Vol. 23, No. 3 Printed in U.S.A.

JOURNAL OF VIROLOGY, Sept. 1977, p. 799-810 Copyright © 1977 American Society for Microbiology

Effect of Interferon on Dimethyl Sulfoxide-Stimulated Friend Erythroleukemic Cells: Ultrastructural and Biochemical Study RONALD B. LUFTIG,* JEAN-FRANCOIS CONSCIENCE, ARTHUR SKOULTCHI, PAUL McMILLAN, MICHEL REVEL, AND FRANK H. RUDDLE Worcester Foundation for Experimental Biology, Shrewsbury, Massachusetts 01545*; Friedrich MiescherInstitut, CH-4002 Basel, Switzerland; Department of Cell Biology, Albert Einstein College of Medicine, Bronx, New York 10461; Weizmann Institute of Sciences, Department of Virology, Rehovot, Israel; Biology Department, Yale University, New Haven, Connecticut 06520

Received for publication 6 January 1977

Treatment of dimethyl sulfoxide-stimulated Friend erythroleukemic cells (clone 745) with mouse interferon (50 U/ml) led to the following changes: (i) a net decrease (40 to 60%) in both the total number of apparently newly synthesized virion particles per cell section and in the average number of cell sections containing one or more virion particles; (ii) a large decrease (80 to 90%) in the number of particles released into the supernatant fluid, as assayed by reverse transcriptase activity; (iii) an initial increase in the number of "immature" or "enveloped A-type" virions followed by an increase in the accumulation of empty, core shell-like particles; and (iv) a decrease in the number of cytoplasmic vacuolar structures, which have been implicated as a major site of virus production and which we show here by serial sectioning to be, in several instances, invaginations of the plasma membrane. The effects on virus production were noticeable 2 h after interferon addition and reached their full extent by 13 h. We conclude from these observations that interferon acts upon the late stage(s) of virion maturation, leading both to a decrease in virion production as well as to the formation of defective particles. In contrast, a small but significant increase in the rate at which globin mRNA and hemoglobin accumulate is observed after interferon treatment. A number of laboratories have used the system of dimethyl sulfoxide (Me2SO)-stimulated cultured erythroleukemic cells as a model for the study of both murine leukemia virus induction (4, 23) and hemoglobin expression (6, 19, 21, 22). The detailed studies of Sato et al. (23) showed that internal cisternae composed of multiple vacuoles (designated "cytoplasmic vacuolar structures" or "CVS") and containing large numbers of C-type virus particles were produced in response to Me2SO stimulation. Increased budding of particles into these cisternae as well as an increase in budding at the plasma membrane were suggested as the sources of increased virion production (23), but a recent study (26) has found that only the former mode could account for increased virion production after Me2SO stimulation. Stimulation of hemoglobin production is accompanied by a large increase in cytoplasmic globin mRNA content, and, although alternative mechanisms cannot be strictly ruled out, increased transcription of the globin genes is

generally regarded as the probable mechanism by which Me2SO exerts its action (19, 21, 22). The concomitant induction of both virus and cellular functions in a single cell led several laboratories to examine the effect of interferon treatment on Me2SO-stimulated erythroleukemic cells (4, 9, 26). These studies basically showed that interferon treatment inhibited virus release, but not hemoglobin synthesis, in stimulated cells. In this investigation, we have attempted to elucidate the mechanism behind these interferon effects by quantitating changes in CVS, mature, and "immature" virions in single and serial thin sections of erythroleukemia cells when interferon is added to Me2SO-treated Friend erythroleukemic cells. Our data strongly suggest that interferon inhibits a late stage of virus production rather than merely virus release. Correlative biochemical studies, performed in addition to the high-resolution electron microscopic examination, to monitor hemoglobin and globin mRNA production after the various treatments show

799

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LUFTIG ET AL.

that interferon is able to increase the rate of Friend cell differentiation. MATERIALS AND METHODS Cell line. All experiments were made with cells grown from clone 745 of the original Friend cell line (6), kindly donated to us by C. Friend (Mount Sinai School of Medicine, New York, N.Y.). Tissue culture and induction conditions. Cells were maintained in Dulbecco modified Eagle medium (Grand Island Biological Co. [GIBCO], Grand Island, N.Y.), supplemented with 10% fetal bovine serum (Flow Laboratories, Inc., Rockville, Md.), 100 U of penicillin (GIBCO) per ml, and 100 Ag of streptomycin (GIBCO) per ml (DVME medium). Appropriate Falcon or Corning plasticware was used, and the cultures were incubated at 370C in a humidified atmosphere of 10% CO2 in air. For experimental purposes, exponentially growing Friend cells were inoculated into medium freshly supplemented with 2 mM glutamine (GIBCO) at a density of 4 x 104 cells/ml, and 50 U of mouse interferon per ml was added. After 24 h, 1.5% (vol/vol) Me2SO was added. The time of Me2SO addition represents day 0. Cultures not receiving interferon or receiving interferon after pretreatment with Me2SO were directly seeded at 105 cells/ml in 1.5% (vol/vol) Me2SO on day 0 under otherwise identical conditions. Preliminary experiments had shown that a combination of 1.5% (vol/vol) Me2SO and 50 U of interferon per ml was a suitable compromise to maximize effects of both agents and minimize cytotoxic effects. Since the quality of the Me2SO was shown to be of importance for eliciting optimal erythroid response,

