1421

0022-1554/90/$3.30The Journal of Histocheznistry and Cytochem.istry

Copyright © 1990 by The Histochemical Society, Inc.Vol. 38, No. 10, pp. 1421-1426, 1990

Printed in USA.

Original Article

Anchorage=dependent Surface Distribution and PartitionDuring Freeze-fracture of Viral TransmembraneGlycoproteins’

M. R. TORRISI,2 A. PAVAN, L. V. LOUI, G. MIGLIACCIO, M. C. PASCALE,

E. COVELLI, A. LEONE, and S. BONATTI

Dipartimento di Medicina Sperimentale, Universith di Roma “La Sapienza,” Roma, Italy (MR7ARLVL), and D:partimento di

Biochimica e Biotecnologie Mediche, II Facolt#{224}di Medicina, Universit� di Napoli, Napoli, Italy (GM,MCREC,AL,SB).

Received for publication December 7, 1989 and in revised form April 30, 1990; accepted May 7, 1990 (9A1856).

We have compared in the same cell type the surface distri-bution and partition in freeze-fractured plasma membranesof Sindbis virus glycoproteins in three different situations:

(i) in permanently transformed cells that express the glyco-proteins as the only viral product; (ii) in cells in which pre-bound viruses were forced to fuse with the plasma mem-brane by low pH treatment; (iii) in virus-infected cells. Wereport here that the viral proteins expressed on the surfaceof transfected cells show a uniform and undustered distri-

bution; conversely, in Sindbis virus-infected cells they ap-pear dustered, regionally distributed, and always associatedwith budding viruses (i.e., interacting with the nudeocap-

Introduction

Immunocytochemical methods, in combination with electron mi-

croscopic techniques, are widely used for analysis ofmembrane struc-

ture, composition, and dynamics. Several methods, including sun-

face replication, are available to visualize the distribution of

membrane components over large areas ofthe cell surfaces, whereas

only a few expose intracellular membranes to immunolabeling. This

is one ofthe advantages ofthe fracture-label method (Pinto da Silva,

1987a,b), which has been mainly used to analyze the partition dur-

ing freeze-fracture of transmembrane proteins on plasma and in-

tracellular membranes (Tornisi and Pinto da Silva, 1984; Pinto da

Silva and Torrisi, 1982; Tornisi and Pinto da Silva, 1982).

Because of the large amount of viral proteins present at their

surfaces and intracellularly, virus-infected cells provide a useful tool

to study the distribution and the behavior on fracture of transmem-brane proteins on both plasma and intracellular membranes. Using

fracture-label and surface replication techniques, viral envelope gly-

coproteins have been immunolabeled on freeze-fractured mem-

1 Supported by grants from Progetto Finalizzato “Oncologia” Consiglio

Nazionale delle Ricerche, Italy.2 Correspondence to: Maria Rosaria Torrisi, Universit#{226} di Roma “La

Sapienza,” Dipartimento di Medicina Sperimentale, Viale Regina Elena,

324, 00161 Roma, Italy.

sid on the cytosolic side of the membrane). Furthermore,the viral proteins expressed on transfected cells or implantedbylow pH-mediated fusion partition during freeze-fracturewith the exoplasmic faces of the cell plasma membranes,whereas an opposite partition is observed in infected cells.These results strongly suggest that in infected cells the duster-ing and the partition with the protoplasmic faces of theplasma membrane depend only on the strong “anchorage”of the glycoproteins to the nudeocapsid. (J Hiscochem

Cytochem 38:1421-1426, 1990)

KEY WORDS: Freeze-fracture immunocytochemistry; Viral proteins;Sindbis virus; Membranes.

