Structural studies of retroviruses: characterization of oligomeric complexes of murine and feline leukemia virus envelope and core components formed upon cross-linking.

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0022-538X/79/04-0157/09$02.00/0

Structural Studies of Retroviruses: Characterization of

Oligomeric Complexes

of Murine and

Feline

Leukemia Virus

Envelope and Core Components

Formed

upon

Cross-Linking

ABRAHAM PINTER* AND ERWIN FLEISSNER

MemorialSloan-KetteringCancer Center, New York, New York 10021 Received forpublication13September1978

Toexamine the protein proximity and subunit organization of type C

retrovi-ruses,preparations of AKRmurine leukemia virus were treated with bifunctional

cross-linkingreagentsandanalyzedby sodium dodecylsulfate-polyacrylamidegel

electrophoresis (SDS-PAGE).Thecross-linkedcomponentsobtained were

char-acterized by imnmunoprecipitation with monospecific antisera against purified

viral proteins,followed by SDS-PAGE analysis both before and after cleavage of

the cross-links.Withtheseprocedures, complexes of both viral envelope and core

components were identified. The major envelope subunit obtained was a large

(apparent molecular weight of 450,000 to 500,000), glycosylated complex,

com-posedoffourtosixgp7O-pl5(E)subunits. Thiscomplex was detected over a

100-fold range of cross-linker concentration and thus seems to represent a particularly

stable viral substructure. The cross-linked complexes of the core proteins

con-sisted of oligomers of p30 dimers, suggesting that the p30 dimer is a basic

structural unit of the viral core. When virionpreparations, which had previously

beendisruptedwith the nonionicdetergent Nonidet P-40, were cross-linked, the

envelope complexwasstill observed, indicating that this structure is stable in the

presence of Nonidet P-40. A similar envelope structurewasobserved for feline

leukemia virus,suggesting that such a complex may be aconserved feature of

oncornavirusstructure.

The type C oncornaviruses possess distinct

core and envelope morphological structures

which have been visualized in the electron

mi-croscope. The presence of surface "knobs"

ap-proximately8.0to10.0nmin diametercovering

theenvelopeof intactvirionshas been

demon-strated afternegative stainingof murine

leuke-mia virus(MuLV) preparationswithuranyl

ace-tateorbyacombination ofphosphotungsticacid

staining followedby freeze-drying (5, 22). Reg-ularly arranged subunits of viral cores, which

possess a diameter of approximately 6.0 nm,

havealsobeenvisualized afterfreeze-dryingand

shadowingof viral coresreleased after various

treatments(22). The MuLVenvelopehas been

showntocontaintwomajor proteincomponents,

a large glycosylated protein of

approximately

70,000daltons, gp70 (1), and asmaller,

nongly-cosylated hydrophobic protein,p15(E)(13);both

arederivedbyproteolytic cleavageofa common

precursor (8, 15, 26, 28). Thetwoenvelope

com-ponents can, underappropriate conditions,form

adisulfide-linked complex, referredto as[gp9O]

(23, 24), demonstrating an integral association

between gp7O and p15(E). The major intemal

structural components of the virus consist of four proteins-p30, p15, p12, and plO-which

are derived by proteolytic processing of the

"gag"geneproduct (7).Tobettercorrelateviral

proteins with the morphological substructures

seen in virions, we have studied the protein proximitiesandspatialarrangements ofproteins inviral substructures by treating virions with

two chemical cross-linking reagents,

dithiobis(succinimidyl proprionate) (DSP) and

1,5-difluoro-2,4-dinitrobenzene (DFDNB).Both

reagents contain two functional groups which

react readily at physiological conditions with

lysine amino groups, thus allowing the cross-linkingofneighboring proteins. DSP possesses

an internal disulfide bond; thus cross-links

formed withthis reagentcanbereadilycleaved

by reduction, facilitating the analysis of the

cross-linked products. These studies have

re-sultedintheidentificationofspecificcomplexes

of both viral envelope and viral core

compo-nents; these complexes may be related to the

morphological substructures which have been

visualizedin the electronmicroscope.

