Development of a transferrin receptor-targeting HVJ-E vector

The development of more effective cancer treatments is anticipated. Tumor-targeted drug delivery is an important strategy in cancer therapy. We have developed an HVJ (hemagglutinating virus of Japan; Sendai virus) envelope (HVJ-E) vector using inactivated Sendai virus. The HVJ-E vector has been observed to target a number of cell lines since its hemagglutinin-neuraminidase (HN) protein recognizes the sialic acids of host cells. Thus, to reduce non-specific binding of the HVJ-E vector, we eliminated HN protein using HN-specific short interfering RNA (siRNA). Then, to further increase its tumor-targeting ability, we constructed HN-depleted HVJ containing the F-transferrin chimeric protein. The modified vectors containing Q-dots demonstrated 32-fold greater tumor-targeting efficiency than wild-type HVJ-E.     

Efficient gene silencing and cell differentiation using siRNA in mouse and monkey ES cells

Small interfering RNA (siRNA) has been widely used for suppressing gene expression in various organisms. Here, we describe efficient methods to suppress target genes (EGFP or Oct4) using siRNA in mouse and monkey ES cells, and differentiation. In mouse ES cells, FACS analysis revealed that EGFP expression was suppressed in 97% of transfected cells at 48 h after transfection. In addition, cells expressed Hand1 and Cdx2, which are the marker genes of trophoblast lineage by the transient suppression of Oct4. In the case of monkey ES cells, highly efficient suppression was achieved in 98% of cells at 96 h post-transfection using the Sendai virus (hemagglutinating virus of Japan, HVJ) envelope as a carrier of siRNA. These efficient transfection methods using synthetic siRNA should contribute to evaluate specific gene function in ES cells and can be used to differentiate ES cells into desired cell lineages.     

HVJ (Sendai virus)-induced envelope fusion and cell fusion are blocked by monoclonal anti-HN protein antibody that does not inhibit hemagglutination activity of HVJ

Two kinds of monoclonal antibodies against HN protein of HVJ were isolated. In competitive binding assay, binding of one of these antibodies to HN protein did not inhibit binding of the other antibody to the same molecule. One of the antibodies, named HN-1 antibody, inhibited hemagglutination activity of HVJ and also blocked neuraminidase activity of the virus when fetuin and Ehrlich ascites tumor cells were used as substrates, but it did not inhibit the activity when neuramine-lactose was used as substrate. The other antibody, HN-2, did not inhibit hemagglutination activity or neuraminidase activity, but blocked HVJ-induced viral envelope-cell fusion, cell-cell fusion and hemolysis. The mechanism by which HN-2 antibody blocked the fusion process is discussed.     

Regulation of mitochondrial morphology by membrane potential,and DRP1-dependent division and FZO1-dependent fusion reaction in mammalian cells

Mitochondria are dynamic organelles that undergo frequent fission and fusion or branching. To analyze the mitochondrial fusion reaction, mitochondria were separately labeled with green or red fluorescent protein (GFP and RFP, respectively) in HeLa cells, and the cells were fused using hemagglutinating virus of Japan (HVJ). The resulting mixing of the fluorescent reporters was then followed using fluorescence microscopy. This system revealed that mitochondria fuse frequently in mammalian cells, and the fusion depends on the membrane potential across the inner membrane. The protonophore, carbonyl cyanide m-chlorophenylhydrazone (CCCP), led to fragmentation of the mitochondria and inhibited the fusion reaction. Removal of CCCP recovered the fusion activity to reform filamentous mitochondrial networks. Analysis of the effects of GTP-binding proteins, DRP1 and two FZO1 isoforms, and the GTPase-domain mutants on the CCCP-induced mitochondrial morphologic changes revealed that DRP1 and FZO1 are involved in membrane budding and fusion, respectively. Furthermore, a HVJ-dependent cell fusion assay combined with RNA interference (RNAi) demonstrated that both FZO1 isoforms are essential and must be acting in cis for the mitochondrial fusion reaction to occur.     

