Membrane Fusion Mediated Targeted Cytosolic Drug Delivery Through scFv Engineered Sendai Viral Envelopes.

Antibody targeted cytoplasmic delivery of drugs is difficult to achieve as antigen-antibody interaction results in the payload being directed to the endosomal compartment. However, Sendai viral envelopes can bring about cytoplasmic delivery due to F-protein mediated membrane fusion. In this study we have generated and fused a recombinant scFv directed to the onco-fetal antigen, the Placental isozyme of Alkaline Phosphatase (PAP) with the trans-membrane and part of the cytoplasmic domain of the Sendai F protein (F(TMC)). Reconstituted virosomes, having both the fusion protein as well as the native F-protein were able to specifically bind and deliver drugs to PAP expressing cells. About 75% of the delivery was cytoplasmic in nature. Hence, this immuno-virosome, which is devoid of the comparatively more toxic HN protein, has the novel ability to combine specific antibody mediated targeting with cytoplasmic delivery. The scFv ensured specific binding to PAP expressing cells, without cross reacting with the other isozymes of alkaline phosphatase. The advantages of cytoplasmic delivery would include reduced degradation and lowered immunogenicity of the payload and carrier. The ubiquitous expression of PAP on a variety of cancers like seminoma, choriocarcinoma, cervical and breast cancers also suggests its potential usefulness in a number of malignancies.

Sustained activation of mitogen-activated protein kinases and activator protein 1 by the hepatitis B virus X protein in mouse hepatocytes in vivo.

Transcriptional activation of diverse cellular genes by the X protein (HBx) of hepatitis B virus (HBV) has been suggested as one of the mechanisms for HBV-associated hepatocellular carcinoma. However, such functions of HBx have been studied using transformed cells in culture and have not been examined in the normal adult hepatocytes, a natural host of HBV. Using an efficient hepatocyte-specific virus-based gene delivery system developed in our laboratory earlier, we studied the HBx action in vivo. We demonstrate that following virosome-mediated delivery of HBx DNA, a large population (>50%) of hepatocytes express the HBx protein in a dose-dependent manner, which induces a significant increase in the activity of extracellular signal-regulated kinases (ERKs) in the livers of HBx-transfected mice. Inhibition of HBx-induced ERK activation following intravenous administration of PD98059, a mitogen-activated protein kinase kinase kinase (MEK) inhibitor, confirmed the requirement for MEK in the activation of ERKs by HBx. Induction of ERK activity by HBx was sustained for up to 30 days. Interestingly, sustained activation of c-Jun N-terminal kinases (JNKs) for up to 30 days was also noted. Such constitutive ERK and JNK activation as a consequence of continued HBx expression also led to sustained stimulation of further downstream events, such as increased levels of c-Jun and c-Fos proteins along with the persistent induction of activator protein 1 binding activity. Taken together, our data suggest a critical role of these molecules in HBx-mediated cell transformation.

An internal segment (residues 58-119) of the hepatitis B virus X protein is sufficient to activate MAP kinase pathways in mouse liver.

The human hepatitis B virus X protein (HBx) is known as a dual-specificity transactivator stimulating the transcriptional machinery in the nucleus and signal transduction pathways in the cytoplasm. HBx-induced activation of mitogen-activated protein kinase (MAPK) signaling cascades is considered to play an important role in hepatitis B virus-mediated hepatocarcinogenesis. Herein, we have identified the regions of HBx that are crucial for activating such signaling cascades in vivo. A truncated mutant incorporating regions C-E (amino acids 58-140) was as effective as the full-length HBx in activating MAPKs and enhancing activator protein-1 binding activity. While deletion of region C (amino acids 58-84) or D (amino acids 85-119) led to a drastic loss of function, region E (amino acids 120-140) was dispensable for the activation of signaling cascades. Overall, these findings provide the first evidence for the requirement of domain 58-119 of HBx in transmitting mitogenic signals to the nucleus in vivo.

Site-specific gene delivery in vivo through engineered Sendai viral envelopes.

Inspite of several stimulating developments in gene therapy, the formulation of a targeted gene delivery "vector" is still far from ideal. We have demonstrated the potential of reconstituted Sendai viral envelopes containing only the fusion glycoprotein (F-virosomes) in targeted delivery of reporter genes to liver cells of BALB/c mouse in vivo. The membrane fusion-mediated high efficiency of gene transfer to liver cells was ascertained following a critical evaluation of the level of the DNA, mRNA, and relevant proteins. Furthermore, the involvement of viral glycoprotein both as a unique natural ligand and as a membrane fusogen could lead to preferential transfection of parenchymal cell types of liver. The integration of transgenes in the mouse chromosomal DNA and its stable expression up to 4 mo after single i.v. administration of this gene carrier has bolstered its efficiency and novelty. Moreover, the F-virosomes did not elicit significant humoral immune response against the fusion protein in the injected animal. The findings reported here open up the possibility for considering "F-virosomes" as a promising "vehicle" for site-specific DNA delivery in gene therapy.

