Effect of lipid composition upon fusion of liposomes with Sendai virus membranes

How the lipid composition of liposomes determines their ability to fuse with Sendai virus membranes was tested. Liposomes were made of compositions designed to test postulated mechanisms of membrane fusion that require specific lipids. Fusion does not require the presence of lipids that can form micelles such as gangliosides or lipids that can undergo lamellar to hexagonal phase transitions such as phosphatidylethanolamine (PE), nor is a phosphatidylinositol (PI) to phosphatidic acid (PA) conversion required, since fusion occurs with liposomes containing phosphatidylcholine (PC) and any one of many different negatively charged lipids such as gangliosides, phosphatidylserine (PS), phosphatidylglycerol, dicetyl phosphate, PI, or PA. A negatively charged lipid is required since fusion does not occur with neutral liposomes containing PC and a neutral lipid such as globoside, sphingomyelin, or PE. Fusion of Sendai virus membranes with liposomes that contain PC and PS does not require Ca2+, so an anhydrous complex with Ca2+ or a Ca2+-induced lateral phase separation is not required although the possibility remains that viral binding causes a lateral phase separation. Sendai virus membranes can fuse with liposomes containing only PS, so a packing defect between domains of two different lipids is not required. The concentration of PS required for fusion to occur is approximately 10-fold higher than that required for ganglioside GD1a, which has been shown to act as a Sendai virus receptor. When cholesterol is added as a third lipid to liposomes containing PC and GD1a, the amount of fusion decreases if the GD1a concentration is low.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetic studies of Sendai virus-target membrane interactions: independent analysis of binding and fusion

Fusion between Sendai virus and liposomes containing phosphatidylethanolamine (PE) and different mole fractions of ganglioside GD1a has been investigated. At different times after mixing the virus and liposomes, the mixture was diluted with a sucrose solution and centrifuged in an airfuge to separate the free and virus-associated liposomes. Since the HN protein of the virus was sensitive to the reducing reagent, inclusion of dithiothreitol in the sucrose solution dissociated the bound but not the fused liposomes. Thus, the kinetics of liposome-virus binding and fusion could be independently measured. The validity of the assay was confirmed by electron microscopic observation of the virus-liposome mixtures. With trypsin-treated Sendai virus, in which the F glycoprotein of the virus had been selectively removed, only virus-liposome binding but not fusion was observed. The kinetic experiments were done under the condition of virus in large excess. Following a very fast initial binding phase, which was completed at the "zero time" of the measurement, the virus-liposome binding followed pseudo-first-order kinetics. The subsequent fusion step was zero order. Judging from the discontinuity in the Arrhenius plots, both binding and fusion events were sensitive to the gel-liquid-crystalline phase transition of the target membrane. The binding rate constants had activation energies between 16 and 23 kcal/mol at temperatures above the transition. They were not sensitive to temperature change at temperatures below the transition. On the other hand, the fusion rate constants were not sensitive to temperature change above the transition, except for 6.3% GD1a liposomes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sendai virus induced leakage of liposomes containing gangliosides

Sendai virus induced liposome leakage has been studied by using liposomes containing a self-quenching fluorescent dye, calcein. The liposomes used in this study were prepared by a freeze and thaw method and were composed of phosphatidylcholine, phosphatidylserine, and phosphatidylethanolamine (1:2.60:1.48 molar ratio) as well as various amounts of gangliosides and cholesterol. The leakage rate was calculated from the fluorescence increment as the entrapped calcein leaked out of the liposomal compartment and was diluted into the media. It was shown that the target liposome leakage was virus dose dependent. Trypsin-treated Sendai virus in which the F protein had been quantitatively removed did not induce liposome leakage, indicating that the leakage was a direct result of F-protein interaction with the target bilayer membrane. The activation energy of this process was approximately 12 kcal/mol below 17 degrees C and approximately 25 kcal/mol above 17 degrees C. Gangliosides GM1, GD1a, and GT1b could serve as viral receptor under appropriate conditions. Liposome leakage showed a bell-shaped curve dependence on the concentration of ganglioside in the liposomes. No leakage was observed if the ganglioside content was too low or too high. Inclusion of cholesterol in the liposome bilayer suppressed the leakage rate of liposomes containing GD1a. It is speculated that the liposome leakage is a consequence of fusion between Sendai virus and liposomes.     

