Major HBV splice variant encoding a novel protein important for infection

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, 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.

Keywords: HBV; HBV in vitro infection; HBV in vivo infection; HBV spliced RNA; HBc(-Cys); HBc(SP1); HepG2-NTCP cells; disulfide bond; hu-FRG mice.