MATERIALS AND METHODS

Viruses.AKRMuLVwasobtained fromproducer

SC-1celLs,andfelineleukemia virus (FeLV)ABwas

obtained from aninfectedfeline lungfibroblast cell 157

on November 10, 2019 by guest

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line. Viruses were radiolabeled by culturing in the

presence of either "C-labeled mixed amino acidsor

3H-labeled glucosamineprecursors,ataconcentration

of 100 uCi/ml, for 18 h, and purified by banding directlyon15to60%sucrosegradients. Concentrated

AKR MuLV, obtainedfrom Electro-Nucleonics Lab-oratory Inc., was labeled with [3H]NaBH4 by the method of Gahmberg and Hakomori (10).

Electrophoresis. Sodium dodecyl sulfate-poly-acrylamide gelelectrophoresis (SDS-PAGE)was

per-formed in 7to21%gradientslabgels by thesystem described by Laemmli (18). Gels were analyzed by

fluorography, accordingtotheprocedure described by Bonner andLaskey (3).Samples analyzed under

non-reducing conditionswereboiledinthepresenceof 1%

SDS for 1 min. Samples analyzed under reducing conditionswereboiled in thepresenceof1% SDS and 1%mercaptoethanol for1min.

Alkylation of virionswith NEM. A stocksolution of10%N-ethylmaleimide (NEM) in acetonitrile was

diluted 1:10 with phosphate-buffered saline (PBS). One volume of the PBS solutionwasthenaddedto9 volumes of the virus sample and incubated atroom

temperaturefor 15min.

Cross-linkingprocedure.Stock solutions of the cross-linking reagentswere prepared in Me2SOata

concentrationof 20 mg/ml. Thesewerethendiluted

with PBStoaconcentration fivefoldgreaterthan the desiredfinal concentration, then further diluted 1:5 with the virussample. DSP reactionswereperformed

in the presence of TN buffer (0.01 M Tris-0.10 M

NaCl); for DFDNB reactions, virus samples prepared inPBSwereused. Reaction timesweretypically 15

minatroomtemperature.Occasionally, unreacted

re-agentwasquenched by the additionof0.1 M

ammo-niumacetatebeforelysis of virions; however, similar results were obtained if thisstep wasomitted. The

concentration ofradiolabeled virions used in these studies wasless than 20 ig/ml. Similar patterns

re-sulteduponcross-linkingof virions ataconcentration of1mg/ml.

Radioimmunoprecipitations. Virionswerelysed

by the addition of NonidetP-40(NP-40) and NaClto

final concentrations of 0.5% and 0.5M, thenincubated with antiseraatafinal concentration of 1:50for1hat 37°C. Immune complexeswerethenpelleted by

treat-mentwith50

id

ofa10%solution ofstaphylococcus A

(Pansorbin, Calbiochem) prepared as described by

Kessler (17). Afterpelletingat3,000rpmfor 10mi,

thestaphylococcus Awaswashedwith 10 mlofhigh salt(1MNaCl,0.5%NP-40,0.02MTris) and10mlof low salt(0.004 M Tris, 0.5% NP-40) buffers, pelleted, and then extracted with 0.1 ml ofSDS-containing bufferat100°Cfor 1min.

Reagents.Goat anti-Rauschergp7O andanti-p30

sera were obtained from R. Wilsnack; rabbit anti-p15(E) was prepared as described (13). DSP and

DFDNB were obtained from Pierce Chemical Co.,

SDS(specially pure)wasobtained from BDH

Chem-icalsLtd.,NEM wasfrom Sigma Chemical Co., and

radiochemicalswereobtained from NewEngland

Nu-clearCorp.

RESULTS

Cross-linking of AKR MuLV with DSP. We havepreviously shown that themajority of

the viral envelope proteins, gp70 and p15(E),

exist in native virionsinthe formofa

noncova-lently linked complex. Upon disruptionofvirions

with NP-40, a covalent disulfide linkage can

forn spontaneously between thetwo proteins,

resulting in the stable gp7O-p15(E) complex

re-ferred to as [gp9O]. In addition, activation of

appropriatethiol residues ofgp70 and p15(E) by

treatmentwith thethiol-specificreagentsNEM

ordithiobis(m-nitropyridine) (DTNP)results in

formation of[gp9O]inintact virions(23, 24) (Fig.

