Fig. 3.

Characterization of the mechanism of CHPV anti-EBOV action. (A) A time-of-addition experiment was performed with EBOV-GP-V to identify the action target for CHPV. CHPV (10 μg/mL) was added at 6 h and 2 h prior to infection (washed away immediately before adding virus), during infection (0 h), and at 2 h, 4 h, 8 h, 12 h and 24 h post-infection. The mock-infected controls mean uninfected HEK293T cells, and the positive controls (PC) were HEK293T cells were infected with pseudovirions in the absence of CHPV. At 2 h post-infection, all of the cells were washed and cultured in complete DMEM. The Gluc activity was tested from the supernatant after a 48 h incubation, and the data are shown as a percentage of control activity (%), a ratio of Gluc activity in the presence of CHPV versus the absence of CHPV. (B, C) Schematic diagram of the evaluation of infection of EBOV–GP-V-bound CHPV. EBOV-GP-V was incubated for 10 min with CHPV, MAb or DMEM at 4 °C. The treated viruses were further concentrated by ultracentrifugation (100,000 g). The infectivity of the pelleted pseudovirions was tested by adding to HEK293T cells, and the Gluc activity after 48 h was shown as a relative ratio when compared with the virus incubated with DMEM. PC: EBOV-GP-V not treated with drug. (D) The same amount of CHPV was ultracentrifuged. The resulting supernatant and the pellet were incubated with EBOV-GP-V and added to HEK293T cells. The Gluc activity after 48 h is shown as a percentage of control activity (%). The results are the mean values ± standard deviations (SD) of three independent experiments. (E) CHPV blocks the attachment of EBOV-GP-V. The same amount of EBOV-GP-Vs (P24, 0.51 ng) and various concentration of CHPV were added to HEK293T cells on ice for 1 h. The sample without pseudovirions or CHPV treatment was used as a negative control. The cells were then washed with cold PBS to remove unbound virus and lysed by RIPA buffer. The viruses attached to cells were monitored by measuring the HIV p24 antigen levels by anti-p24 ELISA.