Major HBV splice variant encoding a novel protein important for infection.
Journal
Journal of hepatology
Journal Volume
80
Journal Issue
6
Start Page
858
End Page
867
ISSN
1600-0641
Date Issued
2024-06
Author(s)
Chung, Chen-Yen
Sun, Cheng-Pu
Tao, Mi-Hua
Wu, Hui-Lin
Wang, Sheng-Han
Zheng, Qing-Bing
Yuan, Quan
Xia, Ning-Shao
Ogawa, Kenji
Nakashima, Kenji
Suzuki, Tetsuro
Abstract
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.
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.
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 (HBc) that lacks the C-terminal cysteine from full-length HBc. In the SHifter assay, HBc 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.
Prior studies unveiled the potential integration of the HBc protein into the HBV capsid. We confirmed the proposal and validated its identity and function during infection.
HBV SP1 RNA encodes a novel HBc protein (HBc) that lacks the C-terminal cysteine from conventional HBc (HBc). HBc 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. We confirmed and validated the identity and function of HBc during infection, building on the current model of HBV particles.
Subjects
HBV
HBV in vitro infection
HBV in vivo infection
HBV spliced RNA
HBc(-Cys)
HBc(SP1)
HepG2-NTCP cells
disulfide bond
hu-FRG mice
SDGs
Type
journal article