Lawrence J B, Cochrane A W, Johnson C V, Perkins A, Rosen C A
Department of Cell Biology, University of Massachusetts Medical Center, Worcester 01655.
New Biol. 1991 Dec;3(12):1220-32.
The impact of the Rev protein of the human immunodeficiency virus type 1 (HIV-1) on RNA transport, intranuclear RNA distribution, and gene expression was examined for two Rev-dependent expression systems by means of fluorescence in situ hybridization, immunofluorescence, S1 nuclease protection, and functional assays. In the pgTat expression system, which utilizes authentic HIV-1 splice signals, unspliced mRNA remained entrapped in the nucleus in the absence of Rev and was exported to the cytoplasm in its presence, consistent with published findings. In the pSVAR expression system, significant levels of mRNA were found in the nucleus and cytoplasm in both the presence and absence of Rev, but only in the presence of Rev was mRNA translated into protein. The presence of cytoplasmic untranslated mRNA in the absence of Rev was demonstrated by in situ hybridization analysis of individual cells as well as by S1 nuclease analysis of cell populations. The results indicate that Rev has the potential to affect translation as well as transport, suggesting the possibility that cellular mechanisms exist whereby the translational efficiency of an mRNA may be affected by the manner in which it is transported from the nucleus. Fluorescence hybridization also provided high-resolution visualization of the intranuclear distribution of RNAs containing the Rev response element. This demonstrated for both expression systems that mRNA was not highly localized in tracks or around the nucleolus in the presence or absence of Rev, a nucleolar protein, but was more widely distributed throughout the nucleus. In pgTat transfectants, HIV-1 RNA often became localized in 5 to 20 discrete large intranuclear clusters in the presence of Rev, the potential significance of which is discussed.
通过荧光原位杂交、免疫荧光、S1核酸酶保护及功能分析等方法，对两种依赖于Rev蛋白的表达系统，研究了人类免疫缺陷病毒1型（HIV-1）的Rev蛋白对RNA转运、核内RNA分布及基因表达的影响。在利用真实HIV-1剪接信号的pgTat表达系统中，未剪接的mRNA在没有Rev时滞留在细胞核内，而在有Rev时则被转运到细胞质中，这与已发表的研究结果一致。在pSVAR表达系统中，无论有无Rev，在细胞核和细胞质中均发现有大量的mRNA，但只有在有Rev时mRNA才能被翻译成蛋白质。通过对单个细胞的原位杂交分析以及对细胞群体的S1核酸酶分析，证实了在没有Rev时细胞质中存在未翻译的mRNA。结果表明，Rev可能会影响翻译及转运，这提示可能存在细胞机制，使mRNA的翻译效率受到其从细胞核转运方式的影响。荧光杂交还提供了含有Rev反应元件的RNA在核内分布的高分辨率可视化图像。这表明，对于这两种表达系统，无论有无Rev（一种核仁蛋白），mRNA在核内均不会高度定位于特定轨迹或核仁周围，而是更广泛地分布于整个细胞核中。在pgTat转染细胞中，在有Rev时HIV-1 RNA常常定位于5至20个离散的大核内簇中，文中对其潜在意义进行了讨论。
