Structural basis of protein synthesis by the hepatitis C virus IRES RNA

描述（由申请人提供）：丙型肝炎病毒（HCV）是一种使用内部核糖体进入位点（IRES） RNA驱动所有病毒蛋白生产的病毒。HCV IRES RNA形成直接结合40S核糖体亚基的特定结构，从而改变亚基的构象。核糖体的这种物理操作似乎是HCV IRES“劫持”翻译机制的关键组成部分。然而，IRES如何完成这些结构变化，它们如何在核糖体中传播，以及如何将诱导结构与典型核糖体构象进行比较仍然未知。此外，越来越多的证据表明，HCV IRES是病毒逃避宿主细胞先天免疫应答策略的一个组成部分，部分原因是在细胞应激过程中，通过替代的真核起始因子（eIF） 2非依赖性途径驱动翻译起始。虽然HCV IRES在非应激细胞中的作用机制已经得到了很好的研究，但在应激细胞中使用的途径却知之甚少。我们试图通过访问替代翻译起始途径以及通过物理操作核糖体来探索IRES操纵宿主细胞机制的能力。我们提出了两个目标：1)确定使HCV IRES在细胞应激条件下运作的IRES结构域、因素和机制；2)确定与核糖体或核糖体亚基结合的HCV IRES RNA的高分辨率结构。为了实现这些目标，我们将结合生物化学、基于细胞培养的检测和结构生物学，以一种综合的方法，专注于以前所未有的细节水平开发HCV IRES功能的新模型。我们将使用的一些方法和工具是在我们实验室开发的，因此是我们实验室独有的。我们的研究有望对IRESs如何发挥作用、哺乳动物核糖体如何运作、核糖体如何被操纵以及在细胞应激期间如何发生翻译等方面做出新的发现。

DESCRIPTION (provided by applicant): Hepatitis C virus (HCV) is an example of a virus that uses an internal ribosome entry site (IRES) RNA to drive production of all the viral proteins. The HCV IRES RNA forms a specific structure that binds directly to the 40S ribosomal subunit and in so doing changes the conformation of the subunit. This physical manipulation of the ribosome appears to be a critical component of the mechanism by which the HCV IRES "hijacks" the translation machinery. However, how the IRES accomplishes these structural changes, how they propagate through the ribosome, and how the induced structures compare to canonical ribosome conformations remains unknown. In addition, there is growing evidence that the HCV IRES is one component of the virus' strategy to evade the host cell's innate immune response, in part by driving translation initiation by alternate, eukaryotic initiation factor (eIF) 2-independent pathways operating during cellular stress. While the mechanism by which the HCV IRES operates in unstressed cells is well-studied, the pathway used in stressed cells is very poorly understood. We seek to explore the ability of the IRES to manipulate the host cell's machinery by accessing alternate translation initiation pathways and also through physical manipulation of the ribosome. We propose two aims: 1) Define the IRES domains, factors, and mechanisms that enable the HCV IRES to operate under conditions of cellular stress, and 2) Determine the high-resolution structure of an HCV IRES RNA bound to a ribosome or ribosomal subunit. To accomplish these aims, we will use a combination of biochemistry, cell culture-based assays, and structural biology in an integrated approach focused on developing new models for HCV IRES function at an unprecedented level of detail. Several of the methods and tools we will use have been developed in our lab and thus are unique to our lab. Our studies promise to contribute new discoveries to how IRESs function, how mammalian ribosomes operate, how ribosomes can be manipulated, and how translation occurs during periods of cell stress.