STUDIES OF MEMBRANE FUSION

Transcription

1 J. Cell Sci. a8, (1977) Printed in Great Britain Company of Biologists Limited 1977 STUDIES OF MEMBRANE FUSION II. FUSION OF HUMAN ERYTHROCYTES BY SENDAI VIRUS S. KNUTTON Department of Biochemistry, St George's Hospital Medical School, Tooting, London SWi-] ore, UK SUMMARY Thin section, negative stain and freeze-fracture electron microscopy have been used to study the fusion of human erythrocytes by Sendai virus. Sendai virus particles are shown to consist of an envelope covered with ~ 12 nm-long projections or spikes which encloses a helical nucleocapsid. The spike complexes are dumbell-shaped and consist of 2 knobs, one ~ 8 nm, the other ~ 14 nm, in diameter connected by a narrow ~ 3 nm-thick stalk, with the larger knob embedded in the lipid bilayer of the viral envelope. Virus particles bind to and agglutinate cells at 4 C; fusion of viral envelopes with the erythrocyte membrane and extensive cell-cell fusion takes place following a brief incubation at 37 C. At 4 C most virus particles are roughly spherical and fractures through the viral envelope reveal concave E fracture faces with ~ 14-nm-diameter intramembrane particles and convex P faces with a complementary arrangement of pits. Incubation at 37 C results in a dramatic change in the structure of the viral envelope of many virus particles. Invaginations of the viral envelope give the virus a convoluted profile and such virus particles are characterized in freeze-fracture replicas by the presence of smooth linear ridges 30 nm wide and up to 05 /tm long on E faces and by a complementary arrangement of linear grooves on P faces. Instead of ~ 14 nm-diameter intramembrane particles on E faces, ~ 8 nm particles are present on both P and E faces. These changes, which only take place when virus particles are bound to erythrocytes, are also accompanied by the disappearance of clearly defined surface spikes. Only virus particles having this altered morphology actually fuse with the erythrocyte membrane. The characteristic 'ridged' morphology of the viral envelope allows specific sites of viral envelope-cell fusion to be identified in freezefracture replicas and successive stages during the incorporation of the viral envelope to be reconstructed. These observations show that viral envelope cell fusion is initiated by a cellmediated temperature-sensitive change in the molecular organization of the viral envelope which allows the particle denuded linear invaginations, possibly lipid bilayer, to interact and fuse with the erythrocyte membrane. Further observations suggest that cell-cell fusion does not occur directly between cells but is achieved by the simultaneous fusion of a virus particle with 2 adjacent erythrocytes. INTRODUCTION The first stage of infection by enveloped paramyxoviruses probably involves fusion of the viral envelope with the cell membrane (Choppin & Compans, 1975). Some enveloped viruses also have cell-cell fusion activity and Sendai virus is the agent most frequently used to promote cell fusion in vitro (Poste, 1972). The fusion of erythrocytes by Sendai virus provides a convenient and relatively simple model system in which to study the mechanism of virus-induced membrane fusion. Several stages of the cell fusion process have been distinguished (Harris, 1970; 13 CEL 28

2 190 S. Knutton Howe & Morgan, 1969; Okada, 1969). Binding of virus to the cell surface and cell agglutination occurs readily at 4 C. Cell surface sialoglycoproteins, which can be correlated with intramembrane particles seen on the P face of fractured erythrocytes, appear to bear the viral receptors (Tillack, Scott & Marchesi, 1972) although membrane gangliosides can also serve as viral receptors (Haywood, 1974). Fusion of viral envelopes with the erythrocyte membrane and cell-cell fusion are temperature dependent and take place following a brief incubation at 37 C. Previous morphological studies have shown direct fusion of viral envelopes with the erythrocyte membrane (Apostolov & Almeida, 1972; Shimizu, Shimizu, Ishida & Homma, 1976) and subsequent diffusion of viral antigens within the plane of the membrane (Bachi, Aguet & Howe, 1972; Okada et al. 1975; Shimizu et al. 1976) but have not shown convincingly whether cell-cell fusion occurs directly between cells or requires an intermediate step involving fusion with a virus particle. It has been proposed that cell-cell fusion is achieved by bridging with interspersed virus particles (Apostolov & Almeida, 1972) although in other studies no virus particles or viral antigens were seen in regions of the cell membrane thought to represent sites of cell-cell fusion (Bachi et al. 1973; Cassone, Caho & Pesce, 1973; Hosaka & Shimizu, 1974)- The freeze-fracture technique, because of the potential for examining extensive areas of the plasma membrane, should be useful in locating specific sites of viral fusion, especially if there are localized regions of altered structure at these sites. It has been proposed that an aggregation of intrinsic membrane proteins is important in membrane fusion (Poste & Allison, 1973) and a clustering of intramembrane particles has been reported to occur during the virally mediated fusion of erythrocytes (Bachi & Howe, 1972; Bachi et al. 1973). In these studies viral antigens seen on the etch face in freeze-etch replicas have been interpreted as representing sites of viral fusion although no evidence for such sites on fracture faces was presented. In an earlier short report (Knutton, 1976) I showed that prior to fusion of the viral envelope with the erythrocyte membrane the virus undergoes a dramatic change in the structural organization of its envelope. In this paper I present new data which demonstrate that specific sites of fusion of viral envelopes with the erythrocyte membrane can be identified in freeze-fracture replicas and that successive stages during the incorporation of viral envelopes into the membrane can be reconstructed. Observations are also presented which suggest that cell-cell fusion is achieved by the simultaneous fusion of a virus particle with 2 adjacent cells. METHODS Cell fusion Fusion of erythrocytes was carried out as described in the preceeding paper (Knutton, Jackson & Ford, 1977), Electron microscopy For freeze-fracture cellsfixedfor 30 min with 3 % glutaraldehyde were washed and infiltrated with 25 % glycerol in Hanks' BSS. Samples were rapidly frozen in freshly melted Freon 22 and fractured at 110 C in a Denton freeze-fracture device. Specimens that were to be etched were

