Naturally occurring mutations in the HBV core N-terminal domain reduce viral replication and HBeAg levels

The N-terminal domain (NTD) of hepatitis B virus (HBV) core region (C region) plays crucial role in its life cycle, while the functional implications of related mutations remain unclear. This study aimed to characterize the emergence of mutations in the HBV C region and investigate the impact of high-frequency C region mutations on HBV life cycle. Three hundred twenty-six HBV samples were collected from 32 blood establishments across 14 provinces in China and subsequently subjected to comprehensive serological analysis, mutational analysis, and assessment of major histocompatibility complex (MHC)-restricted site distribution in C region. The impact of high-frequency C region mutations on HBV replication and expression was further investigated through in vitro experiments. Serological and mutational analysis in genotype B and C revealed a significant association of C region mutations and reduced serum HBV DNA levels and HBeAg negativity. These mutations may be tentatively speculated to be associated with HBeAg seroconversion. We further identified 31 mutations at 29 high-frequency sites in the NTD of C region of genotype B. Notably, mutations V13A, S21T, I27V, S35A, L55I, S87G, I97L, I105L, and P135Q may impair the transcription of HBV DNA into pgRNA. Mutations P5T, E14Q, and S26N may disrupt HBc expression, while S49T and P50A may hinder capsid assembly. Furthermore, C region mutations exhibited differential regulatory effects on HBc and HBeAg expression. This study, leveraging treatment-naïve HBV samples, elucidates and validates the impact of C region mutations on HBV replication and expression, providing a robust foundation for further research.