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Studies of the global translational response to human virus infection

对人类病毒感染的全球转化反应的研究

基本信息

批准号：
8803766
负责人：
John S Parker
金额：
$ 19.38万
依托单位：
CORNELL UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-02-10 至 2017-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8803766
关键词：
A549 Address Amino Acids Bioinformatics Biological Assay Cells Cellular Stress Complement Complex Cytomegalovirus Defense Mechanisms Frequencies Gene Expression Gene Expression Profile Genes Genetic Transcription Genetic Translation Genome Goals Health Herpesvirus 1 Human Human Biology Human Virus Immune Infection Influenza A virus Initiator Codon Investigation Knowledge Libraries Mammalian Orthoreovirus Mediating Messenger RNA Methods Open Reading Frames Outcome Pathogenesis Pathway interactions Pattern Peptides Pharmaceutical Preparations Polyribosomes Protein Isoforms Proteins Proteome Proteomics Protocols documentation RNA Regulation Reovirus Resolution Ribosomes Site Specificity Techniques Terminator Codon Testing Time Transcript Translating Translation Initiation Translational Regulation Translations Viral Viral Genome Viral Proteins Virus Virus Diseases Wit base biological adaptation to stress burden of illness cell type cohort deep sequencing defense response genetic information genome annotation influenzavirus inhibitor/antagonist insight mRNA Transcript Degradation next generation sequencing novel novel strategies novel therapeutics novel vaccines novel virus pathogen polyadenylated messenger RNA polypeptide protein degradation response tandem mass spectrometry tool transcriptome sequencing virology

项目摘要

DESCRIPTION (provided by applicant): To replicate, all viruses must co-opt the host translational machinery to express viral proteins and peptides. Infection activates innate cellular defense and stress responses that also substantially alter cellular transcription, translation, and the resulting proteome. Studies of host gene expression responses to virus infection are incomplete. Virus infection alters the cellular transcriptome, but this often correlates poorly wit changes in protein levels. Similarly, tandem mass spectrometry lacks the sensitivity to fully characterize the proteome. Regulation of protein abundance is predominantly at the level of translation, although mRNA degradation-, protein degradation-, and transcription rates are important factors. A complete description is lacking of which cellular transcripts are preferentialy translated during virus infection and what open reading frames (ORFs) are used. Recent studies of the cellular translatome have indicated a greater than expected complexity, suggesting that there are viral ORFs that remain undiscovered. The overall goal of this project is to determine and quantify which cellular and viral transcripts are being translated and which open reading frames (ORFs) are being decoded at different times post-infection examining three different human viruses -- influenza A virus (IAV), herpes simplex virus 1 (HSV-1), and mammalian orthoreovirus (REOV). Recently developed approaches based on next generation sequencing of total RNA and ribosome-protected fragments of RNA will be used to test two hypotheses: (1) that virus infection induces a cohort of common changes in global cellular translation patterns overlaid by unique changes specific for each virus type and (2) that viral genomes contain more ORFs than have been identified thus far. Two specific aims will address these hypotheses. Aim 1 will determine the host cell translational changes in A549 cells at three different times after infection with IAV, HSV-1, or REOV. To identify changes in translation, the cellular translatome after infection with each virus will be compared to the translatome from uninfected cells. The cellular translatomes of each virus will then be compared to identify common changes and translational patterns. Aim 2 will test the hypothesis that viral genomes contain more ORFs than are currently known. Translation initiation sites will be identified by deep sequencing libraries prepared from ribosome-protected fragments of RNA recovered from cells treated with drugs that fix ribosomes on start codons. The studies will also provide new information about changes in cellular translation during the three different virus infections and will identify common translational responses to infection. In addition, novel virus-encoded ORFs will likely be discovered. Such ORFs may initiate from alternative near- or non-canonical start sites. This information would provide new insights into translational regulation by viruses, and expand our knowledge of basic virology.

描述(申请人提供)：为了复制，所有病毒都必须选择宿主的翻译机制来表达病毒蛋白和多肽。感染激活先天细胞防御和应激反应也极大地改变了细胞转录、翻译和由此产生的蛋白质组。宿主基因表达对病毒感染的反应的研究还不完整。病毒感染改变了细胞转录组，但这通常与蛋白质水平的变化相关性不佳。同样，串联质谱仪缺乏完全表征蛋白质组的灵敏度。蛋白质丰度的调节主要是在翻译水平上，尽管mRNA降解--蛋白质降解--和转录速率是重要的因素。缺乏完整的描述，在病毒感染期间，哪些细胞转录本最好被翻译，以及使用了什么开放阅读框架(ORF)。最近对细胞翻译组的研究表明，其复杂性比预期的要大，这表明有一些病毒开放阅读框仍未被发现。该项目的总体目标是确定和量化哪些细胞和病毒转录本正在被翻译，哪些开放阅读框(ORF)在感染后的不同时间被解码，并检测三种不同的人类病毒--甲型流感病毒(IAV)、单纯疱疹病毒1型(HSV-1)和哺乳动物正病毒(REOV)。最近开发的基于下一代总RNA测序和核糖体保护的RNA片段的方法将用于检验两个假设：(1)病毒感染导致全球细胞翻译模式的一系列共同变化，覆盖着每种病毒类型的独特变化；(2)病毒基因组包含比迄今已确定的更多的ORF。有两个具体目标将解决这些假设。目的1测定A549细胞在感染IAV、HSV-1或REOV后三个不同时间的宿主细胞的翻译变化。为了确定翻译的变化，感染每种病毒后的细胞翻译组将与未感染细胞的翻译组进行比较。然后将对每种病毒的细胞翻译体进行比较，以确定共同的变化和翻译模式。Aim 2将测试病毒基因组包含比目前已知的更多ORF的假设。翻译起始点将通过由核糖体保护的RNA片段制备的深度测序文库来确定，该片段是从用将核糖体固定在起始密码子上的药物处理的细胞中回收的。这些研究还将提供关于三种不同病毒感染期间细胞翻译变化的新信息，并将确定对感染的常见翻译反应。此外，可能还会发现新的病毒编码的开放阅读框。这样的开放阅读框可以从可选的近规范或非规范的起始点开始。这些信息将为病毒的翻译调控提供新的见解，并扩大我们对基础病毒学的知识。