3 Membrane fusion. II 191 frozen in Hanks' BSS without glycerol, fractured at 100 C and etched for up to 2 min, also at 100 C, prior to replication with platinum and carbon. Replicas were cleaned in 5 % sodium hypochlorite, washed in distilled water and examined in a Siemens 101 electron microscope. In freeze-fracture illustrations the encircled arrowhead indicates the direction of shadow. Throughout this paper the recently proposed freeze-etching nomenclature of Branton et al. (1975) is used. For thin sections cells were fixed for 1 h with 3 % glutaraldehyde in 01 M cacodylate buffer ph 74, postfixed for 2 h in 1 % buffered osmium tetroxide and block stained overnight in 2 % aqueous uranyl acetate. Specimens were dehydrated through graded ethanol and propylene oxide solutions and embedded in Epon. Sections were cut with a diamond knife on a Tesla ultramicrotome and stained with uranyl acetate and lead prior to being examined. For negative staining a suspension of virus particles was applied to a grid and negatively stained with 1 % unbuffered uranyl acetate. RESULTS Sendai virus morphology Sendai virus particles harvested following a 72-h infection of embryonated eggs are pleomorphic in both size and shape although most particles are roughly spherical. The majority of particles are nm in diameter although some larger particles up to ~ 600 nm can be seen (Figs. 1-3). Both thin-section (Fig. 1) and negative-stain images (Fig. 2B) reveal a viral envelope, covered with projections or spikes, about 12 nm long, which encloses a helical nucleocapsid. In thin-section images the spikes usually appear as a layer 8 nm thick of fairly uniform electron density separated from the external dense leaflet of the unit membrane structure by a clear space about 4 nm wide (Fig. 1) although the electron-dense layer is sometimes resolved into a row of ~ 8-nm-diameter knobs (Fig. 1, inset). The thin stalks which connect these knobs to the viral envelope are often difficult to resolve in thin-section images (Fig. 1, inset) but the ~ 3-nm-wide stalks are clearly resolved in some negative stain images (Fig. 2 c). Freeze-fractured virus particles reveal internal aspects of their envelope structure. Concave E fracture faces are covered with numerous intramembrane particles ~ 14 nm in diameter while P faces have a complementary arrangement of pits (Fig. 3). The remainder of the fracture face is smooth. There is considerable variability in the numbers of ~ 14-nm intramembrane particles seen on the E face of different fractured virus particles. Interaction of virus particles with the erythrocyte membrane Incubation of erythrocytes with virus at 4 C results in agglutination of the erythrocytes. Freeze-fracture (Fig. 4) and freeze-etch replicas (Fig. 5) reveal virus particles bound to the cell surface. Binding results from interaction of viral spikes with the erythrocyte surface (Fig. 5) and this frequently deforms the erythrocyte causing depressions in the erythrocyte membrane. These are readily seen in freeze-fracture replicas where impressions of the bound virus particles can be seen on both P and E fracture faces (Fig. 4). In some instances the invaginations are such that the fracture plane leaves the erythrocyte membrane and fractures through part of the viral envelope (Fig. 4, arrows). Binding of virus to erythrocytes for 60 min at 4 C does not 13-2