analytical grade Me2SO (Baker's Analyzed Reagent) was redistilled under reduced pressure (750C, 20 mm of Hg) and stored frozen under nitrogen, in small batches, until use. A Coulter Counter (Coulter Electronics, Inc., Hialeah, Fla.) was used for cell counts. Preparation of cell extracts. Friend cells growing in suspension were sampled by low-speed centrifugation of a portion of the culture for 10 min (80 x g; International refrigerated centrifuge, International Equipment Co., Div. of Damon Corp, Needham Heights, Mass.), washed twice with Dulbecco phosphate-buffered saline, and suspended finally in phosphate-buffered saline at a concentration of 108 cells/ml. Nonidet P-40 solution (10%) was added to reach a final concentration of 1.0%, and the cell suspension was gently swirled for a few seconds. Two 5-s bursts of sonic treatment at 20 kHz and 60 W (Biosonik; Bronwill Scientific Inc., Rochester, N.Y.) were then applied, and the lysate was centrifuged at 20,000 x g for 30 min at 4°C. The cell-free supernatant was stored frozen under liquid nitrogen until use. Hemoglobin analysis and benzidine staining. Hemoglobin content of extracts was measured with an assay technique using benzidine. Five to twenty-five microliters of cell extract, depending upon hemoglobin concentration, are mixed with 0.2 ml of a 1% benzidine solution in 90% acetic acid and 0.2 ml of a

J. VIROL.

freshly prepared 1% H202 solution. After 20 min at room temperature, 2 ml of 10% acetic acid are added, and the optical density is read at 515 nm within 0.5 h, against a blank without cell extract. Amounts of hemoglobin are computed by using human hemoglobin (Sigma Chemical Co., St. Louis, Mo.) as a standard and related to the number of cells used to prepare the lysate. Benzidine staining of whole cells was performed in acetic conditions as described by Orkin et al. (16). Positive cells stain deep blue. The preparations were photographed on Polaroid film, and the frequency of benzidine-positive cells was determined on the print, using about 300 cells. Globin mRNA determinations. Methods for isolating cytoplasmic RNA from Friend cells and for preparing globin complementary DNA, as well as the conditions of the hybridization assay, have been described elsewhere (J.-F. Conscience, F. H. Ruddle, A. Skoultchi, and G. J. Darlington, Somatic Cell Genet., in press). Interferon. Mouse interferon was produced in L cells infected with Newcastle disease virus and purified by treatment at pH 2, ammonium sulfate fractionation, and carboxymethyl cellulose chromatography as outlined previously (3). Some of the interferon used in this work was a generous gift of P. Lengyel (Yale University, New Haven, Conn.). Interferon titers were determined on L-cell cultures by measuring the reduction in the yield of vesicular stomatitis virus. One unit of interferon, in this work, is the amount per milliliter that reduces vesicular stomatitis virus yield on L cells by 50%; it corresponded to 10 National Institutes of Health (NIH) mouse reference units, when compared with the NIH standard mouse interferon. The specific activity of the interferon used here ranged from 2 x 107 to 5 x 107 NIH mouse reference units per mg of protein. Reverse transcriptase. Reverse transcriptase was assayed in the supernatant culture medium as described in Lieberman et al. (9). Cell harvest and fixation for electron microscopy. About 5 x 107 cells were sampled by low-speed centrifugation of a portion of the suspension culture, washed twice with phosphate-buffered saline, and pelleted into a small tube at 80 x g. The pellet was then carefully overlaid with a 5% glutaraldehyde solution in 0.15 M sodium cacodylate buffer, pH 7.4, and kept at 0°C for varying intervals (up to 1 month) before embedding. Electron microscope embedding procedure. Glutaraldehyde was removed from the pellets, which were then washed with 0.15 M sodium cacodylate buffer, pH 7.4, postfixed for 1 h in 0.2 M Veronalacetate buffer (pH 7.4) containing 1.0% OSO4, dehydrated through ethanol and propylene oxide, embedded in Epon 812 (Shell Chemical Company, New York, N.Y.), and sectioned by means of a SorvallPorter-Blum MT-2B ultramicrotome (60-nm silver sections). Sections were stained with saturated uranyl acetate and lead citrate. Details are provided in McMillan and Luftig (13). Serial thin sections were collected on slotted grids (Ladd Research Industries, Inc., Burlington, Vt.). Specimens were examined in

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EFFECT OF INTERFERON ON FRIEND CELLS

a Philips EM301 electron microscope. Quantitation of electron micrographs. To obtain a statistically significant estimate for virion production from thin-section electron micrographs of cells, we adopted the following protocol. (i) Micrographs were taken at a magnification of about x2,000, to maximize the number of cells viewed in a single field and also to allow an unambiguous identification of the virus particles for subsequent enumeration. (ii) Negatives were printed at x 3 magnification, and contour lengths of the outer cell surfaces were traced with a planimeter. An estimate, using 25 cells whose outer surfaces were visible on the print, showed an average value of 32 ± 3 cm for the perimeter of a cell section at a total magnification of x 6,300. This value was used to estimate the total number of single cell sections obtained from the totality of sections at each time point, since in many cases only a part of a cell was visible in a field due to the extent of the area available for photography or because of lysing partly on a grid bar. Over different points, viz., at 2 and 5 days post-Me2SO treatment, this value stayed within the range indicated above; 90% of the total cell perimeters available for viewing were in that range, i.e., 29 to 35 cm. (iii) For each print, the number of virions was enumerated and sorted out into the three following classes: external, internal, and budding from the cell surface. In addition, we recorded whether the particles exhibited electron-translucent (A-type) or -dense (C-type) centers. In making such determinations, a number of errors have to be considered: (i) the sampling error, observed between different embeddings for the same experimental point, with a standard deviation of about 23%; (ii) the error in determining the total cell perimeter (12%, as determined by an examination of 100 cell sections); (iii) the error in enumerating the number of virion particles for a given group of micrographs (this error was estimated, from duplicate measurements of the same prints, to be 15%). In addition to these errors, which can be estimated, another source of error in determining the number of virions per cell section arises from the observation of Haguenau (8). This worker has described convincingly how densely stained vesicles containing mucopolysaccharides or collagen can be confused with virions and vice versa. Since the misidentification can be done in both ways, such errors tend to cancel each other over a large series of measurements, but it is impossible to estimate how they affected our experimental points. Based on estimated errors of the type discussed above, our total cumulative error for any experimental sample point (8 = I8X2 + 8,2 + 8a2) is, roughly, 30%.

