Isolation and characterization of receptor sialoglycoprotein for hemagglutinating virus of Japan (Sendai virus) from bovine erythrocyte membrane

Sialoglycoprotein which exhibits inhibitory activity for hemagglutination by Hemagglutinating Virus of Japan (HVJ, Sendai virus) was isolated from the membrane of bovine erythrocytes. Purification steps for this sialoglycoprotein included extraction with lithium diiodosalicylate, phenol partition, precipitation with ethanol, and chromatography on a phosphocellulose column and an SDS-Sepharose CL-4B column. Purified sialoglycoprotein (GP-2) has high specific activity for inhibiting the hemagglutination with HVJ, and a lesser activity for that with Newcastle disease virus, but it does not inhibit the hemagglutination by influenza A virus. Inhibitory activity of GP-2 on hemagglutination by HVJ is 2,500-fold higher than that of fetuin. Liposomes containing a 10,000-fold larger amount of ganglioside mixture of bovine erythrocytes and those containing a 5,000-fold larger amount of each ganglioside of bovine erythrocytes, N-glycolylneuraminosyl-lactosyl ceramide, sialosyllacto-N-neotetraosyl- and sialosyl-lacto-N-norhexaosyl ceramide, had no inhibitory activity toward hemagglutination with HVJ. GP-2 (mol. wt. 250 K daltons) behaved homogeneously in SDS-polyacrylamide gel electrophoresis. It contained 70% carbohydrate and 30% protein, by weight. N-Acetylgalactosamine, N-acetylglucosamine, galactose, sialic acid (N-glycolylneuraminic acid, 96%; N-acetylneuraminic acid, 4%) were identified as carbohydrate components, in molar ratios of 1.0:4.0:5.2:2.9. All the oligosaccharides of GP-2 appeared to be linked to polypeptide chains by alkali-labile O-glycosidic linkages. Sialidase treatment of GP-2 and conversion of sialic acid residue of the glycoprotein to C8 and C7 analogues resulted in the loss of the inhibitory activity on hemagglutination by HVJ. Oligosaccharides isolated by gel filtration after treatment of GP-2 with alkaline borohydride had also lost the ability to inhibit the hemagglutination by HVJ. The above results indicate that isolated sialoglycoprotein is the endogenous receptor in bovine erythrocyte membrane specific to HVJ, and the hydroxy group linked to the 9-carbon atom of sialic acid and probably also the hydrophobic protein moiety are important for the recognition of HVJ attachment.     

Gangliosides as paramyxovirus receptor. Structural requirement of sialo-oligosaccharides in receptors for hemagglutinating virus of Japan (Sendai virus) and Newcastle disease virus

A sensitive assay system for receptor activity of gangliosides to paramyxovirus was developed. This system involves incorporation of gangliosides into neuraminidase-treated chicken erythrocytes (asialoerythrocytes) followed by estimation of virus-mediated agglutination and hemolysis. The asialoerythrocytes coated with I-active ganglioside (Sia alpha 2-3Gal beta 1-4GlcNAc beta 1-3(Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-6)Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc beta 1-Cer) were effectively agglutinated by hemagglutinating virus of Japan (HVJ, Sendai virus). The hemolysis of the asialoerythrocytes mediated by HVJ was restored to the highest level by labeling the cells with gangliosides possessing lacto-series oligosaccharide chains, i.e., I-active ganglioside, N-acetylneuraminosylparagloboside (SiaPG(NeuAc)), and i-active ganglioside (Sia alpha 2-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc beta 1-Cer). The specific receptor activity of ganglioside GD1a possessing a gangliotetraose chain was lower than those of the gangliosides described above. Gangliosides GM3, GD3, GM1a, GD1b, SiaPG(NeuGc) showed little effect on the restoration of HVJ-mediated hemolysis. On infection with Newcastle disease virus (NDV), the highest specific restoration of lysis was found in chicken asialoerythrocytes coated with SiaPG(NeuAc or NeuGc) and GM3(NeuAc or NeuGc), whereas those coated with I-active ganglioside, GD3, GM1a, and GD1b showed very low NDV-mediated hemolysis. The above results indicate that the determinants of receptor for HVJ contain sialylated branched and/or linear lacto-series oligosaccharides carried by I,i-active gangliosides and SiaPG(NeuAc) and sialosylgangliotetraose chain carried by GD1a. The determinants for NDV are carried by SiaPG(NeuAc or NeuGc) containing linear lacto-series oligosaccharide and GM3(NeuAc or NeuGc). The absence of detectable binding of free oligosaccharides obtained from I-active ganglioside and sialoglycoprotein GP-2 isolated from bovine erythrocyte membranes as HVJ receptor (Suzuki, Y., et al. J. Biochem. (1983) 93, 1621-1633; (1984) 95, 1193-1200) indicates that HVJ recognizes the sialooligosaccharides oriented out of the lipid bilayer in the cell membranes where the hydrophobic ceramide or peptide backbone of the receptor is integrated.     