1). Samples of 14C-amino acid-labeled AKR

vir-ionsanalyzedby SDS-PAGE under nonreducing

conditionscontainedgp70andp15(E) (Fig. 1A);

aftertreatmentwithNEM, the gp70 bandisnot

observed, the p15(E) band diminishes in

inten-sity, and the [gp9O]bandappears(Fig. 1B). To

determine whether [gp9O] waspart ofalarger

envelope structure, virions pretreated with

NEMwerereacted with DSP andanalyzed

un-dernonreducingconditions.Atafinal DSP

con-centration of 10,g/ml,newbands with apparent

molecular weightsof 60,000 (a) and 120,000 (b)

wereobserved, along witha more diffuse band

atamolecularweightofapproximately 180,000

(c),andaband ofhigher molecular weight ("x")

(Fig. 10).When theconcentration of

cross-link-A B C D

S

RT

-

gp7O-MD

A

_0

x

- c

-b

0-

P30-p15

(E)-FIG. 1. Cross-linking of "C-amino acid-labeled AKRvirions with DSP. Virus samples were analyzed

bySDS-PAGE under nonreducing conditions after thefollowing treatments: (A) untreated virus; (B) +NEM; (C) +NEM, +DSP (10pg/ml); (D) +NEM, +DSP (100pg/ml). "x" represents the cross-linked envelope component.

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VOL. 30, 1979

mgreagent wasir

icant decrease in

viral protein ban bandsa,b,and c, inmaterialrunnix

To ascertain u

tained gp7O, the

peated with virio

had been specific

dase oxidation,

[3H]NaBH4

(10). tern obtained foi

nonreducing con

served

correspond

ofgp7O and only

ular-weight comj

show thepatterns

10, 100,and1,000

At the lowest DE

ponentwith the

withsome smalli 2B).At 100,ugof the

[gp9O]

has beE itant increase in b

percentageof the

component"x" ai

DSP per ml, alti

mostof theprotei

itX'

bp9g-gp7O

-FIG. 2. Cross-lin Virions, for which NaBH4reductionc (10), were analyze

ducingconditionsc +NEM; (B) +NEA +DSP(100pg/ml);

CROSS-LINKED COMPLEXES OF MuLV AND FeLV 159 icreasedto100l,g/ml,asignif-

larger

aggregateswhichcan no

longer

enterthe i intensity of the monomeric gel

(Fig. 2D).

These

experiments

indicate that

ds was observed, as well asof band "x" containsacross-linked

complex

ofthe

and therewas a

large

increase viral

glycoprotein;

the observations that it is

ng atposition "x"(Fig. 1D). detectedover a100-foldconcentration range of

vhich cross-linked bands con- the

cross-linking

reagent and that

well-charac-above experiments were re- terized

complexes

of smaller sizeare not

readily

)ns in which the glycoprotein observedsuggest that the

large complex

repre--ally labeled by galactose oxi- sents a

particularly

stableviral substructure.

followed by reduction with The size of the

glycosylated complex

was

es-Figure 2A shows the gelpat- timated

by comparison

of its SDS-PAGE

mo-r NEM-treated virions under

bility

with that of the LETSmonomeranddimer

ditions. The major band ob- purifiedfromJLSV9cells

by

immunoprecipita-dsto[gp9O], withafaintband tion with

monospecific

antiserum

prepared

trace amounts ofhigh-molec-

against

human cold insoluble

globulin (25).

The

ponents.

Lanes B, C, and D LETS

protein

monomerhasamolecular

weight

3obtainedaftertreatmentwith of 220,000 and has been shown to exist as a

jug

ofDSPperml, respectively. disulfide-linked dimer

(16).

Such an

analysis

3P concentration, a new com- showed that the cross-linked

complex

has a

mobility of "x" appearsalong

slightly

lower

mobility

than that of the

purified

Dr, more diffuse material (Fig. LETS dimer

(data

not

shown;

see

Fig.