branes and on the unfractured surfaces ofcells infected either with

an RNA virus (Sindbis virus; SV) (Tornisi et al., 1987; Pavan et al.,

1987; Tornisi and Bonatti, 1985) or with a DNA virus (Epstein-Barr

virus, EBV)(Tornisi et al., 1989). In those studies we suggested that

the partition and distribution of the viral envelope glycoproteins

were affected by the interaction with the nucleocapsid, but only

on those membranes where budding occurs (plasma membrane and

inner nuclear membrane for SV and EBV, respectively). In fact, in

SV-infected cells the distribution of the envelope glycoproteins on

the cell plasma membranes was clearly regionalized and associated

with budding figures (Pavan et al., 1987). Similar results were ob-

tamed for the EBV system over the freeze-fractured inner nuclear

membranes of the producing cells (Tornisi et al., 1989). The parti-

tion during fracture of the envelope proteins was also peculiar in

both studies: in fact, in SV-infected cells the viral glycoproteins were

observed to partition with the exoplasmic halfofthe freeze-fractured

intracellular membranes (Torrisi et al. , 1987; Tornisi and Bonatti,

1985), whereas they became associated with the protoplasmic half

during budding at the plasma membrane level (Tornisi and Bonatti,

1985). Similarly, in EBV-producing cells the viral glycoproteins ap-

peared to partition with the protoplasmic halfofthe inner nuclear

membranes at the sites of viral budding (Tornisi et al., 1989).

In the present study, to test our hypothesis on the role of the

nucleocapsid during budding in determining the peculiar distni-

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Figure 1. Surface distribution of Slndbis glycoproteins: the immunolabeling is(a)regionalized, (b)clustered, and almost exclusively associated with budding viruses

(a; arrow in inset) in infected cells, whereas it is dense and uniform over the entire cell surfaces in transfected cells (c,d). Surface replicas. Original magnifications:a x 15,000; Inset x 30,000; b x 17,000; C x 13,000; d x 20,000. Bars = 1 pm; lnsst = 0.5 pm.

1422 TORRISI, PAVAN, LOTTI, MIGLIACCIO, PASCALE, COVELLI, LEONE, BONATFI

bution and partition on fracture of the viral envelope glycopro-

teins, we used cells in which the presence of the viral glycoproteins

on the plasma membranes was not related to budding processes.

Materials and Methods

Cell Culture and Virus Infection. Cultures ofCVl cells were maintainedin plastic tissue culture dishes using Dulbecco’s modified Eagle’s medium

(DMEM) supplemented with 10% fetal calfserum (FCS). Sindbis virus HR

strain was plaqued, grown, and titrated on chick embryo fibroblast cells

as previously described (Pfefferkorn and Hunter, 1963). Subconfluent

monolayers were infected at a multiplicity of 50 plaque-forming units/cell

for 1 hr at 37#{176}Cin PBS containing Ca2� and Mg2� and 1% FCS. After in-

cubation, the medium was replaced with an appropriate volume of mini-

mum essential medium (MEM) containing 5% FBS, and the infection

proceeded for 4 hr.

Cell Transfection. CV! cells growing in DMEM containing 10% FCS

were transfected with pHMT2300 plasmid as detailed previously (Migliac-cio et al., 1989). This plasmid contains the coding region for the entire

El glycoprotein and the 103 carboxy terminal amino acids ofE2 under the

control ofthe human metallothionein ha promoter(Migliaccio cc al., 1989).

Permanently transformed cells were isolated by selection in the presence

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VIRAL TRANSMEMBRANE GLYCOPROTEINS 1423

of G418 after co-transfection with a plasmid carrying the bacterial neomy-

cm gene. Individual clones were screened for surface expression of Sindbis

glycoproteins by indirect immunofluorescence after induction with cad-

mium chloride. The clone used throughout this study, named CVE3, was

shown to express on the cell surface both El and the truncated portion

of E2 (Migliaccio et al., 1989).

Implantation ofSindbis Virus Glycoproteins on the Plasma Membrane

ofCVl Cells. Sindbis virus was purified from the culture medium by pellet-

ing through a sucrose gradient and re-suspended in Tnis-HCI 50 mM, pH

7.4, NaC1 100 mM, EDTA 1 mM at a concentration of 1 mg/mI. One pg

of virus in 50 p1 of binding medium (DMEM containing 0.5% BSA and

20 mM Hepes, buffered at pH 7, was incubated for 2 hr at 4#{176}Cwith 2 x

10� cells. Unbound virus was removed by washing with binding medium,

and then pre-warmed binding medium buffered at pH 5.5 was added for2 mm at 37#{176}Cto allow low pH-mediated fusion (Edwards et al., 1983).

Cells were further incubated in normal medium at 37#{176}Cfor 5 mm.