Occurrence of O-glycosidically peptide-linked oligosaccharides of poly-N-acetyllactosamine type (erythroglycan II) in the I-antigenically active Sendai virus receptor sialoglycoprotein GP-2

Unique high molecular weight (M.W. 4,000-9,000) sugar chains termed erythroglycan II have been obtained from alkali/sodium borohydride digests of I-active asialoglycoprotein derived from sialoglycoprotein GP-2, which was isolated recently from bovine erythrocyte membranes as Sendai virus receptor (Suzuki, Y. et al. (1983) J. Biochem. 93, 1621-1633; (1984) ibid, 95, 1193-1200). It was found that these sugar chains comprise about 40% of total alkali-labile oligosaccharides of asialo GP-2 and contain endo-beta-galactosidase (Flavobacterium keratolyticus)-resistant highly branched and heterogeneous oligosaccharides of poly-N-acetyllactosamine type which are linked O-glycosidically to the peptide backbone through N-acetylgalactosamine. Erythroglycan II also contains endo-beta-galactosidase-susceptible straight terminal polylactosaminyl side chains. A major oligosaccharide released by the enzyme cochromatographed with Gal beta 1-4GlcNAc beta 1-3Gal. Inhibitory activity of Sendai virus-mediated hemagglutination and the receptor activity for the virus were reduced significantly but not completely by the endo-beta-galactosidase. These results indicate that both linear and branched sialosylpolylactosamine sequences in erythroglycan II are important for the reception of the virus into the target cells.     

Reversible susceptibility to natural cell-mediated cytotoxicity observed in altered BHK cells resistant to HVJ (Sendai virus) infection

Altered baby hamster kidney (BHK-R) cells which were subcultured in the continuous presence of HVJ (hemagglutinating virus of Japan--the Sendai strain of parainfluenza 1 virus) showed a high susceptibility to natural cell-mediated cytotoxicity, although BHK-R cells are not transiently or persistently infected with HVJ but contain the restricted amount of sialic acid. By repeated subcultivation of BHK-R cells in growth medium free of HVJ, the sensitivity to natural killer cytotoxicity decreased to the level of normal BHK cells with a counter increase of cellular sialic acid, and the subsequent treatment of the cells with neuraminidase caused a loss of proper sialic acid residues, once again resulting in a significant enhancement of lysis by natural killer cells. In the BHK-R cell system which exhibits a reversible resistance to the interferon action, the enhancing effect induced by interferon on target cell susceptibility to natural killer activity became more pronounced in accord with the recovery of sensitivity to the antiviral action of interferon.     

Fusion of Madin-Darby canine kidney cells by HVJ (Sendai virus): absence of direct association of virus particles with the site of membrane fusion

The process of cell fusion of Madin-Darby canine kidney (MDCK) cells by HVJ (Sendai virus) was investigated to determine whether the HVJ particles were directly associated with the site of membrane fusion. Confluent monolayer cultures of MDCK cells are sealed together by tight junctions on the apices of their lateral membranes, so added virus particles can be adsorbed only to the apical surfaces of the cells. After incubation with HVJ at 37 degrees C for 30 min, the cells still appeared mononucleate and unfused by light microscopy, but electron microscopic examination showed that fusion at the lateral membranes had occurred below the tight junctions. Furthermore, when fluorescein isothiocyanate (FITC)-labeled macromolecules, which cannot pass across the gap junctions, were injected into the cells at this stage, labeled macromolecules were found to diffuse into the adjacent cells. These findings strongly suggest that cell fusion was initiated in the lateral membrane, a region distinct from the site of adsorbed HVJ particles. Thus, the virus particles were not directly associated with the fusion site, but induced fusion of the lateral membranes indirectly.     

