FIG. 4.

Inhibition of HCoV-HKU1 S-pseudotyped virus entry into A549 cells by knockdown of HLA-C expression by siRNAs or by anti-HLA-C antibody. (A) A549 cells were transfected with siRNA1 (lane 1), siRNA2 (lane 2), and siRNA negative control (lane 3) and harvested for RT-PCR after 24 h. (B and C) A549 cells treated by stealth RNAi-1 (lane 1), stealth RNAi-2 against HLA-C (lane 2), and RNAi negative control (lane 3) for 24 h were fixed and surface stained by goat anti-human HLA-C (200 μg/ml; Santa Cruz) (immunofluorescence [IF], 1:100 and 1:150 for flow cytometry) and rabbit anti-goat IgG (H+L) FITC (IF, 1:500 and 1:100 for flow cytometry) and analyzed by immunofluorescent microscopy (Eclipse 80i Nikon) (B) and flow cytometry (FACSCalibur; Becton Dickinson) (C). (D and E) Effects on entry of CoV-HKU1 S pseudotyped virus. The nucleus was stained by DAPI in blue. A549 cells were either transfected with two individual duplexes of siRNAs for 24 h (D) or preincubated with polyclonal HLA-C antibodies at various concentrations as indicated for 1 h at 37°C (E). A total of 40 ng of CoV-HKU1 pseudotyped virus was inoculated onto 1 × 10⁵ treated or untreated A549 cells in 24-well plates and incubated for 1 h at 37°C after three washes and being replenished with fresh medium. Percentage of infection was indicated by GFP expression and determined by flow cytometry. VSV-G pseudotypes were included as positive control.