Interstrain Variation of the Major Internal Structural Component (p30gag) of Two Murine Oncornaviruses: Comparative Immunochemical, Biochemical, and Biophysical Analysis

JOURNALOFVIROLOGY, May 1978,p.522-531 Vol. 26, No. 2 0022-538X/78/0026-0522$02.00/0

Copyright©1978 AmericanSocietyforMicrobiology PrintedinU.S.A.

Interstrain Variation of the

Major Internal

Structural

Component

(p30gag)

of Two

Murine

Oncornaviruses:

Comparative

Immunochemical, Biochemical, and

Biophysical

Analysis

W. NEALBURNETTEt ANDWILLIAM M. MITCHELL*

DepartmentofMedicine, Albert Einstein College ofMedicine, Bronx,New York 10461, andDepartment of

Pathology,

Vanderbilt

University

School

of

Medicine, Nashville, Tennessee 37232*

Received forpublication 3 October1977

The

major

internal structural protein

(p30a9') of the Moloney leukemia virus

and theendogenous Y-1 murine oncornavirus was examined for biochemical and

biophysical manifestations of interstrain

antigenic variation. Although the

two

viral

proteins

share murine

group-specific

antigenic determinants,

the Y-1

virus

p30

appeared

tohaveboth a lower

relative

number of such determinants and

a

decreased

affinity

atthe cross-reactive sites for

Moloney

virus

p30

monospecific

antibodies.

Further, immunological

analysis

indicated the presence of

unique

antigenic sites on the Moloney virusp30 not shared

by

the

analogous

Y-1 virus

molecule. Thetwo

polypeptides copurified

andhad

similar

isoelectric

points (pH

6.2 to

6.3) and sedimentation coefficients

(2.47S).

However, equilibrium

sedimen-tation

yielded

a

significant

mass differencebetween the two

proteins,

28,300

±

600and

31,000

± 300daltons for the

Moloney

and Y-1 virus

molecules,

respec-tively.

Amino acid

analysis indicated

a

concomitant increase

in

total

residues for

the Y-1 virus

p30, although

anumber ofresidues

appeared

tohave

been conserved

between the two viral

proteins.

Conformational

studies

and

hydrodynamic

cal-culations demonstrated marked

secondary

and

tertiary

structural

differences;

with the

Y-1

virus

p30

being

an

asymmetric

prolate

ellipsoid containing

27to

28%

a-helix and

Moloney

virusp30

being

somewhatmore

spherical

and

possessing

an

a-helical

content of 50to 55%.

Two-dimensional

mapping

of

'2II-labeled

tryptic

peptides

of each

p30

suggested

that

considerable

sequence

heterogeneity is

responsible

formany of the

biophysical,

biochemical,

andimmunochemical

dif-ferences in these twoanalogous structural

proteins.

The

provirus

genome

of mammalian

oncor-

reactive determinants

in

the

greatest relative

naviruses

code

for a

number

of

structural

poly-

concentration.

It

does, however,

alsopossess

de-peptides (2) whose

primary

and

secondary

struc-

monstrable

type-specific reactivity,

and

p30

mol-tures

immunologically

define the

interspecies

ecules from various strains

of murine virus

ex-(13, 14,

41),

species-specific (15,

18,

28, 30-33,

36,

hibit

unique antigenic

determinants (42). Unlike

42),

and

type-specific (17,35,39,42,44)

antigenic

the

hemagglutinating

and

interference

proper-reactivities

of various virus strains

in

general,

ties of

gp69/71enV (6,

20), the RNA-dependent

and

analogous proteins

shared among

strains

in DNA

polymerase activity

of the virion reverse

particular.

The

major internal

structural

protein

transcriptase

enzyme

(3,

46),

and the

nucleic

of the murine oncornaviruses is the

approxi-

acid-binding ability

of

p12

(37)

and plO

(12;

M.

mately

30,00-dalton polypeptide,

p309ag

(2,

8, 9,

Schulein,

W. N.

Burnette,

and J. T.

August,

J.

18, 26, 30, 36). The p309ag and the

polypeptides

Virol.,

in

press),

the

p3O

hasasyet no

definitive

p155a5, p125a5,

and

p109a9

are

cleavage products

function,

although

its

involvement

in the process

of a

65,000-dalton

polyprotein

precursor (1, 38,

of

hostrange restriction has

been

suggested (19), 39),

Pr65SaS,

which is itself the

product

of the

and,

because of its

proclivity

for

self-association,

gag

(group-specific

antigen)

gene constituent

(4,

itmay

play

a role in viralcore

assembly

atthe

5) of the

integrated

provirus

genome

(45).

The cell membrane

(8).

p30

hasbeen termed the

group-specific antigen

We

have

previously

described

a number of

(15) because it

possessed

murine-specific

cross-

biophysical

and

biochemical

properties

of

p30

tPresent address: Fred Hutchinson Cancer Research Cen- from the laboratory strain of Moloney murine

ter,Seattle,WA 98104. leukemia

virus

(8).

