HBV infection promotes HBP and increases protein O-GlcNAcylation. (A) Principal component analysis of metabolite profiles obtained using a metabolomics assay in HepG2 cells infected with AdHBV1.3 or AdGFP for 72 h. (B) Heatmap of differentially expressed metabolites subjected to identical treatment conditions as in (A). (C) An overview of the hexosamine biosynthesis pathway (HBP). (D) Relative changes in intermediate metabolites of HBP. (E-F) Relative changes in the levels of UDP-GlcNAc (E) and glucose (F) in HBV-infected HepG2-NTCP cells and HepAD38 cells with tetracycline inducible (Tet-off) HBV expression was determined using the LC-MS/MS targeted metabolomics assay. (G) Immunoblot of total O-GlcNAc from HepG2-NTCP and HepAD38 cells treated for the indicated periods. (H-I) qPCR quantification, n = 3 (H) and immunofluorescence staining (I) of GLUT1 in HepG2-NTCP and HepAD38 cells, DAPI (blue) was used to counterstain nuclei, Scale bar, 10 μm. Data are expressed as the mean ± SD. P values were derived from unpaired, two-tailed Student's t-test in E, F, and H (*P < 0.05,**P < 0.01).