title: Clathrin-dependent entry of severe acute respiratory syndrome coronavirus into target cells expressing ACE2 with the cytoplasmic tail deleted.
abstract: 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.
authors.affiliation: Department of Microbiology and Immunology, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Sendai, 980-8575 Japan.
authors.forename: Yuuki
authors.initials: Y
authors.lastname: Inoue
authors.forename: Nobuyuki
authors.initials: N
authors.lastname: Tanaka
authors.forename: Yoshinori
authors.initials: Y
authors.lastname: Tanaka
authors.forename: Shingo
authors.initials: S
authors.lastname: Inoue
authors.forename: Kouichi
authors.initials: K
authors.lastname: Morita
authors.forename: Min
authors.initials: M
authors.lastname: Zhuang
authors.forename: Toshio
authors.initials: T
authors.lastname: Hattori
authors.forename: Kazuo
authors.initials: K
authors.lastname: Sugamura
mesh_terms: D000085962:Angiotensin-Converting Enzyme 2; D000818:Animals; D019556:COS Cells; D045744:Cell Line, Tumor; D002522:Chlorocebus aethiops; D002746:Chlorpromazine / Q000494:pharmacology; D002966:Clathrin / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D003593:Cytoplasm / Q000201:enzymology; D004705:Endocytosis / Q000187:drug effects; D011992:Endosomes / Q000821:virology; D006801:Humans; D007703:Peptidyl-Dipeptidase A / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000187:drug effects; D045473:Severe acute respiratory syndrome-related coronavirus / Q000187:drug effects / Q000502:physiology*; D017384:Sequence Deletion; D053586:Virus Internalization / Q000187:drug effects
chemical_list: D002966:Clathrin; D034741:RNA, Small Interfering; D007703:Peptidyl-Dipeptidase A; C000705307:ACE2 protein, human; D000085962:Angiotensin-Converting Enzyme 2; D002746:Chlorpromazine