ANALYSIS OF VIRAL TRANSLATION COMPLEXES

病毒翻译复合物的分析

基本信息

批准号：
7646468
负责人：
PETER SARNOW
金额：
$ 14.29万
依托单位：
UNIVERSITY OF CALIFORNIA BERKELEY
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/7646468
关键词：
Affinity Antiviral Agents Binding Binding Proteins Cells Chromatography Complex DNA DNA-Directed RNA Polymerase Dactinomycin Elements Enzymes Family Picornaviridae Goals Hela Cells Hepatitis C Heterogeneity Human poliovirus Indium Individual Internal Ribosome Entry Site Label Ligands Link Mammalian Cell Mass Spectrum Analysis Mengovirus Messenger RNA Methods Modification Nature Nucleotides Object Attachment Outcome Study Peptides Polioviruses Polyribosomes Population Population Heterogeneity Principal Investigator Protein Biosynthesis Proteins RNA Recruitment Activity Ribosomal Proteins Ribosomal RNA Ribosomes Role Specificity Streptavidin System Testing Therapeutic Thiouracil Thiouridine Toxoplasma gondii Translation Initiation Translations Ultraviolet Rays Uracil phosphoribosyltransferase Viral Virus Virus Diseases crosslink insight novel strategies particle reconstitution tripolyphosphate viral RNA

项目摘要

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.

早就知道，真核核糖体本质上是异质的。核糖体某些核糖体蛋白和核糖体的差异存在体现了异质性 RNA，核糖体蛋白和RNA的修饰，非核糖体蛋白与核糖体颗粒。但是，尚不清楚核糖体的异质池是否反映蛋白质生物合成中的不同活性。最近，很明显内部核糖体位于某些病毒mRNA中的入口位点（IRES）可以直接结合并具有高亲和力与哺乳动物 40s亚基。该提案的目的是检查功能上异质的人群是否哺乳动物细胞中存在核糖体的核糖体，以及是否招募不同的核糖体种群受感染细胞中的Picornaviral和丙型肝炎病毒IRES元素。在第一个目标中，构图和将比较来自未感染细胞的总，未结合和多层核糖体亚基的活性通过电喷雾质谱法（ES-MS）从picornavirus感染细胞中分离出来的人。在第二个目的，将检查病毒mRNA复合物中核糖体的存在。具体来说，在表达弓形虫的HeLa细胞中，将产生含硫氨氨酸的病毒RNA 磷酸贝糖基转移酶（UPRT）酶。 UPRT将将硫核转化为硫氨岛5'单磷酸盐核苷酸的三磷酸可以通过病毒RNADEDERTENT选择性地掺入病毒RNA中在放线霉素的情况下，RNA聚合酶D.多硫代标记的RNA将是生物素化，通过链霉亲和素色谱和以ES-MS为特征的相关核糖体分离。该方法还将用于识别与病毒RNA紧密相关的IRES结合蛋白感染细胞。最后，修饰的核糖体在翻译启动的各个步骤中的作用将是在重组的翻译系统中进行了检查。这些研究的结果将揭示哺乳动物细胞中修饰的核糖体，将提供IRES元素募集宿主细胞的见解病毒感染期间的核糖体揭示了抗病毒治疗剂的新潜在靶标。