JOURNAL OF VIROLOGY, OCt. 1968, p. 1133-1146Copyright ( 1968 American Society for Microbiology

Vol. 2, No. 10Prinited int U.S.A.

Ultrastructure of the Surfaces of Cells Infectedwith Avian Leukosis-Sarcoma Viruses

HERBERT R. MORGAN'Itnstitut de Recherches sutr le Cca,cer, Villejuiif 94 Fran7ce, anid the Louis A. Wehle Virus Research Laboratory,

Departmenzt of Microbiology, Uniiversity of Rochester School of Medicin7e anld Denitistry,Rochester, New York 14620

Received for publication 28 June 1968

When stained with ruthenium red (RR), chick embryo cells infected with variousstrains of Rous sarcoma virus (RSV) and with avian leukosis viruses RAV-1 andRAV-3 showed an increase in the layer of acid mucopolysaccharides (AMPS) attheir surfaces as compared with uninfected cells. This increase was most prominentin cells infected with the Fujinami strain of RSV. The layer was resistant to di-gestion with neuraminidase or trypsin but was readily removed by exposure tohyaluronidase. The thickness of this AMPS layer was not correlated with thevarying degree of loss of contact inhibition exhibited by cells infected with thedifferent strains of virus. The staining of the cell envelope with a solution of phos-photungstic and chromic acids (PTA-CR) suggested the presence of glycoproteins.The outer surface of the virions showed the same staining as the cell surface withRR and PTA-CR, and the budding virus particle was seen to incorporate the RRlayer of the cell into its structure. The RR layers of cells and virions appeared tofuse, as did those between virus particles, suggesting that these layers play a rolein the aggregation of virus particles and in their adherence to the surface of the cell.

Previous studies (3) demonstrated that coloniesof transformed cells developing in cultures ofchick embryo fibroblasts infected with BryanRous sarcoma virus (Br-RSV) accumulate acidmucopolysaccharides (AMPS), as indicated byintense staining with the Hale colloidal iron tech-nique. Other investigators (5, 12) showed that in-fection of chick embryo fibroblasts in vitro withRous sarcoma viruses resulted in a significant in-crease in cellular levels of hyaluronic acid synthe-tase and in the production of hyaluronic acid.Since morphological transformation of cells oc-curs after infection with these viruses and isassociated in several with a marked loss of con-tact inhibition of the cells, it was of interest toexamine the surface of infected cells for the pres-ence of mucopolysaccharides. To detect thesematerials, we used the ultrastructural, cytochem-ical method with ruthenium red (RR) staining(8). In addition, the intercellular relationshipswere studied by means of in situ embedding ofthe cultivated cells (9).

Several strains of the avian leukosis-sarcomavirus group were selected for study since thesestrains produce different types of morphological

1 Recipient of a fellowship from the CommonwealthFund.

transformation of cells, which in turn differ inthe degree of loss of contact inhibition exhibited;thus, it was possible to relate the layer of muco-polysaccharides at the surfaces of the cells to thebehavior of the cells in vitro.

MATERIALS AND METHODS

Viruses. All viruses were prepared by removing thesupernatant medium from chick embryo fibroblastcell cultures infected with the various viruses. Thefluids were then stored at -70 C until used. TheBryan strain (Br-RSV) was derived from virus CT-845supplied by W. R. Bryan (National Cancer Institute,Bethesda, Md.), and the morph-r and morph-f strainswere received from H. Temin (University of Wis-consin, Madison) who derived them from the Bryanstandard Rous sarcoma virus. The Fujinami strain(Fu-RSV) was obtained from E. Reich (RockefellerUniversity, New York, N.Y.), and an RAV virus con-taining a mixture of RAV-1 and RAV-3 was obtainedfrom J. Bader (National Cancer Institute, Bethesda,Md.).

Cells anid cell culture. Chick embryo fibroblasts werederived from susceptible embryonated eggs obtainedfrom a flock of chickens maintained by R. E. Lugin-buhl (University of Connecticut, Storrs) and weresupplied by the research resource program of theNational Cancer Institute. All cells were tested foruniform susceptibility to infection with B;-RSV asused in each experiment. After mincing of the embryo

1133

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/j

vi o

n 28

Dec

embe

r 20

21 b

y 93

.126

.11.

218.

