A synonymous mutation in the preS2 region enhances production of infectious hepatitis B virus

Background: Hepatitis B virus (HBV) is classified into at least nine genotypes based on sequence heterogeneity. Clinical and virological characteristics vary among these genotypes, and differences have also been reported among strains within the same genotype. In this study, we aimed to clarify the strain-specific characteristics of patient-derived genotype C (GT-C) strains and identify a synonymous mutation responsible for these characteristics, along with the underlying mechanisms. Materials and methods: HBV molecular clones were constructed from sequences obtained from two chronic hepatitis patients infected with GT-C. To evaluate HBsAg production and infectivity, these molecular clones were transfected into cell cultures, and the characteristics of the generated viruses were assessed. The HBV reporter virus was used to confirm these characteristics and determine the responsible regions. mRNA quantification and mRNA transfection experiments were performed to elucidate the mechanisms underlying high HBsAg production and enhanced infectivity. Results: HBsAg production and infectivity were analyzed in two GT-C strains, GT-C1 and GT-C2. GT-C2 exhibited higher HBsAg production than GT-C1 did, whereas GT-C1 showed greater infectivity. Analysis of chimeric and mutated strains revealed that a synonymous mutation, a3210g, in the preS2 region was responsible for the high HBsAg production of GT-C2. Introducing this mutation into the GT-C1 strain led to increased HBsAg production due to increased HBsAg translation efficiency and further enhanced infectivity. Conclusions: This HBV infection system with both high HBsAg production and high infectivity provides a valuable tool for studying HBV infection and propagation in cell culture and for developing antiviral strategies for HBV infection.