Chan Che Man, Lau Susanna K P, Woo Patrick C Y, Tse Herman, Zheng Bo-Jian, Chen Ling, Huang Jian-Dong, Yuen Kwok-Yung
Department of Microbiology, The University of Hong Kong, Hong Kong.
J Virol. 2009 Jan;83(2):1026-35. doi: 10.1128/JVI.01387-08. Epub 2008 Nov 5.
Human coronavirus HKU1 (HCoV-HKU1) is a recently discovered human coronavirus associated with respiratory tract infections worldwide. In this study, we have identified the major histocompatibility complex class I C molecule (HLA-C) as an attachment factor in facilitating HCoV-HKU1 spike (S)-mediated infection. HCoV-HKU1 S pseudotyped virus was assembled using a human immunodeficiency virus type 1-derived reporter virus harboring the human codon-optimized spike of HCoV-HKU1. We identified human alveolar epithelial A549 cells as the most susceptible cell line among those tested to infection by HCoV-HKU1 S pseudotypes. A549 cells were shown to bind purified soluble HCoV-HKU1 S(1-600) glycopeptide. To search for the functional receptor for HCoV-HKU1, an A549 cDNA expression library was constructed and transduced into the nonpermissive, baby hamster kidney cells line BHK-21. Transduced cells that bind soluble HCoV-HKU1 S(1-600) glycoprotein with C-terminal FLAG were sorted. Sequencing of two independent clones revealed cDNA inserts encoding HLA-C. Inhibition of HLA-C expression or function by RNAi silencing and anti-HLA-C antibody decreased HCoV-HKU1 S pseudotyped virus infection of A549 cells by 62 to 65%, whereas pretreatment of cells with neuraminidase decreased such infection by only 13%. When HLA-C was constitutively expressed in another nonpermissive cell line, NIH-3T3, quantitative PCR showed that the binding of HCoV-HKU1 S pseudotyped virus to cell surfaces was increased by 200-fold, but the cells remained nonsusceptible to HCoV-HKU1 S pseudotyped virus infection. Our data suggest that HLA-C is involved in the attachment of HCoV-HKU1 to A549 cells and is a potential candidate to facilitate cell entry. However, other unknown surface proteins on A549 cells may be concomitantly utilized by S glycoprotein of HCoV-HKU1 during viral entry. Further studies are required to elucidate other putative receptors or coreceptors for HCoV-HKU1 and the mechanism of HCoV-HKU1 S-mediated cell entry.
人冠状病毒HKU1（HCoV-HKU1）是一种最近发现的与全球呼吸道感染相关的人冠状病毒。在本研究中，我们已确定主要组织相容性复合体I类C分子（HLA-C）是促进HCoV-HKU1刺突（S）介导感染的附着因子。使用携带人密码子优化的HCoV-HKU1刺突的1型人类免疫缺陷病毒衍生的报告病毒组装HCoV-HKU1 S假型病毒。我们确定人肺泡上皮A549细胞是测试的细胞系中对HCoV-HKU1 S假型感染最敏感的细胞系。已证明A549细胞可结合纯化的可溶性HCoV-HKU1 S(1-600)糖肽。为了寻找HCoV-HKU1的功能性受体，构建了A549 cDNA表达文库并转导至非允许性的幼仓鼠肾细胞系BHK-21中。对结合具有C末端FLAG的可溶性HCoV-HKU1 S(1-600)糖蛋白的转导细胞进行分选。对两个独立克隆进行测序，揭示了编码HLA-C的cDNA插入片段。通过RNA干扰沉默和抗HLA-C抗体抑制HLA-C表达或功能，可使A549细胞对HCoV-HKU1 S假型病毒的感染降低62%至65%，而用神经氨酸酶预处理细胞仅使这种感染降低13%。当HLA-C在另一个非允许性细胞系NIH-3T3中组成性表达时，定量PCR显示HCoV-HKU1 S假型病毒与细胞表面的结合增加了200倍，但这些细胞对HCoV-HKU1 S假型病毒感染仍不敏感。我们的数据表明，HLA-C参与HCoV-HKU1与A549细胞的附着，并且是促进细胞进入的潜在候选者。然而，HCoV-HKU1的S糖蛋白在病毒进入期间可能同时利用A549细胞上的其他未知表面蛋白。需要进一步研究以阐明HCoV-HKU1的其他假定受体或共受体以及HCoV-HKU1 S介导的细胞进入机制。
