Florez Paola M, Sessions October M, Wagner Eric J, Gromeier Matthias, Garcia-Blanco Mariano A
Duke University Medical Center, Box 3020 (451 Jones), Research Drive, Durham, NC 27710, USA.
J Virol. 2005 May;79(10):6172-9. doi: 10.1128/JVI.79.10.6172-6179.2005.
Mammalian host factors required for efficient viral gene expression and propagation have been often recalcitrant to genetic analysis. A case in point is the function of cellular factors that trans-activate internal ribosomal entry site (IRES)-driven translation, which is operative in many positive-stranded RNA viruses, including all picornaviruses. These IRES trans-acting factors have been elegantly studied in vitro, but their in vivo importance for viral gene expression and propagation has not been widely confirmed experimentally. Here we use RNA interference to deplete mammalian cells of one such factor, the polypyrimidine tract binding protein, and test its requirement in picornavirus gene expression and propagation. Depletion of the polypyrimidine tract binding protein resulted in a marked delay of particle propagation and significantly decreased synthesis and accumulation of viral proteins of poliovirus and encephalomyocarditis virus. These effects could be partially restored by expression of an RNA interference-resistant exogenous polypyrimidine tract binding protein. These data indicate a critical role for the polypyrimidine tract binding protein in picornavirus gene expression and strongly suggest a requirement for efficient IRES-dependent translation.
高效病毒基因表达和传播所需的哺乳动物宿主因子常常难以进行遗传分析。一个典型的例子是反式激活内部核糖体进入位点（IRES）驱动翻译的细胞因子的功能，这种翻译在许多正链RNA病毒中起作用，包括所有微小核糖核酸病毒。这些IRES反式作用因子已在体外进行了深入研究，但它们在体内对病毒基因表达和传播的重要性尚未得到广泛的实验证实。在这里，我们使用RNA干扰来耗尽哺乳动物细胞中的一种这样的因子，即多嘧啶序列结合蛋白，并测试其在微小核糖核酸病毒基因表达和传播中的需求。多嘧啶序列结合蛋白的耗尽导致病毒粒子传播明显延迟，并显著降低脊髓灰质炎病毒和脑心肌炎病毒的病毒蛋白合成和积累。通过表达抗RNA干扰的外源性多嘧啶序列结合蛋白，这些效应可以部分恢复。这些数据表明多嘧啶序列结合蛋白在微小核糖核酸病毒基因表达中起关键作用，并强烈暗示对高效IRES依赖性翻译的需求。
