- Email: [email protected]
Fusion between cell membrane and liposomes containing the glycoproteins of influenza virus
VIROLOGY
104, DL-302
(1980)
Fusion between Cell Membrane and Liposomes Containing Glycoproteins of Influenza Virus R. T. C. HUANG,’ K. WAHN, H. -D. KLENK, institutor
ViroEo&
AND
the
R. ROTT
Justus-L~b~g-U~iv~rs~t~t Giessen, 6900 Giessen, Germany Accepted March 4, 1980
Glycoproteins of influenza virus strains were incorporated into liposomes by a dialysis procedure, using octylglucoside as detergent. Liposomes containing either the cleaved or uncteaved hemagglutinin of the virus were tested for fusion activity with cellutar membranes. Electron microscopic examination as well as microinjection studies revealed that liposomes containing the cleaved hemagglutinin could fuse with cell membranes. In contrast, liposomes containing the uncleaved hemagglutinin were merely adsorbed to the cell surface and fusion occurred only after treatment with trypsin. Native virus particles with the cleaved hemagglutinin could be shown to fuse with liposomes containing cellular receptors of influenza virus. From these results and the known correlation existing between cleavage of hemagglutinin and infectivity of influenza virus, it is suggested that fusion may be an important step in penetration of the nueleocapsid of influenza virus into host cells.
Recently, we succeeded in artificially incorporating glycoproteins of influenza Recent studies revealed that influenza virus into the membrane of liposomes viruses, whether possessing cleaved or un- (Huang et al., 1979). These liposomes procleaved hemagglutinin (HA), were capable vided a system to test the fusion property of adsorbing to cells, but only those con- of viral membranes. We report here the taining the cleaved HA were infectious use of this system to demonstrate the (Klenk et al., 1975, 1977; Lazarowitz and fusion activity of influenza virus. Choppin, 1975; Bosch et ab., 1979). On the basis of these results it was suggested that MATERIALS AND METHODS the HA of influenza virus in the proteolytically cleaved form might contain a Viruses. Influenza virus strains, virus N function which mediated penetration of viral (HavZNeql), fowl plague virus (Restock nucleocapsids into cells. Subsequent studies HavlNl), and A/Victoria/375 (H3N2) were employing physical measurements (Nicolau used. The viruses were grown in chick et ccl., 1978) demonstrated that only those embryos or chick fibroblasts and pu~fied virions containing the cleaved HA could as described before (Chucholowius and alter the fluidity of cell membranes, Experi- Rott, 1972). ments using T-cell-mediated cytotoxicity Viral glycoproteins. Viral glycoproteins tests (Kurrle et al., 1979) further indicated were selectively solubilized by octylglucoside that only those virions with the cleaved (Huang et ah, 1979). Cleaved HA was HA were apparently integrated into the obtained from infectious virions grown in membrane of target cells. The above results chick embryos and uncleaved HA was obpointed to the possibility that the mem- tained from virions grown in chick fibrobrane fusion was an important step for blasts (Klenk et al., 1975). infection. Viral lipids. Viral concentrates containing about 60,000 hemagglutinating units/ml ’ To whom reprint requests should be addressed. were dialyzed against tap water and INTRODUCTION
0042-6822/80/100294-09$02.00/O Copyright All rights
0 1980 by Academic Press, Inc. of reproduction in any form reserved.
294
FUSIONOFRECONSTITUTEDINFLUENZAVIRUSMEMBRANES
lyophilized. The powder obtained was extracted twice with lo-ml portions of chloroform/methanol (2/l). The extracts were pooled and the organic solvents were removed in a rotary evaporator. The lipids which remained were weighed and dissolved in a solution of 20% octylglucoside, the final concentration of lipids in the solution being adjusted to 5%. Cellular lipids. One to two milliliters of packed tissue culture cells were dialyzed in tap water and lyophilized. Cellular lipids were extracted with chloroform/methanol (2/l) as described for viral lipids and dissolved in a solution of 20% octylglucoside. Dansylcerebroside. Dansylcerebroside was used as fluorescent membrane marker to visualize liposomes (Huang et al., 1979). The compound was synthesized as described previously (Huang, 1976). Preparation of viral glycoproteins.
Ziposomes
containing
Liposomes containing the glycoproteins of virus strains were prepared as described previously (Huang et al., 1979). For uv visualization of liposomes, viral lipid was supplemented with 2-5% dansylcerebroside. For microinjection studies, intensive fluorescence could be entrapped in liposomes when 2 mg of fluorescein isothiocyanate or 20 mg of fluorescein isothiocyanate-dextran was included per 10 mg of total lipid present in the mixture before dialysis. Fluorescein isothiocyanate and fluorescein isothiocyanate-dextran (MW 20,000, FD-20) were purchased from Sigma. Trypsinixation of Ziposomes . Proteolytic cleavage of liposomes containing the uncleaved HA was achieved by treating liposomes with trypsin (10 pg/ml) for 15 min at 37”. The liposomes were then freed of trypsin by centrifugation at 16,000 rpm for 1 hr. The pellet was resuspended in phosphate-buffered saline (PBS).
