Reconstitution of fusogenic Sendai virus envelopes by the use of the detergent chaps

Sendai virus envelopes have been a useful tool in studying the mechanism of membrane-membrane fusion and have served as a vehicle for introducing foreign molecules (e.g., membrane proteins) into recipient cells. Reconstituted Sendai virus envelopes are routinely obtained following solubilization of virus particles with Triton X-100. This detergent has a low critical micellar concentration which precludes it from being the best detergent of choice in reconstitution studies. Nevertheless, it has remained in use since other detergents such as sodium deoxycholate and sodium cholate rendered the resultant vesicles inactive. Triton X-100 may be suboptimal for studies of some proteins that need be coreconstituted with the viral envelopes. Thus, alternative advantageous detergents, which retain the envelope fusogenic activity, have been sought. In this study we show that the synthetic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (Chaps) effectively solubilizes the Sendai virions, and that the vesicles formed by simple reconstitution protocols appear structurally and biochemically similar to those obtained with Triton X-100. The resultant vesicles retain functional integrity as assessed in both fusion and hemolysis assays. This protocol seems to be useful in sendai envelope-mediated reimplantation of Fc epsilon receptors into the plasma membranes of rat basophilic leukemia cells.     

Construction of fusogenic vesicles bearing specific antibodies. Targeting of reconstituted Sendai virus envelopes towards neuraminidase-treated human erythrocytes

The cross-linking reagents succinimidyl-4-(p-maleimidophenyl)-butyrate and N-succinimidyl-3-(2-pyridyldithio)-propionate were used to covalently attach antibodies against human erythrocytes to the thiol-containing paraffin, dodecanethiol. The complex formed, dodecanethiol-maleimidophenylbutyrate (or pyridyldithiopropionate)-antibody was inserted into the membranes of reconstituted Sendai virus envelopes. This was achieved by addition of the dodecanethiol-maleimidophenylbutyrate-antibody to a detergent solution (Triton X-100) containing the viral envelope phospholipids and glycoproteins. Removal of the detergent led to the formation of vesicles containing the viral glycoprotein and the dodecanethiol-maleimidophenylbutyrate (or pyridyldithiopropionate)-antibody complexes within the same membrane. Reconstituted Sendai virus envelope-bearing antibodies against human erythrocytes were able to fuse with human erythrocytes (as was reflected by reconstituted Sendai virus envelope-induced hemolysis) from which the natural virus receptors were removed by treatment with neuraminidase. Thus, it appears that anti-human erythrocyte antibodies could substitute for the viral binding protein (hemagglutinin/neuraminidase glycoprotein) in mediating functional binding of the virus particles to the cell plasma membranes. Furthermore, from the results of the present work, it may be inferred that in addition to being the viral-binding protein, hemagglutinin/neuraminidase glycoprotein actively participates in the process of virus-cell fusion.     

Multiple lipid interactions of the Sendai virus fusogenic protein

The membrane topology of the envelope of Sendai virus was investigated using various radioactive photoactivable hydrophobic reagents: 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine and the two phospholipid analogues, 1-palmitoyl-2-(2-azido-4-nitro)benzoyl-sn -glycero-3- phospho[3H]choline and 1-myristoyl-2,12-amino-(4-N-3-nitro-1-azidophenyl)dodecanoyl-sn-glycero- 3-phospho[14C]choline. The hemagglutinin-neuraminidase glycoprotein and the fusogenic (F) glycoprotein were labeled by all three probes, confirming that these proteins are integral components of the viral envelope. The labeled F glycoprotein, composed of the two subunits F1 and F2, was cleaved in situ with trypsin to yield two fragments, F32 (32 kDa) and F19 (19 kDa). F2 was not labeled by any of the probes, suggesting an external location; whereas F19 was labeled by all probes and hence contains the portion of the F glycoprotein which traverses the viral envelope. Fragment F32 reacted both with 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine and with 1-palmitoyl-2-(2-azido-4-nitro)benzoyl-sn-glycero-3-phospho[3H]choline, but not with 1-myristoyl-2,12-amino-(4-N-3-nitro-1-azidophenyl)dodecanoyl-sn-glycero- 3- phospho[14C]choline. This result opens the possibility that the F glycoprotein is formed by a loop-like structure having multiple interactions with viral lipids.     

