Fig 2: HCV infection activates p38a by triggering the TAB1-p38a interaction. (A) Activation effect of HCV infection on p38 in Huh7.5.1 cells. Huh7.5.1 cells were infected with the HCV strain JFH1 at MOIs of 0, 0.1, 1 and 10 for 72 h, and then the total intracellular p38, P-p38 and HCV core protein levels were detected by western blotting. (B) MKK3/6-independent activation of p38 in HCV-infected Huh7.5.1 cells. Huh7.5.1 cells were infected (+) or uninfected (-) with the HCV JFH1 strain. P-p38 and P-MKK3/6 were detected by western blotting. (C) TAB1-p38 interaction in HCV-infected Huh7.5.1 cells. Huh7.5.1 cells were infected (+) or uninfected (-) with JFH1, and the cell lysates were collected for IP with control antibody (IgG) or TAB1 antibody followed by western blotting analysis of proteins as indicated. (D) Interaction between TAB1 and p38a. Cell lysates from HEK293T cells overexpressing Flag-p38a/ß/?/d, and EGFP-TAB1 were subjected to co-IP with anti-Flag antibody, followed by immunoblotting with anti-EGFP and anti-Flag antibodies. (E-G) Effect of TAB1 knockdown on HCV infection-induced p38 activation in Huh7.5.1 cells. TAB1 was knocked down by CRISPR/Cas9 (KD-TAB1) in Huh7.5.1 cells. WT and KD-TAB1 cells were infected with JFH1. P-p38, p38, TAB1 and GAPDH were analyzed by western blotting (E), the fold change of the TAB1 level relative to the GAPDH level was quantified by ImageJ software (F), and the fold change in the phosphorylated p38 level relative to the total p38 level was quantified by ImageJ software (G).

Fig 3: TAB1-mediated p38a activation facilitates for HCV replication. (A-C) Inhibitory effect of p38a knockdown on HCV replication in Huh7.5.1 cells. Huh7.5.1 cells were transfected with control siRNA (NC) or siRNA specific for p38 (p38-RNAi #1 and #2) and infected with the HCV JFH1 strain. The expression levels of p38, P-p38, and the HCV core protein were determined by western blotting (A), and the intracellular (B) and extracellular (C) HCV RNA levels were determined by qRT-PCR. (D-F) Promotion effect of p38a overexpression on HCV replication in Huh7.5.1 cells. Experiments were performed as described above, except the cells were transfected with the control or p38a expression plasmid. (G-I) Effect of p38a on the rescue of HCV replication in p38a-knockdown Huh7.5.1 cells. P38a was knocked down (KD) using CRISPR/Cas9 and p38a was reintroduced into p38a-knockdown cells (KD+p38a). The HCV core protein, p38 and GAPDH were measured by western blotting (G). The intracellular (H) and extracellular (I) HCV RNA levels were measured by qRT-PCR. (J) Abolishing effect of p38a activation site mutation on the promotion of p38a to HCV replication. Flag-p38a and Flag-p38aAF plasmids were expressed in p38a-knockdown Huh7.5.1 cells, and HCV infection was detected. (K) Impaired effect of TAB1 knockdown on HCV replication in Huh7.5.1 cells. WT and KD-TAB1 cells were infected with JFH1, and the HCV core protein was detected by western blotting.

Fig 4: A model of the novel feedforward mechanism boosting HCV infection. HCV infection induces p38a autophosphorylation by triggering the interaction of TAB1 and p38a. Activated p38a in turn catalyzes the phosphorylation of the HCV core protein, thereby facilitating core oligomerization and viral assembly.

Fig 5: Gal-3BP inhibits TAK1-dependent NF-kB activation. (A) HEK293T cells were transfected with NF-?B-luciferase reporter plasmid, along with a vector control, LGALS3BP expression plasmid, or TLR4 expression plasmid. After stimulation with LPS, IL-1ß, or TNF-a for 24 h, NF-?B-dependent luciferase activity was analyzed. (B) HEK293T cells were transfected with a vector control or LGALS3BP expression plasmid and then stimulated with LPS for 3 h. The mRNA expression levels of IL6, TNFA, and IL1B were measured by RT-qPCR. (C) HEK293T cells were transfected with NF-?B-luciferase reporter, TRAF6, TAK1, TAB1, TAB2, and TAB3 vectors, along with a vector control or LGALS3BP expression plasmid. NF-?B-dependent luciferase activity was analyzed at 48 h post-transfection. (D) HEK293T cells were transfected with NF-?B-luciferase reporter, TRAF6, TAB1, TAB2, and TAB3 vectors, along with a vector control or LGALS3BP expression plasmid, and then co-transfected with TAK1. NF-?B-dependent luciferase activity was analyzed at 48 h post-transfection. The results of luciferase assays are expressed as relative luciferase activity compared to each WT cells with NF-?B reporter and control vector. The expressions of the transfected plasmids were confirmed by Western blotting. Data are presented as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, and NS, not significant.