RESULTS Effect of Me2SO on hemoglobin and virion production. Previous studies have shown that Me2SO treatment of Friend erythroleukemic cells stimulates the cells to produce an increased amount of murine leukemia virus over constitutive levels (4, 23). This stimulatory ef-

fect of Me2SO was confirmed, using quantitative electron microscopy, and it was correlated with a concomitant increase in hemoglobin production (Fig. 1). The initial lag in virion induction lasted about 2 days and was followed by a fourfold rise in virion production over the next 3 to 5 days (compare the noninduced [Fig. 2A] and induced [Fig. 2B] cells). Interestingly, there is a striking parallel between the kinetics of virion production and the increase in hemoglobin synthesis. This has suggested that a parallel induction event, affecting, for instance, the control of both viral and cellular genes, occurs. In our quantitation of virions from the thin sections of induced cells (Fig. 1), we only enumerated those virions that had been apparently newly synthesized. This classification is empirical and represents the sum of virions seen budding from the external perimeter of the cell plus the virions located in internal vacuoles. For the former case, the reason for the classification is obvious. In the latter case, an explanation is required. An examination of over 300 single, thin sections of cells at 5 to 7 days postMe2SO addition suggested that the internally located virions represent recently synthesized virion particles or virions in the process of being transported out of the cell. The representative electron micrographs of Fig. 3A and B show evidence supporting these points. Internal virions can be seen either budding into vacuoles or possessing an "enveloped A"-type 10

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6 7 5 4 Days FIG. 1. Effect of Me2SO treatment on virion and hemoglobin production in Friend erythroleukemic cells. Cultures were treated with (--- ) or without ( ) 1.5% (vollvol) Me2SO as described in the text at day 0. Apparent newly synthesized virions (0, *) were enumerated from thin sections of cells, and hemoglobin determinations (0, 0) were made on cell extracts, as described in the text. 0

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morphology characteristic of immature virions (arrowheads). Furthermore, in some instances (arrows) it can be seen that the internal vacuoles lead to the extracellular space or to other CVS, which, in turn, may lead to the extracellular space. These two observations provide evidence supporting the idea that a large number of virions can be synthesized in CVS and then move through passageways or vacuoles into the extracellular space. The greatest error in considering all of these internal virions as "apparently newly synthesized" virions is the extent to which some of them may have been internalized by phagocytosis from the supernatant fluids. This error cannot be presently esti-

mated. In contrast to the above classification, virions associated with the external surface of the cells in a nonbudding state (type C morphology) have not been included in our quantitation. This latter population of tightly associated virions (the cells were washed twice, so loosely associated virions would have been removed) apparently arose from the small percentage of lysed cells, which increased with time in culture. The observation of large numbers of such type C virions close to the cell surface in the vicinity of lysed cells supports this contention. At 3 days, with or without Me2SO, these lysed cells represented 5% of the total cell sections,

and their frequency increased to 20% after 7 days. From three separate experiments with 100 cell sections per data point, we found, on the average, a background value for such apparently non-newly synthesized virions of 3.07 ± 0.21 particles per cell section for each 10% of lysed cells in the sample. Effect of interferon on hemoglobin and globin mRNA production. When Friend cells were exposed simultaneously to 1.5% Me2SO and 50 U of mouse interferon per ml for 6 days according to our standard induction protocol, only a minimal effect on their growth rate was noticed (Fig. 4A). Clearly, the dose of interferon used is well below that needed to produce a significant decrease in proliferating capacity (12). Over 90% of the cells became benzidine positive after 6 days of Me2SO with or without interferon. However, between day 3 and day 5, there was a small, but significant, increase in the number of benzidine-positive cells in the presence of interferon, as compared with cells treated with Me2SO alone (Fig. 4B). This could reflect either a faster mobilization of cells to differentiate in the presence of interferon or a faster rate of hemoglobin production in each cell, allowing them to reach more rapidly 20% of the fully induced hemoglobin content. This threshold has been shown to be the lower limit at which a cell stains positive in the benzidine test (16). This differential rate of increase could

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be seen more clearly when hemoglobin production itself was assayed quantitatively on cell extracts. As seen in Fig. 4C, both a faster rate of accumulation was found, and, in addition,

the fully induced level was slightly higher than in control cultures treated with Me2SO alone. This increase is small, and its extent (1.5- to 2.5-fold) may vary between experiments. Since

804

LUFTIG ET AL.