4 192 S. Knutton

5 Membrane fusion. II Fig. 3. Freeze-fracture replica showing virus particles which have been incubated for 20 min. at 37 C C. Fractured virus particles, which are morphologically identical to virus particles at 4 C, reveal concave E fracture faces covered with intramembrane particles ~ 14 nm in. diameter and convex P faces with a complementary arrangement of pits, x 35500; inset, x Figs Morphology of Sendai virus particles, which are pleomorphic in both size and shape. Most particles are roughly spherical and nm in diameter although some up to ~ 600 nm can be seen. Fig. 1. In thin-section images the viral envelope displays a trilamellar unit membrane structure ~ 8 nm thick and encloses a helical nucleocapsid. An ~ 8-nm layer of electron-dense material separated from the outer dense leaflet of the unit membrane structure by a clear space of ~ 4 nm can be seen in most particles and this electron-dense layer is frequently resolved into individual ~ 8 nm diameter particles (inset). A thin layer of electron-dense material is present in many particles immediately underneath the unit membrane structure (arrow), x ; inset, x Fig. 2. A-c. Negatively stained images of Sendai virus particles. Most virus particles are intact and not penetrated by the negative stain and reveal only the viral envelope which is covered with particles (A). In favourable images the envelope particles or spikes are seen to consist of a knob ~ 8 nm in diameter connected to the envelope by a narrow stalk ~ 3 nm wide (c). Ruptured virus particles penetrated by the negative stain reveal the helical nucleocapsid in addition to the viral envelope (B). A, x 92500; B, c, x

6 S. Rnutton 0-5 pan Fig. 4. Erythrocytes agglutinated with Sendai virus for 30 min at 4 C. In freezefracture replicas impressions of the bound virus particles can be seen on both P and E fracture faces. In many instances the fracture plane deviates from the erythrocyte membrane and fractures through part of the viral envelope (arrows and insets). Erythrocyte membrane and viral envelope intramembrane particles remain randomly distributed during virus binding at 4 C. x 20000; upper inset, x 60000; lower inset, x

7 Membrane fusion. II J Fig. 5. Freeze-etch replica showing part of the P fracture face and the outer ES etch face of an agglutinated erythrocyte. Virus particles are bound to the ES face and in some particles (inset) viral spikes bound to the erythrocyte surface can be seen, x 46000; inset, x result in any change in the distribution of intramembrane particles on either P or E erythrocyte or viral envelope fracture face (Fig. 4). Viral envelope-cell fusion The first noticeable change following incubation of the agglutinated cell suspension at 37 C is a change in the morphology of many virus particles. Although many virus particles remain spherical, the envelopes of others lose their clearly denned spikes and become convoluted with numerous infoldings of the membrane (Fig. 6A) and these infoldings are characterized in freeze-fracture replicas by the presence of linear ridges on the E fracture face (Fig. 6B) and complementary linear grooves on the P face (Fig. 6c). Just how the membrane invaginations give rise to the linear ridges is clearly illustrated in Fig. 6B. These linear ridges and grooves, which are ~ 30 nm wide and up to 0-5 /tm long are always smooth and devoid of intramembrane particles although intramembrane particles ~ 9 nm in diameter are seen on non-ridge regions of the fractured viral envelope. These structural changes of the viral envelope are cell-mediated and only take place when virus is bound to cells; free virus which has been incubated and fixed at 37 C retains the morphology typical of virus bound to cells at 4 C (Fig. 3). Sites of viral fusion with the erythrocyte membrane can be readily recognized in

8 :xpl 5. Knutton freeze-fracture replicas because this characteristic 'ridged' morphology of the viral envelope is maintained during fusion with the cell membrane. Figs. 7 A, B, 8 F, 9 c show viral envelopes which have fused and become completely incorporated into the erythrocyte membrane. The presence of viral envelope 'ridges' also allows different stages during the fusion of the viral envelope to be reconstructed. Figs illustrate reconstructed stages during the fusion of viral envelopes with the erythrocyte membrane as seen on both P and E fracture faces, in cross-fractured cells and in thin sections. Fig. 6. Thin section (A) and freeze fracture replicas (B, C) of virus particles bound to erythrocytes at 4 C and incubated for 1 min at 37 C. Many virus particles are no longer spherical but have a convoluted profile. Several invaginations of the viral envelope (A, arrows) can be seen and the envelope spikes are no longer apparent. In freeze-fracture replicas the invaginations are characterized by the presence of smooth linear ridges ~ 30 nm wide and up to 05 /mi long on E faces (B) and complementary grooves on P faces (c). Intramembrane particles ~ 9 nm in diameter are present on both P and E fracture faces (B, C). A, B, C, x , and 35500, respectively. The initial fusion event appears to involve just two of the linear 'ridges' which are the first elements of the viral envelope to fuse and become incorporated into the erythrocyte membrane (Figs. 8A, 9A, IOA, I I A ) although Fig. 8B may represent an instance in which 3 elements of linear ridge fuse simultaneously. It is not clear from Figs. 8 A and 9A whether a cytoplasmic bridge exists between the virus and the erythrocyte but it is apparent from both cross-fractured (Fig. IOA) and thin-section images (Fig. I I A ) that such a cytoplasmic continuity does, in fact, exist at this initial stage of the fusion process. Subsequent stages appear to involve expansion of this initial fusion event with the successive incorporation of an increasing number of the linear 'ridges' and concomitant enlargement of the cytoplasmic bridge until the entire viral envelope becomes incorporated into the erythrocyte membrane (Fig. 7). Internalization of some viral membrane often appears to take place during the fusion process (Fig. I I B, c). During early stages of the fusion process the fracture plane does not travel around the partially fused virus particle and only those parts of the viral envelope which have become incorporated into and coplanar with the erythrocyte membrane are revealed (Figs. 8A-E, 9A, B). Only when the fusion process is complete is the entire

