Identification of candidate rhinovirus C (RV-C) receptors by gene expression analysis. Homo sapiens

Members of rhinovirus C (RV-C) species are more likely to cause wheezing illnesses and asthma exacerbations compared to other rhinoviruses. The cellular receptor for these viruses was heretofore unknown. We measured gene expression (Human Gene 1.0 ST Array, Affymetrix) in two series of experiments involving cells that were either susceptible or not susceptible to RV-C infection. In one experimental series, susceptible cells included whole sinus mucosal tissue specimens (n = 5), epithelial cell suspension from sinus tissue, and nasal epithelium obtained via brushing, while non-susceptible cells included monolayers of primary undifferentiated epithelial cells and transformed cell lines (n = 5). In a second experimental series, we compared three pairs of undifferentiated and fully differentiated (ALI) sinus epithelial cell cultures. We identified a total of 12 genes upregulated in RV-C susceptible cells (represented by 14 probe sets) encoding proteins localized to plasma membrane, and/or with predicted or functionally demonstrated receptor activity, including members of the Human MHC class II, stomatin, guanine nucleotide-binding, type I cytokine and atypical chemokine receptor and cadherin protein families. Overall design: Sinus and bronchial epithelial tissue samples were obtained from residual surgical specimens. Primary airway epithelial cells were cultured submerged (undifferentiated monolayers) or at air-liquid interface (fully-differentiated). Established cell lines (HeLa, NCI-H358 and WisL) were cultured submerged. Epithelial tissue samples were placed in RNAlater solution (Life Technologies) to stabilize and protect cellular RNA; cultured cells were lysed directly and total RNAs were isolated using TRIzol reagent (Life Technologies). Gene expression was analyzed using the Human Genome 1.0 ST GeneChip arrays (Affymetrix, Santa Clara, CA).

鼻病毒C（rhinovirus C, RV-C）物种相较于其他鼻病毒，更易引发喘息性疾病与哮喘急性加重。此前，这类病毒的功能性细胞受体始终未被发现。我们针对RV-C感染易感与非易感细胞开展了两组实验，检测了细胞的基因表达水平，所用平台为人类基因1.0 ST芯片（Human Gene 1.0 ST Array, Affymetrix）。
在第一组实验中，易感细胞包含完整的鼻窦黏膜组织标本（样本量n=5）、鼻窦组织来源的上皮细胞悬液，以及经刷取获取的鼻上皮细胞；非易感细胞则包括原代未分化上皮细胞单层培养物与转化细胞系（样本量n=5）。
在第二组实验中，我们对比了三对未分化与完全分化（air-liquid interface, ALI）培养的鼻窦上皮细胞。
本研究共鉴定出12个在RV-C易感细胞中表达上调的基因（对应14个探针组），这些基因编码的蛋白或定位于细胞膜，或具备经预测或功能验证的受体活性，涵盖人类主要组织相容性复合体（major histocompatibility complex, MHC）II类家族成员、斯托他汀（stomatin）蛋白家族成员、鸟苷酸结合蛋白家族成员、I型细胞因子家族成员、非典型趋化因子受体家族成员以及钙粘蛋白（cadherin）家族成员。
实验整体设计如下：鼻窦与支气管上皮组织样本均取自手术剩余标本。原代气道上皮细胞采用浸没培养（未分化单层培养）或气液界面（air-liquid interface, ALI）培养的方式实现完全分化。已建立的细胞系（HeLa、NCI-H358及WisL）采用浸没培养方式培养。上皮组织样本置于RNAlater溶液（Life Technologies）中以稳定并保护细胞RNA；培养细胞则直接裂解，使用TRIzol试剂（Life Technologies）提取总RNA。基因表达分析通过人类基因组1.0 ST基因芯片（Human Genome 1.0 ST GeneChip Arrays, Affymetrix, 加利福尼亚州圣克拉拉）完成。