Thermodynamic and phase characterization of phosphatidylethanolamine and ganglioside GD1a mixtures

By employing diphenylhexatriene steady-state fluorescence anisotropy, pyrenedecanoic acid excimer formation, and high sensitivity scanning calorimetry we have demonstrated that the liposomes containing phosphatidylethanolamine (PE) and various mole fractions of ganglioside GD1a had a gel-liquid crystalline phase transition between 15 and 25 degrees C. Calorimetric measurements indicated that these phase transitions were broad and centered between 17 and 21 degrees C. The enthalpy change of the transition was linearly dependent on the ganglioside concentration up to 10.0 mol% and plateaued between 11.4-16.2 mol%. The high enthalpy change (37 kcal/mol of GD1a added into the PE bilayer) indicates the existence of PE-GD1a complex structure in the liposomal membrane. It is proposed that semi-fluid domains containing six PE and one ganglioside molecule are present in the PE-GD1a membranes at temperatures above gel-liquid crystalline phase transition. The Sendai virus induced leakage of PE-GD1a liposomes has been investigated by using an entrapped, self-quenching fluorescent dye, calcein. The leakage rate was dependent on the mole fraction of ganglioside GD1a and was maximal at 6.3 mol%. Arrhenius plots of the leakage rates showed breaks in the 20-25 degrees C temperature range, which correspond to the gel-liquid crystalline phase transition of the target liposomes. These data suggest that the rate of Sendai virus-induced leakage can be regulated via fluidity modulation by changing the PE to GD1a ratio at constant temperatures.     

Reconstitution of lipid vesicles associated with HVJ (Sendai virus) sikes. Purification and some properties of vesicles containing nontoxic fragment A of diphtheria toxin

A mixture of HVJ (Sendai virus) spike proteins, the nontoxic fragment A of diphtheria toxin, lecithin, and cholesterol was solubilized in sucrose solution containing a nonionic neutral detergent. The liposomal vesicles which formed on removal of the detergent by dialysis were purified by gel filtration and centrifugation on a sucrose gradient. The resulting purified vesicles had hemagglutinating activity, hemolytic activity and, after solubilization, the enzymic activity of fragment A. The vesicles had no cell fusion activity. Electron microscopy showed that both the outside and inside of membranes of the vesicles were associated with the spikes. When the vesicles were freeze-fractured, no large aggregates of particles were seen on either face. Such fragment A-containing lipid vesicles (liposomes) with HVJ spikes bound to mamalian cell membrane and released their fragment A into the cytoplasm causing cell death. Neither fragment A-containing liposomes without spikes nor empty liposomes with spikes were toxic.     

Characteristics of Sendai virus receptors in a model membrane

The adsorption of Sendai virus to liposomes of different compositions was studied. Liposomes prepared with only phosphatidylcholine and cholesterol, and liposomes prepared with phosphatidylcholine and cholesterol plus phosphatidic acid or phosphatidyl serine did not adsorb virus. Phosphatidyleholine-cholesterol liposomes containing also stearyl amine or ganglioside did, however, adsorb virus. The ability of the adsorbing liposomes to compete with erythrocytes for virus was measured by hemagglutination inhibition. Liposomes containing ganglioside, but not those containing stearyl amine, inhibited hemagglutination. When the molar ratio of ganglioside N-acetyl neuraminic acid to phosphatidylcholine was less than 0.02, ganglioside liposomes did not inhibit hemagglutination. As the ratio increased from 0.02 to 0.05, the liposomes caused increasing amounts of hemagglutination inhibition, but with further increases in the ratio the hemagglutination inhibition remained constant. It is concluded that gangliosides can serve as Sendai receptors and that a multiplicity of receptors is needed for virus binding.     

Fusion of enveloped viruses with cells and liposomes. Activity and inactivation.

