Bøe S O, Bjørndal B, Røsok B, Szilvay A M, Kalland K H
Department of Microbiology and Immunology, Gade Institute, Bergen High Technology Center, University of Bergen, Norway.
Virology. 1998 May 10;244(2):473-82. doi: 10.1006/viro.1998.9110.
The HIV-1 protein Rev regulates the cytoplasmic levels of incompletely spliced HIV-1 mRNAs. The plasmid pSVc21, which contains a HIV-1 provirus, was introduced into COS cells by transient transfection. Simultaneous detection of HIV-1 RNAs and Rev proteins produced in transfected cells was then performed in order to determine the relative distribution of these two components. HIV-1 RNAs and the Rev protein localized to the same areas of the nucleoplasm, implying that these locations represent sites where Rev interacts with its target RNAs. Using a monoclonal antibody targeted to the splicing factor SC-35 it was demonstrated that the sites where HIV-1 mRNAs and Rev were detected often contained weak anti-SC-35 staining, whereas little RNA and Rev were found in strongly labeled SC-35-containing speckles. The same distribution of HIV-1 RNAs relative to SC-35 was also seen in transfected HeLa cells and in primary human lymphocytes infected with HIV-1 primary isolates. In addition, transiently expressed intron-containing beta-globin RNAs were shown to distribute to weak anti-SC-35 staining in a manner similar to that of HIV-1 RNAs. The findings suggest that Rev and HIV-1 RNAs interact at putative sites of mRNA transcription and splicing.
HIV-1蛋白Rev调节不完全剪接的HIV-1 mRNA的细胞质水平。通过瞬时转染将含有HIV-1前病毒的质粒pSVc21导入COS细胞。然后对转染细胞中产生的HIV-1 RNA和Rev蛋白进行同步检测，以确定这两种成分的相对分布。HIV-1 RNA和Rev蛋白定位于核质的相同区域，这意味着这些位置代表Rev与其靶RNA相互作用的位点。使用针对剪接因子SC-35的单克隆抗体表明，检测到HIV-1 mRNA和Rev的位点通常含有较弱的抗SC-35染色，而在强标记的含SC-35的斑点中几乎没有发现RNA和Rev。在转染的HeLa细胞和感染HIV-1原代分离株的原代人淋巴细胞中也观察到HIV-1 RNA相对于SC-35的相同分布。此外，瞬时表达的含内含子的β-珠蛋白RNA以与HIV-1 RNA相似的方式分布到较弱的抗SC-35染色区域。这些发现表明Rev和HIV-1 RNA在mRNA转录和剪接的假定位点相互作用。