9 Membrane fusion. II 197 B Fig. 7. A, B. Freeze-fracture replicas showing virus particles which have fused and become completely incorporated into the erythrocyte membrane. The presence of the smooth linear grooves on P fracture faces (A) and ridges on E faces (B) allows sites of viral fusion to be recognized in freeze-fracture replicas, A, B, x and 56000, respectively.

10 S. Knutton

11 Membrane fusion. II Figs Selected stages during the fusion of viral envelopes with the erythrocyte membrane as revealed on E fracture faces (Fig. 8), on P fracture faces (Fig. 9), in crossfractured erythrocytes (Figs. 10, 12) and in thin sections (Fig. 11). Initially, 2 elements of smooth viral ' ridge' fuse and become incorporated into the erythrocyte membrane thereby producing a cytoplasmic continuity between the virus and the erythrocyte (Figs. 8 A, 9 A, IOA, I I A). Subsequent stages involve the incorporation of an increasing number of viral ridges and concomitant enlargement of the cytoplasmic bridge (Figs. 8B-E, 9B, IOB, C, I I B, C, 12A, B) until the entire viral envelope has fused and become incorporated into the erythrocyte membrane (Figs. 8 F, 9 c). Figs. 8 F and 9 c are complementary replicas. The arrows indicate the fused viral' ridges'. All x viral envelope revealed on the erythrocyte fracture face (Figs. 7, 8F, 9 c). Stages during the fusion process can also be seen in cross-fractured and thin-sectioned cells and selected stages are illustrated in Figs The presence of viral ridges and grooves and the low density of membrane particles in the viral envelope relative to that of the erythrocyte membrane clearly delineates the viral envelope membrane even when it has become incorporated into the erythrocyte membrane (Fig. 7). Continued incubation at 37 C, however, results in the lateral diffusion of viral 'ridges' within the plane of the membrane away from the initial site of fusion (Figs. 7B, 13B) and diffusion of erythrocyte intramembrane particles into non-ridge regions of the viral envelope (Fig. 13A). Initially there is no diffusion of erythrocyte intramembrane particles into the smooth 'ridge' regions of the viral envelope. This is most clearly seen on the P fracture face where there is a sharp contrast between the smooth linear grooves and the high density of particles lining the grooves (Fig. 9). The fused viral envelopes eventually become indistinguishable from the remainder of the erythrocyte fracture faces as a result of the

12 200 S. Knutton Fig. 13. Freeze-fracture replicas of agglutinated erythrocytes incubated for 10 min (A, B) and 60 min (c) at 37 C C. Lateral diffusion of erythrocyte intramembrane particles into the 'non-ridge' regions of the fused viral envelope (A) and diffusion of elements of smooth linear ridge away from the sites of viral fusion (B) appears to have taken place. After 60 min at 37 C (c) only a very occasional viral ridge is seen (arrow); there is no appreciable aggregation of erythrocyte intramembrane particles. Numerous unfused spherical virus particles can still be seen bound to the now completely haemolysed erythrocytes (c). A, B, C, x 62000, 28000, and 24500, respectively. 13C

13 Membrane fusion. II mmx Fig. 14. Freeze-fracture replica showing the region of close contact between 2 agglutinated erythrocytes. One virus particle has fused and is seen incorporated into the P fracture face of the upper erythrocyte (arrow) and a second virus particle is seen in the initial stages of fusion with the E fracture face of the adjacent erythrocyte (arrowhead), x , disappearance of viral 'ridges' and diffusion of erythrocyte intramembrane particles into the non-ridge regions (Fig. 13 c). Although fusion of viral envelopes with the erythrocyte membrane involves dramatic changes in the structure of the viral envelope no modifications in the organization of the erythrocyte membrane appear to take place and the ~ 9-nm erythrocyte intramembrane particles on both P and E fracture faces are randomly distributed at all stages during virus binding (Fig. 4) and viral envelope-cell fusion (Fig. 7). In some experiments some aggregation of erythrocyte intramembrane particles was seen to take place following a prolonged incubation at 37 C. Cell-cell fusion Incubation of an agglutinated cell suspension at 37 C also results in extensive cell-cell fusion and in as little as 2 min at 37 C many polyerythrocytes have formed. Extensive haemolysis also takes place during cell fusion and after 5-10 min at 37 C

