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

基本信息

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
项目类别：
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

项目摘要

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.

抽象的呼吸道合胞病毒（RSV）是一岁以下儿童住院的主要原因，但没有批准的疫苗或有效的抗病毒治疗。 RSV感染上部和上皮细胞下呼吸道导致幼儿和成人严重的细支气管炎。主要人类的使用支气管上皮培养物（HBEC）使我们能够识别其靶细胞类型（纤毛）和更好在离体模型中了解其发病机理。 HBEC在空气界面上生长，使分化 - 祖细胞将伪造，纤毛和粘液产生的培养物与人类空气相反映的培养物方式。我们发现在HBEC（RSV/HBE）中生产的RSV包含其附件的更大版本（g）糖蛋白（g/hbe; 170 kDa）比在永生细胞中产生的90 kDa形式的糖蛋白（170 kDa）。我们有与HBEC中G蛋白的额外糖基化有关的大小差异一致的证据到HEP-2（永生）细胞。我们还发现，RSV/HBEC对HEP-2细胞的感染率降低了100倍与HBEC相比，基于QRT-PCR定量的病毒基因组相对于表达GFP的感染 RSV。我们将确定HBEC中产生较大G的结构原因，并确定的重要性 G糖基化HBEC感染。结果可以阐明G在体内的作用，改善RSV疫苗生产并允许从临床样本中更有效地隔离RSV，而无需选择修改病毒可以在HEP-2细胞中生长。