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Journal of Virology, January 2009, p. 1026-1035, Vol. 83, No. 2
0022-538X/09/$08.00+0     doi:10.1128/JVI.01387-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Identification of Major Histocompatibility Complex Class I C Molecule as an Attachment Factor That Facilitates Coronavirus HKU1 Spike-Mediated Infection{triangledown}

Che Man Chan,2 Susanna K. P. Lau,1,2,3 Patrick C. Y. Woo,1,2,3 Herman Tse,1,2,3 Bo-Jian Zheng,1,2,3 Ling Chen,4 Jian-Dong Huang,5 and Kwok-Yung Yuen1,2,3*

State Key Laboratory of Emerging Infectious Diseases,1 Department of Microbiology,2 Carol Yu Centre for Infection of The University of Hong Kong, Hong Kong,3 The Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China,4 Department of Biochemistry, The University of Hong Kong, Hong Kong5

Received 2 July 2008/ Accepted 27 October 2008

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 S1-600 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 S1-600 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.

* Corresponding author. Mailing address: Department of Microbiology, 423, University Pathology Building, Queen Mary Hospital, The University of Hong Kong, Hong Kong. Phone: (852) 2855 4892. Fax: (852) 2855 1241. E-mail: hkumicro{at}hkucc.hku.hk

{triangledown} Published ahead of print on 5 November 2008.

Journal of Virology, January 2009, p. 1026-1035, Vol. 83, No. 2
0022-538X/09/$08.00+0     doi:10.1128/JVI.01387-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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