J. CeU Set. a, 71-76 (1967) 71Printed in Great Britain

ELECTRON MICROSCOPIC STUDY OF

PENETRATION OF NEWCASTLE DISEASE

VIRUS INTO CELLS LEADING TO

FORMATION OF POLYKARYOCYTES

N. MEISELMAN,* A. KOHN AND D. DANONThe Weizmarm Institute of Science, Rehovoth, andThe Israel Institute for Biological Research, Ness Ziona, Israel

SUMMARY

Treatment of FL or Lu 106 epithelial cells with Newcastle disease virus (NDV) at an inputmultiplicity of 500 EIDM per cell induces in these cells the formation of polykaryocytes at theend of 2-3 h of contact. Electron micrographs of such NDV-treated monolayers after 2-10 minof incubation show the presence of virions adsorbed to the cell membranes, in vacuoles and withthe viral envelope partly fused with the cell membrane. In polykaryocytes induced by NDV,remnants of cell membranes showing numerous breaks may still be present after 3 h.

INTRODUCTION

Fusion of animal cells in vitro into polykaryocytes by Newcastle disease virus (NDV)added to the cells at high input multiplicity (Kohn, 1965) may be used as a convenienttool for investigation of the events occurring at the cell membrane during adsorptionand penetration of paramyxoviruses.

One of the hypotheses on the mode of penetration of myxoviruses into animal cellsassumes that all viruses, including those viruses with a lipid envelope, enter their hostcells by phagocytosis or pinocytosis (Dales, 1962, 1963, 1965; Dales & Choppin, 1962;Silverstein & Marcus, 1964). The other hypothesis (Adams & Prince, 1957; Cohen,1963; Hoyle & Finter, 1957; Hoyle, 1962; Wecker & Schaefer, 1957) postulates thatthe penetration of myxoviruses into the cytoplasm involves an interaction between thecell membrane and the viral envelope, leading to a reciprocal fusion of these twocomponents and the introduction of the viral nucleoprotein into the cytoplasm, amechanism that would be a reverse of the process of maturation of the virions at thecell surface by 'budding' (Blough, 1964; Dawson, Epstein &' Hummeler, 1965;Morgan, Rose & Moore, 1956; Wyckoff, 1953).

The evidence for the first hypothesis is mainly visual (electron-microscopic), whilethe second is supported by the chemical approach. The present experiments weredesigned to visualize electron-microscopically the sequence of events preceding thefusion of cells in monolayer cultures by NDV.

• Permanent address: C.W. Post College of Long Island University, Greenvale, N.Y.11548, U.S.A.

72 N. Meiselman, A. Kohn and D. Demon

MATERIALS AND METHODS

Two cell lines were used in these experiments: human amnion (FL), and human lung(Lu 106). The cultures were grown in 60-mm polystyrene dishes in Eagle's mediumcontaining 10% calf serum at 37 °C in a CO2 incubator. The cells were grown eitherdirectly on the surface of the dish or on the surface of a 25-mm glass coverslip coatedwith a film of collodion. Clean coverslips were dipped into a 1 -2 % solution of Parlodion(Mallinkrodt Chem.) dissolved in amyl acetate, and air-dried. The collodion-coatedcoverslips were sterilized prior to use by exposing each side to u.v. light (WestinghouseST) for 5 min at a distance of 30 cm.

The HP strain of NDV was used (Kohn, 1955). The virus was grown in the allantoiccavity of 9- to 10-day-old chick embryos. The virus in allantoic fluid was concentratedand purified by 2 cycles of low and 2 cycles of high-speed centrifugation (Spinco L,30000 rev/min, 1 h). The final pellets were resuspended in 1:50 volume of phosphate-buffered saline and stored at — 60 °C.

Two to 3 days after seeding the cells, when confluent monolayers were formed,the nutrient medium was removed from the dish, and 0-2 ml of virus suspension,calculated to contain about 500 EID (chick embryos infective dose)^ per cell, weredropped on to the centre of the dish. The cultures were then incubated at 37 °C for 2,10, 30, 60 and 180 min before fixation. Cultures kept in contact with the virus forperiods exceeding 30 min were fed with 4 ml of nutrient medium added at the end ofthe first \ h. The extent of cell contact and the formation of polykaryocytes weredetermined by optical microscopy on replicate cultures fixed in absolute methanol andstained with Giemsa.