FIG. 4. Growth rate, frequency of benzidine-positive cells, and amount of hemoglobin per cell in Friend erythroleukemic cells grown in the presence of 1.5% Me2SO, with and without 50 U of interferon per ml. Culture and assay conditions are described in the text. (A) Growth rate; (B) percent benzidine-positive cells; (C) micrograms of hemoglobin per cell. (0) Culture with interferon; (0) control cultures in Me2SO only; (E) cultures not treated with Me2SO, either with or without interferon. The latter cultures display a growth rate similar to the MeS$O-treated cultures, between 1 and 3% benzidine-positive cells throughout and only a minimal amount of hemoglobin.

J. VIROL.

pendent (9); 5 U of interferon sufficed to produce a 50% decrease in the level of reverse transcriptase in the medium 18 h after the addition of the antiviral substance, whereas 50 U produced a >80% decrease. Using quantitative electron microscopy, we attempted to follow this effect by measuring the number and type of virions synthesized between 2 and 16 h after addition of 50 U of interferon per ml to Friend cells that had been pretreated with 1.5% Me2SO for 3.5 days. It is important to note that in such short-term kinetic experiments, the effect of 50 U of interferon per ml on cell growth was negligible. The most striking observation, which we noted by the electron microscopic measurements defined in the first section, was a rapid decrease in the total number of newly synthesized particles associated with the cells (Fig. 3C through E; compare to Fig. 3A and B). When quantitated over 50 to 100 cell sections, we observed about a 60% decrease in the average number of virus particles per cell section (Fig. 5A); there was also a parallel decrease (40 to 50%) in the number of cell sections exhibiting any virion. The decrease in cell-associated virions was already marked after 2 h and increased to its maximum at about 5 to 6 h after interferon addition (Fig. 5A). Accumulation of extracellular Friend virus in the culture medium, measured by reverse transcriptase activity, ceased between 5 and 10 h after interferon, causing close to a 90% reduction in extracellular virions (Fig. 6). Although it may be difficult to compare data obtained by such different techniques, these observations suggest that virion release in the medium is not inhibited before virion

the amount of hemoglobin per cell goes through a maximum, the effect of interferon after 6 to 8 days is variable. In further experiments, both globin mRNA production, as assayed by molecular hybridization of total cytoplasmic RNA with globin complementary DNA, and hemoglobin were assayed in the same samples. It was noted (Table 1) that when hemoglobin was elevated, an inTABLE 1. Amounts of hemoglobin and globin crease in mRNA was also noticed, although its mRNA in cell extracts from cultures treated with and magnitude was usually lower. The results con- without interferon, in the presence or the absence of firm the observation of Lieberman et al. (9) that Me2SO a there is a small but significant increase in heAmt after 4 days moglobin production after interferon treatthat this In demonstrate ment. addition, they jg of hemo- mRNA in toTreatment increase is paralleled by a similar increase in globin per tRalcytopaglobin mRNA. cell (x 10) mic RNA (%) Effect of interferon on virion production 0.017 6:1 from Me2SO-stimulated cells. Interferon addi- None 0.110 22.2 Me2SO tion to Friend cells stimulated by Me2SO in- 1.5% 0.160 46.9 1.5% Me2SO and interferon duces the antiviral state, as evidenced by a (50 U/mi) strong inhibition of vesicular stomatitis virus 2.2 0.012 None replication in the cells: 5 U of mouse interferon Interferon 0.009 1.9 produced in Friend cells a 50% reduction of 1.5% Me2SO(50 U/mi) 0.075 26.7 vesicular stomatitis virus yield, and 50 U pro- 1.5% Me2SO and interferon 0.090 35.4 (50 U/mi) duced a 50-fold decrease in vesicular stomatitis virus yield. The decrease in leukemia virus a Hemoglobin and globin mRNA assays were done as production from Friend cells treated with described in the text. Cultures were split 1:5 into fresh Me2SO and interferon was similarly dose de- medium at day 4, as described in the text.

VOL. 23, 1977

EFFECT OF INTERFERON ON FRIEND CELLS

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treatment and by threefold at later change FIG. 5. Quantitation ofF changes inin virion produc. . times. This increase m immature virions sugtion of MeS$O-stimulated cells after interferon treatment. (A, or top graph) Average number of total gests that a late stage of virus maturation is virions per cell section, xwith (0) and without (0) affected by interferon and causes the profound interferon treatment. In t)his total we have included inhibition in virus release. The genetic identity virions on the external surface, as well as the appar- of these enveloped A-type particles with Friend ently newly synthesized o. nes, e.g., budding and in- virus remains, of course, to be firmly estabternally located virions. IThis was done so that the lished, to confirm that these are precursors to overall effect on total virioin production by interferon the mature C-type virions. could be observed. If we u)ant to account only for the An additional observation, however, supapparently newly synthesi zed virions, then a value of ' . . t 1.38 + 0.40 virions per c-ell section should be sub- ports the idea that a maturation step in vision tracted from each ofthe da tta points. This latter value assembly is impaired by interferon treatment. is the average plus or min the standard deviation, We noted that 12 to 16 h after interferon, there for the number ofexternal virions per cell section over was an increase in the average number of the time of the experimentet. As can be seen by the empty shell particles in the cell (Fig. 7A and B) magnitude of the error, tthis value, which we have from 0.15 per cell section in Me2SO-stimulated attributed mostly to the piresence of lysed cells in the cultures to a value of 0.5 by 16 h after interferon about the population, remains relallively unchanged over the treatment. Since these particles duration of the experimen,t. Not shown on this graph virion size (diameter, 60 nm) is the parallel decrease in the fraction of cell sections shells in thin sections, they may represent with 2.1 virion. after intherferon treatment. The retspective fractions are 0.48, 0.39, 0.31, 028, and 0.32 at 0, 2, 5,13, and 16 h post-interferon addition at day 50 3.5. (B, or bottom graph). Average number of en- cr R.T. level on day 4 veloped A-type virions per cell section with (A) and 40 without (A) interferon treatment. The zero points in Of) ro (A) and (B) are averages of the values found for 0._ 30 MeSO-stimulated cells at days 3 and 4. These are up Control + DMSO 0the values we would expect for Me2SO-treated cells at E 20 3.5 days posttreatment, the starting point of the interferon treatment. Each data point represents an C.) I0 average value taken over 50 to 100 cell sections.

