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Journal of Virology, August 2007, p. 8722-8729, Vol. 81, No. 16
0022-538X/07/$08.00+0     doi:10.1128/JVI.00253-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Clathrin-Dependent Entry of Severe Acute Respiratory Syndrome Coronavirus into Target Cells Expressing ACE2 with the Cytoplasmic Tail Deleted{triangledown}

Yuuki Inoue,1 Nobuyuki Tanaka,1,4* Yoshinori Tanaka,1 Shingo Inoue,3 Kouichi Morita,3 Min Zhuang,2 Toshio Hattori,2 and Kazuo Sugamura1

Department of Microbiology and Immunology,1 Department of Infectious and Respiratory Diseases, Internal Medicine, Tohoku University Graduate School of Medicine, Sendai 980-8575, Japan,2 Department of Virology, Institute of Tropical Medicine, Nagasaki University, Nagasaki 852-8523, Japan,3 Division of Immunology, Miyagi Cancer Center Research Institute, Natori 981-1293, Japan4

Received 6 February 2007/ Accepted 7 March 2007

The penetration of various viruses into host cells is accomplished by hijacking the host endocytosis machinery. In the case of severe acute respiratory syndrome coronavirus (SARS-CoV) infection, viral entry is reported to require a low pH in intracytoplasmic vesicles; however, little is known about how SARS-CoV invades such compartments. Here we demonstrate that SARS-CoV mainly utilizes the clathrin-mediated endocytosis pathway for its entry to target cells by using infectious SARS-CoV, as well as a SARS-CoV pseudovirus packaged in the SARS-CoV envelope. The SARS-CoV entered caveolin-1-negative HepG2 cells, and the entry was significantly inhibited by treatment with chlorpromazine, an inhibitor for clathrin-dependent endocytosis, and by small interfering RNA-mediated gene silencing for the clathrin heavy chain. Furthermore, the SARS-CoV entered COS7 cells transfected with the mutant of ACE2 with the cytoplasmic tail deleted, SARS-CoV receptor, as well as the wild-type ACE2, and their entries were significantly inhibited by treatment with chlorpromazine. In addition, ACE2 translocated into EEA1-positive early endosomes immediately after the virus attachment to ACE2. These results suggest that when SARS-CoV binds ACE2 it is internalized and penetrates early endosomes in a clathrin-dependent manner and that the cytoplasmic tail of ACE2 is not required for the penetration of SARS-CoV.

* Corresponding author. Mailing address: Department of Microbiology and Immunology, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Sendai, 980-8575 Japan. Phone: 81-22-717-8096. Fax: 81-22-717-8097. E-mail: n-tanaka{at}mail.tains.tohoku.ac.jp

{triangledown} Published ahead of print on 23 May 2007.

Journal of Virology, August 2007, p. 8722-8729, Vol. 81, No. 16
0022-538X/07/$08.00+0     doi:10.1128/JVI.00253-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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