295
and liposomes were prepared by the dialysis procedure in the same manner as described for liposomes coated with viral glycoproteins. Interaction of viral glycoprotein-coated Ziposomes with tissue culture cells. Mono-
layers of tissue culture cells were dissociated with EDTA-phosphate buffer containing 0.2% trypsin as usual and washed three times with PBS. Interaction of liposomes with cells was initiated by mixing liposome suspensions containing about 250 hemagglutinating units with approximately lo6 dissociated cells suspended in 2 ml of PBS. The reaction was allowed to continue for various lengths of time at room temperature or at 37”. For reasons stated under Results, a final standard reaction period of 30 min at room temperature was adapted. Microinjection. Approximately lo6 dissociated chick embryo fibroblasts were suspended in 1 ml of PBS and mixed with liposomes possessing about 250 hemagglutinating units and containing either fluorescein isothiocyanate or fluoresceinlabeled dextran. The mixture was observed under an uv microscope for 1 hr at 15-min intervals. Occurrence of microinjection was characterized by the appearance of orange yellow fluorescence in the cytoplasm. Interaction of liposomes containing myxovirus receptors with influenza virus. One-
tenth milliliter of a milky suspension of cellular receptor-coated liposomes was mixed with two drops of a virus concentrate containing about 3000 hemagglutinating units and allowed to react for 30 min at room temperature. The sample was then negatively stained for electron microscopy. Electron microscopy. For negative contrasting, samples were stained with 2% uranyl acetate (pH 4.5) or 2% phosphotungstate (pH 7). For observation of Preparation of Ziposomes containing the thin sections, samples in suspension were cellular receptors of in$luenxa virus. About fixed with 1.25% glutaraldehyde, post0.5 ml of packed chick embryo fibroblasts fixed with 1% osmium tetroxide, dehywas solubilized in 2 ml of a 10% solution drated, and embedded in epoxy resin of octylglucoside and centrifuged at 16,000 (Luft, 1961). Thin sections were stained rpm for 1 hr in a Sorvall centrifuge, using with 2% uranyl acetate and Reynolds the SS-34 rotor. Twenty milligrams of total lead (Reynolds, 1963) in the conventional cellular lipids were added to the supernatant manner.
HUANGETAL.
296 RESULTS
whole length of contact. Fusion was not observed even in cases where several liposomes were seen to be adsorbed to the cell (Fig. 2a). In previous work it was reported that Glycoproteins were solubilized from in- trypsin activation of noninfectious virions fluenza virus strains grown in different was associated with transformation of HA hosts. With all virus strains used, HA was from uncleaved to cleaved forms (Klenk present in the cleaved form when viruses et at., 1975, 1977; Lazarowitz and Choppin, were grown in chick embryos. To obtain 1975; Bosch et al., 1979). We therefore uncleaved HA, virus N was grown in chick treated liposomes containing the uncleaved HA of virus N with trypsin in the same fibroblasts. Incorporation of glycoproteins into liposomes was carried out as described manner as for activation of virions to see previously (Huang et al., 1979). Liposomes whether fusion activity could then be obprepared by this method contained HA tained with these liposomes. Figures 3a-c as well as neuraminidase in their mem- show that these liposomes indeed acquired branes. In all cases, whether cleaved or the full activity of fusion after treatment uncleaved HA was used, the protein to lipid with trypsin. Liposomes prepared in the absence of ratio of liposomes was approximately 0.1, a ratio usually obtained when the same viral glycoproteins did not adsorb to cells method of reconstitution was used (Huang and showed no fusion capacity. et al., 1979). Studies It was found by electron microscopic Microinjection observation that fusion occurred rapidly In addition to electron microscopic obbetween dissociated chick embryo fibro- servation, we also obtained another eviblasts and liposomes containing the cleaved dence confirming the fusion activity of HA of any influenza virus strains used. influenza virus by microinjeetion experiWithin 15 min after reaction of cells with ments. Fluorescence substances, such as liposomes at room temperature, different fluorescein isothiocyanate or fluoresceinstages of fusion initiated at areas of con- labeled dextran, were trapped in liposomes tact could be detected (Figs. la, b). Arrows containing the cleaved HA. After mixing indicate positions where plasma membrane these liposomes with chick fibroblasts, the and liposomal membrane fused. When the progress of injection was monitored microreaction was continued for 30 min at room scopically by watching the d~fusion of temperature, most of the liposomes could fluorescence into the cytoplasm of cells, be seen to fuse with cells (Figs. lc, d). If which was characterized by the appearance the temperature was raised to 37” during of orange-yellow fluorescence in the cytoreaction of cells with liposomes, inter- plasm. Injection was usually detectable mediate stages of fusion were no longer within a few minutes at room temperature detected, presumably because fusion had and became intensified during a 1-hr realready gone to completion at this tempera- action period (Fig. 4a). Under the same ture. In subsequent experiments, the re- conditions, almost no fluorescence was deaction period of 30 min at room tempera- tected in the cytoplasm when liposomes ture was therefore adopted as the standard containing the uncleaved HA of virus N condition for electron microscopic observa- were reacted with cells (Fig. 4b). Trypsin tion of fusion. treatment of liposomes with the uncleaved No fusion was seen when cells and HA visibly enhanced the intensity of liposomes with the uncleaved HA were fluorescence injected into the cytoplasm, reacted under the same condition. Figures again confirming the importance of pro2a-c show that liposomes containing un- teolytic cleavage for membrane fusion. cleaved HA could be tightly adsorbed to cells. In most cases areas of contact be- ~nter~~t~a~ of ~~~~~e~~~ Vims with Liposwnes ~o~~ai~~ng cellular Receptors tween liposomes and cells were rather extended, but the membranes of liposomes Since the above evidence of fusion was and cells were clearly separate along the obtained in a reconstituted viral mem-
FUSION
a
OF RECONSTITUTED
INFLUENZA
VIRUS
MEMBRANES
b
FIG. I.. Fusion of chick embryo fibroblasts with liposomes containing the cleaved HA of virus N. (a, b) Fusion after 15-min reaction at room temperature. (c, d) Fusion after 30-min reaction at room temperature. Bars represent 0.25 pm. Arrows indicate positions of fusion
297
298
HUANG
ET AL.