MORGAN

tissues and treatment with trypsin, the cells were cul-tured in plastic petri dishes according to techniquespreviously described (10).To infect cells derived from a single chick embryo

with the various strains of viruses, we used between2 X 10 and 3 X 104 focus-forming units per 100-mmplastic petri dish and a dose of RAV which produceda solid resistance of infected cells to superinfection byBr-RSV 7 days after infection. After 1 week, all cellswere subcultured in prescription bottles, Leightontubes, and 100-mm plastic petri dishes containingcover slips. The cultures were observed daily untilmorphological transformation and formation of fociwere clearly visible in those strains producing thesechanges; then (about 2 weeks after infection) ap-propriate cultures were selected for histological andultrastructural examination. Uninfected cells werehandled in the same manner. Some infected cultureswere examined up to 33 days after infection, at whichtime almost all cells had developed the characteristicmorphology of viral infected cells. i-Xi

Histological examination. Cover slips were removed,fixed with neutral buffered Formalin, and stained withMay-Grunwald Giemsa dye.

Electron microscopy. The method described byMartinez-Palomo et al. (9) for preparation and ex-amination of cells was employed. Cells in prescriptionbottles and Leighton tubes were fixed in situ with2.5% glutaraldehyde-cacodylate buffer solution (pH7.3) for 1 hr at 4 C. Postfixation was carried out witha solution of 2% osmium tetroxide in cacodylatebuffer for 3 hr at room temperature. The RR dye wasadded to both fixatives at a concentration of 50mg/100 ml. The cells were then dehydrated in increas-ing concentrations of acetone and were embedded insitu in tubes or bottles with a layer of Epon 2 to 3 mmthick. After polymerization at 60 C, the glass con-tainers were broken and the layer of Epon containingthe cells was detached after a brief immersion in boil-ing water. The cells were located with the aid of amicroscope, and suitable colonies were selected; thesecolonies were then cut out in blocks, approximately2 by 3 by 1 mm, and were oriented to obtain verticalsections through the colonies. The sections werestained with lead citrate (Reynolds) for 10 min.

In other instances, cells were scraped from the glasssurface into a 1.6% solution of glutaraldehyde inSorenson's buffer kept for 15 min at 4 C; then thecells were embedded in glycol methacrylate accordingto the method of Leduc and Bernhard (7) and weresubsequently polymerized with ultraviolet irradiation.

Sections were prepared from the cell button andwere stained with an aqueous solution containing 1%phosphotungstic and 10% chromic acids according tothe method described by Rambourg (1 1).

Sections were examined with a Siemens Elmiskop Ielectron microscope.Enzyme digestions. Cell cultures were rinsed with

0.85% saline and were treated with (i) a solutioncontaining 300 National Formulary units of hyaluroni-dase (Wyeth) per ml in 0.85% saline or (ii) a solutioncomprised of 500 units (number of micrograms ofN-acetyl neuraminic acid liberated at 37 C from acida--l-glycoprotein) of neuraminidase (Behringwerke) in

a sodium acetate-acetic acid buffer (pH 5.5) contain-ing 0.9 g of NaCl and 0.1 g of CaC12 per 100 ml for 3hr at 37 C. In both instances, digestion was con-tinued until the cells began to detach from the glasssurface.

For trypsin digestion, cells were fixed for 1 hr with2.5% glutaraldehyde-cacodylate buffer solution,rinsed repeatedly with cacodylate buffer, and thenexposed to a 0.25% solution of trypsin (Difco) inphosphate-buffered saline (pH 7.3) for 2 hr at 37 C.

In all instances, the cells were then carefully rinsedwith cacodylate buffer and were processed as de-scribed for RR staining.

RESULTS

Giemsa and RR stains. Normal chick embryofibroblasts grew in typical patterns (Fig. 1) withoccasional overlapping of cells (Fig. 1 and 2) andpossessed a layer of dark material at the cellsurface when stained with RR; this materialvaried in thickness and density over the cell sur-face (Fig. 2, lOa).

