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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

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.

Published ahead of print on 23 May 2007.

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