5), and

DSP perml, essentially allof thuswehave

assigned

itanapparentmolecular

ancross-linked,witha concom-

weight

of450,000to500,000.

and "x"(Fig.2C). Asignificant Characterization of DSP-cross-linked

radioactivity isstillpresent as components by immunoprecipitation. To

ftertreatmentwith

1,000,ug

of determine the

composition

of the cross-linked iough under these conditions, components obtained after DSP

treatment,

inhas been cross-linkedtostill cross-linkedvirionswere

lysed

withNP-40,and

thecross-linked

complexes

wereisolatedby

im-A

B

C D munoprecipitation with monospecific antisera

againstindividual viralproteins.Thisprocedure

hasthe

advantage

thatit allows the

analysis

of

3 -^"': individual

complexes

evenincaseswhere there

may beseveraldifferentcross-linked

complexes

of similar size. Thecomponentsof the

complexes

arethusidentifiedby their

antigenic reactivity

as well as by their SDS-PAGE mobility after

reduction.

Figure

3 shows the SDS-PAGE

pattern

ob-tained for

'4C-amino

acid-labeled AKR virions

beforeandafter

cross-linking

with 100 ,Lgof DSP

perml. In this

experiment,

the NEMtreatment

wasomitted. The

pattern

obtained after

cross-linking

wassimilartotheonedescribedearlier; almost all of the gp70 and most ofthe

p15(E)

have been

cross-linked,

andthereisa

significant

reduction in theintensityof the

p30

band. The

60,000-and

120,000-molecular-weight

bandsare readily apparent, and there is a considerable

amount of

high-molecular-weight

material

which is morediffuse.Uponreaction with

anti-gp70 serum, very little radioactivity is

precipi-tated; this suggests that the DSP treatment

kingofAKRMuLVglycoproten3s.

resulted in a considerable modification of the gp70 had been labeled by[3H]-*

zfter

galactoseoxidaseoxidation

gp7O

molecule

with

aconsequent loss of

immu-,d by SDS-PAGE under nonre- noreactivity. A faint band is observed for the

zfterthefollowingtreatments: (A) anti-gp70

precipitate (Fig.

3A). In the unreduced

, +DSP (10pg/mI); (C) +NEM, sample, it has the

mobility

of

"x";

after

reduc-(D) +NEM, +DSP

(1,OX

pg/ml).

tion, the disulfide bonds of the

cross-linking

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[image:3.501.55.246.366.607.2]

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UNREDUCED REDUCE AKR AKR A

DSP

a,.

gp70- £

p30--p 15(E)-p15,p12 -p'0

-AitA.

B C D AKR A B c D

f.

_w

a

[image:4.501.60.456.72.347.2]

S

FIG. 3. Immunoprecipitation of DSP cross-linkedcomponents. "C-aminoacid-labeledvirionswere

cross-linked with100pg/mlof DSP, lysed with 0.5% NP-40 and0.5MNaCI, and treated with: (A) anti-gp7O serum;

(B) anti-p15(E) serum; (C) anti-p30serum; (D) normalgoatserum.Immunecomplexeswerecollected with

staphylococcusAand, after washing, weredissolved inSDS-containing buffer and analyzed both with and

withoutadding mercaptoethanol. The bandlabeled RT has been characterizedas reversetranscriptaseby virtueof its identical mobility with that of thepolypeptideimmunoprecipitated with monospecific antiserum

topurifiedreversetranscriptase.

reagentarecleaved, andaband withthe

mobil-ity of free gp7O is observed. The sample istoo

dilutetodetectanyadditionalcomponents

pres-ent.

Reactionwith antisera top15(E) resulted in the immunoprecipitation ofconsiderablymore

material(Fig. 3B); apparently the immunoreac-tivity of p15(E) ismoreresistanttomodification. Upon analysis under nonreducing conditions,a

large diffuse band, similartothat recognized by the anti-gp7O serum, is observed. After

reduc-tion, this band is showntoconsist of onlytwo

majorcomponents, gp70and p15(E), indicating thatalarge complex of gp7O and p15(E) is

sta-bilized by DSP treatment. This complex does

not contain any p15, p12, or pLO; however a

slightamountof p30 isobservedafter reduction (Fig. 3B). It isnotclearwhether this is dueto

some trapping of cross-linked p30 aggregates with the cross-linked envelope complexes or

whether this reflects actual cross-linking

be-tweenp30 and the envelopecomponents.Inany

case,the relativeamountof p30 observed in this sample isso low that this does not appear to

representastructurally significant interaction.