Foci of fibroblasts infected with Br-RSVshowed characteristic, rounded, dark cells (Fig.3) piled up in multiple layers (Fig. 3-5) withmany long cell processes (Fig. 5). The RR layerat the cell surface was heaviest at the free surfacesin the foci, as the dye did not penetrate well intothe mass of cells. The RR layer was slightly moreprominent in infected (Fig. lOb) than in controlcells (Fig. lOa). Intercellular contacts of the typeknown as close junctions were seen; in these junc-tions, the RR staining layer between the cells wascontinuous (Fig. 5). Foci of cells infected for 2weeks showed the same type of RR layer as thosefrom cultures infected for as long as 33 days.Since only cells in foci of transformation wereselected for study, the possibility of examininguninfected cells in the culture was eliminated andcells at the upper surface of a colony were used topermit maximal interaction of the RR stain withthe free cell surface.

Cells infected with morph-r-RSV developedsharp, round foci of piled up cells (Fig. 7) withmany processes (Fig. 6) at the cell surface, whichwas covered with an RR layer (Fig. 6, lOc) some-what more prominent than that seen in controlcells; cells infected with morph-f-RSV showedtypical, fusiform cells in foci, with overlapping ofcells (Fig. 8) but without the formation of mul-tiple layers. Contacts between cells infected withmorph-r-RSV were of the type characterized asclose junctions (Fig. 6). The RR layer of morph-f-RSV-infected cells (Fig. lOd) was as prominentas that seen with the other two strains of virus;however, few cell processes were seen and theywere not as long.The Fu-RSV-infected cells became rounded and

produced multilayered, dense foci (Fig. 9) in

1134 J. VIROL.

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/j

vi o

n 28

Dec

embe

r 20

21 b

y 93

.126

.11.

218.

VOL. 2, 1968 SURFACES OF VIRUS-INFECTED CELLS 1135

4W-?4 *' os s >,rS * _

-~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~-

4!, C X s X ' s. 4 -e t &> M

-i'- ; g F _,;:' 'ow':E St

IN~~~~~~~~~~~~~~~~~~~~~I"

.%.~~~~~~~~~~~~~~~~~4

FiG. I. Mooae cutr of noma chc emry fibroblsts with ocsnal ovrapig cls. Gesa

AL.

stain.X 362F q0.FIG.'2.Normalchickembryofibroblastsshowing overlapping of cells and ruthenium red stained layers whichartccmn

7w~~~~~l: *- r

*:++ D: ... .Wl.

.4@S: ' .. * . 'ill ?' AP14f'ist,aing. X.320:FIG.>2. Noma chc emry firbat showiSXg ovrappi. Aqfetsad rtheu re ,...,Stai .e laes which

aemrpriiettathfreclsufc.Rteiuedstain.X3020.0

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/j

vi o

n 28

Dec

embe

r 20

21 b

y 93

.126

.11.

218.

MORGAN

which the cells showed many processes whichinterdigitated; this was also seen in cells infectedwith Br-RSV and morph-r-RSV. In Fu-RSV-infected cells, the RR layer was denser and muchthicker than in uninfected cells or in cells infectedwith the other strains of virus (Fig. lOe).The infection of cells with a mixture of RAV-1

r.

f}. < A

and RAV-3 produced no obvious changes in cellmorphology (Fig. 11); the cells remained as amonolayer with occasional overlapping. The RRlayer of the cells was similar to that found oncells infected with various strains of RSV exceptFu-RSV (Fig. 12).

Virus particles were seen in all infected cul

0

:No*:w X .

P"~~~t^S^,4.

+A r

X . ^ . :. .. .: ' . , W_ . . .

-~~~~~~~~~~~~~~~~~-o

FIG. 3. Co/olil) oj'click embryo.fibroblasts ilnfected with Br-RSV showinzg piled uip cells. Giemsa stanil X 320.FIG. 4. Vertical sectionz throuigh a coloniy of cells infected with Br-RSV demonzstratinzg loss of conitact inihibitionz

and the layer of ruthenium red staining material at the cell surfaces. Ruthenium reds:ain. X 12.000.

1136 J. VIROL.

'k. jII j,:i, -

41-",,

:,-- ivAel

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/j

vi o

n 28

Dec

embe

r 20

21 b

y 93

.126

.11.

218.

VOL. 2, 1968 SURFACES OF VIRUS-INFECTED CELLS 1137

< < f ;, e t > j * > t f tO.91 ~lis'.t A,i..11I #1:WI)*,* J(.-'lxl._l!N- ,.'.; A1,e-

.I

5

'A-

'P tw.4%t *,s\.-4-zw eS>uaa ; Cv*.7

FIG. 5. Vertical sectiont through a colonty of cells inifected with Br-RSV showing characteristic lonlg processesextending.from the cells anld intercellular conitacts characterized as close junctions (arrow). Rutheniunm red stain.X 6,000.

