Hassel B A, Zhou A, Sotomayor C, Maran A, Silverman R H
Department of Cancer Biology, Cleveland Clinic Foundation, OH 44195.
EMBO J. 1993 Aug;12(8):3297-304. doi: 10.1002/j.1460-2075.1993.tb05999.x.
2-5A-dependent RNase is the terminal factor in the interferon-regulated 2-5A system thought to function in both the molecular mechanism of interferon action and in the general control of RNA stability. However, direct evidence for specific functions of 2-5A-dependent RNase has been generally lacking. Therefore, we developed a strategy to block the 2-5A system using a truncated form of 2-5A-dependent RNase which retains 2-5A binding activity while lacking RNase activity. When the truncated RNase was stably expressed to high levels in murine cells, it prevented specific rRNA cleavage in response to 2-5A transfection and the cells were unresponsive to the antiviral activity of interferon alpha/beta for encephalomyocarditis virus. Remarkably, cells expressing the truncated RNase were also resistant to the antiproliferative activity of interferon. The truncated RNase is a dominant negative mutant that binds 2-5A and that may interfere with normal protein-protein interactions through nine ankyrin-like repeats.
2-5A依赖性核糖核酸酶是干扰素调节的2-5A系统中的终末因子，被认为在干扰素作用的分子机制以及RNA稳定性的总体调控中均发挥作用。然而，2-5A依赖性核糖核酸酶特定功能的直接证据通常较为缺乏。因此，我们开发了一种策略，利用一种截短形式的2-5A依赖性核糖核酸酶来阻断2-5A系统，该截短形式保留了2-5A结合活性但缺乏核糖核酸酶活性。当截短的核糖核酸酶在鼠细胞中稳定高水平表达时，它可防止因转染2-5A而导致的特定rRNA切割，并且这些细胞对α/β干扰素针对脑心肌炎病毒的抗病毒活性无反应。值得注意的是，表达截短核糖核酸酶的细胞对干扰素的抗增殖活性也具有抗性。截短的核糖核酸酶是一种显性负性突变体，它结合2-5A，并且可能通过九个锚蛋白样重复序列干扰正常的蛋白质-蛋白质相互作用。
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