Protease-mediated entry via the endosome of human coronavirus 229E - PubMed (original) (raw)

Protease-mediated entry via the endosome of human coronavirus 229E

Miyuki Kawase et al. J Virol. 2009 Jan.

Abstract

Human coronavirus 229E, classified as a group I coronavirus, utilizes human aminopeptidase N (APN) as a receptor; however, its entry mechanism has not yet been fully elucidated. We found that HeLa cells infected with 229E via APN formed syncytia when treated with trypsin or other proteases but not in a low-pH environment, a finding consistent with syncytium formation by severe acute respiratory syndrome coronavirus (SARS-CoV). In addition, trypsin induced cleavage of the 229E S protein. By using infectious viruses and pseudotyped viruses bearing the 229E S protein, we found that its infection was profoundly blocked by lysosomotropic agents as well as by protease inhibitors that also prevented infection with SARS-CoV but not that caused by murine coronavirus mouse hepatitis virus strain JHMV, which enters cells directly from the cell surface. We found that cathepsin L (CPL) inhibitors blocked 229E infection the most remarkably among a variety of protease inhibitors tested. Furthermore, 229E infection was inhibited in CPL knockdown cells by small interfering RNA, compared with what was seen for a normal counterpart producing CPL. However, its inhibition was not so remarkable as that found with SARS-CoV infection, which seems to indicate that while CPL is involved in the fusogenic activation of 229E S protein in endosomal infection, not-yet-identified proteases could also play a part in that activity. We also found 229E virion S protein to be cleaved by CPL. Furthermore, as with SARS-CoV, 229E entered cells directly from the cell surface when cell-attached viruses were treated with trypsin. These findings suggest that 229E takes an endosomal pathway for cell entry and that proteases like CPL are involved in this mode of entry.

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Figures

FIG. 1.

FIG. 1.

Syncytium formation of 229E-infected HeLa cells induced by trypsin and other protease treatment. (A) HeLa cells infected with 229E were cultured for 2 days and then treated or left untreated with trypsin (Try; 100 μg/ml) at RT for 5 min. Cells were cultured for an additional 2 h and observed for syncytium formation and viral antigen via phase-contrast (PC) and fluorescent (FA) microscopy, respectively. (B) Cells infected (Infect) with 229E or mock infected (Mock) were cultured for 2 days and treated with 100 μg/ml of trypsin (Try) or DMEM adjusted to pH 5.0 at RT for 5 min. After 2 h of culture, cells were fixed with formaldehyde and stained with crystal violet. Cells without any treatment were shown as controls (None). (C) Syncytium formation of cells infected with 229E was induced by treatment with various proteases. HeLa cells infected with 229E and cultured for 2 days were treated with a variety of proteases at RT for 5 min and cultured for an additional 2 h. Cells were fixed and stained as described above. The following concentrations were employed for treatment: for proteinase K, 8 μg/ml; for elastase, 2 mg/ml; for thermolysin, 200 μg/ml; for dispase, 1 unit/ml; for papain, 0.19 unit/ml; for chymotrypsin, 2 mg/ml; and for collagenase, 1 mg/ml. Protease (−) indicates the absence of protease.

FIG. 2.

FIG. 2.

Western blotting analysis of the 229E S protein after treatment with trypsin. (Left) 229E virions concentrated as described in Materials and Methods were treated with trypsin (lane 1, 0 μg/ml; lane 2, 50 μg/ml; lane 3, 100 μg/ml) at RT for 5 min. (Right) Also, 229E-infected and mock-infected HeLa cells were treated with trypsin (lanes 1, 0 μg/ml; lanes 2, 250 μg/ml; lanes 3, 500 μg/ml). The resultant samples were analyzed by Western blotting using anti-229E S antibodies raised with a synthetic peptide corresponding to the C-terminal region of the S protein. Uncleaved S protein and cleaved S2 protein are indicated by black and white arrowheads, respectively.

FIG. 3.

FIG. 3.

Characterization of HeLa cells permissive for 229E infection. (A) HeLa cells infected with 229E and cultured for 1 day in the presence or absence of trypsin (Try; 5 μg/ml) in DMEM containing 10% TPB were fixed and stained as described above. The syncytia formed, indicated by arrows, were observed under microscopy as plaques. (B) To evaluate the plaque assay developed using HeLa cells and trypsin, 229E virion solutions at ca. 120 PFU/50 μl were diluted by twofold steps and inoculated into 3 wells of HeLa cells prepared in 24-well plates. The relationship between the virus dilution and the plaque number is depicted. (C) HeLa cells prepared in 96-well plates were treated with anti-human APN sera at 4°C for 45 min and then allowed to be adsorbed with ca. 150 PFU of 229E at 4°C for 45 min in the presence of antiserum. Next, those cells were cultured for 1 day in the presence of the antiserum. The infected cells were counted under fluorescence microscopy after cells had been stained with antibody (Ab) against 229E and secondary fluorescein isothiocyanate-labeled anti-rabbit serum. Cells in which infection was blocked by APN antibody were compared to those without APN antibody treatment.