A 45,000-M(r) glycoprotein in the Sendai virus envelope triggers virus-cell fusion.

Sendai virus envelopes devoid of hemagglutinin-neuraminidase but containing the fusion protein (F-virosomes) were prepared. F-virosomes exhibited discernible serine protease activity at neutral pH. Electrophoretic analysis of the protein profile of the F-virosomes under nonreducing conditions, by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing, led to the identification of a previously unknown glycoprotein with a relative molecular weight of 45,000 (45K protein) associated with the F protein. The identity of the 45K protein, as distinct from F protein, was established by Western blot analysis with F- and 45K-specific antibodies. This 45K protein forms a nexus with the F protein through noncovalent hydrophobic interactions, as proved by its sensitivity to urea treatment, and it is essential for the proteolytic activity of the F-virosomes as well as for the fusion of the viral envelope with host cell membrane. N-terminal sequence analysis (first 11 amino acids) of this protein showed strong homology (> 90%) to flavivirus NS3 serine proteases but no similarity to any of the Sendai viral proteins. On the basis of the N-terminal sequence, oligonucleotides were designed corresponding to the sense and antisense DNA sequences. Dot blot hybridization and primer extension with these oligonucleotides with the viral and the host genome confirmed the host origin of this protein. Further, the limited proteolytic digestion of the target membrane resulted in significant inhibition of viral fusion with it. On the basis of these results, we postulate a model for the molecular mechanism of F protein-induced membrane fusion, which may provide a rationale for other paramyxoviruses.

Novel gene delivery to liver cells using engineered virosomes.

We have demonstrated for the first time that the reconstituted Sendai viral envelopes containing only the fusion protein (F-virosomes) are efficient vehicles for the delivery of foreign genes specifically into human hepatoblastoma cells (HepG2) in culture. The membrane fusion-mediated entry of CAT (chloramphenicol acetyl transferase) gene into the cells was confirmed and the amount delivered to various subcellular fractions was quantitated. The dose dependence and kinetics of expression of biologically active CAT protein in HepG2 cells was measured. The CAT expression level in F-virosome-mediated delivery was significantly higher than that of Lipofectin or liganded proteo-liposome-mediated gene transfer. This kind of targeted delivery by means of membrane fusion induced by viral envelope glycoprotein may have wide applications to various gene transfer strategies both in vitro and in vivo.

Dilation of the influenza hemagglutinin fusion pore revealed by the kinetics of individual cell-cell fusion events.

We have monitored kinetics of fusion between cell pairs consisting of a single influenza hemaglutinin (HA)-expressing cell and a single erythrocyte (RBC) that had been labeled with both a fluorescent lipid (Dil) in the membrane and a fluorescent solute (calcein) in the aqueous space. Initial fusion pore opening between the RBC and HA-expressing cell produced a change in RBC membrane potential (delta psi) that was monitored by a decrease in Dil fluorescence. This event was followed by two distinct stages of fusion pore dilation: the flux of fluorescent lipid (phi L) and the flux of a large aqueous fluorescent dye (phi s). We have analyzed the kinetics of events that occur as a result of transitions between a fusion pore (FP) and a solute permissive fusion pore (FPs). Our data are consistent with a fusion pore comprising six HA trimers.

F protein induced fusion of Sendai viral envelopes with mouse teratocarcinoma cells through Le(x)-Le(x) interaction.

The efficiency of membrane fusion between reconstituted Sendai viral envelopes containing only the fusion protein (F-virosomes) and the plasma membrane of mouse teratocarcinoma cells (F9) in culture was assessed using an assay based on the relief of self-quenching of a lipid probe incorporated in the F-virosomes. The potential of F-virosomes was also evaluated for a targeted cytosolic delivery of lysozyme to F9 cells. [125I]Lysozyme entrapped into F-virosomes was taken to examine its fusion-mediated transfer to the F9 cells. Target specificity of the F-virosomes was confirmed by the interaction between the terminal Le(x) moiety (Gal beta 1-->4(Fuc alpha 1-->3)GlcNAc) of F protein and the Le(x) determinant on the membrane of F9 cells. Incubation of the loaded F-virosomes with cells led to fusion-mediated delivery, as inferred from the ability of cells to internalize lysozyme in the presence of azide (a potent inhibitor of endocytosis). These results suggest that carbohydrate-carbohydrate interaction is strong enough for target cell recognition followed by phospholipid bilayer melding induced by fusion glycoprotein of Sendai virus.

