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Journal of Virology, January 2009, p. 712-721, Vol. 83, No. 2
0022-538X/09/$08.00+0     doi:10.1128/JVI.01933-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Protease-Mediated Entry via the Endosome of Human Coronavirus 229E{triangledown}

Miyuki Kawase, Kazuya Shirato, Shutoku Matsuyama, and Fumihiro Taguchi*

Laboratory of Respiratory Viral Infections, Department of Virology III, National Institute of Infectious Diseases, Musashi-Murayama, 208-0011 Tokyo, Japan

Received 15 September 2008/ Accepted 21 October 2008

Human coronavirus 229E, classified as a group I coronavirus, utilizes human aminopeptidase N (APN) as a receptor; however, its entry mechanism has not yet been fully elucidated. We found that HeLa cells infected with 229E via APN formed syncytia when treated with trypsin or other proteases but not in a low-pH environment, a finding consistent with syncytium formation by severe acute respiratory syndrome coronavirus (SARS-CoV). In addition, trypsin induced cleavage of the 229E S protein. By using infectious viruses and pseudotyped viruses bearing the 229E S protein, we found that its infection was profoundly blocked by lysosomotropic agents as well as by protease inhibitors that also prevented infection with SARS-CoV but not that caused by murine coronavirus mouse hepatitis virus strain JHMV, which enters cells directly from the cell surface. We found that cathepsin L (CPL) inhibitors blocked 229E infection the most remarkably among a variety of protease inhibitors tested. Furthermore, 229E infection was inhibited in CPL knockdown cells by small interfering RNA, compared with what was seen for a normal counterpart producing CPL. However, its inhibition was not so remarkable as that found with SARS-CoV infection, which seems to indicate that while CPL is involved in the fusogenic activation of 229E S protein in endosomal infection, not-yet-identified proteases could also play a part in that activity. We also found 229E virion S protein to be cleaved by CPL. Furthermore, as with SARS-CoV, 229E entered cells directly from the cell surface when cell-attached viruses were treated with trypsin. These findings suggest that 229E takes an endosomal pathway for cell entry and that proteases like CPL are involved in this mode of entry.

* Corresponding author. Mailing address: Laboratory of Viral Respiratory Infections, Department of Virology III, National Institute of Infectious Diseases, Musashi-Murayama, 208-0011 Tokyo, Japan. Phone: 81-42-561-0771, ext. 3533. Fax: 81-42-567-5631. E-mail: ftaguchi{at}nih.go.jp

{triangledown} Published ahead of print on 29 October 2008.

Journal of Virology, January 2009, p. 712-721, Vol. 83, No. 2
0022-538X/09/$08.00+0     doi:10.1128/JVI.01933-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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