The fusion of viruses with cells and liposomes is reviewed with focus on the analysis of the final extents and kinetics of fusion. Influenza virus and Sendai virus exhibit 100% of fusion capacity with cells at pH 5 and pH 7.5, respectively. On the other hand, there may be in certain cases, a limit on the number of virions that can fuse with a single cell, that is significantly below the limit on binding. It still remains to be resolved whether this limit reflects a limited number of possible fusion sites, or a saturation limit on the amount of viral glycoproteins that can be incorporated in the cellular membrane, like the case of virus fusion with pure phospholipid vesicles, in which the fusion products were shown to consist of a single virus and several liposomes. Both viruses demonstrate incomplete fusion activity towards liposomes of a variety of compositions. In the case of Sendai virus, fusion inactive virions bind essentially irreversibly to liposomes. Yet, preliminary results revealed that such bound, unfused virions can be released by sucrose gradient centrifugation. The separated unfused virions subsequently fuse when incubated with a "fresh" batch of liposomes. We conclude, therefore, that the fraction of initially bound unfused virions does not consist of dective particles, but rather of particles bound to liposomes via "inactive" sites. Details of the low pH inactivation of fusion capacity of influenza virus towards cells and liposomes are presented. This inactivation is caused by protonation and exposure of the hydrophobic segment of HA2, and affects primarily the fusion rate constants. Some degree of inactivation also occurs when virions are bound to cellular membranes.     

Sendai virus membrane fusion: time course and effect of temperature, pH, calcium, and receptor concentration

The conditions that optimize Sendai virus membrane fusion with liposomes have been studied. No fusion occurs in the absence of ganglioside receptors. Maximum fusion occurs when the molar ratio of ganglioside GD1a to phospholipid is 0.02 or greater. The amount of fusion at 37 degrees C increases with time up to at least 6.5 h. The rate of fusion increases from the lowest temperature tested, 10 degrees C, to 40 degrees C. Above 43 degrees C the amount of fusion decreases because of thermal inactivation of the viral proteins. There is a broad pH maximum between pH 7.5 and pH 9.0. At both ends of the pH range the amount of fusion increases and exceeds that found in the physiologic pH range. Neither ethylenediaminetetraacetic acid nor Ca2+ changes the amount of membrane fusion. The optimal conditions for membrane fusion of Sendai virus membranes with liposomes are the same as the optimal conditions for fusion with host cells and with red blood cells. Since the liposomes contain no proteins, the optimal conditions for Sendai virus membrane fusion must be determined by the viral proteins and be mostly independent of the nature or presence of the host proteins.     

Irreversible unfolding of the neutral pH form of influenza hemagglutinin demonstrates that it is not in a metastable state

The thermal denaturation of the proteins of influenza virus has been measured by differential scanning calorimetry in the presence and absence of lipids as a function of scan rate. We have applied theories of irreversible thermodynamics to obtain the activation energy. In the presence of liposomes of dioleoylphosphatidylcholine with the ganglioside, GD(1a), the denaturation temperature of the hemagglutinin protein is lowered. This lowering of thermal stability is also reflected in the temperature dependence of the circular dichroism spectra. Quasi-elastic light scattering confirms that liposomes containing GD(1a) interact with the virus and inhibit the growth in the size of the particle as a function of temperature. Although the virus can fuse with the liposomes at higher temperatures, the enthalpy change for this process is not detectable. Our results also demonstrate that the compact folded structure of the influenza hemagglutinin protein is not a kinetically trapped metastable high-energy form.     

Active function of membrane receptors for enveloped viruses : I. Specific requirement for liposome-associated sialoglycolipids, but not sialoglycoproteins, to allow lysis of phospholipid vesicles by reconstituted Sendai viral envelopes

Phospholipid liposomes composed of phosphatidylcholine (PC) and cholesterol (chol), bearing the sialoglycoprotein glycophorin (GP), are able to effectively bind Sendai virus particles, but not to be lysed by them. Incorporation of gangliosides (gangl) into the above phospholipid vesicles (yielding liposomes composed of PC/chol/gangl/GP), although not increasing their ability to interact with Sendai virions, rendered them susceptible to the viral lytic activity. This was inferred from the ability of the virus to induce release of carboxyfluorescein (CF) upon interaction at 37 degrees C with liposomes composed of PC/chol/gangl/GP. Lysis of liposomes required the presence of the two viral envelope glycoproteins, namely the hemagglutinin/neuraminidase (HN) and the fusion (F) polypeptides, and was inhibited by phenylmethyl sulfonylfluoride (PMSF), dithiothreitol (DTT) and trypsin, showing that virus-induced lysis of PC/chol/gangl/GP liposomes reflects the fusogenic activity of the virus. Incubation of Sendai virus particles with liposomes containing the acidic phospholipid dicetylphosphate (DCP) but lacking sialic acid containing receptors, also resulted in release of the liposome content. Lysis of these liposomes was due to the activity of the viral HN glycoprotein, therefore not reflecting the natural viral fusogenic activity. Fluorescence dequenching studies, using fluorescently labeled reconstituted Sendai virus envelopes (RSVE), have shown that the viral envelopes are able to fuse with neutral, almost to the same extent, as with negatively charged liposomes. However, fusion with negatively charged liposomes, as opposed to fusion with neutral liposomes, was mediated by the viral HN glycoprotein and not by the viral fusion polypeptide.     

