Localization of viral structural proteins in the cytoplasm and nucleus of Rous-associated virus-2-infected chicken embryo fibroblasts.

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Vol.44,No.2 JOURNALOFVIROLOGY, Nov. 1982, p. 658-665

0022-538X/82/110658-08$02.00/0

Localization of Viral Structural Proteins in

the

Cytoplasm

and

Nucleus of Rous-Associated Virus-2-Infected Chicken Embryo

Fibroblasts

PAULA J. ENRIETTOt* ANDRAYMOND L. ERIKSON

DepartmentofPathology, School ofMedicine, UniversityofColorado HealthScienceCenter,Denver, Colorado 80262

Received 5May1982/Accepted 26 July 1982

The cellular location of viral structural proteinswascarried outby

immunohis-tochemistry and by cell fractionation. Antibody against the structural protein p27

wasused inimmunohistochemical reactionstodemonstrate thepresenceof viral

proteins in thecytoplasm and nucleus of Rous-associated virus 2-infected chicken

cells. Localization in the nucleuswasfoundoverheterochromatic regions; in the

cytoplasm itwasfound indiscrete particulatestructures.These observationswere

extended incellfractionation studies in which cytoplasmic and nuclear fractions

wereimmunoprecipitated with antibody against the viral structural proteins.

In an effort to better understand the

interac-tions of the viral structural proteins of avian leukosis sarcoma viruses with the host cell, a

study was initiated to localize these proteins within the cell. The structural proteins of the virus are synthesized from35S viral RNA as a

polyprotein precursor Pr76, which is then

cleaved to give the mature structural proteins found in the virusp27, p12, p15, and p19 (fora

review, see reference 10). The location ofthese

proteins withinthevirusparticle has been stud-ied (17), and it has been

proposed

that p19

makes up the inner coat ofthe virion and

p27

andp15 thecore

shell,

andthat

p12

isa constitu-ent of the

ribonucleoprotein

core ofthe virion

(2). Several functions have been

assigned

to

these

proteins.

Ithas been

postulated

that

p19,

a

phosphoprotein (11), binds

specifically

to viral

RNA,

suggesting

a

regulatory

function

(15).

In

addition, p15

has been shown to be a protease

whichcancleavePr76saeto

give

risetothe viral

structuralproteins (9, 25).

The site ofsynthesis ofPr76 in the host cell

has been studied by Purchio et al.

(22),

who

showed that

85%

of Pr76 was synthesized on

free polyribosomes, whereas 15% was synthe-sized on membrane-bound polysomes. It has

been postulated that some oftheprocessing of

Pr76 occurs at the membrane, since agents

known to affect membrane protein interactions

block the cleavageof Pr76 invitro

(24).

Tofurtheranalyzetheinteraction of theviral

structural proteins with the host cell, and

per-hapsidentifysitesofsynthesisorprocessing,we

tPresent address: Imperial Cancer Research Fund, Lin-coln'sInnFields,London WC2A3PX, England.

began by carrying out ultrastructural

localiza-tion of the viral structural proteins by using

antibody against them in immunohistochemical

reactions. Cell fractionations of labeled cells

were thencarriedout, followedby

immunopre-cipitation with antibody against the structural

proteins.

MATERIALS ANDMETHODS

Cells and viruses.Chicken embryo fibroblasts (CEF)

wereobtained from10-to 11-day old chicken embryos

(Spafas, Inc.) andweregrown in IMEMZO with zinc

and insulin (Associated Biomedical Systems, Inc.)

containing 10%tryptosephosphate and5%calf serum.

Cellswere platedat adensity of 8 x 105on a 60-mm

petri dish, infected with an appropriate dilution of

stock Rous-associated virus 2 (RAV-2) or

Schmidt-Ruppin D (a generous gift of H. Hanafusa), and

maintained in culture with 10% tryptose

phosphate-5% calfserum.

Preparation ofantibodies.Antibodyagainstthe viral

internal structural protein p27 purifiedby

polyacryl-amide gel electrophoresis was prepared as described

previously(21).

Tumor-bearing rabbit serum which recognizes the

viral internal structuralproteins was prepared as

de-scribedby Brugge and Erikson (3).

