Biomedical subjects

A Loyter

Publications and source records attributed to A Loyter.

At least 55 records · Page 3Linked to original sources

PubMed1987

Transient expression of the plasmid pCaMVCAT in plant protoplasts following transformation with polyethyleneglycol.

Petunia and carrot protoplasts have been transformed with the plasmid pCaMVCAT by the use of polyethyleneglycol (PEG) as a facilitator. Transformation was revealed by the appearance of the chloramphenicol-acetyl transferase (CAT) enzyme within the transformed cells. Maximal activity of the CAT enzyme was detected within 15 h following transformation, while after 60 h, its activity was significantly reduced, indicating transient expression of the CAT gene. The efficiency of transformation was highly dependent on the presence of CaCl2 in the transformation system, was stimulated by non-functional carrier DNA and was independent on the molecular weight (MW) of PEG used.

Acetyltransferases↗

PubMed1987

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.

Erythrocyte Membrane↗

PubMed1987

[The relationship between Ca++ ions and a protein specific to cromolyn in the degranulation of mast cells and basophils in the rat].

Previous research has shown that cromolyn (disodium cromoglycate) binds specifically to the membrane of rat mast and basophil leukemia (RBL) cells, forming a ternary complex with Ca++, blocking of the Ca++ results in inhibition of histamine release upon immunologic triggering. The specific cromolyn binding protein (CBP) has been isolated by using its high affinity form cromolyn. Specific monoclonal anti CBP antibodies have been obtained in mice and polyclonal anti-cromolyn antibodies in rabbits. These antibodies have been used for further purification and characterization of the CBP. Experiments on RBL cell lines have shown that CBP is essential for Ca++ influx and histamine liberation upon immunologic triggering by these cells. Variant CBP deficient RBL do not take up Ca++ and do not degranulate in response to immunologic triggering but their ability to respond normally can be induced by incorporating CBP into their membranes with help of Sendai virus carrier vesicles. This shows that CBP plays a crucial role for the RBL cells Ca++ influx and histamine release following IgE crosslinking.

Animals↗

PubMed1986

Membrane vesicles containing the Sendai virus binding glycoprotein, but not the viral fusion protein, fuse with phosphatidylserine liposomes at low pH.

Membrane vesicles containing the Sendai virus hemagglutinin/neuraminidase (HN) glycoprotein were able to induce carboxyfluorescein (CF) release from loaded phosphatidylserine (PS) but not loaded phosphatidylcholine (PC) liposomes. Similarly, fluorescence dequenching was observed only when HN vesicles, bearing self-quenched N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylethanolamine (N-NBD-PE), were incubated with PS but not PC liposomes. Thus, fusion between Sendai virus HN glycoprotein vesicles and the negatively charged PS liposomes is suggested. Induction of CF release and fluorescence dequenching were not observed when Pronase-treated HN vesicles were incubated with the PS liposomes. On the other hand, the fusogenic activity of the HN vesicles was not inhibited by treatment with dithiothreitol (DTT) or phenylmethanesulfonyl fluoride (PMSF), both of which are known to inhibit the Sendai virus fusogenic activity. Fusion was highly dependent on the pH of the medium, being maximal after an incubation of 60-90 s at pH 4.0. Electron microscopy studies showed that incubation at pH 4.0 of the HN vesicles with PS liposomes, both of which are of an average diameter of 150 nm, resulted in the formation of large unilamellar vesicles, the average diameter of which reached 450 nm. The relevance of these observations to the mechanism of liposome-membrane and virus-membrane fusion is discussed.

Animals↗

PubMed1986

Fusion of Sendai virus with negatively charged liposomes as studied by pyrene-labelled phospholipid liposomes.