Efficient introduction of contents of liposomes into cells using HVJ (Sendai virus)

The conditions for efficient introduction of the contents of liposomes into cells were examined using fragment A of diphtheria toxin (DA) as a marker; one molecule of DA can kill a cell when introduced into the cytoplasm. Liposomes containing DA (DA liposomes) were toxic to cells treated with HVJ (Sendai virus) at 4 degrees C just before exposure to DA liposomes at 37 degrees C, but were not toxic to untreated cells. This toxicity was temperature-dependent. DA outside of liposomes was not toxic to HVJ-treated cells. Results also showed that liposomes could fuse with HVJ at 37 degrees but not at 4 degrees C and that liposomes preincubated with HVJ at 37 degrees C could associate with cells. DA liposomes preincubated with HVJ at 37 degrees C were highly toxic to cells. This toxicity was dependent on the duration of preincubation with HVJ and the dose of HVJ. When plasmid DNA coded herpes simplex virus thymidine kinase was trapped in liposomes and fused with Ltk- cells with HVJ, the thymidine kinase activity was expressed in about 10% of the cells. These data show that naked liposomes fuse efficiently with cells with HVJ and that the contents of the liposomes can be introduced into the cytoplasm 100-10 000 times more efficiently by treatment of the cells or liposomes with HVJ.     

Studies on the interaction of Sendai virus with liposomal membranes. Sendai virus-induced agglutination of liposomes containing glycophorin

Liposomes constituted with the major sialoglycoprotein of human erythrocytes, glycophorin, were used as models for studies on cell-virus interactions. Liposomes composed of egg yolk phosphatidylcholine, cholesterol and glycophorin were found to interact with the paramyxovirus HVJ to form aggregates. The aggregation process was temperature dependent: it was maximal at 0 degrees C and decreased with increase of the incubation temperature. The activity of viral neuraminidase is also temperature dependent, and it increases with increase of the incubation temperature; release of N-acetylneuraminic acid was negligible at 0 degrees C. Shift-up of the incubation temperature immediately cancelled HVJ-induced agglutination of liposomes. Viruses attached to liposomes seemed to be released into the supernatant when the 'virus-liposome' complex formed at 0 degrees C was incubated at 37 degrees C, possibly as a result of breakdown of the 'binding' site by neuraminidase. The characteristics of the interaction of HVJ with liposomes containing glycophorin appeared to be phenomenologically similar to those of HVJ-cell interaction.     

Kinetic analysis of fusion of hemagglutinating virus of Japan with erythrocyte membrane using spin-labeled phosphatidylcholine

HVJ* (hemagglutinating virus of Japan containing spin-labeled phosphatidylcholine in its envelope around 10 mol %) was adsorbed onto erythrocytes or erythrocyte ghosts at various doses, and the ESR spectrum of the virus-cell system was measured at 37 degrees C. The peak-height increase for the HVJ*-ghost system was satisfactorily analyzed on the basis of envelope fusion by a first-order kinetic equation with two different rate constants. The rate constant was obtained as k1 = 0.84 min-1 and k2 = 0.011 min-1, independent of the virus dose. The fraction of virus fused at the rate constant k1 decreased with the dose. However, the average number of fast-fusing viruses per cell was nearly independent of the dose, and the value was one to two. The peak-height increase in the HVJ*-erythrocyte system was caused by both envelope fusion and phospholipid exchange catalyzed by the virus-induced hemolyzate. At lower doses, where the virus-induced hemolysis was small and, therefore, the rate of phospholipid exchange was small, the peak-height increase could be analyzed by the same kinetic equation with nearly the same rate constant value for k1 as that for HVJ*-ghosts. However, the k2 was larger than that for HVJ*-ghost, owing to the additional transfer by phospholipid exchange.     