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production decreases, and there does not seem, therefore, to be a direct impairment of virus release. These early released reverse transcriptase particles could be defective, as suggested by Pitha et al. (20). It may be puzzling that at later times, when virus release is almost completely inhibited, cells still contained one-third to one-half of the original amount of viral particles. Electron microscopic analysis of these virions showed, however, that their morphological characteristics are very different from those in Me2SO-stimulated cells not treated by interferon. The most evident morphological change was,

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either aberrant or incomplete byproducts of virion assembly. Large numbers of these empty shell particles, as well as enveloped A-type particles with elongated aberrant-appearing internal electron-dense material (Fig. 7B and C), were also seen in cells to which interferon was added at the time of Me2SO addition and maintained continuously for 4 days. These bizarre forms have been observed in other oncornavirus cell lines, and it was suggested that they result from a block in virus assembly (7). These results, taken together, suggest that after interferon treatment a defect appears in the assembly of virions, which explains the reduced number of total virions seen per cell section and the increase in abnormal forms. This conclusion is also in line with the fact that, by polyacrylamide gel electrophoresis, the amount of core protein p30 is only slightly reduced in interferon-treated Friend cells (9). Synthesis of some viral components continues after interferon treatment, but virus assembly and maturation seems impaired. Further work was, therefore, concentrated on the study by electron microscopy of virus formation and budding in the cell and the role internal cisternae or CVS (23) play in this process. The role of CVS in virion production before and after interferon treatment of Me2SOstimulated Friend erythroleukemic cells. The CVS are a rather novel feature caused by Me2SO stimulation of Friend erythroleukemic cells, and they have been implicated as a major site of virion production (23, 26). We have made the following two observations. (i) The CVS probably represent, to a significant extent, invaginations of the plasma mem-

brane and are not exclusively separate cytoplasmic organelles. In serial thin sections of cells after Me2SO stimulation, CVS (Fig. 2B, 3A and B), in every instance where they could be followed, led to either another CVS or to the external surface. Some of the CVS also appeared fused to other, perhaps phagolysosomal, vacuoles. If some of these vacuoles arose from part of the Golgi region (e.g., the GERL area [15]), then the fusion of CVS to the vacuoles could eventually lead to the release of viruses through movement of fused CVS vesicles to the outer surface. (ii) The increased membrane surface in Me2SO-stimulated cells created by the formation of CVS seems to provide an increased surface for the budding and, hence, production of virus. Specifically, we found that, by 3 days after Me2SO stimulation, there was a constant, about twofold increase in both the number of cell sections with one or more CVS and the total number of CVS per cell section, relative to unstimulated cells. This ratio was maintained for up to 7 days poststimulation. This observation, taken together with the frequent budding of virions into CVS (Fig. 3), is consistent with the suggestion that there is a correlation between an increased number of CVS and an increased yield of virions. We then examined the effect of interferon treatment on the CVS (Fig. 8). By 13 to 16 h after addition of interferon to cells that had been treated for 3.5 days with 1.5% Me2SO, there was a 1.5- to 2-fold decrease in the number of CVS per cell section (Fig. 8, light boxes) but not significant change in the average number of cells with at least one CVS (Fig. 8, dark

VOL. 23, 1977

EFFECT OF INTERFERON ON FRIEND CELLS

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0

6

7

Interferon on day 3 2 after DMSO (mo)

FIG. 8. Quantitation of CVS in DVME, Me2SO, and Me2SO-interferon-treated cells. The average number of cell sections with CVS is given by the solid lines, and the average total number of CVS per cell section is represented by dashed lines.

boxes). It thus appears that the effects of interferon on CVS are parallel to the effects we have seen on total virion production. These data are most consistent with the notion that the surface area available for virus production decreases rapidly after interferon treatment, leading to a decrease in the total number of virions exported in the vacuolar structures. This phenomenon is coupled with a defect in virus maturation, leading to more immature forms. A unified model to explain both phenomena would be the decreased synthesis of a viral protein that normally becomes located within the membrane of CVS and is needed for virus assembly; this product would be rapidly depleted after interferon treatment. If this is the case, then this would represent a novel action of interferon in a viral system. In most other cases (29), interferon inhibits the production of almost all viral proteins. DISCUSSION Friend virus induction by Me2SO and inhibition by interferon. Our results demonstrate that in the presence of Me2SO intracellular virus production in Friend cells is stimulated in parallel to hemoglobin production. This stimulation was measured in two ways, (i) increase in the number of virus particles per cell section and (ii) increase in the number of cell sections exhibiting at least one virion. The two measurements combined indicate a 2.5- to 5-fold stimulation of virus production for a given pop-