HN glycoprotein of HVJ (Sendai virus) enhances the selective cytotoxicity of diphtheria toxin fragment A-containing liposomes on subacute sclerosing panencephalitis virus-infected cells

Fragment A of diphtheria toxin-containing liposomes (naked liposomes) selectively kill subacute sclerosing panencephalitis virus-infected cells (SSPE cells) (Exp cell res 132 (1981) 259) [10]. Fragment A-containing liposomes associated with either hemagglutinating and neuraminidase (HN) or fusion (F) glycoprotein of HVJ (Sendai virus) were prepared. These liposomes did not kill normal cultured cells. Fragment A-containing liposomes associated with HN protein were much more cytotoxic than naked liposomes containing fragment A to SSPE cells. Their cytotoxicity to the SSPE cells was influenced by the duration of incubation and the amount of HN protein. Fragment A-containing liposomes associated with F protein had about the same cytotoxicity on SSPE cells as had naked liposomes containing fragment A. Fragment A-containing liposomes associated with wheat germ agglutinin (WGA) were also prepared, but these also had the same toxicity as naked liposomes containing fragment A. The effects of monoclonal antibodies against HN protein on the cytotoxicity on SSPE cells of fragment A-containing liposomes associated with HN were studied. The significance of these results with regard to the actions of HN protein and possible reasons for the selective killing of SSPE cells are discussed.     

Effect of cytolytic infection on maintenance of resistance to HVJ (Sendai virus) in an altered BHK cell culture

Altered baby hamster kidney (BHK-R) cells were serially cultured in the continuous presence of hemagglutinating virus of Japan (HVJ). These cells showed a distinct resistance to superinfection with the homologous HVJ. This resistance of BHK-R cells gradually disappeared after serial passages in the presence of ultraviolet-irradiated HVJ particles which lost infectivity but still preserved hemagglutinating and neuraminidase activities. When BHK-R cells were serially cultured in the presence of a temperature-sensitive mutant of HVJ at non-permissive temperature, the cells also lost the resistance. The resistance of BHK-R cells remained unchanged, even after prolonged incubation in virus-free maintenance medium under the conditions of no cell division. It was suggested that killing of virus-sensitive cells, which were generated during cell proliferation, was required for maintenance of the resistance.     

Role of neuraminidase in the morphogenesis of influenza B virus.

When ts7, a temperature-sensitive (ts) mutant of influenza B/Kanagawa/73 virus, infected MDCK cells at the nonpermissive temperature (37.5 degrees C), infectious virus was produced at very low levels compared with the yield at the permissive temperature (32 degrees C) and hemagglutinating activity and enzymatic activity of neuraminidase (NA) were negligible. However, viral protein synthesis and transport of hemadsorption-active hemagglutinin to the cell surface were not affected. When the cell lysate was treated with bacterial NA, hemagglutinating activity was recovered but infectivity was not, even after further treatment with trypsin. It was found that ts7 was defective in transport of NA to the cell surface and formation of virus particles. Analysis of the genomes of non-ts recombinants obtained by crossing ts7 and UV-inactivated B/Lee showed that ts7 had the ts mutation only in RNA segment 6 coding for NA and the glycoprotein NB. Nucleotide sequence analysis of the RNA segment revealed that ts7 had four amino acid changes in the NA molecule but not in NB. We suggest that assembly or budding of influenza B virus requires the presence of NA at the plasma membrane, unlike influenza A virus.     

Rapid delivery of small interfering RNA by biosurfactant MEL-A-containing liposomes

The downregulation of gene expression by RNA interference holds great potential for genetic analysis and gene therapy. However, a more efficient delivery system for small interfering RNA (siRNA) into the target cells is required for wide fields such as cell biology, physiology, and clinical application. Non-viral vectors are stronger candidates than viral vectors because they are safer and easier to prepare. We have previously used a new method for gene transfection by combining cationic liposomes with the biosurfactant mannosylerythritol lipid-A (MEL-A). The novel MEL-A-containing cationic liposomes rapidly delivered DNA (plasmids and oligonucleotides) into the cytosol and nucleus through membrane fusion between liposomes and the plasma membrane, and consequently, enhanced the gene transfection efficiency. In this study, we determined the efficiency of MEL-A-containing cationic liposomes for siRNA delivery. We observed that exogenous and endogenous protein expression was suppressed by approximately 60% at 24 h after brief (30 min) incubation of target cells with MEL-A-containing cationic liposome/siRNA complexes. Confocal microscopic analysis showed that suppression of protein expression was caused by rapid siRNA delivery into the cytosol. We found that the MEL-A-containing cationic liposomes directly delivered siRNA into the cytoplasm by the membrane fusion in addition to endocytotic pathway whereas Lipofectamine™ RNAiMax delivered siRNA only by the endocytotic pathway. It seems that the ability to rapidly and directly deliver siRNA into the cytosol using MEL-A-containing cationic liposomes is able to reduce immune responses, cytotoxicity, and other side effects caused by viral vectors in clinical applications.     

