Bachelerie F, Rodriguez M S, Dargemont C, Rousset D, Thomas D, Virelizier J L, Arenzana-Seisdedos F
Unité d'Immunologie Virale, Institut Pasteur, Paris, France.
J Cell Sci. 1997 Nov;110 ( Pt 22):2883-93. doi: 10.1242/jcs.110.22.2883.
De novo synthesized IkappaBalpha accumulates transiently in the nucleus where it inhibits NF-kappaB-dependent transcription and reduces nuclear NF-kappaB content. A sequence present in the C-terminal domain of IkappaBalpha and homologous to the HIV-1 Rev nuclear export signal (NES) has been recently defined as a functional NES conferring on IkappaBalpha the ability to export IkappaBalpha/NF-kappaB complexes. Rev utilises its RNA-binding activity and NES sequence to promote specifically the transport of unspliced and monospliced viral RNAs to the cytoplasm. The object of this work was to determine if nuclear IkappaBalpha could interfere with Rev-dependent transport of viral RNA from the nucleus to the cytoplasm. We report that accumulation of IkappaBalpha in the cell nucleus blocks viral replication. This effect could be dissociated from the capacity of IkappaBalpha to inhibit NF-kappaB-DNA-binding activity and required a functional IkappaBalpha NES motif. Indeed, mutation of the NES abrogated the capacity of IkappaBalpha to inhibit Rev-dependent mechanisms involved in the replication of either wild-type or NF-kappaB-mutated HIV-1 molecular clones. Nuclear accumulation of a reporter protein tagged with a nuclear localization signal (NLS) and fused to the IkappaBalpha NES motif (NLS-PK-NES) was sufficient to inhibit HIV-1 replication at a post-transcriptional level by specifically blocking the expression of a Rev-dependent gene. Furthermore, in cells pulsed with TNF, a treatment which favors nuclear accumulation of newly synthesized IkappaBalpha, NLS-PK-NES expression promoted sustained accumulation of nuclear NF-kappaB lacking DNA-binding activity. This NES-mediated accumulation of inactive nuclear NF-kappaB is likely the consequence of interference in the IkappaBalpha-mediated export of NF-kappaB. These findings indicate that IkappaBalpha and Rev compete for the same nuclear export pathway and suggest that nuclear accumulation of IkappaBalpha, which would occur during normal physiological cell activation process, may interfere with the Rev-NES-mediated export pathway of viral RNAs, thus inhibiting HIV-1 replication.
新合成的IκBα在细胞核中短暂积累，在那里它抑制NF-κB依赖的转录并降低细胞核中NF-κB的含量。IκBα C末端结构域中存在的一段与HIV-1 Rev核输出信号（NES）同源的序列最近被定义为功能性NES，赋予IκBα输出IκBα/NF-κB复合物的能力。Rev利用其RNA结合活性和NES序列来特异性促进未剪接和单剪接病毒RNA向细胞质的转运。这项工作的目的是确定细胞核中的IκBα是否会干扰Rev依赖的病毒RNA从细胞核到细胞质的转运。我们报告IκBα在细胞核中的积累会阻断病毒复制。这种效应可以与IκBα抑制NF-κB-DNA结合活性的能力分开，并且需要功能性的IκBα NES基序。事实上，NES的突变消除了IκBα抑制参与野生型或NF-κB突变的HIV-1分子克隆复制的Rev依赖机制的能力。标记有核定位信号（NLS）并与IκBα NES基序融合的报告蛋白（NLS-PK-NES）在细胞核中的积累足以通过特异性阻断Rev依赖基因的表达在转录后水平抑制HIV-1复制。此外，在用TNF脉冲处理的细胞中，这种处理有利于新合成的IκBα在细胞核中的积累，NLS-PK-NES表达促进了缺乏DNA结合活性的细胞核NF-κB的持续积累。这种NES介导的无活性细胞核NF-κB的积累可能是干扰IκBα介导的NF-κB输出的结果。这些发现表明IκBα和Rev竞争相同的核输出途径，并表明在正常生理细胞激活过程中会发生的IκBα在细胞核中的积累可能会干扰Rev-NES介导的病毒RNA输出途径，从而抑制HIV-1复制。
