Nomoto A, Tsukiyama-Kohara K, Kohara M
Department of Microbiology, University of Tokyo, Japan.
Princess Takamatsu Symp. 1995;25:111-9.
In vitro translation experiments involving reticulocyte and HeLa cell lysates have demonstrated that translation initiation on hepatitis C virus (HCV) RNA occurs by entry of ribosomes to the internal sequence (internal ribosomal entry site [IRES]) within the 5' noncoding region (5'NCR). Specific binding factors that bind to the HCV IRES were examined by UV cross-linking tests. A protein p57 (polypyrimidine tract binding protein [PTB]), known to be a binding factor that binds to picornavirus IRES, was found to bind to the HCV IRES. To investigate functions of this PTB in IRES-dependent translation initiation, hyperimmune antibodies to PTB (anti-PTB) were prepared by immunizing rabbits with purified recombinant PTB produced in E. coli. Anti-PTB IgGs were purified and used to deplete PTB in a cell-free protein synthesis system prepared from HeLa cells. In vitro translation experiments using PTB-depleted lysates suggested that host factor dependency of IRES is different in individual IRESs. As we showed previously, function of the IRES typical of group II HCV is more efficient than the IRES typical of group I HCV in a cell-free protein synthesis system from HeLa cells. Recombinant HCV IRESs were constructed and tested for their function in the in vitro system. Most recombinant IRESs showed an initiation activity of the more efficient typical group II IRES level or higher. This observation, together with the fact that group I HCV is the most common isolate of HCV worldwide, suggests that the efficiency of the IRES function of HCV may be generally suppressed in nature, and that the replication ability of HCV that permits its persistent infection in humans may require a delicate balance at a molecular level.
涉及网织红细胞和HeLa细胞裂解物的体外翻译实验表明，丙型肝炎病毒（HCV）RNA的翻译起始是通过核糖体进入5'非编码区（5'NCR）内的内部序列（内部核糖体进入位点[IRES]）来实现的。通过紫外线交联试验检测了与HCV IRES结合的特异性结合因子。发现一种已知与微小RNA病毒IRES结合的结合因子——蛋白质p57（多聚嘧啶序列结合蛋白[PTB]）能与HCV IRES结合。为了研究该PTB在IRES依赖性翻译起始中的功能，用在大肠杆菌中产生的纯化重组PTB免疫兔子，制备了针对PTB的超免疫抗体（抗PTB）。纯化抗PTB IgG并用于在由HeLa细胞制备的无细胞蛋白质合成系统中消耗PTB。使用耗尽PTB的裂解物进行的体外翻译实验表明，不同的IRES对宿主因子的依赖性有所不同。正如我们之前所表明的，在来自HeLa细胞的无细胞蛋白质合成系统中，II型HCV典型的IRES功能比I型HCV典型的IRES更有效。构建了重组HCV IRES并在体外系统中测试其功能。大多数重组IRES表现出更有效的典型II型IRES水平或更高的起始活性。这一观察结果，连同I型HCV是全球最常见的HCV分离株这一事实，表明HCV的IRES功能效率在自然界中可能普遍受到抑制，并且HCV在人类中持续感染所允许的复制能力可能在分子水平上需要微妙的平衡。
