algumas contribuições para o desenvolvimento da fisiologia celular em Portugal

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A Common Mechanism for Influenza Virus Fusion Activity and Inactivation

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 °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 °C, but was also detected at 0 °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. © 1993, American Chemical Society. All rights reserved

Kinetic modeling of Sendai virus fusion with PC-12 cells. Effect of pH and temperature on fusion and viral inactivation

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 octadecylrhodamine, a fluorescent non-exchangeable probe. The results were analysed with a mass action kinetic model which could explain and predict the kinetics of virus2013cell fusion. When the temperature was lowered from 37°C to 25°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°C to 25°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°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°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°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