FIG. 2. Absence of fusion between chick embryo fibroblasts and liposomes containing the uncleaved HA of virus N. Liposomes were reacted with cells for 30 min at room temperature. (a) Liposomes adsorbing to chick fibroblasts, but resulting in no fusion. (b, c) Tightly associated membranes of iiposomes and cells without fusion. Bars represent, 1 pm in a and 0.5 pm in b and c.
FUSION
OF RECONSTITUTED
INFLUENZA
VIRUS
MEMBRANES
b
FIG. 3. Trypsin activation of fusion between chick embryo fibroblasts and liposomes containing the uncleaved hemagglutinin of virus N. Liposomes containing the uncleaved hemagglutinin of virus N were treated with trypsin (see Materials and Methods section) and reacted with cells for 30 min at room temperature. (a-e) show that these liposomes acquired fusion activity after treatment with trypsin. Bars represent 0.5 pm. Arrows indicate fusion.
299
300
HUANG ET AL.
FIG. 4. Interaction of fluorescein-dextran-loaded liposomes with chick embryo fibroblasts. Fluorescein-dextran-loaded liposomes containing viral glycoproteins were reacted with cells for 30 min at room temperature. (a) Liposomes containing the cleaved HA injected fluoresceindextran into cells. (b) Liposomes containing the uncleaved HA did not inject fluorescein-dextran into cells.
FUSION
OF RECONSTITUTED
INFLUENZA
VIRUS
MEMBRANES
301
influenza virus. The concept is in agreement with earlier electron microscopic observation by Morgan and Rose (1968) and Hoyle et al. (1962), who also suggested a fusion process for the entry of influenza virus into the cell. It seems to be in contrast, however, to the concept of viropexis (Fazekas de St. Groth, 1948; Dales and Choppin, 1962; Dourmashkin and Tyrrell, 1974) as an alternative model of influenza virus penetration according to which intact particles were thought to be taken up by engulfment into the cell. However, if this were the regular mode of penetration, the inner viral components would still be surrounded by the viral envelope and the membrane of cytoplasmic vesicles. Therefore, the fusion process described here would seem to explain more plausibly how the viral nucleocapsid could find its way into the cytoplasm. The reason why fusion between viral enDISCUSSION velope and cellular membranes has been Electron microscopic studies showed that difficult to demonstrate electron microliposomes containing cleaved HA could fuse scopically might be that it takes place very with cell membranes. Liposomes with un- rapidly under physiological conditions. In cleaved HA were adsorbed on the cell sur- the studies described in this paper, emface, but fusion occurred only after in vitro ploying relatively large liposomes, fusion treatment with trypsin. This membrane was completed within 30 min even at room interaction was followed by injection of temperature. liposomal contents into the cytoplasm, as There is no doubt that proteolytic shown by staining of cytoplasm with cleavage of HA is a prerequisite for the fluorescent substances incorporated into fusion activity of influenza viruses. It the liposomes. Since liposomes can be seems possible that the hydrophobic segconsidered as an artificial system, it was of ment of HA2, which becomes exposed by interest to see whether the native virus the proteolytic cleavage of HA, is mediatcould also fuse with membranes. From ing fusion of the two tightly associated studies using virus particles and liposomes membranes. The situation here looks with cellular receptor of influenza viruses, very much like that existing in parait could be demonstrated that the envelopes myxoviruses, where the expression of of intact virions were inserted into the fusion activity is associated with proteoliposomal membrane. Such liposomes were lytic cleavage of Fo into two F glycoproagain able to fuse with the membrane of teins (Homma and Ohushi, 1973; Scheid and tissue culture cells. Hence, it could be Choppin, 1974; Nagai et al., 1976). The inferred that an event equivalent to virus common biological role of the HA of penetration had occurred. influenza viruses and F glycoprotein of These results together with data ob- paramyxoviruses has become even more tained from physical measurements (Nicolau apparent by the recent findings of Gething et al., 1978), T-cell-mediated cytotoxicity et al. (1978) that HA2 of influenza viruses tests (Kurrle et al., 1979), and the correla- and Fl glycoprotein of paramyxoviruses tion existing between infectivity and possess an N-terminal structure, very cleavage of HA (Klenk et al., 1975; similar in its amino acid sequence and its Lazarowitz and Choppin, 1975) indicate extreme hydrophobicity. It can be concluded from above results that fusion of viral envelope with cellular membrane is involved in the penetration of that influenza viruses and paramyxoviruses brane and cell system, another system was further designed in which we mixed virions with liposomes containing the cellular receptors of influenza virus to see whether native virions also possess fusion activity. For this purpose, chick fibroblasts were solubilized with octylglucoside and the soluble components, which contained receptors of influenza virus, were incorporated into liposomes as described in the previous section. It was found that such liposomes were aggregated immediately by influenza viruses, indicating the presence of virus receptors in their membranes. Electron microscopic observation showed that viral spikes had become incorporated into liposomal membrane (not shown). Such liposomes resulting from interaction of vii-ions and liposomes could in turn fuse with cells in a similar way as shown in Figs. 1 and 3 for reconstituted viral membrane.
