INTERACTION OF POLYPYRIMIDINE TRACT-BINDING PROTEIN WITH THE 5'-NONCODING REGION OF THE HEPATITIS-C VIRUS-RNA GENOME AND ITS FUNCTIONAL REQUIREMENT IN INTERNAL INITIATION OF TRANSLATION
INTERACTION OF POLYPYRIMIDINE TRACT-BINDING PROTEIN WITH THE 5'-NONCODING REGION OF THE HEPATITIS-C VIRUS-RNA GENOME AND ITS FUNCTIONAL REQUIREMENT IN INTERNAL INITIATION OF TRANSLATION
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DOI：
10.1128/jvi.69.10.6367-6375.1995
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发表时间：
1995-10-01
影响因子：
5.4
通讯作者：
SIDDIQUI, A
中科院分区：
文献类型：
--
作者：
ALI, N;SIDDIQUI, A
人类丙型肝炎病毒 (HCV) RNA 基因组的翻译起始是通过内部核糖体以帽独立方式进入 5' 非编码区 (5'NCR) 进行的。 HCV 5'NCR 的内部核糖体进入位点先前已被定义为涵盖几乎整个 5'NCR。在这里，我们通过 UV 交联测定报告了 5'NCR 内三个不同区域的聚嘧啶束结合蛋白 (PTB) 的相互作用。所有三个区域都包含共有的多嘧啶束基序。重组 PTB 在 5'NCR 内多个位点相互作用的证据基于使用 5'NCR 突变体作为竞争剂以及通过突变体 RNA 的直接 UV 交联。此外，HeLa 核提取物中的 PTB 异构体与 HCV 5'NCR 相互作用，如 UV 交联复合物与抗 PTB 血清的免疫沉淀所示。翻译裂解物中 PTB 的免疫耗竭表明 HCV RNA 翻译起始过程中对 PTB 的功能要求。将纯化的 PTB 添加到免疫耗竭的裂解物中并不能恢复由 HCV 5'NCR 介导的翻译，表明需要在免疫耗竭期间去除的 PTB 相关因子。
Initiation of translation of the human hepatitis C virus (HCV) RNA genome occurs by internal ribosome entry into the 5' noncoding region (5'NCR) in a cap-independent manner. The internal ribosome entry site of the HCV 5'NCR has been previously defined to encompass almost the entire 5'NCR. Here we report the interaction of polypyrimidine tract-binding protein (PTB) at three distinct regions within the 5'NCR by UV cross-linking assays. All three regions contain a consensus polypyrimidine tract motif. The evidence for the interaction of recombinant PTB at multiple sites within the 5'NCR is based on the use of 5'NCR mutants as competitors and by direct UV cross-linking of the mutant RNAs. Furthermore, the PTB isomers from HeLa nuclear extracts interact with the HCV 5'NCR, as shown by immunoprecipitation of a UV cross-linked complex with anti-PTB serum. Immunodepletion of PTB from translation lysates suggested the functional requirement for PTB during translation initiation of the HCV RNA. Addition of purified PTB to immunodepleted lysates did not restore translation mediated by the HCV 5'NCR, indicating the requirement of PTB-associated factors that were removed during immunodepletion.