14 202 S. Knutton Figs. 15, 16. Freeze-fracture replicas (Fig. 15) and thin-section images (Fig. 16) showing virus particles {sv) in the process of fusing with 2 adjacent erythrocytes (i, 2) simultaneously. In the freeze-fracture replicas viral ridges can be seen to have fused with the 2 adjacenterythrocyte membranes (arrows). In thin-section images fusing virus particles are identified by the presence of the membrane invaginations (arrowheads) and the circular profiles of cross-sectioned nucleocapsid material (we). Fig. 15 A, x 32500; Fig. i5b, x 82500; Fig. I6A, x 70000; Fig. I6B, x

15 Membrane fusion. II //m 1 "SB 17B Fig. 17. A, B. Freeze-fracture replicas showing later stages of cell-cell fusion. Internalization of some erythrocyte membrane (arrowheads) takes place during the 'unfolding' of the fused membranes which results in the formation of a single spherical polyerythrocyte. A, x 24500; B, x virtually all cells have haemolysed (Fig. 13c). At earlier times (Figs. 6-12, 14-17) there is a mixture of both intact, partially lysed and completely haemolysed cells. Agglutination results from cross-linking of cells by virus particles and incubation at 37 C results in extensive viral envelope-cell fusion. Since there does not appear to be any change in the structure of the erythrocyte membrane during virus-cell fusion but only in the structure of the viral membrane it would seem likely that cell-cell fusion requires an intermediate step involving fusion of a virus particle with two

16 204 Knutton adjacent erythrocytes. Fig. 14 illustrates an example of virus-cell fusion which might possibly lead to cell-cell fusion. Fig. 14 shows part of the P and E fracture faces at a region of close contact between 2 agglutinated erythrocytes. One virus particle has fused and is seen incorporated into the P fracture face of the upper erythrocyte (Fig. 14, arrow) while a second virus is seen at the initial stages of fusion with the adjacent erythrocyte (Fig. 14, arrowhead). Either of these virus-cell fusion events could involve fusion of the virus with both cells simultaneously. En face fractures, however, cannot reveal fusion of a virus particle with 2 adjacent erythrocytes but such fusion events are revealed in replicas of cross-fractured cells (Fig. 15) and in thin sections (Fig. 16). In both instances fusion of single virus particles with 2 adjacent erythrocytes appears to have taken place. Virus particles can be recognized both by the presence of viral 'ridges' (Figs. 15, 16) and in thin sections by the circular profiles of cross-sectioned nucleocapsid material (Fig. I6B). Subsequent events in cell-cell fusion appear to involve cell swelling and an 'unfolding' of the fused membranes so as to produce a single spherical polyerythrocyte. At these later stages of cell-cell fusion during cell swelling the formation of intercellular bridges and internalization of some erythrocyte membrane is frequently seen (Fig. 17). DISCUSSION In this paper I have demonstrated that specific sites of fusion of Sendai virus particles with the erythrocyte membrane can be pin-pointed in freeze-fracture replicas and that successive stages during the incorporation of viral envelopes into the membrane can be reconstructed. Virus-cell fusion requires a specific cell-mediated temperature-sensitive reorganization of the viral envelope and cell-cell fusion appears to require fusion of a virus particle with 2 adjacent erythrocytes simultaneously. No specific reorganization of the erythrocyte membrane appears to be necessary for either virus-cell or cell-cell fusion. A diagrammatic representation of possible stages during the fusion of a Sendai virus particle with the erythrocyte membrane is shown in Fig. 19, p Viral envelope reorganization The thin-section and freeze-fracture observations are consistent with the viral envelope being composed of a lipid bilayer. Fractured virus particles reveal concave E fracture faces with intramembrane particles ~ 14 nm diameter. There is now strong evidence that intramembrane particles represent, at least in part, protein or protein aggregates intercalated into the membrane lipid bilayer (Branton & Deamer, 1972; Hong & Hubbell, 1972). Sendai virus particles possess two membrane glycoproteins which constitute the two different types of envelope spike (Homma, Tozawa, Shimizu & Ishida, 1975; Shimizu, Shimizu, Kohama & Ishida, 1974). The ~ 14 nm E-face particles therefore probably represent portions of one or both types of spike glycoprotein complex embedded in the viral envelope. The combined freeze-fracture and negative-stain images indicate that the viral spikes are dumbell-shaped complexes consisting of a knob ~ 14 nm in diameter embedded in the lipid bilayer and a stalk