Sendai virus particles fuse with negatively charged liposomes but not with vesicles made of zwitterionic phospholipids. The liposome-virus fusion process was studied by dilution of the concentration-dependent excimer-forming fluorophore 2-pyrenyldodecanoylphosphatidylcholine contained in the liposomes by the viral lipids. The data were analyzed in the framework of a mass action kinetic model. This provided analytical solutions for the final levels of probe dilution and numerical solutions for the kinetics of the overall fusion process, in terms of rate constants for the liposome-virus adhesion, deadhesion and fusion. This analysis led to the following conclusions: At neutral pH and 37 degrees C, only 15% of the virus particles can fuse with the phospholipid vesicles, although all the virions may aggregate with the liposomes. The rate constants for aggregation, fusion and deadhesion are of the orders of magnitude of 10(7) M-1 X s-1, 10(-3) s-1 and 10(-2), s-1, respectively. The fraction of active virus increases with temperature. At acidic pH, both the fraction of 'fusable' virus and the rate of fusion increase markedly. The optimal pH for fusion is 3-4, where most of the virus particles are active. At higher pH values, an increasing fraction of the virus particles become inactive, probably due to ionization of viral glycoproteins, whereas at pH values below 3.0 the fusion is markedly reduced, most likely due to protonation of the negatively charged vesicles. While only 15% of the virions fuse with the liposomes at pH 7.4 and 37 degrees C, all the liposomes lose their content (Amselem, S., Loyter, A. Lichtenberg, D. and Barenholz, Y. (1985) Biochim. Biophys. Acta 820, 1-10). We therefore propose that release of entrapped solutes is due to liposome-virus aggregation, and not to fusion. Both trypsinization and heat inactivation of the virus particles inhibit not only the fusion process but also the release of carboxyfluorescein. This demonstrates the obligatory role of viral membrane proteins in liposome-virus aggregation. Reconstituted vesicles made of the viral lipid and the hemagglutinin/neuraminidase (HN) glycoprotein fuse with negatively charged liposomes similar to the intact virions. This suggests that the fusion of virions with negatively charged vesicles, unlike the fusion of the virus with biological membranes, requires only the HN and not the fusion glycoprotein.

Calcium↗

PubMed1986

Use of reconstituted Sendai virus envelopes for fusion-mediated microinjection of double-stranded RNA: inhibition of protein synthesis in interferon-treated cells.

Poly(I).poly(C) molecules were trapped with reconstituted Sendai virus envelopes when added to the reconstitution system. A quantitative estimation indicated that about 10% of the added poly(I).poly(C) remained associated with the fusogenic viral envelopes. About 50% of the associated poly(I).poly(C) were found to be RNAase A resistant, enclosed within the viral envelopes. Incubation of loaded viral envelopes with HeLa or L-cells resulted in strong inhibition of protein synthesis, indicating fusion-mediated microinjection of the enclosed poly(I).poly(C). Introduction of poly(I).poly(C) into cultured cells by the use of reconstituted Sendai virus envelopes was as efficient as the introduction of these polynucleotides using the calcium phosphate coprecipitation technique. The inhibition of protein synthesis in L-cells but not in HeLa cells was dependent upon pretreatment with interferon. Incubation of poly(I).poly(C)-loaded viral envelopes with interferon-treated variant cells of the NIH 3T3 line, which possess a very low amount of RNAase L, resulted in only 25% inhibition of protein synthesis, compared to 85% inhibition observed in L-cells.

Animals↗

PubMed1986

Protein blot analysis of virus receptors: identification and characterization of the Sendai virus receptor.

Receptors for Sendai virions in human erythrocyte ghost membranes were identified by virus overlay of protein blots. Among the various erythrocyte polypeptides, only glycophorin was able to bind Sendai virions effectively. The detection of Sendai virions bound to glycophorin was accomplished either by employing anti-Sendai virus antibodies or by autoradiography, when 125I-labeled Sendai virions were used. The binding activity was associated with the viral hemagglutinin/neuraminidase (HN) glycoprotein, as inferred from the observation that the binding pattern of purified HN glycoprotein to human erythrocyte membranes was identical to that of intact Sendai virions. No binding was observed when blots, containing either human erythrocyte membranes or purified glycophorin, were probed with the viral fusion factor (F glycoprotein). Active virions competed effectively with the binding of 125I-labeled Sendai virions (or purified HN glycoprotein), whereas no competition was observed with inactivated Sendai virus. The results of the present work clearly show that protein blotting can be used to identify virus receptors in cell membrane preparations.