Antibody Preparation. An anti-Sindbis antiserum was prepared in rabbitagainst purified Sindbis virus grown on chick embryo fibroblast cells. The

immunoglobulin G fraction was obtained by Na-sulfate precipitation and

DEAE-cellulose column and was pre-adsorbed against formaldehyde.fixeduninfected BHK cells by two incubations (30 mm, 37#{176}C).The IgG fraction

was then stored at 4#{176}Cin aliquots at a concentration of 10 mg/mI. This

C

Figure 2. Partition during freeze-fracture of Sindbis glycoproteins in transfected cells: the immunolabeling is confined to the exoplasmic faces (C), and uniformas over the unfractured cell surface (a); protoplasmic faces are virtually unlabeled (b). Original magnifications: a x 9000; b x 34,000; c x 24,500. Bars: a =

1 pm; b,c = 0.5 pm.

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1424 TORRISI, PAVAN, LOTfl, MIGLIACCIO, PASCALE, COVELLI, LEONE, BONATTI

antibody does not react with any cell proteins, as shown by immunofluo-rescence microscopy, but specifically recognizes the Sindbis envelope gly-

coproteins at all stages of maturation, as determined by indirect immuno-

precipitation (data not shown).

Surface Immunolabeling. All cells were washed three times in PBS, pH

7.4, and fixed with 1% glutaraldehyde in the same buffer (25#{176}C,30 mm).

The cells were then incubated in anti-Sindbis spike antibodies (0.5 mg/mI)in PBS for 1 hr at 4’C, washed extensively, and labeled for 3 hr at 4#{176}C

with colloidal gold (prepared by the citrate method) conjugated with pro-

tein A (Pharmacia; Uppsala, Sweden) (Slot and Geuze, 1981).

Fracture Immunolabeling. Cells were fixed in 1% glutaraldehyde in

PBS (30 mm at 25#{176}C)andembedded in 30% bovine serum albumin cross-linked by glutaraldehyde. The resulting gels were sliced into small pieces,impregnated in 30% glycerol in PBS, and frozen in Freon 22 cooled by

liquid nitrogen. Frozen gels were fractured in liquid nitrogen by repeatedcrushing with a glass pestle, thawed in 1% glutaraldehyde/30% glycerol

in PBS, gradually deglycerinated, and washed twice in PBS before im-

munolabeling as above.

Thin Sectioning. Labeled gel fragments and isolated cells were post-

fixed in 1% osmium tetroxide in Veronal acetate buffer, pH 7.4, for 2 hr

at 4#{176}C,stained with uranyl acetate (5 mg/ml), dehydrated in acetone, andembedded in Epon 812. Thin sections were examined unstained or post-

stained with uranyl acetate and lead hydroxide.

Surface Replication. Labeled cell monolayers on coverslips were de-hydrated in a series ofethanol washes and air-dried. Platinum-carbon replicas

were obtained in a Balzers BAF 300 freeze-etching apparatus (Balzers AG;

Liechtenstein) by shadowing the cell surface with platinum-carbon evapo-

ration. The replicas were cleaned overnight in household bleach, washed

extensively in distilled water, and examined in a Philips CM1O (Philips Elec-

tron Instruments; Eindhoven, The Netherlands).

Results and Discussion

Surface Distribution of Sindbis Glycoproteins on

Transfected Cells

The distribution of SV glycoproteins over the plasma membranes

ofCVE3 cells was analyzed on both thin sections and surface replicas

of the immunolabeled cells. The results were compared with par-

allel observations of Sindbis-infected CV1 cells, 4-5 hr post

infection.

In SV-infected CV1 cells the immunolabeling was mostly in small

clusters and associated with budding viruses (Figures la, ib, and

3a). The strict relationship ofthe surface immunolabeling and the

presence of nucleocapsids beneath the plane of the membrane was

p ‘�:�v .

rr���: (�tt� ...

� � /

rL*��-- !.�� .;�-�:‘ �--

;��:��;; � � ��ci#{176} � ___

Figure a Partition during freeze-fracture of Sindbis glycoproteins in infected cells: the immunolabeling is confined to the protoplasmic faces (C) and is clustered,co-localized with underlying nucleocapsids (C, arrow) and regionalized as over the unfractured cell surface (a,b). Ewplasmic faces (d), where mature virions bud-ded from the cell surface and trapped in the gel matrix are visible (d, arrowhead), are unlabeled. Original magnifications: a x 15,000; b x 74,300;c x 39,200;d x 35,000. Bars: a,b = 1 pm; c,d = 0.5 pm.

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ing nucleocapsids in the cytoplasm (b, arrowhead). After fracture, the immunolabeling is associated with the exoplasmic face, as shown by complementary fracture

faces in close apposition (C, arrow). Where the virions are not fused with the plasma membrane (C, arrowhead), immunolabeling is absent over both fracturefaces. Original magnifications: a x 35,000; b x 33,800; c x 15,600. Bars = 0.5 pm.

