Comparison of lipophilic proteins in murine and avian oncornaviruses

Vol. 100, No. 3,198l June

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS Pages 1173-1182

16, 1981

COMPARISON OF LIPOPHILIC

PROTEINS IN MURINE AND AVIAN ONCORNAVIRUSES.

Fraqois

AUDUBERT and Marianne

SEWEL.

LABORATOIRE DE CARCINOLOGIE EXPERIMENTALE GROUPE DE RECHERCHE no8 du CNRS

INSTITUT GUSTAVE-ROUSSY 16 bis,

Received

March

24,

avenue Paul Vaillant-Couturier 94800 VILLEJUIF, FRANCE

1981

SUHARY This report deals with the occurence of lipophilic proteins in avian and murine oncornaviruses. In these compounds the protein moieties are firm1.y though not covalently bound to the phospholipid moieties. This linkage may contribute to the solubility of these compounds in orqanic solvents, which provides a mean to separate them from other proteins. With an acidic chloroform-methanol mixture, proteins of an apparent molecular weight of 25.000 and 10-17.000 daltons are extracted from Rous Sarcoma Virus and from Rous associated Virus, and proteins of apparent M.W. of 12-22.000 daltons are extracted from the Friend Virus Complex. Immunoprecipitation with specific antisera shows that the lipophilic material contains avian ~27, murine ~15, pl5 E and probably pl2 E and that murine gp69/71, ~10 and ~12 are not lipophilic. Some phospholipids remain attached to the lipophilic proteins after extraction ; they are mainly phosphatidylserine and phosphatidylinositol. A model descrihinq the organisation of the protein constituents in virions based on the known properties of oncornavirus proteins and our results is proposed. INTRODUCTION Lipophilic lipids

proteins

: lipids

are

solvents.

These

solvents

in the

proteolipids phobic

proteins

aminacids

sources

Forest

exception

naviruses.

have

Virus

(SFV)

fl\,

are known

as in aqueous

they

compounds

resemble rich

several

Stomatitis

bonds.

components enveloped Virus

the

in h.ydro-

and hydrophobic

from

and

in organic

as well

to be membrane

Vesicular

proteins

fVSV\

f21. viru(41,

Rous associated virus (RAV 21(51 and bacteriophage PM2 proteins of SFV have been shown to be fragments of glycoproteins and not complete structural viral proteins. With

of RAV 2, no lipophilic However,

and Lees

been extracted f3),

both extraction

solvents

by electrostatic

lipids

proteins

their

In man-y respects

detergents.

various

contain

after

in organic

by Folch to

Influenza Virus, (61. The lipophilic sylated memhrane the

of

which

proteins

are soluble

and linked

from

compounds

the

described

Lipophilic ses : Semliki

to

presence

first

Proteolipids

are

bound

several

of

the

proteins structural

have proteins

been described of these

for viruses

oncorhave

0006-291X/81/111173-IO$Ol.OO/O 1173

Cop.vright 8 1981 b.v Academic Press, Inc. All rights of reproduction in any form reserved.

Vol. 100, No. 3,198l

BIOCHEMICAL

been shown to and gp69/71 et al.

murine

of

murine

(101.

"core

may shed

is hydrophobic. proteins

33,5

Thus,

hydrophobic

(71.

been

described p30 as core differ,

p27 contains

the

to

murine

be

murine

however,

at

lipids

and avian

regions the (avian

viral pl9,

~15 as internal

(111.

in

amino-acid

their

pl5

to Reynolds

proteins

48 % non polar

pl5E,

hydrophobic located

of viral

shell

COMMUNICATIONS

According

p30 contains shown

or at the vicinity

authors

proteins

avian

RESEARCH

degrees. Murine

have

pl5El

Other

shell"

only

The avian

aminoacids,

the

structural

viral

and

composimurine

~31

% (121.

Evidence

of

light

on their

investigated

in varying

p27 and murine

the

BIOPHYSICAL

or moderatel~y

gp69/71,

avian

: while

contains

pl9

these

(murine pI51

proteins,

have

avian

Some

membrane

tion

are strongly

f8),

(91.

be h.ydrophobic

AND

the

the

lipophilic relation

eventual

nature

of

the

viral

with lipophilic

the

membrane,

properties

and that

of viral

proteins is why we

proteins.