Ferritin-conjugated anti-NDV globulin was added to the cultures in some experi-ments in order to facilitate recognition of virions and permit identification of viralantigens in the electron micrographs. Following a saline wash of the culture incubatedwith NDV, ferritin-conjugate was added for 10 min prior to fixation. Preparation ofthe ferritin-conjugated antibody was carried out by the method of Singer (1959)as modified by Morgan et al. (1961). The antibody was prepared by bi-weekly injectionof stock NDV mixed with Freund's adjuvant intramuscularly into guinea-pigs, andbleeding them 2 weeks after the last injection. The antibody titre, as measured byinhibition of haemagglutination, was 1:640.

In some experiments, the cells were removed from the Petri dish by scraping themoff with rubber. The cells were then resuspended in phosphate-buffered saline,centrifuged, and fixed as a pellet. In most experiments the cells were grown on col-lodion membranes. Fixation was done by addition of 2 ml of fixative deposited directlyon the culture. The cell monolayers were fixed for 1 h at 4 °C with either 2 % glutar-aldehyde followed for 30 min by 2% OsO4 in phosphate-buffered saline, or directlyby 2% OsO4 in o-i M phosphate buffer for 1 h. After fixation, the cells on the coverglass were gently rinsed in cold phosphate buffer. After scratching the edges of thecoverslip with a razor blade to cut the film of collodion, the membrane was floated freewith the aid of fine forceps, the flotation buffer serving as the final wash. The filmbearing the cells was then lifted from the wash solution so that it draped into folds to

Electron microscopy of NDV entry 73

form a curtain-like strand and this was immediately passed through a 2% gelatinesolution at a temperature just above the point of gelification, to prevent unfolding ofthe film during dehydration and embedding. The fibre-like strand was passed 2 or 3times through gelatin, allowing each coat to dry slightly. This technique, inspired bythe method of Dr J. A. Murphy (personal communication) for orienting red blood cellsfor cross-sections, produced a fairly rigid strand which could then be oriented verti-cally or horizontally in the embedding capsule. Dehydration in graded acetones andembedding in Vestopal W were carried out according to Ryter & Kellenberger (1958).Sections were cut with glass knives on a Danon Ultramicrotome and examined in anRCA EMU 2 electron microscope.

RESULTS

Both the human amnion cells (FL) and lung cells (Lu 106) respond to NDV added tothem at high input multiplicity, with the formation of polykaryocytes (Kohn, 1965;Okada, 1962). As seen in phase-contrast or in Giemsa-stained cultures, coalescence ofthe cytoplasm into multinuclear cells begins after about 1 h, the 'fusion' reachingmaximum about 3 h after addition of the virus. Lu 106 form larger polykaryocytes thanFL cells, with fusion indices (average number of nuclei per cell) of 8 compared with 3,respectively (Kohn & Adler, 1966).

When, after 3 h, the NDV-treated cell cultures were covered for 2-3 min with asolution of O-OO2 M ethylenediaminetetra-acetic acid (EDTA), the monolayers of unfusedcells separated into individual cells, while the polykaryocytes retained the nucleargrouping (Fig. 1 A-D).

Following adsorption of the virus to the cells, changes were observed with theelectron microscope in both the FL and the Luio6 cultures, similar to those describedby Okada (1962) for the effect of HJV virus on Ehrlich's ascites tumour cells, byHarris, Watkins, Ford & Schoefl (1966) for parainfluenza virus on cell cultures ofvarious species, and by Holmes & Choppin (1966) for parainfluenza virus SV 5 onbaby hamster kidney cells. There is an increasing activity of the cell surfaces treatedwith NDV, as evidenced by the appearance and increase in size of microvilli. Cellstreated with NDV and sectioned transversely to the surface plane showed an abun-dance of microvilli on their surface. When the cells were already in contact prior toaddition of the virus, the adjacent cell membranes were fairly regular, and microvilliwere less numerous or absent. Following the addition of virus, extensive interdigita-tion of microvilli of adjacent cells was observed. Cytoplasmic bridges were found inareas of high activity. InLU 106 cells an increase in the number of cytoplasmic bridgeswas observed with prolongation of incubation after addition of the virus, but completedisappearance of traces of membranes between cells was rarely found, even after3 h of incubation (Figs. 11, 12), when complete cell fusion was seen in the light micro-scope (Fig. 1 B, D). Transverse or near transverse sections through cell monolayers(provided by the known orientation of collodion membrane in the fixed and embeddedstrand) showed that what would appear as fusion in the optical microscope is probablya shingle-like overlap of adjacent cells, which would tend to obscure a distinct line ofdemarcation between adjacent cells.

