Dual defects in HBsAg antigenicity and secretion caused by the C138R mutation in the S gene trigger occult hepatitis B virus infection.

BACKGROUND: Occult hepatitis B virus infection (OBI) carries high risk of transmissibility and pathogenicity. Mutations in the S gene are considered major contributors to OBI development. Our previous study identified the C138R mutation in the S gene of OBI patients. This study aimed to investigate the impact of the C138R mutation on HBsAg function and its mechanisms leading to OBI. METHODS: Full-length hepatitis B virus (HBV) plasmids (HBV 1.3, genotype B) and mutant plasmids (HBV C138R) were constructed, along with HA-tagged wild-type (sWT) and mutant (sC138R) S protein expression plasmids. The effects of the C138R mutation on HBsAg antigenicity, secretion, and dimer formation were evaluated through in vitro cell transfection and in vivo hydrodynamic tail vein injection in mice. RESULTS: The C138R mutation markedly reduced intra- and extracellular HBsAg levels without affecting HBV transcription, replication, or other viral protein expression. HA-tag assays indicated that this reduction resulted from impaired antigenicity. Several commercial ELISA kits results and HA levels in cell supernatants demonstrated that the C138R mutation also impaired HBsAg secretion. Similarly, the C107R mutation significantly decreased both HBsAg antigenicity and secretion. Non-reducing WB assays and Alphafold 3-based structural predictions revealed that the C138R mutation lead to the formation of aberrant HBsAg dimers. These findings were confirmed in the HBV mouse model. CONCLUSION: The C138R mutation contributed to OBI by simultaneously impairing HBsAg antigenicity and secretion. Disruption of the disulfide bond between aa107 and aa138, resulting in the formation of structurally abnormal dimers, represented the primary mechanism underlying HBsAg dysfunction caused by this mutation.