Fig. 2: HBsAg enhances TBK1 dimerization but inhibits IRF3 activation.

A Immunoblot analysis of phosphorylated (p-)TBK1, IRF3, and p62 in THP-1 cells transfected with a vector or Flag-HBs for 24 h and then stimulated with LPS, poly (I:C), or HT-DNA. B Immunoblot analysis of (p-) TBK1, IRF3, and p62 in THP-1 cells infected with SeV (MOI of 1) for the indicated times. C IP and immunoblot analysis of endogenous TBK1 and IRF3 in HepG2-hNTCP cells infected with HBV. D Huh7 cells were transfected with increasing amounts of a plasmid encoding Flag-HBs for 24 h and then infected with SeV (MOI of 1) for 8 h. IP and immunoblot analysis of endogenous TBK1 and IRF3. E Huh7 cells were transfected with increasing amounts of a plasmid encoding Flag-HBs for 36 h, followed by immunoblot analysis of TBK1 dimerization with native PAGE. F Huh7 cells were transfected with increasing amounts of a plasmid encoding Flag-HBs and then infected with SeV. Immunoblot analysis of IRF3 dimerization with native PAGE. G Huh7 cells were transfected with increasing amounts of a plasmid encoding Flag-HBs and IRF3-5D for 36 h. Immunoblot analysis of IRF3 dimerization with native PAGE. H Huh7 cells were transfected with increasing amounts of a plasmid encoding Flag-HBs and then infected with SeV, and immunoblot analysis of IRF3 in nuclear (Nucle) and cytoplasmic (Cyto) fractions. GAPDH served as a cytoplasmic control. LaminA/C served as a nuclear protein control. I Immunoblot analysis of TBK1 in WT and TBK1 knockout Huh7 cells. J Schematic representation of TBK1 structural domains and critical amino acid sites. KTBK1 knockout Huh7 cells transfected with plasmids expressing HA-TBK1 (WT), GPF-TBK1 (WT), or its mutants (GFP-TBK1^EE, TBK1^HIF, TBK1^EE+HIF, TBK1^S172A, TBK1^K30R+K401R), and along with or without Flag-HBs. IP analysis of TBK1 dimerization. All experiments were repeated at least three times with consistent results.