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

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

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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（由申请人提供）：要复制，所有病毒都必须选择宿主翻译机械以表达病毒蛋白和肽。感染激活先天的细胞防御和压力反应也大大改变了细胞转录，翻译和由此产生的蛋白质组。对病毒感染的宿主基因表达反应的研究不完整。病毒感染改变了细胞转录组，但这通常会使蛋白质水平的机智变化不佳。同样，串联质谱法缺乏对完全表征蛋白质组的灵敏度。尽管mRNA降解，蛋白质降解和转录速率是重要因素，但蛋白质丰度的调节主要是在翻译水平上。缺乏完整的描述，这些细胞转录本在病毒感染过程中是最佳翻译以及使用了哪些开放式阅读框（ORF）。对细胞翻译组的最新研究表明，比预期的复杂性要大，这表明有些病毒ORF仍然未被发现。该项目的总体目的是确定和量化哪些细胞和病毒转录本正在翻译中，哪些开放式阅读框（ORF）在感染后的不同时间被解码，检查了三种不同的人类病毒 - 流感病毒（IAV），病毒（IAV），单纯疱疹病毒1（HSV-1）（HSV-1），以及哺乳动物或哺乳动物或哺乳动物或哺乳动物或哺乳动物或哺乳动物（Reov）。最近开发的方法是基于总RNA的下一代测序和核糖体保护的RNA的片段将用于检验两个假设：（1）病毒感染引起了全球细胞翻译模式中的一系列共同变化，被特定于每种病毒类型的独特变化和（2）所含有更多ORF的独特变化所覆盖。两个具体的目标将解决这些假设。 AIM 1将在感染IAV，HSV-1或REOV后三个不同的时间确定A549细胞的宿主细胞平移变化。为了识别翻译的变化，将感染每种病毒感染后的细胞翻译组与未感染细胞的翻译组进行比较。然后将比较每种病毒的细胞翻译，以识别常见的变化和翻译模式。 AIM 2将检验以下假设：病毒基因组含有比目前已知的更多ORF。翻译起始位点将通过从核糖体保护的片段中制备的深层测序库来鉴定，这些ro核糖体保护的片段是从用固定核糖体在起始密码子上固定核糖体的细胞的RNA的片段来鉴定的。这些研究还将提供有关三种不同病毒感染过程中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，可能会发现新型病毒编码的ORF。此类ORF可以从替代或非规范起始站点启动。这些信息将提供有关病毒翻译调节的新见解，并扩展我们对基本病毒学的了解。