Analysis of the anti-p30 immunoprecipitate undernonreducingconditions(Fig. 3C) demon-stratesthe presence ofasmall amount ofp30, along with considerable amounts of the

cross-linked bands at molecular weights of 60,000, 120,000, and 180,000, andmore diffusematerial

ofhigher molecular weight. The disproportion-ately low recognitionofmonomericp30appears

to reflect the loss ofimmunoreactivity due to modificationbytheDSP; apparentlythiseffect

is diminished for the higher oligomers. After

reduction, the major component presentisp30; minoramountsof three otherproteinsareseen as well. In order ofdecreasing size, these have

been assigned by virtue of their mobilities as

reversetranscriptase, unreduced p30 dimer,and

actin. Theco-precipitationofreverse

transcrip-tasewithp30has beenpreviouslyobserved(14), and thus isnotanindicationthat the two

com-ponentswere cross-linked. Actin has also been

observedtobea contaminantof viral

immuno-precipitates, and thus it is not clear whether there isanactualp30-actincross-linkedcomplex

_RT

- gp7O -(P3)2

-a

- p30

- p15(E)

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CROSS-LINKED COMPLEXES OF MuLV AND FeLV 161 formed by DSP treatment.

Treatment with nonimmiune serum did not

result in the precipitation of any labeled com-ponents (Fig. 3D), indicating that nonspecific adsorption of the cross-linked components to staphylococcus A is not occurring.

DSP treatment of NP-40-solubilized vir-ions. Recent studies have indicated that certain

MuLV morphological structures can survive

NP-40 treatment (9, 31). To study the MuLV

substructures which exist in the presence of

non-ionic detergent, AKR virions were disrupted

with 0.5% NP-40 and subsequently cross-linked

with 100

fig

of DSP per ml. Cross-linking of

intact virions results in the pattern described

earlier; gp7O and p15(E) are cross-linked into a large complex, and p30 oligomers are formed (Fig. 4B). DSP treatment of NP-40-lysed virions still results in the cross-linking of gp7O and

p15(E); however the p30 oligomers do not form,

thusallowingtheclearresolution of the envelope

complex (Fig.4C). A similar effect is observed

for virions pretreated with NEM (Fig. 4C').

These results indicate that the large gp7O-pl5(E)

A B C A' B' C'

_-, _f_ a _ Sto

_~~~~~~i

[image:5.501.53.244.326.560.2]

--dslo.91

[gp9go]

gp70

-p15(E)

FIG. 4. DSP cross-linking of AKR virions dis-rupted with NP-40. "4C-amino acid-labeled virions analyzed bySDS-PAGE undernonreducing condi-tions after the following treatments: (A)

uncross-linkedvirions; (B) cross-linked with 100jpgof DSP perml; (C) disruptedwith 0.5% NP-40for15minat

roomtemperaturefollowedby cross-linking with 100

pgofDSPperml. Samples A',B' and Cwere

pre-treated with0.1% NEM before theother manipula-tions to convert gp7O to thegp7O-pl5(E) disulfide-linkedcomplex [gp90].

complex is quitestable in the presence of non-ionic detergent, whereas the p30 complexes are not.Itisbelievedthatnonionicdetergentsbind

primarily to hydrophobic regions of proteins

(12); thus thedissociation of the p30 complexes

by NP-40 suggests that the p30 associationsare

duemainly tohydrophobic interactions.

Cross-linking of AKR virions with DFDNB. To further characterize the subunit composition of native oncornaviruses, intact preparations of AKR MuLV were treated with DFDNB, a bifunctional cross-linking reagent

which reactsreadily with protein amino groups,

resulting in theformation of cross-links with a

maximum span of 0.5 nm. Since this span is

considerablyshorterthan that of DSP

(approx-imately1.1nm),DFDNBis useful as aprobe for

protein associations of closer proximity than

those detectedby DSP. For thisparticular

ex-periment,aviruspreparationat aconcentration

of 1 mg/mlwas used, and the proteinpattern

wasdeterminedby staining thepolyacrylamide

gelswithCoomassieblue. The patterns observed

upon cross-linking were independent of virus

concentration,andsimilar results were obtained

for more dilute, radioactively labeled

prepara-tions.