Animals↗

PubMed1986

The use of circular dichroism to study conformational changes induced in Sendai virus envelope glycoproteins. A correlation with the viral fusogenic activity.

Fusion of Sendai virus envelopes with erythrocyte membranes or with phosphatidylcholine/cholesterol liposomes requires the presence of the two viral glycoproteins, namely the hemagglutinin/neuraminidase (HN) and the fusion (F) polypeptides. Membrane vesicles bearing only the HN or the F glycoprotein (HN or F vesicles) or a mixture of both do not possess fusogenic activity. These results clearly show that in order to be fusogenic, the two viral envelope glycoproteins must be present within the same membrane, thus indicating their mutual interaction. Circular dichroism studies revealed that the conformation of the viral glycoproteins in reconstituted viral envelopes or in HN-F vesicles (vesicles formed by co-reconstitution of the HN and F glycoproteins) is different from that of the conformation of these glycoproteins in either HN or F vesicles or in a mixture of both. It has been observed that the mean residue ellipticity as measured at 222 nm (theta 222) of the viral glycoproteins in reconstituted Sendai virus envelopes (RSVE) is lower by about 75% than the value observed for these glycoproteins in isolated HN or F vesicles. Treatment of RSVE or of HN-F vesicles with inhibitors of the viral fusogenic activity such as phenylmethylsulfonyl fluoride, proteolytic enzymes, or incubation at 70 degrees C caused a substantial conformational change in the viral glycoproteins. The theta 222 of unfusogenic RSVE or unfusogenic HN-F vesicles is very close to that observed for a mixture of HN and F vesicles. It is proposed here that in order to be fusogenic, the viral envelope glycoproteins must possess a certain conformation which exists only when they are present within the same membrane.

Cell Fusion↗

PubMed1986

Fusion of fluorescently labeled Sendai virus envelopes with living cultured cells as monitored by fluorescence dequenching.

Fluorescently labeled (bearing N-4-nitrobenzo-2-oxa-1,3-diazole-phosphatidylethanolamine (N-NBD-PE)) reconstituted Sendai virus envelopes (RSVE) were used to study fusion between the viral envelopes and cultured living cells such as lymphoma, Friend erythroleukemia cells (FELC) and L cells. Incubation of fusogenic viruses with the above cell lines resulted in a relatively high degree (40-45%) of fluorescence dequenching. On the other hand, incubation of unfusogenic (trypsin or phenylmethylsulfonylfluoride (PMSF)-treated) RSVE with these cells led to very little (6-9%) fluorescence dequenching. The degree of fluorescence dequenching was linearly correlated to the surface density of the virus-inserted N-NBD-PE molecules. Fluorescence photobleaching recovery experiments showed that fusion of fluorescent RSVE with FELC resulted in an infinite dilution of the fluorescent molecules in the recipient cell membranes. The fluorescent probe 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (N-NBD-Cl) was covalently attached to envelopes of intact Sendai virions without significantly impairing their biological activity. Incubation of fluorescently labeled, intact Sendai virions with cultured cells resulted in about 20% fluorescence dequenching. The present data clearly indicate that fluorescently labeled Sendai virions can be used for a quantitative estimation of the degree of virus-membrane fusion.

Animals↗

PubMed1986

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

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

Cholesterol↗

PubMed1985

Fusion of Sendai virions with phosphatidylcholine-cholesterol liposomes reflects the viral activity required for fusion with biological membranes.

