Local translation and viral infection in the airway epithelium

气道上皮的局部翻译和病毒感染

• 批准号: 10736284
• 负责人: Wellington V. Cardoso
• 金额: $ 65.23万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-06-09 至 2028-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10736284
• 关键词: Active Sites; Address; Adult; Air; Airway Disease; Apical; Biological Products; Cell Differentiation process; Cells; Centrioles; Chimeric Proteins; Cilia; Cytoplasm; Cytoplasmic Granules; Data; Defect; Defense Mechanisms; Enzymes; Epithelium; Event; Fluorescent in Situ Hybridization; Human; Infant; Influenza A virus; Inhalation; Knowledge; Label; Liquid substance; Lung; Lung diseases; Mammalian Cell; Mediating; Messenger RNA; MicroRNAs; Morbidity - disease rate; Morphology; Mucociliary Clearance; Mus; Names; Pathogenesis; Pathologic; Pathway interactions; Peptide Initiation Factors; Phenotype; Play; Poly(A)-Binding Proteins; Production; Protein Biosynthesis; Proteins; Public Health; Puromycin; RNA immunoprecipitation sequencing; Reporting; Respiratory Syncytial Virus Infections; Respiratory System; Respiratory Tract Infections; Respiratory physiology; Respiratory syncytial virus; Ribonucleoproteins; Ribosomal Proteins; Role; Severe Acute Respiratory Syndrome; Signal Transduction; System; Testing; Translating; Translations; Trinucleotide Repeats; Viral Proteins; Virus Diseases; airway epithelium; cilium biogenesis; coronavirus disease; long-term sequelae; mRNA Transcript Degradation; mutant; novel; pathogen; pathogenic virus; polypeptide; progenitor; programs; recruit; respiratory; respiratory virus; ribosome profiling; single molecule; transcriptome

Project Summary - Abstract Multiciliated cells (MCCs) are key components of the airway epithelium playing a major role in mucociliary clearance, the first line of lung defense against inhaled pathogens. MCCs are well-known targets of viral pathogens, including influenza A, SARS-COVID2 and respiratory syncytial virus (RSV), often resulting in severe respiratory conditions with long-term sequelae and morbidity. Still there are major gaps of knowledge on the mechanisms by which these pathogens trigger airway disease. MCCs require production and apical localization of a large number of proteins essential for the assembly of hundreds of cilia per cell during multiciliogenesis. The mechanisms that allow efficient production and local translation of these proteins in MCCs are still poorly understood. There is evidence that during viral infection, the host’s cellular translation machinery is hijacked to produce viral proteins for replication. How local translation is established in MCCs and targeted in viral infection will be studied in this proposal. Our preliminary studies revealed a striking expression of translation initiation factors (eIFs), ribosomal proteins and nascent polypeptides colocalized with miRNAs, Trinucleotide repeat- containing 6a (TNRC6a) and Argonaute 2 (AGO2) in immature MCCs undergoing multiciliogenesis. These signals were concentrated in not yet reported apical cytoplasmic granules, which we named as Localized Translation granules (LT granules). Surprisingly, unlike other granules traditionally associated with miRNA function, no enzymes required for mRNA degradation were detected in LT granules. Instead, LT granules were highly active sites of protein translation. Notably, disrupting Tnrc6a expression, disturbed local translation and resulted in defective multicilia formation, a phenotype we also observed in RSV-infected MCCs. Here we will test the hypotheses that i) components of the miRNA pathway recruit subsets of mRNAs and the translation machinery to LT granules for local efficient large-scale protein synthesis during MCC differentiation; ii) disruption of this local translation program is a key determinant in the pathogenesis of RSV infection. Thus, we propose to identify mechanisms that target mRNAs (Aim 1) and recruit the translation machinery (Aim 2) to LT granules in MCCs, and determine how RSV disrupts these mechanisms to infect the human airway epithelium (Aim 3). The knowledge generated from these studies will significantly advance our understanding of the mechanisms of local translation in multiciliogenesis and the pathogenesis of RSV infection in the respiratory tract. RELEVANCE TO PUBLIC HEALTH: Multiciliated cells (MCCs) are crucial components of the defense mechanisms of the lung. Viral infections in the immature epithelium of conducting airways can have devastating effects in infants or adults with compromised respiratory function. Novel information generated from these studies will advance our understanding of how proper ciliogenesis is regulated, and how viral infections, such as RSV disrupt cilia formation in immature respiratory tract.

项目摘要 - 摘要 多纤毛细胞 (MCC) 是气道上皮的关键组成部分，在粘液纤毛中发挥着重要作用 间隙，肺部抵御吸入病原体的第一道防线。 MCC 是众所周知的病毒攻击目标 病原体，包括甲型流感、SARS-COVID2 和呼吸道合胞病毒 (RSV)，通常会导致严重的 具有长期后遗症和发病率的呼吸系统疾病。仍然存在重大知识空白 这些病原体引发气道疾病的机制。 MCC 需要生产和根尖定位 多纤毛发生过程中每个细胞数百个纤毛组装所必需的大量蛋白质。这 允许这些蛋白质在 MCC 中高效生产和局部翻译的机制仍然很薄弱 明白了。有证据表明，在病毒感染期间，宿主的细胞翻译机器被劫持 产生用于复制的病毒蛋白。如何在 MCC 中建立本地翻译并针对病毒感染 将在本提案中进行研究。我们的初步研究揭示了翻译起始的惊人表达 因子 (eIF)、核糖体蛋白和新生多肽与 miRNA、三核苷酸重复共定位 正在经历多纤毛发生的未成熟 MCC 中含有 6a (TNRC6a) 和 Argonaute 2 (AGO2)。这些 信号集中在尚未报道的顶端细胞质颗粒中，我们将其命名为局部化 翻译颗粒（LT颗粒）。令人惊讶的是，与传统上与 miRNA 相关的其他颗粒不同 功能，在 LT 颗粒中没有检测到 mRNA 降解所需的酶。相反，LT 颗粒是 蛋白质翻译的高活性位点。值得注意的是，破坏 Tnrc6a 表达、扰乱局部翻译和 导致多纤毛形成缺陷，我们也在 RSV 感染的 MCC 中观察到了这种表型。这里我们将测试 假设 i) miRNA 通路的组成部分招募 mRNA 子集和翻译 LT 颗粒机械，用于 MCC 分化过程中局部高效大规模蛋白质合成； ii) 破坏 这种本地翻译程序的缺失是 RSV 感染发病机制的关键决定因素。因此，我们建议 确定靶向 mRNA（目标 1）并招募翻译机制（目标 2）到 LT 颗粒的机制 MCC，并确定 RSV 如何破坏这些机制来感染人类气道上皮（目标 3）。这 这些研究产生的知识将极大地促进我们对局部机制的理解 多纤毛发生的翻译和呼吸道 RSV 感染的发病机制。 与公共卫生的相关性：多纤毛细胞 (MCC) 是防御的重要组成部分 肺的机制。传导气道的未成熟上皮中的病毒感染可能具有毁灭性的后果 对呼吸功能受损的婴儿或成人的影响。这些研究产生的新信息 将增进我们对如何调节正确的纤毛发生以及病毒感染（例如RSV）如何进行的理解 破坏未成熟呼吸道中纤毛的形成。