CP1 blocks viral attachment but not viral entry into Marc-145 cells. (A and B) In an attachment assay, cells were cooled at 4°C for 30 min, and then challenged with CH-1a (MOI =0.01) in the presence of various concentrations of CP1 or YPD (control) for 3 h at 4°C. Viral RNA transcription (A) and protein levels (B) were analyzed. (C, D, E, and F) In an entry assay, the kinetics of the antiviral activity of CP1 against CH-1a were evaluated with time-of-addition assays. Cells were challenged with CH-1a (MOI =0.01) for 3 h at 4°C and then incubated at 37°C in the presence of CP1 or YPD (control) for 6 h. CP1 was added at 0, 2, or 4 h (the time point at which the cells were switched to 37°C was set to 0 h). The cells were then rinsed and incubated for another 24 h at 37°C. The inhibitory effects were determined when CP1 was added at 0 (C and D), 2 (E), of 4 h (F) after the cells were shifted to 37°C. Data are the results of three independent experiments (means ± SE). Significant differences compared with the YPD-treated control group are denoted: *P <0.05, **P <0.01, or ***P <0.001.