In this

report

we compare

522

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VOL. 26, 1978 ONCORNAVIRUS p30"a9 INTERSTRAIN VARIATION 523

many of

those

properties

with the p30 of the Y- the slices wereminced;and theproteins were

eluted

1

N-ecotropic endogenous murine

oncornavirus overnight at

370C

in a buffer containing 50 mM

(9)

to

elucidate the

primary and secondary struc- NH4HCO3(pH8.3), 0.05%sodiumdodecyl sulfate,and

tural

characteristics

responsible for the

antigenic

1 mMphenylmethylsulfonyl fluoride. Proteinrecovery

variation exhibited

by

this

evolutionarily

con- wasroutinely 80 to 90% by thisprocedure. The eluants

served

polypeptide.

were

centrifuged briefly

toremove

gel

debris,

KCl

was

addedtothe supernatants to a final concentration of 0.2 M,and, after 15min onice, the precipitates were MATERIALS AND METHODS

collected

by

centrifugation

(7).

The

precipitates

were

washed once with ice-cold acetone containing 0.1 N

Celis and viruses. Moloney leukemia virus was HCI, washed again with acetone alone, and dried.

propagatedinhigh-passage-levelSwissmouseembryo They were suspended in20

Al

of 0.5 M sodium

phos-cells maintained in Eagle minimal essential medium phate (pH 7.5)containing 0.1% sodium dodecyl sulfate, with 10% fetal calfserum.Murine adrenal carcinoma heated for2 min at 100°C, and allowed to cool. The cells(clone Y-1, ATCCno.CCL-79) producing the N- proteinpreparationswerethen radioiodinated by the tropic Y-1 virus were grown in Ham F-10 medium chloramine T method (21), and free radioiodine was containing15% fetal calfserumand 2.5% horse serum. removed from the quenched reaction mixtures by pas-Virus was harvested and purified as previously de- sageovercolumns(0.8by14.5cm) ofSephadex G-25

scribed (9). superfine equilibratedinabuffer of 1.0M

Tris-hydro-Antisera. Heterospecific goat anti-Moloney leu- chloride (pH 8.6) with 0.1% sodium dodecyl sulfate. kemia virus (1S-166) and goat anti-AKR virus (2S- Reduction and S-aminoethylation were performed by 296) sera were obtained through the courtesy of R. modificationsofpreviously published procedures (10, Wilsnack, Huntingdon Laboratories, Md. Monospe- 11).Briefly, theradioiodinated proteins from the G-cific antiseraagainst p30wereobtainedby hyperim- 25-excluded volumes (1 ml each) were reduced by munization of rabbits with the purified protein (see addingdithiothreitolto afinal concentration of 0.1 M,

below) incompleteFreundadjuvant followedbytwo flushing the reaction vessels with N2, capping them,

fortnightly boosters in incomplete Freund adjuvant. and incubating them for3 h at 370C. The reduced

Goat anti-rabbit immunoglobulin G was purchased proteinpreparationswerethenS-aminoethylated by fromCalbiochem(LaJolla, Calif.). thesequential addition ofone10-,lI portion and two 5-p30 Purification. 5-p30was purifiedunder nonde- ,ul portions ofethyleneimine (ICN Pharmaceuticals, naturing conditions fromcorepreparations of virus by Inc.)at10-min intervals underaN2 atmosphere. A

50-phosphocellulose ion exchange andSephadex G-100 jig quantity of carrier bovine immunoglobulin was

(Pharmacia) gel filtration chromatography as de- addedtoeachpreparation (16),and the mixtures were

scribedbyStrand andAugust (43).Column effluents precipitatedat4°Cwith 10%trichloroaceticacid.The

were monitored for relative fluorescence at 287 precipitates were collected by centrifugation and

nm/348nm. washed twicewithice-coldacetone.Thedried

precip-Gelelectrophoresis.Sodiumdodecylsulfate-poly- itateswere

resuspended

in50

,lt

of50mM NH4HCO3

acrylamide gelelectrophoresiswasperformed in cylin- (pH 8.3), 1 ,ug of tolylsulfonyl phenylalanyl

chloro-drical 10%polyacrylamide gelsaspreviouslydescribed methyl ketone-treated trypsin (Worthington

Bio-(8). chemicals) was added to each suspension, and the

Radioiodination.Purifiedp30wasradioiodinated preparationswereincubated for3to4hat370C.An

forradioimmunoassaybythechloramine T method of additional 1,ug oftrypsinwasaddedtoeach

prepara-Hunter(21)andchromatographedonSephadexG-25 tion,and incubationwascontinuedovernight.

Diges-superfinetoremoveunbound radioiodine. tionwasroutinelyfoundtobemorethan 90%complete

Immunodifusion

andradioimmunoassay. Im- as measured

by

inclusion on

Sephadex

G-25. After

munodiffusion wasperformed in 1% Difco agarcon- briefcentrifugationtoremoveinsolublematerial,the

taning100mMNaCl,1mM

EDTA,

and10mMTris-

samples

were

spotted

atonecornerof

Polygram

Cel

hydrochloride(pH 7.2) for24 to 72hatroomtemper- 300plastic-backedcellulose sheets(Brinkman

Instru-atureand thenfor24hat4°C. Thegelswerewashed ments). The sheetswerewetted withapH3.5buffer

extensivelyinphosphate-buffered saline and stained ofpyridine-aceticacid-water(1:10:89)and

electropho-withCoomassie brilliant blue R-250. Competition ra- resed under Varsol

(Savant

Instruments, Inc.)for

ap-dioimmunoassay was performed as described by proximately30 min at1,000Vwith acid fuchsinas a