HUANG ET AL.
302
may initiate infection by a common mecha- HOMMA, M., and OHUCHI, M. (1973). Trypsin action on the growth of Sendai virus in tissue culture nism, namely fusion of viral envelope and cells. III. Structural differences of Sendai viruses cellular membranes. It is, however, not grown in eggs and tissue culture cells. clear why influenza viruses do not cause HOYLE, L., HORNE, R. W., and WATERSON, A. P. fusion of cells as is known to be a property (1962). The structure and composition of the of paramyxoviruses. It seems possible myxoviruses. III. The interaction of influenza that enveloped viruses in general, including virus particles with cytoplasmic particles derived those not hitherto known to be fusogenic, from normal chorioallantoic membrane cells. Virology 17, 533-542. may fuse with their host cells. The presently described liposome system would HUANG, R. T. C. (1976) Labeling of animal cells with fluorescent dansylcerebroside. Z. Naturforsch. seem to provide a useful tool for the study 31c, 737-740. of such processes. ACKNOWLEDGMENTS This work was supported by the Sonderforschungsbereich 47 of the Deutsche Forschungsgemeinschaft. The technical assistance of Miss Heidrun Will is greatly appreciated. Note added in proof. In a recent study (H. U. Koszinowski, H. Allen, M. I. Gething, M. D. Waterfield, and H. -D. Klenk, 1980, J. Exp. Med. 151, 945958) it has been reported that reconstituted influenza envelopes containing cleaved hemagglutinin could fuse only in the presence of Sendai virus. It should be pointed out that these liposomes were prepared by a different procedure, and that they showed distinct differences in size and lipid composition if compared to the liposomes analyzed in the present study. This may indicate that structure and composition of the liposomes are also important for fusing capacity.
REFERENCES BOSCH, F. X., ORLICH, M., KLENK, H. -D., and ROTT, R. (19’79).The structure ofthe hemagglutinin, a determinant for the pathogenicity of influenza virus. Virology 95, 197-207. CHUCHOLOWIUS,H. -W., and ROTT, R. (1972). A new method for purification of myxoviruses by zonal centrifugation with two different sucrose density gradients. Proc. Sot. Exp. Biol. Med. 140, 245247.
DALES, S., and CHOPPIN, P. W. (1962). Attachment and penetration of influenza virus. Virology 18, 489-493.
DOURMASHKIN,R. R., and TYRRELL, D. A. (1974). Electron microscopic observation on the entry of influenza virus into susceptible cells. J. Gen. Viral. 24, 129-141. FAZEKAS DE ST. GROTH, S. (1948). Viropexis, the mechanism of virus infection. Nature (London) 162, 294-295.
GETHING, M. J., WHITE, J. M., and WATERFIELD, M. D. (1978). Purification of the fusion protein of Sendai virus. Analysis of the NH,-terminal sequence generated during precursor activation. Proc. Nat. Acad. Sci. 1JSA 75. 2737-2740.
HUANG, R. T. C., WAHN, K., KLENK, H. -D., and ROTT, R. (1979). Association of the envelope glycoproteins of influenza virus with liposomesModel study on viral envelope assembly. Virology 97, 212-217.
KLENK, H. -D., ROTT, R., and ORLICH, M. (1977). Further studies on the activation of influenza virus by proteolytic cleavage of the hemagglutinin. J. Gen. Viral. 36, 151-161. KLENK, H. -D., ROTT, R., ORLICH, M., and BL~DORN,J. (1975). Activation of influenza viruses by trypsin treatment. Virology 68, 426-429. KURRLE, R., WAGNER, H., ROLLINGHOFF,M., and ROTT, R. (1979). Influenza virus-specific T cellmediated cytotoxicity: Integration of the virus antigen into the target cell membrane is essential for target cell formation. Eur. J. Zmmunol. 9, 107-111. LAZAROWITZ, S. G., and CHOPPIN, P. W. (1975). Enhancement of the infectivity of influenza A and B viruses by proteolytic cleavage of the hemagglutinin polypeptide. Virology 68, 440-454. LUFT, J. H. (1961). Improvement in expoxy resin embedding methods. J. Biophys. Biochem. Cytol. 9, 409-414. MORGAN, C., and ROSE, H. M. (1968). Structure and development of viruses as observed in the electron microscope. VIII. Entry of influenza virus. J. Viral.
2, 925-936.
NAGAI, Y., KLENK, H. -D., and ROTT, R. (1976). Proteolytic cleavage of the viral glycoproteins and its significance for the virulence of Newcastle disease virus. Virology 72, 494-508. NICOLAU, C., KLENK, H. -D., REIMAN, A., HILDEBRAND, K., and BAUER, H. (1979). Molecular events during the interaction of envelopes of myxo- and RNA-tumor viruses with cell membranes. A 270 MHz ‘H nuclear magnetic resonance study. Biochim. Biophys. Acta 511, 83-92. REYNOLDS, E. S. (1963). The use of lead citrate at high pH as an electronopaque stain in electron microscopy. J. Cell Biol. 17, 208-212. SCHEID, A., and CHOPPIN, P. W. (1974). Identification of biological activities of paramyxovirus glycoproteins. Activation of cell fusion, hemolysis and infectivity by proteolytic cleavage of an inactive precursor protein of Sendai virus. Virology 57, 475-490.
