ROLE OF INTRACELLULAR TRAFFIC IN HIV INFECTION

人类免疫缺陷病毒(艾滋病毒)和其他逆转录病毒的生命周期依赖于核运输。一旦进入细胞质，HIV RNA就会被逆转录成双链DNA，然后进入细胞核。其他逆转录病毒调节蛋白也进入细胞核执行其功能，病毒转录本被输出到细胞质。病毒调节蛋白REV和TAT被认为是HIV包膜基因转录和核外运输的关键调节因子。我们正在使用HIV rev蛋白在体外检查核蛋白的进出口过程。REV蛋白活跃地穿梭在核仁和细胞质之间，并介导未剪接逆转录病毒RNA的过早输出。利用洋地黄素渗透培养的细胞，我们建立了一种体外实验，用于研究HIV Rev.等分子的核转运和核仁积累。HIV rev等核穿梭蛋白的定位受核进出口相对速度的控制。我们还开发了一种使用绿色荧光蛋白标记的REV与激素诱导的输入信号(REV/Gr/GFP)融合的核出口体外检测方法。我们研究了HIV Rev在体外和活细胞中的核转运和核仁积累的要求。这些数据表明，从核仁解离是一个依赖于ATP的过程。这种受调控的核仁定位可能反映了一个更广泛的现象，因为它已经在参与细胞凋亡和细胞周期控制的其他核仁蛋白中观察到。我们已经描述的系统应该允许识别受调控的核仁靶向的要求。

The life cycle of the human immunodeficiency virus (HIV) and other retroviruses is dependent upon nuclear transport. 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 to the cytoplasm. The viral regulatory proteins, rev and tat, have been identified as key modulators of the transcription and transport of HIV envelope mRNA out of the nucleus. We are examining the processes of nuclear protein import and export in vitro using the HIV rev protein. The rev protein actively shuttles between nucleolus and cytoplasm and mediates premature export of unspliced retroviral RNAs. Using digitonin permeabilized cultured cells, we developed an in vitro assay for studying both nuclear transport and nucleolar accumulation of molecules such as HIV Rev. The localization of nuclear shuttling proteins such as HIV rev is controlled by the relative rates of nuclear import and export. We also have developed an in vitro assay for nuclear export using the green fluorescent protein-labelled rev fused to a hormone-inducible import signal (Rev/Gr/GFP). We examined the requirements for nuclear transport and nucleolar accumulation of HIV Rev in vitro and in living cells. The data suggest that dissociation from the nucleolus is an ATP-dependent process. This regulated nucleolar localization may reflect a more widespread phenomenon since it has been observed for other nucleolar proteins involved in apoptosis and cell cycle control. The system we have described should allow the identification of the requirements for regulated nucleolar targeting.