Strand and August (42); unlabeled p30 competed migration marker. The sheetswere dried and chro-againstradioiodinated p30for antibodies inamono- matographedatascendingright anglestothe direction

specificantiserum dilution sufficienttobindapproxi- ofelectrophoresis inanatmosphere-equilibrated tank

mately50% of thetrichloracetic acid-precipitablein-

containing

a

pH

5.3 buffer of n-butanol-acetic putradioiodinatedprotein.Precipitationwaseffected acid-water-pyridine (15:3:12:12). The sheets were bytheaddition of goat anti-rabbitimmunoglobulin G, again dried,and autoradiography wasperformed by

and theprecipitatewaswashedextensivelytoremove exposuretoKodakno-screenmedicalX-rayfilm.

nonspecific radioactivity. Radioactivityin theprecip- Other biochemical and biophysical analyses.

itate was determinedby gamma counting. Extinctioncoefficient determinations, liquid column

Peptideanalysis.Y-1andMoloney leukemiavirus andpolyacrylamide gelisoelectricfocusing,

sedimen-p30 bands, each containing approximately 5 ug of tation equilibrium andvelocity analysis, amino acid

protein,were cutfrom driedCoomassiebrilliant blue- analysis, and far-UV circular dichroism were per-stained sodium dodecyl sulfate-polyacrylamide gels; formedaspreviouslydescribed(8).

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524 BURNETTE AND MITCHELL J. VIROL.

RESULTS

5

Purity of

the protein

preparations.

Frac-

0

x

tionation

of the

solubilized

core

proteins

from

the

Moloney and Y-1 viruses

on

phosphocellu-

z

40

lose

with

a

continuous

gradient

of

0 to 1.0

M

+

KCI revealed

a

major

peak for both viruses

at

about 0.2 M

(Fig.

1).

When these

peaks

were 3

subsequently

rechromatographed

by gel

filtra- ! 30

tion,

a

single

homogeneous

peak

was

observed

w

for each

preparation

in

the

30,000-molecular-

z

weight region of the column effluent

(Fig. 2).

' 20

-Electrophoresis

of

these

peak

fractions

in the l

presence

of sodium

dodecyl

sulfate

demon-

°

strateda

single

bandfor each

p30 preparation

at J l

about

30,000

daltons

(Fig. 3),

although

it

is

ap- w 10

parent

that the

Moloney

virus

p30

migrates

P

slightly

faster, and thus

has a

lower

molecular

-J

mass,

than the

corresponding

Y-1 virus

protein.

Isoelectric

focusing

in

a

sucrose-ampholyte

col-

20 40 60

80

umn

(Fig. 4)

yielded

an

isoelectric

point

of

ap-

FRACTION

NUMBER

proximately pH

6.3

for

both viral

proteins.

The

FIG 2.

Purification of

the

major

structuralprotein

minor peak

seen as a

shoulder

at

about

pH

7

of

_ofteMoloney

theI

2. P _and

anYmno

_Y-1

_murine

e

oncrnaviruses

_{oncornaviruses}

_by may be

analogous

to the

heterogeneously

molecular sieve

chromatography.

The peak fractions

charged species of p30 observed

by

Oroszlanet elutingfrom

phosphocellulose

at

approximately

0.2

al.

(33)

in

complement

fixationassays

of isoelec-

M KCI were concentrated by precipitation with

tric-focusing

column effluents.

High-resolution

(NHJ)2S04

at 75%saturation and

chromatographed

inparallel columnsof Sephadex

G-1X00

inabuffer of

1.0M

NaCl,

1 mM EDTA, and 10mM

N,N-bis(2-If,

I I | I |

.hydroxyethyl)-2-aminoethanesulfonic

acid (pH

6.5).

O Moloney leukemia virusprotein (A);Y-1virusprotein

x

().

Z40_

30-

_

I.O

0~~~~~~

S

_

_-

C0

BOVINE

ALBUMIN-o-

J

z

(67,000)

w

20C

2 6 1 0

w 0

U.

