the

stackinggel) as described by Maurer and Allen (11)

and modified by Tegtmeyer et al. (17). Electrophore-siswasfor12 to 18hat12.5mA.

Preparation and chromatography of tryptic

pep-tides.The methods used for elution of proteins from

acrylamide gels, trypsin treatments, and

subse-quentanalysis of peptides by ion-exchange

chroma-tographyweremodified from those described by Fey

and Hirt (5) and Vogt et al. (19).

Bands of labeled T Ag proteins identified by auto-radiography or fluorography (2) were cut out from acrylamide gels, and the proteins were eluted with

0.1 M (NH4)2CO3-0.1% sodium dodecyl sulfate, pH

8.6,by shaking at 37°C for 48 to 72 h. The eluates

wereclarified by centrifugation at 25,000 rpm in the

Spinco SW 27.1 rotor for 40 min at 23°C. The

solu-tionswere lyophilized, and the material was

resus-pendedin 2 mlof water. Radiolabeled protein was

precipitated with 25% trichloroacetic acid in the

presenceofhuman serum albumin (50 ,ug/ml) at0°C

for 16 h. Theprecipitates werecollected by

centrifu-gation at 14,000 x g for 20 min, washed once in

acetoneandresuspended in 0.2 ml of 0.1 M NaOH.

Aftera secondtrichloroacetic acid precipitation and

acetone wash, the protein was washed once in

di-ethyl ether and resuspended in 0.2 ml of 0.1 M

NH40H. The protein samples were then precipitated

with25% trichloroacetic acid, washed once in

ace-tone to quantitatively remove the sodium dodecyl

sulfate, and resuspended in 0.1 ml of ice-cold, fresh

performic acid prepared by incubating 1.9 ml of

formic acid and0.1 mlof 30% H202 for1 hat 23°C.

Oxidation of proteinsby performic acid was carried

outfor 1 h at 0°C, and the reaction was stoppedby

the addition of 1 ml of water. Theprotein samples

were lyophilized andresuspended in 1ml of 0.05 M

(NH4)2CO3, pH 8.6, and lyophilized a second time.

Trypsin (TPCK treated; Worthington Biochemicals

Corp.) was added at afinal concentration of310 ,ug/

ml toproteinsamplesin 0.2ml of 0.05M (NH4)2CO3,

pH 8.6, and incubated for 4 h at 37°C. The same

amounts oftrypsin were added, and the mixtures

wereincubated foranadditional 4-hperiodat37°C.

The trypsindigestionswerestoppedwith1mlof 0.01

M acetic acid, and the peptides were lyophilized

twice from 0.01 M acetic acid and resuspended in

0.5 ml of water-acetic acid-formic acid-pyridine

(2,354:1,264:350:32, pH 1.9). The peptide solutions

wereclarifiedby centrifugation at 8,000 xg for10

minat 23°C and applied toa 40-by0.8-cmcolumnof

P-type Chromobeads (Technicon Chemicals)

equili-bratedinthe above solutionatpH1.9.

Chromatog-raphywascarriedout at60°C,and thepeptideswere

eluted withanexponentially increasing

concentra-tion ofpyridine from 0.1 to 2M. The pyridine

gra-dient wasapproximately linearinpH from1.9 to4.5

and madebyconnecting threemixingchambers,the

first two containing 210 ml of the solution at pH

1.9and the third containing210ml of thesolutionat

pH 4.5 (water-pyridine-acetic acid-formic acid,

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PEPTIDES OF SV40 AND BKV TUMOR ANTIGENS 321

663:158:140:39). Fractions(2.5

ml)

were

collected and

evaporatedto

dryness

inan80'Coven.

The

peptides

were resuspended in 0.4 ml of 0.01 M HCl

and

counted for radioactivity in 10 ml of counting

cocktail (Toluene-Triton X-100-water-fluor,

279:-150:50:21).

RESULTS

Chromatography

of

methionine-labeled

tryptic

peptides of SV40 and

BKV

T

Ag's from

lytically infected cells. In

a

subsequent

manu-script

(submitted

for

publication),

we

will

re-port

that the

molecular

weights of the

largest

forms

of SV40 and

BKV T

Ag from

extracts

of

lyrically infected cells

are

both

approximately

97,000,

as

determined by

acrylamide gel

elec-trophoresis. Because these proteins have

one or more

antigenic

determinants

in common

(15),

we

expected

them

to

have

some

similar

amino

acid

sequences,

and

possibly

common

tryptic

peptides. To examine this

possibility,

[3H]-methionine-labeled

SV40 T

Ag and

[35S]-methionine-labeled BKV

T

Ag

were

prepared

by

immunoprecipitation from

extracts

of

pro-ductively infected

cells, using

the

homolo-gous

hamster anti-T

serum.