Functional modification of Sendai virus by siRNA

Sendai virus (hemagglutinating virus of Japan; HVJ) is a negative-strand RNA virus with robust fusion activity, and has been utilized for gene transfer and drug delivery. Hemagglutinin-neuraminidase (HN) protein on the viral membrane is important for cell fusion, but causes agglutination of red blood cells. HN-depleted HVJ has been desired for in vivo transfection in order to improve safety. Here, we succeeded in producing HN-depleted HVJ using HN-specific short interfering RNA (siRNA). Viral production was not affected by the siRNA. HN protein was markedly decreased in the new HVJ, while other viral proteins were retained. Consequently, the hemagglutinating activity was substantially reduced and infection activity was suppressed. When the HN-depleted HVJ was mixed with cultured cells and the mixture was centrifuged for 10min at 2000xg, the modified HVJ recovered its infectivity to approximately 80% of wild HVJ. However, infectivity was abolished in the presence of anti-F antibody. Moreover, transfection of FITC-labeled oligodeoxynucleotides using the modified HVJ was also recovered by centrifugation. Thus, the HN-depleted HVJ produced using siRNA technology will be applicable to a delivery vector.     

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.     

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.     

The improved efficient method for introducing macromolecules into cells using HVJ (Sendai virus) liposomes with gangliosides

Macromolecules such as DNA and RNA can be entrapped within liposomes associated with gangliosides by reverse-phase evaporation. When these liposomes are incubated with HVJ2 (Sendai virus), they deliver their contents into cultured cells efficiently. More than 95% cells of a Ltk- cell line (thymidine kinase-deficient cells) transiently expressed thymidine kinase activity by thymidine kinase gene transfer using HVJ liposomes with gangliosides. Stable transformants could be obtained efficiently from various cell lines by use of HVJ liposomes containing the neoR gene. The neo+ transformants were obtained at frequencies of about 0.2-1.0, 0.06-0.25, and 0.06-0.1% in monolayers of L, CHO-Kl, and HeLa-S3 cells, respectively. Moreover, in Ehrlich ascites tumor cells which grow in suspension, the frequency was more than 0.01%. On introduction of plasmid pTK4 into Ltk- cells, about 0.5-1.0% TK+ transformants were obtained. Cosmid DNA containing the neoR gene (about 45 kbp) was also introduced into L cells by this method and neo+ transformants were obtained at a frequency of 0.1%. When rat liver mRNA was introduced into L cells by HVJ liposomes with gangliosides, immunoprecipitation studies showed that the L cells secreted rat albumin and some other proteins into the cultured medium. Moreover, using erythrocyte membrane vesicles containing IgM that had been incubated with HVJ empty liposomes with gangliosides, the IgM could be introduced into all the L cells.     

Growth of poliovirus in HeLa cells persistently infected with HVJ (Sendai virus)

The growth of poliovirus in a HeLa cell culture persistently infected with the hemagglutinating virus of Japan (HVJ, the Sendai strain of parainfluenza 1 virus) (HeLaHVJ) was studied. Plaques produced by poliovirus on HeLaHVJ cell monolayers were hazier, smaller and fewer than those on HeLa cells. HeLaHVJ cells were indistinguishable from normal HeLa cells with respect to adsorption rate and penetration efficiency of poliovirus. Extracellular yields of poliovirus in HeLaHVJ cells were lower, and the cytopathic changes were less than those in normal HeLa cells, while cell-associated virus growth in HeLaHVJ cells was nearly equal to that in HeLa cells. HeLaHVJ cells responded more effectively to the action of magnesium chloride, which facilitates virus release from infected cells, resulting in an cytopathic effects. No reduction in poliovirus yield could be detected in HeLa cells acutely infected with HVJ. The relationship between the inhibition of the release of poliovirus from HeLaHVJ cells and the persistent infection of the cells with HVJ is discussed.     

Early antibodies specific for the neutralizing epitope on the receptor binding subunit of the lymphocytic choriomeningitis virus glycoprotein fail to neutralize the virus

Lymphocytic choriomeningitis virus (LCMV) is a murine arenavirus whose glycoprotein consists of a transmembrane subunit (GP-2) and a receptor-binding subunit (GP-1). LCMV-neutralizing antibodies (nAbs) are directed against a single site on GP-1 and occur 1 month after the infection of cytotoxic-T-lymphocyte (CTL) deficient mice. In wild-type mice, however, CTLs control early infection, and weak nAb titers emerge very late (after 70 to 150 days) if at all. Production of recombinant GP-1 in native conformation enabled us to study the emergence of GP-1-binding antibodies directed against the neutralizing epitope. By combining binding and neutralization assays, we correlated the development of binding antibodies versus nAbs in wild-type and CTL-deficient mice after infection with different LCMV doses. We found that wild-type mice developed GP-1-specific antibodies already by day 8 after exposure to high but not low doses, demonstrating that naive GP-1-specific B cells were infrequent. Furthermore, the induced antibodies bound to the neutralizing GP-1 epitope but failed to neutralize the virus and therefore were of low affinity. In CTL-deficient mice, where massive viremia quickly levels initial differences in viral load, low and high doses induced low-affinity non-neutralizing GP-1-binding antibodies with kinetics similar to high-dose-infected wild-type mice. Only in CTL-deficient mice, however, the GP-1-specific antibodies developed into nAbs within 1 month. We conclude that LCMV uses a dual strategy to evade nAb responses in wild-type mice. First, LCMV exploits a "hole" in the murine B-cell repertoire, which provides only a small and narrow initial pool of low-affinity GP-1-specific B cells. Second, affinity maturation of the available low-affinity non-neutralizing antibodies is impaired.     