Differences in dispersion of influenza virus lipids and proteins during fusion.

Digitally enhanced low-light-level fluorescence video microscopy and immunochemical staining were used to examine influenza virus envelope lipid and protein redistribution during pH-induced fusion. Video microscopy was performed using viruses labeled with either the lipid analogue octadecylrhodamine B (R18) or fluorescein isothiocyanate (FITC) covalently linked to envelope proteins. Viruses were bound to human red blood cells, and the pattern and intensity of fluorescence were monitored for 30 min while cell-virus complexes were perfused with pH 7.4 or 4.8 media at temperatures either above or below 20 degrees C. R18 showed complete redistribution and dequenching by 30 min at all incubation temperatures, confirming reports that viral fusion occurs at subphysiological temperatures. FITC-labeled protein showed spatial redistribution at 28 degrees C but no change at low temperature. Electron microscopy observations of immunochemical staining of viral proteins confirmed both that protein redistribution at 37 degrees C was slower than R18 and the failure of movement within 30 min at 16 degrees C. Video microscopy monitoring of RNA staining by acridine orange of virus-cell complexes showed redistribution to the RBCs at all temperatures but only after low pH-induced fusion. The results are consistent with differential dispersion of viral components into the RBC and the existence of relatively long-lived barriers to diffusion subsequent to fusion pore formation.

Restricted movement of lipid and aqueous dyes through pores formed by influenza hemagglutinin during cell fusion.

The fusion of cells by influenza hemagglutinin (HA) is the best characterized example of protein-mediated membrane fusion. In simultaneous measurements of pairs of assays for fusion, we determined the order of detectable events during fusion. Fusion pore formation in HA-triggered cell-cell fusion was first detected by changes in cell membrane capacitance, next by a flux of fluorescent lipid, and finally by flux of aqueous fluorescent dye. Fusion pore conductance increased by small steps. A retardation of lipid and aqueous dyes occurred during fusion pore fluctuations. The flux of aqueous dye depended on the size of the molecule. The lack of movement of aqueous dyes while total fusion pore conductance increased suggests that initial HA-triggered fusion events are characterized by the opening of multiple small pores: the formation of a "sieve".

Effect of substitution of hemagglutinin-neuraminidase with influenza hemagglutinin on Sendai virus F protein mediated membrane fusion.

Recombinant virosomes containing fusion protein (F) of Sendai virus and the envelope glycoproteins hemagglutinin (HA) and neuraminidase (NA) of influenza virus within the same membrane were prepared. Such hybrid vesicles were found to hemolyse red blood cells both at pH 7.4 and pH 5.0. Hemolysis induced by hybrid vesicles was much higher than seen with F-virosomes in the presence of WGA, but was about two-fold less than the hemolysis caused by F,HN-virosomes. Reconstituted influenza virus envelopes and F-virosomes failed to induce hemolysis at pH 7.4. Using a fluorescence probe-based lipid mixing fusion assay, hybrid virosomes were found to fuse with cultured HeLa cells both at pH 7.4 as well as pH 5.0. The data indicate that the presence of Sendai virus HN protein in the virosomal membrane is not absolutely essential for the virosome cell fusion process.

Fusion-mediated microinjection of lysozyme into HepG2 cells through hemagglutinin neuraminidase-depleted Sendai virus envelopes.

The potential of reconstituted Sendai viral envelopes containing only the fusion protein (F-virosomes) was evaluated for a targeted cytosolic delivery of lysozyme to human hepatoblastoma cells (HepG2) in culture. 125I-Lysozyme loaded into F-virosomes was used to monitor its fusion-mediated transfer to the HepG2 cells. Using fusion assay based on the transfer of water soluble probe, we have demonstrated the existence of aqueous connection between F-virosomes and target cells. Target specificity of the F-virosomes was ensured by the strong interaction between terminal beta-galactose moiety of F protein and the asialoglycoprotein receptor on the membrane of HepG2 cells. Incubation of the loaded F-virosomes with cells resulted in fusion-mediated injection, as inferred from the ability of cells to internalize lysozyme in the presence of azide (an inhibitor of the endocytotic process). Binding as well as fusion of the F-virosomes to HepG2 cells was solely mediated by the F protein. Introduction of 125I-lysozyme into the HepG2 cells was confirmed by selective accumulation of acid and antibody-precipitable radioactivity in the cytosolic compartment. The structural integrity of the internalized lysozyme was also assessed. The potential usefulness of F-virosomes with defined specificities as biological carrier for both in vitro and in vivo cytosolic delivery of macromolecules and drugs has been established.