Neither

of these

T

Ag proteins

was

precipitated

in

the

presence

of

normal hamster

serum.

The incorporation of

methionine

radioactivity into these proteins

was

optimized

by

labeling

infected cells

at

the

times

of maximal T

Ag

synthesis

(20

to25

h

and

75 to

85 h

for infections with

SV40

and

BKV,

respectively)

(data

not

shown) and by

adding

the

chymotrypsin inhibitor

TPCK

to

the

cells

either

at

the

beginning

of the

labeling

period

14

r_112A ®

0

ax

it

|

ll

6~8

Al~

C 6II

~ '~ Hi Alt

4

~l

(for SV40 infections)

or near

the

end

of

the

labeling

period (for BKV infections). The

pre-cipitated SV40

and

BKV

T Ag proteins

were

preparatively

fractionated

on

acrylamide gels,

and those

corresponding

to a

molecular weight

of 97,000

were

eluted

from the

gels. The

pro-teins were

digested

with trypsin

as

described

in

Materials

and Methods,

and their

methionine-labeled peptides

were

compared by

ion-ex-change chromatography

on

columns of

chromo-beads. Figure

1

shows

a

characteristic

20-peak

elution profile (each peptide

is

numbered) for

the

methionine

tryptic

peptides of SV40 T Ag

(dashed lines). The solid

lines in Fig. 1

indicate

the

elution profile

of the BKV-specific methio-nine tryptic

peptides and

its characteristic 21

peaks.

Both of these tryptic

peptide profiles

were

reproducible

from experiment to experi-ment

and consisted

of major and minor

peaks.

Figure 1

shows that

at least seven

of

the

3H-labeled SV40-specific peptides coeluted with

35S-labeled

BKV-specific

tryptic

peptides.

These

pairs of

coeluting peptides

chromato-graphed

at

the

positions

labeled

3, 7, 10, 13, 14, 17,

and

19(Fig. 1)

and represented

25 to 30%

of

the

total methionine

radioactivity

present in

peak

fractions

only.

Other

SV40-specific

pep-tides eluted within

a

single

fraction

of

a

BKV-specific

peptide

(e.g.,

peptides

labeled

2

and 15)

and

were not

included

in

such

a

calculation.

Since

ona

totally random basis

one or

possibly

two

of the SV40-

and BKV-specific

peptides

which

coeluted from the column could have the

sameionic

charge but different

amino

acid

se-quences,

these

seven

pairs

of coeluting

peptides

,, I , ,, I, ,,,I 25

20_Cn

N

15 9

e X

,

15

.11

:,,

A~~~~~~~~~~~~~~~~~~

,A0

150

FRACTION NUMBER

FIG. 1. Chromobead ion-exchange chromatography of methionine-labeled tryptic peptides of SV40 and BKV T Ag'sfrom lytically infected cells. [3H]methionine-labeled SV40 (22 to 23.5 h postinfection) and [35S]methionine-labeledBKV (85 to 86.5hpostinfection)TAg'swereprepared by immunoprecipitation from

extractsof

lytically

infected cellsasdescribed in the text. The bands correspondingto TAgproteins with

molecular weights of97,000 wereexcisedfrom preparativeacrylamide gels. Theproteins wereelated from the

gels and treated withtrypsin.SV40- and BKV-specificT Agpeptideswereanalyzed together by ion-exchange

chromatography onP-type Chromobeads. Peptides were elated with a linear pHgradient from1.9 to 4.5

consisting ofexponentiallyincreasingconcentrations ofpyridine, and the fractions wereevaporatedtodryness

andcounted for3Hand35S radioactivities. Symbols: ( )35S-labeled tryptic peptides of BKV T Agfrom

infectedcells; (---) 3H-labeled tryptic peptides ofSV40 T Ag from infected cells.

VOL. 24, 1977

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represent

a maximum estimate for the number

of

identical methionine

tryptic

peptides

be-tween the T

Ag's

of these two papovaviruses.