VIRAL TRANSMEMBRANE GLYCOPROTEINS 1425

confirmed by thin section images (Figure 3b). In addition, both

budding viruses and the associated immunolabeling were region-

alized, being concentrated at the cell periphery and along cell

processes (Figure la). All these findings fully confirmed the obser-

vations made previously in SV-infected baby hamster kidney cells

(BHK) (Pavan et al., 1987). In contrast, the immunolabeling in

CVE3 cells was dense and uniform over the entire cell surface, as

clearly shown on both surface replica and thin section images

(Figures ic, id, and 2a). In addition, the gold particles were always

individual and unclustered, similarly to the immunolabeling ob-

served in SV-infected BHK cells at early times post infection, be-

fore the onset of virus maturation (Pavan et al. , 1987). This last

finding suggests that the clustering of glycoproteins observed in

infected cells since the beginning of viral budding does not de-

pend on lateral interactions between the glycoproteins.

Partition on Fracture of Sindbis Glycoproteins on

Transfected Cells

In fracture-label experiments, thin sections of both protoplasmic

and exoplasmic portions of plasma membranes revealed the typi-

cal aspect of interrupted unit membrane segments. This appears

to be due to reorganization of the fracture monolayers into inter-

rupted bilayer structures, as discussed elsewhere (Pinto da Silva,

1987a,b).

We analyzed the partition on fracture of Sindbis glycoproteins

expressed on the plasma membrane of CVE3 cells. The immu-

nolabeling over the exoplasmic portions appeared significant

and uniformly distributed (Figure 2c), whereas protoplasmic faces

were virtually unlabeled (Figure 2b). Parallel fracture-label experi-

ments on infected CV1 cells showed an opposite partition: the im-

munolabeling was confined to the protoplasmic portion and was

usually clustered and co-localized with underlying nucleocapsids

(Figure 3c); the exoplasmic faces, where mature vinions budded from

the surfaces could be observed, were virtually unlabeled (Figure

3d). These results on infected CV1 cells confirmed our previousobservations on BHK cells (Tornisi and Bonatti, 1985). Therefore,

these results suggest that in the absence of the strong anchorage

provided by the nucleocapsids during budding (Fuller, 1987; Si-

mons and Garoff 1980), SV glycoproteins partition on all cell mem-

branes with the exoplasmic portion (Tornisi et al. , 1989; Torrisi and

Bonatti, 1985).

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1426 TORRISI, PAVAN, LOTFI, MIGUACCIO, PASCALE, COVELLI, LEONE, BONATFI

Edwards), Mann E, Brown DT (1983): Conformational changes in Sindbis

Partition on Fracture ofSindbis Glycoproteins

Implanted on the Cell Surfaces

To further support the hypothesis that the nucleocapsid anchorage

is responsible for the change in partition of the viral glycoproteins

observed on the plasma membrane of infected cells, we decided

to implant a full complement of SV glycoproteins on the plasma

membrane of recipient cells. The implantation ofSV glycoproteins

was achieved by low pH-mediated fusion of viruses pre-bound at

4#{176}Cto the cell plasma membrane (Edwards et al., 1983). On the

cell surfaces, a significant amount of randomly implanted proteins

were present, overlying nucleocapsids in the cytoplasm. Part of the

bound viruses did not fuse with the membranes and remained at-

tached to the surfaces (Figures 4a and 4b). After fracture, the im-

munolabeling appeared associated with the exoplasmic faces and

absent over the protoplasmic faces, as clearly shown where the com-plementary inner and outer leaflets ofthe plasma membranes re-

mained in close apposition (Figure 4c). Portions of the exoplasmic

faces, which correspond to surface areas where viruses did not fuse,

as shown by intact vinions bound on the extracellular side of themembrane and trapped in the gel matrix, were all unlabeled (Fig-

ure 4c).We conclude, therefore, that SV glycoproteins partition with

the protoplasmic faces only as a consequence of interactions withthe nucleocapsids during the budding process. Lateral interactionsamong the glycoproteins or interactions between the glycoproteins

and underlying cytoskeletal elements might occur on the plasma

membrane (Fuller, 1987) but are not primarily responsible for the

partition with the protoplasmic face.

Acknowledgments

w� thank M, Giuseppe Lucania for excellent technical assistance.

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