807

ulation. Thus, increased virus production in the cell is the primary cause for the increased release of virus in the extracellular compartment observed by Lieberman et al. (9), using reverse transcriptase and p30 determinations in the culture medium. Strikingly, we find here that this increased virus production is accompanied by an increased number of CVS per cell as well as an increase in the number of cell sections exhibiting at least one CVS. Interferon treatment inhibits Friend virus production in Me2SO-stimulated Friend cells, while the induction of hemoglobin synthesis continues (9, 25). This may be rather surprising, in view of the fact that hemoglobin synthesis and virion production are simultaneously stimulated by Me2SO, and suggests that interferon and Me2SO affect different cellular mechanisms. More puzzling is the observation, made first by Lieberman et al. (9) and confirmed here, that interferon can enhance the effect of Me2SO by stimulating Friend cells to produce more hemoglobin and hemoglobin mRNA. This effect is, primarily, an acceleration of the differentiation process, which is complete at an earlier time in the interferon-treated culture. Interferon seems to be able to favor the expression of a differentiation program in the cell, as found also in lymphocytes (12). It is not clear whether this is linked to the decreased rate of cell proliferation or the inhibition of RNA tumor virus production, which appears as the major effect of interferon on Friend cells. Many studies have been concerned with the mechanism by which interferon inhibits the production of oncornaviruses in chronically infected cell lines (1, 5, 9, 20, 26). An important feature of the present study is the kinetic measurements of the changes in the morphology, localization, and amount of viral particles in the first few hours after interferon addition, which were carried out by quantitative electron microscopy of the Friend cells. Taking cells that have been stimulated by Me2SO to produce high amounts of virus, we find that the interferon rapidly decreases the number of virus particles per cell and the number of cells exhibiting one or more viral particles. Very rapidly also, we observe an increase in the number of immature, enveloped A-type particles, followed by an increase in the number of core shell particles and aberrant virions. In the presence of interferon, the number of CVS per cell decreases, whereas the number of cells exhibiting at least one CVS remains unchanged. There is also a decrease in the number of CVS with multiple virions. Budding activity remains unaffected or even slightly ele-

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LUFTIG ET AL.

vated after interferon. The effect of interferon on the amount of mature virus released in the medium, measured by the reduction in reverse transcriptase activity, appears more profound than that on cell-associated virus but also appears to occur less rapidly. From the greater decrease in extracellular virus than in cell-associated virus, it was suggested that virus release through the membrane is affected by interferon (1, 5). If this latter model were valid for the Friend cell system, we would have observed (i) an increase, at least transiently, in intracellular virions and (ii) an earlier decrease of extracellular virions, compared with intracellular virions. Actually, we observed just the opposite in both cases, which seems to rule out, at least in this system, that only virus release is affected by interferon through a direct effect of interferon on the cell membrane. Kinetic considerations indicate that the decrease in the total number of newly formed particles per cell section is a primary effect of interferon. This decrease in virus production does not, however, appear to result from a general inhibition of viral protein synthesis, as is found in other virus cell systems (28, 29; E. Yakobson, C. Prives, J. Hartman, E. Winocour, M. Revel, Cell, in press, 1977). This is shown by the fact that the amount of intracellular viral core protein p30 and its rate of labeling by [35S]methionine remains practically the same in interferon-treated and untreated cells (9, 20). There is, however, also no increase in the amount of viral protein, as would be expected from a pure block of virus release. The amount of viral RNA was also found not to increase after interferon treatment of Friend cells, even under conditions where the number of enveloped A particles increases (18). A faster turnover of the viral products could account for these results. The data shown here clearly demonstrate that the viral particles remaining in the cells after interferon treatment and, presumably, made while the antiviral state is established have a highly abnormal morphology, as compared with those in normal Me2SO-stimulated Friend cells. Immature, A-type particles form a large fraction (35%) of the total, as compared with 7% in the absence of interferon. Moreover, abnormal budding forms and empty viral shells become apparent in sections of interferontreated cells, suggesting that a defect in assembly and maturation of the virions is responsible for most of the reduction in virus formation. This conclusion is in agreement with that of Pitha et al. (20) and, more recently, Chang et

J. VIROL.

al. (2), who found that oncornaviral particles released from interferon-treated cells are noninfectious. In our hypothesis, impairment of Friend virus maturation could result from the decreased synthesis of a special class of late viral RNA or protein product. As mentioned earlier, this latter point would be a rather novel effect of interferon; however, there is some precedent for such an occurrence, i.e., the finding that synthesis of individual viral proteins is affected to varying degrees by interferon treatment was already seen in poxvirus-infected cells (17). Another possibility is that there is a deficiency in the cleavage of some essential polypeptide needed for closure of the virion bud and maturation of the virions (14, 27). A quantitation of all viral products would be needed to demonstrate this mechanism, but the data suggest that the missing component is likely to be a viral product that becomes associated with the cell membrane. Our electron microscopic data demonstrating a decrease in the number of CVS also support the idea of a decrease in membrane surface available for internal virion production. Role of CVS in virion production. We have shown by serial thin sections that the CVS near the perimeter ofthe cell are, in many instances, invaginations of the plasma membrane or are contiguous with other CVS (11; Luftig and McMillan, unpublished observations), thus building an elaborate and intricate network of membrane passageways. This observation is in contrast to the results of Sato et al. (23), who found by using lanthanum staining that the CVS were vacuoles. The CVS further inside the cell could not be followed through enough serial sections to determine if they were also invaginations; thus, they may be true vacuoles, which have formed by endocytosis, or they may be lysosomal vesicles formed by Golgi-mediated synthesis. The observations of free vacuoles emerging both from the cell and in lysed cells (unpublished observations) support such a contention. The possibility of lysosomal action occurring in the vesicles is supported by the observation that the virions in these vacuoles are mostly of the C, or mature, type. In contrast, the vacuoles appearing near the surface contain A-type, or immature, virions. Such a differential localization of types of virions has already been reported for Eveline cells chronically infected with Friend leukemia virus (11) and JLS-V9 (Moloney leukemia virus) (R. B. Luftig, E. P. Bedigian, M. P. Paskind, and R. A. Weinberg, J. Cell Biol., 67:250a, 1975). Based on these considerations, we propose the model

