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 env介导的细胞膜融合是基于 HIV-1 env表达细胞和CD 4阳性细胞之间的荧光染料 相邻细胞，通过荧光视频显微镜监测。 该试验表明，术语细胞融合和合胞体 形成不能互换使用。细胞融合与合胞体 形成遵循不同的动力学。细胞融合在15分钟开始。 90 min时达到平台期，而合胞体的形成首先出现在 在2小时时，并且在5-6小时时达到平台。此外， 在不利于合胞体的条件下（即细胞比率）， 形成（类似于体内情况）。 该试验用于重新检查粘附所起的作用， 分子LFA-1/ICAM-1在HIV-1 env介导的细胞融合过程中的作用。LFA-1 - 缺陷型B细胞系（来自LAD患者）和T细胞（产生的 在我们的实验室通过化学诱变）在研究中使用。 发现LFA-1粘附分子在细胞凋亡过程中不起作用。 HIV-1 env介导的细胞融合的早期事件，但确实有助于 随后的事件导致合胞体形成。 在一个单独的项目中，我们研究了人类B细胞系之间的融合 表达HIV-1 env的细胞。我们发现了一种融合机制， 由CD 4-gp 120相互作用启动，但被IG大大增强 B细胞上的CD 4受体，只要它们与细胞表面的CD 4结合区结合， GP 120信封这项研究为选择性地 表达抗gp 120 Ab受体的B细胞的融合和消除。 该试验将用于筛选可能阻断早期阶段的药物， HIV-1细胞膜融合。

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 HIV-1 env-expressing cells and CD4-positive adjacent cells, monitored by fluorescence video microscopy. This assay demonstrated that the terms cell fusion and syncytia formation can not be interchangeably used. Cell fusion and syncytia formation followed different kinetics. Cell fusion started at 15 min. and reached plateau at 90 min., while syncytia formation first appeared at 2 hr, and reached a plateau at 5-6 hrs. Furthermore, fusion occurred under conditions (i.e. cell ratios) which did not favor syncytia formation (similar to the in vivo situation). This assay was used to re-examine the role played by the adhesion molecules LFA-1/ICAM-1 during HIV-1 env-mediated cell fusion. LFA-1 - deficient B cell lines (from patients with LAD), and T cells (generated in our laboratory by chemical mutagenesis) were used in the study.It was found that the LFA-1 adhesion molecules do not play a role during the early events of HIV-1 env-mediated cell fusion, but do contribute to later events leading to syncytia formation. In a separate project, we studied the fusion between human B cell lines end HIV-1 env-expressing cells. We identified a fusion mechanism which is initiated by CD4-gp120 interactions, but greatly enhanced by the Ig receptors on B cells, providing they bind to the CD4-binding-region on the gp120 envelope. This study provides a mechanism for the selective fusion and elimination of B-cells expressing anti-gp120 Ab receptors. This assay will be used to screen drugs which may block early stages in HIV-1 cell membrane fusion.