MATERIAL AND METHODS Virus production and labeling. Rous Sarcoma Virus (RSVl (strainSchmidt-Ruooin. D. London] and RAVE came from Dr Viaier's laboratorv. RSV (Strain Prague; subgroup C, ts mutant LA 3351 came from'Dr. Wyke's laboiatory. Friend Virus (FVl complex fSpleen Focus Forming Virus and Lymphatic Leukemia Helper Virus.1 were isolated from the 745A clone of Friend cells, given by Dr. C. Friend to Dr. Lacour's laboratory. Primary chick embryo cells were explanted from 11 da.y old lymphomatosis free embr,yos. The cultures were infected in Falcon plastic bottles with either RSV or RAV at a multiplicity of = 0.1 and incubated in Eagle's minimal medium containing 10 % tryptose phosphate broth, and 10 % newborn calf serum. The cells were maintained at 37' and trypsinized when they reached conf1uenc.y. After 7-9 days, the medium of semiconfluent cultures was replaced by the labeling medium : when proteins were to be labeled, Eagle's minimum medium consaining 7 % newborn calf serum, I % dimethyl sulfoxide, lacking valine, and 5 &i/ml of 1 HI valine were used. When phospholipids were to be labeled, Eagle's minimal medium containing 50 % of the normal amount of phosph@e, 2 % dialyzed calf serum, 1 % dimethyl sulfoxide, and 5 &i/ml of [ P] phosphate without carrier were used. DMSO was used to enhance virus release as recomended by VOGT et al. f261. Cells were incubated for 48 h, supernatant was collected at 12 h intervals and sto ed at +4'. Friend cells were seeded at 10 5 cells/ml in Eagle's minimal medium lacking valine, containing 10 % fetal calf serum and 1.8 % dimethy sulfoxide and incubated at 37' until the cells reached a density of 8-10x10 cells/ml. Then 5 uCi/ml of ?H lvaline were added and the cells were incubated for 24 h. When phospholipids were to be labeled, the cells were-grown in Eagle's minimal medium containing 10 % fetal calf ser y and 1.8 % dimethyl sul xide ; when cells/ml, 5 uCi/ml of l 59 Plphosphate the cells reached a density of 8-10x10 without carrier were added, and the cells were incubated for 24 h. When FV glycoproteins were to be labeled, 5 &i/ml of [3H] qlucosamine were added to the normal medium, cells were incubated for 24 h and harvested as above. Virus purification and extraction of lipophilic proteins. Four to 600 ml of supernatants were concentrated to a volume 25 - 50 ml in an Amicon chamber on' Diaflo membranes and the concentrate was clarified by centrifugation for 10 min at I2.000 g. The resulting supernatant was layered on a douhle sucrose cushion f60 % and 15 %, W/V sucrose in NTE buffer - NaCl 0.1 M, Tris-HCl 0.01 , EDTA 0.001 pH 7.21 and centrifuged for 90 min at 70.000 g. The interphase between thi I5 % and the 60 % sucrose was collected,