104, DL-302
(1980)
Fusion between Cell Membrane and Liposomes Containing Glycoproteins of Influenza Virus R. T. C. HUANG,’ K. WAHN, H. -D. KLENK, institutor
ViroEo&
AND
the
R. ROTT
Justus-L~b~g-U~iv~rs~t~t Giessen, 6900 Giessen, Germany Accepted March 4, 1980
Glycoproteins of influenza virus strains were incorporated into liposomes by a dialysis procedure, using octylglucoside as detergent. Liposomes containing either the cleaved or uncteaved hemagglutinin of the virus were tested for fusion activity with cellutar membranes. Electron microscopic examination as well as microinjection studies revealed that liposomes containing the cleaved hemagglutinin could fuse with cell membranes. In contrast, liposomes containing the uncleaved hemagglutinin were merely adsorbed to the cell surface and fusion occurred only after treatment with trypsin. Native virus particles with the cleaved hemagglutinin could be shown to fuse with liposomes containing cellular receptors of influenza virus. From these results and the known correlation existing between cleavage of hemagglutinin and infectivity of influenza virus, it is suggested that fusion may be an important step in penetration of the nueleocapsid of influenza virus into host cells.
Recently, we succeeded in artificially incorporating glycoproteins of influenza Recent studies revealed that influenza virus into the membrane of liposomes viruses, whether possessing cleaved or un- (Huang et al., 1979). These liposomes procleaved hemagglutinin (HA), were capable vided a system to test the fusion property of adsorbing to cells, but only those con- of viral membranes. We report here the taining the cleaved HA were infectious use of this system to demonstrate the (Klenk et al., 1975, 1977; Lazarowitz and fusion activity of influenza virus. Choppin, 1975; Bosch et ab., 1979). On the basis of these results it was suggested that MATERIALS AND METHODS the HA of influenza virus in the proteolytically cleaved form might contain a Viruses. Influenza virus strains, virus N function which mediated penetration of viral (HavZNeql), fowl plague virus (Restock nucleocapsids into cells. Subsequent studies HavlNl), and A/Victoria/375 (H3N2) were employing physical measurements (Nicolau used. The viruses were grown in chick et ccl., 1978) demonstrated that only those embryos or chick fibroblasts and pu~fied virions containing the cleaved HA could as described before (Chucholowius and alter the fluidity of cell membranes, Experi- Rott, 1972). ments using T-cell-mediated cytotoxicity Viral glycoproteins. Viral glycoproteins tests (Kurrle et al., 1979) further indicated were selectively solubilized by octylglucoside that only those virions with the cleaved (Huang et ah, 1979). Cleaved HA was HA were apparently integrated into the obtained from infectious virions grown in membrane of target cells. The above results chick embryos and uncleaved HA was obpointed to the possibility that the mem- tained from virions grown in chick fibrobrane fusion was an important step for blasts (Klenk et al., 1975). infection. Viral lipids. Viral concentrates containing about 60,000 hemagglutinating units/ml ’ To whom reprint requests should be addressed. were dialyzed against tap water and INTRODUCTION
0042-6822/80/100294-09$02.00/O Copyright All rights
0 1980 by Academic Press, Inc. of reproduction in any form reserved.
294
FUSIONOFRECONSTITUTEDINFLUENZAVIRUSMEMBRANES
lyophilized. The powder obtained was extracted twice with lo-ml portions of chloroform/methanol (2/l). The extracts were pooled and the organic solvents were removed in a rotary evaporator. The lipids which remained were weighed and dissolved in a solution of 20% octylglucoside, the final concentration of lipids in the solution being adjusted to 5%. Cellular lipids. One to two milliliters of packed tissue culture cells were dialyzed in tap water and lyophilized. Cellular lipids were extracted with chloroform/methanol (2/l) as described for viral lipids and dissolved in a solution of 20% octylglucoside. Dansylcerebroside. Dansylcerebroside was used as fluorescent membrane marker to visualize liposomes (Huang et al., 1979). The compound was synthesized as described previously (Huang, 1976). Preparation of viral glycoproteins.
Ziposomes
containing
Liposomes containing the glycoproteins of virus strains were prepared as described previously (Huang et al., 1979). For uv visualization of liposomes, viral lipid was supplemented with 2-5% dansylcerebroside. For microinjection studies, intensive fluorescence could be entrapped in liposomes when 2 mg of fluorescein isothiocyanate or 20 mg of fluorescein isothiocyanate-dextran was included per 10 mg of total lipid present in the mixture before dialysis. Fluorescein isothiocyanate and fluorescein isothiocyanate-dextran (MW 20,000, FD-20) were purchased from Sigma. Trypsinixation of Ziposomes . Proteolytic cleavage of liposomes containing the uncleaved HA was achieved by treating liposomes with trypsin (10 pg/ml) for 15 min at 37”. The liposomes were then freed of trypsin by centrifugation at 16,000 rpm for 1 hr. The pellet was resuspended in phosphate-buffered saline (PBS).