Cellfractionations.Preparations ofclean nucleifree

ofcytoplasmic material were made in the following manner.Cellswerewashed threetimeswithcold STE

(0.15MNaCl, 0.05MTris [pH 7.2],0.0001 MEDTA),

after whichtheywere removed from theplate by brief

treatmentwith0.05% trypsininsalinD(0.05% trypsin,

0.1% glucose, 0.15 M NaCl, 0.001 M KCl, 0.1 M

sodium phosphate buffer). After trypsinization, cells

werewashed twice incold STE, pelleted at 2,000 rpm

for5min,andlysedin 0.01 MTris-hydrochloride(pH

7.2)0.15MNaCl-0.5% NonidetP-40withblendingat

high speed inaVortex mixer forapproximately15 s.

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LOCALIZATION OF VIRAL PROTEINS 659

Nuclei were pelletedfrom the lysate by centrifugation

at5,000 rpmfor 5 min. The cytoplasmic fractionwas

removed and kept onice; nucleiwerewashedtwo to

three times with lysisbuffer to remove allcytoplasmic

material.Nuclei preparationsweremonitoredateach

step by light microscopy. Washed nucleiwere then

lysed in RIPA (13) lysis buffer (0.01 M

Tris-hydrochlo-ride [pH 7.2], 0.15 M NaCl, 1% Triton X-100, 1%

deoxycholate,0.1% sodiumdodecyl sulfate[SDS]).A

general proteaseinhibitor, Trasylol (FBA

Pharmaceu-ticals, New York, N.Y.), was added to nuclei lysis

buffer andRIPAlysis bufferto afinal concentration of

1% (1). Cytoplasmic fractionswereadjustedtoRIPA

lysis conditions. All stepswerecarriedout at4°C.

Radiolabeling cells and immunoprecipitations. RAV-2-infected CEF were radiolabeled and

immunoprecipi-tatedessentially as previously described (3).Analysis

of immunoprecipitates wascarriedout on10%

SDS-polyacrylamide gels as described by Laemmli (14).

Gels were then soakedfor 30 min in a1 Msolution of

salicylic acid (4),dried,and exposed at-70°C (Kodak

X-Omat X-rayfilm).

Preparations of antibody forimmunohistochemistry.

Antibody against the viral internal structural protein

p27 was used for immunohistochemical staining reac-tions. Immunoglobulin G (IgG) fractions of normal and immune sera made as described previously (23) were absorbed with normal CEF which had been fixed in

acetone for 10 min and washed three times in

phos-phate-buffered saline (PBS) (0.15 M NaCl, 0.0042 M

KCI, 0.01 M sodium phosphate buffer [pH 7.2], 5 x

10-4M MgCl2, 9 x 10-4 M CaCl2). Absorption was

carried outovernightat4°C, and absorbed serawere

clarified at 100,000 x gfor 30 min. Absorbed antisera

were used in staining reactions at dilutionsof 1:16 in

PBS-10% sucrose.

Preparation of horseradish peroxidaseconjugatesof

IgG. Sheep anti-rabbit IgG was kindly provided by

AntonioMartinez-Hernandez(Universityof Colorado

Health ScienceCenter, Denver) andwasconjugatedto

horseradishperoxidase bythemethodof Nakane and

Kawaoi(18).

Fixation and staining procedure. RAV-2-infected

CEFweregrownon35-mmpetri dishes andfixed for

20minat4°C with 2% paraformaldehyde in PBS. After

three washings in PBS-100o sucrose (each wash, 10

min), cells were incubated in absorbed normal or

immune serum for 3 h at room temperature, washed

threetimes inPBS-10%sucrose, andincubated for 1 h

insheep anti-rabbit IgG labeled with horseradish

per-oxidase. After postfixing in 2% glutaraldehyde in

PBS-10%sucroseandwashing inPBS-10%sucrose,

the cells were incubated for 30 min in 7x 10-5M

3,3'-diaminobenzidene in 0.05 M Tris buffer (pH 7.2)

(DAB) plus10% sucrose and thenin DABplus 0.05%

H202 for5 min. Cellswereosmicatedin 1%OS04in

0.01 M phosphate buffer afterthree washes in PBS-10% sucrose and were dehydrated in graded ethanols andembedded inEpon-aralditeasdescribed

previous-ly (16). Poprevious-lymerized blockswere thinsectioned,

exam-inedon aPhillips201 electronmicroscope operatingat

80kV, and photographed.