Effects of pH on the binding of Clostridium botulinum type E derivative toxin to gangliosides and phospholipids

Abstract Clostridium botulinum type E derivative toxin directly bound to gangliosides G_T1b, G_D1a, and G_Q1b but not to G_M1 or G_D1b at pH 5.0 or above, At the same pH values, it bound to negatively charged phospholipids but not to noncharged ones. At pH 4.0, it bound to any of gangliosides and phospholipids including G_M1, G_D1a, and non-charged phospholipids. It bound to ceramide, a hydrophobic component of ganglioside and also to sphingomyelin, a phospholipid containing a ceramide moiety, only at pH 4.0. It bound to ceramide and sphingomyelin less firmly than to other phospholipids at pH 4.0. We assume that botulinum toxin adheres to the neural cell surface mainly by sialic acid-specific and charge-dependent binding possibly aided by nonspecific hydrophobic(toxin)-hydrophobic(lipids, mainly phospholipids) interaction.     

The interaction of Sendai virus with negatively charged liposomes: virus-induced lysis of carboxyfluorescein-loaded small unilamellar vesicles

The interaction of Sendai virus with small, unilamellar vesicles, lacking virus receptors and loaded with self-quenched 6-carboxyfluorescein, was studied. Sendai virions induced release of carboxyfluorescein from vesicles composed of negative charged phospholipids, despite the fact that they did not contain virus receptors. Preliminary experiments indicate that the carboxyfluorescein release is accompanied by mixing of the virus and liposome lipids and their entrapped contents, suggesting liposome-virus fusion. No release of carboxyfluorescein was observed with vesicles containing only phosphatidylcholine. The rate of virus-induced carboxyfluorescein release was temperature dependent; the lytic activity of the virus was greatly enhanced above 25 degrees C. This effect was not due to a thermal phase transition of the lipids in either the lipid vesicles or the virions. Virus-induced carboxyfluorescein release was inhibited by the presence of calcium ions in the medium and of cholesterol in the lipid vesicles. It increased with increasing concentrations of either the lipid vesicles or the virions. pretreatment of virions with increasing concentrations of three different proteolytic enzymes (trypsin, chymotrypsin and proteinase) inhibited the virus' ability to cause release of carboxyfluorescein from negatively charged liposomes. Inhibition of the viral lytic activity was also observed after virions were incubated above 56 degrees C.     

Use of the Airfuge for analysis and preparation of receptors incorporated into liposomes: Studies with the receptor for immunoglobulin E

A Beckman Airfuge has been employed for studying the interaction between lipids and the receptor for immunoglobulin E (IgE). For analytic experiments, samples were applied underneath a discontinuous sucrose gradient. After a 30-min centrifugation in a fixed-angle rotor, liposomes floated toward the top of the gradient whereas unincorporated receptor-IgE complexes remained at the bottom of the tube. Liposomes with incorporated receptors were also efficiently separated in the ACR-90 preparative rotor. These methods of "Airfuge flotation" can provide useful adjuncts to more traditional methods for density-gradient centrifugation especially when rapid analysis of small samples is desired.     