295
and liposomes were prepared by the dialysis procedure in the same manner as described for liposomes coated with viral glycoproteins. Interaction of viral glycoprotein-coated Ziposomes with tissue culture cells. Mono-
layers of tissue culture cells were dissociated with EDTA-phosphate buffer containing 0.2% trypsin as usual and washed three times with PBS. Interaction of liposomes with cells was initiated by mixing liposome suspensions containing about 250 hemagglutinating units with approximately lo6 dissociated cells suspended in 2 ml of PBS. The reaction was allowed to continue for various lengths of time at room temperature or at 37”. For reasons stated under Results, a final standard reaction period of 30 min at room temperature was adapted. Microinjection. Approximately lo6 dissociated chick embryo fibroblasts were suspended in 1 ml of PBS and mixed with liposomes possessing about 250 hemagglutinating units and containing either fluorescein isothiocyanate or fluoresceinlabeled dextran. The mixture was observed under an uv microscope for 1 hr at 15-min intervals. Occurrence of microinjection was characterized by the appearance of orange yellow fluorescence in the cytoplasm. Interaction of liposomes containing myxovirus receptors with influenza virus. One-
tenth milliliter of a milky suspension of cellular receptor-coated liposomes was mixed with two drops of a virus concentrate containing about 3000 hemagglutinating units and allowed to react for 30 min at room temperature. The sample was then negatively stained for electron microscopy. Electron microscopy. For negative contrasting, samples were stained with 2% uranyl acetate (pH 4.5) or 2% phosphotungstate (pH 7). For observation of Preparation of Ziposomes containing the thin sections, samples in suspension were cellular receptors of in$luenxa virus. About fixed with 1.25% glutaraldehyde, post0.5 ml of packed chick embryo fibroblasts fixed with 1% osmium tetroxide, dehywas solubilized in 2 ml of a 10% solution drated, and embedded in epoxy resin of octylglucoside and centrifuged at 16,000 (Luft, 1961). Thin sections were stained rpm for 1 hr in a Sorvall centrifuge, using with 2% uranyl acetate and Reynolds the SS-34 rotor. Twenty milligrams of total lead (Reynolds, 1963) in the conventional cellular lipids were added to the supernatant manner.
HUANGETAL.
296 RESULTS
whole length of contact. Fusion was not observed even in cases where several liposomes were seen to be adsorbed to the cell (Fig. 2a). In previous work it was reported that Glycoproteins were solubilized from in- trypsin activation of noninfectious virions fluenza virus strains grown in different was associated with transformation of HA hosts. With all virus strains used, HA was from uncleaved to cleaved forms (Klenk present in the cleaved form when viruses et at., 1975, 1977; Lazarowitz and Choppin, were grown in chick embryos. To obtain 1975; Bosch et al., 1979). We therefore uncleaved HA, virus N was grown in chick treated liposomes containing the uncleaved HA of virus N with trypsin in the same fibroblasts. Incorporation of glycoproteins into liposomes was carried out as described manner as for activation of virions to see previously (Huang et al., 1979). Liposomes whether fusion activity could then be obprepared by this method contained HA tained with these liposomes. Figures 3a-c as well as neuraminidase in their mem- show that these liposomes indeed acquired branes. In all cases, whether cleaved or the full activity of fusion after treatment uncleaved HA was used, the protein to lipid with trypsin. Liposomes prepared in the absence of ratio of liposomes was approximately 0.1, a ratio usually obtained when the same viral glycoproteins did not adsorb to cells method of reconstitution was used (Huang and showed no fusion capacity. et al., 1979). Studies It was found by electron microscopic Microinjection observation that fusion occurred rapidly In addition to electron microscopic obbetween dissociated chick embryo fibro- servation, we also obtained another eviblasts and liposomes containing the cleaved dence confirming the fusion activity of HA of any influenza virus strains used. influenza virus by microinjeetion experiWithin 15 min after reaction of cells with ments. Fluorescence substances, such as liposomes at room temperature, different fluorescein isothiocyanate or fluoresceinstages of fusion initiated at areas of con- labeled dextran, were trapped in liposomes tact could be detected (Figs. la, b). Arrows containing the cleaved HA. After mixing indicate positions where plasma membrane these liposomes with chick fibroblasts, the and liposomal membrane fused. When the progress of injection was monitored microreaction was continued for 30 min at room scopically by watching the d~fusion of temperature, most of the liposomes could fluorescence into the cytoplasm of cells, be seen to fuse with cells (Figs. lc, d). If which was characterized by the appearance the temperature was raised to 37” during of orange-yellow fluorescence in the cytoreaction of cells with liposomes, inter- plasm. Injection was usually detectable mediate stages of fusion were no longer within a few minutes at room temperature detected, presumably because fusion had and became intensified during a 1-hr realready gone to completion at this tempera- action period (Fig. 4a). Under the same ture. In subsequent experiments, the re- conditions, almost no fluorescence was deaction period of 30 min at room tempera- tected in the cytoplasm when liposomes ture was therefore adopted as the standard containing the uncleaved HA of virus N condition for electron microscopic observa- were reacted with cells (Fig. 4b). Trypsin tion of fusion. treatment of liposomes with the uncleaved No fusion was seen when cells and HA visibly enhanced the intensity of liposomes with the uncleaved HA were fluorescence injected into the cytoplasm, reacted under the same condition. Figures again confirming the importance of pro2a-c show that liposomes containing un- teolytic cleavage for membrane fusion. cleaved HA could be tightly adsorbed to cells. In most cases areas of contact be- ~nter~~t~a~ of ~~~~~e~~~ Vims with Liposwnes ~o~~ai~~ng cellular Receptors tween liposomes and cells were rather extended, but the membranes of liposomes Since the above evidence of fusion was and cells were clearly separate along the obtained in a reconstituted viral mem-
FUSION
a
OF RECONSTITUTED
INFLUENZA
VIRUS
MEMBRANES
b
FIG. I.. Fusion of chick embryo fibroblasts with liposomes containing the cleaved HA of virus N. (a, b) Fusion after 15-min reaction at room temperature. (c, d) Fusion after 30-min reaction at room temperature. Bars represent 0.25 pm. Arrows indicate positions of fusion
297
298
HUANG
ET AL.
