Major HBV splice variant encoding a novel protein important for infection

Background & Aims HBV expresses about 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 Chronic hepatitis B patient serum, hu-FRG mice, HepG2-NTCP cells used for HBV infection; SHifter assays and cryo-EM. Results We found the infectivity of splicing-deficient HBV in hu-FRG mice was decreased 100-1000 folds compared with that of the wild-type. Another mutant, A487C, which loses the most abundant spliced RNA (SP1), was also severely impaired in infectivity. SP1 hypothetically encodes a novel protein HBcSP1 (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% versus conventional HBc, as well as in the serum of chronic hepatitis B patients, but not in A487C. When infection was conducted with a shorter incubation time of 4-8 hours at lower PEG concentrations in HepG2-NTCP cells, the entry of the A487C mutant was significantly slowed. SP1 cDNA complementation of the A487C mutant succeeded in rescuing its infectivity in the hu-FRG mice and HepG2-NTCP cells. Moreover, cryo-EM revealed a disulfide bond between HBc cysteine 183 and 48 in the HBc intradimer of A487C capsid, leading to a locked conformation that disfavored viral entry in contrast to capsid of the wild-type. 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.