Figure 5.

P38α directly interacts with the N-terminus of the HCV core protein. (A) The interaction of endogenous phosphorylated p38 with the HCV core protein in Huh7.5.1 cells. Huh7.5.1 cells were infected with JFH1, and cell lysates were prepared for co-IP followed by western blotting. IgG antibody was used for IP of cellular lysates with HCV infection as the control group. P-p38 antibody was used for IP of cellular lysates with or without HCV infection as the experimental group. (B) The interaction of exogenous p38α with the HCV core protein in HEK293T cells. HEK293T cells were cotransfected with the Flag-HCV core protein expression plasmid and the p38α expression plasmid and cell lysates were prepared for co-IP followed by western blotting. (C) GST pulldown analysis of the direct interaction of p38α and the HCV core proteins. The GST-HCV core and His-p38α proteins were expressed in E. coli and purified. The purified proteins were incubated with Ni-NTA beads and the bound proteins were identified by immunoblotting with anti-His and anti-GST antibodies. (D) Colocalization of the p38α and HCV core proteins in HCV-infected Huh7.5.1 cells. Cells were fixed in 4% paraformaldehyde, and immunofluorescence staining was performed using an anti-P-p38 antibody (red) and anti-HCV core antibody (green). Cells were counterstained with DAPI to label nuclei (blue). Scale bar, 10 µm. (E) Schematic illustration of the full-length HCV core protein (WT) and the N-terminal or C-terminal truncated core protein constructs. WT: 1-191; N1: 1-152; N2: 1-123; N3: 1-111; C1: 24-191; C2: 38-191; C3: 58-191. (F) The interaction of p38α with the N- or C-terminal region of the HCV core protein. HEK293T cells were cotransfected with the truncated Flag-core protein expression plasmid and HA-p38α expression plasmid. Cell lysates were prepared for co-IP followed by western blotting of HA and Flag.