74 N' Meiselman, A. Kohn and D. Demon

As to the fate of the virus added to the cell monolayers at high input multiplicity, itsadsorption to the cell surface and the microvilli could be well seen in electron micro-graphs of both ferritin-labelled and non-labelled preparations 2 and 10 min after itsaddition to the cells (Figs. 2, 5). In the io-min preparations, virions were seen in 4different topographical locations: adsorbed to the surface of the cell (Fig. 2); partlyengulfed by a fold of the membrane (Figs. 6, 7); inside a vacuole-like structure(Figs. 8-10); and in a state which looked as if the viral envelope were partly fused withthe cell membrane (Fig. 4A, B). The virions in vacuoles were either in fairly openspaces (Fig. io), which perhaps represented crypts rather than vacuoles, or weresurrounded by the vacuolar membrane (Fig. 9).

After incubation for 30 min or more, virions were rarely seen outside the cells, andthere was no evidence of their presence inside the cells.

DISCUSSION

Electron micrographs of sections of cell monolayers treated with NDV at high inputmultiplicity and incubated for short periods (2-10 min) showed the presence of intactvirions either on the cell membrane, or partly engulfed, or inside the vacuole. Thesefindings would indicate that at least one method of entry of NDV into the cell is byphagocytosis. Phagocytosis itself cannot provide an explanation for a course of eventsleading to cell fusion, since NDV does not induce fusion of HeLa cells, known to bestrongly phagocytic (Musgay & Weibel, 1962; Dr R. Oren, personal communication;Silverstein & Marcus, 1964). Since, however, virions were also observed partly fusedwith the cell membrane, one must consider the possibility of virus entry by fusion ofthe viral envelope with the cell membrane; in this case the viral nucleoprotein wouldbe directly released into the cytoplasm.

For the virus to start synthetic activity by using cell enzymes and ribosomes for itsown production it has to be uncoated first, i.e. freed of its envelope. Even if the virusis engulfed in a vacuole it is still in effect outside the cell and the problem of crossingthe barrier of the membrane forming the vacuole remains. Crossing of the membrane,be it outer membrane of the cell or the vacuolar membrane inside the cell, may beachieved by fusion of the viral envelope with the cell membrane, a state which wasactually seen in some electron micrographs (Fig. 4), or by fusion of a lysosome with aphagocytic vacuole to form a phagosome, which subsequently will let leak into thecytoplasm the products of the disintegration of the virus, such as infective nucleicacid.

If one considers the possibility that the entry of myxoviruses and paramyxo-viruses into their host cells is a reverse of the process of their maturation, then buddingof influenza virus (Wyckoff, 1953) or of NDV (Blough, 1964) through the outer cellmembrane, or budding of encephalitic arboviruses (Ota, 1965) into cell vacuoles, wouldrepresent the reverse of the process of fusion either directly on the cell membrane orinside the cell with the vacuolar membrane.

In the course of infection of cell monolayers by syncytial viruses, such as paramyxo-viruses or herpes virus, the cells fuse into giant multinuclear cells (Okada, 1958, 1962).

Electron microscopy of NDV entry 75

The fusion 'from within' usually starts with the formation of a cytoplasmic bridgebetween the infected and non-infected cell (Roizman, 1962), which then widens so asto permit the movement of nuclei from one cell into the other. This course of eventsmay be thought to be the result of encounter of budding virions with adjacent neigh-bouring cell membranes, which by fusing with the virion envelope would open achannel between the two cells. There is some evidence (Dawson et al. 1965) that theagent in these viruses inducing fusion is an enzyme located in the lipoprotein envelopeof the virus, since various proteolytic and lipolytic agents which selectively destroythis property of the virus may be without effect on other measurable properties, forexample, infectivity (Kohn, 1965; Kohn & Adler, 1966).