Characterization of the fusogenic properties of Sendai virus: kinetics of fusion with erythrocyte membranes

A novel fluorescence assay [Hoekstra, D., De Boer, T., Klappe, K., & Wilschut, J. (1984) Biochemistry 23, 5675-5681] has been used to characterize the fusogenic properties of Sendai virus, using erythrocyte ghosts and liposomes as target membranes. This assay involves the incorporation of the "fusion-reporting" probe in the viral membrane, allowing continuous monitoring of the fusion process in a very sensitive manner. Fusion was inhibited upon pretreatment of Sendai virus with trypsin. Low concentrations of the reducing agent dithiothreitol (1 mM) almost completely abolished viral fusion activity, whereas virus binding was reduced by ca. 50%, indicating that the fusogenic properties of Sendai virus are strongly dependent on the integrity of intramolecular disulfide bonds in the fusion (F) protein. Pretreatment of erythrocyte ghosts with nonlabeled Sendai virus inhibited subsequent fusion of fluorophore-labeled virus irrespective of the removal of nonbound virus, thus suggesting that the initial binding of the virus to the target membrane is largely irreversible. As a function of pH, Sendai virus displayed optimal fusion activity around pH 7.5-8.0. Preincubation of the virus at suboptimal pH values resulted in an irreversible diminishment of its fusion capacity. Since virus binding was not affected by the pH, the results are consistent with a pH-induced irreversible conformational change in the molecular structure of the F protein, occurring under mild acidic and alkaline conditions. In contrast to virus binding, fusion appeared to be strongly dependent on temperature, increasing ca. 25-fold when the temperature was raised from 23 to 37 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cotransfection of nucleic acid segments by Sendai virus envelopes

It is reported here that Sendai virus envelopes (SVE) can be used to transfect multiple copies of DNA segments of different varieties and size. This capability further increases the usefulness of SVE. In addition, the ability to simultaneously transfect multiple copies of different genome segments promises to be a powerful tool in the field of molecular biology. The simultaneous transfection of NEO gene and cytomegalovirus immediate early antigen gene was successfully done. Sendai virus envelopes (SVE)1 have been used successfully to study carcinogenesis of Epstein-Barr virus (1, 2). SVE have been shown to have a large carrying capacity (3) for the microinjection of macromolecules into target cells. SVE are hollow vesicles constructed from the viral proteins hemagglutinin HN and fusion factor F.     

Reconstitution of the fusogenic activity of vesicular stomatitis virus.

Enveloped virus glycoproteins exhibit membrane fusion activity. We have analysed whether the G protein of vesicular stomatitis virus, reconstituted into liposomes, is able to fuse nucleated cells in a pH-dependent fashion. Proteoliposomes produced by octylglucoside dialysis did not exhibit cell fusion activity of the G protein. However, by making use of n-dodecyl octaethylene monoether (C12E8) as the solubilizing agent and by removal of the detergent in two steps, we were able to produce fusogenic G protein liposomes. These G protein liposomes fuse to the BHK-21 cell surface at pH 5.7-6.0 with an efficiency of fusion comparable with that of the parent virus. Physical and chemical analysis revealed that the fusogenic liposomes exhibited a protein to lipid weight ratio of 0.67 and showed an average diameter of 130 nm.     

A new method for reconstitution of highly fusogenic sendai virus envelopes

A new way for reconstituting highly fusogenic Sendai virus envelopes is described. As opposed to previously described methods, in the present one the detergent (Triton X-100) is removed by direct addition of SM-2 Bio-beads to the detergent solubilized mixture of the viral phospholipids and glycoproteins, thus avoiding the long dialysis step. The vesicles obtained in the present work resemble, in their composition, size and features, envelopes of intact Sendai virus particles. The present method allows the enclosure of low and high molecular weight material within the reconstituted viral envelopes.     