VOL. 23, 1977

_+@-s~

EFFECT OF INTERFERON ON FRIEND CELLS

depicted in Fig. 9 to account for the enhanced production of Friend leukemia virus in Friend erythroleukemic cells after Me2SO stimulation. This model is adapted from a schematic of amphibian neurulae epithelial cell development made by L6fberg (10). What we propose is that the Me2SO-stimulated Friend cells form frequent invaginations, into which the virus can "bud." These are near the plasma membrane outer surface, and virions budding from them can be released into the external space. In some instances, such invaginations gradually endocytose, leading to formation of an internal cytoplasmic vacuole which is moved further into the cell by microtubules. In time, this vacuole leaves the cell, but in its life in the vacuole, the virion is exposed to endogenous or exogenous proteases and nucleases. This treatment would cause collapsing of the internal viral nucleoid, leading to formation of a C-type particle. Of course, the data on which this model is based are only morphological and are subject to a degree of misinterpretation. As discussed, the degree of phagocytosis is an unknown here and certainly accounts for a percentage of the viri-

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FIG. 9. Model of Friend virus morphogenesis. This model takes into account the observation that viruses are not only produced at the external surface of the cell, but that they are also produced at the CVS. We propose that Me2SO stimulates murine leukemia virus-infected cells to form frequent invaginations, into which virus particles can bud. Such budding sites start out on or near the plasma membrane; then, as the plasma membrane invaginates, the sites gradually endocytose, leading to the formation of internal cytoplasmic vesicles which can be moved further into the cell by microtubules. In time, such vesicles may or may not leave the cell; however, the immature, enveloped A-type virions in these vesicles are eventually exposed to lysosomal enzymes, which cause a collapsing of their internal structure, leading to the formation of mature, C-type virions.

809

ons seen in cytoplasmic vacuoles. Such phagocytic vacuoles would be expected not to leave the cells, but to fuse with lysosomes, leading to phagolysosomes, which would appear as dense bodies in an electron microscope. However, we believe this dead-end pathway cannot be the only explanation for the CVS, since (i) in many instances, with different murine cell lines, vacuoles can be seen in the act of emptying their contents to the extracellular space (this often consists of a sequential array of C-type particles attached to what was the inside of the vacuole membrane), and (ii) most of the CVS-containing vacuoles appear clear, and the virions in them appear undegraded, which is not what we would expect for phagolysosomes. Another approach to study this process is by the method of Steinman et al. (24), using horseradish peroxidase as a marker to determine the rate of ongoing pinocytosis with time after Me2SO stimulation and virus production; if the model in Fig. 9 is correct, there should be a large increase in pinocytotic activity after Me2SO stimulation. Furthermore, the observed decrease in number of CVS (Fig. 8) observed after interferon treatment should also be able to be quantitated by this independent procedure. By these methods we can begin to determine what role, if any, is played by the decreased internalization of membrane as a mechanism whereby interferon inhibits MuLV virus production. In conclusion, the ability of interferon to cause a decrease in virion production can be said to be linked to both a loss of some maturation factor (whose production would be inhibited by interferon) and to a decrease in membrane surface available for virus production. The very rapid response to interferon, which may be faster than in other virus-host systems because the Friend cells used here are rapidly growing, suggest that the postulated maturation factor is rate limiting in virus production. Identification of the component missing after interferon treatment should be very useful for understanding the mechanism of virion assembly. ACKNOWLEDGMENTS This research was supported by U.S. Public Health Service (PHS) grants P30-12708 and CA-15573 from the National Cancer Institute (NCI) (R.B.L.). J.F.C was supported by a PHS international research fellowship (no. 5F05TW2213-02, from the Fogarty International Center). A.S. is supported by PHS grant CA-16368 from the NCI and F.R. is supported by PHS grant GM09966 from the National Institute of General Medical Sciences. In addition, the support by Israel-U.S.A. Science Foundation and the German Science Fund (GSF, Munich) is gratefully acknowledged by M.R.

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J. VIROL .

