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. 2020 Jan 17;9(1):33. doi: 10.3390/antibiotics9010033

Figure 1.

Figure 1

BmKDfsin3 inhibits HCV replication in vitro under noncytotoxic concentrations. (A) Amino acid sequence of BmKDfsin3. There are six cysteine residues forming three pairs of disulfide bonds. Cysteine residues are labeled with yellow, basic residues with blue, and acidic residues with red. (B,C) Concentration-dependent inhibition of BmKDfsin3 on HCV infection in Huh7.5.1 cells. Huh7.5.1 cells were preincubated with the BmKDfsin3 at different concentrations for 1 h and then infected with HCV J399EM at an multiplicity of infection (MOI) of 0.1. After 72 h, cells were collected, and intracellular HCV core protein levels were analyzed by western blotting (B) and intracellular HCV RNA was analyzed by qPCR (C). The J399EM was derived from the JFH-1 virus (HCV genotype 2a strain) by insertion of eGFP into the HCV NS5A region. (D) Inhibition of BmKDfsin3 on extracellular virus particles of Huh7.5.1 cells. Huh 7.5.1 cells treated with or without BmKDfsin3 (5 μM) were infected by J399EM for 72 h, and then supernatant was collected and used to incubate naïve Huh7.5.1 cells for 72 h. Virus in Huh 7.5.1 cells were observed by immunofluorescence microscope. HCV, green. 4’,6-diamidino-2-phenylindole (DAPI), blue. Scale bar, 100 μm. (E) The statistics of the fluorescence ratio of extracellular virus particles as described in (D). (F) Cytotoxicity of BmKDfsin3 to Huh7.5.1 cells by the MTT assay. BmKDfsin3 was dissolved in the medium and the medium without BmKDfsin3 was used as a negative control in all experiments. The internal control of subfigure (C) was glyceraldehyde-phosphate dehydrogenase (GAPDH). ***, p < 0.001. Data represented the mean ± standard deviation (SD) of at least three independent experiments.