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. 2020 Oct 30;10(26):12223–12240. doi: 10.7150/thno.50992

Figure 7.

Figure 7

Disruption of the p38α-core interaction inhibits HCV assembly. (A) A schematic of HCV single-cycle replication and secondary infection assays. (B, C) SB203580 treatment within 24 h (HCV single-cycle) did not affect the biosynthesis of the HCV core protein (B) and RNA (C). Huh7.5.1 cells were infected with J399EM at an MOI of 1, and the cells were then treated with 10 µM SB203580 before collection as described in (A). (D, E) Inhibitory effect of SB203580 treatment on HCV assembly according to the HCV secondary infection assay. Huh7.5.1 cells were infected with J399EM at an MOI of 1 and then treated with 10 µM SB203580 before collection as described in (A). Intracellular and extracellular viral particles from the above samples (B) were collected and incubated with naïve cells for 72 h to determine the infectious viral titers. After 72 h, the cellular lysates were identified by immunoblotting (D). The infected cells were subjected to immunofluorescence staining with NS5A (green) and DAPI (blue). The immunofluorescence was examined at 200× magnification (E). (F-H) Attenuation effect of SB203580 on the p38α-core interaction. HEK293T cells were preincubated with or without 10 µM SB203580 for 1 h and then transfected with Flag-core and HA-p38α expression plasmids for 24 h. Cell lysates were subjected to co-IP analysis (F). Purified GST-core and His-p38α proteins treated with or without SB203580 (G) and CN-peptide (HCV core N-terminal peptide) (H) were incubated with Ni-NTA beads, and the bound proteins were identified by immunoblotting. (I, J) Effect of the inhibition of the CN-peptide on HCV replication in Huh7.5.1 cells. J399EM-infected Huh7.5.1 cells were treated with the CN-peptide for 72 h. The intracellular expression level of the HCV core protein was determined by western blotting (I). The extracellular infectious virus titer was measured by an end-point dilution assay (J). (K, L) Inhibitory effect of CN-peptide on HCV replication in PHHs. PHHs were infected with the HCV JFH1 strain and incubated with the CN-peptide for 72 h. The expression level of the HCV core protein was determined by western blotting (K), and the intracellular HCV RNA level was measured by qRT-PCR (L). (M, N) Inhibitory effect of SB203580 on HCV replication in PHHs. PHHs were infected with JFH1 at an MOI of 5 and then incubated with 10 µM SB203580 for 72 h. The expression level of the HCV core protein was determined by western blotting (M), and the intracellular HCV RNA level was measured by qRT-PCR (N).