Construction of Full-Length HBV Plasmids and Site-Directed Mutagenesis

Plasmid DNA containing a full genome copy of the HBV genome was constructed in-house. Briefly, HBV full genome was amplified from phenol-chloroform DNA isolated from a high viral load clinical sample (>5 × 10⁸ HBV copies/mL; genotype C) with high-fidelity Phusion DNA polymerase (New England Biolabs, Whitby, Canada). HBV FG P1 and P2 primers for amplification (Supplementary Table S1) were derived from Günther et al. (1995). PCR amplicons were purified from a 0.9% agarose gel followed by digestion with HindIII (New England Biolabs) and ligated with T4 DNA ligase into the cloning vector pUC19 which was similarly prepared. Ligations were transformed into TOP10 E. coli chemically competent cells (Invitrogen, Carlsbad, United States) and successful clones were identified and confirmed via PCR and sequencing, respectively, with standard M13 primers thus generating the pUC19-HBV.wt plasmid (Supplementary Table S1).

Generation of G1896A and A1762T/G1764A mutants were achieved using the QuikChange II site-directed mutagenesis kit (Agilent Technologies, Santa Clara, United States), as per manufacturer’s protocol using the pUC19-HBV.wt and mutagenic primers (Supplementary Table S1). Purified plasmids (pUC19-HBV.g1896a and pUC19-HBV.dmut) were isolated from selected clones and subsequently sequenced in-house to confirm the mutagenesis.