17 Membrane fusion. II 205 ~ 3 nm wide and a knob ~ 8 nm in diameter external to the lipid bilayer. Although there are 2 chemically different types of envelope spike in Sendai virus only one type of spike morphology has been observed suggesting that both types are structurally similar. This is supported by the studies of Shimizu et al. (1974), who have isolated and purified the Sendai virus spikes and have shown that both types are dumbellshaped although these workers have described minor morphological differences between the 2 types of spike. The layer of electron-dense material located immediately beneath the unit membrane structure probably represents the viral M protein which is thought to be located on the inside surface of the envelope (Choppin & Compans, 1975). A diagrammatic representation of the proposed viral envelope structure taking into account the freeze-fracture data is shown in Fig. 18. HANA+ Fig. 18. Diagrammatic representation of the molecular organization of the Sendai virus envelope. Both types of envelope spike (HANA+, HANA~) are dumbell-shaped and consist of particles ~ 8 and ~ 14 nm in diameter connected by an ~ 3-nm-wide stalk; the ~ 14 nm diameter particle is embedded in the lipid bilayer core of the membrane. The viral M protein (m) is thought to form a continuous layer of protein bound to the cytoplasmic side of the membrane. HA, haemagglutinin; NA, neuraminidase. Incubation of an agglutinated cell suspension at 37 C results in a dramatic change in the molecular organization of the envelope of many bound virus particles. Virus particles assume a convoluted profile with linear infoldings of the membrane and the characteristic envelope spikes are no longer apparent. In freeze-fracture preparations membrane changes involve a loss of the ~ 14-nm E face particles, the appearance of ~ 9-nm particles on both P and E faces and the formation of smooth linear E face ridges and P face grooves. Although the biochemical changes taking place are not known at present, the morphological observations suggest a complete reorganization of the membrane spike glycoprotein complexes resulting in the formation of specialized regions of the viral envelope devoid of these membrane proteins. These changes in viral envelope structure are both cell-mediated and temperaturesensitive. Binding of virus to cells involves one type of viral spike which bears both haemagglutinin and neuraminidase activities (Shimizu et al. 1974); the second type of viral spike is thought to be involved in the fusogenic properties of the virus (Scheid & Choppin, 1974)- It is not known what triggers the changes in molecular organization of the viral envelope when the temperature is raised to 37 C although the disappear- 14 CEL 2S

18 206 S. Knutton ance of all the 14-nm intramembrane particles and all of the viral spikes indicates that both types of spike complexes are involved in the membrane changes. Virus-cell fusion Only virus particles having the convoluted morphology are capable of fusion with the erythrocyte membrane. If the spherical unconvoluted particles did fuse and become incorporated into the erythrocyte membrane, aggregates of the large ~ 14 run membrane particles should certainly have been detected on the erythrocyte E fracture face. The virus particles which remain spherical and never fuse with the erythrocyte membrane must either be defective in some way or alternatively they may not interact with the erythrocyte membrane in a way that leads to a triggering of the membrane reorganization. The characteristic 'ridged' morphology of the viral envelope allows specific sites of viral fusion to be recognized in freeze-fracture replicas and successive stages during incorporation of the viral envelope to be reconstructed. Although the freeze-fracture observations provide new insights into the process of virus-cell fusion the nature of the membrane fusion mechanism in molecular terms is still unclear. Membrane fusion probably occurs between regions of perturbed lipid bilayer (Ahkong, Fisher, Tampion & Lucy, 1975) and requires that the 2 membranes be brought into close molecular contact so that interaction and fusion can take place. The cell-mediated changes in viral envelope structure appear to represent a viral specialization which has evolved to allow this process of membrane fusion, and thus infection, to take place efficiently. The smooth membrane 'ridges', which appear to be the first elements of the viral envelope to fuse and become incorporated into the erythrocyte membrane, probably represent areas of protein-denuded lipid bilayer (Deamer & Branton, 1967). Although no gross changes in the organization of the erythrocyte membrane have been detected during virus-cell fusion, specific localized changes actually at the site of fusion might give rise to complementary regions of particle-denuded erythrocyte membrane. Unfortunately such changes would be difficult to detect because the initial fusion event has already taken place by the time the sites of viral fusion can be recognized in freeze-fracture replicas. The close molecular contact of the 2 adjacent membranes necessary for fusion requires that the repulsive electrostatic forces which tend to keep the membranes apart be overcome. The removal of negatively charged sialic acid residues close to the site of fusion by the viral neuraminidase would be expected to reduce the local electrostatic repulsion. Furthermore, calculations by Poste (1972) have shown that close contact of membranes would be favoured by a membrane surface with a low (< o-i /tm) radius of curvature; the viral envelope invaginations provide just such a membrane surface. Following the initial fusion event in which 2 segments of linear ridge fuse with the erythrocyte membrane, successive stages lead to the complete incorporation of the viral envelope into the erythrocyte membrane. Viral fusion is known to cause temporary damage to the permeability barrier of the cell membrane (Okada et al. 1975; Pasternak & Micklem, 1974) and the resultant osmotic swelling which takes place (Knutton et al. 1977) might be expected to provide further if not a major impetus for

