Physical, morphological, and biochemical alterations in the

Prop. Natl. Acad. Sci. USA Vol. 75, No. 4, pp. 1859-1863, April 1978 Cell Biology

Physical, morphological, and biochemical alterations in the membrane of AKR mouse cells after interferon treatment (interferon action/plasma membrane/intramembranous particles/glycoproteins)

ESTHER H. CHANG, FRANCIS T. JAY, AND ROBERT M. FRIEDMAN Laboratory of Experimental Pathology, National Institute of Arthritis, Metabolism, and Digestive Diseases, National Institutes of Health, Bethesda, Maryland 20014

Communicated by C. B. Anfinsen, January 20, 1978

ABSTRACT Interferon treatment of AKR,C- cells was followed by the establishment of an antiviral state and apparently concomitant morphological, physical, and biochemical alterations of the cell plasma membrane. The density of the plasma membrane was significantly altered, and the concentration of some plasma membrane glycoproteins and the number of intramembranous particles observed in freeze-fracture electron micrographs were increased. A parallel increase in the concentration of intramembranous particles and the resistance to viral infection during interferon treatment as well as their parallel decrease upon removal of interferon suggests a relationship between the particle density and the establishment of the antiviral state.

Work from several laboratories has implicated cell surface alterations in the mechanism of action of interferon. Mouse interferon enhances the expression of cell surface histocompatibility antigens in L 1210 cells (1). Treatment of L cells with interferon resulted in their having a higher electrophoretic mobility toward the anode (2). Exposure of L-cell membranes to interferon led to a decrease in their capacity to bind the polypeptide hormone thyrotropin, and the binding of cholera toxin was also altered in membranes from interferon-treated cells (3, 4). These changes could have been due either to interferon-induced alterations in the plasma membrane or to competition between interferon and the other ligands for a common binding site. In addition, interferon treatment of cells infected with RNA tumor viruses caused an unusual inhibition, which may be related to interferon-induced changes in the plasma membrane. In some systems, murine leukemia virus appeared to accumulate at the surface of interferon-treated cells (5, 6); This resulted in a decreased production of the virus and an increase in the intracellular concentration of viral p30 antigen (7, 8). In other systems, virus particle production in interferon-treated cells was quite close to that in untreated cells, but the infectivity of the particles was markedly decreased (9-11). Moloney murine leukemia virus produced in interferon-treated TB cells contained a much higher concentration of a glycoprotein with a molecular weight of 85,000 (gp 85) than did normal virus (12). Because gp 85 may be a precursor of gp 69/71 and is a membrane-associated protein, it is possible that this alteration may be related to an inhibition of the cleavage of the membraneassociated gp 85; therefore, the appearance of excess gp 85 in virus produced in interferon-treated cells may also be related to an alteration in the plasma membrane. In the present study, we have investigated alterations in the plasma membrane of uninfected mouse cells after their treatment with interferon. We observed an increase in the buoyant The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

