Wild-type human coronaviruses prefer cell-surface TMPRSS2 to endosomal cathepsins for cell entry

Abstract

Human coronaviruses (HCoVs) enter cells via two distinct pathways: the endosomal pathway using cathepsins to activate spike protein and the cell-surface or early endosome pathway using extracellular proteases such as transmembrane protease serine 2 (TMPRSS2). We previously reported that clinical isolates of HCoV-229E preferred cell-surface TMPRSS2 to endosomal cathepsin for cell entry, and that they acquired the ability to use cathepsin L by repeated passage in cultured cells and were then able to enter cells via the endosomal pathway. Here, we show that clinical isolates of HCoV-OC43 and -HKU1 preferred the cell-surface TMRRSS2 to endosomal cathepsins for cell entry, similar to HCoV-229E. In addition, the cell-culture-adapted HCoV-OC43 lost the ability to infect and replicate in air-liquid interface cultures of human bronchial tracheal epithelial cells. These results suggest that circulating HCoVs in the field generally use cell-surface TMPRSS2 for cell entry, not endosomal cathepsins, in human airway epithelial cells.

Keywords: Air-liquid interface culture; Entry; Human bronchial tracheal epithelial cells; Human coronavirus.

Fig. 1

Replication of laboratory and clinical isolates of HCoV-OC43 in HBTE-ALI cultures. Identical amounts (10⁸ copies) of laboratory (VR-1558) and clinical (SGH-36/2014) isolates of HCoVOC43 were inoculated onto a) HCT-8 or b) HBTE-ALI cells. After incubation for 4 h, the cells were washed with DMEM twice and incubated at 34 °C. After 1 day of incubation, the cells were collected and used for analysis of virus entry. After 3 days of incubation, supernatants of HCT-8 cells and cell washes of HBTE-ALI were collected and used for analysis of viral replication. Data were expressed as log copies of RNA per well (n = 6 for HCT-8, n = 3 for HBTE-ALI).

Fig. 2

Entry inhibition assay. Before virus inoculation, cells were treated with EST or camostat (10 μM) for 1 h. Then, 10⁷ to 10⁸ copies of the viruses were inoculated onto HBTE-ALI cultures and incubated in the presence of inhibitors. Cell viability after inhibitor treatment was determined using CellTiter-Glo and a GloMAX luminometer (a) (n = 3). Five HCoV-OC43 isolates [b) VR-1558, c) SGH-36/2014, d) SGH-61/2014, e) SGH-06/2015, and f) SGH-65/2016] and two HCoV-HKU1 isolates [g), SGH-15/2014 and h) SGH-18/2016] were examined. After incubation for 4 h, the cells were washed twice and incubated at 34 °C. After 1 day of incubation, the cells were collected and viral entry was calculated by quantifying subgenomic RNA containing nucleocapsid protein via real-time RT-PCR. DMSO was used as an inhibitor control. Data are shown as log copies RNA per well. (n = 3).

Fig. 2

Entry inhibition assay. Before virus inoculation, cells were treated with EST or camostat (10 μM) for 1 h. Then, 10⁷ to 10⁸ copies of the viruses were inoculated onto HBTE-ALI cultures and incubated in the presence of inhibitors. Cell viability after inhibitor treatment was determined using CellTiter-Glo and a GloMAX luminometer (a) (n = 3). Five HCoV-OC43 isolates [b) VR-1558, c) SGH-36/2014, d) SGH-61/2014, e) SGH-06/2015, and f) SGH-65/2016] and two HCoV-HKU1 isolates [g), SGH-15/2014 and h) SGH-18/2016] were examined. After incubation for 4 h, the cells were washed twice and incubated at 34 °C. After 1 day of incubation, the cells were collected and viral entry was calculated by quantifying subgenomic RNA containing nucleocapsid protein via real-time RT-PCR. DMSO was used as an inhibitor control. Data are shown as log copies RNA per well. (n = 3).

Fig. 3

Entry inhibition assay in HCT-8 cells. a) TMRPSS2 expression in HBTE-ALI and HCT-8 cells. Total RNAs were extracted by TRIzol reagent. The mRNA expression of TMPRSS2 was determined by real-time PCR; the data are presented as relative values to GAPDH expression (n = 4). A virus entry inhibition assay was performed using HCT-8 cells with b) VR-1558 and c) SGH-36/2014 according to the legend for Fig. 2. The inhibitor concentrations used were 10 or 100 μM. All data are shown as relative virus entry (%) as compared with that in the control case (DMSO treatment) (n = 4).

Fig. 4

Alignment of the amino acid sequence of the S protein of HCoV-OC43. The alignment was constructed using GeneDoc software. The amino acid sequence from 655 to 1029 of VR-1558 is shown. The accession numbers of the sequences are as follows: VR-1558, NC_005147; SGH-36/2014, LC315646; SGH-61/2014, LC315647; SGH-06/2015, LC315648; and SGH65-2016, LC315649.