Sendai virus envelopes were reconstituted after solubilization of intact virions with either Triton X-100 or octylglucoside. Envelopes obtained from Triton X-100, but not from octylglucoside solubilized virions, were hemolytic and promoted cell-cell fusion. Fluorescence dequenching studies [using N-4-nitrobenzo-2-oxa-1,3-diazole phosphatidylethanolamine-bearing viral envelopes] revealed that both preparations fused with negatively charged phospholipids. Fusion with phosphatidylcholine (PC)/cholesterol (chol) liposomes was promoted only by the hemolytic viral envelopes. Fluorescence dequenching studies, using intact virions bearing octadecylrhodamine B chloride, revealed that intact virions fused with PC/chol as well as with negatively charged phospholipids. Only fusion with PC/chol liposomes was inhibited by phenylmethylsulfonyl fluoride and dithiothreitol, reagents which are known to block the viral ability to fuse with biological membranes.

Cholesterol↗

PubMed1985

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

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

Calcium↗

PubMed1985

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.

Cholesterol↗

PubMed1985

Fusion between Sendai virus envelopes and biological membranes. The use of fluorescent probes for quantitative estimation of virus-membrane fusion.

The fluorescent probes, N-4-nitrobenzo-2-oxa-1,3-diazole-phosphatidylethanolamine and lissamine-rhodamine-B-sulfonylphosphatidylethanolamine, were inserted at the appropriate surface density into membranes of reconstituted Sendai virus envelopes, thus allowing transfer of energy between the fluorescent probes. In addition, only the fluorescent molecule N-4-nitrobenzo-2-oxa-1,3-diazole-phosphatidylethanolamine was inserted into the viral envelopes, resulting in self-quenching. Incubation of fluorescent, reconstituted Sendai virus envelopes with human erythrocyte ghosts resulted in either reduction in the efficiency of energy transfer or in fluorescence dequenching. No reduction in the efficiency of energy transfer or fluorescence dequenching was observed when fluorescent, reconstituted Sendai virus envelopes were incubated with glutaraldehyde-fixed or desialized human erythrocyte ghosts. Similarly, no change in the fluorescence value was observed when nonfusogenic, reconstituted Sendai virus envelopes were incubated with human erythrocyte ghosts. These results clearly show that reduction in the efficiency of energy transfer or dequenching is due to virus-membrane fusion and not to lipid-lipid exchange. Incubation of reconstituted Sendai virus envelopes, carrying inserted N-4-nitrobenzo-2-oxa-1,3-diazolephosphatidylethanolamine, with cultured cells also resulted in a significant and measurable dequenching. However, incubation of nonfusogenic, fluorescent reconstituted Sendai virus envelopes with hepatoma tissue culture cells also resulted in fluorescent dequenching, the degree of which was about 50% of that observed with fusogenic, fluorescent reconstituted viral envelopes. It is therefore possible that, in addition to virus-membrane fusion, endocytosis of fluorescent viral envelopes results in fluorescence dequenching as well.

Calcium↗

PubMed1985

Use of virus-attached antibodies or insulin molecules to mediate fusion between Sendai virus envelopes and neuraminidase-treated cells.

Anti-human erythrocyte antibodies or insulin molecules were covalently coupled to the glycoproteins (the hemagglutinin/neuraminidase and the fusion polypeptides) of Sendai virus envelopes with N-succinimidyl 3-(2-pyridyldithio)propionate and succinimidyl 4-(p-maleimidophenyl)butyrate as cross-linking reagents. Reconstituted Sendai virus envelopes, bearing covalently attached anti-human erythrocyte antibodies or insulin molecules, were able to bind to but not fuse with virus receptor depleted human erythrocytes (neuraminidase-treated human erythrocytes). Only coreconstitution of Sendai virus glycoproteins, bearing attached anti-human erythrocyte antibodies or insulin molecules with intact, untreated viral glycoproteins, led to the formation of fusogenic, targeted reconstituted Sendai virus envelopes. Binding and fusion of reconstituted Sendai virus envelopes, bearing anti-human erythrocyte antibodies or insulin molecules, with neuraminidase-treated human erythrocytes were blocked by the monovalent fraction, obtained after papain digestion of immunoglobulins, made of anti-human erythrocyte antibodies or free insulin molecules, respectively. The results of this work demonstrate an active role of the viral binding protein (hemagglutinin/neuraminidase polypeptide) in the virus membrane fusion process and show a novel and efficient method for the construction of targeted, fusogenic Sendai virus envelopes.

