Altered structure and activity of the RSV attachment glycoprotein produced in primary human airway cells

原代人气道细胞中产生的 RSV 附着糖蛋白的结构和活性发生改变

• 批准号: 9911262
• 负责人: Tiffany King
• 金额: $ 3.33万
• 依托单位: RESEARCH INST NATIONWIDE CHILDREN'S HOSP
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-27 至 2021-05-15
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9911262
• 关键词: 1 year old; A549; Adult; Age; Air; Antiviral Agents; Apical; Attenuated; Biology; Bronchiolitis; C-terminal; Cancer cell line; Cause of Death; Cell Culture Techniques; Cell Line; Cells; Child; Clinical; Elderly; Enzymes; Epithelial Cells; GTP-Binding Proteins; Glycoproteins; Goals; Hela Cells; Heparan Sulfate Proteoglycan; Hospitalization; Human; In Vitro; Infection; Interleukin-13; Knowledge; Label; Laboratories; Link; Liquid substance; Lower respiratory tract structure; Mass Spectrum Analysis; Methionine; Methods; Modeling; Modification; Molecular Weight; Mucins; Mucous body substance; Mutate; N-terminal; Nose; Pathogenesis; Patients; Pattern; Physiologic pulse; Play; Polysaccharides; Proteins; Quantitative Reverse Transcriptase PCR; Respiratory Syncytial Virus Vaccines; Respiratory System; Respiratory syncytial virus; Respiratory syncytial virus RSV G glycoprotein; Role; Sampling; Site; Structure; Testing; Vaccine Production; Vaccines; Vero Cells; Viral; Viral Antigens; Viral Genome; Virus; Virus Diseases; Vulnerable Populations; airway epithelium; base; bronchial epithelium; cell immortalization; cell type; drug development; experimental study; glycoprotein G; glycosylation; glycosyltransferase; improved; in vivo; infant death; inhibitor/antagonist; neoplastic cell; neutralizing antibody; novel; pathogen; prevent; receptor; stem cells; sugar; transcriptome sequencing; vaccine development

ABSTRACT Respiratory syncytial virus (RSV) is the leading cause of hospitalization of children under one year of age, but there are no approved vaccines or effective anti-viral treatments. RSV infects epithelial cells in the upper and lower respiratory tracts causing severe bronchiolitis in young children and adults. The use of primary human bronchial epithelial cultures (HBEC) has enabled us to identify its target cell type (ciliated) as well as better understand its pathogenesis in an ex vivo model. HBEC are grown at an air-liquid interface allowing differentia- tion of progenitor cells into pseudostratified, ciliated and mucus-producing cultures that mirror the human air- ways. We have found that RSV produced in HBEC (RSV/HBE) contains a much larger version of its attachment (G) glycoprotein (G/HBE; 170 kDa) than the “classical” 90 kDa form produced in immortalized cells. We have evidence consistent with this size difference being due to extra glycosylation of the G protein in HBEC compared to HEp-2 (immortalized) cells. We have also found that RSV/HBEC is 100-fold less infectious for HEp-2 cells compared to HBEC, based on qRT-PCR quantified viral genomes relative to the infectivity of GFP-expressing RSV. We will identify the structural reason for the larger G produced in HBEC and determine the importance of G glycosylation for infection of HBEC. The results may elucidate the role of G in vivo, improve RSV vaccine production and allow much more efficient isolation of RSV from clinical samples without selection for modified viruses able to grow in HEp-2 cells.

摘要 呼吸道合胞病毒（RSV）是一岁以下儿童住院的主要原因， 目前还没有批准的疫苗或有效的抗病毒治疗方法。RSV感染上皮细胞， 下呼吸道引起严重的细支气管炎的幼儿和成人。使用初级人类 支气管上皮细胞培养物（HBEC）使我们能够确定其靶细胞类型（纤毛），以及更好地 了解其在离体模型中的发病机制。HBEC在空气-液体界面生长，允许差异化生长。 将祖细胞培养成假复层、纤毛和产生粘液的培养物，这些培养物反映了人类的空气- 的方式我们发现在HBEC中产生的RSV（RSV/HBE）含有更大版本的其附着物， (G)与在永生化细胞中产生的“经典”90 kDa形式相比，在细胞中产生的糖蛋白（G/HBE; 170 kDa）的浓度更高。我们有 与HBEC中G蛋白的额外糖基化相一致的证据表明， HEp-2（永生化）细胞。我们还发现RSV/HBEC对HEp-2细胞的感染性低100倍 与HBEC相比，基于qRT-PCR定量的病毒基因组相对于表达GFP的 RSV。我们将确定HBEC中产生较大G的结构原因，并确定以下因素的重要性： HBEC感染的G糖基化。这一结果为阐明G在体内的作用，改进RSV疫苗提供了实验依据 并且允许从临床样品中更有效地分离RSV，而无需选择修饰的 能够在HEp-2细胞中生长的病毒。