Immunofluorescence microscopy. Human fibroblasts were treated with an anti-CD13 antibody and a fluorochrome-conjugated secondary antibody on ice and incubated at 37°C for 0 min (a to c), 10 min (d to f), 30 min (g to i), or 60 min (j to l). After fixation, the cells were labeled with an anti-caveolin-1 antibody. Labeling for caveolin-1 was seen as patches in all samples (b, e, h, and k). Labeling for CD13 was observed evenly on the cell surface without warming (a); after 10 min of incubation at 37°C, most of the labeling showed a linear alignment (d); after 30 min at 37°C, CD13 showed clustering, and colocalization with caveolin-1 became apparent (g to i; arrows); and after 60 min at 37°C, CD13 and caveolin-1 were colocalized extensively (j to l; arrows). The cells were treated with the anti-TfR antibody and the secondary antibody in the same manner as that described above. Labeling for TfR was observed evenly on the cell surface without warming (m to o), and even after 60 min, TfR and caveolin-1 did not show colocalization (p to r). Bar = 50 μm.

Electron microscopy of human fibroblasts bound to HCoV-229E. (A) The cells were treated with HCoV-229E on ice and incubated at 37°C for 0 h (a and b) or 3 h (c to g) before fixation. At 0 h, most viruses bound to the flat portion of the plasma membrane (a and b). After 3 h of incubation at 37°C, viruses localized at or within 150 nm of the orifices of caveolae (c to f) were observed frequently. In contrast, clathrin-coated pits (g) were not observed near the virus particles. Cross-bridge structures of high electron density were observed between the virus particles and the plasma membrane (a, inset of b [arrows]). Caveolae are shown by arrowheads (c). Bar = 100 nm. (B) Quantification of HCoV-229E associated with caveolae. The proportion of HCoV-229E localized at or within 150 nm of the orifices of caveolae was calculated by the use of electron micrographs. The proportion increased significantly by 3 h of incubation at 37°C (29.4 versus 2.2%) (chi-square test for independence; P < 0.0001).

Effect of MβCD on intracellular entry of HCoV-229E. The data shown are from RT-PCRs of HCoV-229E RNA and G3PDH mRNA. The amount of the PCR template was normalized by using G3PDH in two different PCR cycles. The cells were incubated in the presence (+) or absence (−) of MβCD and MβCD-cholesterol (cholesterol) before being bound to HCoV-229E on ice and then incubated for 0 or 3.5 h at 37°C. Trypsinization was done to remove virus particles bound to the cell surface for an estimation of the virus amount that entered the cell. Without the virus treatment, HCoV-229E RNA was not detected (lane 1). For cells kept on ice, the amounts of HCoV-229E RNA were equivalent in all samples, but trypsinization decreased the amount greatly (lanes 2 to 4, no trypsinization; lanes 5 to 7, after trypsinization). After incubation at 37°C for 3.5 h, the intensity of the band was significantly lower for MβCD-treated cells than for control cells or cells that were treated sequentially with MβCD and MβCD-cholesterol (lanes 8 to 10).

Identification of CD13 as a major DRM fraction of human fibroblasts. (A) Total lysates and DRMs of human fibroblasts were subjected to SDS-PAGE (7 to 16% gel) and silver staining. Eighteen bands (160, 125, 107, 97, 86, 70, 58, 49, 46, 41, 38, 35, 31, 29, 28, 22, 21, and 16 kDa) were detected (arrow and arrowheads). Two peptides (peptides 1 and 2) obtained from the prominent 160-kDa band (arrow) were subjected to amino acid sequencing. In peptide 1, 8 of 10 amino acids agreed with amino acids 231 to 239 of CD13, and in peptide 2, all 12 amino acids agreed with amino acids 924 to 935 of CD13. (B) Fractions obtained by sucrose density gradient ultracentrifugation of the total cell lysate treated with Triton X-100 were subjected to immunoblotting for CD13, TfR, caveolin-1, and α-tubulin. CD13 and caveolin-1 were enriched in DRMs, but TfR and α-tubulin were detected in the bottom fraction.

Immunofluorescence microscopy of human fibroblasts bound to HCoV-229E. Optical sections obtained by laser confocal scanning microscopy were stacked. The cells were treated with HCoV-229E on ice and incubated at 37°C for 0 h (a to c and m to o), 1 h (d to f), or 3 h (g to l and p to r). After fixation, the cells were labeled with an anti-HCoV-229E or anti-CD13 antibody in combination with an anti-caveolin-1 antibody. Labeling for caveolin-1 was always seen as patches (b, e, h, k, n, and q). Labeling for HCoV-229E was seen as randomly distributed dots in cells kept on ice (a); after 1 h of incubation at 37°C, HCoV-229E showed some linear alignment, and colocalization of HCoV-229E and caveolin-1 was seen occasionally (d to f; arrows); and after 3 h at 37°C, the colocalization of HCoV-229E and caveolin-1 became prominent (g to l; arrows). The colocalization of HCoV-229E and caveolin-1 in small clusters was often observed (g to i; arrowheads). Labeling for CD13 was seen as randomly distributed dots at 0 h (m) but showed extensive colocalization with caveolin-1 after 3 h of incubation at 37°C (p to r; arrows). Bar = 50 μm.

Effect of MβCD on binding and redistribution of HCoV-229E. The cells were bound to HCoV-229E on ice after being preincubated with nothing, with MβCD alone, or sequentially with MβCD and then an MβCD-cholesterol complex (cholesterol). (A) Western blot analysis with anti-HCoV-229E antiserum. Fifty-kilodalton bands were observed in all samples of cells bound to HCoV-229E (lanes 1 to 3) but were not detected without the virus treatment (lanes 4 to 6). (B) Degree of colocalization of HCoV-229E and caveolin-1. The ratio of colocalization after 3 h of incubation at 37°C was 23.8% for the control, whereas it was significantly lower for cells treated with MβCD (6.3%). The ratio recovered to the control level when cells treated with MβCD were replenished with cholesterol by the addition of an MβCD-cholesterol complex (22.4%) (Mann-Whitney U test; P < 0.0001). The results are representative of three experiments.

Effect of caveolin-1 knockdown on binding and intracellular entry of HCoV-229E. The cells were not transfected (−) or were transfected with 80 nM control siRNA (cont) or 5 to 80 nM caveolin-1 siRNA and then were incubated at 37°C for 24 h. Forty-eight hours after subculturing, the cells were bound to HCoV-229E on ice and processed immediately or incubated at 37°C for 48 h. (A) Immunoblotting of total lysates of cells incubated on ice with anti-HCoV-229E, anti-caveolin-1, and anti-G3PDH antibodies. The amounts of the cell lysates were normalized with G3PDH. The amount of caveolin-1 decreased in a dose-dependent manner, and the expression of the HCoV-229E N protein was equivalent in all samples. (B) Immunofluorescence microscopy of cells transfected with 80 nM control siRNA (a to c) and caveolin-1 siRNA (d to f). Cells were bound to HCoV-229E on ice and incubated at 37°C for 48 h. In cells transfected with the caveolin-1 siRNA, labeling for caveolin-1 decreased (e), and the number of cells showing cytoplasmic HCoV-229E labeling was also reduced (d and f). Bar = 50 μm. (C) Proportion of cells showing cytoplasmic labeling of HCoV-229E. The proportion was significantly lower in cells transfected with 5, 20, or 80 nM caveolin-1 siRNA (52.0, 42.8, and 40.1%, respectively) than in nontransfected cells (72.2%) or cells transfected with a control siRNA (73.0%) (chi-square test for independence; P < 0.0001).