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运输。 一旦进入细胞质，HIV RNA 就会逆转录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发挥其功能，并且病毒转录物被输出到细胞质。 病毒调节蛋白 rev 和 tat 已被确定为 HIV 包膜 mRNA 转录和转运出细胞核的关键调节剂。我们正在利用 HIV rev 蛋白在体外研究核蛋白导入和导出的过程。 rev 蛋白在核仁和细胞质之间主动穿梭，并介导未剪接的逆转录病毒 RNA 的过早输出。使用洋地黄皂苷透化培养细胞，我们开发了一种体外测定方法，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输入和输出的相对速率控制。 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 与激素诱导输入信号 (Rev/Gr/GFP) 融合。我们检查了体外和活细胞中 HIV Rev 的核运输和核仁积累的要求。数据表明从核仁解离是一个 ATP 依赖性过程。这种受调节的核仁定位可能反映了一种更广泛的现象，因为在参与细胞凋亡和细胞周期控制的其他核仁蛋白中也观察到了这种现象。我们描述的系统应该能够识别受监管的核仁靶向的要求。

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.