Immunoprecipitation of BKV T Ag from

transformed cell

lysates. A

recent report (3)

suggested that T Ag from SV40-transformed

cells is larger than the T Ag from SV40-infected

monkey cells. In

our

hands,

however, these two proteins are

indistinguishable

in

molecular

weight

(97,000;

submitted

for

publication).

To compare

the

sizes

of

BKV

T

Ag's

from infected

and transformed cells, T Ag

was

immunopre-cipitated from labeled

extracts

of

BKV-trans-formed hamster cells

(Fig. 2).

A

labeled

protein

significantly larger

than the 97K form of

SV40

T

Ag

(Fig. 2a) wasspecifically

immunoprecipi-tated from

the extracts (Fig.

2c).

The size of this protein was estimated to be 113,000 daltons in ouracrylamide gels, using as molecular weight

standards

ovalbumin (45,000), bovine serum

a

b

C

c

----113K

97 K

albumin (68,000), and phosphorylase A (94,000) (Fig. 3). The immunoreactive 113K protein was not precipitated in the control reaction with normal serum (Fig. 2b). Labeled proteins with molecular weights of 103,000

and

94,000, as

well

assmaller-sized proteins, were also specifically

immunoprecipitated

with anti-$KV T serum (see

Fig.

4).

In

this experiment,

larger

amounts

of labeled BKV T

Ag, immunoprecipitated from

transformed hamster

cells,

were

subjected

to

acrylamide gel electrophoresis.

In

other

prepa-rations

of T Ag from BKV-transformed cells,

a 97K

form of T Ag

was

also

observed (data

not

shown).

Chromatography

of

methionine-labeled

tryptic peptides of BKV

T Ag isolated from

transformed

cells. In view of

the

differences

in molecular weight of the BKV T Ag's from

transformed

and

lyrically

infected cells,

me-thionine-labeled

tryptic peptides of BKV T Ag from transformed cells were compared with the

peptides of SV40

T

Ag from lyrically infected

cells. [3H]methionine-labeled SV40 T Ag

(97,000

daltons) and [35S]methionine-labeled

BKV T

Ag

(113,000

daltons)

were

prepared

by immuno-precipitation

and acrylamide

gel

electrophore-0

-j

[image:4.504.60.246.286.531.2]

02

FIG. 2.Immunoprecipitation ofTAg from

BKV-transformed hamster cells. BKV-transformed cells

werelabeled with[35S]methionine for1.5h when the cellswereatapproximately80%of confluence. Total cellextractswerepreparedand incubatedin the

pres-enceofanti-BKVTserumornormal hamsterserum

asdescribedin thetext.Labeledprecipitated proteins

weresubjectedtoelectrophoresis onacrylamide gels

and detected in thegel by autoradiography. (a)SV40 97K TAgmarkerprepared by immunoprecipitation fromextractsof[35S]methionine-labeledmonkeycells

infectedwithSV40.(b)Labeledproteins precipitated

from extracts of BKV-transformed cells, using

nor-mal hamsterserumand(c) anti-BKVTserum.

3 4 5 6 7 8 9 10

DISTANCE MIGRATED(cm)

FIG. 3. Molecular weight estimate of BKV T Ag

from transformed cells. BKV T Ag prepared by

im-munoprecipitationfrom transformed cells using

anti-BKVTserum was subjected to acrylamide gel

elec-trophoresis in thepresence of unlabeled ovalbumin

(45,000daltons), bovineserumalbumin (68,000

dal-tons),and phosphorylaseA(94,000daltons) as

pro-tein markers. Unlabeled proteins were detected by

staining thegel with Coomassie brilliant blue, and

labeledTAgwasdetectedby exposing the dried gelto

X-ray film (autoradiography). The distance the

marker proteinsmigratedintothegelwasplottedas

afunction of the logarithm of their molecular weight.

The arrow indicates the position in the gel ofthe

largestTAg protein immunoprecipitated from

BKV-transformed cells. From thelinear relationship

ob-tained, the molecular weight ofBKV TAgwas

esti-matedtobe 113,000.

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Ag

shown

in

Fig.

1 was

absent

or present

in

smaller

amounts

in

the

tryptic

peptides of T Ag

from BKV-transformed cells (Fig. 5). This

re-sult did

not

significantly alter the proportion of

methionine counts in

coeluting SV40- and

BKV-specific

peptides.

113 K

103 K

94 K

FIG. 4. Detection of 103K and 94K T Ag proteins

from BKV-transformed cells. A sample of T Ag

im-munoprecipitated from [35S]methionine-labeled

BKV-transformed cells wassubjected to

electropho-resisforalonger period oftimethanwasdoneinFig.

