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 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.
Pedroso de Lima
M C
MC
Center for Cell Biology, Faculty of Medicine, University of Coimbra, Portugal.
Ramalho-Santos
J
J
Martins
M F
MF
Pato de Carvalho
A
A
Bairos
V
V
Nir
S
S
D000818:Animals; D002462:Cell Membrane / Q000382:microbiology; D004705:Endocytosis; D019286:Endopeptidase K; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008561:Membrane Fusion; D016716:PC12 Cells / Q000378:metabolism / Q000382:microbiology*; D010222:Parainfluenza Virus 1, Human / Q000187:drug effects / Q000502:physiology*; D051381:Rats; D012697:Serine Endopeptidases / Q000494:pharmacology; D013696:Temperature; D014357:Trypsin / Q000494:pharmacology
D012697:Serine Endopeptidases; D014357:Trypsin; D019286:Endopeptidase K