FIG. 6. Cellular processes of chick fibroblasts infected with morph-r-RSV having manty intercellular contactsof the type idenitified as close juncetiolns (arrows). Ruttheniu(m red stahil. X 60,000.

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/j

vi o

n 28

Dec

embe

r 20

21 b

y 93

.126

.11.

218.

MORGAN

:I

4

...4:

Iri"!$ f,

xAk:. i.,:.21

<<.,'^-, .#. 4aR t ..* .'

i-.~ ~~

10*̂ .t

8

*:. F

.... .:,

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

h. Ak

Mi!i

..9

rr

...X

...

sI ;

...

Vss,g

SS.

**,S

*t **: mV4..41

_ F

FIG. 7. Cells infected with morph-r-RSV forming a clump of roulnded cells. Giemsa stainl. X 320.FIG. 8. Cells infPcted wvit/i morp/i-f'RSV. The characteristic fusiform cells show some overlappilng. Giemsa

staini. X 320.FIG. 9. Colony, of roainided cells ilfected with Fa-RS V (arrow) in a cu/ltore of' chick embryo fibroblasts. Giemsa

staini. X 320.

:7,.U

..,

:S

4k

:4.'''

:W pz>S:

A,.bM

:.''s+.

1 138 J. VIROL.

A:

.xk ::

:.... 'i

At';

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/j

vi o

n 28

Dec

embe

r 20

21 b

y 93

.126

.11.

218.

VOL. 2, 1968 SURFACES OF VIRUS-INFECTED CELLS

4a

4L

b

y~~~~~~~.7.. &4:; A"~~~~~~~~~~~~~~~~ -A-,'A

4.^:..V

ta-ffv.c*

iS O

N

Ait'".4 . ..Il

G *.

.1

I!

.4 ..p 'k.zlil , -41,

.,. i

a

V

F P..S.

ts,c: , < W,1rwS^.O'AizI-W40V

**~~~~~~~~ t~~~~~~~~

FIG. 10. Electron micrographs illustrating the ruthenium red layer at the upperfree surface of (a) normal cellsanid of cells infected with (b) Br-RSV, (c) morph-r-RSV, (d) morph-f-RSV, and (e) Fu-RSV. Ruthenium red stain.X 60,000.

1139

.4

_lU ~l.I

sVR=--

I

-11.

14

. .

VF5I..-,L -MA A_ A,

t

.k

>. ~~~~~~~~~~~1-

IL

ilI

.

I .1.11

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/j

vi o

n 28

Dec

embe

r 20

21 b

y 93

.126

.11.

218.

1140 MORGAN J. VIROL.

i7.7M A:

411,--. V-'N NI&jfe

El;. 7.7f- 6i

M:A 1W. -is..

..suf:

2v

4,`7

R Ali

Al

'10.5-Zi

Ns 4 :.!.'..1.2i;i.,PE L :f :.:sE4

;Z.

fs,

ZZ`:::.:JF s:.

q-- Wwa "PIP!

Nk,

Zs N.

osf NW

AM. 'IfisIf

Wt

-AL

"Ra

W

7

.jNN4

.....g; !IN 7

,4-7] R4k

.77 -J. .31 ,j

..Ar

M-mfi

e,1TV Jf 0

.-A

AfE, .7iki,..... ... T

AJz

7Sllmsm.'g W4-A

T..lw

i. V

12

FiG. I 1. Cells inl.cted with RA V-] aizd RA V-3. Giemsa staiii. X 320.Fic,. 12. Cells i0ected with RA V-I aiid RA V-3. Ruthenium red staiii. X 60,000.

tures, but not in normal cell cultures, and their GMA and stained with PTA-CR showed a dismorphology is described below. continuous layer of material at the surface of

Phosphotungstic-chromic acid (PTA-CR) stain, normal (Fig. 13) and infected (Fig. 14) cells; thisExamination of the btittons of cells embedded i,,i discontinuous layer probably resulted from

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/j

vi o

n 28

Dec

embe

r 20

21 b

y 93

.126

.11.