Transmembrane phospholipid motions induced by F glycoprotein in hemagglutinating virus of Japan.

Transfer of phospholipid from the envelope of hemagglutinating virus of Japan (HVJ) to erythrocyte (RBC) membrane and the virus-induced transfer of phospholipid between RBC membranes were studied using spin-labeled phosphatidylcholine (PC). The transfer of PC from membranes labeled densely with PC to unlabeled membranes was followed by the peak height increase in the electron spin resonance spectrum. The two kinds of transfer reactions took place very rapidly as reported previously. To obtain further details, the transfer reactions were studied with HVJ, HVJ inactivated by trypsin, HVJ harvested early, HVJ grown in fibroblast cells, the fibroblast HVJ activated by trypsin, influenza virus, and glutaraldehyde-treated RBCs. The results demonstrated that the viral F glycoprotein played a crucial role in the transmembrane phospholipid movements as well as in the fusion and hemolysis of RBCs. The transfer from HVJ to RBC's occurred partially through an exchange mechanism not accompanying the envelope fusion. This was shown by a decrease in the exchange broadening of the electron spin resonance spectrum of released spin-labeled HVJ (HVJ) and also by an increase in the ratio of PC to viral proteins incorporated into RBC membranes. HVJ modified RBC membrane so as to be able to exchange its phospholipids with those of inactive membranes such as fibroblast HVJ, influenza virus, glutaraldehyde-treated RBC'S, and phosphatidylcholine vesicles. HVJ affected the fluidity of RBC membranes markedly, the environments around PC being much fluidized. The virus-induced fusion was discussed based on close apposition of the membranes by HANA proteins and on the destabilization and fluidization of RBC membranes by F glycoproteins.     

Complementation between avirulent Newcastle disease virus and a fusion protein gene expressed from a retrovirus vector: requirements for membrane fusion.

The cDNA derived from the fusion gene of the virulent AV strain of Newcastle disease virus (NDV) was expressed in chicken embryo cells by using a retrovirus vector. The fusion protein expressed in this system was transported to the cell surface and was efficiently cleaved into the disulfide-linked F1-F2 form found in infectious virions. The cells expressing the fusion gene grew normally and could be passaged many times. Monolayers of these cells would plaque, in the absence of trypsin, avirulent NDV strains (strains which encode a fusion protein which is not cleaved in tissue culture). Fusion protein-expressing cells would not fuse if mixed with uninfected cells or uninfected cells expressing the hemagglutinin-neuraminidase (HN) protein. However, the fusion protein-expressing cells, if infected with avirulent strains of NDV, would fuse with uninfected cells, suggesting that fusion requires both the fusion protein and another viral protein expressed in the same cell. Fusion was also seen after transfection of the HN protein gene into fusion protein-expressing cells. Thus, the expressed fusion protein gene is capable of complementing the virus infection, providing an active cleaved fusion protein required for the spread of infection. However, the fusion protein does not mediate cell fusion unless the cell also expresses the HN protein. Fusion protein-expressing cells would not plaque influenza virus in the absence of trypsin, nor would influenza virus-infected fusion protein-expressing cells fuse with uninfected cells. Thus, the influenza virus HA protein will not substitute for the NDV HN protein in cell-to-cell fusion.     

Restitution of hemagglutinating activity to spikeless particles of HVJ (Sendai virus) by glycoprotein components of Newcastle disease virus.