10

CYToCHRioMEC--

ma s

U

a

>

~~~~~~~~~(12,4

00)

-aJ

20

406080 100

1 2

34 56

FRACTION NUM BER FIG. 3. Sodiumdodecyl

sulfate-polyacrylamide

gel

FIG. 1. Ion-exchange chromatographic fractiona- electrophoresis ofthemajorstructuralproteinofthe tion oftheproteinsof Moloneyand Y-1 murineon- Moloney and Y-1 murine oncornaviruses. Electro-cornaviruses. Moloneyleukemiavirusand Y-1 virus phoresis was performed in 10%polyacrylamidegels weredisruptedin Triton X-100, the soluble proteins in thepresenceof 0.1% sodium dodecyl sulfate and 3

were appliedtoparallelcolumnsofWhatman P-11 Murea asdescribedin thetext.

(1)

and(6), molecular

phosphocellulose, andp30waselutedwithalinear weightmarkers; (2) wholedisrupted Moloney

leuke-gradient of0 to 1.0 MKCI in 10 mM N,N-bis(2- miavirus; (3) purified Moloney virusp30;(4)purified

hydroxyethyl)-2-aminoethanesulfonic acid (pH 6.5), Y-1virusp30;(5) wholedisruptedY-1virus.Protein

and1 mMEDTA.Moloneyleukemia virusproteins bandswerestained withCoomassiebrilliantblue

R-(A); Y-1 virusproteins(0). 250.

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VOL. 26, 1978 ONCORNAVIRUS p30gag INTERSTRAIN VARIATION 525

<,

zT

115.0

~ ~ ~ ~ ~ 80z 6

p

LL0

10 20 30 40 50

FRACTION NU MBER

FIG. 4. Columnisoelectric focusing of the purified

major structural protein of the Moloney and

Y-l1

,/\ 26

murine oncornaviruses. The viral proteins were fo- \

cused in a 1.2%XampholytepH gradient of 3.5 to 10at/\

4°C. The pHgradient was stabilized with a 0 to40%o4

sucrose gradient to prevent convection, and focusing n3)

was at 500 Vfor the time indicated. Moloney leukemia

pH(A.viu poein 268

hC0;Ylvrsprti,9

;

(g B P39W

focusing in a

polyacrylamide-ampholyte

matrix

_________________

(not

shown) demonstrated a difference of about

FG.5Im

ndifsoaalisfthmune

0.1 pH unit between the isoelectric pointsofthe

spcFIc.

crs-eatvt

ImndfsonalsOf

Mooethe

mn

-

iusie

two e . w p30

protens.

Immunodiffusion

wasperformed as

de-being the slightly more basic component. No scribed in the text with the purified viralcomponents.

evidence of minor speciesin these gelswas ob- JS-166,goat anti-Moloney virus serum; 2S-296, goat

served.

Thus, the minor charge

heterogeneity

anti-AKR virus serum. Gels were stained with

Coo-obtained with column isoelectric focusing may massie brilliant blue R-250. (A) Immunodiffusion

secondary to self-association (8) at the higher plate; (B) illustration ofimmunodiffusion plate to

concentrations required

for analysis. highlight spur.

Immunological

analysis.

In

immunodiffu-sion (Fig. 5), the

Yim andite Moloney

virus p30

polypeptides

extensively cross-reacted with

both_______________

anti-Moloney

virus

and

anti-AKRvirushetero- _

specific

sera. The

presence

of a small spur

in the

Z a

precipitin

band

pointing

toward the Y-1 p30 wellm 80

with the anti-Moloney

virus

serum

and the ap-

FIG. 5 I

parent

absence

of

spurs in the

cross-reaction 60 _ a YI rus

with the

antiserum

directed against AKR virus,

5

p \ x

a

virus very

similar

to

Y-1 (9), indicated the j; X 40 - w \

presence

of

detemninants

on the

Moloney

virus s 2 goa

p30 not shared by

the

Y-1 virus

protein.

In XiAR20 _ \

competition

radioimmunoassay

with

monospe-lateXto

cific

anti-Moloney

virus p30 serum, the

differ-ences

between the

two

proteins became more '

l-5

loo

o-obvious

(Fig. 6). Although

Y-1 p30 could

COMnyiuNGPROTEINmg)

com-pete

with

Moloney p30, indicative of

the

group-specific

and type-specific cross-reactivity ob- FIG. 6. Analysis of the antigenic

determinants

of

served inimmunodiffusion, the

displacement

of the p30 of Moloney and Y-1 murine oncornaviruses.

ed

c

peition

c thed

slo

Competition radioimmunoassay was performed with

t sa final dilution of monospecific anti-Moloney virus

relative

to the homologousMoloney

virus

pro-

p30serum

50%4

sufficient to precipitate approximately tein competition, and the failure of the

Y-1

Of theradioiodinated

Moloney

p30 intheassay. The

polypeptide

to compete fully for all the antibody amount ofcompetingproteinis asindicated.Moloney

species

in the

anti-Moloney

p30

serum

popula-

virus

p30

(-);

Y-1 virus p30

().

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526

BURNETTE AND MITCHELL J. VIROL.

tion

clearly

demonstrated unique

determinants formed in the analytical ultracentrifuge, with

present on

the

Moloney virus p30 that

were

not

initial loading concentrations and ionic strength

exposed

on

the

analogous

Y-1

protein.

favoring the monomer. Under these

conditions

Amino acid

composition.

The amino acid the

s'20w

for both the Y-1 and

Moloney virus

p30

compositions of the Moloney virus and the Y-1

proteins

was

found

to

be

2.46 to

2.47S

(data

not

virus

p30 polypeptides

are

presented

inTable 1.

shown). However,

high-speed,

meniscus-deple-The best integral fit of the recovery data was

tion

equilibrium sedimentation revealed

a

sig-obtained with three residues of histidine

for the nificant

difference

in the mass

of

the

two

pro-Y-1

and four residues for the Moloney

virus p30. teins

(Fig. 7),

confirming the

initial observation

Because of its

greater

apparent mass, the Y-1 in sodium

dodecyl

sulfate-polyacrylamide

gels.

virus

polypeptide

was found to

contain

more Using partial specific volumes of 0.719 ml/g for

residues than

the Moloney protein. However, Moloney virus p30 and 0.722 ml/g for Y-1

p30

the

total number

oflysine and alanine residues (determined from amino acid analysis) for the

appeared

tohavebeenconserved, and histidine calculation of weight average molecular

weights

and

methionine were actually in excess in Mo- and extrapolating to zero concentration, we

ob-loney

virus

p30.

Two to three half-cystine resi-

tained

molecular weights of

28,300

+ 600 forthe

dues

were

assigned

toboth

polypeptides

since in Moloney virus protein and 31,000 ± 300 forthe

each

casethe calculated value wasfound to be Y-1

p30.

nonintegral.

On thebasis of the mass and

velocity

param-Sedimentation

analysis. Although p30

ex- eters, a

frictional ratio relative to a perfectly

hibits

a

marked

proclivity

for

concentration-de-

spherical ellipsoid could

be

calculated.

A

ratio

of

pendent self-association (8), sedimentation

ve- 1.38 for Moloney

virus p30 and 1.45 for Y-1

p30

locity and molecular weight analysis

can

be per-

corresponded to axial ratios of about 5 and

6,

TABLE1.Amino acidcompositionof the major internal structural protein of the Y-1 andMoloney oncornavirus

Apparentresidues per mol ofprotein'

Aminoacid Y-1 virusprotein Moloneyvirus

protein(

Aminoacid_