2,andthe dried gelwasexposedtoX-rayfilm fora

period oftime necessary to visualize minorprotein

bands.Inthisautoradiogram, bands of BKV T Ag

proteins with molecular weights of 103,000 and

94,000 as well as smaller TAg proteins were

de-tected.

sis. The proteins were digested with trypsin, and the resulting peptides were subjected to

ion-exchange chromatography on columns of

Chromobeads (Fig. 5). The elution pattern of

the BKV-specific peptides from transformed

cell TAg (solid lines) was remarkably similar

to the peptide pattern of the corresponding T Ag protein from lyrically infected cells (cf. solid lines ofFig. 1). All but one of the SV40- and

BKV-specific peptides that coelutedwhen BKV

TAg was prepared from productively infected

cells also eluted together when the T Ag was

obtained from BKV-transformed cells (Fig. 5, peptides 3, 10, 13, 14, 17, and 19). The

BKV-specific peptide eluting with peptide7ofSV40T

DISCUSSION

Inthis report, we have

shown that

BKV T

Ag

from

transformed hamster cells

was

signifi-cantly larger

(113,000

daltons)

than

the

corre-sponding T Ag isolated from lyrically infected

human

cells

(97,000

daltons).

In

another

line of

BKV-transformed hamster

cells, the T Ag

iso-lated

was

also approximately

113,000

daltons

in

size.

On the other

hand, the largest

species

of T

Ag

isolated from

two

different lines of

SV40-transformed cells (11A8 hamster cells and SV80

human

cells) had the

same

molecular

size as

SV40 T

Ag from

lyrically infected cells

(97,000

daltons; submitted

for

publication).

This result is in agreement

with

those of Tegtmeyer et al. (16)

and Ito

et

al.

(8),

who have found

no

differ-ences in

the size of

SV40

or

polyoma

T

Ag

isolated from lyrically infected and transformed

cells. Carroll and Smith (3) and Ahmad-Zadeh

et

al. (1)

have

reported,

on

the

other

hand,

that

the

molecular weight of SV40

T

Ag

is

smaller

in

lyrically infected cells than

in

acutely infected

nonpermissive or

transformed cells. At the

presenttime, we

do

not

have

a

reasonable

ex-planation for the difference

insize

of the

BKVT

Ag's from transformed cells

(113,000

daltons)

and

infected human cells

(97,000

daltons) and

why this molecular weight difference

was

ob-served for

BK

T

Ag's

and

not

for SV40 T Ag's

(16;

our

observations)

or

polyoma T Ag's

(8).

In

addition

to

the

113K T

Ag

protein,

smaller

species of

T

Ag

were

isolated from

BKV-trans-formed hamster cells, including three proteins

with

molecular

weights of

103,000, 97,000,

and

94,000.

In some

experiments,

the 103K

or

97K

protein

was

the

predominant

species of T Ag

isolated from BKV-transformed cells. The

var-ious

molecular weight classes

of BKV

T

Ag

either

may represent

degradation products of

the 113K form

or

they

may

each be

gene

prod-ucts

of

different viral DNA molecules

inte-grated

within

the host chromosome. We believe

that the formerexplanation is more likely since

the

pattern of

immunoprecipitated

T Ag pro-teins

from

the same

BKV-transformed cells

was

somewhat

variable from

experiment to

ex-periment and

since the protease

inhibitor

TPCK

inhibited the formation

of the

lower-molecular-weight

T Ag proteins

(data

not

shown).

The

relationship between

the SV40-

and

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324

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FRACTION NUMBER

FIG. 5. Ion-exchange chromatography of methionine-labeled tryptic peptides ofSV4O T Ag fromlytically

infected cells and BKV T Ag from transformed cells. [3H]methionine-labeled SV4O T Ag from infected

monkey cells and [35S]methionine-labeled BKV T Ag from transformed cells were prepared by immuno-precipitation with anti-T serum as described in the text. The bands corresponding to T Ag proteins of 97K

(SV4O) and 113K (BKV)daltons, respectively, were cut out of acrylamide gels, and the proteins were eluted

from the gels and treated withtrypsin. 3H- and 35S-labeled peptides were analyzed together by ion-exchange

chromatography. Symbols: ( ) 35S-labeled trypticpeptides of BKV T Ag from transformed cells;

(----3H-labeled tryptic peptides ofSV4OT Ag fromlyticallyinfected cells.