Treatment of intact virions with 100 ,ug of

DFDNBperml resulted in the loss of thegp7O

band and the appearance of

higher-molecular-weight material which possesses a similar mo-bility to that of the envelope complex formed

with DSP (Fig. 5C).There does not appear to

be adetectable decrease in theintensityof the

p30 band, and bands corresponding tothe p30

oligomersarenotpresent.DFDNBtreatmentof

virus which had beenpretreatedwith NEM also

resulted in the cross-linking of gp7O and the

resolution of a large complex (Fig. 5D). This

band appears slightly larger than the

corre-spondingbandobserved in laneC;this may be

an indication that thecomplexformedwithout

NEM treatment contains less than the

maxi-mum number of gp7O or p15(E) constituents.

For the NEM-treated virions, DFDNB

treat-mentdoesresult inadecrease in theintensityof

the p30 band, indicatingthat cross-linkinghas

occurred; however, the p30 intermediates

de-tected after DSP trea,tment are not observed.

Thismay indicatethat NEM treatmentinduces

achangeinconformation of thep30 molecules,

whichallowstheoccurrenceoflarge-scale

cross-linking by DFDNB. Figure 5E shows the gel

patterns

obtainedafter DSPcross-linking.The p30 dimer,tetramer, and hexameraredetected, and a broadband,encompassingthemobilities

of thecross-linkedenvelopecomplexesresolved

inlanesCandD,isobserved.

VW4..

4WAIP

4..

VOL. 30, 1979

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162 PINTER AND FLEISSNER

A E

i-E

TS

--v

dirner

!gp9O} C

qp7O- *_

1 e

e

31-IC

FIG. 5. AKR Coomassie blue

alyzedbySDS-1 afterthefollowi

(B)virions treat linked with IOC

treated with 0.19 100pg ofDFDJ with

100,pgofD

dimer" is an in

grates with pur

nonreducingcoi not observed a monomericLE2

3 C D E Cross-linking of FeLV. The studies

de-scribed above have been performed with the

_

, -t-~ mAKR strain of

MuLV;

-

-w-x

~~Rauscher

and

Moloney

virions

(data

not

shown).

To determine whether these structures are

pres-ent in retroviruses of otherspeciesaswell, the

DSP

cross-linking

experiments were repeated

witharepresentative strain of FeLV. Upon

SDS-PAGEanalysis of '4C-amino acid-labeled FeLV,

the overallpattern obtained was similar to that

observed with AKRMuLV,althoughthe

abso-lute mobilities of the variousproteinswere

dif-ferent. As was the case forMuLV, analysisunder

nonreducing

conditions demonstrated the

pres-enceof free gp7O(Fig. 6B andC),whereas NEM

treatment resulted in the formation of a

disul-fide-linked gp7O-pl5(E) complex (Fig. 6D).

Upon treatment with increasing amounts of

DSP, the intensity of the [gp9O] band

dimin-ished, as did that of the core components. The

onlynewcomponentresolvedwas alarge band

withsimilarmobilitytothat of the cross-linked

virions cross-linked with DFDNB. MuLV envelope complex (Fig. 6G).

Cross-link-staining pattern of AKR virions an- ing of

[3H]glucosamine-labeled

FeLV resultedin

PAGE undernonreducing conditions, the appearance of a band of identical

mobility,

[ng treatments:(A) untreatedvirions; * * *

tdwith 0.1%NEM; (C) virions cross-

proving

that

thls

does factrepresent the

cross-)pg of DFDNB per ml; (D) virions linkedFeLV envelope

complex

(Fig. 6J and K).

%0

NEMfollowedby cross-linking with Thus, it appears that a similar envelope complex

NBper ml; (E) virions cross-linked exists for FeLV as for

MuLV,

suggesting

that

'SP

per mL (The bandlabeled "LETS thismay be auniversal feature ofmammalian

%purity found in virions which comi- leukemia virus structure. Cross-linking of the

ifiedLETSprotein analyzed under FeLV p27 apparently occurred, although

inter-rtditions.Uponreduction, this band is mediate-sized oligomers were not detected.