Efficient interaction of nonpolar lipids with intermediate filaments of the vimentin type

Based on the finding that vimentin isolated and purified from cultured mammalian cells is heavily contaminated by neutral lipids, the binding of a series of radioactively labeled nonpolar lipids to pure, delipidated vimentin was investigated. Employing gel permeation chromatography of the complexes on Sephacryl S-300, cholesterol, cholesteryl fatty acid esters and mono-, di- and triglycerides were found to efficiently associate with vimentin. These compounds also showed a strong tendency to bind to vimentin filaments. While the non-alpha-helical head piece of vimentin did not interact with neutral lipids under the above assay conditions, the alpha-helical rod domain was highly active. When cholesterol or 1,2-dioleoyl-glycerol was incorporated into phospholipid vesicles, the affinity of the liposomes for vimentin filaments was considerably increased. However, in sucrose density gradient equilibrium centrifugation the filament-vesicle adducts were only stable when the liposomes contained negatively charged phospholipids. These results suggest that the association of intermediate filaments with lipid vesicles is initiated by interaction of the arginine-rich N-termini of their subunit proteins with the negatively charged vesicle surface and stabilized by partial insertion of the protein molecules into the lipid bilayer, particularly at those sites where immiscible, nonpolar lipids create defects in phospholipid packing. Very likely, nonpolar lipids play a significant role in the interaction of intermediate filaments with natural membrane systems.     

Membrane fusion activity of influenza virus. Effects of gangliosides and negatively charged phospholipids in target liposomes

Fusion of influenza virus with liposomes composed of negatively charged phospholipids differs from fusion with biological membranes or zwitterionic liposomes with ganglioside receptors [Stegmann, T., Hoekstra, D., Scherphof, G., & Wilschut, J. (1986) J. Biol. Chem. 261, 10966-10969]. In this study, we investigated how the kinetics and extent of fusion of influenza virus, monitored with a fluorescence resonance energy-transfer assay, are influenced by the surface charge and the presence of receptors on liposomal membranes. The results were analyzed in terms of mass action kinetic model, providing separate rate constants for the initial virus-liposome adhesion, or aggregation, and for the actual fusion reaction. Incorporation of increasing amounts of cardiolipin (CL) or phosphatidylserine (PS) into otherwise zwitterionic phosphatidylcholine (PC)/phosphatidylethanolamine (PE) vesicles results in a gradual shift of the pH threshold of fusion to neutral, relative to the pH threshold obtained with PC/PE vesicles containing the ganglioside GD1a, while also the rate of fusion increases. This indicates the emergence of a fusion mechanism not involving the well-documented conformational change in the viral hemagglutinin (HA). However, only with pure CL liposomes this nonphysiological fusion reaction dominates the overall fusion process; with pure PS or with zwitterionic vesicles containing CL or PS, the contribution of the nonphysiological fusion reaction is small. Accordingly, preincubation of the virus alone at low pH results in a rapid inactivation of the viral fusion capacity toward all liposome compositions studied, except pure CL liposomes. The results of the kinetic analyses show that with pure CL liposomes the rates of both virus-liposome adhesion and fusion are considerably higher than with all other liposome compositions studied.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biochemical and biophysical properties of purified phospholipid vesicles containing bovine heart cytochrome c oxidase

Liposomes containing bovine heart cytochrome c oxidase (COV) prepared by the cholate dialysis technique were purified from those devoid of the enzyme using discontinuous sucrose density ultra centrifugation to eliminate interference in proton-pumping assays. This technique was also used to purify liposomes containing cytochrome c oxidase depleted in subunit III (COV-III), a COX enzyme preparation with altered subunit structure, to assess if the technique could be applied to COX enzymes in which structural and functional changes have occurred. Upon discontinuous sucrose density ultra gradient ultracentrifugation, either COV or COV-III were separated into two bands. Liposomes devoid of enzyme sedimented into the 12% sucrose layer, whereas enzyme-containing liposomes (pCOV or pCOV-III) were found in the 13% sucrose layer. The yield of both pCOV or pCOV-III was greater than 60% (based on heme aa(3) content), suggesting a similar distribution of cytochrome c oxidase (COX) and subunit III-depleted enzyme (COX-III) in the purified liposomes. The number of COX or COX-III molecules per phospholipid vesicle in purified fractions was estimated to be two. Removal of subunit III (M(r)=29,918) from COX resulted in a 30% decrease in electron transfer activity (either in COV-III or pCOV-III) when compared with COV and pCOV, respectively. Both pCOV and pCOV-III exhibited low endogenous proton permeability, as assessed by possessing high respiratory control ratios (14 and greater) and by having similar valinomycin concentration dependencies for stimulation of electron transfer activity in the presence of saturating amounts of CCCP. COV-III and pCOV-III exhibited a 39-44% decrease in proton-pumping activity when compared with COV and pCOV. These results showed that the separation of COX containing liposomes from those lacking enzyme by sucrose density gradient centrifugation can be used to characterize the biophysical properties of these liposomes.     