BKV-specific T Ag's

was

studied by

comparing

their methionine-containing

tryptic

peptides by

ion-exchange

chromatography. A

quantitative

estimate

for the

degree

of

homology

between

the

T

Ag's of

BKV

and SV40

wasnot

entirely

possible

since

only

methionine-containing

pep-tides

were

examined.

Figures 1

and

5

show that

although

six or seven

of

the SV40- and

BKV-specific peptides had

very

similar

or

identical

ionic charges, the majority were clearly differ-ent

from

one

another. Further

experiments are necessary to

show whether

the coeluting pairs

of

peptides with similar

ionic

charges also had

similar

molecular

weights.

Although

we

identi-fied

20

SV40- and

21

BKV-specific tryptic

pep-tides, several appeared

as minor

labeled peaks

in

the chromatograms,

and, consequently,

it wasnot

known whether each

peptide peak

rep-resented

aseparate

and unique

peptide

of each

T

Ag protein

or

whether

some

of the

minor

peptides

were

products

of

a

contaminating

pro-tease

activity

in

the

TPCK-trypsin.

Neverthe-less, the peptide profiles ofthe SV40 and BKV T

Ag

proteins were quite

reproducible

from

experiment

toexperiment

and

were

essentially

equivalent

to a

peptide "fingerprint" of each

T

Ag

molecule. Our

interpretation of

only partial

homology

between the

amino

acid

sequences

of

the

SV40 and

BKV T

Ag's

is

compatible

with

results

of T.

Kelley and co-workers

(personal

communications)

that extensive

homology

was

found between

the

early

regions

of the genomes of

SV40 and

BKV

when

the

hybridization

ofthe twoDNAs was

performed under

relatively

non-stringent conditions.

We must

point

out that

when

hybridization of SV40 and

BKV

DNAs

is

done under standard reaction

conditions, little

homology

can

be detected

in

the

early regions of

the respective viral

genomes

(10;

P.

Howley,

unpublished data).

Generally,

whenever

two proteins

have similar

amino

acid

sequences,

the DNA molecules that

encode

them have

a

much

smaller degree of

homology

when tested

by

standard DNA-DNA

hybridization

condi-tions. For

example,

rabbit and

duck

hemoglo-bins

share

70%

of

their amino acid

sequences

(4),

yet

the

hemoglobin

complementary DNAs

hybridize with

one

another

to an extentof only 5%(18).

By

comparing Fig. 1

and

5, it was apparent

that the methionine

peptides

of

T

Ag

from

BKV-infected human cells (Fig.

1) were very

similar

to

those

of

BKV T

Ag

from

transformed

cells (Fig. 5). The major

peptide peaks

in

the

profile shown

in

Fig.

1were

also

present in

the

pattern

shown

in

Fig.

5.

The

differences

ob-served between the

two BKV T

Ag peptide

patterns were

relatively

small and

involved

only

what

we

considered

minor

peptides.

For

example,

the

BKV-specific

peptides eluting

at

position

7

and

just after

position

15

(fraction

178) in

Fig.

1 were

either

absent

or

found

in

reduced

amounts at

the

same

positions

in

the

peptide profile

of

T

Ag

from

BKV-transformed

cells

(Fig.

5). A

peptide

in T

Ag

from

trans-formed cells

that

eluted

just before

peptide

17

(fraction

213, Fig. 5) was

absent

from BKV T

Ag

of

lyrically

infected cells (Fig. 1). The total difference between the methionine

Tryptic

pep-tides of

BKVT

Ag's

from these

cells

appeared

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PEPTIDES OF SV40 AND BKV TUMOR ANTIGENS 325

to

be

too

small

to account for the difference in

molecular weight of these

proteins (113,000 ver-sus 97,000).

It

is

unlikely that this

size

differ-ence is

due

to

macromolecules other than

pep-tides,

since T Ag

from

BKV-transformed cells

does

not appear tocontain carbohydrate or

nu-cleic acid

moieties

(unpublished observations).

One

possible explanation

is

that the

extra amino

acid

sequences

of

T

Ag from

BKV-trans-formed cells (equivalent

to 16,000

daltons) lack

methionine residues. To test this possibility, we are in

the

processof analyzing all of the tryptic

peptides

in

the

BKV

T Ag's from

lyrically

in-fected and

transformed cells by labeling

T

Ag

with a mixture

of amino

acids.

