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Background & Aims: Persons with chronic HBV infection coinfected with HIV experience accelerated progression of liver fibrosis compared to those with HBV monoinfection. We aimed to determine whether HIV and its proteins promote HBV-induced liver fibrosis in HIV/HBV-coinfected cell culture models through HIF-1α and TGF-β1 signaling., Methods: The HBV-positive supernatant, purified HBV viral particles, HIV-positive supernatant, or HIV viral particles were directly incubated with cell lines or primary hepatocytes, hepatic stellate cells, and macrophages in mono or 3D spheroid coculture models. Cells were incubated with recombinant cytokines and HIV proteins including gp120. HBV sub-genomic constructs were transfected into NTCP-HepG2 cells. We also evaluated the effects of inhibitor of HIF-1α and HIV gp120 in a HBV carrier mouse model that was generated via hydrodynamic injection of the pAAV/HBV1.2 plasmid into the tail vein of wild-type C57BL/6 mice., Results: We found that HIV and HIV gp120, through engagement with CCR5 and CXCR4 coreceptors, activate AKT and ERK signaling and subsequently upregulate hypoxia-inducible factor-1α (HIF-1α) to increase HBV-induced transforming growth factor-β1 (TGF-β1) and profibrogenic gene expression in hepatocytes and hepatic stellate cells. HIV gp120 exacerbates HBV X protein-mediated HIF-1α expression and liver fibrogenesis, which can be alleviated by inhibiting HIF-1α. Conversely, TGF-β1 upregulates HIF-1α expression and HBV-induced liver fibrogenesis through the SMAD signaling pathway. HIF-1α small-interfering RNA transfection or the HIF-1α inhibitor (acriflavine) blocked HIV-, HBV-, and TGF-β1-induced fibrogenesis., Conclusions: Our findings suggest that HIV coinfection exacerbates HBV-induced liver fibrogenesis through enhancement of the positive feedback between HIF-1α and TGF-β1 via CCR5/CXCR4. HIF-1α represents a novel target for antifibrotic therapeutic development in HBV/HIV coinfection., Impact and Implications: HIV coinfection accelerates the progression of liver fibrosis compared to HBV monoinfection, even among patients with successful suppression of viral load, and there is no sufficient treatment for this disease process. In this study, we found that HIV viral particles and specifically HIV gp120 promote HBV-induced hepatic fibrogenesis via enhancement of the positive feedback between HIF-1α and TGF-β1, which can be ameliorated by inhibition of HIF-1α. These findings suggest that targeting the HIF-1α pathway can reduce liver fibrogenesis in patients with HIV and HBV coinfection., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Background & Aims: HBV expresses more than 10 spliced RNAs from the viral pregenomic RNA, but their functions remain elusive and controversial. To address the function of HBV spliced RNAs, we generated splicing-deficient HBV mutants and conducted experiments to assess the impact of these mutants on HBV infection., Methods: HepG2-NTCP cells, human hepatocyte chimeric FRG mice (hu-FRG mice), and serum from patients with chronic hepatitis B were used for experiments on HBV infection. Additionally, SHifter assays and cryo-electron microscopy were performed., Results: We found the infectivity of splicing-deficient HBV was decreased 100-1,000-fold compared with that of wild-type HBV in hu-FRG mice. Another mutant, A487C, which loses the most abundant spliced RNA (SP1), also exhibits severely impaired infectivity. SP1 hypothetically encodes a novel protein HBc SP1 (HBc -Cys ) that lacks the C-terminal cysteine from full-length HBc. In the SHifter assay, HBc SP1 was detected in wild-type viral particles at a ratio of about 20-100% vs. conventional HBc, as well as in the serum of patients with chronic hepatitis B, but not in A487C particles. When infection was conducted with a shorter incubation time of 4-8 h at lower PEG concentrations in HepG2-NTCP cells, the entry of the A487C mutant was significantly slower. SP1 cDNA complementation of the A487C mutant succeeded in rescuing its infectivity in hu-FRG mice and HepG2-NTCP cells. Moreover, cryo-electron microscopy revealed a disulfide bond between HBc cysteine 183 and 48 in the HBc intradimer of the A487C capsid, leading to a locked conformation that disfavored viral entry in contrast to the wild-type capsid., Conclusions: Prior studies unveiled the potential integration of the HBc -Cys protein into the HBV capsid. We confirmed the proposal and validated its identity and function during infection., Impact and Implications: HBV SP1 RNA encodes a novel HBc protein (HBc SP1 ) that lacks the C-terminal cysteine from conventional HBc (HBc -Cys ). HBc SP1 was detected in cell culture-derived HBV and confirmed in patients with chronic infection by both immunological and chemical modification assays at 10-50% of capsid. The splicing-deficient mutant HBV (A487C) impaired infectivity in human hepatocyte chimeric mice and viral entry in the HepG2-NTCP cell line. Furthermore, these deficiencies of the splicing-deficient mutant could be rescued by complementation with the SP1-encoded protein HBc SP1 . We confirmed and validated the identity and function of HBc SP1 during infection, building on the current model of HBV particles., (Copyright © 2024 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)