Identification and characterization of the cell surface 70-kilodalton sialoglycoprotein(s) as a candidate receptor for encephalomyocarditis virus on human nucleated cells.

The attachment of encephalomyocarditis (EMC) virus to human nucleated cells susceptible to virus infection was examined with HeLa and K562 cell lines. Both cell types showed specific virus binding competitively blocked by unlabeled virions. The number of binding sites for EMC virus on HeLa and K562 cells were approximately 1.6 x 10(5) and 3.5 x 10(5) per cell, respectively, and dissociation binding constants were 1.1 and 2.7 nM, respectively. Treatment of cells with cycloheximide after pretreatment with trypsin eliminated EMC virus attachment, suggesting that the virus-binding moiety is proteinaceous in nature. Digestion of cells, cell membranes, and sodium deoxycholate-solubilized cell membranes with proteases or neuraminidases or treatment of cells with lectins demonstrated that the EMC virus-cell interaction is mediated by a sialoglycoprotein. Proteins with a molecular mass of 70 kDa were isolated from detergent-solubilized cell membranes of both HeLa and K562 cells by EMC virus affinity chromatography. The purified proteins, as well as their 70-kDa-molecular-mass equivalents detected in intact surface membranes of HeLa and K562 cells, specifically bound EMC virus in a virus overlay protein blot assay, whereas membranes from nonpermissive K562 D clone cells did not. Western immunoblot analysis with glycophorin A-specific antibody confirmed that the identified 70-kDa binding site on K562 cells is not glycophorin A, which is the EMC virus receptor molecule on virus-nonpermissive human erythrocytes (HeLa cells do not express glycophorin A). These results indicate that EMC virus attachment to permissive human cells is mediated by a cell surface sialoglycoprotein(s) with a molecular mass of 70 kDa.     

The role of extracellular calcium ions in HVJ (Sendai virus)-induced cell fusion

The biochemical and biophysical roles of extracellular calcium ions in HVJ (Sendai virus)-induced cell fusion were studied. (1) Various kinds of cell, such as Ehrlich ascites tumor cells, mouse melanoma cells (B16-CW1 cells) and human epidermoid carcinoma cells (KB cells), could fuse in Ca2+-free medium containing a cheletor, glycoletherdiaminetetraacetic acid, in the same way as in Ca2+-containing medium. (2) The ATP content in Ehrlich ascites tumor cells decreased rapidly when the cells were treated with the virus in Ca2+-free medium but not in Ca2+-containing medium. (3) Intracellular adenine nucleotides leaked out into the reaction medium when the cells were treated with the virus in Ca2+-free medium but not in Ca2+-containing medium. (4) On addition of the virus, O2 consumption of Ehrlich ascites tumor cells decreased in Ca2+-free medium, but not in Ca2+-containing medium. (5) HVJ (Sendai virus) did not affect production of lactate by Ehrlich ascites tumor cells in both Ca2+-free medium and Ca2+-containing medium. These observations suggest that the role of extracellular Ca2+ in virus-induced cell fusion is to maintain the ATP and other intracellular metabolite contents at normal levels instead of triggering the fusion reaction itself.     