RESULTS

Localization ofinternal structural proteins by

immunohistochemistry. Localization was carried

outby the procedure described in Materials and

Methods by using the IgG fraction from serum

containing antibody against the viral structural protein p27 or normal serum preabsorbed as described above. The specificity of the serum

used was determined by immunoprecipitating

total cell extracts of RAV-2 and CEF labeled with [35S]methionine. Anti-p27 IgG precipitated predominantly Pr76 (which contains p27 anti-genic determinants) and p27, whereas normal

rabbit IgG precipitated no detectable proteins

(datanotshown).

RAV-2-infected CEF and normal CEF were

grown, fixed, and stained. After the embedded

cells were sectioned, they were examined by

electron microscopy.

As canbeseeninFig. 1,RAV-2-infected CEF

showed two major stainingpatterns, one

cyto-plasmic,asexpected, and theother nuclear.The

nuclear stainingwasobserved in approximately

85% of the cells examined and appeared to be

concentrated over a heterochromatic region

withinthenucleus that may represent the

nucle-olus. Twoexamples ofthe nuclear staining

pat-tern can be seen in Fig. 1. Diffuse nuclear

staining was also seen, but on examination at

high magnificationit wasfoundtobeparticulate,

with noobvious structure.

Inadditiontothe nuclearstaining observed, a

diffuse cytoplasmic staining was seen which

showed little localizationto structural elements

(Fig. 1). At higher magnification, the staining

was found to be localized in discrete particles

withinthe cytosolwhich ranged insize from 12

to36 nmindiameter (Fig. 2).

Control staining reactions were carried out

(Fig. 3). RAV-2-infected cells stained with ab-sorbed normal IgG and uninfected cells stained with absorbed anti-p27 showednospecific stain-ing and little background stainstain-ing in either the

cytosolorthenucleus.

Immunoprecipitation of cell fractions. To

con-firm the localization ofaviral structuralprotein

to the nucleus, cellfractionations were carried

out. In addition, itwas of interestto determine whether the protein(s) localized to the nucleus

represented only Pr76orp27,orif both could be foundthere, sincetheantibodyusedfor

staining

recognized both.

RAV-2-infected CEF were labeled with

[35S]methionine,

fractionated into cytoplasmic

and nuclear fractions, and immunoprecipitated

withanantiserumwhich had a high titer against

all the viral structural proteins and pp6Osr'.

Analysis ofthe immunoprecipitates was carried

outby SDS-polyacrylamide gel electrophoresis.

Inall cases, thenucleiweremonitored by

phase-contrastmicroscopyforcytoplasmic

contamina-tion. Ascanbe seen inFig.4a, thepredominant protein precipitated from the nuclear fraction

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.4.

r~~~~~~~~~~~~~~~~~~~~Ar

4*1~~~~~4

4.

FIG. 1. Electronmicrographsof cells stainedwithabsorbed immuneserum.RAV-2-infected CEFwerefixed

in 2%p-formaldehydeand stained with theIgGfraction ofanti-p27 absorbed with normal CEF. The second

antibodyused in these experimentswas sheepanti-rabbitIgGlabeled withhorseradishperoxidase. (a)Arrows

indicate staining over a heterochromatic area ofthe nucleus. Diffuse cytoplasmic staining can also be seen.

Magnification, x24,684. (b) Arrows showreactionproductwithin thenucleus overheterochromaticareas. In

addition, diffusecytoplasmic stainingcanbe seen.Magnification, x20,401.

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a,''

<',

'"'';-ik ;

4

^ r. 9. 1

*-6Z

AL~~~~~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ '

4~~~~~~~~~~~~~~4

cytplsmManiiction

x41,400

..

(@: ors~~~~~~~

FI.

.Hih-aniiatonpotmirgrps

f

yopamicsann.RV2ifce

E eefxd

stiedn

peaedfrelcrn

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ecibdi tetxt

a Arw

idctepriclt

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el.Mgiiain

3,0.()Hge

manfiG.io

2.

Hg-agenification

phthbomicrogaph

sof

c

in

topasi staining.

RAV-2-stinfced CEFti

were

fixhnted

cytoplasm. Magnification, x410,400.