19 Membrane fusion. II 207 completion of the fusion process which results in complete incorporation of the viral envelope into the erythrocyte membrane. Although it cannot be proved that the reconstructed stages of virus-cell fusion illustrated in Fig. 8A-F are, indeed, a temporal sequence, they do clearly represent definite stages during the fusion of individual virus particles and do constitute what appears to be a logical sequence of events which would lead to the complete incorporation of the viral envelope into the erythrocyte membrane. These proposed stages during virus-cell fusion, are summarized diagrammatically in Fig. 19. Fig. 19. Diagrammatic representation of stages during the fusion of a Sendai virus particle with the erythrocyte membrane, A, at 4 C viral envelope spikes bind to receptors on the erythrocyte membrane (em); nc, nucleocapsid. B, warming to 37 C C results in a rearrangement of the envelope spike complexes and the formation of linear invaginations (») of the viral envelope devoid of membrane proteins. C, the initial fusion event involves fusion of 2 of the smooth linear invaginations (i) with the erythrocyte membrane creating a cytoplasmic bridge between erythrocyte and virus. Fusion and incorporation of increasing numbers of the membrane invaginations enlarges the cytoplasmic bridge (D) until the entire viral envelope (ve) has fused and become incorporated into the erythrocyte membrane. E, continued incubation at 37 C results in lateral diffusion of viral envelope components within the plane of the erythrocyte membrane (F). The 'ridged' membrane morphology which is essential for fusion is temporarily maintained following fusion. The smooth 'ridges' appear to diffuse away from the original sites of fusion but these eventually disappear; erythrocyte intramembrane 14-2

20 208 S. Knutton particles also diffuse into the non-ridge regions of the viral envelope so that after 30 min at 37 C, regions of fused viral envelope become indistinguishable from the remainder of the erythrocyte fracture face. These observations are consistent with other studies which have shown that viral antigens, presumably the envelope spike glycoproteins, undergo lateral diffusion within the plane of the membrane following fusion (Bachi et al. 1973; Okada et al. 1975; Shimizu et al. 1976). Poste & Allison (1973) have suggested that an aggregation of intramembrane particles is important in membrane fusion. Bachi et al. (1973) have reported that a virus-induced aggregation of erythrocyte intramembrane particles takes place during cell fusion although these authors only show intramembrane particle aggregation after 10 min at 37 C. Since extensive virus-cell and cell-cell fusion takes place during the first few minutes of incubation at 37 C when there is no detectable aggregation of intramembrane particles, an aggregation of intramembrane particles does not appear to be an essential condition for membrane fusion. Elgsaeter & Branton (1974) have shown that removal of sialic acid from intact erythrocytes enhances intramembrane particle aggregation in ghosts made from such cells. Virus-induced intramembrane particle aggregation, therefore, may be the combined result of the action of the viral neuraminidase and erythrocyte haemolysis. The extensive particle aggregation observed by Bachi et al. (1973) is possibly a reflexion of the high virus:cell ratio used by these workers. The freeze-fracture technique has revealed membrane specializations in other membrane systems where membrane fusion occurs (Satir, Schooley & Satir, 1973). During mucocyst discharge in Tetrahymena specializations of both the plasma membrane and of the mucocyst membrane allow the co-ordinated process of membrane fusion and mucous secretion to take place. These membrane specializations differ markedly from those seen during fusion of Sendai virus although both types of membrane specialization presumably represent different systems which have evolved to allow the interaction and fusion of 2 membranes. It is not surprising, however, that the membrane specializations reported here which result in viral infection appear to be specializations of the viral envelope only. Cell-cell fusion Extensive cell-cell fusion occurs under the same conditions as for virus-cell fusion. Instances in which virus particles are seen fusing with 2 adjacent erythrocytes are frequent enough following the incubation of an agglutinated cell suspension for up to 2 min at 37 C to suggest strongly that such cases of viral fusion do, indeed, initiate cell-cell fusion events. The possibility that direct cell-cell fusion also occurs, however, cannot be ruled out although no instances of direct fusion of adjacent erythrocytes has been observed in these studies. Temporary damage to the cells permeability barrier (Okada et al. 1975; Pasternak & Micklem, 1974) and the resultant osmotic swelling (Knutton et al. 1977) is probably the main driving force which results in an 'unfolding' of the fused membranes so as to produce large polyerythrocytes. Internalization of surface membrane which may include the fused viral envelope and the formation of intercellular bridges are frequently seen in partially fused cells and appear to occur as a result of osmotic swelling. Cytoplasmic bridges of the type