FIG. 4.

FIG. 4.

Effect of lysosomotropic agents on 229E infection. (A) HeLa cells in 24-well plates were treated with 100 nM of Baf from 1 h before infection to 4 h postinfection (early) or from 4 to 24 h after 229E infection (late). Virus titers were determined for cells at 24 h postinfection. HeLa cells were infected at an MOI of 1. (B) HeLa-AC cells prepared in 24-well plates were treated with 1,000 nM of Baf or 50 mM NH4Cl for 1 h at 37°C. Then, cells were infected with 1 × 105 PFU of virus (229E, MHV-JHM, or MHV-2) in DMEM containing each agent and incubated at 34°C (229E) or 37°C (MHV) for 1 h. After being washed once, cells were cultured with DMEM containing each agent for 3 h, and then medium was changed to DMEM without agent. Virus titers were examined by plaque assay in cells at 24 h (229E) or 10 h (MHV) after infection. (C) HeLa-A cells prepared in 96-well plates were treated with different concentrations of Baf or NH4Cl for 1 h; infected with ca. 500 IU of pseudotyped VSV bearing 229E S (solid line with circle), SARS-CoV S (broken line with square), or VSV G protein (dotted line with triangle); and cultured for 24 h in the presence of each agent. Then, GFP-positive cells were photographed by Keyence fluorescence microscopy and counted. The percentage of infection was calculated as follows: (GFP-positive cell number in the presence of agent)/(GFP-positive cell number in the absence of agent) × 100. Cr, control.

FIG. 5.

FIG. 5.

Effects of protease inhibitors on pseudotyped virus infection. HeLa-A cells pretreated with different concentrations of protease inhibitors for 1 h were infected with ca. 500 IU of pseudotyped VSV bearing 229E S (solid line with circle); SARS-CoV S (broken line with square) or VSV G protein (dotted line with triangle) and cultured at 37°C for 24 h in the presence of protease inhibitors. The percentage of infection was determined as described for Fig. 4C. MDL, MDL28107.

FIG. 6.

FIG. 6.

Effect of CPL knockdown by siRNA on pseudotype infection (A and B) and digestion of 229E S by CPL (C). (A) HeLa-A cells were treated with siRNA for CPL (lane 1) and control nonsense RNA (lane 2) or were left untreated (lane 3) and cultured for 4 days. Amounts of CPL expressed in those cells were examined by Western blotting. The arrowhead indicates the position of CPL. (B) HeLa-A cells transfected with CPL siRNA and cultured for 4 days (black columns) were infected with ca. 500 IU of pseudotyped VSV with 229E S, SARS-CoV S, or VSV G protein. Their infection rates (GFP-positive cell numbers) were compared to those for cells transfected with control RNA (gray columns). (C) 229E virions concentrated as described in Materials and Methods were treated with 36 μg/ml of CPL (lane 2) or CPB (lane 3) or were mock treated (lane 1) at 37°C for 30 min in a buffer at pH 5.0, and S protein was analyzed by Western blotting using anti-229E S antibodies as described in the Fig. 2 legend. As a control, virions were also treated (lane 5) or mock treated (lane 4) with 50 μg/ml of trypsin at 37°C for 30 min in a buffer at pH. 70 and analyzed by Western blotting. M, molecular mass.

FIG. 7.

FIG. 7.

Infection with 229E S-bearing pseudotyped VSV from cell surfaces. HeLa-A cells prepared in 96-well plates were treated with 100 nM Baf for 1 h at 37°C and then allowed to adsorb VSV bearing 229E S, SARS-CoV S, or VSV G protein on ice for 30 min. The plate was left at RT for 10 min, and adsorbed virus was treated with 100 μg/ml trypsin for 5 min. Then, those cells were cultured for 24 h at 37°C, and GFP-positive cell numbers were calculated as described above (Baf positive, Try positive). GFP-positive cell numbers were also determined for the groups of cells treated with Baf alone (Baf +, Try −) or trypsin alone (Baf −, Try +) or maintained as untreated control cells (Baf −, Try −). The number of GFP-positive cells in each group is shown as a percentage in comparison with that of GFP-positive cells without any treatment.

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