ROLE OF INTRACELLULAR TRAFFIC IN HIV INFECTION

细胞内运输在 HIV 感染中的作用

• 批准号: 6162051
• 负责人: J A HANOVER
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6162051
• 关键词: HIV envelope protein; HIV infections; antigen presentation; corticosteroid receptors; green fluorescent proteins; human immunodeficiency virus; intracellular transport; messenger RNA; nucleolus; protein sequence; recombinant proteins; virus RNA; virus antigen; virus protein

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.

病毒成分穿过核膜发生在许多步骤中 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 成双链DNA，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其功能， 病毒转录本被输出到细胞质。 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 mRNA 的转录和转运出细胞核。我们 重点研究了 HIV 的 rev 蛋白。 嵌合蛋白已被 生成由与绿色荧光蛋白偶联的 rev 组成。 已开发出使用透化细胞的体外系统 检查具有这种靶向的蛋白质的核仁靶向 信号。 我们观察到向核仁的转运是一种主动的 该过程需要 GTP 和细胞内 Ca+2 储存。 此外 向其核仁运输的转速是从细胞核主动输出的。 虽然介导 rev 核输出的特定信号已被 人们对蛋白质输出的机制知之甚少。 到 检查这个核输出事件我们构建了一个嵌合蛋白 由与绿色荧光蛋白 (GFP) 耦合的 rev 组成。 这 嵌合体还含有糖皮质激素的类固醇结合结构域 受体从而可以精确控制核输入。 添加 激素导致核运输；去除激素开始 导出但不允许后续导入。 我们已经能够使用 这种在活细胞中进行核输出测定以确定要求 用于核出口。 此外，该测定可以在体外进行 添加洋地黄皂苷以透化质膜后。这 因此，测定可以鉴定所需的可溶性因子 用于体外核输出。