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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 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% 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-Cys 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 (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. [Display omitted] • The infectivity of splicing-deficient HBV was significantly decreased compared to WT HBV in hu-FRG mice. • SP1 RNA encodes HBcSP1, which is found in 10 to 50% of nucleocapsids in patients with CHB and cell line-derived WT HBV. • The splicing-deficient mutant A487C, lacking HBcSP1, reduces the efficiency of viral entry kinetics. • Disulfide bonds between Cys48 and Cys183 in the A487C capsid led to a locked conformation that disfavored viral entry. [ABSTRACT FROM AUTHOR]

Background & Aims Absence or low anti-HBV surface antibody (anti-HBs) is associated with an increased risk of HBV reactivation in patients with lymphoma and resolved HBV infection receiving rituximab-containing chemotherapy. Quantification of anti-HBV core antibody (anti-HBc) is a new marker associated with the natural history and treatment response of chronic HBV infection. This study investigated whether baseline anti-HBc and anti-HBs levels may better predict HBV reactivation. Methods We prospectively measured the HBV DNA levels of patients with lymphoma and resolved HBV infection receiving rituximab–cyclophosphamide, hydroxydaunorubicin, vincristine, and prednisolone-based chemotherapy and started an antiviral therapy upon HBV reactivation, defined as a greater than 10-fold increase in HBV DNA compared with previous nadir levels. Anti-HBs and anti-HBc were quantified by a double-sandwich assay. Receiver-operating-characteristic-curve analysis was used to determine the optimal baseline anti-HBc/anti-HBs levels for predicting HBV reactivation. Results HBV reactivation occurred in 24 of the 197 patients enrolled, with an incidence of 11.6/100 person-years. For the 192 patients with enough serum samples for analysis, low anti-HBs (<56.48 mIU/ml) and high anti-HBc (≥6.41 IU/ml) at baseline were significantly associated with high risk of HBV reactivation (hazard ratio [HR] 8.48 and 4.52, respectively; p  <0.01). The multivariate analysis indicated that (1) patients with both high anti-HBc and low anti-HBs at baseline (36 of 192 patients) had an HR of 17.29 for HBV reactivation (95% CI 3.92–76.30; p  <0.001), and (2) HBV reactivation may be associated with inferior overall survival (HR 2.41; 95% CI 1.15–5.05; p  = 0.02). Conclusions Baseline anti-HBc/anti-HBs levels may predict HBV reactivation in these patients with lymphoma and help optimize prophylactic antiviral therapy for high-risk patients. Lay summary In this study, we identified a subgroup of patients with lymphoma and resolved hepatitis B virus infection that had a high risk of hepatitis B virus reactivation after receiving rituximab-containing chemotherapy. These findings will help optimize a preventive strategy, especially in hepatitis B virus endemic regions with limited healthcare resources. Clinical trial number: NCT 00931229. [ABSTRACT FROM AUTHOR]