FIG. 2. Absence of fusion between chick embryo fibroblasts and liposomes containing the uncleaved HA of virus N. Liposomes were reacted with cells for 30 min at room temperature. (a) Liposomes adsorbing to chick fibroblasts, but resulting in no fusion. (b, c) Tightly associated membranes of iiposomes and cells without fusion. Bars represent, 1 pm in a and 0.5 pm in b and c.
FUSION
OF RECONSTITUTED
INFLUENZA
VIRUS
MEMBRANES
b
FIG. 3. Trypsin activation of fusion between chick embryo fibroblasts and liposomes containing the uncleaved hemagglutinin of virus N. Liposomes containing the uncleaved hemagglutinin of virus N were treated with trypsin (see Materials and Methods section) and reacted with cells for 30 min at room temperature. (a-e) show that these liposomes acquired fusion activity after treatment with trypsin. Bars represent 0.5 pm. Arrows indicate fusion.
299
300
HUANG ET AL.
FIG. 4. Interaction of fluorescein-dextran-loaded liposomes with chick embryo fibroblasts. Fluorescein-dextran-loaded liposomes containing viral glycoproteins were reacted with cells for 30 min at room temperature. (a) Liposomes containing the cleaved HA injected fluoresceindextran into cells. (b) Liposomes containing the uncleaved HA did not inject fluorescein-dextran into cells.
FUSION
OF RECONSTITUTED
INFLUENZA
VIRUS
MEMBRANES
301
influenza virus. The concept is in agreement with earlier electron microscopic observation by Morgan and Rose (1968) and Hoyle et al. (1962), who also suggested a fusion process for the entry of influenza virus into the cell. It seems to be in contrast, however, to the concept of viropexis (Fazekas de St. Groth, 1948; Dales and Choppin, 1962; Dourmashkin and Tyrrell, 1974) as an alternative model of influenza virus penetration according to which intact particles were thought to be taken up by engulfment into the cell. However, if this were the regular mode of penetration, the inner viral components would still be surrounded by the viral envelope and the membrane of cytoplasmic vesicles. Therefore, the fusion process described here would seem to explain more plausibly how the viral nucleocapsid could find its way into the cytoplasm. The reason why fusion between viral enDISCUSSION velope and cellular membranes has been Electron microscopic studies showed that difficult to demonstrate electron microliposomes containing cleaved HA could fuse scopically might be that it takes place very with cell membranes. Liposomes with un- rapidly under physiological conditions. In cleaved HA were adsorbed on the cell sur- the studies described in this paper, emface, but fusion occurred only after in vitro ploying relatively large liposomes, fusion treatment with trypsin. This membrane was completed within 30 min even at room interaction was followed by injection of temperature. liposomal contents into the cytoplasm, as There is no doubt that proteolytic shown by staining of cytoplasm with cleavage of HA is a prerequisite for the fluorescent substances incorporated into fusion activity of influenza viruses. It the liposomes. Since liposomes can be seems possible that the hydrophobic segconsidered as an artificial system, it was of ment of HA2, which becomes exposed by interest to see whether the native virus the proteolytic cleavage of HA, is mediatcould also fuse with membranes. From ing fusion of the two tightly associated studies using virus particles and liposomes membranes. The situation here looks with cellular receptor of influenza viruses, very much like that existing in parait could be demonstrated that the envelopes myxoviruses, where the expression of of intact virions were inserted into the fusion activity is associated with proteoliposomal membrane. Such liposomes were lytic cleavage of Fo into two F glycoproagain able to fuse with the membrane of teins (Homma and Ohushi, 1973; Scheid and tissue culture cells. Hence, it could be Choppin, 1974; Nagai et al., 1976). The inferred that an event equivalent to virus common biological role of the HA of penetration had occurred. influenza viruses and F glycoprotein of These results together with data ob- paramyxoviruses has become even more tained from physical measurements (Nicolau apparent by the recent findings of Gething et al., 1978), T-cell-mediated cytotoxicity et al. (1978) that HA2 of influenza viruses tests (Kurrle et al., 1979), and the correla- and Fl glycoprotein of paramyxoviruses tion existing between infectivity and possess an N-terminal structure, very cleavage of HA (Klenk et al., 1975; similar in its amino acid sequence and its Lazarowitz and Choppin, 1975) indicate extreme hydrophobicity. It can be concluded from above results that fusion of viral envelope with cellular membrane is involved in the penetration of that influenza viruses and paramyxoviruses brane and cell system, another system was further designed in which we mixed virions with liposomes containing the cellular receptors of influenza virus to see whether native virions also possess fusion activity. For this purpose, chick fibroblasts were solubilized with octylglucoside and the soluble components, which contained receptors of influenza virus, were incorporated into liposomes as described in the previous section. It was found that such liposomes were aggregated immediately by influenza viruses, indicating the presence of virus receptors in their membranes. Electron microscopic observation showed that viral spikes had become incorporated into liposomal membrane (not shown). Such liposomes resulting from interaction of vii-ions and liposomes could in turn fuse with cells in a similar way as shown in Figs. 1 and 3 for reconstituted viral membrane.