Spikeless particles of HVJ (Sendai virus) lacking in hemagglutinating (HA) activity were obtained by enzymatic digestion of virions with trypsin followed by centrifugation through a sucrose gradient. When they were mixed with glycoprotein components of Newcastle disease virus (NDV) obtained by treatment of purified virions with deoxycholate (DOC), the mixture showed hemagglutination reaction, which was inhibited by anti-NDV serum, but not by anti-HVJ serum. Sedimentation profile of the HA active agents was then examined by centrifugation of the mixture of spikeless particles of HVJ (labeled with 3H-uridine) and glycoproteins of NDV (labeled with 14C-amino acid mixture). The results showed that the peak of HA activity had both of the radioactivities, and that the sedimentation rate of the HA was faster than that of spikeless HVJ but slower than that of intact HVJ. Electron micrographs of such HA active structures showed that they were morphologically closely similar to intact virion of HVJ, although they had neither hemolytic activity nor infectivity. The mixture of spikeless HVJ and glycoproteins of HVJ or NDV which were removed from virions by proteolytic enzymes, on the other hand, did not show any detectable hemagglutinating activity.     

Cellular delivery of siRNA mediated by fusion-active virosomes

RNA interference is expected to have considerable potential for the development of novel specific therapeutic strategies. However, successful application of RNA interference in vivo will depend on the availability of efficient delivery systems for the introduction of small-interfering RNA (siRNA) into the appropriate target cells. This paper focuses on the use of reconstituted viral envelopes ("virosomes"), derived from influenza virus, as a carrier system for cellular delivery of siRNA. Complexed to cationic lipid, siRNA molecules could be efficiently encapsulated in influenza virosomes. Delivery to cultured cells was assessed on the basis of flow cytometry analysis using fluorescently labeled siRNA. Virosome-encapsulated siRNA directed against Green Fluorescent Protein (GFP) inhibited GFP fluorescence in cells transfected with a plasmid encoding GFP or in cells constitutively expressing GFP. Delivery of siRNA was dependent on the low-pH-induced membrane fusion activity of the virosomal hemagglutinin, supporting the notion that virosomes introduce their encapsulated siRNA into the cell cytosol through fusion of the virosomal membrane with the limiting membrane of cellular endosomes, after internalization of the virosomes by receptor-mediated endocytosis. It is concluded that virosomes represent a promising carrier system for cellular delivery of siRNA in vitro as well as in vivo.     

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.     

Mouse C2 myoblast cells resist HVJ (Sendai virus)-mediated cell fusion in the proliferating stage but become capable of fusion after differentiation

To investigate the mechanism of myoblast fusion, we attempted to prepare artificial myotubes of mouse C2 myoblast cells using the hemagglutinating virus of Japan (HVJ, Sendai virus). Proliferating C2 cells showed strong resistance to HVJ-mediated cell fusion and remained morphologically unchanged even though massive numbers of virions adsorbed onto their surface. They showed no membrane disruption, which occurs in the early stage of cell fusion induced by HVJ. These observations suggest that proliferating C2 cells are resistant to HVJ-mediated cell fusion. However, upon induction of differentiation, C2 cells gradually became capable of fusion induced by HVJ and then even generated heterokaryons with Ehrlich ascites tumor cells. When differentiated C2 cells that had become fusion-sensitive were treated with HVJ in the presence of EDTA, they did not fuse but degenerated, suggesting that their cell membranes were transiently disrupted by interaction with HVJ. These results suggest that the cell membranes of myoblasts change to a fusion-capable state during the process of differentiation.     

Cellular Delivery of siRNA Mediated by Fusion-Active Virosomes

RNA interference is expected to have considerable potential for the development of novel specific therapeutic strategies. However, successful application of RNA interference in vivo will depend on the availability of efficient delivery systems for the introduction of small-interfering RNA (siRNA) into the appropriate target cells. This paper focuses on the use of reconstituted viral envelopes (“virosomes”), derived from influenza virus, as a carrier system for cellular delivery of siRNA. Complexed to cationic lipid, siRNA molecules could be efficiently encapsulated in influenza virosomes. Delivery to cultured cells was assessed on the basis of flow cytometry analysis using fluorescently labeled siRNA. Virosome-encapsulated siRNA directed against Green Fluorescent Protein (GFP) inhibited GFP fluorescence in cells transfected with a plasmid encoding GFP or in cells constitutively expressing GFP. Delivery of siRNA was dependent on the low-pH-induced membrane fusion activity of the virosomal hemagglutinin, supporting the notion that virosomes introduce their encapsulated siRNA into the cell cytosol through fusion of the virosomal membrane with the limiting membrane of cellular endosomes, after internalization of the virosomes by receptor-mediated endocytosis. It is concluded that virosomes represent a promising carrier system for cellular delivery of siRNA in vitro as well as in vivo.     

