ROLE OF INTRACELLULAR TRAFFIC IN HIV INFECTION

穿过核膜的病毒成分在许多步骤中发生在 人类免疫缺陷病毒(HIV)和其他病毒的生命周期 逆转录病毒。一旦进入细胞质，HIV RNA就会被逆转录 变成必须进入细胞核的双链DNA。其他逆转录病毒 调节蛋白也进入细胞核来执行它们的功能， 病毒转录本被输出到细胞质中。病毒式传播 蛋白质，rev和tat，已被确定为关键的调节因子。 HIV包膜基因的转录和核外转运。我们 都把重点放在了HIV的rev蛋白上。一种嵌合蛋白已经被 产生由连接到绿色荧光蛋白的rev组成。 一种使用渗透化细胞的体外系统已经被开发出来 研究核仁靶向具有这种靶向作用的蛋白质 信号。我们观察到，向核仁的运输是一种活跃的 需要GTP和细胞内Ca+2存储的过程。此外 到它的核仁运输，rev是从细胞核主动输出的。 虽然调解REV核出口的特定信号一直是 人们对蛋白质输出的机制知之甚少。至 检查这一核出口事件我们构建了一个嵌合蛋白 由连接到绿色荧光蛋白(GFP)的REV组成。这个 嵌合体还含有糖皮质激素的类固醇结合结构域。 因此，可以对核进口进行精确控制。添加 荷尔蒙的减少导致核运输；荷尔蒙的去除启动了 导出，但不允许后续导入。我们已经能够使用 这项核出口分析在活细胞中确定的要求 用于核出口。此外，该检测可以在体外进行。 加入洋地黄素后，使质膜通透。这个 因此，化验可以鉴定所需的可溶性因子 用于体外核出口。

Viral components cross the nuclear membrane occurs at numerous steps in the life cycle of the human immunodeficiency virus (HIV) and other retroviruses. Once in the cytoplasm, HIV RNA is reverse-transcribed into double-stranded DNA which must enter the nucleus. Other retroviral regulatory proteins also enter the nucleus to perform their function, and viral transcripts are exported out to the cytoplasm. The viral proteins, rev and tat, have been identified as a key regulators of the transcription and transport of HIV envelope mRNA out of the nucleus. We have focused on the rev protein of HIV. A chimeric protein has been generated consisting of rev coupled to the green fluorescent protein. An in vitro system using permeabilized cells has been developed to examine the nucleolar targeting of proteins bearing such targeting signals. We have observed that transport to the nucleolus is an active process requiring both GTP and intracellular Ca+2 stores. In addition to its nucleolar transport rev is actively exported from the nucleus. While a specific signal mediating nuclear export of rev has been identified the mechanism of protein export is poorly understood. To examine this nuclear export event we have constructed a chimeric protein consisting of rev coupled to the green fluorescent protein (GFP). The chimera also contains the steroid binding domain of the glucocorticoid receptor thus allowing precise control over nuclear import. Addition of hormone leads to nuclear transport; removal of hormone initiates export but does not allow subsequent import. We have been able to use this nuclear export assay in living cells to determine the requirements for nuclear export. In addition, the assay can be performed in vitro after addition of digitonin to permeabilize the plasma membrane. The assay thus allows identification of soluble factors which are required for nuclear export in vitro.