FIG 6

CEACAM5 recombinant protein and siRNA knockdown of CEACAM5 inhibit MERS-CoV entry. (A and B) MERS-CoV at an MOI of 0.1 was preincubated with human recombinant proteins at the indicated concentrations for 1 h at 37°C. After the preincubation, the protein-virus mixture was added to Calu3 cells (A) or Huh7 cells (B) for 2 h at 37°C. The cell lysates were subsequently harvested for qPCR analysis. Human recombinant DPP4 and human recombinant IgG were included as positive and negative controls, respectively. Results from the human recombinant DPP4-, human recombinant CEACAM5-, and control human recombinant IgG-treated samples were compared with those of the mock-treated samples. (C) Huh7 cells were treated with CEACAM5 protein for a total of 3 h during preincubation and virus inoculation (−1 to 2) or after virus inoculation (2 to 5). MERS-CoV entry was assessed by qPCR. (D) Huh7 cells were treated with 100 nM gene-specific or scrambled siRNA for two consecutive days. (E and F) Summary of the reduction of surface CEACAM5 and DPP4 expression. (G) siRNA-treated Huh7 cells were subjected to MERS-CoV infection at an MOI of 1 for 2 h at 37°C. The cell lysates were subsequently harvested for qPCR analysis, and the result was normalized to those of the mock-treated cells. The results from the DPP4 siRNA-, CEACAM5 siRNA-, and scrambled-siRNA-treated samples were compared with those of the mock-treated samples. The data are represented as means and standard deviations of the results of three independent experiments. Statistical analyses were carried out using Student's t test. Statistical significance is indicated by the asterisks (P < 0.05).