Expression and functional characterization of bluetongue virus VP2 protein: role in cell entry

Segment 2 of bluetongue virus (BTV) serotype 10, which encodes the outer capsid protein VP2, was tagged with the S-peptide fragment of RNase A and expressed by a recombinant baculovirus. The recombinant protein was subsequently purified to homogeneity by virtue of the S tag, and the oligomeric nature of the purified protein was determined. The data obtained indicated that the majority of the protein forms a dimer and, to a lesser extent, some trimer. The recombinant protein was used to determine various biological functions of VP2. The purified VP2 was shown to have virus hemagglutinin activity and was antigenically indistinguishable from the VP2 of the virion. Whether VP2 is responsible for BTV entry into permissive cells was subsequently assessed by cell surface attachment and internalization studies with an immunofluorescence assay system. The results demonstrated that VP2 alone is responsible for virus entry into mammalian cells. By competition assay, it appeared that both VP2 and the BTV virion attached to the same cell surface molecule(s). The purified VP2 also had a strong affinity for binding to glycophorin A, a sialoglycoprotein component of erythrocytes, indicating that VP2 may be responsible for BTV transmission by the Culicoides vector to vertebrate hosts during blood feeding. Further, by various enzymatic treatments of BTV-permissive L929 cells, preliminary data have been obtained which indicated that the BTV receptor molecule(s) is likely to be a glycoprotein and that either the protein moiety of the glycoprotein or a second protein molecule could also serve as a coreceptor for BTV infection.     

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.     

Noncytotoxic ribonuclease, RNase T1, induces tumor cell death via hemagglutinating virus of Japan envelope vector.

Several ribonucleases, including onconase and alpha-sarcin, are known to be toxic to tumor cells. On the other hand, although its structure is related to that of alpha-sarcin, RNase T1 is noncytotoxic because of its inability to internalize into tumor cells. In this study, we internalized RNase T1 into human tumor cells via a novel gene transfer reagent, hemagglutinating virus of Japan (HVJ) envelope vector, which resulted in cell death. This cytotoxicity was drastically increased by pretreatment of HVJ envelope vector with protamine sulfate, and was stronger than that of onconase, which is in phase III human clinical trials as a nonmutagenic cancer chemotherapeutic agent. Furthermore, internalized RNase T1 induced apoptotic cell death programs. Because its cytotoxicity is unfortunately not specific to tumor cells, it cannot at present be developed as an anticancer drug. However, we believe that RNase T1 incorporated in HVJ envelope vector will be a unique anticancer drug if HVJ envelope vector can be targeted to tumor cells.     

Abnormal blood-group-Ss-active sialoglycoproteins in the membrane of Miltenberger class III, IV and V human erythrocytes.

1. We have studied the inherited changes occurring in the sialoglycoproteins of membranes from erythrocytes of type Miltenberger Class III (Mi.III), Miltenberger Class IV (Mi.IV) and Miltenberger Class V (Mi.V) by using sodium dodecyl sulphate/polyacrylamide gel electrophoresis and lactoperoxidase radioiodination. 2. Mi.III erythrocytes lack the normal blood-group-Ss-active sialoglycoprotein but contain an unusual s-active sialoglycoprotein of higher apparent molecular weight. A similar abnormal S-active sialoglycoprotein appears to occur in Mi.IV erythrocytes. 3. The Mi.V condition is associated with the hemizygous absence of both the normal blood-group-MN-active sialoglycoprotein and the normal Ss-active sialoglycorprotein. However, a new sialoglycoprotein component is present in these cells that has properties characteristic of both the MN-active and Ss-active sialoglycoproteins. 4. Our results suggest that the new sialoglycorportein present in Mi.V erythrocytes is a hybrid of the normal MN sialoglycoprotein and an s-active sialoglycoprotein that has properties similar to the s-active sialoglycoprotein found in Mi.III erythrocytes. We suggest that the unusual Mi.V sialoglycoprotein is derived from chromosomal misalignment with unequal crossing-over between the genes for the MN- and Ss-active sialoglycoproteins in a manner similar to that which gives rise to haemoglobin Lepore. 5. Further studies of S-s-erythrocytes confirm that these cells lack normal Ss-active sialoglycoprotein, but contain an unusual component that shows some of the properties of the normal Ss-active sialoglycoprotein. 6. Analysis of erythrocytes of type Mk/Mi.III confirms that, in addition to the known hemizygous lack of the MN-active sialoglycoprotein, the Mk condition is also associated with a loss of the Ss-active sialoglycoprotein. 7. In order to facilitate discussion of the complex changes that occur in these variant erythrocytes, a new unified nomenclature is used for the erythrocyte sialoglycoproteins.