Critical Role of the 36-Nucleotide Insertion in Hepatitis B Virus Genotype G in Core Protein Expression, Genome Replication, and Virion Secretion

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Abstract
Frequent coinfection of hepatitis B virus genotype G with genotype A suggests that genotype G may require genotype A for replication or transmission. In this regard, genotype G is unique in having a 12-amino-acid extension in the core protein due to a 36-nucleotide insertion near the core gene translation initiation codon. The insertion alters base pairing in the lower stem of the pregenome encapsidation signal, which harbors the core gene initiator, and thus has the potential to affect both core protein translation and pregenomic RNA encapsidation. Genotype G is also unusual for possessing two nonsense mutations in the precore region, which together with the core gene encode a secreted nonstructural protein called hepatitis B e antigen (HBeAg). We found that genotype G clones were indeed incapable of HBeAg expression but were competent in RNA transcription, genome replication, and virion secretion. Interestingly, the 36-nucleotide insertion markedly increased the level of core protein, which was achieved at the level of protein translation but did not involve alteration in the mRNA level. Consequently, the variant core protein was readily detectable in patient blood. The 12-amino-acid insertion also enhanced the genome maturity of secreted virus particles, possibly through less efficient envelopment of core particles. Cotransfection of genotypes G and A did not lead to mutual interference of genome replication or virion secretion. Considering that HBeAg is an immunotolerogen required for the establishment of persistent infection, its lack of expression rather than a replication defect could be the primary determinant for the rare occurrence of genotype G monoinfection.