1174

Vol. 100, No. 3,198l

8lOCHEMlCAL

AND

8lOPHYSlCAL

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diluted to 25 ml in NTE buffer, and this procedure was repeated once. The resultinq interphase was collected, diluted 4 times with NTE buffer containing 4 % calf serum and precipitated with an equal volume of saturated neutralized (NH \ SO The mixture was ?eft overnight at 4', centrifuged for 10 min at 12.006 $ a& the precipitate was dissolved in NTE. An aliquot was used to check the density and the homoqeneity of the virus preparation on a sucrose gradient (15 - 50 % sucrose in NTE). The material was shown to sediment in a single peak at a density characteristic of the virus fl.16 g/ml). Another method of virus purification was also used : the peak of a sucrose qradient was collected fl5-50 % sucrose in NTE buffer\ and pelleted by centrifugation for 60 min. at 70 000 g. Such preparations tended to contain less gp 69/71 (FV1 or gp 85 (RSV, RAVE than virus preparations precipitated with ammoniumsulfate. An aliquot of the virus preparation was stored at - 20” and used for the determination of the viral proteins, phospholipids, and the extraction of lipophilic proteins. Lipophilic proteins were extracted with acidic chloroform-methanol as described elsewhere (l.21. Phospholipids were extracted with neutral chloroform-methanol by the method described by FOLCH et al. (141, as modified by Bligh and Dyer (15). SDS Pol.vacrylamide Gel Electrophoresis (PAGE), Immunoprecipitation and Thin Layer Chromatography (TLC) The apparent molecular weight of viral proteins d of the lipophilic proieins extracted from the virus was determined by & - 13 % polyacrylamide slab gel electrophoresis as described by Laernnli fl61. Label was detected by fluoroqraphy as described by Bonner and Laskey (171. The fluoroqraphs were read with a Vernon automatic recording spectrophotemeter. M.W. of proteins was determined by cwarison with commercial standards run on the same gel. The standards were [ Cl labeled : phosphorylase b (97.500 daltons\ ; serum albumine (69.000 daltonsj ; ovalbumine (46.000 daltonsl ; carbonic anhydrase (30.000 daltonsl ; lactoglobulin A fl8.400 daltonsj ; cytochrome C fl2.300 daltonsl ; insuline (5.800 daltons). For immunoprecipitation, lipophilic proteins were dissolved in RIPA-BSA (NaCl 0.15 M, Triton X 100 1 %, SDS 0.1 %, TRIS 0.01 M pH 7.2, DOC I %, EDTA 0.001 M, Trasylol 1 %, bovine serum albumin 5 ug/lJ, mixed with 3 ~1 of the pertinent antiserum, incubated for 30 min. at O', centrifuged for 3 min. at 2000 g. The precipitate was washed twice with RIPA-BSA and once with RIPA. Electrophoresis sample buffer was added to the pellet and the mixture was heated for 3 min. at lOO', centrifuged for 3 min. at 2000 g and the supernatant was applied to the gel. When chicken antiserum was used, the RIPA was made in 1M NaCl. TLC was performed on thin layer silica qel 60 plated (Merck) in 2 dimensions : first in chloroform-methanol-water *f65 : 25 : 4 bv Vol.1 and then in n-butanol-acetic acid-water (3 :1 : 1 by vol.). The plates were dried and a Kodak RoyalX-Omat Film RPL2 was applied. Radioactive spots were located by radioautoqraphy, scraped off, and the radioactivity was determined in a scintillation counter by Tcherencov radiation. Phospholipids were identified by cochromatography with commercial standards, and the standards were revealed by either a rhodamine or a fluoresceine spray. To distinguish phosphatidylserine from phosphatidylinositol, the spot containing the mixture of these phospholipids was reextracted from the silica powder with methanol and chromatographed in 2 dimensions accordinq to Schneider (18). The dried plates were autoradiographed, the spots localized, scraped off, and the radi0activit.y determined as above. The commercial standards used were : phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, l,ysolecithin, cardiolipin, phosphatidic acid and phosphatidylglycerol. Material. Media were purchased from FLohio and Eurobio. Radioactive precursors came frome C.E.A., Saclay. Acrylamide and N,N'-methylene-bisacrylamide were purchased from Eastman-Kodak, dimethylsulfoxide from Fluka. All other chemicals were either Merck or Carlo Erba mrest grade. Labeled standard proteins came from NEN. Commercial phospholipid standards were purchased from

1175

Vol. 100, No. 3,198l

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

: Lipophilic proteins of 13H] valine labeled ---------lipophilic. proteins extracted from this kF pitate of lipophilic proteins . . . . . . ...* Upper panel

FV. a) Whole virusvirus. c) Immunopreci: antimurine pl5 antiantiserum. Insert, from left to of lipophilic proteins from ; lipophilic proteins of FV.

serum. Lower panel : anti RauscherLeukemiavirus right : total viral proteins ; imnunoprecipitate FV with anti-Rauscher leukemia virus antiserum

General Biochemicals, Chicago. Anti murine ~15, ~12, and pI0 (goat) and anti Rauscher leukemia virus (rhesus monkey) antisera came from NIH, Bethesda. Anti avian p27 (rabbit) and anti RSV (chicken) antisera were a gift from Dr Vigier. Inactivated St. aureus was a gift from Dr. Huppert. RESULTS Both

phospholipids

form-methanol phosphate

labeled of

contained

the less

virus [32Pl than

(phosphoproteins, Fig. lipophilic

and proteins

purified

and 13H 1 valine.

of 132Pl ration

from

virus

were

extracted

preparations,

with

as witnessed

acidic

chloro-

by the

presence

10 % of the 13Hl valine and 1 % of the [32Pl are extracted by acidic chloroform-methanol. The migAbout

labeled

material

on TLC showed

1 % phosphorylated

material

that

other

the than

acid

extract

phospholipids

RNA\. 1 shows

proteins

the extracted

electrophoretic from

profile the

1176

virus

and

of

Friend

of

the

Virus,

of

immunoprecipitate

the

Vol. lOO,No.3,1981

8lOCHEMlCAL

AND

origin

;;gihil;