~~~~~~~~~~~~~~~~~Observed

(Y-1)

Avg or extrapo- Assumed compo- Avgorextrapo- Assumed compo- minusMoloney lated value sition lated value sition

Lysine... 16.7 17 17.3 17 0

Histidine.3.0 3 4.0 4 -1

Arginine.31.2 31 28.6 29 +2

Tryptophanc.3.8 4 3.0 3 +1

AsparticAcid.29.1 29 26.9 27 +2

Threonine .18.0 18 14.5 15 +3

Serine .17.0 17 15.8 16 +1

Glutamic Acid.51.2 51 44.5 45 +6

Proline.20.1 20 16.3 16 +4

Glycine.18.3 18 16.0 16 +2

Alanine .14.4 14 13.6 14 0

Half-cystined.2.6 3(2) 2.3 2(3) (0-1)

Valine .9.2 9 7.5 8 +1

Methionined.2.0 2 3.2 3 -1

Isoleucine.6.6 7 4.8 5 +2

Leucine.35.0 35 27.8 28 +7

Tyrosine... 6.3 6 5.3 5 +1

Phenylalanine 5.6 6 5.9 6 0

Calculatedmolwt 33,400 30,000

"Calculated with histidineastheintegral residue. Analyseswereperformedin

triplicate

onsamples dialyzed extensivelyagainstdistilledwater,lyophylized,andhydrolyzedat110°Cfor20,40, and 72 h, respectively, in 6 NHCIcontaining0.01%(vol/vol) phenoltopreservetyrosineresidues. Therecovery values ofthreonineand serinewereobtainedbyextrapolationof the recoveries from the varioushydrolysistimestozerotime.Valine, leucine, and isoleucine valuesweretakenastheirmaximum recoveriesfrom the timed HCIhydrolyses. The

remaining residuerecoveryvalueswereobtained fromanaverage of the timedHCIhydrolyses.

bDataforMoloneyvirus

p30

taken fromBurnette etal.(8).

cTryptophananalyzed after hydrolysisat115°Cfor 22 hin4Nmethanesulfonic acid.

dHalf-cystine determinedascysteic acid and methionineasthesulfone byperformic acidoxidation

followed

by20hof HCIhydrolysis.

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VOL. 26, 1978 ONCORNAVIRUS p305&S INTERSTRAIN VARIATION 527

respectively,

assuming

a

hydration value of

0.3

ordered

secondary

structure as

reported

previ-g

of

water per g

of protein. These ratios indicate

ously

for

the

Moloney

virus

p30

alone (8),

that,

although both

are

apparently asymmetric

namely,

a

resolution of the

patterns into

a

min-ellipsoids, the Y-1 p30 is

a more

elongated glob-

imum number of

gaussian

curves to

achieve

a

ular molecule.

best fit of the data and a

least-squares

fit of

Conformational

analysis.

Figure

8

illus-

ellipticity

to a

linear combination

of

the

possible

tratesthe

major conformational differences be-

structural contributions.

