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.

FIG. 1.

Effects of chlorpromazine and MBCD on pseudoviruses and SARS-CoV infection. (A and B) HepG2 cells were treated with the indicated doses of chlorpromazine or MBCD and then infected with SARS-CoV(HIV), VSV(HIV), or A-MLV(HIV) for overnight. The effects of chlorpromazine and MBCD on the infectivity of each pseudovirus were evaluated by measuring the luciferase activities. The columns represent the mean values of triplicate experiments; bars indicate maximum values. (C) HepG2 cells treated with the indicated doses of chlorpromazine were infected with SARS-CoV (Vietnam/NB-04/2003) for 24 h, and their expressions of viral RNA were measured by RT-PCR.

FIG. 2.

Effects of CHC-specific siRNA on pseudoviruses and SARS-CoV infection. (A) HepG2 cells were transfected with the CHC-specific siRNA or the control siRNA and analyzed by immunoblotting with anti-CHC or anti-tubulin monoclonal antibody after 48 h of transfection. (B) The cells were treated with 2.5 mM MBCD for 30 min and then infected with each pseudovirus. The infectivities of the pseudoviruses are represented as luciferase activities. The experiment was performed in triplicate. (C) The cells were also infected with SARS-CoV (Vietnam/NB-04/2003) for 24 h, and their expressions of viral RNA were measured by RT-PCR.

FIG. 3.

Receptor activity of ACE2 mutants for pseudoviruses and SARS-CoV infection. (A) COS7 cells were transiently transfected with ACE2-wt, ACE2-Δtail, ACE2-Δtail-TM, ACE2-sol or control plasmids, and after 48 h of transfection their lysates were tested by immunoblotting with anti-ACE2 or anti-tubulin monoclonal antibody. (B and C) The transfected cells were infected with SARS-CoV(HIV) (B) or SARS-CoV (Vietnam/NB-04/2003) (C). Their luciferase activities were measured in triplicate, and their expressions of viral RNA were measured by RT-PCR.

FIG. 4.

Effects of chlorpromazine on pseudovirus infection to cells expressing ACE2-Δtail. COS7 cells were transiently transfected with ACE2-wt or ACE2-Δtail and then treated with the indicated amounts of chlorpromazine. Subsequently, the cells were infected with SARS-CoV(HIV). Their luciferase activities were measured in triplicate.

FIG. 5.

Immunohistochemical localization of ACE2 after pseudovirus infection. HepG2 cells were cultured in the FCS-free medium to induce ACE2 on cell surfaces and incubated with concentrated SARS-CoV(HIV) for 10 min at 37°C. They were then stained for ACE2 and EEA1 by immunofluorescence.

FIG. 6.

Dependence on acidic environment for infection by the pseudoviruses. COS7 cells transiently transfected with ACE2-wt, ACE2-Δtail, or control plasmids were treated with 20 mM NH₄Cl and then infected with SARS-CoV(HIV) and VSV(HIV). Their infectivities were evaluated by measuring the luciferase activity. The experiment was performed in triplicate.