FUSION OF ENVELOPED VIRUSES WITH CELLS AND LIPOSOMES - ACTIVITY AND INACTIVATION

The fusion of viruses with cells and liposomes is reviewed with focus on the analysis of the final extents and kinetics of fusion. Influenza virus and Sendai virus exhibit 100% of fusion capacity with cells at pH 5 and pH 7.5, respectively. On the other hand, there may be in certain cases, a limit on the number of virions that can fuse with a single cell, that is significantly below the limit on binding. It still remains to be resolved whether this limit reflects a limited number of possible fusion sites, or a saturation limit on the amount of viral glycoproteins that can be incorporated in the cellular membrane, like the case of virus fusion with pure phospholipid vesicles, in which the fusion products were shown to consist of a single virus and several liposomes. Both viruses demonstrate incomplete fusion activity towards liposomes of a variety of compositions. In the case of Sendai virus, fusion inactive virions bind essentially irreversibly to liposomes. Yet, preliminary results revealed that such bound, unfused virions can be released by sucrose gradient centrifugation. The separated unfused virions subsequently fuse when incubated with a fresh batch of liposomes. We conclude, therefore, that the fraction of initially bound unfused virions does not consist of dective particles, but rather of particles bound to liposomes via inactive sites. Details of the low pH inactivation of fusion capacity of influenza virus towards cells and liposomes are presented. This inactivation is caused by protonation and exposure of the hydrophobic segment of HA2, and affects primarily the fusion rate constants. Some degree of inactivation also occurs when virions are bound to cellular membranes. © 1990 Humana Press Inc

INTERACTION OF CLATHRIN WITH LARGE UNILAMELLAR PHOSPHOLIPID-VESICLES AT NEUTRAL PH - LIPID DEPENDENCE AND PROTEIN PENETRATION

The interaction of clathrin with large unilamellar vesicles of various lipid compositions has been examined at neutral pH. Clathrin induces leakage of contents of vesicles that contain the acidic phospholipid phosphatidylserine. Leakage is greatly enhanced by the presence of a relatively minor amount of cholesterol, but is inhibited by phosphatidylcholine. Resonance energy transfer measurements between tryptophan residues of the protein and a fluorescent lipid analog, N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)phosphatidylethanolamine incorporated into the liposomal bilayer, suggests a dynamic interaction of clathrin with the bilayer at neutral pH. This interaction includes a (partial) penetration of the protein into the lipid bilayer, as revealed by hydrophobic photoaffinity labeling with 3-(trifluoromethyl)-3-(m-[I-125]iodophenyl)-diazirine. The interaction of clathrin with lipid vesicles at neutral pH is inhibited when the protein is pretreated with trypsin or with the reducing agent dithiothreitol, suggesting that structural requirements govern clathrin-membrane interaction at these conditions. The physiological relevance of the present observations in light of vesiculation and endosomal maturation is discussed