One rinding worthy of note in the present work is that polykaryocytes formed byNDV in Lu 106 cells, which in an optical microscope appear to be completely fused,still show in electron micrographs considerable amounts of membranous structuresseparating nuclei of the cell forming the polykaryocyte. The extent of such membranesin virus-induced polykaryocytes is much greater in Lu 106 cells than in FL cells. Someevidence of the persistence of cell membranes in parainfluenza-induced polykaryo-cytes is also brought out in electron micrographs of Harris et al. (1966).

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BLOUGH, H. A. (1964). Role of the surface in the development of myxoviruses. In CellularBiology of Myxovirus Infection (ed. G. E. W. Wolstenholme & J. Knight), pp. 120-143.CIBA Symposia, London: Churchill.

COHEN, A. (1963). Mechanism of cell infection. I. Virus attachment and penetration. InMechanisms of Virus Infection (ed. W. Smith), pp. 135-190. New York: Academic Press.

DALES, S. (1962). An electron microscopic study of the early association between two mammalianviruses and their hosts. J. Cell Biol. 13, 303-322.

DALES, S. (1963). The uptake and development of vaccinia virus in strain L cells followed withlabelled viral DNA. J. Cell Biol. 18, 51-72.

DALES, S. (1965). Penetration of animal viruses into cells. Progr. med. Virol. 7, 1-43.DALES, S. & CHOPPIN, P. W. (1962). Attachment and penetration of influenza virus. Virology

18, 489-491.DAWSON, C. R., EPSTEIN, M. A. & HUMMELER, K. O. (1965). Cytochemical and electron micro-

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HARRIS, H., WATKINS, J. F., FORD, C. E. & SCHOEFL, G. I. (1966). Artificial heterokaryons ofanimal cells from different species. J. Cell Sci. I, 1-30.

HOLMES, K. V. & CHOPPIN, P. W. (1966). On the role of the response of the cell membrane indetermining virus virulence. Contrasting effects of the parainfluenza virus SV 5 in two celltypes. J. exp. Med. 1x4, 501-520.

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76 N. Meiselman, A. Kohn and D. Danon

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KOHN, A. & ADLER, A. (1966). The possible chemical nature of cell-fusion inducing factor(FIF) of Newcastle disease virus. IXth Int. Congr. Microbiol., Moscow, p. 465.

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{Received 7 October 1966)

Journal of Cell Science, Vol. 2, No. 1

Fig. 1. Light-microscopy of NDV-induced fusion. All cells were treated with EDTA(0-002 M) for 2 min before fixation in methanol and staining with Giemsa. 1 A, Lu 106cells, control; IB, Lu 106 cells, fused by NDV, after 3 h. x 125. ic , FL cells, control;1 D, FL cells fused by NDV, 3 h after addition of virus, x 360.Fig. 2. Ferritin-labelled NDV adsorbed to FL cell surface, 2 min after addition ofvirus; v, virion. x 40000.Fig. 3. Ferritin-labelled NDV on microvilli (mi) of FL cells, after 2 min. x 46000.Fig. 4 A, B. Partial fusion of NDV (v) with FL cell membrane, 10 min after additionof virus, x 40000.

N. MEISELMAN, A. KOHN AND D. DANON (Facing p. 76)

Journal of Cell Science, Vol. 2, No. 1

Figs. 5-8. NDV (t>) in surface folds of Lu 106 cell membranes, after 10 min. Figs. 5,7, 8 are x 50000; Fig. 6 x 40000.Fig. 9. NDV (v) in a tightly fitting vacuole in Lu 106 cells. Note double membrane,x 50 000.

Fig. 10. Ferritin-labelled NDV in vacuole in FL cells, after 10 min. x 46000.

N. MEISELMAN, A. KOHN AND D. DANON

Journal of Cell Science, Vol. 2, No. 1

M' • > ' • - •

n "

Figs. 11, 12. Cell membranes in adjacent cells in cell monolayers of Lu 106 treatedwith NDV at high input multiplicity.

Fig. 11. Control, no virus added. Note completeness of separating membrane(m); n, nucleus, x 18400

Fig. 12. LU 106 cells, 3 h after addition of NDV (500 E I D ^ per cell). Noteremnants of cell membranes (m). x 47000.

N. MEISELMAN, A. KOHN AND D. DANON