Antibodies↗

PubMed1985

Affinity targeting of Sendai virions to desialized human erythrocytes using hybrid antibody molecules.

F(ab') fragments obtained from anti-Sendai virus antibodies were chemically coupled to F(ab') fragments obtained from anti-human red blood cell antibodies (anti-hRBC-Ab). This led to the formation of hybrid antibody molecules (anti-SV-anti-hRBC(F(ab')2) each of whose F(ab') fragment possessed different binding specificity. The anti-SV(F(ab'] part of the hybrid molecule interacted specifically with Sendai virus particles, while the anti-hRBC(F(ab'] part interacted with the surface of hRBC. These hybrid antibodies were able to mediate binding and fusion of SV to hRBC, from which the virus receptors were removed by treatment with neuraminidase (desialized hRBC). Neither anti-SV-anti-SV(F(ab')2) nor anti-hRBC-anti-hRBC(F(ab')2) possessed the same ability. Thus, it is shown that soluble, hybrid antibody molecules can effectively mediate functional binding of Sendai virus to virus-receptor-depleted cells.

Antibodies, Viral↗

PubMed1985

Sendai virus envelope glycoproteins become laterally mobile on the surface of human erythrocytes following fusion.

Fluorescence photobleaching recovery has been employed to study the lateral mobility of the Sendai virus envelope glycoproteins (HN, neuraminidase/hemagglutinin protein (HN) fusion protein (F) on the surface of human erythrocytes. Our results indicate that the two viral glycoproteins are laterally immobile on the cell surface prior to fusion, and become mobile during the fusion process. The two fused glycoproteins are mobilized to the same extent (diffusion coefficients of 3.1-3.3 X 10(-10) cm2/sec with mobile fractions of 0.53-0.57 for both HN and F). Their mobilization is blocked under conditions that allow virus adsorption and hemagglutination, but not virus-cell or cell-cell fusion. These findings suggest a possible role for the lateral diffusion of the viral glycoproteins in the mechanism of cell-cell fusion, enabling them to perturb the membranes of adjacent cells and lead to cell-cell fusion.

Cell Fusion↗

PubMed1985

Targeting of loaded Sendai virus envelopes by covalently attached insulin molecules to virus receptor-depleted cells: fusion-mediated microinjection of ricin A and simian virus 40 DNA.

Insulin molecules were covalently attached to detergent-solubilized Sendai virus envelope glycoproteins (HN and F polypeptides) by the use of the crosslinking reagent succinimidyl 4-(p-maleimidophenyl)butyrate (SMPB). Reconstitution of modified viral glycoproteins (carrying covalently attached insulin) together with unmodified viral glycoproteins resulted in the formation of "fusogenic" viral envelopes bearing insulin molecules. Reconstitution of such fusogenic viral envelopes in the presence of ricin A or simian virus 40 (SV40) DNA resulted in the formation of viral envelopes bearing insulin molecules and loaded with ricin A or SV40 DNA. Such viral envelopes were able to bind to hepatoma tissue culture cells (HTCC) from which Sendai virus receptors were removed by treatment with neuraminidase. Incubation of viral envelopes loaded with ricin A with virus receptor-depleted HTCC resulted in fusion-mediated injection of the toxin, as inferred from inhibition of protein synthesis and decrease in cell viability of the microinjected cells. Fusion-mediated injection of SV40 DNA was inferred from the appearance of SV40 tumor antigen in microinjected cells. Binding and fusion of the loaded viral envelopes to neuraminidase-treated HTCC was mediated solely by the virus-associated insulin molecules. Addition of free insulin molecules inhibited binding of the viral envelopes and, consequently, the microinjection of ricin A and SV40 DNA.

Carcinoma, Hepatocellular↗