The activity of membranes reconstituted from HVJ envelope proteins and lipids to induce hemolysis and fusion between liposomes and erythrocytes

A simple method for preparation of lipid-free envelope proteins (HN protein and F protein) of HVJ (Sendai virus) was developed. Reconstituted 'envelopes' were then prepared from envelope proteins and various lipids by the detergent dialysis method, and the activity to induce hemolysis and fusion between liposome and erythrocyte was studied. Lipid-free envelope protein aggregates could induce hemolysis and liposome-erythrocyte fusion. The activity was however greatly augmented by incorporation of envelope proteins into membrane of viral total lipids. Hemolytic and fusogenic activity was somewhat augmented by incorporation of envelope proteins into dipalmitoylphosphatidylcholine/cholesterol (1:1, molar ratio) and dimyristoylphosphatidylcholine/cholesterol (1:1), though the augmentation was lower than that observed with viral total lipids. When 'envelopes' were reconstituted with the proteins and viral total lipids supplemented with phosphatidylethanolamine, two kinds of 'envelopes' were prepared; one was permeable to Dextran (Mr 75000) and hemolytic, and the other was impermeable to Dextran and nonhemolytic. The latter acquired hemolytic activity after subjection to freezing and thawing, and its barrier function was lost concomitantly. The study suggests that envelope proteins (HN protein and F protein) could function without lipids but their activity was greatly influenced by not only the composition of additional lipids but also mode of arrangement of components on the reconstituted membranes.     

A spin-label study on fusion of red blood cells induced by hemagglutinating virus of Japan.

Fusion of red blood cells (RBC) induced by hemagglutinating virus of Japan (HVJ) has been studied using a phosphatidylcholine spin label. The spin label was readily incorporated and diffused into the lipid bilayer portion of the viral envelope. The exchange broadening in the electron spin resonance (ESR) spectrum of densely labeled virus disappeared rapidly when the virus was mixed with RBC at 37 degrees. The spectrum gradually approached that of the host cell spin labeled with the phosphatidylcholine label. The results directly indicate transfer and intermixing of phospholipid molecules between the viral envelope and RBC membrane. The transfer reaction was strongly dependent on temperature. No transfer was observed at lower temperatures where the virus adsorbed to the cell and caused aggregation but no hemolysis and fusion. The transfer rate remained negligibly small until 19 degrees and increased rapidly between 25 and 30 degrees. The virus-induced hemolysis showed similar temperature dependence. The transfer rate was greatly reduced under inhibitory conditions of fusion: glutaraldehyde treatment of RBC, trypsin treatment of HVJ, or the presence of concanavalin A. Only slight transfer was observed from fusion-inactive influenza virus to RBC. The transfer was greatly enhanced by the help of HVJ. The close parallelism suggests that the transfer and intermixing are necessary steps to the cell fusion. The transfer rate was dependent on fluidity of the host cell membrane and independent of the viral dose. The virus-induced transfer of phospholipid molecules between RBC's was also detected by the spin label. Its temperature dependence was quite similar to that for the virus-to-cell transfer. The intercellular transfer was nearly proportional to the viral dose.     

Inhibition of measles virus replication and RNA synthesis by actinomycin D

Measles virus replication and RNA synthesis in Vero cells are inhibited by actinomycin at concentrations which inhibit cellular RNA synthesis. Drug present from the 2nd to the 24th hr post infection inhibited infectivity but not hemagglutinating activity or cell fusion. Infectivity was much less sensitive to drug added during the second 24-hr period, and 52S RNA was labeled and incorporated into virions during this later time interval.