I-;;;ihilic

proteins

obtained

with

anti-murine This

virus

antiserum.

membrane

preparation dalton.

daltons. antiserum

were

with pl5E

reacted proteins

the

contained

preparation

p27 antiserum

of apparent

proteins

have

material

were

whole

M.W.

have

antisera

identifies or

virus

as

10-15.000

~27. peak

quantity The

same

69/71, these

~15.

leukemia the virus

and

12-

M.W. of

12-22.000

proteins

as being,

proteins,

there

from

glucosamine

[3Hl

with The

30.000

When anti-p12

The virus

and anti-p10

was no precipitate. labeled

virus,

at 10-15.000 is

no

lipophilic

of the

corresponding

were

proteins,

obtained

or RAV 1 were

compared

shown).

I177

with

there

is some

25.000

dalton

with lipophilic

corresponding but

antibodies when

poly-

The lipophilic

Immmunoprecipitation the

immunoprecipitate

of the

contains

daltons. In addition

daltons.

identifies

the lipophilic with 11 anti-

preparation

19 and 10-15.000

antiserum

There

results

RSV fPrague-Cl (not

37, 25,

diffusely

avian

~27

~151.

an apparent

M.W. of 25 and 15 Kdalton.

and anti-RSV

dalton

insufficient

antiserum.

M.W. 70,

migrating

: anti

and reacts

and possibly

proteins

extracted

---

and B1 anti-Rauscher

apparent

pl2E

panel

virus

no label.

an apparent

anti-p27

protein

viruses

. . . . . . . . . . Upper fchickenj.

pl2E

lipophilic

RSV fSRJ. from this

extracted

from

of

the

and B) anti-RSV

peptides

from

labeled

COMMUNICATIONS

Fig. 2 shows the electrophoretic profile of RSV, of extracted from this virus and of the immunoprecipitate

proteins

both

valine oroteins

pl5E,

and either with

When lipophilic

z K daltons

derived

lipophilic

The reaction ~15,

is

fgp69/71,

RESEARCH

%

antiserum

polypeptides

The

respectivel.y,

pl5

serum

proteins

contains

$

lipophilic proteins : anti RSV antiserum

panel

Al

viral 22.000

of

S

of r3Hl Lipophilic

b\

----c\ Itmnunoprecipitate antiserum. Lower

BIOPHYSICAL

this

lipophilic the

might

in the

proteins

the

be due to an

anti-RSV

proteins total

to

serum.

extracted of

these

Vol. 100, No. 3,198l

w

8lOCHEMlCAl

: Lipophilic oroteins from labeled RSV fSRJ. Whole virus

E3Hlivaline

labeled

The [32P]

lipophilic

labeled

M.W. 12.000

and 20.000

Most of the

label

same

rate

as

lipophilic

free

extracted

from

When virus lipophilic

was purified

the

extractability

corresponding In

(extracted

behind (Fig.

3)

L~H]

valine

virus

migrates

were

table with

known

Neither

to

of apparent

as free same

at the

coincides

with

All

label

cells.

the

phospholipids. and pelleting,

as

those

fnot modif~y

and pI9.

marker,

band

labeled

proteins

shown).

viral nor

from

Therefore,

antigens,

their

the

extracted does

capacity

1 the neutral

distribution

relative

distribution

of

chloroform-methanol1

of

phospholipids in Material

the

phospholipids

and in lipophilic

in proteins

1

(percent and Methods).

cpm

determined

as

described

-

FV

SR-RSV

Sphinqomvelin Phosphatidylserine Phosphatidylinositol Phosphatidylcholine Phosphatidylethanolamine Others x

x Lysolecithin, XX Less than

not

to react

antisera.

TABLE Relative

to

pl2

blue

centrifugation

the

lipophilic

in 2 bands

phosphoproteins

preparations of

compared

.

the bromophenol

from

by ammoniumsulfate,

the

RSV (SRJ

migrates the

by gradient

extracted precipitated

precipitation

preparation

slightly

COMMUNICATIONS

----------.

presumably

labeled

RESEARCH

13HJ valine labeled f[ *PI\ labeled

phospholipids.

proteins

afmnoniumsulfate

with

virus

extracted

[32P]

8lOPHYSlCAl

proteins

daltons,

migrates

proteins

affect

AND

cardiolipin,

Whole Virus

lipophilic proteins

4.5 3.8

*El

13.8 30.3 37.8 5.1

34.0 19.4 15.u 3.5

phosphatidyl

1 %.