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a

At

oh

a

FIG. 3. Electronmicrographsofstainingcontrols. RAV-2-infected andnormal CEFwerefixed, stained,and

preparedfor electron microscopy as described in the text. (a) RAV-2-infected CEF stained with normal IgG

preabsorbed as described in thetext todetect nonspecific staining. Magnification, x24,684. (b) Normal CEF

stainedwithanti-p27 preabsorbedas describedinthetext todeterminethe level ofnonspecificbackgroundin

normal cells.Magnification, x24,684.

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nuc _b

SR-D

cyto nuc

Pr-76-

-Pr

76-

p27-1 2

pp60-3 4

_-35k

1 2 3 4 T7

FIG. 4. Cell fractionationand immunoprecipitation of RAV-2- andSchmidt-RuppinD(SR-D)-infectedCEF.

In eachcase,cellswerelabeled with [35S]methionine, fractionated, andimmunoprecipitatedasdescribed in the

text.Theywereanalyzed by SDS-polyacrylamide gel electrophoresisfollowed by fluorography. (a)

Autoradio-gramof RAV-2-infected CEF cytoplasmic (cyto)and nuclear (nuc)fractions precipitated with immune serum

from tumor-bearing rabbits (tracks 1 and 3)ornormal serum(tracks 2and4).(b) Autoradiogram of

Schmidt-Ruppin D-infectedCEF cytoplasmic (cyto) and nuclear (nuc) fractions withimmuneserumfrom tumor-bearing

rabbits (tracks1and3)ornormalserum(tracks2 and4). Molecular-weightmarkersare[35S]methionine-labeled

T7.

was Pr76; Pr76 and p27 were precipitated from

the cytoplasmic fraction.

To determine whether the observation of viral

structuralproteins in the nucleuswasrestricted

to RAV-2 infections (a leukosis virus), CEF

infected with asarcoma virus, Schmidt-Ruppin

D, were labeled with [35S]methionine,

fraction-ated, and immunoprecipitated. Ascanbeseenin

Fig. 4b, the predominant protein precipitated in

the nucleuswas again Pr76.

To rule out thepossibility that the structural

proteins precipitated from the nucleus were

boundnon-specificallytothe nuclei during their

isolation,areconstitution experimentwasdone.

RAV-2-infected CEF labeled with

[35S]methi-oninewerefractionatedasbefore,as were

unla-beledinfected cells. The total labeled

cytoplas-micfraction wasthen mixed with the unlabeled

nuclearfraction for 15minat4°C. The unlabeled

nucleiwerethen pelleted and washedoncewith

lysis buffer, aswerelabeled nuclei. The labeled

cytoplasm, the labeled nuclei, and unlabeled

nuclei that had been mixed with labeled

cyto-plasm were then immunoprecipitated with

tu-mor-bearing rabbitserum.AscanbeseeninFig.

5, Pr76 and p27 could be precipitated from the

labeledcytoplasmic fraction from

RAV-2-infect-ed CEF. The predominant protein precipitated

from thelabeled nuclear fraction was Pr76; no

detectable Pr76orp27wasprecipitable from the

unlabeled nuclear fraction which had been

mixed with labeledcytoplasm.

This experiment seemed to indicate that the

Pr76 precipitable from the nuclear fraction of

RAV-2-infected CEFwas notaconsequence of

nonspecific binding of that proteintothe nuclei

during this isolation procedure. Instead, it

ap-peared that Pr76was aspecificcomponentof the

nuclear fraction.

Pr76- _ t 80K

_ l_

~~ _

W

-35K

p27-1 2 3 4 5 6 T7

FIG. 5. Reconstitutionexperiment withcytoplasm

and nucleus. Parallel platesof RAV-2-infected CEF

were fractionated intocytoplasmic and nuclear frac-tions afteroneplatewaslabeled for 2 h with 150,uCiof

[35S]methionine. Labeledcytoplasm was mixed with

unlabelednuclei for15minat4°C. After the unlabeled

nuclei were washed once, three fractions were

immunoprecipitated. Lanes 1 and 4, labeled

cyto-plasm; lanes 2 and 5, labeled nuclei; lanes 3 and 6,

unlabeled nuclei mixed with labeled cytoplasm and

washedoncewith RIPA. Lanes1to3,tumor-bearing

rabbit serum; lanes 4 to 6, normal rabbit serum.