218.

SURFACES OF VIRUS-INFECTED CELLS

*si.

13*4

tZ .*.v

...a.>_ .e,,.t. ;'.il': .. ,*' '.,ts{W* 4 W saAb.. . st+. 6 _S. s3s_n t t

,;¢ ,If.S_ .. < ,t,: .-e:

1So . iaS..*tF

_, w.*

r4

04:~ ~ ~ 4

tt

*

..f,

r,_

FIG. 13. Normal cells. Phosphotungstic-chromic acid stain. X 30,000.FIG. 14. Cells infected with Fu-RSV. Phosphotungstic-chromic acid stain. X 30,000.

trauma to the cell surface when the cells were re- normal cells somewhat (Fig. 15a, b) but had nomoved from the glass substrate in the preparation effect on the RR layer of cells infected with Fu-of the cell buttons for examination. RSV (Fig. 15d, e) and other strains of RSV.Enzyme treatment. Neuraminidase reduced the Hyaluronidase markedly reduced the RR layer

thickness and density of the RR-staining layer of of normal fibroblasts (Fig. 15c) and almost en-

VOL. 2, 1968 1 141

.t:::i3

.-- I ..,-

.ivll_, ---.

..",zdMLi.- -r

K. I

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/j

vi o

n 28

Dec

embe

r 20

21 b

y 93

.126

.11.

218.

MORGAN

JY-r g1

U ..

A

44,

b

"I

Ih~4

C

d

-OR

e

t

f

FIG. 15. Electroni micrographs illustratintg the actioni of variolus enzzymes oii nzormal cells anid oln cells infectedwith Fu-RSV. Normal cells: (a) conitrol, (b) nieuraminiidase, and (c) hiyalurolnidase. Cells inifected with Fu-RSV:(d) control, (e) nieuraminiidase, anid (f) hyalulronzidase. Ruthenlium red staini. X 60,000.

tirely removed the thick RR layer from cells in-fected with Fu-RSV (Fig. 15f). Trypsin did notaffect the RR layer of glutaraldehyde-fixed normal

cells or the RR layer of cells infected with morph-r-RSV, although the enzyme penetrated the cellsand disrupted intracellular structures.

Im

a lL*.-W i--jamd,

J V IROL.1142

.4 I. ,

-4*-. 1:...- -.h.

-4; I

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/j

vi o

n 28

Dec

embe

r 20

21 b

y 93

.126

.11.

218.

SURFACES OF VIRUS-INFECTED CELLS

f..*ile16

Ji1111.1:s

A

ML \

17 ..

w~~~s_

SF'':f

.R.

i&, ,:

:.11

II.:.p*:'. : - .: 3P' ''-' ''jllllE # t:S F - .XB . ,w ?>. .g::e5. ''.-i :> . y ' t .. /' ...... .'''

FIG. 16. Viruts particle (arrow) budding from surface of cell inifected with Fu-RSV. Ruthienium red stain. X90,000.

FIG. 17. Two virus particles (arrow) at the surface of a cell iniftcted with Fu-RSV. Ruthelnium red stain. X90,000.

Virus. With all strains of RSV studied and withRAV-1 and RAV-3, the virus particles were sur-rounded with a thick layer ofRR staining material(Fig. 17 and 18). As the virus was seen to budfrom the cell surface (Fig. 16), the RR layer was

incorporated as part of the viral envelope. Largeclumps of virus particles were frequently seen(Fig. 18), with the RR layers of the virions closelypacked together. These clumps and single virusparticles were often attached to the cell surfaces,

1143VOL. 2, 1968

i r."

r:'-.

*1 t.o

:X.;.. I-WI wfi,4

ALa

Ll.

N'ON.z

i%;.: 'v.,6

aI :.

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/j

vi o

n 28

Dec

embe

r 20

21 b

y 93

.126

.11.

218.

MORGAN

with the RR layer of the virus and the cell form-ing a continuous structure (Fig. 17 and 18). Theviral RR layer was not affected by treatment withneuraminidase and the virions remained attachedto the cells; however, in cultures treated withhyaluronidase the abundant virus particles dis-appeared, suggesting that the enzyme had re-leased them from the cells and that they had dis-persed and were washed from the cultures whenthe enzyme solution was removed. Virus particlessedimented from the enzyme solution by centrifu-gation and stained with RR possessed no darkouter layer.The virus particles in the GMA sections stained

with PTA-CR were embedded in a stained matrixin clumps which usually adhered closely to thesurfaces of cells, with continuity of the viral andcellular envelope material (Fig. 19).

