Bink Hugo H J, Hellendoorn Koen, van der Meulen Jannes, Pleij Cornelis W A
Leiden University, Leiden Institute of Chemistry, Gorlaeus Laboratories, Einsteinweg 55, 2300 RA Leiden, The Netherlands.
Proc Natl Acad Sci U S A. 2002 Oct 15;99(21):13465-70. doi: 10.1073/pnas.202287499. Epub 2002 Oct 2.
The 5' UTR of turnip yellow mosaic virus RNA contains two conserved hairpins with internal loops consisting of C.C and C.A mismatches. In this article, evidence is presented indicating that the 5' proximal hairpin functions as an encapsidation initiation signal. Extensive mutagenesis studies on this hairpin and sequencing of virus progeny showed a clear preference for C.C and C.A mismatches within the internal loop. The importance of these mismatches lies in their pH-dependent protonation and stable base pair formation. Encapsidation efficiency was found to be severely affected for several mutants lacking the protonatable mismatches in the internal loop of the 5' proximal hairpin. Furthermore, gel mobility-shift assays were performed with various RNA hairpins and empty capsids with a hole. Protonatable hairpins containing C.C and/or C.A pairs were found to bind specifically to the interior of the protein shell under acidic conditions (pH 4.5) in the presence of spermidine. Based on these results we propose that this binding of protonated cytosines to the coat protein of turnip yellow mosaic virus may represent a new motif in RNA-protein interactions.
芜菁黄花叶病毒RNA的5'非翻译区包含两个保守的发夹结构，其内部环由C.C和C.A错配组成。在本文中，有证据表明5'近端发夹作为衣壳化起始信号发挥作用。对该发夹进行的广泛诱变研究以及病毒子代的测序显示，内部环内明显偏好C.C和C.A错配。这些错配的重要性在于它们的pH依赖性质子化和稳定碱基对的形成。发现对于5'近端发夹内部环中缺乏可质子化错配的几个突变体，衣壳化效率受到严重影响。此外，用各种RNA发夹和有孔的空衣壳进行了凝胶迁移率变动分析。发现在亚精胺存在下，含有C.C和/或C.A对的可质子化发夹在酸性条件（pH 4.5）下特异性结合到蛋白质外壳内部。基于这些结果，我们提出质子化胞嘧啶与芜菁黄花叶病毒衣壳蛋白的这种结合可能代表RNA-蛋白质相互作用中的一个新基序。
