Cheung Paul, Zhang Mary, Yuan Ji, Chau David, Yanagawa Bobby, McManus Bruce, Yang Decheng
Department of Pathology, University of British Columbia, Vancouver, Canada.
Virus Res. 2002 Dec;90(1-2):23-36. doi: 10.1016/s0168-1702(02)00138-7.
Translation initiation of the coxsackievirus B3 (CVB3) RNA occurs by internal ribosomal entry. The internal ribosomal entry site (IRES) of the virus has been mapped to the 5' untranslated region (5' UTR) of the genome. As well, the 5' UTR has been suggested to play roles in determining the tissue tropism and infectivity of the virus. In this study, we investigated interactions between HeLa cell protein extracts and radiolabeled RNA of CVB3 5' UTR by competitive UV cross-linking. We have observed a number of proteins that specifically interact with the three sub-cloned regions of the 5' UTR. In particular, the molecular weights of five of these proteins resemble those of the eukaryotic translation initiation factors 4A, 4B and 4G, as well as the La autoantigen and the polypyrimidine tract binding protein. Based on this data, we focused on the interaction of the 5' UTR with the La autoantigen, which was purified by the glutathione-S-transferase affinity method. We have confirmed the highly specific interaction of the La autoantigen with the 5' UTR sequence nt 210-529. The core IRES (nt 530-630) and nt 1-209 also appear to bind to the La protein at moderate and weak affinities, respectively. A functional role of the La autoantigen in translation initiation is suggested.
柯萨奇病毒B3（CVB3）RNA的翻译起始通过核糖体内部进入发生。该病毒的核糖体内部进入位点（IRES）已被定位到基因组的5'非翻译区（5'UTR）。此外，5'UTR被认为在决定病毒的组织嗜性和感染性方面发挥作用。在本研究中，我们通过竞争性紫外光交联研究了HeLa细胞蛋白提取物与CVB3 5'UTR放射性标记RNA之间的相互作用。我们观察到许多与5'UTR的三个亚克隆区域特异性相互作用的蛋白质。特别是，其中五种蛋白质的分子量与真核翻译起始因子4A、4B和4G以及La自身抗原和多嘧啶序列结合蛋白的分子量相似。基于这些数据，我们重点研究了5'UTR与通过谷胱甘肽-S-转移酶亲和法纯化的La自身抗原之间的相互作用。我们已经证实了La自身抗原与5'UTR序列nt 210-529之间的高度特异性相互作用。核心IRES（nt 530-630）和nt 1-209也分别以中等和弱亲和力与La蛋白结合。这表明La自身抗原在翻译起始中具有功能作用。