A

tabulation

of

the

ro-tween

the

analogous

Moloney and Y-1 virus p30

tational

strengths

of the resolved

gaussian bands

proteins

as

determined

by far-UV circular di-

by the former method is presented in Table

2,

chroism. We have utilized

two

independent

although both

procedures

gave

closely

compa-methods for

estimating the

type

and

amount

of

rable results. While both

polypeptides have

1

I

I

I

In6 - 600 ll l

I

0~~~~~~~

0.05 0.10 0.15 0.20 0.25 /

Ci(OPTICAL

DENSITY AT 280nm) E

0

E E 10

B.

~~~~~~~~~~~~~~B

300 EU

0~~~~~~~~~~~~2

0.05 0.10

Ci

(OPTICAL DENSITYAT280nm) 200 210 220 230 240 250

FIG. 7. Plotsof l/molecular weight versus initial WAVELENGTH (nm) loading concentration in analytical ultracentrifuge

cellfor the major internal structuralproteinof the FIG. 8. Far-UV circular dichroic spectra of the

Moloneyand Y-1 murine oncornaviruses.High-speed majorinternal structuralprotein oftheMoloneyand

sedimentation equilibrium wasperformed asprevi- Y-1 murine oncornaviruses. Circular dichroismwas

ously described (8), and the molecular weights at performed in 0.5-mmpath length fused-silica cells various initialprotein concentrations wereextrapo- from 250nm to200 nmin0.2Mborate(pH8.0) and latedto zero concentration by the method ofleast 0.15Mpotassiumfluoride. (A)Moloneyvirusprotein,

squares. (A) Moloney leukemia virusprotein; data 360 pg/ml. (B) Y-1 virusprotein, 350 ug/ml. [e]',

takenfromBurnetteetal. (8). (B) Y-1 virus protein. Reducedmeanresidueellipticity.

TABLE 2. Meanresidue rotational strengths of the far- UV circular dichroic bands of the major internal structuralprotein of Moloney leukemia virus and Y-1 virus

Virusprotein and

XOa

[OImaxb

Rc Transition Conformation %Conformation'

Y-1

223 nm -10,500 -5.81 n-X* a-helix 28

215nm -3,500 -0.83 n-S7*

fl-structure

11

Moloneyleukemia

224 nm -17,000 -10.88 n-7T* a-helix 52

216rum -3,000 -0.80 n-f* fl-structure 10

192nm +41,000 +17.25 IT-IT* a-helix 50

A., Wavelengthofcircular dichroictransition.

b

[6]max,

Maximummeanresidue

ellipticity.

R, Rotational strength in cgs units, x

10-,

of each resolved gaussian band determined as previously

described(8).

dBasedon acomparison of the resolved gaussianband rotationalstrengths calculatedaspreviously described (8).

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528 BURNETTE AND MITCHELL J. VIROL.

about 10 to 14%

fl-structure,

Moloney

p30

ex-

hindered,

and

proline-adjacent arginyl

and

lysyl

hibits

an

exceptional

50 to 55%

a-helicity

(8), bonds thatare not

hydrolyzed by trypsin.

whereas the Y-1

p30 is predicted

to possess a

significantly

lower 27 to

28% a-helix. The

re- DISCUSSION

mainder

of

the

structure is

presumably

ape- The

principal physical

and

chemical

charac-riodic.

teristics

of

Moloney leukemia

virus

p30

and the

Tryptic

peptide

analysis.

Two-dimensional

endogenous

Y-1

virus

p30

are

summarized

in

peptide

mapping

(Fig.

9) demonstrates that the

Table 3. For

analogous proteins

from two

strains

Y-1

and

Moloney

virus

p30 proteins share

two of murine

oncornavirus possessing

group-spe-major

radioiodinated

tryptic

peptides.

However,

cific

cross-reactivity, they display

a

surprising

the

Y-1

virus

p30

appearstopossess

five

unique

dissimilarity.

Although

they share

common

iso-major iodinated

peptides, and the

Moloney

leu-

electric points

and

sedimentation

coefficients,

kemia virus

p30,

three.

In

view of

this

observed

distinct

differences exist

in

molecular

weight,

sequence

heterogeneity,

it is not

surprising

that amino acid

composition, tryptic peptides,

and

the two

proteins exhibit

significant type-specific

conformation.

The

conformational disparity

ob-antigenic variation

and

secondary

structural

dis- served by

circular dichroic analysis

is, in

con-parity.

junction

with the

peptide maps,

the most

signif-Assuming that

each

radioiodinated

tyrosine

icant

interstrain variation detected

as an

expla-residue is

located

on a separate

peptide,

the

nation of the

observed antigenic

heterogeneity.

number of labeled

major

peptides corresponds

The

helical

differences

are most likely due to

rather well

to

the amino acid

analyses.

The

presence

of minor

peptides that

amount to

less

than

10%

of

the total

radioactivity

may be the

TABLE

3.

Summary of the

physical

and chemical

result of a number of

secondary

reactive proc- properties of the major internal structural protein of

esses,

such

as

diiodination of

tyrosine

and for-

Moloney leukemia virus and

Y-1

virus

mation

of

monoiodohistidine

(22),

incomplete

S-

Majorstructuralprotein

aminoethylation

of

cysteinyl

residues

(23, 24)

Property Moloney

leuke-and other chemical modifications

(27),

arginyl

miaS

virusav

and lysyl residues that are

conformationally

Extinction coefficient

(E1cm,28)

13.7 9.5C

Absorption maximum

(nm) 280-281 279

Isoelectricpointd 6.2 6.3

Molecular

weighte

28,300

±600

31,000

±300

Partial

specific

volume

o G

~~~~~~~~~~~(ml/g)f

0.1...0.71 0.722

,_. ,.m Sedimentation

coeffi-s.

_

952

,

,--'cient (so,w)

Z..;

2.47 2.47

Diffusion coefficient

(D2w)

(cm2/s)....

7.33xi07 6.69x 10-v a

-,

,-~~,-:_

- -

~Frictional

ratio

(f/fW)

