1. Major HBV splice variant encoding a novel protein important for infection.
- Author
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Chung CY, Sun CP, Tao MH, Wu HL, Wang SH, Yeh SH, Zheng QB, Yuan Q, Xia NS, Ogawa K, Nakashima K, Suzuki T, and Chen PJ
- Subjects
- Humans, Animals, Mice, Hep G2 Cells, RNA Splicing, Mutation, RNA, Viral genetics, RNA, Viral metabolism, Cryoelectron Microscopy, Hepatitis B virus genetics, Hepatitis B, Chronic virology
- Abstract
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, HBcSP1 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 (HBcSP1 ) that lacks the C-terminal cysteine from conventional HBc (HBc-Cys ). HBcSP1 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 HBcSP1 . We confirmed and validated the identity and function of HBcSP1 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.)- Published
- 2024
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