LITERATURE CITED

1. Ahmad-Zadeh, C., B. Allet, J. Greenblatt, and R. Weil. 1976.Twoforms ofsimian-virus-40-specificT-antigen inabortive andlytic infection. Proc. Natl.Acad. Sci. U.S.A. 73:1097-1101.

2. Bonner, W. M.,and R. A. Laskey. 1974. A film detec-tionmethodfortritium-labeled proteins and nucleic acidsinpolyacrylamide gels. Eur. J. Biochem. 46:83-88.

3. Carroll,R.B., and A. E. Smith. 1976. Monomer molec-ular weight of T antigen from simian virus 40-in-fected and transformed cells. Proc. Natl. Acad. Sci. U.S.A. 73:2254-2258.

4. Dayhoff, M.0.(ed.). 1972. Atlas of protein sequence and structure, vol. 5, D53-D54. NationalBiomedical ResearchFoundation,Washington, D.C.

5. Fey, G., and B. Hirt. 1974. Fingerprints of polyoma virus proteinsand mouse histones.Cold Spring Har-bor Symp. Quant. Biol. 39:235-241.

6. Gardner, S. D., A. M. Field, D. V.Coleman, and B. Hulme. 1971. Newhuman papovavirus (B.K.) iso-latedfrom urineafter renal transplantation. Lancet i:1253-1257.

7. Howley, P. M., M. F. Mullarkey, K. K. Takemoto, and M. A.Martin. 1975.Characterization of human papo-vavirus BKDNA. J. Virol. 15:173-181.

8. Ito,Y., N. Spurr, and R. Dulbecco. 1977.

Characteriza-tion of polyoma virus T antigen. Proc. Natl. Acad. Sci.U.S.A. 74:1259-1263.

9. Jonsson,S., and G. Kronvall. 1974. Theuseofprotein A-containing Staphylococcus aureus asasolid phase anti-IgG reagent in radioimmunoassays as exempli-fied in the quantitation of a-fetoprotein in normal humanadult serum. Eur. J.Immunol. 4:29-33. 10. Khoury, G., P. M. Howley, C. Garon, M. F. Mullarkey,

K. K. Takemoto, and M. A. Martin. 1975.Homology andrelationship between the genomes of papovavi-ruses, B. K. virus and simian virus 40. Proc. Natl. Acad. Sci. U.S.A. 72:2563-2567.

11. Maurer, H. R.,and R. C. Allen. 1972. Useful buffer and gel systems for acrylamide gel electrophoresis. Z. Klin.Chem. Klin. Biochem. 10:220-225.

12. Mullarkey, M. F., J. F. Hruska, and K. K.Takemoto. 1974.Comparison of two human papovaviruses with simian virus 40by structural protein and antigenic analysis.J. Virol.13:1014-1019.

13. Shah, K. V., H. L. Ozer, H. N. Ghazey, and T. J. Kelley, Jr. 1977.Common structural antigen of papo-vavirusesof the simian virus 40-polyoma subgroup. J.Virol.21:179-186.

14. Takemoto, K. K.,and M. A. Martin. 1976. Transforma-tionof hamsterkidney cells byBKpapovavirus DNA. J.Virol. 17:247-253.

15. Takemoto, K. K., and M. F. Mullarkey.1973. Human papovavirus, BK strain: biologicalstudiesincluding antigenicrelationshipto simian virus40. J. Virol. 12:625-631.

16. Tegtmeyer, P., K. Rundell, and J. K. Collins. 1977. Modification of simian virus 40 protein A. J. Virol. 21:647-657.

17. Tegtmeyer, P., M. Schwartz, J. K. Collins, and K. Rundell. 1975.Regulation of tumor antigen synthesis bysimian virusgene A. J.Virol. 16:168-178.

18. Verna, I. M.,G. F. Temple, H. Fan, and D. Baltimore. 1972. In vitro synthesis of DNA complementary to rabbitreticulocyte 10S RNA. Nature (London)New Biol.235:163-167.

19. Vogt, V. M., R. Eisenman, andH.Diggelmann. 1975. Generation of avianmyeloblastosisvirusstructural proteins to proteolytic cleavage of precursor poly-peptide.J.Mol. Biol. 96:471-493.

20. Wright, P. J., and G. DiMayorca. 1975. Virion poly-peptidecompositionof the human papovavirus BK: comparison with simian virus40andpolyomavirus. J.Virol. 15:828-835.

VOL. 24, 1977