Kinetics of fusion with cells of reconstituted Sendai virus envelopes lacking hemagglutinin-neuraminidase.

The fusion potential of reconstituted Sendai virus envelopes containing only the F protein (F-virosomes) has been assessed. F-virosomes and F,HN-virosomes were prepared by solubilization of the intact virus in Triton X-100 followed by its removal using SM-2 biobeads. Viral envelopes containing HN whose disulphide bonds were irreversibly reduced (HNred) were also prepared by treating the envelopes with dithiothreitol followed by dialysis. Both F-virosomes and F,HNred-virosomes hemolysed red blood cells in the presence of wheat germ agglutinin. The rates and extent of hemolysis induced by these virosomes were, however, significantly lower than that induced by F,HN-virosomes. Using a fluorescence probe based membrane mixing fusion assay, F- and F,HNred-virosomes were found to fuse with cultured HeLa cells in the presence of wheat-germ agglutinin. A direct comparison of the fusion activity of F,HN-virosomes and F-virosomes was made by using desialylated HepG2 cells as target containing the asialoglycoprotein receptor (ASGP-R) that binds to a terminal beta-galactose moiety of F protein. A 2- to 3-fold enhancement in the fusion rate when HN was included in the viral envelope was observed. Based on the kinetic data, a model for fusion of paramyxo-virosomes with HepG2 cells is proposed.

Reconstituted Sendai virus envelopes as biological carriers: dual role of F protein in binding and fusion with liver cells.

We have assessed the potential of reconstituted Sendai viral envelopes containing only the fusion protein (F-virosomes) as biological carriers for the delivery of drugs and macromolecules. [125I]lysozyme entrapped in F-virosome is used to study its distribution in various organs of Balb/c mouse in vivo as a function of dose and time. F-virosomes injected intravenously are rapidly cleared from circulation. A major percentage (55-60%) of vesicle contents is delivered to liver at 15 min after injection, showing thereby the liver to be the major site for the accumulation of vesicles. Uptake of virosomes by liver is found to reach a near saturation level at a dose of 0.5 mg F-protein associated with virosomes. In competition studies, the inhibitory effect of asialofetuin on the uptake of F-virosomes suggests the involvement of asialoglycoprotein receptor in its recognition by hepatic parenchymal cells. Incorporation of asialoganglioside-GM1 in the F-virosomes enhanced the uptake by about 1.6-fold. The observed specific interaction of hepatic receptor with F-protein containing a terminal galactose moiety is further supported by degalactosylation of F-virosomes with hard-shelled clam exoglycosidase. The uptake of degalactosylated F-virosomes by liver is found to be significantly reduced. The subcellular radioactivity profile in liver cells exhibits a considerable decrease in cytosolic localisation of the degalactosylated F-virosomal contents with a concomitant increase in their accumulation in lysosomal/mitochondrial fraction as compared to the untreated virosomes. Trypsinized and heat-treated F-virosomes also reflect similar subcellular distribution profile as that of degalactosylated virosomes. Moreover, F-virosomes are able to interact and deliver [125I]lysozyme to the HepG2 cells in culture in the presence of a potent inhibitor of endocytotic process. These results indicate the involvement of specific binding of F-proteins with hepatic receptors followed by their fusion with the membrane of liver cells in the delivery of [125I]lysozyme. The findings reported here open up the possibility of using F-virosomes with defined specificity as fusogenic vehicles for efficient delivery of drugs and biologically active macromolecules both in vivo and in vitro.

Targeted delivery of hygromycin B using reconstituted Sendai viral envelopes lacking hemagglutinin-neuraminidase.

