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We have studied the importance of hydrophobic interactions in the fusion activity of Sendai virus. Viral fusion activity towards model lipid membranes was monitored using a fluorescence dequenching assay. Fusion was dependent on the composition of the target membrane and was more rapid and extensive at low pH. Dehydrating agents such as dimethylsulfoxide and dimethylsulfone greatly enhanced the initial rate and extent of the fusion process, the effect of dimethylsulfone doubling that of dimethylsulfoxide. However, dehydrating agents had a less pronounced stimulatory effect at low pH. Using the fluorescent probe ANS (1-aminonaphthalene-8-sulfonate) we have shown that low pH by itself involves a marked increase in the viral envelope hydrophobicity, probably resulting in the observed lower stimulation of viral fusion activity by dehydrating agents. Therefore, there seems to be a strong correlation between viral envelope hydrophobicity and viral fusion activity.

We have studied the role of hydrophobic interactions in the fusion activity of two lipid enveloped viruses, influenza and Sendai. Using the fluorescent probe ANS (1-aminonaphtalene-8-sulfonate) we have shown that low-pH-dependent influenza virus activation involves a marked increase in the viral envelope hydrophobicity. The effect of dehydrating agents on the fusion activity of both viruses towards model lipid membranes was studied using a fluorescence dequenching assay. Dehydrating agents such as dimethylsulfoxide and dimethylsulfone greatly enhanced the initial rate of the fusion process, the effect of dimethylsulfone doubling that of dimethylsulfoxide. The effect of poly(ethylene glycol) on the fusion process was found to be dependent on the polymer concentration and molecular weight. In general, similar observations were made for both viruses. These results stress the importance of dehydration and hydrophobic interactions in the fusion activity of influenza and Sendai viruses, and show that these factors may be generally involved in membrane fusion events mediated by many other lipid enveloped viruses.

The fusion of influenza virus (A/PR/8/34 strain) with PC-12 cells was monitored by a fluorescence assay, and the results were analyzed with a mass-action model which could explain and predict the kinetics of fusion. The model accounted explicitly for the reduction in the fusion rate constant upon exposure of the virus to low pH, either for the virus alone in suspension or for the virus bound to the cells. When the pH was lowered without previous viral attachment to cells, an optimal fusion activity was detected at pH 5.2. When the virus was prebound to the cells, however, reduction of pH below 5.2 resulted in enhanced fusion activity at the initial stages. These results were explained by the fact that the rate constants of both fusion and inactivation increased severalfold at pH 4.5 or 4, compared to those at pH 5.2. At pH 5.2, lowering the temperature from 37 to 20 or 4 degrees C resulted in a decrease in the fusion rate constant by more than 30- or 1000-fold, respectively. Inactivation of the virus when preincubated in the absence of target membranes at pH 5 was found to be rapid and extensive at 37 degrees C, but was also detected at 0 degrees C. Our results indicate a strong correlation between fusion and inactivation rate constants, suggesting that the rate-limiting step in viral hemagglutinin (HA)-mediated fusion, that is, rearrangement of viral glycoproteins at the contact points with the target membrane, is similar to that involved in fusion inactivation.

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-[125I]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.

We have studied the fusion activity of Sendai virus, a lipid-enveloped paramyxovirus, towards a line of adherent cells designated PC-12. Fusion was monitored by the dequenching of octadecyl-rhodamine, a fluorescent non-exchangeable probe. The results were analysed with a mass action kinetic model which could explain and predict the kinetics of virus-cell fusion. When the temperature was lowered from 37 degrees C to 25 degrees C, a sharp inhibition of the fusion process was observed, probably reflecting a constraint in the movement of viral glycoproteins at low temperatures. The rate constants of adhesion and fusion were reduced 3.5-fold and 7-fold, respectively, as the temperature was lowered from 37 degrees C to 25 degrees C. The fusion process seemed essentially pH-independent, unlike the case of liposomes and erythrocyte ghosts. Preincubation of the virus in the absence of target cell membranes at neutral and alkaline pH (37 degrees C, 30 min) did not affect the fusion process. However, a similar preincubation of the virus at pH = 5.0 resulted in marked, though slow, inhibition in fusion with the fusion rate constant being reduced 8-fold. Viral preincubation for 5 min in the same acidic conditions yielded a mild inhibition of fusogenic activity, while preincubation in the cold (4 degrees C, 30 min) did not alter viral fusion activity. These acid-induced inhibitory effects could not be fully reversed by further viral preincubation at pH = 7.4 (37 degrees C, 30 min). Changes in internal pH as well as endocytic activity of PC-12 cells had small effect on the fusion process, thus indicating that Sendai virus fuses primarily with the plasma membranes.