STUDIES OF HIV-1 ENV-MEDIATED MEMBRANE FUSION AND SYNCYTIA FORMATION

我们开发了一种灵敏的检测方法来检测HIV-1的早期阶段 基于再分布的Env介导的细胞膜融合 相邻细胞膜之间的荧光染料，由 荧光视频显微镜。这个测试证明了膜 表达gp120/41的细胞与CD4+细胞的融合可以在 没有形成联轴节的条件。总的来说，核聚变发生了 合胞体形成早于合胞体形成，细胞比率不利于 合胞体的形成(类似于体内的情况)。还有，细胞膜 融合(但不是联会)对机械力敏感。 本实验用来确定黏附分子LFA-1的作用 在HIV-1env介导的细胞膜融合和合胞体的形成。CD_4- LFA-EBV转化的两种白细胞黏附缺陷B细胞系 (LAD)患者，感染表达gp120/41的重组疫苗 (HIV-1 IIIB)，并与人T细胞系的CD4+亚克隆共培养 CEM是由化学诱变产生的，并表达 正常或低水平的LFA-1。研究发现，LFA-1(低)T细胞 与LFA-1+相比，克隆形成的合胞体要小得多和少得多 回复亚克隆。相反，这两个亚克隆与 表达LFA-1-B细胞的gp120/41 荧光染料。此外，抗LFA-1的单抗， 减少合胞体形成的数量，但对膜没有影响 核聚变。这些数据表明：1.黏附分子LFA-1具有 在HIV-1包膜介导的细胞早期事件中不起关键作用 膜融合，但导致合胞体过程较慢 队形。不能使用融合和合胞体形成这两个术语 可以互换。这种化验方法可用于检测可能 阻断HIV-1依赖的细胞融合。

We have developed a sensitive assay to examine the early stages of HIV-1 env-mediated cell membrane fusion which is based on redistribution of fluorescent dyes between membranes of adjacent cells, monitored by fluorescence video microscopy. This assay demonstrated that membrane fusion between gp120/41 expressing cells and CD4+ cells, can occur under conditions where no synctytia are formed. In general, fusion took place earlier than syncytia formation and at cell ratios which did not favor syncytia formation (similar to the situation in vivo). Also, cell membrane fusion (but not synctia) were sensitive to mechan- ical forces. This assay was used to determine the role of the adhesion molecule LFA-1 in HIV-1 env-mediated cell membrane fusion and syncytia formation. CD4- LFA- EBV transformed B-cell lines from two leukocyte adhesion deficient (LAD) patients, were infected with reombinant vaccinia expressing gp120/41 (HIV-1 IIIB), and co-cultured with CD4+ subclones of the human T cell line CEM, which were generated by chemical mutagenesis and express either normal or low levels of LFA-1. It was found that the LFA-1 (low) T cell clone formed much smaller and fewer syncytia compared to a LFA-1+ revertant subclone. In contrast, both subclones fused equally well with the gp120/41 expressing LFA-1- B cells as monitored by redistribution of fluorescent dyes. Furthermore, monoclonal antibodies agains LFA-1, reduced the number of syncytia formed but had no effect on membrane fusion. These data demonstrate that: I. The adhesion molecule LFA-1 does not play a crucial role in the early events of HIV-1 envmediated cell membrane fusion but contributes to the slower process of syncytia formation. II. The terms fusion and syncytia formation cannot be used interchangeablly. III. This assay can be used to test drugs which may block HIV-1 dependent cell fusion.