1.38 1.45

o

Amino acid

residuesh

259 290

I'--',_a-Helicity (%).50 27

/3-Structure (%). 10-13 11-14

Major radioiodinated tryptic peptides

(shared/unique) 2/3 2/5

aDataonMoloneyvirus

p30

taken fromBurnette

<|

~~~~~~~~~~~~~~~et

al.(8).

ELECTROPHORESIS bDeterminedboth bydryweightmeasurementand

[image:7.509.268.458.317.545.2]

synthetic boundary formation.

FIG. 9. Two-dimensional trypticpeptideanalysis cDeterminedbydryweightmeasurementonly.

ofthe Y- 1andMoloneyleukemiavirusp30proteins. d Difference estimatedon thebasis offocusing in Thepurifiedproteins wereiodinated,

S-aminoethyl-

polyacrylamidegels.

ated,digestedwithtrypsin,andmappedonthin-layer eDeterminedbyhigh-speedsedimentation

equilib-sheetsasdescribed in the text.

Electrophoresis

was rium.

in the horizontal plane and

chromatography

was fCalculatedfrom amino acid

composition.

vertical (ascending). Symbols:

0,

shared

major

pep- g Calculated from sedimentation

equilibrium

and tides;,, shared minorpeptides;

0,

unique

major

velocity data.

Moloney viruspeptides;

*0,

unique major Y-1 virus h Basedontimed

hydrolysis

and aminoacid

recov-p30 peptides. ery data.

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[image:7.509.67.255.366.560.2]

VOL. 26, 1978 ONCORNAVIRUS p309ag INTERSTRAIN VARIATION 529

the

presence

of amino acid sequences and par-

kemia virus

(W.

N.

Burnette,

S.

Duttagupta, and

ticular residues (e.g., proline) that

act as

helix-

R. Soeiro, manuscript

in

preparation).

Such

in-breakers

at

critical

regions

in

the

Y-1 virus p30.

tragenic

exclusion could account for the

approx-These sequentially different

and/or

additional

imately

9% difference in molecular

weight

seen

residues in the Y-1

polypeptide

henceinterfere between theN-tropic Y-1 virus p30 and the

p30

with the formation of

a-helix

as

observed

inthe from the

NB-tropic Moloney

virus used in our

Moloney

virus

molecule.

How the

conforma-

studies. Intramolecular recombination

has been

tional differences affect the

tertiarystructure of

previously

described in

oncornaviruses

by Wyke

these

proteins is

not

known, although the hydro-

et

al. (47)

for

transformation-dependent,

tem-dynamic studies indicate

someminorratio

dif-

perature-sensitive

mutantsof the Rous sarcoma

ferences.

Steric hindrance

or otherfactors due

virus

genome.

to

regional conformational

differences about the The sequence

heterogeneity

observed in these

immunologically

cross-reactive sites

may con-

investigations is also compatible with

point

mu-tribute

to

the

observed decrease

in

antibody

tations.

Such

point mutations would give rise

to

affinity for the

Y-1

virus

p30 molecule.

helix-forming amino acid residues and

sequences

On

a

biological level,

the

Moloney leukemia

and thus

to

the

increased

helicity of the Moloney

virus

and the

Y-1

endogenous

virus

also

differ virus p30, since the 9% mass

exclusion

probably

greatly. The Moloney virus is highly oncogenic

cannot

fully

account

for the

22 to

28%

difference

in vivo

and

displays high

titer

infectivity

with

in

helicity between the proteins from the

endog-NB-tropic host

range in

vitro

(C. Riggin,

W. N. enous

and

exogenous

viruses. Further, point

mu-Burnette, and W.

Mitchell,

unpublished

data). tations

could also be the

source

of the

mass

Since the

laboratory strain

of

Moloney leukemia

difference

by leading

to

altered

processing and

virus arose

from

an

endogenous murine

virus

cleavage

of the

p30 from the

gag gene

polypro-(24, 25) as a

result

of

the

application

ofevolu-

tein precursor. If altered processing

were the

tionary

pressures

designed

to

select for enhanced

case, one

might

expect a

concomitant

mass

infectivity

and

oncogenicity,

it is

reasonable

to

change

in

those

polypeptides (e.g., pl10a9) that

expect

these

pressures to

be

reflected

in

both

are

contiguous with the p30agin the polyprotein.

the

structure

and

functional

nature

of

its

gen-

Alternate cleavage of the

precursor

for Rauscher

omic

products. The

nature

of the variations that

leukemia virus

envelope glycoproteins is thought

give rise

to

the

immunochemical differences, for

to

be the

source

of

the

twocomponents

of

the

example,

can

be ascribed

to

the chemical and

gp69/71efv complex (W. N. Burnette and J.

T.

physical disparities noted in this study. Con-

August,

Fed.

Proc.

35:1736).

versely, the conservation

of the intrinsic charge A

functional

approach

to

the effect of

evolu-and

general

shape between the p30 molecules

tionary

pressure on

the

major internal structural

argues

for

an

important functional

role, such

as

protein

component

of mammalian

oncornavi-molecular association

(8) and subviral

core

self-

ruses

has

notyet

been

possible,

since

the

func-assembly.

tions of this

polypeptide

have

yet to

be

estab-Our data

suggest

considerable

sequence het-

lished.

However, investigations

to

elucidate the

erogeneity

between the

major

structural

pro-

functions of viral

p30,

particularly

in

relation

to

teins of these

two

viruses, although

Oroszlan

et

its

possible

role

in

virion

self-assembly

and host

al.

(29) have described

only

a

single

amino

acid

range

restriction,

are

currently

in

progress.

difference in the first

24

N-terminal residues of

the

p30

proteins

from six different

murine on- ACKNOWLEDGMENTS

comaviruses. Itispossiblethat theN-terminus Thisstudy was supported by PublicHealthServiceGrant

