Danieli T, Pelletier S L, Henis Y I, White J M
Department of Cell Biology, University of Virginia Health Sciences Center, Charlottesville 22908, USA.
J Cell Biol. 1996 May;133(3):559-69. doi: 10.1083/jcb.133.3.559.
In this study we tested the hypothesis that fusion mediated by the influenza virus hemagglutinin (HA) is a cooperative event. To so this we characterized 3T3 cell lines that express HA at nine different defined surface densities. HA densities ranged from 1.0 to 12.6 x 10(3) HA trimers/microns2 as determined by quantitative fluorescent antibody binding. The lateral mobility and percent mobile fraction of HA did not vary significantly among these cells, nor did the contact area between HA-expressing cells and target RBCs. The fusion reaction of each HA-expressing cell line was analyzed using a fluorescence dequenching assay that uses octadecylrhodamine (R18)-labeled RBCs. For each cell line we measured the lag time preceding the onset of fusion, the initial rate of fusion, and final extent of fusion. The final extent of fusion was similar for all cell lines, and the initial rate of fusion as a function of HA surface density displayed a Michaelis-Menten-type dependence. However, the dependence of the lag time preceding the onset of fusion on HA surface density was clearly sigmoidal. Kinetic analysis of the data for the reciprocal lag time vs HA surface density, by both a log/log plot and a Hill plot, suggested that the observed sigmoidicity does not reflect cooperativity at the level of formation of HA aggregates as a prerequisite to fusion. Rather, the cooperativity of the process(es) that occur(s) during the lag time arises at a later step and involves a minimum of three, and most likely four, HA trimers. A model is proposed to explain HA cooperativity during fusion.
在本研究中，我们检验了流感病毒血凝素（HA）介导的融合是一个协同事件的假说。为此，我们对在九种不同定义的表面密度下表达HA的3T3细胞系进行了表征。通过定量荧光抗体结合测定，HA密度范围为1.0至12.6×10³个HA三聚体/微米²。这些细胞中HA的侧向迁移率和可移动部分百分比没有显著差异，表达HA的细胞与靶红细胞之间的接触面积也没有显著差异。使用用十八烷基罗丹明（R18）标记的红细胞的荧光猝灭测定法分析每个表达HA的细胞系的融合反应。对于每个细胞系，我们测量了融合开始前的延迟时间、融合的初始速率和融合的最终程度。所有细胞系的融合最终程度相似，融合的初始速率作为HA表面密度的函数呈现米氏类型依赖性。然而，融合开始前延迟时间对HA表面密度的依赖性明显呈S形。通过对数/对数图和希尔图对倒数延迟时间与HA表面密度的数据进行动力学分析表明，观察到的S形并不反映作为融合前提的HA聚集体形成水平的协同性。相反，在延迟时间内发生的过程的协同性出现在稍后的步骤，并且涉及至少三个，最可能是四个HA三聚体。提出了一个模型来解释融合过程中HA的协同性。