Fusion of Sendai virions or reconstituted Sendai virus envelopes with liposomes or erythrocyte membranes lacking virus receptors

Incubation of intact Sendai virions or reconstituted Sendai virus envelopes with phosphatidylcholine/cholesterol liposomes at 37 degrees C results in virus-liposome fusion. Neither the liposome nor the virus content was released from the fusion product, indicating a nonleaky fusion process. Only liposomes possessing virus receptors, namely sialoglycolipids or sialoglycoproteins, became leaky upon interaction with Sendai virions. Fusion between the virus envelopes and phosphatidylcholine/cholesterol liposomes was absolutely dependent upon the presence of intact and active hemagglutinin/neuraminidase and fusion viral envelope glycoproteins. Fusion between Sendai virus envelopes and phosphatidylcholine/cholesterol liposomes lacking virus receptors was evident from the following results. Anti-Sendai virus antibody precipitated radiolabeled liposomes only after they had been incubated with fusogenic Sendai virions. Incubation of N-4-nitrobenzo-2-oxa-1,3-diazole-labeled fusogenic reconstituted Sendai virus particles with phosphatidylcholine/cholesterol liposomes resulted in fluorescence dequenching. Incubation of Tb3+-containing virus envelopes with phosphatidylcholine/cholesterol liposomes loaded with sodium dipicolinate resulted in the formation of the chelation complex Tb3+-dipicolinic acid, as was evident from fluorescence studies. Virus envelopes fuse efficiently also with neuraminidase/Pronase-treated erythrocyte membranes, i.e. virus receptor-depleted erythrocyte membranes, although fusion occurred only under hypotonic conditions.     

Epstein-Barr virus co-reconstituted with Sendai virus envelopes infects Epstein-Barr virus-receptor negative cells

Epstein-Barr virus (EBV) was co-reconstituted with Sendai virus envelopes. The reconstituted "hybrid' virus could bind and penetrate into EBV-receptor negative cells. Using this approach, T-cell-derived human and mouse leukemia cells, human T-lymphocytes and mouse spleen cells were successfully infected as judged by the induction of EBV-determined antigens and stimulation of DNA synthesis. The T-cell-derived human leukemia line Molt-4, that can absorb EBV but without virus penetration, could be also infected by the reconstituted EBV.     

Fusogenic properties of Sendai virosome envelopes in rat brain preparations

Sendai virosomes were characrerized with respect to their ability to bind to, fuse with, and introduce substances into several rat brain preparations. Encapsulation efficiency for Sendai virosomes was enhanced but binding to cerebral cortical P₂ preparations was attenuated by addition of bovine brain phosphatidylcholine during reconstitution. A higher percentage of Sendai virosomes than phosphatidylcholine liposomes appeared to bind to, fuse with and subsequently deliver [¹⁴C]sucrose into osmotically labile pools of the P₂ preparation. Fusogenic activity was estimated by measuring dequenching of fluorescently labelled N-NBD-phosphatidylethanolamine. More virosomally encapsulated [¹⁴C]sucrose was bound to the P₂ fraction than introduced into osmotically labile organelles, and the fraction of vesicles undergoing fusion was intermediate between these two values. Non-encapsulated [¹⁴C]sucrose did not bind to and was not taken up by the P₂ fraction in a quantifiable manner. Virosomal envelopes also bound to primary cultures of rat brain neurons and glia in an apparently saturable manner. Addition of increasing amounts of the adenoassociated virus-derived vector pJDT95 increased encapsulation efficiency, and virosomes reconstituted in the presence of 60 g DNA retained most of their binding activity (5.4% of total label) compared to those containing [¹⁴C]sucrose alone (8.4%). These data indicate that Sendai virosomes may be useful in the delivery of substances into brain-derived tissues, potentially for the modulation of gene expression and neurotransmission.     