1178

Whole Virus

lipophilic proteins

12.1

x::

15.6

68.4

29.7 42.3

31.5

glycerol,

phosphatidic

1 j

acid.

virus (ex-

Vol. 100, No. 3,198l

tracted

with

The major

BIOCHEMICAL

acidic

phospholipids

of both

lit

lipophilic

proteins

sitol,

also

from

viruses)

proteins

these

extracted

from

extracted

FV are contain

COMMUNICATIONS

is

summarized.

enriched

from

amount

of

virus

In this

chick

embryo

are en-

respect

cells

in phosphatidylserine

a high

and phos-

avian

and in phosphatidylinositol.

proteins

from

but

RESEARCH

RSV and FV are phosphatidylcholine

Lipophilic

in ohoshatidylserine

resemble

BIOPHYSICAL

chloroform-methanol

phatidylethanolamine. riched

AND

(13).

they

Lipophi-

and phosphatid.ylino-

phosphatidylethanolamine.

DISCUSSION Lipophilic hydrophobic,

proteins,

though

lipophilic

nature

membrane lipids

in order could

procedure. solvents

is the

nents,

lead

to

link

to the

shown that

~15,

in vivo

comigrate

before

proteins.

gp69/71

glucosamine organic

solvents

derived

from

is extracted

lioophilic

virus

are

cellular

lipophilic,

and no material with

components

with

because

is p30 lipophilic

comigrates

reqions

organic

the

viral

~30.

contaminating

to

extraction

with

organic compo-

is a ribonu-

partition

into

solvents

fl9).

because

these

the

comigrating

the

affinity the

; pl0

material with

proteins viral

from

the

material

However

viral

[3Hl gp69/71

extracted

lipophilic

the virus

the

We have

corresponding

no labeled

: no labe?ed

the

I).

Both

with

immunoprecipitation

not

Neither

(Table

has h.ydrophobic

pl2E

durinq

pl5E and plZE are membrane

phosphoproteintl.1).

virus

insure

in or near

extracted

phospholipids

be

should to

; however,

material

gp69/71,

and p30

and after is

protein

specific

and probab1.v

labeled

is extracted.

the

lipids,

be located

and lipids

an internal

pl5E

also

of proteins

proteins,

when the

with be sufficient

phospholipids

with

and pl2 phase

The.y should

that

viral

interact

may not

with

is hydrophobic

cleoprotein

to

linkage

shown

associated

pl5

aqueous

a protein.

murine

order

characteristic

of

We have

Amongst

in

this

with

proteins

preparation

may also

be present. Amongst membrane surface

proteins

the

material

p27 before

and after

serum

or anti-RSV

highly

improbable

that

cellular

origin.

None of

grates lipophilic.

with

viral The

proteins,

gp85,

pI2

coreshell.

extracted

pl5 (11,

with

12).

the

material

extracted

gp37,

pl9

comigrating

II79

solvents with

protein or ~12.

pl0

protein

We have found

organic

antiserum

broad peak

probably,

and p27 have a high

itmnunoprecipitation As this

the

and,

to be an internal

is known

aminoacids

gp37

and seem to be located

lipophilic

antiserum.

rather

gp85,

are hydrophobic

or the

(141,

of non polar

: labeled

viral

viral

virion

aminoacids

a low content

lipophilic the

avian

; ~15 and pl9

of either

of non polar have

the

with

the

and to

that

p27 is with

anti-p27 with

organic these

content

anti-

on chicken

comiqrating

Therefore

at the

comigrates

either

was produced with

are

p27

solvents proteins

plO-pl5

it

is

is

of

comiare

group

not may

Vol. 100, No. 3,198l

BIOCHEMICAL

AND

BIOPHYSICAL

TABLE 2 Molecular weight, location, and properties of according to the litterature ; comparison Columns 1 and 2 refer to the review article LOCATION

PROTEIN M.W.in parentheses determined ly SDS-PAGE

RSV, with of

RESEARCH

COMMUNICATIONS

of TV, PAV. and RSV proteins experimental results. Eisenman and Vogt (11).