21 Membrane fusion. II 209 illustrated in Fig. 17 have been considered to represent sites of cell-cell fusion and an absence of virus or viral antigens at such sites has been interpreted as evidence for direct cell-cell fusion (Bachi et al. 1973). If, as this and other studies (Apostolov & Almeida, 1972) suggest, cell-cell fusion is initiated by viral fusion then extensive cytoplasmic bridge formation occurs at a relatively late stage when cell-cell fusion is virtually complete. The absence of labelled viral antigens at such sites, therefore, is not surprising. The author is grateful to Mrs Diane Jackson for expert technical assistance and to the Cancer Research Campaign forfinancialassistance. REFERENCES AHKONG, Q. F., FISHER, D., TAMPION, W. & LUCY, J. A. (1975). Mechanisms of cell fusion. Nature, Lond. 253, APOSTOLOV, K. & ALMEIDA, J. D. (1972). Interaction of Sendai (HVJ) virus with human erythrocytes: a morphological study of haemolysis cell fusion. J. gen. Virol. 15, BACHI, T., AGUET, M. & HOWE, C. (1973). Fusion of erythrocytes by Sendai virus studied by immuno-freeze-etching. J. Virol. 11, BACHI, T. & HOWE, C. (1972). Fusion of erythrocytes by Sendai virus studied by electron microscopy. Proc. Soc. exp. Biol. Med. 141, BRANTON, D., BULLIVANT, S., GILULA, N. B., KARNOVSKY, M. J., MOOR, H., MUHLETHALER, D., NORTHCOTE, D. H., PACKER, L., SATIR, B., SATIR, P., SPETH, V., STAEHELIN, L. A., STEERE, R. L. & WEINSTEIN, R. S. (1975). Freeze-etching nomenclature. Science, N.Y. 190, BRANTON, D. & DEAMER, D. W. (1972). Membrane structure. Protoplasntatologia II/E/i: CASSONE, A., CAHO, R. & PESCE, C. D. (1973). Interaction of Sendai virus with human erythrocytes. II. The fusion reactions. Boll. Inst. Sieroter. Milan 52, CHOPPIN, P. W. & COMPANS, R. W. (1975). Reproduction of paramyxoviruses. In Compreliensive Virology, vol. 4 (ed. J. Fraenkel-Conrat & R. R. Wagner), pp New York and London: Plenum Press. DEAMER, D. W. & BRANTON, D. (1967). Fracture planes in an ice-bilayer model membrane system. Science, N.Y. 158, ELGSAETER, A. & BRANTON, D. (1974). Intramembrane particle aggregation in erythrocyte ghosts. I. The effects of protein removal. J. Cell Biol. 63, HARRIS, H. (1970). Cell Fusion. Oxford: Clarendon Press. HAYWOOD, A. M. (1974). Characteristics of Sendai virus receptors in a model membrane. J. molec. Biol. 83, HOMMA, M., SHIMIZU, K., SHIMIZU, Y. K. & ISHIDA, N. (1976). On the study of Sendai virus hemoly8is. I. Complete Sendai virus lacking hemolytic activity. Virology 71, HONG, K. & HUBBELL, W. L. (1972). Preparation and properties of phospholipid bilayers containing rhodopsin. Proc. natn. Acad. Sci. U.S.A. 69, HOSAKA, Y. & SHIMIZU, Y. K. (1974). Cell fusion by the antigen-antibody complex of Sendai virus studied by electron microscopy. J. gen. Virol. 24, HOWE, C. & MORGAN, C. (1969). Interactions between Sendai virus and human erythrocytes. J. Virol. 3, KNUTTON, S. (1976). Changes in viral envelope structure preceding infection. Nature, Lond. 264, KNUTTON, S., JACKSON, D. & FORD, M. (1977). Studies of membrane fusion. I. Paramyxovirus-induced cell fusion. A scanning electron-microscope study. J. Cell Sci. 28, OKADA, Y. (1969). Factors in fusion of cells by HVJ. Curr. Top. Microbiol. Immun. 48, OKADA, Y., KOSEKI, I., KIM, J., MAEDA, Y., HASIMOTO, T., KANNO, Y. & MATSUI, Y. (1975). Modification of cell membranes with viral envelopes during fusion of cells with HVJ (Sendai virus). Expl Cell Res. 93,

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