Sendai virus envelopes can mediate Epstein-Barr virus binding to and penetration into Epstein-Barr virus receptor-negative cells

Epstein-Barr virus (EBV) receptor-negative cells were treated with UV-inactivated Sendai virus (SV) or with reconstituted SV envelopes having a low hemolytic activity and then assayed for EBV binding or for susceptibility to EBV infection. EBV binding was assessed by using both unlabeled and fluoresceinated EBV preparations. It was found that SV or SV envelope treatment renders these cells able to bind EBV. Various experiments were performed to clarify the mechanism of this SV-induced binding. The EBV receptor-negative 1301 cells were treated with SV either at 0°C or at both 0 and 37°C successively and then examined for EBV binding at 0°C. It was thus found that when SV treatment was performed exclusively at 0°C, the target cells showed higher fluorescence intensity after their incubation with fluoresceinated EBV. In addition, Clostridium perfringens neuraminidase treatment of 1301 cells did not induce any EBV binding to these cells. These data indicate that EBV binding is not due to the disturbance of the cell membrane by SV envelope fusion or to the uncovering of EBV binding sites on the cells after the enzymatic action of SV neuraminidase. Moreover, bound EBV was partly eluted from SV-treated 1301 cells at 37°C, and the treatment of EBV with C. perfringens neuraminidase inhibited its SV-mediated binding. These data indicate that EBV binds to the hemagglutinin-neuraminidase of SV on the target cell surface and that a fraction of the bound EBV becomes irreversibly associated with the SV-treated cell membrane. Our data also show that EBV can penetrate into 1301 cells which have incorporated SV envelopes into their membrane, as demonstrated by the induction of the EBV-determined nuclear antigen by B95-8 EBV in SV envelope-treated 1301 cells.     

Cytotoxicity acquired by ribosome-inactivating proteins carried by reconstituted Sendai virus envelopes

Association of the ribosome-inactivating proteins (RIPs): pokeweed antiviral protein (PAP), gelonin, Momordica charantia inhibitor (MCI), with reconstituted Sendai virus envelopes (RSVE) was obtained without detectable loss of activities either of RIPs or of viral envelope glycoproteins. RIPs are inactive towards intact cells, but, once encapsulated in RSVE, they become cytotoxic. The concentration of RSVE-associated PAP, which causes 50% inhibition of protein synthesis by Friend erythroleukemic cells, is 0.5 ng/ml. Substances capable to inhibit the viral activities block the acquired cytotoxicity of RIPs associated to RSVE.     

Reconstitution of functional influenza virus envelopes and fusion with membranes and liposomes lacking virus receptors.

Reconstituted influenza virus envelopes were obtained following solubilization of intact virions with Triton X-100. Quantitative determination revealed that the hemolytic and fusogenic activities of the envelopes prepared by the present method were close or identical to those expressed by intact virions. Hemolysis as well as virus-membrane fusion occurred only at low pH values, while both activities were negligible at neutral pH values. Fusion of intact virions as well as reconstituted envelopes with erythrocyte membranes--and also with liposomes--was determined by the use of fluorescently labeled viral envelopes and fluorescence dequenching measurements. Fusion with liposomes did not require the presence of specific virus receptors, namely sialoglycolipids. Under hypotonic conditions, influenza virions or their reconstituted envelopes were able to fuse with erythrocyte membranes from which virus receptors had been removed by treatment with neuraminidase and pronase. Inactivated intact virions or reconstituted envelopes, namely, envelopes treated with hydroxylamine or glutaraldehyde or incubated at low pH or 85 degrees C, neither caused hemolysis nor possessed fusogenic activity. Fluorescence dequenching measurements showed that only fusion with liposomes composed of neutral phospholipids and containing cholesterol reflected the viral fusogenic activity needed for infection.     

Fusion between Sendai virus envelopes and biological membranes as monitored by energy transfer methods

Chlorophyll a and chlorophyll b have been inserted into reconstituted envelopes of Sendai virus particles. Fluorescence measurements indicated a high efficiency of energy transfer between the two chlorophyll molecules due to their close proximity in the viral envelope. Fusion of reconstituted, pigmented virus envelopes with various biological cell membranes at 37 degrees C resulted in a significant decrease in the yield of energy transfer. Reduction in the efficiency of energy transfer was temperature and time dependent, and was also dependent upon the ratio between the reconstituted Sendai virus envelopes (donor) and recipient cells (acceptor). No reduction in the efficiency of energy transfer was observed when non-fusogenic, reconstituted viral envelopes were incubated with cell membranes.     