density of the plasma membranes of interferon-treated cells. This increase was not correlated with any qualitative alterations in the composition of the protein or glycoproteins of the plasma membranes. Possibly accounting for the shift in density, however, was the finding that in the interferon-treated cells the two major glycoproteins of the plasma membrane were significantly increased in concentration. Because the molecular weights of these glycoproteins resembled those of glycophorin and band 3 which are thought to make up the intramembranous particles seen in freeze-fracture analysis of erythrocyte membranes (13), we also investigated whether the increased physical density of the plasma membranes of interferon-treated AKR mouse cells' was correlated with an increased concentration of intramembranous particles per unit area. Our findings indicated that interferon-treated cells did indeed contain a significantly higher concentration of intramembranous particles than are normally present in AKR cells. We also demonstrated that interferon treatment of AKR,C- cells led to the establishment of an antiviral state and a concomitant alteration of the cell plasma membrane. MATERIALS AND METHODS Cell and Viruses. AKR,C- (AKR-2B), a mouse embryo cell line kindly provided by W. P. Rowe (National Institute of Allergy and Infectious Diseases), was grown in monolayers on McCoy's modified 5a medium (Grand Island Biological Co., Grand Island, NY) supplemented with 10% heat inactivated fetal calf serum (GIBCO), gentamycin (50 ,g/ml), streptomycin (100 ,gg/ml), penicillin (100 units/ml), and Fungizone (25 ,g/ml). Murine leukemia viral gs (p30) antigen and reverse transcriptase activity were not expressed in the cells or in their culture fluid (7, 14). Ly cells were a strain of mouse cells originally obtained from J. Youngner, University of Pittsburgh, School of Medicine; these cells were grown in monolayers in Eagle's medium with 10% fetal calf serum (GIBCO). Vesicular stomatitis virus, plaque-purified three times, was grown in VERO cells. Interferon and Interferon Assays. The mouse interferon used was prepared by the method of Ogburn et al. (15) by K. Paucker, Pennsylvania College of Medicine, Philadelphia, PA. The preparation had a specific activity of at least 2 X 107 mouse interferon reference units per mg of protein. Interferon was assayed by either a plaque inhibition or a cytopathic effect inhibition assay in Ly cells. In either assay, vesicular stomatitis virus was used as the challenge virus. Preparation and Density Analysis of Plasma Membrane. AKR cell monolayers were treated with interferon at 100 Abbreviations. Pi/NaCl, phosphate-buffered saline; Mr, molecular weight; PF, protoplasmic face (inner fracture); EF, endoplasmic face (outer fracture); NaDodSO4, sodium dodecyl sulfate.

1859

1860

Cell Biology: s0

Chang et al.

1j43%1

45%

Proc. Nati. Acad. Sci. USA 75 (1978)

