ANALYSIS OF VIRAL TRANSLATION COMPLEXES

人们早就知道，真核细胞的核糖体在本质上是异质性的。核糖体 异质性的例证是某些核糖体蛋白和核糖体的不同存在。 RNA，核糖体蛋白和RNA的修饰，以及非核糖体蛋白与 核糖体颗粒。然而，目前尚不清楚核糖体的异质性池是否反映了 蛋白质生物合成中不同的活性。最近，有一点变得明显，即内部核糖体 位于某些病毒mRNAs中的进入位点(IRES)可以直接与哺乳动物结合，并与哺乳动物具有高亲和力 40s亚基。这项提议的目的是检查功能异质性的种群 在哺乳动物细胞中是否存在核糖体，以及不同的核糖体群体是否被招募到 微小核糖核酸病毒和丙型肝炎病毒在感染细胞中的IRES元件。在第一个目标中，构图和 将比较未感染细胞的总核糖体亚基、游离核糖体亚基和多聚体结合核糖体亚基的活性。 用电喷雾-质谱仪(ES-MS)从小核糖核酸病毒感染细胞中分离得到。在 第二个目的，将检查病毒信使核糖体复合体中是否存在改变的核糖体。具体来说， 含硫尿苷的病毒RNA将在表达弓形虫尿嘧啶的HeLa细胞中产生 磷酸核糖转移酶(UPRT)。UPRT将硫尿嘧啶转化为硫尿嘧啶5‘单磷酸 其三磷酸可以通过依赖于病毒的RNA选择性地掺入病毒RNA的核苷酸 在放线菌素D存在下的RNA聚合酶多聚体硫代标记的RNA 生物素化，链霉亲和素层析分离，相关核糖体用ES-MS表征。 该方法还将用于鉴定与病毒RNA紧密连锁的IRES结合蛋白 被感染的细胞。最后，修饰的核糖体在翻译起始的各个步骤中的作用如下 在重组的翻译系统中进行了审查。这些研究的结果将揭示 在哺乳动物细胞中修饰核糖体，并将提供IRES元件招募宿主细胞的洞察力 病毒感染过程中的核糖体，揭示了抗病毒治疗的新潜在靶点。

It has been long been known that eukaryotic ribosomes are heterogeneous in nature. Ribosome heterogeneity is exemplified by the differential presence of certain ribosomal proteins and ribosomal RNAs, modification of ribosomal proteins and RNA, and association of non-ribosomal proteins with ribosomal particles. However, it remains unknown whether the heterogeneous pools of ribosomes reflect distinct activities in protein biosynthesis. More recently, it has become clear that the internal ribosome entry sites (IRES) located in certain viral mRNAs can bind directly and with high affinity to mammalian 40S subunits. The aims of this proposal are to examine whether functionally heterogeneous populations of ribosomes exist in mammalian cells and whether distinct ribosome populations are recruited to picornaviral and hepatitis C viral IRES elements in infected cells. In the first aim, the composition and activity of total, unbound and polysome-bound ribosomal subunits from uninfected cells will be compared to those isolated from picornavirus infected cells by electrospray mass spectrometry (ES-MS). In the second aim, the presence of altered ribosomes in viral mRNA-complexes will be examined. Specifically, thiouridine-containing viral RNA will be generated in HeLa cells expressing the Toxoplasma gondii uracil phosphoribosyltransferase (UPRT) enzyme. UPRT will convert thiouracil to thiouridine 5' monophosphate nucleotides whose triphosphates can be selectively incorporated into viral RNA by the viral RNAdependent RNA polymerase in the presence of actinomycin D. Polysomal thio-labeled RNA will be biotinylated, isolated by streptavidin chromatography and associated ribosomes characterized by ES-MS. This method will also be used to identify IRES-binding proteins that are tightly linked to the viral RNA in infected cells. Finally, the roles of modified ribosomes in various steps of translation initiation will be examined in reconstituted translation systems. The outcome from these studies will reveal roles of modified ribosomes in mammalian cells and will provide insights by which IRES elements recruit host cell ribosomes during viral infection, revealing new potential targets for antiviral therapeutics.