Poly(aspartic acid)-dependent fusion of liposomes bearing the quaternary ammonium detergent [[[(1,1,3,3-tetramethylbutyl)cresoxy]ethoxy]ethyl] dimethylbenzylammonium hydroxide.

Addition of the quaternary ammonium detergent [[[(1,1,3,3-tetramethylbutyl)cresoxy]ethoxy]ethyl] dimethylbenzylammonium hydroxide (DEBDA[OH]) and the fluorescent probes N-(7-nitro-2-1,3-benzoxadiazol-4-yl)phosphatidylethanolamine and N-(lissamine rhodamine B sulfonyl)phosphatidylethanolamine (N-NBD-PE and N-Rh-PE, respectively) to liposomes composed of phosphatidylcholine (PC) and cholesterol (chol) resulted in the formation of fluorescently labeled liposomes bearing DEBDA[OH]. Incubation of the anionic polymer poly(aspartic acid) (PASP) with such liposomes resulted in strong agglutination, indicating an association between the negatively charged PASP and the positively charged liposome-associated DEBDA[OH]. Addition of PASP to a mixture of fluorescently labeled and nonlabeled liposomes, both carrying DEBDA[OH], resulted in a significant increase in the extent of fluorescence, namely, fluorescence dequenching. The degree of the fluorescence dequenching was dependent upon the ratio between the nonfluorescent and the fluorescent liposomes, upon the temperature of incubation, and upon the amount of DEBDA[OH] which was associated with the liposomes. Electron microscopic observations revealed that large liposomes were formed upon incubation of liposomes bearing DEBDA[OH] with PASP. The results of the present work strongly indicate that the fluorescence dequenching observed is due to a process of PASP-induced liposome-liposome fusion.

Animal viruses are able to fuse with prokaryotic cells. Fusion between Sendai or influenza virions and Mycoplasma.

Sendai and influenza virions are able to fuse with mycoplasmata. Virus-Mycoplasma fusion was demonstrated by the use of fluorescently labeled intact virions and fluorescence dequenching, as well as by electron microscopy. A high degree of fusion was observed upon incubation of both virions with Mycoplasma gallisepticum or Mycoplasma capricolum. Significantly less virus-cell fusion was observed with Acholeplasma laidlawii, whose membrane contains relatively low amounts of cholesterol. The requirement of cholesterol for allowing virus-Mycoplasma fusion was also demonstrated by showing that a low degree of fusion was obtained with M. capricolum, whose cholesterol content was decreased by modifying its growth medium. Fluorescence dequenching was not observed by incubating unfusogenic virions with mycoplasmata. Sendai virions were rendered nonfusogenic by treatment with trypsin, phenylmethylsulfonyl fluoride, or dithiothreitol, whereas influenza virions were made nonfusogenic by treatment with glutaraldehyde, ammonium hydroxide, high temperatures, or incubation at low pH. Practically no fusion was observed using influenza virions bearing uncleaved hemagglutinin. Trypsinization of influenza virions bearing uncleaved hemagglutinin greatly stimulated their ability to fuse with Mycoplasma cells. Similarly to intact virus particles, also reconstituted virus envelopes, bearing the two viral glycoproteins, fused with M. capricolum. However, membrane vesicles, bearing only the viral binding (HN) or fusion (F) glycoproteins, failed to fuse with mycoplasmata. Fusion between animal enveloped virions and prokaryotic cells was thus demonstrated.

Osmotic swelling allows fusion of sendai virions with membranes of desialyzed erythrocytes and chromaffin granules.

Sendai virus particles are able to fuse with Pronase-neuraminidase-treated human erythrocyte membranes as well as with vesicles obtained from chromaffin granules of bovine medulla. Fusion is inferred either from electron microscopic studies or from the observation that incubation of fluorescently labeled (bearing octadecyl Rhodamine B chloride) virions, with right-side-out erythrocyte vesicles (ROV) or with chromaffin granule membrane vesicles (CGMV), resulted in fluorescence dequenching. Fusion of Sendai virions with virus receptor depleted ROV was observed only under hypotonic conditions. Fusion with virus receptor depleted ROV required the presence of the two viral envelope glycoproteins, namely, the HN and F polypeptides. A 3-fold increase in the degree of fluorescence dequenching (virus-membrane fusion) was also obtained upon incubation of Sendai virions with CGMV in medium of low osmotic strength. This increase was not observed with inactivated, unfusogenic Sendai virions. The results of the present work demonstrate that, under hypotonic conditions, fusion between Sendai virions and biological membranes does not require the presence of specific receptors. Such fusion is characterized by the same features as fusion with and infection by Sendai virions of living cultured cells.

A possible involvement of virus-associated protease in the fusion of Sendai virus envelopes with human erythrocytes.

A proteolytic activity is shown to be associated with relatively purified preparations of intact Sendai virus particles or with their reconstituted envelopes which are vesicles containing mainly the viral glycoproteins. Intact Sendai virus as well as reconstituted Sendai virus envelopes have been shown to be able to hydrolyze various protein molecules such as the human erythrocyte membrane polypeptide designated as band 3 and soluble polypeptides such as histone and insulin B-chain. The results of the present work raise the possibility that a direct correlation exists between the virus-associated proteolytic activity and the ability of the virions to lyse cells, to fuse with their membranes, and to promote cell-cell fusion. Inhibitors of proteolytic enzymes such as phenylmethylsulfonyl fluoride, tosyllysinechloromethylketone and tosylamidephenylethylchloromethylketone, or combinations thereof, inhibit the virus-associated proteolytic activity concomitantly with inhibition of its hemolytic and fusogenic activities. Electron microscopic studies showed that the various inhibitors did not affect the binding ability of the virus preparations. The possible involvement of a protease in the process of virus-membrane fusion is discussed.

Viral and non-viral induced fusion of pronase-digested human erythrocyte ghosts.

Human erythrocyte ghosts but was able to fuse only iso-human erythrocyte ghosts. Iso- and hypo-human erythrocyte ghosts were incubated with the proteolytic enzyme pronase under isotonic (iso-human erythrocyte ghosts) or hypotonic (hypo-human erythrocyte ghosts) conditions. Gel electrophoresis and electron microscope (freeze-etching) studies revealed that most of the erythrocyte membrane polypeptides were hydrolyzed by pronase under hypotonic conditions. Sendai virus readily agglutinated both pronase-digested iso-human erythrocyte ghosts and hypo-human erythrocyte ghosts were fused by the non-viral fusogenic agent glyceromonooleate. Freeze-etching studies revealed that during fusion the membranes of pronase-digested human erythrocyte ghosts are intermixed.

Fusion of intact human erythrocytes and erythrocyte ghosts.

Sendai virus is able to induce the fusion of human erythrocytes. Bivalent cations or ATP are not essential for polyerythrocyte formation. High fusion indices were obtained when Sendai virus was added to cells incubated in the presence of both EDTA and iodoacetic acid. Human erythrocyte ghosts prepared by gradual hemolysis still retain the potential to undergo virus-induced fusion. Fusion of human red blood cells without the addition of viruses was obtained by incubation of erythrocytes at pH 10.5 in the presence of Ca(++) (40 mM) or by addition of phospholipase C Clostridium perfringens preparations to cells previously agglutinated or polylysine.