Precipitateswereanalyzed by SDS-polyacrylamide gel

electrophoresis, fluorography, and autoradiography. R AV-2

cyto

VW

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664 ENRIETTO AND ERIKSON

Quantitation of Pr76 in cytoplasmic and

nucle-ar fractions of RAV-2-infected CEF. To

deter-mine the proportion of Pr76 in each fraction,

labeled RAV-2-infected CEF were fractionated

and immunoprecipitated in antibody excess to

precipitate allof the structural protein present. The bands corresponding to Pr76 were cut from

thegel and counteddirectlyin scintillation fluid.

After subtraction of background counts, it was

calculated thatapproximately 6% of the Pr76 in RAV-2-infected cells was precipitable from the nuclear fraction.

DISCUSSION

Inthis series of experiments, itwasfound that viralstructural proteins could be localized to the

nucleus of CEF infected with avian leukosis

sarcoma virus. The predominant protein found

inthe nucleus byimmunoprecipitationwas Pr76;

however, because of the relative proportions of

Pr76 andp27 in thecytoplasmic fraction,it is not

possible to rule out the presence of p27 inthe nucleus also. It should be said, however, that

intermediate cleavage products of Pr76 (p66,

p60) were never observed in the nucleus by

immunoprecipitation.

A nuclear phase for the viral structural

pro-teins of avian leukosis sarcoma virus has not

been described before. However, regulatory roleshave been proposed for theviralstructural

proteins by several investigators. It has been

suggested that a virus-specific protein is

re-quired for integration of proviral DNA during virusreplication(6, 7, 12), and such a model has

been describedin the murine system to explain

theabrogation of

Fv_lb

restriction (8). In

addi-tion, it has been suggested that a viral protein

might be involved in the regulation of the

splic-ing of the5'terminalsequence of the genome to

subgenomic mRNA (5, 7).

Athird regulatory role could beimagined for viralstructuralproteinsin theselection of newly transcribed viral RNA destined to become

ge-nomic RNA in budding virions. The fact that viral structural proteins have been shown to

bindviral RNA andassociatewith it in thevirion

(2, 15) indicates arole for structural

proteins

in

virusmorphogenesis atanother level.

Anindication that the viral structuralproteins

may have adifferent role inthe cytoplasmand

nucleus comes from preliminary pulse-chase

experiments. When Pr76 inthe

cytoplasm

and

nucleus arefollowedduring variouspulse-chase

conditions, the turnover time of cytoplasmic

Pr76 appears to be longer than that ofnuclear

Pr76, suggesting adifferent roleforthe nuclear

structuralproteins.

The second

pattern

ofstaining observed was

cytoplasmicanddiffuseinnature.In

addition,

it

wasfoundthatboth Pr76 andp27were

immuno-precipitable from the cytoplasm of infected

cells. Thepresenceof viral structuralproteins in

the cytoplasm wasexpected; however, we were

surprised to find such a diffuse pattern. No

evidencewas seenfor specificassociationof the

viralstructural proteinswithinternal membrane

systems of the host cell or the plasma

mem-brane. Thismayreflect thelimits ofdetection of this technique, since it has been reported that

only 15% of the Pr76 is synthesized on

mem-brane-bound polysomes(22). Some localization at the plasma membrane was also expected; however,werarelysawbudding viruswhere an

accumulation of viral proteins wouldhavebeen

more likely. Again, this may reflect the limita-tions of the technique employed.

Thelocalizationthat was seen athigh magnifi-cation appeared in discrete particles, which

rangedin size from 12 to 36 nm. Thepossibility

existed that these structures represented

poly-somes in the process of translating viral 35S

mRNAintoPr76.This seemsunlikely, sincethe

size of the average ribosome is approximately

200 nm(20). Theseparticlesmay instead

repre-sent viral protein complexes or protein:RNA complexes which will, with maturation, bud from the surface of the cell.

In summary, localization of viral structural

proteins Pr76 and p27 wascarried out, with the finding that a portion of Pr76 is located in the

nucleus oftheinfected cell. Itisnotcompletely clear whether cleavage products of Pr76 are

presentinthenucleus, amatterwhich could be

clarified by using monospecific antisera of

high

titeragainst theseproteins.

ACKNOWLEDGMENTS

Theadvice andhelp of Alan S. Jones and P.K.Nakaneis gratefullyacknowledged.

This workwassupportedby Public Health Service grants CA 15828 and CA 21117 from the National Institutes of Health. P.J.E. wastherecipientofapredoctoral fellowship (training grant CA 09157) from the National Institutes of Health.

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