HUANG ET AL.
302
may initiate infection by a common mecha- HOMMA, M., and OHUCHI, M. (1973). Trypsin action on the growth of Sendai virus in tissue culture nism, namely fusion of viral envelope and cells. III. Structural differences of Sendai viruses cellular membranes. It is, however, not grown in eggs and tissue culture cells. clear why influenza viruses do not cause HOYLE, L., HORNE, R. W., and WATERSON, A. P. fusion of cells as is known to be a property (1962). The structure and composition of the of paramyxoviruses. It seems possible myxoviruses. III. The interaction of influenza that enveloped viruses in general, including virus particles with cytoplasmic particles derived those not hitherto known to be fusogenic, from normal chorioallantoic membrane cells. Virology 17, 533-542. may fuse with their host cells. The presently described liposome system would HUANG, R. T. C. (1976) Labeling of animal cells with fluorescent dansylcerebroside. Z. Naturforsch. seem to provide a useful tool for the study 31c, 737-740. of such processes. ACKNOWLEDGMENTS This work was supported by the Sonderforschungsbereich 47 of the Deutsche Forschungsgemeinschaft. The technical assistance of Miss Heidrun Will is greatly appreciated. Note added in proof. In a recent study (H. U. Koszinowski, H. Allen, M. I. Gething, M. D. Waterfield, and H. -D. Klenk, 1980, J. Exp. Med. 151, 945958) it has been reported that reconstituted influenza envelopes containing cleaved hemagglutinin could fuse only in the presence of Sendai virus. It should be pointed out that these liposomes were prepared by a different procedure, and that they showed distinct differences in size and lipid composition if compared to the liposomes analyzed in the present study. This may indicate that structure and composition of the liposomes are also important for fusing capacity.
REFERENCES BOSCH, F. X., ORLICH, M., KLENK, H. -D., and ROTT, R. (19’79).The structure ofthe hemagglutinin, a determinant for the pathogenicity of influenza virus. Virology 95, 197-207. CHUCHOLOWIUS,H. -W., and ROTT, R. (1972). A new method for purification of myxoviruses by zonal centrifugation with two different sucrose density gradients. Proc. Sot. Exp. Biol. Med. 140, 245247.
DALES, S., and CHOPPIN, P. W. (1962). Attachment and penetration of influenza virus. Virology 18, 489-493.
DOURMASHKIN,R. R., and TYRRELL, D. A. (1974). Electron microscopic observation on the entry of influenza virus into susceptible cells. J. Gen. Viral. 24, 129-141. FAZEKAS DE ST. GROTH, S. (1948). Viropexis, the mechanism of virus infection. Nature (London) 162, 294-295.
GETHING, M. J., WHITE, J. M., and WATERFIELD, M. D. (1978). Purification of the fusion protein of Sendai virus. Analysis of the NH,-terminal sequence generated during precursor activation. Proc. Nat. Acad. Sci. 1JSA 75. 2737-2740.
HUANG, R. T. C., WAHN, K., KLENK, H. -D., and ROTT, R. (1979). Association of the envelope glycoproteins of influenza virus with liposomesModel study on viral envelope assembly. Virology 97, 212-217.
KLENK, H. -D., ROTT, R., and ORLICH, M. (1977). Further studies on the activation of influenza virus by proteolytic cleavage of the hemagglutinin. J. Gen. Viral. 36, 151-161. KLENK, H. -D., ROTT, R., ORLICH, M., and BL~DORN,J. (1975). Activation of influenza viruses by trypsin treatment. Virology 68, 426-429. KURRLE, R., WAGNER, H., ROLLINGHOFF,M., and ROTT, R. (1979). Influenza virus-specific T cellmediated cytotoxicity: Integration of the virus antigen into the target cell membrane is essential for target cell formation. Eur. J. Zmmunol. 9, 107-111. LAZAROWITZ, S. G., and CHOPPIN, P. W. (1975). Enhancement of the infectivity of influenza A and B viruses by proteolytic cleavage of the hemagglutinin polypeptide. Virology 68, 440-454. LUFT, J. H. (1961). Improvement in expoxy resin embedding methods. J. Biophys. Biochem. Cytol. 9, 409-414. MORGAN, C., and ROSE, H. M. (1968). Structure and development of viruses as observed in the electron microscope. VIII. Entry of influenza virus. J. Viral.
2, 925-936.
NAGAI, Y., KLENK, H. -D., and ROTT, R. (1976). Proteolytic cleavage of the viral glycoproteins and its significance for the virulence of Newcastle disease virus. Virology 72, 494-508. NICOLAU, C., KLENK, H. -D., REIMAN, A., HILDEBRAND, K., and BAUER, H. (1979). Molecular events during the interaction of envelopes of myxo- and RNA-tumor viruses with cell membranes. A 270 MHz ‘H nuclear magnetic resonance study. Biochim. Biophys. Acta 511, 83-92. REYNOLDS, E. S. (1963). The use of lead citrate at high pH as an electronopaque stain in electron microscopy. J. Cell Biol. 17, 208-212. SCHEID, A., and CHOPPIN, P. W. (1974). Identification of biological activities of paramyxovirus glycoproteins. Activation of cell fusion, hemolysis and infectivity by proteolytic cleavage of an inactive precursor protein of Sendai virus. Virology 57, 475-490.