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Major HBV splice variant encoding a novel protein important for infection.
- Source :
-
Journal of hepatology [J Hepatol] 2024 Jun; Vol. 80 (6), pp. 858-867. Date of Electronic Publication: 2024 Feb 07. - Publication Year :
- 2024
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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.<br />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.<br />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 <superscript>SP1</superscript> (HBc <superscript>-Cys</superscript> ) that lacks the C-terminal cysteine from full-length HBc. In the SHifter assay, HBc <superscript>SP1</superscript> 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.<br />Conclusions: Prior studies unveiled the potential integration of the HBc <superscript>-Cys</superscript> protein into the HBV capsid. We confirmed the proposal and validated its identity and function during infection.<br />Impact and Implications: HBV SP1 RNA encodes a novel HBc protein (HBc <superscript>SP1</superscript> ) that lacks the C-terminal cysteine from conventional HBc (HBc <superscript>-Cys</superscript> ). HBc <superscript>SP1</superscript> 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 <superscript>SP1</superscript> . We confirmed and validated the identity and function of HBc <superscript>SP1</superscript> during infection, building on the current model of HBV particles.<br /> (Copyright © 2024 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)
Details
- Language :
- English
- ISSN :
- 1600-0641
- Volume :
- 80
- Issue :
- 6
- Database :
- MEDLINE
- Journal :
- Journal of hepatology
- Publication Type :
- Academic Journal
- Accession number :
- 38336347
- Full Text :
- https://doi.org/10.1016/j.jhep.2024.01.037