PROPERTIES

RBSULTS

MURINE p30

(31.000)

Core

pl2

(12.000)

Internal

pl5

(15.000)

Hydrophobic non polar

shell

regions (8). Low amino acids (12)

Phosphoprotein chloroform-methanol SDS-PAGE with Moderately phosphatidyl chemical SDS-PAGE

Internal

(II). ~12

Membrane

Disulfide R protein

pl5

E (17.000)

Membrane

pl0

(10,000)

Internal

Strongly hydrophobic (8). Disulfide linked to gp70 (23). Basic ribonucleoprotein (11). Insoluble in chloroform-methanol Moderately hydrophobic (8). glycosylated (11).

Membrane, spikes

linked ? (24).

to

gp

70

Not

lipophilic

Not

lipophilic

in

hydrophobic (8). Links to ethanolamine in situ by agents (10) - Migrates in with pl2 (22).

E (12.000)

(69-71.000)

of

Insoluble in (19). migrates (22).

~12

gp70

content

Lipophilic

(23)

Lipophilic

(?)

Lipophilic (19).

Not

lipophilic

Not

lipophilic

AVIAN

. .

p27 Dl9

(25.000) (21.000)

High content of non polar amino acids (12) IMaior uhosuhoorotein : binds snecificallv to RNA (11). Links to phosphatidyl ethanolamine in situ by chemical agents (10). Hydrophobic (8). Hydrophobic, proteolytic activity (11). Migrates in SDS-PAGE with ~12 (22).

I

pl5

(12.000)

Internal

p12

(14.000)

Internal

‘lo

*. .

Core shell I Internal

Basic ribonucleoprotein, phospharylated (11). with pl5 (22). Weakly

glycosylated,not

~l~‘~~~(25)

Membrane

gp85

(70.000)

Membrane, knobs

Major

gp37

(32.000)

Membrane , intramembranous

Glycosylated by disulfide

correspond cellular

proteins believed

to pl0 components In table and our

(?)

and ~15.

weakly Migrates

always

in

SDS-PAGE

observed

(22)

Not

lipophilic

I Lipophilic

Not

glycosylated

(11).

; linked to bonds (11).

However,

this the

material

virus

gp85

could

also

Lipophilic

avian

"core

1180

lipophilic

Not

lipophilic

from

preparation.

2 we list the properties and location of the structural that a viral results : It appears from these results the

Not

be derived

shell"

p27 is either

attached

(?)

lipophilic

1

contaminating

to be internal,

Lipophilic !

viral protein to the

(?)

Vol. 100, No. 3,198l

8lOCHEMlCAL

Fia. 4 : Model I?%%-properties some of these lipophilic

phospholipids has

the

for

to

(33.5 %j,

viral

membrane

these

phospholipids

the

relationship

viral

proteins

lope.

Lipophilic

The

content to the

presence

site

might

sults,

with

becomes

hasis

of

nature

of

them or else

attached

of

the

we have in Fiq.

in

the

membrane

known

constructed

these

lipophilic

to them

difference

~2'7 (48 %l proteins

proteins

connections virus

ma,y and

p30

regarding

for

the

viral

Virus,

properties

of for

these

in phosphatiin phosphasurround lipophilic

phospholipids. could

soluble

oncornaviruses avian

enve-

also

core,

also

retrovirus

that

viral

phospholipids

of

proteins

to

Forest

the

Alternatively,

association

Lipophilic

a model

proteins these

proteins. the

suggests

with

are enriched

that

affinity for

(201

the

of

between

suggests

membrane. viral

aminoacids

have

lipophilic

evidence

as the 62p of Semliki

: this

FV lipophilic

a special

of

polar

lipophilic

to whole

finding

have

Combining

shown

it

the

membrane.

compared

presented

viral

attachement (21).

non

not

a difference phospholipids

This

have the

so that

p30 is

and phosphatidylinositol,

authors

and coextracted

in vivo

to be internal

proteins

implantation

function

in viral

of

believed

tidylethanolamine.

with

murine

and may indicate

their

proteins

COMMUNICATIONS

extraction.

be related

the

RESEARCH

avian and murine oncornavirus constructed on of structural viral proteins and the lipophilic nroteins demonstrated in the present work. proteins non lipophilic proteins.

The correspondinq

d,ylserine

8lOPHYSlCAL

of

of the

an affinity

during

AND

avian play and

Several

and murine a role serve

in organic proteins and the

pl5

in the

a similar solvents and our

re-

FV complex,

4.

REFERENCES 11 21 31 41 5)

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