teins. Cell 4:31-36. LITERATURE CITED 1. Billiau, A., V. G. Edy, H. Sobis, and P. De Sommer. 15. Novikoff, P. M., A. B. Novikoff, N. Quintana, and J. J. Hauw. 1971. Golgi apparatus, GERL, and lysosomes 1974. Influence of interferon on virus particle syntheof neurons in rat dorsal root ganglia, studied by thick sis in oncornavirus carrier line. Evidence for a direct section and thin section cytochemistry. J. Cell Biol. effect on particle release. Int. J. Cancer 14:335-340. 2. Chang, E. H., M. W. Myers, P. K. Y. Wong, and R. M. 50:859-886. Friedman. 1977. Inhibitory effect of interferon on a 16. Orkin, S., F. I. Harosi, and P. Leder. 1975. Differentiats mutant of Moloney leukemia virus. Virology tion in erythroleukemic cells and their somatic hybrids. Proc. Natl. Acad. Sci. U.S.A. 72:98-102. 77:625-635. 3. Content, J., B. Lebleu, A. Zilberstein, H. Berissi, and 17. Osterhoff, J., M. Jager, C. Jungwirth, and G. Bode. M. Revel. 1974. Mechanisms of the interferon-in1976. Inhibition of poxvirus-specific functions induced duced block of mRNA translation in mouse L-cells: in chick-embryo fibroblasts by treatment with homolreversal of the block by transfer RNA. FEBS Lett. ogous interferon. Eur. J. Biochem. 69:535-543. 41:125-130. 18. Ostertag, W., T. Cole, T. Crozier, G. Gaedicke, N. 4. Dube, S. K., I. B. Pragnell, N. Kluge, G. Gaedicke, G. Kluge, J. Kind, C. Krieg, G. Roesler, G. Steinheider, B. Weimann, and S. K. Dube. 1973. Viral involveSteinheider, and W. Ostertig. 1975. Induction of enment in differentiation of erythroleukemic mouse and dogenous and of spleen focus-forming viruses during dimethyl-sulfoxide-induced differentiation of mouse human cells, p. 485-513. In S. Nakahara et al. Differerythroleukemia cells transformed by spleen focusentiation and control of malignancy of tumor cells. forming virus. Proc. Natl. Acad. Sci. U.S.A. 72:1863University of Tokyo Press, Tokyo. 1867. 19. Ostertag, W., H. Melderis, G. Steinheider, N. Kluge, 5. Friedman, R. M., E. H. Chang, J. M. Ramseur, and M. and S. Dube. 1972. Synthesis of mouse hemoglobin W. Myers. 1975. Interferon-directed inhibition of and globin nRNA in leukemia cell cultures. Nature chronic murine leukemia virus production in cell cul(London) New Biol. 239:231-234. tures: lack of effect on intracellular viral markers. J. 20. Pitha, P. M., W. P. Rowe, and M. N. Oxman. 1976. Virol. 16:569-574. Effect of interferon on exogenous, endogenous, and 6. Friend, C., W. Scher, J. G. Holland, and T. Sato. 1971. chronic murine leukemia virus infection. Virology Hemoglobin synthesis in murine virus-induced leu70:324-338. kemia cells in vitro: stimulation of erythroid differen- 21. Ross, J., J. Gielen, S. Packman, Y. Ikawa, and P. tiation by dimethylsulfoxide. Proc. Natl. Acad. Sci. Leder. 1974. Globin gene expression in cultured U.S.A. 68:378-382. erythroleukemia cells. J. Mol. Biol. 87:697-714. 7. Hackett, A. J., and S. S. Sylvester. 1972. Cell line 22. Ross, J., Y. Ikawa, and P. Leder. 1972. Globin messenderived from Balb/3T3 that is transformed by murine ger RNA induction during erythroid differentation of leukemia virus: a focus assay for leukemia virus. cultured leukemia cells. Proc. Natl. Acad. Sci. Nature (London) New Biol. 239:164-167. U.S.A. 69:3620-3623. 8. Haguenau, F. 1973. "Viruslike" particles as observed 23. Sato, T., C. Friend, and E. deHarven. 1971. Ultrastrucwith the electron microscope, p. 391-398. In A. J. tural changes in Friend erythroleukemia cells Dalton and F. Haguenau (ed.), Ultrastructure of treated with dimethylsulfoxide. Cancer Res. 31:1402animal viruses and bacteriophages. Academic Press 1417. Inc., New York. 24. Steinman, R. M., J. M. Silver, and Z. A. Cohn. 1974 9. Lieberman, D., Z. Voloch, H. Aviv, U. Nudel, and M. Pinocytosis in fibroblasts. J. Cell Biol. 63:949-969. Revel. 1974. Effects of interferon on hemoglobin syn- 25. Swetly, P., and W. Ostertag. 1974. Friend virus release thesis and leukemia virus production in Friend cells. and induction of hemoglobin synthesis in erythroleuMol. Biol. Rep. 1:447-451. kemia cells respond differently to interferon. Nature 10. Lofberg, J. 1974. Apical surface topography of invagi(London) 251:642-644. nation and non-invaginating cells. A scanning trans- 26. Ussery, M. A., R. Ramirez-Mitchell, and B. A. Harmission study of amphibian neurulae. Dev. Biol. desty. 1976. Inhibition of Friend murine leukemia 36:311-329. virus production by low-ionic-strength medium. J. 11. Luftig, R. B., P. N. McMillan, and D. P. Bolognesi. Virol. 17:453-461. 1974. An ultrastructural study of C-type virion as- 27. Vogt, V. M., R. Eisenman, and H. Diggelmann. 1975. sembly in mouse cells. Cancer Res. 34:3303-3310. Generation of AMV structural proteins by proteolytic 12. Lundahl, P., P. Leary, and I. Gresser. 1973. Enhancecleavage of a precursor polypeptide. J. Mol. Biol. ment by interferon of surface antigens on L1210 cells. 96:471-493. Proc. Natl. Acad. Sci. U.S.A. 70:2785-2788. 28. Wiebe, M. E., and W. K. Joklik. 1975. The mechanism 13. McMillan, P. N., and R. B. Luftig. 1975. Preservation of inhibition of reovirus replication by interferon. of membrane ultrastructure with aldehyde or imidate Virology 66:229-240. fixatives. J. Ultrastruct. Res. 52:243-260. 29. Yamazaki, S., and R. R. Wagner. 1970. Action of inter14. Naso, R. B., L. J. Arcement, and R. B. Arlinghaus. feron: kinetics and differential effects on viral func1975. Biosynthesis of Rauscher leukemia viral protions. J. Virol. 6:421-429.