ROLE OF INTRACELLULAR TRAFFIC IN HIV INFECTION

人类免疫缺陷病毒（HIV）和其他逆转录病毒的生命周期依赖于核转运。 一旦在细胞质中，HIV RNA被逆转录成双链DNA，其必须进入细胞核。 其他逆转录病毒调节蛋白也进入细胞核发挥功能，病毒转录物被输出到细胞质。 病毒调节蛋白rev和达特已被鉴定为HIV包膜mRNA转录和转运出核的关键调节剂。我们正在使用HIV rev蛋白研究体外核蛋白输入和输出的过程。rev蛋白在细胞核和细胞质之间穿梭，并介导未剪接的逆转录病毒RNA的过早输出。使用毛地黄皂苷透化培养细胞，我们开发了一种体外试验，用于研究核转运和核仁积累的分子，如HIV Rev。核穿梭蛋白，如HIV Rev的本地化是由核进口和出口的相对速率控制。 我们还开发了一种体外核输出检测方法，使用绿色荧光蛋白标记的Rev融合到可诱导的输入信号（Rev/Gr/GFP）。我们研究了在体外和活细胞中HIV Rev的核运输和核仁积累的要求。这些数据表明，从核仁的解离是一个ATP依赖的过程。这种受调节的核仁定位可能反映了一种更普遍的现象，因为它已被观察到其他核仁蛋白参与细胞凋亡和细胞周期控制。我们所描述的系统应该允许确定的要求，调节核仁靶向。

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.