This

nd a band which

comigrates

with may be a reflection of the differences in the

rS proteinappears)

14C-aa

A B C D E

x

-

[gP9o]-gp7O- fl

p

27-_e a a

se

m

e

pl5(E)-FIG. 6. Cross-linking of FeLV proteins withDSP. "C-amino acid-labeled (A-G) and [3H]glucosamine-labeled(H-K)FeLVanalyzed bySDS-PAGE under reducing (A)and nonreducing(B-K)conditions afterthe

followingtreatments:(A and B) untreated virus; (C)+1%iodoacetamide; (D) +0.1% NEM;(E) +0.1%NEM, +DSP(4 pg/ml); (F) +0.1% NEM, +DSP(20 pg/ml); (G) +0.1% NEM, +DSP(100pg/ml); (H)+0.1%NEM; (I)

+0.1%NEM, +DSP (10 pg/ml); (J) +0.1%NEM, +DSP (100pg/ml);(K) +0.1% NEM, +DSP(1,000pg/ml).

F G

a

3H-gln

H J K

SW

dik-J. VIROL.

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CROSS-LINKED COMPLEXES OF MuLV AND FeLV 163

primary structures ofp27and p30, or may

ac-tuallyindicate a difference between the

struc-turalarrangementof the majorcoreproteins of

the twoviruses.

DISCUSSION

The cross-linking experiments described in

this paper demonstrate thepresence of distinct

associations between leukemia virus envelope

components and between viral core proteins.

Treatment ofMuLVwith thereversible

cross-linking reagent, DSP, results in the formation of alarge complex (component "x") which is

gly-cosylated (Fig. 2) and can be

immunoprecipi-tated by both anti-gp70 and anti-pl5(E) sera.

SDS-PAGE oftheimmunoprecipitatedcomplex after reduction demonstrates that the only

com-ponents present inappreciableamountsaregp7O

andp15(E) (Fig.3).Thiscomplexisformed both

withnativevirionsand with virionswhich have

been treated with NEM, and which therefore

contain the gp70-p15(E) disulfide-linked

com-plex. The acylationrate ofproteins byDSP is

veryrapid, with ahalf-lifeof less than 1 min at

neutral pH (19); thus it doesnot appearlikely

that the formationof"x" requireslateral

diffu-sionofcomponents in theviralmembrane. The

envelope complex is observed over a greater than 100-fold concentration range of DSP; in

addition, an apparently identical complex is

formed aftertreatment of virions with a second

cross-linking reagent, DFDNB, which has a

shorterspan (0.5nmcompared with1.1nmfor

DSP)anddifferent functionalgroups thanDSP (Fig. 5). These results indicate that gp7O and

p15(E) exist in theviralmembrane in theform

of a specific structural complex, which

corre-sponds to the component "x" observed in gels after stabilizationwith thecross-linkingagents.

The fact that theenvelope complexsurvives in

the presence of NP-40 (Fig. 4)is a further

dem-onstrationof the

stability

of thecomplex.

Theapparent sizeof theenvelope complexas

determined by SDS-PAGE, usingtheLETS

di-mer asa molecular-weight marker, is

approxi-mately450,000to

500,000

(Fig. 5). However,the

cross-linked structure ofthe complex could

re-sult in an anomalousmigrationrate. It has

re-centlybeenreported thatcross-linked

proteins

with molecular weights larger than 50,000

mi-grate in SDS-Laemmli gels asifthey were 17 to

25%largerthan themolecular weights calculated

fromtheirpolypeptide composition (21). By

us-ing the larger value as the limit of error, a

minimumi

molecular weight of 360,000 can be

calculated for thecross-linkedcomplex,

suggest-ing that it is composed of at least four

gp7O-p15(E) components; by calculating a molecular

weight directly from the observed mobility of

the complex, a maximum value of six subunit

componentsisobtained. The demonstrationthat

asimilar complex ispresent on FeLVindicates

that this structure is a conserved feature of

retroviruses. Since the size of the complex is

large enough to account for the 8- to 10-nm

projectionsobservedonthe surface ofMuLV in

the electron microscope, it is attractive to

hy-pothesize that this complexactuallycorresponds

totheindividualknobsofthe viralenvelope.

The majorcross-linked core components

de-tected after DSP treatment of intact MuLV

consistedofp30 oligomers. Thep30dimer,

tet-ramer, andhexamer wereresolvedand

charac-terizedbyimmunoprecipitation (Fig. 3) andby

two-dimensional gel electrophoresis, in which

thefirstdimension wasrununder nonreducing

conditions and the second dimension was run

after reduction (data not shown). Larger

com-plexes were also formed, but these were not

resolved in the gels and formed abroad band

nearthe top of the gel.