Hygromycin B was encapsulated in reconstituted Sendai viral envelopes containing only the fusion (F) protein (F-virosomes). Incubation of loaded F-virosomes with cultured HepG2 cells resulted in fusion mediated delivery of hygromycin B to the cell cytoplasm, as was inferred from inhibition of DNA synthesis. Binding of the F-virosomes to HepG2 cells was mediated by the interaction of terminal beta-galactose residues of fusion protein with asialoglycoprotein receptor on HepG2 cells, subsequently leading to fusion between the two membranes. The cytotoxic effect of hygromycin B enclosed in F-virosomes was comparable with that of F,HN-virosomes containing both hemagglutinin-neuraminidase (HN) and F protein and F,HNred-virosomes containing HN whose disulfide bonds were irreversibly reduced (HNred). Hygromycin B loaded fusogenic liposomes were prepared by coreconstituting the viral envelope containing only fusion protein with exogenous lipids. These fusogenic liposomes were found to be more active than F-virosomes at the same fusion protein concentrations.

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.

Single cell fusion events induced by influenza hemagglutinin: studies with rapid-flow, quantitative fluorescence microscopy.

Fusion of individual human erythrocytes to fibroblasts expressing the influenza virus hemagglutinin Cells were attached to coverslips fitted in a specially designed flow chamber mounted on a microscope stage, and fusion was triggered by rapid acidification to pH less than 5.2. Fusion between single cell pairs was monitored by a fluorescence increase due to redistribution of fluorescent dyes between either membrane or cytoplasmic compartments of fusing cells. The single cell fusion events were broadly heterogenous in lag times, rise times, and overall shape of the curves. Lag times obtained with a water-soluble dye were within the range obtained with a water-soluble dye were within the range obtained with the membrane-bound fluorophores, (10-160 s). Fusion was both all-or-nothing and irreversible, in that once dye redistribution in any cell commenced, it completed, regardless of pH. Short pulses of pH 4.9 for 6-10 s led to about half of the cell pairs fusing, but pulses greater than 14 s were as effective as constant low pH. Pulses that were too short to trigger fusion did not partially activate nor deactivate the fusion process, as shown by the ability of a second acidification to cause fusion of the same cells, with similar lag times. These results indicate that the overall hemagglutinin-mediated fusion process is composed of at least two stages, one required for commitment of the hemagglutinin to a fusogenic state that is pH-dependent and a maturation stage that is pH-independent.

Observation of single influenza virus-cell fusion and measurement by fluorescence video microscopy.

We have used intensified video fluorescence microscopy and digital image processing to observe and quantitate influenza virus (A/PR8/34/H1N1) fusion to human erythrocyte membranes. Viruses labeled with the lipid probe octadecylrhodamine B (R18) were seen to undergo fluorescence dequenching and eventual disappearance after exposure to pH levels known to induce virus-cell membrane fusion. Quantitative intensity measurements of single individual particles were possible. From these fluorescence data it has been possible to calculate the fraction of R18 dye molecules transferred from the virus to the cell. The redistribution of the lipid probe upon fusion at pH 5.0 had a t1/2 of 46 s, longer than expected for a free-diffusion model. The R18 loss was approximately twice as fast at pH 5.0 as at pH 5.1. No obvious delay until the start of fluorescence dequenching was observed after the pH changes, suggesting that activation processes are faster than the time resolution, 1-5 s, of the current method.

Initial stages of influenza hemagglutinin-induced cell fusion monitored simultaneously by two fluorescent events: cytoplasmic continuity and lipid mixing.

We have monitored the mixing of both aqueous intracellular and membrane-bound fluorescent dyes during the fusion of human red blood cells to influenza hemagglutinin-expressing fibroblasts using fluorescence spectroscopy and low light, image-enhanced video microscopy. The water-soluble fluorescent dye, N-(7-nitrobenzofurazan-4-yl)taurine, was incorporated into intact human red blood cells. The fluorescence of the dye in the intact red blood cell was partially quenched by hemoglobin. The lipid fluorophore, octadecylrhodamine, was incorporated into the membrane of the same red blood cell at self-quenching concentrations (Morris, S. J., D. P. Sarkar, J. M. White, and R. Blumenthal. 1989. J. Biol. Chem. 264: 3972-3978). Fusion, which allowed movement of the water-soluble dye from the cytoplasm of the red blood cell into the hemagglutinin-expressing fibroblasts, and movement of octadecylrhodamine from membranes of red blood cell to the plasma membrane of the fibroblasts, was observed by fluorescence microscopy as a spatial relocation of dyes, and monitored by spectrofluorometry as an increase in fluorescence. Upon lowering the pH below 5.4, fluorescence increased after a delay of about 30 s at 37 degrees C, reaching a maximum within 3 min. The kinetics, pH profile, and temperature dependence were similar for both fluorescent events measured simultaneously, indicating that influenza hemagglutinin-induced fusion rapidly establishes bilayer continuity and exchange of cytoplasmic contents.