Hemagglutinin-neuraminidase enhances F protein-mediated membrane fusion of reconstituted Sendai virus envelopes with cells.

Reconstituted Sendai virus envelopes containing both the fusion (F) protein and the hemagglutinin-neuraminidase (HN) (F,HN-virosomes) or only the F protein (F-virosomes) were prepared by solubilization of the intact virus with Triton X-100 followed by its removal by using SM2 Bio-Beads. Viral envelopes containing HN whose disulfide bonds were irreversibly reduced (HNred) were also prepared by treating the envelopes with dithiothreitol followed by dialysis (F,HNred-virosomes). Both F-virosomes and F,HNred-virosomes induced hemolysis of erythrocytes in the presence of wheat germ agglutinin, but the rates and extents were markedly lower than those for hemolysis induced by F,HN-virosomes. Using an assay based on the relief of self-quenching of a lipid probe incorporated in the Sendai virus envelopes, we demonstrate the fusion of both F,HN-virosomes and F-virosomes with cultured HepG2 cells containing the asialoglycoprotein receptor, which binds to a terminal galactose moiety of F. By desialylating the HepG2 cells, the entry mediated by HN-terminal sialic acid receptor interactions was bypassed. We show that both F-virosomes and F,HN-virosomes fuse with desialylated HepG2 cells, although the rate was two- to threefold higher if HN was included in the viral envelope. We also observed enhancement of fusion rates when both F and HN envelope proteins were attached to their specific receptors.     

Determination of the internal volume of reconstituted Sendai virus envelopes by quenching of calcein fluorescence

Sendal virus envelopes (SVE) were isolated from Sendal virus particles by Triton X-100 solubilization and ultracentrifugation. The envelopes were reconstituted in the presence of the fluorescent dye calcein by gradual removal of the detergent with Bio-beads SM-2. The internal volume of reconstituted Sendal virus envelopes (RSVE) was determined by quenching the fluorescence of calcein with cobalt (II) ions. The internal volume of RSVE was found to be proportional to the initial SVE protein concentration in the recon-stitution mixture, reaching about 18% of the total volume with 5.6 mg of SVE protein per ml. When radiolabelled cloned Epstein-Barr virus DNA fragment was included in the reconstitution mixture, the proportion of DNA associated with the vesicles much exceeded the trapping volume, indicating adsorption of DNA to the internal surface of RSVE. These deter-minations will allow optimization of the use of RSVE as gene-transfer vehicles.     

Glycoproteins of Sendai virus (HVJ) have a critical ratio for fusion between virus envelopes and cell membranes

The biological activity of two glycoproteins, hemagglutinin and neuraminidase (HN) and fusion (F) proteins, of Sendai virus (HVJ) were studied using purified proteins. The proteins were purified by chromatography on DEAE and CM cellulose in the presence of Nonidet P-40 (NP40). The glycoproteins were reconstituted at various ratios of F to HN into lipid vesicles containing fragment A of diphtheria toxin. The association of HN and F proteins with the vesicles was confirmed by electron microscopy and sucrose density gradient centrifugation. The cytotoxic activity of vesicles containing fragment A on fusion with L cells was determined by measuring colony formation of the cells. It was found that for maximum cytotoxic activity of the vesicles, there was an optimal ratio of F to HN of two. This suggests that HN is not merely the initial binding site to the cell surface, and that interactions between HN and F proteins on the virus surface may be important for the biological activities of these proteins on the cells.     

Biological properties of plaque-size variants of Sendai virus

Large (RL)-and small (RS)-plaque variants of Sendai virus were isolated in culture of LLCMK2 cells in the presence of trypsin and their biological properties were determined. The RL variant was more virulent to mice than the RS variant. The RL variant had a higher growth rate than the RS variant in multiple-step growth in the presence of trypsin, but the two variants had an almost equal growth rate in its absence. Restoration of hemolytic activity in cleavage of the F protein of the RL variant were achieved by milder trypsin treatment than was needed for the RS variant.