Fig. 3: Viperin mediates HBc localization on GAL9-positive puncta.

a Viperin interacts with HBc and GAL9. NanoBRET analysis was performed on HepG2 cells expressing indicated HT proteins and NL-conjugated HBc (n = 3 experiments, mean ± SD). ****P < 0.0001, two-tailed unpaired t-test. b GAL9 can bind HBc in the presence of viperin. Recombinant HBc and GAL9 were incubated with or without viperin and subjected to in vitro pull-down assay using anti-HBc antibody. c Viperin colocalization with GAL9 and HBc. Confocal microscopic imaging of HepG2 cells expressing GFP-GAL9, HT-viperin and HA-HBc. Nuclei were stained with DAPI. Scale bar, 10 µm. Another view of the cell image is shown in Supplementary Fig. 4b. d, e Viperin depletion decreases the interaction between HBc and GAL9. HepG2 transduced with control- (Ctrl) or viperin-targeting siRNA were transfected with vectors encoding HA-HBc and GFP-GAL9. Cell lysates were precipitated with anti-HA antibody, followed by immunoblotting (d). Viperin knockdown was confirmed by RT-PCR (e). The mean ± SD of two independent determinations is plotted. **P = 0.0059, two-tailed unpaired t-test. f GAL9 and HBc bind to different domains of viperin. Immunoprecipitation assays of HepG2 cells expressing truncated viperin mutants and HA-HBc or GFP-GAL9. Cell lysates were precipitated with anti-HA or anti-GFP antibodies, followed by immunoblotting. g Viperin depletion attenuates HBc localization on GAL9-positive puncta. HepG2 transduced with control- (Ctrl) or viperin-targeting siRNA were transfected with vectors encoding HA-HBc and GFP-GAL9. Nuclei were stained with DAPI. The graph on the right is the percentage of HBc-puncta-forming cells (n = 50 cells examined over two independent experiments, mean ± SD). *P = 0.0313, two-tailed unpaired t-test. Scale bar, 10 µm. Another view of the cell image is shown in Supplementary Fig. 4e. h HepG2.15.7 cells transduced with control- (Ctrl) or viperin-targeting siRNA were treated with IFN-β (1000 U/mL) for 24 h. Cells were stained with anti-GAL9 and anti-HBc antibodies (n = 50 cells examined over two independent experiments, mean ± SD). **P = 0.0063, two-tailed unpaired t-test. Scale bar, 10 µm. Another view of the cell image is shown in Supplementary Fig. 4g. Immunoblots and micrographs are representative of experiments with similar results (n ≥ 2). Source data are provided as a Source Data file.