Fusion of Sendai virus with human HL-60 and CEM cells: different kinetics of fusion for two isolates.
The kinetics of fusion of Sendai virus (Z strain) with the human promyelocytic leukemia cell line HL-60, and the human T lymphocytic leukemia cell line CEM was investigated. Fusion was monitored by fluorescence dequenching of octadecylrhodamine (R-18) incorporated in the viral membrane. For one virus isolate (Z/G), the overall rate of fusion (at 37 degrees C) increased as the pH was lowered, reaching a maximum at about pH 5, the lowest pH tested. For another isolate (Z/SF) the rate and extent of fusion were lower at pH 5 than at neutral pH. Lowering the pH from neutral to 5 after several minutes of incubation of either isolate with HL-60 cells resulted in an enhanced rate of fluorescence dequenching. Nevertheless, experiments utilizing NH4Cl indicated that fusion of the virus with cells was not enhanced by the mildly acidic pH of the endosome lumen. Analysis of the kinetics of fusion by means of a mass action model resulted in good simulation and predictions for the time-course of fusion. For the isolate which showed maximal fusogenic activity at pH 5, the rate constant of fusion (approx. 0.1 s-1) at neutral pH was in the range found previously for virus-liposome fusion, whereas the rate constant of adhesion was close to the upper limit for diffusion-controlled processes (1.4.10(10) M-1 s-1). However, for the other isolate (Z/SF) the rate constant of fusion at neutral pH was very small (less than 0.01 s-1), whereas the rate constant of adhesion was larger (greater than or equal to 2.10(10) M-1 s-1). Lowering the temperature decreased the fusion rate. Experiments involving competition with excess unlabeled virions indicated that not all binding sites for Sendai virus on HL-60 cells are fusion sites. The virus fusion activity towards HL-60 cells at neutral pH was not altered significantly by pre-incubation of the virus at pH 5 or 9, in contrast to earlier observations with liposomes and erythrocyte ghosts, or results based on erythrocyte hemolysis or cell-cell fusion.
Pedroso de Lima
M C
MC
Cancer Research Institute, University of California, San Francisco.
Nir
S
S
Flasher
D
D
Klappe
K
K
Hoekstra
D
D
Düzgüneş
N
N
D000643:Ammonium Chloride / Q000494:pharmacology; D002460:Cell Line; D004910:Erythrocyte Membrane / Q000502:physiology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D015473:Leukemia, Promyelocytic, Acute; D015458:Leukemia, T-Cell; D008081:Liposomes; D008561:Membrane Fusion / Q000187:drug effects; D010222:Parainfluenza Virus 1, Human / Q000502:physiology*; D013050:Spectrometry, Fluorescence; D013696:Temperature
D008081:Liposomes; D000643:Ammonium Chloride