Surface topography of sulfatide and gangliosides in unilamellar vesicles of dipalmitoylphosphatidylcholine

The property of the dyes, acridine orange and methylene blue, to exhibit metachromatic changes upon binding to negatively charged groups that are within a defined spatial separation was employed to study the lateral and transverse topography of sulfatide and gangliosides GM1 and GD1a mixed with dipalmitoylphosphatidylcholine (DPPC) in unilamellar vesicles. The spectral changes of the dyes in the presence of liposomes containing anionic glycosphingolipids (GSLs) (hypochromism and frequency shift) are typical of polyanionic lattices while minor changes are found for neutral lipids. The metachromatic changes are abolished by the presence of Ca2+ in the external medium. The proportion of anionic GSLs accessible to the dyes on the external surface of the liposomes is greater as the GSLs are more complex (sulfatide less than GM1 less than GD1a) and as its proportion in the mixture decreases. The number of molecules of anionic GSLs that are laterally distributed on the external surface in a position favorable for the formation of dye dimers (at intermolecular distances not exceeding 1 nm) is greater for sulfatide than for ganglioside. This is correlated to the greater intermolecular distances and delocalization in ganglioside-, compared to sulfatide-containing interfaces. The experimental values indicate that the mixture with DPPC of any of the anionic GSLs studied behaves as if it was more enriched in the GSLs compared to the proportions of the whole mixture.     

Virus Meets Liposome

Abstract

Sendai virus was the first virus to encounter liposomes. Gangliosides when incorporated into liposomes act as Sendai virus receptors even at 0–4°C. When receptor-containing liposomes are incubated with virus at 37°C, they envelop the virus. At 37°C liposomes also fuse with Sendai virus membrane.

Virus binding initially involves weak adhesion, which may allow the virus to “browse” the cell, and which is followed by adhesion strengthening. MicrogrΔpHs of Sendai virus fusion with liposomes after one minute at 37°C indicate that fusion occurs at the very curved leading edge of the region of the liposome enveloping virus. A model of fusion is proposed that emphasizes the role of the curvature and membrane tension in this localized region of “host” membrane. The curvature assists close approach and destabilizes the outer monolayer. The proposed intermediates are consistent with the “stalk” hypothesis.     

Characterization of human sperm plasma membrane: glycolipids and polypeptides

The plasma membranes from ejaculated human spermatozoa were removed by nitrogen cavitation (600 PSI for 10 min) and isolated by centrifugation followed by a discontinuous sucrose density gradient centrifugation. Glycolipid analysis of the plasma membrane revealed a three-fold enrichment in gangliosides: GM3 and GD1a/GD1b and neutral glycolipids: globoside and sulfatide as compared to that of whole human sperm. Two dimensional electrophoresis of human sperm plasma membranes revealed about 75 polypeptides. Several of these polypeptides were similar in migration and in display of shape and color to that found in boar sperm plasma membranes.     

Herpesvirus sylvilagus I. Polypeptides of virions and nucleocapsids.

Herpesvirus sylvilagus was propagated in juvenile cotton tail rabbit kidney cells and purified from the cytoplasmic fraction of the infected cells. The purification procedure included zonal centrifugation through a 5 to 30% dextran t-10 gradient, followed by equilibrium centrifugation in a 5 to 50% potassium tartrate gradient. H. sylvilagus formed one band after centrifugation through the tartrate gradient at a density of 1.22 g/cm3. Contamination of the purified virus preparation by cellular proteins was less than 0.2% as determined by the removal of radioactivity from an artificially mixed sample containing [35S]methionine-labeled control cells and nonlabeled infected cells. H. sylvilagus nucleocapsids were isolated from infected cell nuclei and purified by sedimentation through a 36% sucrose cushion, followed by equilibrium centrifugation in 5 to 50% tartrate gradient. Forty-four polypeptides ranging in molecular weight from 18,000 to 230,00 were resolved when [35S]methionine-labeled enveloped H. sylvilagus was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Seventeen polypeptides found within the enveloped virus were also identified with the nucleocapsid. Six additional nucleocapsid polypeptides han no counterparts within the enveloped virus. The major polypeptide within both the virus and the nucleocapsid had a molecular weight of 150,000.