DISCUSSION

When used in combination with osmium te-troxide, the RR stain which has a particular affin-ity for highly polymerized acidic polysaccharidesproduces a heavy black layer at the surface ofcells containing such substances (8). The dye isknown to combine readily with polymerizedhyaluronic acid and chondroitin sulfate but notwith sialic acid (J. H. Luft, personal communica-tion). Thus, it is a good stain for the study of thesurface layer of cells infected with Rous sarcomaviruses, since this layer has been shown to containAMPS belonging to the hyaluronic acid-chon-droitin sulfuric acid group (3). The easy removalof this layer by hyaluronidase and its resistance todissolution by neuraminidase and trypsin indicatethat hyaluronic acid-containing AMPS are re-sponsible to a large extent for the RR staining.Since hyaluronidase appears to have a slightlygreater effect on RSV-infected cells than onnormal cells and the AMPS layer of infected cellsis totally resistant to treatment with neuramini-dase, differences in the AMPS layer of normaland RSV-infected cells may exist. Chemicalanalyses (6) have shown that the hyaluronic acidisolated from Rous sarcoma tissues has a smallerparticle size than the hyaluronic acid isolatedfrom normal tissues. Furthermore, the hyaluronicacid isolated from Rous sarcoma tissues producedincreased spreading of dye in tissues and increasedcapillary permeability when injected locally,whereas the hyaluronic acid from normal tissuesdid not (6). Therefore, it is possible that theAMPS layer on the tumor cells may be differentfrom that on normal cells, even though Ishiomotoet al. (5) were unable to detect differences betweenthe hyaluronic synthetase derived from RSV-infected or uninfected cells cultured in vitro.

This surface layer is probably the same as theamorphous material, which was thought to con-tain AMPS, previously described at the surface ofRous sarcoma tumor cells in electron microscopicstudies (4). The staining of the layer with PTA-CRsuggests that it also contains glycoproteins (11).

In these investigations, no correlation was ob-served between the thickness of the RR layer andthe characteristic social behavior of cells infectedin vitro with the various strains of RSV or leu-kosis viruses. RSV strains such as Br-RSV andmorph-r-RSV, which induce the formation ofdense foci of cells exhibiting a prominent loss ofcontact inhibition, had an RR layer of the samethickness as cells infected with morph-f-RSV orRAV-1 and RAV-3, which showed little or noloss of contact inhibition. It is of interest to note,however, that the Fu-RSV infected cells whichproduced the most significant increase in AMPSafter infection in vitro (12) also had the densestand thickest layer of AMPS at the cell surface.

These findings with the RSV strains are atvariance with the results obtained by Defendi andGasic (2) and Martinez-Palomo et al. (9); theseinvestigators found a correlation between an in-crease of AMPS at the surface of cells trans-formed by infection with polyoma, SV40, oradeno-12 viruses and their loss of contact inhibi-tion. The situation between these DNA virusesand the ribonucleic acid leukosis-sarcoma virusesmay be different and the increase in surfaceAMPS is not necessarily related to the loss of con-tact inhibition in all cases.The development of long processes at the sur-

faces of RSV-infected cells was most common inthose cells found in the multilayered foci and isprobably correlated with the increased surfaceactivity of these cells as they migrated over oneanother to form the foci.The intercellular contacts between both normal

and virus-infected cells involved the formation ofclose junctions (intermediate junctions and tightjunctions were not observed) and no essentialdifferences in the numbers or nature of these celljunctions was seen. The differences in cell contactsobserved by Martinez-Palomo et al. (unpublisheddata) in hamster cells transformed with SV40virus or adenovirus 12, where the tight junctionsseen in controls had practically disappeared inthe transformed cultures, could not be studied inthis investigation since no tight junctions wereobserved in either control or infected cells.The outer envelope of the virus particles showed

the same RR staining and reaction to PTA-CRas did the cell surface. This was expected since thebudding virus particle (Fig. 16) was seen to in-corporate the RR layer of the cell into its struc-

1144 J. VIROL.

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/j

vi o

n 28

Dec

embe

r 20

21 b

y 93

.126

.11.