48%I 50%

155%160%

40%

~~~~0.8

E

0.6

D

Table 1. Effect of interferon on the distribution of AKR,C- cell membrane into the two density fractions

%A2o in:

0.40 interferon

units/ml hr. Plamamembranes2frominterferon 0.4 for016-18

h.ol

Control Interferon, 100 units/ml * Low density, p - 1.216/1.231. t High density, p 1.231.

0.2

-

in te rfe ro n

treated or control cells were prepared by the Tris/Mg method of Warren and Glick (16). Monolayers were washed with phosphate-buffered saline

(Pi/NaCl),

scraped, and washed

twice in cold P1/NaCl and once in 50 mM Tris.HCl, pH 7.5. The cell pellet was resuspended in 50 mM Tris.HCl and 1/20 volume of 100 mM MgCl2 was added. The cells were Dounce homogenized after 10

mmn;

this yielded

>80%o

cell disruption. The

homogenate was sedimented at 1380 X g for 15

mo

through

gradient made in the same Tris/Mg buffer; the crude membrane was collected from the 35% and 40% sucrose bands and washed in the same buffer. The a discontinuous sucrose density

membrane preparation was analyzed by redistribution at 100,000 X g for 90 mi in a second discontinuous sucrose density gradient containing layers of 40,43,45,48, 50,55, and 60% sucrose prepared by diluting (vol/vol) a 60% (wt/wt) sucrose stock solution. The gradients were scanned at 280 nm by using an ISCO UA-4 UV monitor with a type 6 optical unit. When the incorporation of [d4C]glucosamine into these membrane fractions was studied, these membrane fractions were pelleted at 6000 X g for 15 min resuspended in Tris/Mg buffer, and assayed for protein content by the method of Lowry et al. (17); the trichloroacetic acid-precipitable radioactivity was estimated by liquid scintillation.

[c4CtGlucosamine Incorporation. Interferon-treated (100 units/ml) or control AKR cell monolayers were labeled with [W4C]glucosamine (2.5 ouCi/mf,4250 mCi/mmol, New England Nuclear, Bostor, MA) for 8 hr before plasma membrane preparation. Labeling medium (McCoy's modifiedsa) containing 0.06% glucose was used. Polyacrylamide Gel Electrophoresis of Fractionated

['4C]glucosamin2Ela-

Plasma Membrane Proteins. Purified

beled plasma membrane fractions

were subjected to sodium

(NaDodSO4)/polyacrylamide gel electrophoresis and followed by fluorography. The 7.5% gels, prepared according to Laemmli (18), were run on a Hoefer slab gel apparatus (Hoefer Scientific, San Francisco, CA) for 61/2 hr at 10 mA. Gels were stained with Coomassie brilliant blue R to locate the molecular marker proteins: f3-galactosidase (130,000), phosphorylase A (95,000), bovine serum albumin (68,000), and ovalbumin (43,000). After destaining, the gels were impregnated with 2,5-diphenyloxazole (New England Nuclear, Boston MA), dried, (19), and exposed to Kodak X-OMAT x-ray film. Preparation of Cells for Freeze-Fracture. Samples collected

dodecyl sulfate

Low density*

High densityt

77 40

23 60

at various times were washed five times with Pi/NaCl (pH 7.4) and fixed in 2.5% glutaraldehyde in P1/NaCl for 20 min at room temperature. After three washings with Pi/NaCl, the cells were suspended in 30% glycerol in Pi/NaCl, and droplets of the sample were put on 3-mm gold planchets for rapid freezing first in Freon and then in liquid nitrogen. The samples were freeze-fractured and then shadowed with platinum/carbon in a Balzar freeze-etching apparatus (Balzar High Vacuum Corp. Santa Ana, CA). The replicas were cleaned with Clorox, washed with distilled water, picked up on 0.25% Formvar-coated grids, and viewed in a Philips EM 200 electron microscope. Intramembranous particles in 20 random 1-Mum2 fields were counted in each experiment. Five such experiments were performed for statistical analysis (P 0

o

°

C

0/

or an event resulting from the antiviral state. 0

4

8

16 24 48 Duration of interferon treatment, hr

We thank Raymond Carter for his excellent technical assistance. 96

FIG. 5. Effect of varying the duration of interferon treatment on the time course of increase of intramembranous particle density (A) and establishment of antiviral activity (B) tested against vesicular stomatitis virus. Broken lines represent the samples after interferon removal.

density because such a change would also increase the protein-to-lipid ratio in the plasma membrane. Evidence obtained from numerous studies suggested that glycoprotein complexes associated with band 3 and glycophorin are the major glycoproteins of intramembranous particles in the erythrocyte system (13, 23-27). The apparent Mrs of the major glycoproteins of AKR cell plasma membrane (about 100,0(0 and 45,000) resembled those of band 3 and glycophorin when they were analyzed on acrylamide gel under our conditions. An increase of about 2-fold in the uptake of [14C]glucosamine with a corresponding increase of glycoprotein concentration in the isolated denser membrane fractions after interferon treatment might reflect an increase in glycoprotein insertion in the plasma membrane. Because interferon increases the concentration of these two glycoproteins in AKR cells, it seemed reasonable to check whether an increase in intramembranous particles was correlated with the alterations found in plasma membrane glycoproteins. These AKR cell glycoproteins, therefore, may correspond to the glycophorin and band 3 complexes in the erythrocyte system because morphological studies showed that there was an increase in intramembranous particle counts after interferon treatment. Branton (28) first suggested that freeze-fracture splits the lipid bilayer of plasma membranes and that the 7- to 8-nm globular units revealed by this technique actually resided intramembranously. These intramembranous particles have since been reported on the freeze-fracture faces of cell membranes from bacteria, animals, and plants and from membranes of most intracellular organelles (for review, see ref. 22). The parallel increase in the density of intramembranous particles and the resistance to viral infection during interferon treatment as well as their coincident decrease upon removal of interferon suggest a relationship between the intramembranous particle density and establishment of the antiviral state. Based on the results of previous studies, several laboratories have suggested that interferon treatment inhibits murine leukemia virus at a late stage(s) in virus replication (5-11). In view of the relationship between glycoproteins and intramembranous particles, the block of virus production by interferon may involve an alteration of membrane-associated glycoproteins. This may in turn result in an impairment of virus release through

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