Tne

formationofonly

theeven-numberedoligomersindicates the

sta-bility ofthe p30 dimerandsuggests that it is a

basic subunit of the viral core. Ourobservation

that thep30 complexes are notcross-linked in

thepresence of NP-40 suggests that

hydropho-bic forces playa rolein the self-association of

p30 molecules; thisis consistent with the

dem-onstratedpresence ofhydrophobicsitesonp30

(20,27).It isinterestingtocompare theseresults

with those ofBurnette et al. (4), who

demon-strated thatpurified p30ofMoloney MuLV

self-associates into multimeric forms at increasing concentrations. At the highest concentration

tested,the observed molecularweightfor

p30

as determined by sedimentation velocity analysis wasapproximately120,000,anda

computer-sim-ulated analysis of the data suggested a

mono-mer-octomerequilibrium.Thesep30 complexes

mayberelatedtothe6-nmcorestructureswhich

have beenvisualized in theelectronmicroscope

(22).

Cross-linked complexes of the other major componentsofthe viralcore,

p15,

p12,andplO, were not detected under conditions which

re-sulted inextensivecross-linkingof theenvelope

components and ofp30.This was notdueto a

lack of accessible amino groups on these

pro-teins,since wehaveobserved thataradioactive

analog of DSP, N-succinimidyl

[2,3-3H]pro-prionate(Amersham Corp.),caneffectivelylabel

all of the core proteins (data not shown). In

addition, after treatmentofvirions withDSPat

100

ltg/ml,

p15

can nolongerbe

immunoprecip-itated with anti-15 sera, indicating that it is

heavilysubstituted with DSP molecules.Alack

ofcross-linking isnotconclusive evidencethat

molecularassociations donotexist,since it may

VOL. 30, 1979

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merely indicate that the necessary functional groups are not arranged in the proper spatial

orientation to be cross-linked by a particular

reagent. Nonetheless, the fact that the smaller

core components are not readily cross-linked

may bean indication thatthey donotexist as

specific multiprotein complexes in virions.

Structural studies of otherenveloped viruses,

performed with the sameorrelated cross-linking

reagents, have demonstrated the existence of

heterocomplexes between virion core and

enve-lope components. Treatment ofSemliki Forest

viruswithdimethyl suberimidate resulted in the

cross-linking of viral envelope components to

cores (11). Cross-linking of vesicular stomatitis

virus withDSP and related reagents resulted in

thedetection of complexesformed between the

viral glycoprotein, G, and the membrane protein,

M(6), and also betweenthe Gprotein and the

nucleocapsid protein (21). Such

heterocom-plexes between envelope and core components

of retroviruses were not detected in ourstudy.

In particular it is of interest that cross-linked

complexes of p15 and theenvelopeproteins were

notobtained(Fig.3B), since p15 has been shown

topossesspropertiesofamembrane-associated

protein (2).Although failure todetect such

in-teractionsbythis type ofastudyis not

conclu-sive, it does indicate an increased probability

that such interactions are not present. It has

beendemonstrated that the envelope and core

components of MuLV aresynthesizedvia

sepa-rateclasses of mRNA's (29); thus they must find

their waytotheviralassembly site,theplasma

membrane, byindependent routes. Since

retro-viralproteins are relatively minorcomponents

ofinfectedcells,there mustbe anefficient and

specific interaction between the viral envelope

andcoreproteinswhich results in their

associa-tion in the presence ofalargeexcessofcellular

material. Recently it has been shown that the

assembly of budding virus particles can occur

with uncleaved core precursor Pr7O (30). It is

interestingto speculate that the association of

viral core and envelope components at the

plasma membrane requiresthe uncleaved core

precursor and that, in extracellular particles

which contain the cleaved core proteins, these

interactions are not retained. A possible

ap-proach toward resolving this question would be

tocross-linkbudding virus particles and to look

for thepresence ofenvelope-core complexes in

either solubilized membranes or subsequently

released virusparticles.

ACKNOWLEDGMEENTS

This workwas supported by grants CA-02214 from the National Cancer Institute and VC-222 from the American CancerSociety.

We thankJudyLieman-Hurwitz and Anne Swartout for providing excellent technical assistance.

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