isthemosthighlyconservedportionofthemol- CA 14792 from the National Cancer Institute and bythe

ecule and

may

be the

portion responsible

for the

American Cancer Society.

species-specific

_{specirthees-specific immuhanologic}

immunological cross-reactivity.

_{whicrss- heati}

Theinvaluable assistance of Leslie

Holladay

intheanalysis

of thecircular dichroic datais

gratefully

acknowledged. The Nevertheless, the mechanism by which the het- iodinated peptideanalysis procedure was developed jointlyby

erogeneityarises is notcurrently understood.On W.N.B. and MarkKrantz in theDepartment ofMolecular

astructurallevel, unequal crossingoverwithin Biology, Albert Einstein College ofMedicine, Bronx, New

the p30 gene during recombinational events York.Quantitative optical density scans of thetrypticpeptide

couldhaveledto the exclusionof genetic infor- mapswereprovided byW.Lutin,VanderbiltUniversity.

mation from the

endogenous virus

that is not

LITERATURE

CITED

essential,

and

perhaps inimical,

to exogenous 1. Arcement,

L.

J., W.

L.

Karshin, R. B. Naso, G.

viral infectivity

and

oncogenicity

functions.

This Jamjoon, and R. B.

Arlinghaus.

1976.Biosynthesis

isparticularly intriguing with respect to recent ofRauscher leukemia viralproteins:presence ofp30

observationsof massvariationinp30molecules and envelopep15 sequences in precursorpolypeptides.

inhostangerevrtants o Moloneyleukemia Virology69:763-774.

in host range revertants of Moloney leukemia 2. August, J. T.,D. P. Bolognesi, E. Fleissner, R.V.

virus

(19)

and hostrange clones of Friend leu-

Gilden, and

R. C.

Nowinski. 1974.

A proposed

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530 BURNETTE AND MITCHELL J. VIROL

menclature for the virion proteins of oncogenic RNA Weir(ed.), Handbook of experimental immunology. F. viruses.Virology 60:595-601. A.DavisCo., Philadelphia.

3. Baltimore, D. 1970. RNA-dependent DNA polymerase 22. Lamoureux,G., P. R. Carnegie, and T. A. McPherson. in virionsof RNA tumour viruses. Nature (London) 1967. Experimental allergic encephalomyelitis-226:1209-1211. propertiesofaniodinatedencephalitogenicpolypeptide. 4. Baltimore, D. 1974. Tumor viruses. Cold Spring Harbor Immunochemistry4:273-281.

Symp.Quant.Biol. 39:1187-1200. 23. Lindley,H. 1956. Anewsyntheticsubstrate fortrypsin 5.Barbacid, M., J. R. Stephenson, and S. A. Aaronson. and itsapplicationto thedetermination of the amino-1976. gag Geneofmammalian type-C RNA tumour acid sequence of proteins. Nature (London) viruses. Nature(London)262:554-559. 178:647-648.

6. Bilello, J. A., M. Strand, and J. T. August. 1977. 24. Moloney, J. B. 1960. Biological studies on a lymphoid Expression of viralenvelope glycoproteinand transfor- leukemia virus extracted from sarcomaS37.I.Origin mationgenes in cells transformedby a defective kirsten and introductory investigations. J. Natl. Cancer Inst. murine sarcoma virus. Virology77:233-244. 24:933-951.

7. Bray,D., and S. M. Brownlee. 1973.Peptidemapping 25. Moloney, J. B. 1966. A virus-inducedrhabdomyosarcoma of proteins from acrylamide gels. Anal. Biochem. ofmice.Natl.Cancer Int. Monogr.22:139-142. 55:213-221. 26. Nowinski, R. C., E.Fleissner,N. H.Sarkar, and T. 8. Burnette, W.N.,L.A.Holladay,andW. M. Mitchell. Aoki. 1972. Chromatographic separationandantigenic 1976.Physicaland chemicalpropertiesofMoloneymu- analysis of proteins of theoncornaviruses. II. Mamma-rineleukemia virusp30protein:amajorcorestructural lianleukemia-sarcomaviruses. J.Virol.9:359-366. componentexhibitinghighhelicity and self-association. 27. Offord, R. E.1966.Electrophoreticmobilitiesofpeptides J.Mol. Biol. 107:131-143. onpaper and their use in thedetermination of amide 9. Burnette,W. N., C. H.Riggin, and W. M. Mitchell. groups. Nature (London)211:591-593.

1974.Physicalandchemicalpropertiesofanoncorna- 28. Oroszlan, S., D. Bova, R. J. Huebner, and R. V. virusassociatedwithamurine adrenal carcinoma cell Gilden. 1972. Major group-specific protein of rat type line.J. Virol. 14:110-115. Cviruses. J.Virol. 10:746-750.

10. Chen, K. C. S., T. J. Kindt, and R. M. Krause. 1975. 29. Oroszlan, S., T. Copeland, M. R. Summers, G. Smy-Primary structure of the L chain fromarabbit homo- thers, and R. V. Gilden. 1975.Amino acid sequence geneousantibodytostreptococcal carbohydrate. I. Pu- homology of mammalian type C RNA virus major in-rification ofantibodyandsequence determination of temalproteins. J. Biol. Chem.250:6232-6239. peptidesfroma-cnymotrypticandthermolyticdigests. 30. Oroszlan,S., C. L. Fisher, T. B. Stanley, and R. V. J.Biol. Chem. 250:3280-3288. Gilden. 1970. Proteins of the murine C-type RNA 11. Cole, R. D. 1967.S-aminoethylation.MethodsEnzymol. tumorviruses: isolation of a group-specific antigen by

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12. Davis,J.,M.Scherer, W. P.Tsai,and C.Long. 1976. 31. Oroszlan,S., C. Foreman, G. Kelloff, and R. V. Gil-Low-molecular-weightRauscher leukemia virusprotein den. 1971. Thegroup-specific antigen and other struc-withpreferential bindingforsingle-strandedRNA and tural proteins of hamster and mouse C-type viruses. DNA.J. Virol. 18:709-718. Virology 43:665-674.

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NB-tropic murine leukemiavirusesderivedfrom a B- 39. Stephenson,J.R., S. R.Tronick, and S.A.Aaronson. tropic virus ofBALB/c have altered p30. J. Virol. 1974.Analysisoftypespecific antigenicdeterminants 21:309-318. of twostructuralpolypeptidesof mouseC-typeviruses. 20. Hunsmann,G.,V.Moennig,L.Pister,E.Seifert,and Virology58:1-8.

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