仙台病毒与人类HL-60和CEM细胞的融合：两种分离株的不同融合动力学。

Fusion of Sendai virus with human HL-60 and CEM cells: different kinetics of fusion for two isolates.

作者信息

Pedroso de Lima M C, Nir S, Flasher D, Klappe K, Hoekstra D, Düzgüneş N

机构信息

Cancer Research Institute, University of California, San Francisco.

出版信息

Biochim Biophys Acta. 1991 Dec 9;1070(2):446-54. doi: 10.1016/0005-2736(91)90085-m.

DOI:10.1016/0005-2736(91)90085-m

PMID:1662538

Abstract

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.

摘要

研究了仙台病毒（Z株）与人早幼粒细胞白血病细胞系HL-60以及人T淋巴细胞白血病细胞系CEM的融合动力学。通过掺入病毒膜中的十八烷基罗丹明（R-18）的荧光猝灭来监测融合。对于一种病毒分离株（Z/G），随着pH值降低，融合的总体速率（在37℃下）增加，在约pH 5（测试的最低pH值）时达到最大值。对于另一种分离株（Z/SF），在pH 5时融合的速率和程度低于中性pH值时。将任何一种分离株与HL-60细胞孵育几分钟后，将pH从中性降至5会导致荧光猝灭速率增强。然而，利用氯化铵的实验表明，内体腔的微酸性pH值并未增强病毒与细胞的融合。通过质量作用模型分析融合动力学，对融合的时间进程进行了良好的模拟和预测。对于在pH 5时显示出最大融合活性的分离株，中性pH值下的融合速率常数（约0.1 s-1）处于先前发现的病毒-脂质体融合的范围内，而粘附速率常数接近扩散控制过程的上限（1.4×10¹⁰ M⁻¹ s⁻¹）。然而，对于另一种分离株（Z/SF），中性pH值下的融合速率常数非常小（小于0.01 s-1），而粘附速率常数较大（大于或等于2×10¹⁰ M⁻¹ s⁻¹）。降低温度会降低融合速率。涉及与过量未标记病毒粒子竞争的实验表明，HL-60细胞上仙台病毒的并非所有结合位点都是融合位点。与脂质体和红细胞血影的早期观察结果或基于红细胞溶血或细胞-细胞融合的结果相反，在pH 5或9下预孵育病毒后，中性pH值下病毒对HL-60细胞的融合活性没有明显改变。