218.

VOL. 2, 1968 SURFACES OF VIRUS-INFECTED CELLS 1145

__ _

is.~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

A195.aFIG. 18. Aggregationz of virlus particles at the suirface of a cell inifected with Br-RSV. Riuthe,uuirnm red staini. X

90,000.FIG. 19. Viruts particles in clumps at the surface of a cell ilnfrcted with Fit-RS V. Plhospluotuntigstic-chromic acid

staini. X 30,000.

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/j

vi o

n 28

Dec

embe

r 20

21 b

y 93

.126

.11.

218.

MORGAN

ture, which thus contained AMPS and glycopro-teins. The viral envelope appeared to merge withthe cellular envelope to which the virions werefrequently attached, suggesting that these en-velopes play a role in this attachment as well asin the formation of large clumps of virus particleswhich were frequently seen in the infected cul-tures. The relationship between the viral andcellular envelopes was the same when stainedwith RR or PTA-CR. Since most of the virionsin the cultures were present in aggregates, theimportance of releasing the virus particles fromthe cells and dispersing them before attemptingto assay the virus content of fluids or attemptingto prepare stock virus from infected tissue cul-tures is obvious.

ACKNOWLEDGMENTS

The author is indebted to W. Bernhard for adviceand counsel and to Paul Tournier for providingfacilities for the tissue culture work. The excellenttechnical assistance of J. Burglen and A. Viron is alsoacknowledged.

This investigation was supported by Public HealthService research grant RICA5208 from the NationalCancer Institute, and by a grant from the Louis A.Wehle Foundation and the Centre de Recherches surles Lymphoma Malins, Lausanne, Switzerland.

LITERATURE CITED

1. Abercrombie, M., and E. J. Ambrose. 1964. Thesurface of cancer cells. Cancer Res. 22:525-548.

2. Defendi, V., and G. Gasic. 1963. Surface muco-polysaccharides of polyoma virus transformedcells. J. Cellular Comp. Physiol. 62:23-31.

3. Erichsen, S. J., J. Eng, and H. R. Morgan. 1961.Comparative studies in Rous sarcoma withvirus, tumor cells and chick embryo cells trans-

formed in vitro by virus. I. Production ofmucopolysaccharides. J. Exptl. Med. 114:435-439.

4. Heine, U., G. De The, H. Ishiguro, and J. W.Beard. 1962. Morphologic aspects of Roussarcoma virus elaboration. J. Natl. CancerInst. 29:211-223.

5. Ishimoto, I., H. M. Temin, and J. L. Strominger.1966. Studies of carcinogenesis by avian sar-coma viruses. II Virus-induced increase inhyaluronic acid synthetase in chicken fibro-blasts. J. Biol. Chem. 241:2052-2057.

6. Kimoto, E., and M. R. Shetlar. 1965. Propertiesof Rous sarcoma hyaluronic acid. Proc. Soc.Exptl. Biol. Med. 118:627-631.

7. Leduc, E. H., and W. Bernhard. 1967. Recentmodifications of the glycol methacrylate em-bedding procedure. J. Ultrastruct. Res. 19:196-199.

8. Luft, J. H. 1966. Fine structure of capillary andendocapillary layer as revealed by rutheniumred. Federation Proc. 25:1773-1783.

9. Martinez-Palomo, A., and C. Brailovsky. 1968.Surface layer in tumor cells transformed byadeno-12 and SV 40 viruses. Virology 34:379-382.

10. Morgan, H. R. 1965. Antibodies for Rous sar-coma virus in fowl, animal and human popula-tions of East Africa. I. Antibodies in domesticchickens and wildfowl. J. Natl. Cancer Inst.35:1043-1C45.

11. Rambourg, A. 1967. Detection des glycoproteinesen microscopie electronique; coloration de lasurface cellulaire et de l'appareil de Golgi parun melange acide chromique-phosphotungstic.Compt. Rend. 265:1426-1428.

12. Temin, H. M. 1965. The mechanism of carcino-genesis by avian sarcoma viruses. I. Cell mul-tiplication and differentiation. J. Natl. CancerInst. 35:679-693.

1146 J. VIROL.

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/j

vi o

n 28

Dec

embe

r 20

21 b

y 93

.126

.11.

218.