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: 10302872
• 负责人: Wyatt ALLEN MILLER
• 金额: $ 17.48万
• 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 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10302872
• 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; 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; Vaccines; 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; response; ribosome profiling; tool; transcriptome sequencing; vaccine candidate; 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RNAs(SfRNAs)，构成病毒基因组~500核苷酸3‘非翻译区的大部分。这些新的病毒RNA最近被证明与许多宿主蛋白相互作用，并抑制先天免疫系统某些基因的翻译(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的突变株如此温和，因此有望成为疫苗候选。然而，缺乏关于sfRNA如何在全球范围内影响细胞mRNAs翻译的图景。2014-16年寨卡病毒(ZIKV)的流行导致了因宫内接触ZIKV而导致的小头症和其他发育和神经疾病的频繁病例，令人震惊。除了抑制免疫反应外，ZIKV sfRNA还抑制脆性X智力低下蛋白(FMRP)，FMRP是神经发育的关键翻译调节因子。因此，了解ZIKV sfRNA对宿主翻译的全球影响可能有助于未来理解该病毒如何操纵宿主并导致疾病的研究。方法：我们将使用核糖体轮廓转换方法(RiboSeq)来采样细胞中整个mRNAs群体的翻译水平，以确定在sfRNA存在的情况下，单独或在复制ZIKV的背景下，哪些基因在翻译上上调或下调。RiboSeq是高通量信使核糖核酸测序(RNAseq)的一种改进，它只揭示了受翻译核糖体保护的mRNAs片段。从给定的信使核糖体保护片段中读取的次数与该信使核糖体被翻译的活跃程度成正比。这种信息量很大的方法仅少量应用于黄病毒，据我们所知，从未具体确定sfRNA的影响，sfRNA是一种已知的翻译调节因子。我们还将开发新的统计方法，以改进目前不完美的方法，以计算治疗反应中mRNAs翻译效率变化的重要性。最后，我们将使用各种生物信息学工具来确定其翻译效率类似地受到sfRNA影响的mRNAs的共同结构特征。预期结果。识别其翻译受sfRNAs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，这些结果可能会为规范翻译提供新的途径。这项研究可以为针对ZIKV和其他黄病毒的疫苗或抗病毒药物的合理设计提供信息，并通过更好地了解RNA对蛋白质合成的控制，从而有益于人类健康。