Global effects of flavivirus sfRNA on translation determined by ribosome profiling

通过核糖体分析确定黄病毒 sfRNA 对翻译的整体影响

• 批准号: 10418800
• 负责人: Wyatt ALLEN MILLER
• 金额: $ 21.64万
• 依托单位: IOWA STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-06-04 至 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10418800
• 关键词: 3' Untranslated Regions; 5' Untranslated Regions; Address; Affect; Americas; Antiviral Agents; Basic Science; Binding; Cell Line; Cell Nucleus; Cell physiology; Cells; Clinical Trials; Data Analyses; Dengue; Dengue Virus; Detection; Development; Disease; Epidemic; Exposure to; FMR1; Fetus; Flavivirus; Future; Gene Expression; Gene Expression Regulation; Genes; Genetic; Genome; HIV; Health; Host Defense; Human; Immune response; Infection; Innate Immune System; Integration Host Factors; Interferons; Invaded; Knowledge; Messenger RNA; Methods; Microcephaly; Modification; Molecular; Mothers; Natural Immunity; Neurodevelopmental Disorder; Neurologic; Nucleotides; Outcome; Parasites; Pathogenicity; Pathway interactions; Pattern; Persons; Polyribosomes; Population; Positioning Attribute; Protein Biosynthesis; Proteins; Quantitative Reverse Transcriptase PCR; RNA; RNA chemical synthesis; RNA replication; Regulation; Research; Ribosomes; Role; Sampling; Severity of illness; Specific qualifier value; Statistical Methods; Structure; Testing; Therapeutic; Transcript; Transfection; Translating; Translational Regulation; Translations; Untranslated RNA; Vaccine Design; Viral Genome; Virus; Virus Diseases; Virus Replication; West Nile virus; Yellow fever virus; ZIKA; ZIKV infection; Zika Virus; bioinformatics tool; developmental disease; female reproductive system; genomic RNA; immunoregulation; improved; in utero; mRNA sequencing; mutant; neonatal infection; nervous system disorder; novel; pathogen; rational design; response; ribosome profiling; tool; transcriptome sequencing; vaccine candidate; vaccine development; vector mosquito; viral RNA; viral genomics; virology

Flaviviruses include many serious pathogens such as Zika, dengue, West Nile, and yellow fever viruses. In infected cells these viruses produce abundant, noncoding, short flavivirus RNAs (sfRNAs) that comprise most of the ~500 nucleotide 3’ untranslated region of the viral genome. These novel viral RNAs have been shown recently to interact with numerous host proteins and inhibit translation (protein synthesis) of certain genes of the innate immune system. Thus, virus strains that produce high levels of sfRNA are more pathogenic, and mutants that produce no sfRNA are so mild as to be promising vaccine candidates. However, a picture of how sfRNA globally affects translation of cellular mRNAs is lacking. The Zika virus (ZIKV) epidemic of 2014-16 resulted in frighteningly frequent cases of microcephaly and other developmental and neurological disorders caused by exposure to ZIKV in utero. In addition to inhibiting the immune response, ZIKV sfRNA inhibits Fragile X Mental Retardation Protein (FMRP), a key translational regulator of neurological development. Thus, this knowledge of the global effects of ZIKV sfRNA on host translation could contribute to future research on understanding how this virus manipulates the host and causes disease. Method: We will employ the transformative method of ribosome profiling (RiboSeq) to sample the level of translation across the entire population of mRNAs in the cell in order to identify genes that are translationally up- or down-regulated in the presence of sfRNA, either alone, or in the context of replicating ZIKV. RiboSeq - a modification of high-throughput mRNA sequencing (RNAseq) - reveals only the fragments of mRNAs that are protected by translating ribosomes. The number of reads of ribosome-protected fragments from a given mRNA is proportional to how actively that mRNA is translated. This highly informative method has been applied only sparingly to flaviviruses and, to our knowledge, never to determine specifically the effects of sfRNA, a known translational regulator. We will also develop new statistical methods to improve on current imperfect approaches for calculating significance of changes in translational efficiency of mRNAs in response to a treatment. Finally, we will use a variety of bioinformatics tools to identify common structural features of mRNAs whose translation efficiencies are similarly affected by sfRNA. Expected Outcomes. Identification of genes whose translation is affected by sfRNAs will reveal potential genes and genetic pathways that facilitate - or defend against – ZIKV infection. These can be the focus of future studies by us and others to determine their role and regulation in virus infection, the immune response, or possibly neurodevelopmental disorders. Moreover, these results may reveal new ways of regulating translation. This research can benefit human health by informing rational design of vaccines or antivirals targeting ZIKV and other flaviviruses, and by providing better understanding of control of protein synthesis by RNA.

黄病毒包括许多严重的病原体，如寨卡病毒、登革热病毒、西尼罗河病毒和黄热病病毒。在感染的细胞中，这些病毒产生丰富的非编码短黄病毒RNA（sfRNA），其包含病毒基因组的约500个核苷酸的3'非翻译区的大部分。这些新的病毒RNA最近已被证明与许多宿主蛋白质相互作用，并抑制先天免疫系统的某些基因的翻译（蛋白质合成）。因此，产生高水平sfRNA的病毒株更具致病性，而不产生sfRNA的突变体是如此温和，以至于成为有希望的疫苗候选物。然而，缺乏sfRNA如何全面影响细胞mRNA翻译的图片。2014年至2016年的寨卡病毒（ZIKV）流行导致了由子宫内暴露于ZIKV引起的小头畸形和其他发育和神经系统疾病的频繁病例。除了抑制免疫应答外，ZIKV sfRNA还抑制脆性X智力迟钝蛋白（FMRP），这是神经发育的关键翻译调节因子。因此，ZIKV sfRNA对宿主翻译的整体影响的知识可能有助于未来研究了解这种病毒如何操纵宿主并导致疾病。方法：我们将采用核糖体谱分析（RiboSeq）的转化方法来对细胞中整个mRNA群体的翻译水平进行采样，以鉴定在sfRNA存在下单独或在复制ZIKV的背景下特异性上调或下调的基因。RiboSeq -高通量mRNA测序（RNAseq）的一种改进-仅显示受翻译核糖体保护的mRNA片段。来自给定mRNA的核糖体保护片段的读段数与该mRNA翻译的活跃程度成比例。这种信息量很大的方法仅少量应用于黄病毒，据我们所知，从未专门用于确定sfRNA（一种已知的翻译调节因子）的作用。我们还将开发新的统计方法，以改善目前不完善的方法，用于计算mRNA翻译效率变化对治疗的反应。最后，我们将使用各种生物信息学工具来识别其翻译效率受到sfRNA类似影响的mRNA的共同结构特征。预期成果。鉴定其翻译受sfRNA影响的基因将揭示促进或防御ZIKV感染的潜在基因和遗传途径。这些可能是我们和其他人未来研究的重点，以确定它们在病毒感染，免疫反应或可能的神经发育障碍中的作用和调节。此外，这些结果可能会揭示新的方式来调节翻译。这项研究可以通过为靶向ZIKV和其他黄病毒的疫苗或抗病毒药的合理设计提供信息，并通过更好地理解RNA对蛋白质合成的控制，从而有益于人类健康。