SARS coronavirus entry into host cells through a novel clathrin- and caveolae-independent endocytic pathway

Abstract

While severe acute respiratory syndrome coronavirus (SARS-CoV) was initially thought to enter cells through direct fusion with the plasma membrane, more recent evidence suggests that virus entry may also involve endocytosis. We have found that SARS-CoV enters cells via pH- and receptor-dependent endocytosis. Treatment of cells with either SARS-CoV spike protein or spike-bearing pseudoviruses resulted in the translocation of angiotensin-converting enzyme 2 (ACE2), the functional receptor of SARS-CoV, from the cell surface to endosomes. In addition, the spike-bearing pseudoviruses and early endosome antigen 1 were found to colocalize in endosomes. Further analyses using specific endocytic pathway inhibitors and dominant-negative Eps15 as well as caveolin-1 colocalization study suggested that virus entry was mediated by a clathrin- and caveolae-independent mechanism. Moreover, cholesterol- and sphingolipid-rich lipid raft microdomains in the plasma membrane, which have been shown to act as platforms for many physiological signaling pathways, were shown to be involved in virus entry. Endocytic entry of SARS-CoV may expand the cellular range of SARS-CoV infection, and our findings here contribute to the understanding of SARS-CoV pathogenesis, providing new information for anti-viral drug research.

Figures

Figure 1

SARS-CoV receptor ACE2 translocates from the plasma membrane to cytoplasmic compartments following treatment with spike protein. (A) In HEK293E-ACE2-GFP cells, ACE2 is primarily located on the cell surface. (B) When HEK293E-ACE2-GFP cells were treated with the S1190-Fc protein (for 3 h at 37 °C), ACE2-GFP was internalized from the cell surface. (C) When HEK293E-ACE2-GFP cells were treated with Fc protein alone (for 3 h at 37 °C), no translocation of the SARS-CoV receptor ACE2 was observed. (D) Spike protein colocalizes with ACE2-GFP in cytoplasmic vesicles after 3 h incubation at 37 °C. Spike-Fc protein was probed with Alexa-568 goat anti-human IgG. (E) ACE2 receptor recycling was observed after a 14-h incubation with spike-Fc at 37 °C; few green vesicles were visible in the cells after 14 h. (F) HEK293E-ACE2-GFP cells were treated with NH4Cl before they were incubated with S1190-Fc protein. ACE2 was trapped in cytoplasmic vesicles, even after 14 h. Scale bar: 20 μm.

Figure 2

SARS-CoV spike-bearing pseudoviruses can enter cells via endocytosis. (A) When HEK293E-ACE2-GFP cells were treated with spike-bearing pseudovirus for 3 h, translocation of the ACE2 receptor was observed. (B) When HEK293E-ACE2-GFP cells were treated with spike minus pseudovirus (VSV-G pseudovirus) for 3 h, no translocation of the ACE2 receptor was observed. (C) Colocalization of SARS-CoV spike protein and ACE2-GFP in cytoplasmic vesicles (3 h after treatment with spike-bearing pseudovirus). Spike protein was probed with primary antibody and then detected with Alexa-568 goat anti-mouse secondary antibody. (D) Twelve hours after treatment with spike-bearing pseudovirus, HEK293E-ACE2-GFP cells showed few cytoplasmic vesicles. (E) After chloroquine treatment, cytoplasmic vesicles were detected, even at 12 h after spike-bearing pseudovirus infection. (F) NH4Cl, chloroquine, and bafilomycin A1 treatments inhibit viral GFP expression. Vero E6 cells were mock treated or pretreated with 50 mM NH4Cl, 100 μM chloroquine, or 80 nM bafilomycin A1 for 1 h before pseudovirus infection. After 48 h, the cells were lysed and GFP was measured as described in the Materials and Methods section. Double asterisks indicate a significant difference from controls (P < 0.01, t-test). Error bars represent the SD of three independent experiments. (G-I) Alexa 594 transferrin (G) and EEA1 (H) colocalized in Vero E6 cells after a 1-h incubation at 37 °C. EEA1 was detected with its specific antibody followed by Alexa 488 secondary antibody. (J-L) Vero E6 cells were infected with spike-bearing pseudovirus for 1 h before they were fixed and immunolabeled with primary antibodies specific for SARS-CoV spike protein (J) and the early endosome marker EEA1 (K). These two proteins were found to colocalize. Spike protein was detected by Alexa 568 secondary antibody, while EEA1 was detected with Alexa 488 secondary antibody.

Figure 3

The effect of CPZ treatment and siRNA knockdown of clathrin on SARS-CoV entry. Vero E6 cells were mock treated (A and C) or pretreated with CPZ (10 μM) (B and D), and then incubated with Alexa 594 transferrin (A and B) or spike-bearing pseudovirus (C and D) for 1 h at 37 °C. Spike protein was detected with anti-spike antibody, followed by Alexa 488 secondary antibody. (E) Vero E6 cells were treated with the indicated amount of chlorpromazine (CPZ) before they were incubated with GFP-spike-bearing pseudovirus. Virus infectivity was measured using a spectrofluorometer at 60 h post-infection (see details in Materials and methods). Virus entry was not inhibited by this treatment. (F) HEK293 cells engineered to express ACE2-Myc were treated with siRNA specific for clathrin HC. Western blotting revealed that the siRNA markedly reduced the level of clathrin HC. Cells were then treated with GFP-spike-bearing pseudoviruses, and the infectivity was measured using a spectrofluorometer at 48 h post-infection. The siRNA treatment did not inhibit virus entry. Error bars represent the SD of three independent experiments.

Figure 4

SARS-CoV entry into cells expressing a dominant-negative mutant of Eps15. Vero E6 cells were transfected with the GFP-tagged construct Eps15 D3Δ2 (A-C and G-I) or EΔ95/295 (D-F and J-L), 48 h later, cells were incubated with Alexa 594 transferrin (A-F) or infected with spike-bearing pseudovirus (G-L) for 1 h at 37 °C. Expression of Eps15 proteins was monitored by GFP fluorescence. Spike-bearing pseudovirus was detected using anti-spike antibody, followed by incubation with Alexa-568 anti-mouse antibodies.

Figure 5

SARS-CoV entry into Vero E6 cells in the absence of caveolae-mediated endocytosis. Vero E6 cells were pretreated with nystatin (A, D, G), MβCD (B, E, H) or filipin (C, F, I), and were then incubated with Alexa-594-CTB (A-C) or spike-bearing pseudovirus (D-F and G-I). The spike-bearing pseudovirus in (D-F) was detected using anti-spike antibody (after 1 h incubation at 37 °C), followed by Alexa-488 anti-mouse antibodies, while in (G-I), pseudovirus was observed by monitoring GFP expression. Virus infectivity was measured using a spectrofluorometer at 60 h post-infection. (J-L) Colocalization of caveolin-1 (K) and spike-bearing pseudovirus (J) was not observed (after 1 h incubation at 37 °C). Spike-bearing pseudovirus was detected using anti-spike antibody, followed by Alexa-568 anti-mouse antibodies, while caveolin-1 was detected using its specific primary antibody followed by Alexa-488 secondary antibody. Double asterisks indicate a significant difference from controls (P < 0.01, t-test). Error bars represent the SD of three independent experiments.